Naunyn-Schmiedeberg's Arch Pharmacol (1991) 343 (Suppl.): R1-R132 002812989100017F 1 CHARACTERIZATION OF THE MOUSE-MAS ONCOGENE R. Metzger, B. Bunnemann and K. Fuxe

R1

3

PROTO-

The mas on¢ogen encodes a protein with 7 transmembrane domains. Besides Its mitogenlc activity mas expression In mammalian cell culture confers anglotensin responsiveness. Therefore. the mas protein has been discussed as a possible anglotensln receptor. To study the regulatory mechanisms Involved In the production of the mas oncogene and to reveal Its primary structure, we have cloned the mouse mas gene, From a genomlc mouse library we were able to Isolate four independent positive clones by filter hybridization to human genomlc DNA. The Identification of these clones by restriction mapping and Southern hybridization revealed 3 clones with an extended 3'-region and 1 clone with a corresponding long 5'- promotor region. One clone designated Zmas 12 was subcloned into the Bluescript vector and sequenced using the dldeoxynucleotide method. The mouse mas gene has no Introns and shares 93.6% and 84.3% homology to the rat and human DNA sequences respectively. The alignment of the amino acid sequences shows high conservation of hydrophobic and high divergency in hydrophilic amlnoterminal domains. The tissue distribution of the mas protoo oncogene transcript In NMRI mice as measured by RNaseprotection assay shows high expression regions In the brain as well as In testis and kidney. Only weak but detectable amounts were found in heart, liver and adrenal gland. These results suggest a possible role of the mas oncogene as an Angiotensin receptor predomlnantely located In the central nervous system. The cloning of the mouse mas gene provides an Important precondition to Investigate the regulation of this receptorprotein under physiological and pathophlslologlcal conditions In further detail. German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, Im Neuenhelmer Feld 366, D-6900 Heidelberg, FRG

AUTOCRINE GROWTH FACTOR EXPRESSION IN C H E M I CALLY AND ONCOGENE-INDUCED HEPATOCARCINOGENESIS M. ll6hne, K. llirsch-Ernst, S. Zieroth, and G. F. Kahl kutocrine growth factor production may be involved in many processes of malignant growth. We investigated the expression of transforming growth factor-~ (TGF-~) and insulin-like growth factor I (IGF-I) as potent hepatotrophic mitogens in two model systems of hepatocarcinogenesis by Northern blot analysis and in situ hybridization techniques. In the rat hepatocyte resistance model the development of liver foci and diploid hepatocytes was induced by 2-acetylaminofluorene (AAF) treatment for 4 weeks after partial hepatectomy and diethyl nitrosamine injection. Transgenic mice carrying the SV40 early region and human growth hormone sequences as transgenes in inverse orientation driven by the metallothionein promoter develop hepatic nodules progressing to hepatocellular carcinomas. In AAF-treated rats and in transgenie mice significant expression of IGF-I restricted to the hepatocyte was observed. High TGF-~ expression was seen in some~-glutamyl transpeptidase-negative nodular areas in rat liver and in normal livers of. transgenic mice, but not in nodules or tumors. The scheme of carcinogen initiation followed by tumor promotion results in similar changes of growth factor expression as in oncogene-induced liver preneoplasia in the transgenic mouse system. IGF-I and TGF-~ may act as secondary oncogenic factors in malignant transformation of the hepatocyte. Institut f6r Pharmakologie und Toxikologie, Universit~t G6ttingen, Rober t-Koch-Strafe 40, D-3400 G6ttingen, F.R.G.

2

4

EARLY TRANSIENT DECREASE IN e-mye AND e-myb mRNA LEVELS IN ACLACINOMYCIN A TREATED FRIE/~D ERYTHROLEUKEMIA CELLS. A. Schaefer~ K. Lingelbach, C.A. Schmldt*, S. Schr~der and A. Dressel

PENTOSANPOLYSULFATE INHIBITS KAPOSI-FGF DEPENDENT TUMOR GROWTH A. Wellstein, G. Zugmaier, J.A. Califano III, S. Palk, and Marc E. Lippman

Chemical inducers of the differentiation are known to induce an early transient decrease in c-mye and c-myb mRNA levels in murlne e~ythroleukemla cells. Therefore, we investigated the early effect of the differentiationinducing anthracyclln antitumor antibiotic, aclaclnomyeln A, on n-myc and e-myb mRNA levels in Friend erythroleukemla cells, llne F4-6, using Northern blot analysis. Aclaelnomyein A, 200 ng/ml induced a rapid deerease in both e-mye and e-myb m~l~A levels during the first few hours after treatment I e-myc and e-myb m/LNA levels returned to prelncubatlon levels within 12 hours. Adriamyeln, 15 ng/ml, which is highly active at inhibiting cell proliferation in F4-6 cells without inereaslng differentlatlonj did not induce a similar early deerease in e-myc and e-myb mRNA levels. These results suggest that the transient decrease in c-myc and e-myb mRNA levels may be an early differentiationspeclfle effect of aclaelnomyeln A.

A neoangiogeulc response is critical for the unrestricted growth of solid tumors beyond a few millimeters in diameter. Release of adequate growth stimulating activity from tumor cells is obviously required for the stimulation of blood vessel growth and blockade of such stimulatory activity should repress tumor growth at microscopical level. To test this hypothesis and to study appropriate inhibitors, we used a cell line (SW-13) engineered to express Kaposi's sarcoma derived fibroblast growth factor (K-FGF). This cell construct (SW-13/K-FGF) grows into highlyvascularized subcutaneous tumors in animals due to paracrine and autocrine growth stimulation by secreted, authentic K-FGF and hence can serve as a defined tumor angiogenesis model (WELLSTEIN et al. (1990): Cell Growth and Diff 1:63-71).

*Immunbiochemisches Laboratorium, Universlt~tskllnlkum Rudolf Virchow, Berlin.

RESULTS: /n v/ire studies: Different polysulfates were studied for their selective inhibition of K-FGF-induced growth in in vitro assays. Human breast cancer ceils growing independently from FGF served as controls. Suramin and detransulfate showed slightly selective inhibition of FGF-dependent growth (3and 5-fold respectively), whereas heparin was inactive. The heparin analogue pentosanpolysulfate (PPS), however, was fully inhibitory for FGF-dependent growth and > 1,000-fold selective for this pathway. Inhibitory effects on SW13/K-FGF cells were fully reversible by an excess of added FGF. Furthermore, the concentration-response curve of PPS in human umbilical vein endothelial cells stimulated by K-FGF was indistinguishable from that in the SW-13/KFGF cells, animal studies: Daily i.p. injections of PPS (20 mg/kg) were tolerated well by athymic nude mice and prevented growth of subcutaneous SW-13/K-FGF tumor xenografts. In contrast, heparin treatment of the animals showed no effect on tumor growth.

Abt.f.Allgemelne Toxikologle, Universit~t Hamburg, und Fraunhofer Instltut f~r Toxlkologie und Aerosolforschung, Grindelallee 117, D-2000 Hamburg 13, F.R.G.

We conclude that PPS will be a useful tool to elucidate the significance of FGFs in vitro and in vivo and appears to be a prototype for the development of tumoricidal therapy based on growth factor targeted tumor therapy.

This work was supported schaft.

by Deutsche

Forsehungsgemeln-

V.T. Lombardi Cancer Center and Dep. of Pharmacology Georgetown University, Washington DC 20007

82

5 MORPHOLOGICAL ALTERATIONS I N D U C E D BY M I C R O I N J E C T I O N C. M o h r and M. L a u x

OF X E N O P U S O O C Y T E S OF p21 v ~ 4 ~ ~ ° ~

It has b e e n s u g g e s t e d that the small GTPbinding protein rho, a substrate of Clostridium botulinum C3 ADP-ribosyltransferase, is i n v o l v e d in the regulation of c y t o s k e l e t o n e l e m e n t s of m a m m a l i a n c e l l s (Paterson et al. (1990) J. Cell. Biol. Iii:i001) H e r e we r e p o r t a b o u t studies on the effects of the constitutively active recombinant p21 v a ~ 4 - r ~ o k in Xenopus oocytes. Microinjection of p21 rbo into oocytes induced dramatic morphological c h a n g e s w i t h r e d i s t r i b u t i o n of p i g m e n t s from the animal pole resulting in spotted oocytes. This e f f e c t of p21 rh° p r o t e i n was regulated by progesterone in a dosedependent manner, whereas prior ADPribosylation of the p21 r~° blocked its biological activity. About 30 min after microinjection, p21 ~b° was a s s o c i a t e d w i t h the p l a s m a m e m b r a n e . M e m b r a n e a s s o c i a t i o n of p21 rb° and its biological activity were i n h i b i t e d by l o v a s t a t i n . The d a t a i n d i c a t e that p21 ~b° is i n v o l v e d in the regulation cytoskeletal elements of Xenopus oocytes and s u g g e s t that membrane a t t a c h m e n t and b i o l o g i c a l a c t i v i t y d e p e n d on p o l y i s o p r e n y l a t i o n of the rho p r o t e i n . R u d o l f - B u c h h e i m - I n s t i t u t fur P h a r m a k o l o g i e , F r a n k f u r t e r Str. 107, D - 6 3 0 0 G i e ~ e n

7 TRANSCRIPTIONAL ACTIVATION BY CYCLIC AMP THROUGH CELL TYPE-/PROMOTER-DEPENDENT MECHANISMS W. Knepel, J. Chafitz, and J.F. Habener The 5'-flanking region of the rat glucagon gene contains, from nucleotide -291 to -298, a sequence, TGACGTCA, which mediates cAMP-responsiveness in several genes (cAMP-responsive element, CRE). However, because of non-permissive bases surrounding the CRE octamer, the glucagon CRE does not confer cAMP-responsiveness to an inert heterologous promoter in placental JEG cells that do not express the glucagon gene. This study shows that glucagon gene expression is activated by cAMP-dependent protein kinase A in a glucagon-expressing pancreatic islet cell line (InR1-G9). In transient transfection experiments using 350 base pairs of the 5'-flanking region of the rat glucagon gene fused to a chloramphenicol acetyltransferase reporter gene, cotransfection of an expression vector coding for the catalytic subunit of protein kinase A enhanced reporter activity about 4fold, whereas cotransfection of a mutant expression vector, encoding an inactive catalytic subunit, did not. Cyclic AMP induced glucagon gene transcription in another pancreatic islet cell line, HIT. By 5'-deletion, internal deletion and oligonucleotide cassette insertion this study shows that the glucagon CRE confers protein kinase A responsiveness to rat glucagon gene transcription. The glucagon CRE, at its native position, mediates protein kinase A induction as effectively and with the same sensitivity as the somatostatin or chorionic gonadotropin alpha CREs as shown by mutating the CRE contextual sequences within the rat glucagon gene 5'-flanking region. The in vitro DNase I footprinting assay revealed that the protein kinase A - dependent transactivator protein CREB-327, bacterially expressed, binds to the rat glucagon CRE. Various in vitro binding assays demonstrated multiple glucagon CRE-binding proteins in nuclear extracts of InR1-G9 cells. These data raise the possibility that the effect of cAMP on gene expression may differ depending on the cell type and/or linked promoter, a variability created at least in part by a multiplicity of CRE-binding proteins. Harvard University Medical School, Lab. Molecular Endocrinology, and Howard Hughes Medical Institute, Wellman 3, Boston, MA 02114, USA

6 LESION-INDUCED CHANGES IN EXPRESSION OF THE CHOLECYSTOKININ GENE IN RAT BRAIN CORTEX: STUDIES ON THE MECHANISM OF ACTION C. Olenik, A. Lais, A. Holland and D.K. Meyer Injuries to rat cortex cause an increase in the expression of the cholecystokinin and somatostatin genes in the ipsilateral hemisphere. The concentration of cholecystokinin-mRNA (CCK-mRNA) is significantly increased by 137% 24 hours after the injury. Forty-eight and 72 hours after the lesion enhancements by 190% are observed, while 6 days after cortex injury the levels have again reached control values. Cortex lesions have also been described to enhance the immunoreactivity of the immediate early gene (lEG) c-fos in rat cortex (Dragunow and Robertson, Brain Res. 455:295, 1988). This finding is of interest, since it has been shown that the c-fos protein can form complexes with other proteins coded for by IEGs and that the complex formed with c-jun protein can affect the transcription of the proenkephalin gene (Sonnenberg et al., Science 246:1622, 1989). In the present study, it was investigated whether a cortex injury also increased the levels of c-fos- and c-jun-mRNA and whether the timecourse of these changes corresponded to that of CCK-mRNA. One hour after a cortex injury, levels of c-fos-mRNA were enhanced by 184%. However, after 4 h levels were again in the control range. Surprisingly, a second enhancement (by 324%) was seen 24 h after cortex injury which had vanished 24 h later. In contrast, the levels of c-jun-mRNA did not increase until 24 h after the cortex lesion (by 45%), but stayed enhanced (by 104%) for the next 24 h. Thus, concentrations of both IEGs were increased 24 h after the injury, at a time at which also increased levels of CCK-mRNA were observed. The results are in agreement with the hypothesis that the complex formed by c-fos and c-jun protein may act as a "third messenger", and thus may be involved in the enhanced expression of the CCK-gene which is induced by cortex lesions. Department of Pharmacology, University of Freiburg, Hermann Herderstr. 5, D-7800 Freiburg, FRG.

8 I N F L U E N C E O F A D N A - C R O S S L I N K I N G A G E N T WITH AFFINITY TO T H E A N D R O G E N R E C E P T O R O N THE M O U S E M A M M A R Y T U M O R VIRUS H O R M O N E R E S P O N S E E L E M E N ~ IN M A M M A R Y C A R C I N O M A CELLS. F.J.BUSCH', A.B.C. C A T O ~, G. EISENBRAND" i- (2-Chloroethyl)-l-nitrosocarbamoyl- L- alanine-dihydrotestosterone-17-ester (CNC- ala-DHT) is an antitumor drug with affinity to the androgen receptor displaying high effectiveness in experimental m a m m a r y carcinomas. Interaction with the steroid hormone response element (HRE) was studied in T 47-D cells stably cotransfected with a construct containing m o u s e m a m m a r y tumor virus long terminal repeat s e q u e n c e s (MMTV-LTR) linked to the chloramphenicol acetyltranferase (CAT) g e n e and a chimeric construct containing the S V 40 promotor linked to the neomycin resistance gene. S e q u e n c e s within the LTR region of M M T V confer androgen inducibility to the M M T V - L T R promotor. CAT-activity and the ability of the c o m p o u n d to induce transcription at the M M T V - L T R region in Sl-nuclease mapping H e r e measured. After incubation with C N C - a l a - D H T (10"~M; 15 rain) there was a marked d e c r e a s e of expression at the M M T V L T R promotor in the CAT-assay. A simultaneous treatment with a n equimolar dose of DHT resulted only in a slight inc r e a s e in CAT-activity. The results obtained in the CAT a s s a y agree with m e a s u r e m e n t s of amounts of R N A accumulated at the M M T V - L T R promotor after treatment with the various compounds. Trans.qription at the M M T V LTR promotor w a s inhibited b y 10"~M C N C - a I ~ - D H T and there w a s no substantial competition with i0" M DHT. In contrast, incubation with a mixture of CNC-ala and DHT (10"~M; 15 rain) led to ove~induction of M M T V transcription in comparison to 10"~M DHT alone. ~_Iithese effects w e r e not o b s e r v e d at the S V 40 promotor which was u s e d as an internal control. Our results suggest a specific interaction b e t w e e n C N C - a l a - D H T and the LTR region or with the h o r m o n e receptor. Iun~versit~tKaisersl.,F~ Le~ensm~ittelchemie/Umwelttox., P.O.B.3049~ D-6750Kaiserslastern,FRG. ~Kernf0rsch.Zent.Krh,, Inst.f.Genetiku.T0xik01. v. Spaltst0ffen,P.0.B.3640, D 7500-Karlsruhe:FRG

R3 9 3' NONTRANSLATED REGION AFFECTS EXPRESSION OF/91 AND .82 SUBUNIT ISOFORMS OF THE MOUSE NA,K-PUMP IN XENOPUSOOCYTES S. GIoor1, S. Kr6ner2, and G. Schmalzing2

Expression of/91 subunit isoforms of the Na,K-pump of various species including mouse raises Na,K-pump activity in Xenopus oocytes (Schmalzing et al., submitted for publication). Since these cells contain (~ subunits which are nonfunctional because they lack a/9 subunit (Geering et al. AJP 257, C851, 1989), the rise of Na,K-pump activity is likely to result from the assembly of exogenous/91 subunits with excess (z subunits. Here we report that the number of Na,K-pumps of oocytes increased also after injection of cRNA specific for the/9 2 subunit isoform of the Na,K-pump of the mouse identified recently as adhesion molecule on glia (AMOG, Gloor et el. JCB 110, 165, 1990). In an attempt to examine which part of the/9 subunit is important for e//9 assembly, we injected cRNAs encoding deletion mutants into oocytes. Control cRNA for the ~e1 subunit containing 207 bases of the 3' end in addition to the 912 bases of the coding region and 35 bases of the 5' nontranslated end (cDNA hydrolyzed with Barn HI) raised the ouabain binding capacity 1.7fold. Unexpectedly, cRNA derived from cDNA hydrolyzed with Mae I containing the complete coding region but only 26 bases of the 3' end had almost no effect on ouabain binding, although the cRNA was correctly translated in the reticulocyte lysate system. Deletion of 3' nontranslated bases did not affect stability of the/91 subunit-specific cRNA in the oocyte as assessed by Northern blot analysis 4h after injection, cRNA comprising the complete coding region together with 64 or 158 bases of the 3' end (cDNA hydrolyzed with Spe I or Dra I, respectively) raised ouabain binding in oocytes approximately 1.3fold. The 3' nontranslated region was also important for the expression of the mouse/~2 subunit (AMOG) in oocytes, since deletion of 35 bases from the 3' end of the cRNA abolished its effect on ouabain binding. 1Neurobiologie, ETH ZSrich, Switzerland, and 2Max-Planck-lnstitut for Biophysik, Frankfurt/M, FRG.

10 UBIQUINONE ~ INCORPORATEDI~ PHOSPHATIDYLCHOLINE LIPOSOMES: C-METHOXYLAND H-NMREXPERIMMENTS L. Michaelis and K.E. Wirth Ubiquinone-]O is a lipid-soluble redox carrier located in the inner mitochondrial membrane. Its functioning is of vital importance for any aerobic energy transduction. The protonmotive ubiquinone cycle (respiratory chain) due to Mitchell makes specific predictions about the location and T~tion of this substance. C-NMR spectra of labelled ubiquinone in solution show only one methoxyl peak. When the ubiquinone is incorporated into sonicated liposomes two methoxyl peaks are resolved. As the portion of ubiquinone is increased from 4 molt to 12 molt the relative amplitude of the high f i e l d peak grows rapidly. The two peaks cannot simply result from the different methoxyl groups and must be due to two different environments for the ubiquinone. A series of experiments gave evidence for a power relationship between the areas. This suggests that the high-field component may originate from tetrameric ubiquinone aggregates. Similar results are seen with deuterium-labelled ubiquinone. Some trivalent Lanthanide ions influence the resonance of adjacent nuclei. Peak broadening may occur, and decreases with the inverse square of separation. Pseudo-contact shifts decrease with the inverse cube. Praseodymium ions bind to the phosphate groups of l i p i d molecules forming the outer leaflet of the bilayer. They shift the resonances from the choline N-methyl groups to the glycerol CH~. In addition, the ions alter the bulk magnetic susceptibility of the medium and hence shift the resonance position of all peaks. I t can be concluded that the ubiquinone ring is not accessible from the aqueous phase. There must be at least two different resonance positions for the ubiquinone methoxyl groups. Membrane curvature (sonication time) has a negligibly small effect on the resonance positions, Institute of Pharmacology, Heinrich-Heine-University, Moorenstr. 5, D-4000 D~sseldorf

11 LIPOPHILICITY MEASUREMENT BY CHROMATOGPJ%PHY K. Belsner and M. Pfeifer

REVERSED-PHASE LIQUID

He modified a recently published method using reversed phase HPLC for measuring the l i p o p h i l i c i t y of organic molecules (Minnick et a l . , J. Chromatograph. 461, 1989, 177). As mobile phases we used: A. 20 mM HOPS-buffer in H20, 0.15% (v/v) n-decylamine, pH 7.4 adjusted with 1M NaOH; B: 94.75Z (v/v) MeOH, 5Z (v/v) of a solution of 3~ diethylamine in water adjusted to pH 7.4 with HC], 0.25~ (v/v) n-octanol. Log(k'), the HPLC retention index is determined at different methano] concentrations by changing the relative amounts of A and B. Log(kw) is the extrapolated value of log(k') for I00~ water content of the eIuent. Extrapolation was allowed because the correlation between log(k') and the volume fraction of methanol was l i n e a r . Of 41 substances tested, 34 had a correlation coefficient r ! 0.999, for 7 r was > 0.99 and for 3 r exceeded 0.9. The HPLC data of a group of aIkylbenzenes, 4-alkylanilines, phenones, mioflazine, R 56865 (N-[l-[4-(4-fIuorophenoxy)-butyl].-4-piperidinyl]-N-methyl-2-benzothiazolamine), sabeluzole, propranolol, flunarizlne, lidoflazine, lidocain, cinnarizine, verapamil and diltiazem were measured and regressions calculted for log(PaDD) versus log(kw) (PaDD = pK. corrected octano]-water-'partition coefficient). A linear regression of log(PaDD) versus log(kw) resulted in a correlation coefficieht of r = 0.9B7 (n=30). The method is discussed as a substitute for the octanol-water p a r t l t i t i o n determination. Janssen Research Foundation, Raiffeisenstr. 8, D-4040 Neuss 21, F.R.G.

12 BINDING TO ACTIVATED CHARCOAL (AC) OF 8-ADRENOCEPTOR BLOCKING AGENTS (B-BL) CORRELATES WITH MOLECULAR WEIGHT, BUT NOT WITH HYDROPHOBICIYg{. D. Hellenbrecht and S. Eiermann _

_

Nonspecific cardiovascular toxicity of B-BL correlates with hydrophobicity (octanol-buffer partition coefficients at pH 7.0 = Papp, c.f. Hellenbrecht + Enenkel, Drug Res 34:980-83,1984). AC is given clinically as an adsorbent in the treatment of intoxications, but the structural requirements of drug binding to AC are not well understood. We measured the adsorbability of 14 B-BL (20 mg%) at pH 7.4 by constructing linear Langmuir isotherms for the monolayer binding capacity, cmax, either expressed as milligrams or as mmoles of drug bound per Ig of AC. - The log Papp of nadolol (-2.1), acebutolol (-0.70), propranolol (0.73) and of 11 amino- or ring-alkylated K~-compounds (c.f. Hellenbrecht et el, Eur J Pharmacol 29: 223-25, 1974) correlated linearly with the mg of cmax values of the 36 experiments with the B-BL (tab. IA), but not with the respective molar amounts of drug adsorption (tab. IB). Tab.l: Linear regression equations and statistical significance p y=cmax b= c= P6 A) mg/g AC = 11.0 log Papp + 171 0.000001 B) mmoles/g AC 0.0064 log Papp + 0.68 0.665 C) mmoles/g AC - 0.0024 mol wt + 1.27 0.000001 Instead, there was a very close negative relationship of mmoles of drug adsorption with the resp. molecular weights (mol wt) of the compounds (tab. IC). Binding isotherms at pH 4.0 and 1.2 revealed only 20-30% lower values of cmax. The conclusion from in vitro experiments is that B-BL adsorb to AC irrespectively of hydrophobicity and that pH of the medium is not limiting for drug adsorption onto AC. Supp by grant of Dr Paul + Cilli Weill foundation Dr med D Hellenbrecht, Centre Pharmacology, Univ Clin, Theodor Stern Kai 7, 6000 Frankfurt/M, F R G

R4 13

15

POTENTIAL ERRORS IN OONVENTIONAL SINGLE-DOSE PHARMAOOKINETIO STUDIES M. Weiss

DRUG MONITORING IN HUMAN BLOOD SERUM USING CONTINUOUS FLOW FAB/MS W.Lenhart*, Ch.Siethoff*, T.M.L6ffler #

The i n a d e q u a t e a s s u m p t i o n of i n s t a n t a n e o u s mixing o f d r u g i n a homogeneous b l o o d p o o l f o l l o w i n g bolus intravenous injection ( s a m p l i n g and e l i m i n a tion f r o m t h e same c o m p a r t m e n t ) l e a d s t o e r r o r s in the estimation of clearance (CL), steady-state volume of distribution ( V s s ) and mean d i s p o s i t i o n r e s i d e n c e t i m e (MDRT). U s i n g a p h y s i o l o g i c a l l y realistic recirculation model of drug disposition i t i s shown t h a t t h e s e e r r o r s a r e due t o ( i ) t h e neglect of the transit time of drug molecules through the sampling tissue (from the arterial to the peripheral venous site) and, (ii) the underestimation of the area under the concentration-time c u r v e as a consequence o f t h e n e g l e c t

of the initial concentration

impulse

(first

passage Of drug a t t h e r e f e r e n c e point). The effect of (i) l e a d s t o an o v e r e s t i m a t i o n o f MDRT (and c o n s e q u e n t l y of Vss) for drugs with relatively s h o r t MDRT and r e l a t i v e l y high tissue binding. The o v e r e s t i m a t i o n o f OL r e s u l t i n g from (ii) has t o be t a k e n i n t o a c c o u n t f o r a l l highly extracted drugs. While the error ( i ) can be e x c l u d e d by arterial s a m p l i n g , t h e r e a r e p r a c t i c a l constraints (experimental d e s i g n and d a t a e v a l u a in case (ii). tion) In conclusion, it appears difficult to determine the intrinsic v a l u e s o f CL and Vss ( d e f i n e d in terms of tissue v o l u m e s and e q u i l i b r i u m partition coefficients) by s i n g l e - d o s e studies u n l e s s OL/Q 99% ~3C) were analyzed by lmc N1VIR spectrospopy as described recently [C. O. Meese and P. Fischer, Z. Naturfor~ch. 45c, 139 (1990)]. The radioactivity measurements and the NMR method both demonstrate a biphasin kinetics (t½ - 6h and 20h) of urinary excretions. During the 0-8h period mainly unchanged CMC was eliminated, whereas after 8h metabolites of CMC, recently identified as thiodiglycolic acid, and its sulphoxide, and a novel metabolite of yet unknown structure (that accounts for about 5% of the dose administered) were determined in the samples. In addition, a slight but significant increase in radioactivity in the exhaled breath, according to 5-10% of the dose admin;stered, was registered after 8h that ceased after 24-30h. Since at least 70% of the dose administered has been recovered and no significant amounts ( > 1-2%) of cystelnyl sulphoxides were detected in the urines, a polymorphic sulphoxidation of CMC in respect to these sulpho:ddes can be excluded.

Supported by the Robert Bosch Foundation Stuttgart 1Dr. Margarmte Fiscbem-Boseh-Insfitnt fiir Klinische Phurmakologie, D-7000 Stuttgart 50, AuerbacbstraBe 112, 2Abt. Kllnische Chemle, Robert-BoschKrankenhans, D-7000 Stuttgart 50, AunrbachstraSe 110

c a r b o n y l g r o u p b e a r i n g s u b s t r a t e s in m a n y m a m m a l i a n a n d n o n m a m m a l i a n t i s s u e s . In a d d i t i o n to x e n o b i o t i c a l i p h a t i c and aromatic aldehydes and ketones the carbonyl reductases i n v o l v e d in t h i s r e a c t i o n m e t a b o l i z e a v a r i e t y of p h y s i o logical substrates including prostaglandins and steroids. In p r e v i o u s i n v e s t i g a t i o n s we p u r i f i e d a n d c h a r a c t e r i z e d a c a r b o n y l r e d u c i n g e n z y m e from m o u s e l i v e r m i c r o s o m e s u s i n g t h e k e t o n e m e t y r a p o n e (MP) a s a s u b s t r a t e . Moreover, we d e m o n s t r a t e d t h a t M P - r e d u c t i o n o c c u r s in c u l t u r e d c e l l s of c o n t i n u o u s cell l i n e s t h u s g i v i n g r i s e to t h e s u g g e s t i o n t h a t carbonyl reductases are constitutive enzymes, which play an i m p o r t a n t r o l e in n o r m a l cell p h y s i o l o g y . In t h e p r e s e n t s t u d y we c h u r a c t e r i z e d M P - r e d u c i n g e n z y m e s in h u m a n l i v e r m i c r o s o m a l a n d c y t o s o l i c f r a c t i o n s u s i n g (1) different cosubstrates, (2) d i a g n o s t i c i n h i b i t o r s a n d (3) antibodies against the MP-reductase from m o u s e l i v e r microsomes: We f o u n d NADPH to be a more p o t e n t c o s u b s t r a t e t h a n NADH in b o t h f r a c t i o n s . Of t h e i n h i b i t o r s t e s t e d o n l y d i c o u m a r o l in m i c r o s o m e s a n d i n d o m e t h a c i n in c y t o s o l c o n s i d e r a b l y decreased enzyme activity, whereas quercitrin, barbiturate a n d h e x o b a r b i t a l c a u s e d o n l y a w e a k i n h i b i t i o n . However, in microsomes 5a-dihydrotestosterone (5c(-DHT) was the s t r o n g e s t i n h i b i t o r , w h e r e a s in c y t o s o l i t h a d no e f f e c t . Immunological crossreaction w a s f o u n d in h u m a n l i v e r microsomes with the antibody against the mouse liver enzyme indicating structural homologies between the m i c r o s o m a l e n z y m e s of t h e t w o s p e c i e s . In c o n c l u s i o n , MP-reduction was s h o w n to o c c u r in m i c r o s o m e s a n d c y t o s o l of h u m a n l i v e r b e i n g m e d i a t e d b y c a r b o n y l r e d u c t a s e s . S t r u c t u r a l h o m o l o g i e s seem to e x i s t b e t w e e n t h e M P - r e d u c t a s e s from m o u s e a n d h u m a n l i v e r m i c r o s o m e s a n d 5¢(-DHT is s u p p o s e d to be t h e p h y s i o l o g i c a l s u b s t r a t e o f t h e m i c r o s o m a l enzyme, Dept. P h a r m a c o l . , P h i l i p p s - U n i v e r s i t y ,

Lahnberge, 355 Marburg

38 REVISED PROTOCOLFOR CARBOCYSTEINEPHENOTYPINGIN MAN A. Kfipfer*, F. Gerber, E. Manser, I. Turner, J.R. Idle

40

Carbocysteine (CC) exhibits polymorphic metabolism in man and the underlying deficiency of organosulfur metabolism is visible by the absence of the main CC metabol i t e (CCM) in poor metabolizer (PM) phenotypes. Several methodological d i f f i c u l t i e s have been encountered e.g, with the originally used paper chromatography (PC) and we therefore have revised the protocol for phenotype determinations as follows: CC was administered in form of capsules or syrup (750 mg p.o.) and urines were collected from 0-8 h and from 8-16 h. Two population studies (Swiss, n=106 and United Kingdom, N=60) were carried out. For CC and CCM analysis, urine was applied to silicagel TLC plates and developed in 1-butanol/ water/cone, acetic acid/butylacetate (24/10/10/4 by vol/vol). Then the TLC spots were visualized using potassium iodoplatinate solution (200 mg/L) in acetone. The Rf values for CC and CCM were 0.28 and 0.43, respectively. For peak quantification, a Shimadzu dual wavelength flying spot model CS 9000 TLC scanner (reflection mode at 510 nm) was used. The results of TLC analysis were also veryfied using the previously proposed PC method. After CC capsules and after CC syrup, the 0-8 h urine sample contained mainly CC whereas CCM was the main organosulfur compound in the 8-16 h urine collection. For statistical analysis, the CC over CCM metabolic ratio (MR) was calculated. The frequency distribution histograms showed bimodal distributions with an antimode at MR=17 in b o t h population studies. In the Swiss study, 7.5% PM (95~ confidence limits of 3.4% to 13.4%) were identified and in the British study, I0~ (g5% confidence limits of 3.8~o to 19%) were PM. Therefore, polymorphic CC metabolism occurs in Swiss and British populations and extended urine collections for proper MR calculations are required. *Dept. of Clinical Pharmacology, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland

In the isolated mucosa of guinea-pig intestine the conjugation of xmnobietics administered to the lumlnal or blood side of the preparation takes place in different compartments (I). Comparative experiments with p-nitroanisol (PNA) and its metebolite p-nitrophenol (PNP) formed by oxidative demethylmtioe now indiemte thmt the latter reaction is restricted mainly to the luminal compartment. I) p-Nitrophenyl sulfate (PNP-S) originating from PNA appears in the luminal solution irrespective of the side of PNA administration. However, only after luminal administration of PNP is its metabolite PNP-S released almost completely to the lumlnal side, whereas PNP-S from blood side PNP Is equally distributed to both sides. Z) For the formation of PNP-S from PNA mainly luminal 35S is used Irrespective of the sid~sof PNA administration, whereas PNP-S from PNP is formed with luminal S after luminal, and with S from both aides after bleed side PNP administration. 3) Selective damage of the luminal compartment by a hypmrtonic preincubation with 2 M sodium cyciohexanesuifamate reduces PNA metabolism to 5 % of the control value irrespective of the side of administration, whereas sulfoconjugation of bleed side PNP decreases only to 50 %. Looking for a merpholegieel correlate of theme funmtional compartments, isolated mucosae were examined histologically after luminal or blood slde hypertonic incubation. Luminal hypertoniclty resulted in s severe damage or even less of the villus epithelium while blood side hypertonicity was mainly destructive to the epithelium of the crypts suggesting a corresponding localization of the lumlnal and blood side compartment.

COMPARTMENTATO I N OF INTESTINAL DEALKYLATION AND SULFOCONJUGATION: COMPARATIVEINVESTIGATIONSWITHp-NITROANISOLANDp-NITROPHENOL F. Lauterbach,G. Ulrich-Merzenioh and G. H. RUM*

(q) La~terbach, F., Schorn, M., Sprakties, G. and Sued, R. B.; Progress in Pharmacology and Clinlcal Pharmacology 7/.__22,231 - 2~2 (1989). Supported by a grant from the M£mSstmr for W£ssensmhmft and Tmrsmh~mg des Landes Nerdrhein-Westfalmn (IV A 6 - 40102387) Arbeitsgruppe Biochemische Pharmakologie, Abtellung fur Pharmakmlogie and Toxtkolmgie and Inmt£tut for Pathologie*~ Ruhr-Univermit~t~ D-4630 BOCHUM

Rll 41

PARACETAMOL GLUCURONIDATION BY HUMAN LIVER PHENOL UDP-GLUCURONOSYLTRANSFERASE A. Forster, M. Brfick, M. El Mouelhi, S. Fournel-Gigleux', G. Siest% B. BurchelP and K.W. Bock Paracetamol glucuronidation has recently been found to be significantly increased in heavy smokers and m patients treated with phenobarbital-type inducers (Beck et al., Eur. J. Clin. Pharmacol. 31, 677-683, 1987). Three routes were followed to identify human liver UDP-glucuronosyltransferase (UGT) isozymes responsible for paracetamol glucuronidation: (I) Solubilized microsomal UGTs were separated by chromatefocusing. Three fractions exlfibiting marker UGT activities could be separated, (a) estriol UGT (pI 7.4), (b) 4-methylumbelliferone UGT (pI 6.6) and (c) 4-aminobiphenyl UGT (pI 6.2). Paracetamol UGT eluted at pI 6.6. However, due to instability of enzyme activity the isozyme could not be further purified. (2) UGT activities were studied in V79 cells containing integrated human liver phenol UGT = HLUGP1 (Harding et al. Prec. Natl. Acad. Sci. USA 85, 8381-8385, 1988). These cells expressed 4-methylumbelliferone and paracetamol UGT activities at levels comparable to those found in human liver homogenates. (3) DNA sequencing of genomic DNA fragments(350 bp from the translation start site, using primers synthesized according to the sequence of HLUGP1) indicated that human phenol UGT appears to be conserved in this region. However, some variation was observed. For example, in one DNA fragment 3 nucleotide exchanges (not leading to amino acid changes) were noted. The results suggest that human liver phenol UGT is the major enzyme responsible for paracetamol glucuronidation in human liver. Institute of Toxicology, Univers,ty of Tiibingen (D-7400 Tiibingen, WilhelmstraBe 56, Germany), 'Centre du M~dicament, Nancy (France) and bNinewells Hospital and Medical School, Dundee (UK)

42 DERMAL ESTER CLEAVAGE

IN THE ISOLATED PERFUSED

RABBIT EAR B. Henr~kus

Rabbit

and H. Kampffmeyer

ears were single-pass

protein-free

steady-state procaine, 151-1~i, following

rate

(apparent Vma x) during

and methylsalicylate 1989) was determined

19

from the effluent

acid moiety was measured by

after extraction with ethyl acetate.

The rate of hydrolysis the ectanol/perfusion

seems to be governed by buffer coefficient.

Whether the penetration the cell,

2-chloro-

(Xenobiotica,

dermal or arterial drug application.

The 4-aminobenzoic h.p.l.e,

perfused with

condition of procaine,

of the substrate

or the enzymic hydrolysis

by the lipophilicity

iDto

is dominated

will be shown in further

experiments. About

10% of the q-aminobenzoic

to be 4-acetamSdobenzoic

Walther-Straub-lnstitut Toxikologie

acid was shown

acid.

for Pharmakologie

und

der Ludwig-Maximilians-Universit~t

Nu~baumstrssse

26, 8000 M~nchen

2.

CONSTITUENTS IN THE ISOLATED PERFUSED RAT LIVER C. Egen-Sehwind, R. Eckard, and F.H. Kemper Further studies on pharmacokinetics of garlic (Allium sativum L.) constituents showed that after oral application of vinyldithiins these compounds could be detected in serum, liver, kidney, and fat tissue. Other garlic constituents or metabolites were not found after administration of fresh garlic or lyophilized garlic powder even when these preparations were administered in high dosages. Therefore the isolated perfused rat liver was used to study the metabolism of different garlic preparations as well as of garlic constituents in an isolated organ. Single pass studies were carried out using a modified KrebsHenseleit buffer as perfusion medium, after liver passage the medium was collected in fractions. As viability parameters the enzyme activities of GOT and GPT and potassium concentration in the perfusate as well as the interpretation of tissue section of the liver were used. Additionally the perfusate was analyzed by HPLC and GC-MS to find garlic constituents or metabolites. To study the .metabolism, the test substances were solved or dispersed in the pexfusion medium and infused in the flowing medium with a rate of 1 ml/min. The administration of garlic powder in different dosages revealed that allicin, the main constituent of garlic, could be detected in the perfusate only after a dose of 200 mg garlic powder~rain over 50 rain. After application of lower dosages allicin was degradated during the first liver passage. Diallyl disulfide was identified as metabolite of allicin in the perfusate as well as in the liver and in the bile which was confirmed by studies with isolated allicin. Furthermore ajoenes and vinyldithiins were investigated in this model but no metabolites could be identified. Institut ffir Pharmakologie und Toxikologie der Westffilischen Wilhelms-Universitfit Mfinster, DomagkstraBe 12, D-4400 Mfinster, FRG

44 PENICILLIN BINDING IN BETA-LACTAMASE PRODUCING STAPHYLOCOCCI: PROTECTIVE EFFECT OF POLIDOCANOL H. Keppeler, A. G~rke, B. Thielen, and W. Bruns The beta-lactamase dependent penicillin resistance of S. aureus can be greatly reduced by polidocanol (PDO), a dodecyl polyethyleneoxid ether (Bruns et al. 1985, Antimicrob. Agents Chemother. 27, 632-639). PDO does not affect the activity of beta-lactamase, but the induced synthesis of this enzyme is inhibited by PDO-concentrations which are not inhibitory for bacterial cell growth.

buffer solution.

The hydrolysis

43 STUDIES ON DIVERSE GARLIC PREPARATIONS AND GARLIC

We studied the consequences of this effect on the binding of ~H-benzylpenicillin (3H-BP) to the penicillin-binding proteins (PBPs) of resistant staphylococci. Induced cells of S. aureus S 108 grown with and without PDO were incubated with various concentrations of 3H-BP. The cytoplasmic membranes were isolated and subjected to SDS-PAGE and fluorography. Penicillin binding to the PBPs of PDO-free cells was detectable only with high concentrations of 3H-BP (100 nmol/ml). In presence of PDO (50 ~g/ml), however, binding of 3H-BP to the PBPs was about 10-fold increased. This is c o n s i s t e n t with the 90% inhibition of beta-lactamase synthesis found under these conditions. The strong PDO-effect on beta-lactamase synthesis and, on the other hand, the lacking inhibition of bacterial growth can probably be explained by preferential interaction of PDO with membrane associated protein synthesis as it has been assumed for the minocycline-inhibited beta-lactamase synthesis (Chopra and Anderson 1985, J. Antimicrob. Chemother. 16, 17-21). The results of own experiments4Point in this direction. The incorporation of I C-serine into membrane proteins was stronger inhibited by PDO than that into cytoplasmic proteins. Institut f~r Pharmakologie der Universit~t zu K~in, Gleuelerstra~e 24, D-5000 K~in 41

R12 45

47

EBSELEN INHIBITS PLATELET ACTIVATION BY MODULATION OF INTRACELLULAR CALCIUM RELEASE B. Briine

INFLUENCE OF ASPIRIN AND ESCULETIN ON THE UTILIZATION OF ASCORBIC ACID AND TOCOPHEROL DURING PLATELET AGGREGATION; R. Pella, a n d A. L u d e s

Ebselen (PZ 51; 2-phenyl-l.2-benzoisoselenazol-3(2H)-one) is a selenoorganic compound with anti-inflammatory properties. The reported glutathione peroxidase (GSH-Px)-like activity is thought to be responsible for the pharmacological activity of ebselen. We investigated the effect of ebselen on aspirin-treated platelets. Ebselen inhibits platelet aggregation caused by several commonly used agonists like thrombin or U46619 a prostaglandin endoperoxide analogue. The observed inhibitory activity followed a sigmoidal doseresponse curve with" IC50 values around 10 IxM (for both agonists; stimulated with threshold concentrations). Mobilization of intracellular calcium represents a critical parameter for platelet aggregation. Therefore the effect of ebselen on the inositol 1,4,5-trisphosphate lIP3) releasable calcium was studied. Ebselen in a concentration-dependent fashion (IC50:14±4 IxM; mean±SD, n=3) inhibits a cytosolic calcium increase in fura-2 loaded platelets after stimulation with 0.1 U/ml thrombin. In additional experiments we studied the effect of ebselen on calcium release using microsomal vesicles. Low concentrations of ebselen (1-5 IxM) added 2 min before the IP 3 stimulus lIP3; 31xM) reduces the calcium release completely. The described effects of ebselen on calcium homeostasis may contribute to the anti-inflammatory pharmacodynamic properties of this compound.

We quantitated the amounts of ascorbic acid (Vit. C) and tocopherol (Vit. E) using HPLC with coulometric electrochemical detection. Our results confirm the described high concentrations in the human platelets a n d s h o w a direct relationship between the degree of platelet aggregation and the Vit. C and E utilized by washed platelets. The mean conc e n t r a t i o n of vit. C in washed human platelets was 13.8 ± 2.4 (SEM) mg/g protein and the concentration of Vit. E 31.5 ± 3.2 (SEM) ~g/g protein. We found a significant correlation between the net loss of Vit. C and E from platelets and the degree of platelet aggregation induced by 1 ~M arachidonic acid and i0 mU/ml thrombin. In experiments with platelet rich plasma we measured that vit. C and E are not released into the plasma during platelet aggregation. Results after 1 minute with washed platelets: Vit. C (% of 0 s) control Aspirin Esculetin A r a c h i d o n i c acid 64 ± 4 92 ± ii 67 ± i0 Thrombin 74 ± 14 69 ,± 17 61 ± 6 Vit. E (% of 0 s) A r a c h i d o n i c acid 14 ± 1 65 ± 6 15 ± 2 Thrombin 24 ± 4 21 ± 8 25 ± 6 A s p i r i n is known as inhibitor of platelet cycloxygenase and esculetin of platelet lipoxygenase, i0 ~M esculetin had no effect on the reduced vitaminlevels. The inhibition of the cycloxygenase pathway by i00 ~M aspirin leads to a lowered utilization of ascorbic acid and tocopherol after arachidonic acid stimulation. Both vitamins seem to act as m o d u l a t o r s of the production of hydroperoxides and appear to serve some definite metabolic function in platelet aggregation in vivo.

Department of Biology, University of Konstanz, Universit~itsstr. 8-10, D-7750 Konstanz, F.R.G.

Department of Biology, University Postfach 5560,D-7750 Konstanz, FRG

46 EBSELEN AFFECTS THE INOSITOL (1,4,5)-TRISPHOSPHATE INDUCED CALCIUM RELEASE FROM HUMAN PLATELET MICROSOMES BY INHIBITION OF INOSITOL (I,4,5)TRISPHOSPHATE BINDING. S. Dtmmeler a n d V. Ullrlch The seleno-organie c o m p o u n d ebselen (PZ 51; 2 - p h e n y l - l , 2 benzisoselenazol-3-(2H)-one) is known to exhibit antiInflammatory activity. This property s e e m s to be related to t h e glutathtone peroxidase (GSH-Px)-like activity as well a s to t h e inhibition of lipoxygenase reactions. We have investigated t h e effect of low concentrations of ebselen on the [3H]inositol 1,4,5t r i s p h o s p h a t e lIP3) binding a n d the IP3 induced Ca 2+ release from h u m a n platelet microsomes. Using platelets m l e r o s o m e s ebselen a s well a s other SH-reagents like reactive disulfides (2,2'-dithiodipyridine), p-chloromercurobenzoyl sulfonate (CMBS) a n d N-ethylmalelmide (NEM) displaced the specific [ H]-IP 3 binding in a concentration-dependent fashion. Halfm a x i m a / inhibition w a s observed at 5.0±1.2 IxM ebselen (mean±SD; n=4). A maximally effective concentration of NEM (200 ~tM) showed 70±8.5 % (mean±SD, n=3) displacement of the specific IP 3 binding whereas 2,2'-dithiodipyridine at a concentration of 6 ~tM revealed about 40±7 % displacement (mean±SD, n=3). A commonly u s e d concentration of i mM CMBS displaced nearly all of b o u n d radioactive IP3. Since essential SH groups have been demonstrated at the IP 3sensitive c h a n n e l a n d reducing agents like dlthiothrettol (lmM), a l m o s t completely reversed the Inhibition of both, t h e IP 3 binding a s well as t h e IP 3 induced calcium release from mierosomes, we conclude a n interference of ebselen with critical sulfhydryl groups. D e p a r t m e n t of Biology, University of Konstanz, Universit&tsstr. 8-10, 7750 Konstanz, Germany.

of

Konstanz,

48 E F F E C T OF T H I M E R O S A L ON THE Ca2+-HOMEOSTASIS IN HUMAN NEUTROPHH.S A. Kottig The normal Ca2+-signal in human neutrophils is biphasic after receptor stimulation with the chemoattractant fMLP. The first phase is due to the release of intracellular calcium during the PI-response, whereas the second phase of the signal is dependent on extracellular calcium. A preincubation with the organic mercury compound Thimerosal significantly increases this second phase. We could now demonstrate by means of Mn2+-quench of the fura-2 signal that this effect of Thimerosal is due to an increased Ca2+-influx. The different receptor agonists of human neutrophils or different concentrations of one agonist differ in their ability to stimulate Ca2+-influx. Only with thos e stimuli that evoke a Ca 2+ -influx by themselves there was an amplifying effect of thimerosal on the second phase of the calcium signal. It seems that Thimerosal is only able to increase an already existing Ca2+-influx but not to activate one by itself. By means of a voltage-sensitive fluorescent dye we measured the effect of Thimerosal on the membrane potential. Thimerosal did not alter the membrane potential of resting cells but significantly reduced dep,olarisation after receptor stimulation. There are no voltage-gated Ca'~+-channels in human neutrophils. Nevertheless, the weaker depolarisation can explain the increased Ca2+-infiux, because it amplifies the free enthalpy for the entry of this divalent cation. With a superimposed artificial depolarisation by the ionophore Gramicidin it was possible to completely inhibit the Thimerosal-induced increased Ca2+-influx. University of Konstanz, LS V. Ullrich, Am GieBberg, W-7750 Konstanz, FRG

R13 49

51

THE USE OF MTT COLORIMETRIC ASSAY TO MEASURE RPE CELL PROLIFERATION N. B0tz, M.Bresgen, P.Wiedemann, and K.Heimann

ON REGIOSPECIFIC CONJUGATIONS OF VARIOUS GLUCURONOSYLTRANSFERASES

In v i t r o q u a n t i f i c a t i o n of cell p r o l i f e r a t i o n is an important step in screening cytotoxic and mitogenic substances. For this purpose c o l o r i m e t r i c assays, e s p e c i a l l y the MTT dye reduction have been shown to be a useful technique. In this study we adjusted the rapid and simple MTT c o l o r i metric assay to test the mitogenic potency of b-FGF, EGF, and TNF-alpha on r e t i n a l pigment epithelium (RPE) cells in vitro. Our results show a l i n e a r relationship between MTT formazan production and the cell number obtained by Coulter cell counting at a range from 5.000 to 100.000 RPE cells per w e l l . Increasing f e t a l c a l f serum (FCS) concentrations lead to a r i s i n g dye development r e f l e c t i n g the induced cell p r o l i f e r a t i o n . The mitogens b-FGF and EGF do not cause RPE cell p r o l i f e r a t i o n at concentrations from 0,1 to 100 ng/ml. On the contrary TNF-alpha shows a marked mitogenic a c t i v i t y for this cell line even at a concent r a t i o n of I ng/ml. Although b-FGF and EGF did not stimulate RPE cell p r o l i f e r a t i o n in this study, they may promote growth when assayed in combination with other agents. In Summary, our adaption of the MTT dye reduction technique provides a rapid, sensitive, and reproducible device for detecting mitogenic a c t i v i t y on RPE cells in vitro.

The aim of the present investigation was to characterize and to compare the glucuronidation pattern of all available phenols and of the main dihydrodiols of the polycyclic aromatic hydrocarbons (PAH) phenanthrene, chrysene, and picene. These metabolites were conjugated by five partially purified glucuronosyltransferase enzyme forms (GT) with different substrate specificities. As expected, the activity of the GTs to glucuPAH decreased with inronidate h y d r o x y l a t e d creasing number of benzene rings from phenanthrene to picene. Compared to the phenols the dihydrodiols in general w e r e poor s u b s t r a t e s or no substrates at all (picene), except of the phenanthrene-Kregion-9,10-dihydrodiol. A differing regioselective glucuronidation could be obtained with the phenanthrene and the chrysene phenols: whereas those GTs with main activity towards testosterone and androsterone preferentially conjugated the K-region phenols the GT form with main activity towards morphine preferred the non-K-region phenols 2- and 3-OHphenanthrene and 3-OH-chrysene. With picene this difference could only be seen in parts, in this case the K-region-5-OH-picene was the best substrate for all GT enzyme forms. In conclusion the specific activities of various GT forms towards different OH-derivatives of PAH show distinct regioselectivities.

Abteilung fur Netzhaut- und Glask~rperchirurgie, Universit~ts-Augenklinik K~In, J0seph-Stelzmann-Stra~e 9, 5000 K~In 41, FRG Mit UnterstUtzung der DFG (Wi 880/3-I) und der Retin0vitStiftung.

C.Augustin,

HYDROXY-PAH

BY

A.Schmoldt

Institut f~r Rechtsmedizin der Universit~t Hamburg, Butenfeld 34, 2000 Hamburg 54

50

PURIFICATION AND CHARACTERIZATION OF A RAT LIVER SULFOTRANSFERASE ACTIVATING BENZYLIC ALCOHOLS TO MUTAGENS A. C z i c h , H . - J . M a r t u s , N. Enders, H. Thomas , S. Hornhardt and H.R. G l a t t .

The m e t a b o l i c

activation

of benzylic alcohols

to

e l e c t r o p h i l i c s u l f a t e e s t e r s and t h e i r i n t e r a c t i o n w i t h c r i t i c a l c e l l u l a r n u c l e o p h i l e s such as DNA was shown in several s t u d i e s . However, very l i m i t e d i n f o r m a t i o n is a v a i l a b l e about the i n v o l v e d s u l f o t r a n s f e r a s e s . We now have p u r i f i e d and s t a b i l i z e d a sulfotransferase capable o f act i v a t i n g v a r i o u s b e n z y l i c a l c o h o l s t o mutagens. F r a c t i o n s were analyzed f o r c o n j u g a t i n g a c t i v i t y toward dehydroepiandrosterone and, in p a r t , f o r the c a p a b i l i t y o f a c t i v a t i n g b e n z y l i c a l c o h o l s in b a c t e r i a l mutagenicity assays. A r a p i d p r o cedure was developed f o r p r e p a r a t i o n o f the enzyme from l i v e r o f female Sprague-Dawley r a t , i n v o l v i n g anion exchange chromatography, concent r a t i o n w i t h aquacide I and chromatography on a h y d r o x y l a p a t i t e column. The r e s u l t i n g p u r i t y was >90~, the p u r i f i c a t i o n f a c t o r was 186 and the molecular weight amounted t o 29 kD. The enzyme activated 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene and

9-hydroxymethylanthracene

t o mutagens.

Expressed

per unit dehydroepiandrosterone-conjugating activity, o y t o s o l and p u r i f i e d enzymes showed comparable r e s u l t s . The p u r i f i e d enzyme was f u r t h e r c h a r a c t e r i z e d w i t h regard t o k i n e t i c parameters, pH optimum, and the i n f l u e n c e o f i n h i b i t o r s o r a c t i v a t o r s o f known s u l f o t r a n s f e r a s e s . A f t e r complete p u r i f i c a t i o n , an antibody was r a i s e d . U n i v e r s i t y o f Mainz, I n s t i t u t e o f T o x i c o l o g y , D6500 Mainz, FRG

52 MECHANISM BASED INHIBITION OF HUMAN LIVER CYTOCHROME P-450 BY SEVERAL SYNTHETIC STEROIDS R. B6cker and H. Lepper It could recently be demonstrated that the progestogenic gestodene inhibits the nifedipine oxidase (formation of the pyridine) as well as the hydroxylation of ethinylestradiol (EE) on human liver microsomes in vitro (F.P.Guengerich, 1990, Chem.Res.Tox.3,363). The hydoxylation of EE as the nifedipine oxidation are known to be catalyzed by closely related enzymes or by the same enzyme (F.P.Guengerich, 1988, Mol. Pharmacol. 33,500). Several synthetic steroids (including gestodene, norethisterone, desogestrel, 3-ketodesogestrel, and EE) have now been investigated on 15 different human liver microsomal preparations for their potency to inhibit the nifedipine-oxidation and the ethinylestradiol-hydroxylation in vitro. Liver microsomes were preincubated with various concentrations of the steroids for up to 240 minutes in presence of a NADPH generating system. After a tenfold dilution of the microsomal preincubation mixture nifedipine (and some other closely related dihydropyridines) and EE were used as test substrates for the determination of the residual enzymatic activity. Gestodene was the most potent inhibiting synthetic steroid of the above mentioned reactions. Preliminary experiments with the same synthetic steroids on primary rat liver hepatocytes showed a comparable inhibitory effect of the steroids at the dihydropyridine oxidation reaction. It can be concluded that by the metabolism of the synthetic steroids in vitro an inhibition is caused on the isolated microsomal P-450 fraction as on the intact hepatocytes. Lehrstuhl f0r Toxikologie und Pharmakologie Universit&tsstraBe 22, D8520 Erlangen

R14 53

55

ARE 7-DIALKYLAMINO-4-METHYL-COUMARINS SUITABLE AS SUBSTRATES OF THE MURINE COUMARIN 7-HYDROXYLASE ?

EFFECT OF SMOKING ON BIOTRANSFORMATION ENZYME ACTIVITIES IN HUMAN PLACENTA, FETAL LIVER AND KIDNEY S.J. Wallner, F. Siegers*, F. Klink* and C.-P. Siegers

T e g t m e i e r M. L e g r u m W Coumarin and its 7-alkoxy derivatives (such as herniarin, 7 - e t h o x y c o u m a r i n a n d s c o p a r o n e ) a r e w i d e l y u s e d a s model s u b s t r a t e s to m e a s u r e m o n o o x y g e n a s e a c t i v i t i e s . The O - d e a l k y l a t i o n f o r m i n g t h e f l u o r e s c e n t u m b e l l i f e r o n e is c a t a l y z e d m a i n l y b y t w o c y t o c h r o m e P - 4 5 0 i s o z y m a s . One is a h i g h m o l e c u l a r form i n d u c e d b y 8 - m e t h y l c h o l a n t h r e n e (MC) a n d s u l m a z o l e , t h e o t h e r is c h a r a c t e r i z e d b y i t s low m o l e c u l a r w e i g h t (48.5 kDa) a n d i t s i n d u c i b l i t y b y s e l e c t i v e C o h inducers such as cobalt and N-containing heteroaromates and b y p h e n o b a r b i t a l (PB). The l a t t e r i s o z y m e is c a l l e d c o u m a r i n 7-hyroxylase (Coh) or C y p I I A S - t y p e II. This study investigated 7-dimethylamino-4-methyl-coumarin and 7-diethylamino-4-methyl-coumarin a s s u b s t r a t e s of t h e coumarin 7-hydroxylase. The compounds used as laser dyes a r e known a s " C o u m a r i n 3 1 1 " a n d " C o u m a r i n 1", w h i l e t h e f r e e a m i n e is " C o u m a r i n 120". I n c u b a t i o n of t h e s u b s t r a t e (10 -4 M) w i t h c y t o c h r o m e P - 4 5 0 (10-n M) a t 87°C u p to 30 rain l e a d s to t h e m o n o a l k y l a m i n e s and 7-amino-4-methyl-coumarin as visualized by thin layer c h r o m a t o g r a p h y on s i l i c a gel 60 F=~. In t h e a c i d i f i e d a s s a y t h e m o n o a l k y l a m i n e s c a n be q u a n t i f i e d f l u o r i m e t r l c a l l y u s i n g t h e w a v e l e n g t h p a i r 3 6 5 nm a n d 4 5 5 nm f o r e x c i t a t i o n a n d emission, respectively, Experiments with microaomes of d i f f e r e n t l y p r e t r e a t e d NMRI mice r e v e a l t h a t PB d o u b l e s t h e f o r m a t i o n r a t e of t h e m o n o a l k y l a m l n e s , w h e r e a s MC d e c r e a s e s it. Among C o h - i n d u c a r s o n l y c o b a l t s l i g h t l y e n h a n c e s t h e N m o n o d e a l k y l a t l o n r a t e of t h e n e w s u b e t r a t e s . T h e s e o b s e r vations are true also for 7-diethylamino-4-CF3-coumarin ( ' C o u m a r i n 152") a s s u b s t r a t e . T h e r e s u l t s i n d i c a t e t h a t a t l e a s t t h e N - m o n o d e a l k y l a t i o n of t h e s u b s t r a t e s t e s t e d is c a t a l y z e d a t a m i n o r d e g r e e b y t h e Coh. In c o m p a r i s o n t o t h e 7 - a l k o x y c o u m a r i n s t h e s e new s u b s t r a t e s do n o t o f f e r a d e c i s i v e a d v a n t a g e w i t h r e g a r d t o t h e i r s e l e c t i v i t y i n r e c o g n i z i n g a n i n d u c t i o n of t h e Coh. Dept. Pharmacol.~ P h i l i p p s - U n i v e r s i t y ,

The influence of cigarette smoking on the glutathione (GSH) and malondialdehyde (MDA) contents as well as GSH-dependent enzymes (GSH-S-trensferase towards CDNB, GSH-peroxidase towards tbutylhydroperoxide, GSH-S-epoxide transferase towards 1,2-epoxy-3(pnitro-phenoxy)-propene)) was studied in maternal and fetal tissues of pregnant women. In smokers (n=5-10) continuing smoking during the whole pregnancy (5-40 cigarettes/die) a diminished GSH-content and GSH-S-aryltransferase activity was found in placentas at term, placentas of spontaneous abortion (week 12-28 of pregnancy), fetal livers and kidneys as compared to non-smokers (n=11-21). The GSH-peroxidase activities were not different between both groups, whereas the MDAcontents tended to be higher in fetal liver and kidney of smoking women. Concerning microsomal enzyme activities there was a 50% increase in dimethylhydrazine demethylase activities in postpartum placental tissue and 30% increase of ethoxyresorufin deethylase activities in fetal liver of smokers. The most important findings of our investigations were the decrease in the GSH-conjugating enzyme system in placenta} and fetal tissues of smoking women indicating a requirement of this defense system against the toxic constituents of cigarette smoke, in previous investigations smoking did not alter the activities of GSH-S-transferases in the placenta and fetal tissues (Drug Metab. Dispos. 9, 472, 1981; Xenobiotica 12, 543, 1982). Institute of Toxicology and Department of Gynecology and Obstetrics*, Medical University of L0beck, Ratzeburger Allee 160, D-2400 L0beck, FRG

Lahnberge, 355 Marburg

54 IN VITRO METABOLISMOF ETHYLCHLORIDE BY F-344 RAT LIVER AND B6C3FI MOUSELIVER MICROSOMES N. Fedtke, R. Hamphoff-KShler, T. Reinert, H.-J. Wiegand

56 LAURIC ACID METABOLISM IN RABBIT LUNG: MICROSOMAL KINETIC BEHAVIOR AND ANTIBODY INHIBITION Schulze, J. and Richter, E.

Two-year inhalation of 15,000 ppm ethyl chloride (ECL) induced uterine carcinomas in B6C3F1 mice but not in F-344 rats. The mechanism of this species and sex spec i f i c tumor induction is not known, but metabolic differences might play an important role. The in vitro metabolism of ECL was investigated using micresomes isolated from male and female F-344 rats and B6C3F1mice. Headspace gas chromatographywas used to determine the rate of the ECL metabolism in microsomal suspensions containing 12.5 mg microsomal protein and 250 ~mol/l ECL in the liquid phase. Under t h e s e conditions ECL was metabolized in a CO-inhibited and NADPH-dependent reaction. The micresomal a c t i v i t y of untreated animals towards ECL was about 1.5 times higher in mice than in rats (male rats 719, female rats 806, male mice 1304, female mice 1211 pmol ECL/min/mg). The gas-chromatograms obtained with complete incubation mixtures shewedan additional peak not present in the gas phase of the CO-inhibited or NADPH-lacking incubations. This peak was tentatively identified as acetaldehyde. The rate of ECL-metabolism was not enhanced comparing microsomes from untreated animals with microsomes from animals treated with methylcholanthrene (MC) or phenobarbital (PB). The data suggest that cytochrome P-450 catalyzed oxidation may be important in ECL-metabolism under in vivo conditions, in part resulting in the formation of acetaldehyde. However, the main MC- and PB-inducible cytochrome P-45D subfamilies P-450IIA and P-450IIB seem not to be involved in the microsomal ECL-metabolism. Hence, the possible induction of the ECL-metabolism by ECL i t s e l f acting upon the ethanol inducible subfamily P-450IIE is currently investigated.

Laurie acid (LA) is a medium chain fatty acid with strong detergent properties. For degradation mammals have evolved a specific and effective system of LA metabolizing enzymes (Group Cyp450IVA). In rat and rabbit liver and kidney up to 6 different isozymes have been identified, whereas in lung no data are available yet. Masters et al. (Rev. Biochem. T0xieol. 1985, p. 74) have suggested that in rabbit lung Cyp450IVBI is responsible for LA hydroxylation in 11- and 12-position. We therefore investigated the kinetic parameters of LA hydroxylation in rabbit liver, lung and kidney microsomes and tried to selectively inhibit LA metabolism in rabbit lung mierosomes by inhibitory antibodies. In all three organs tested the kinetic of LA-hydroxylation is similar. The concentration range tested was 1 - 400 umole LA; due to its detergent properties LA inhibited its own metabolism at concentrations over 100 umoles. It appears to be a one enzyme system with an apparent K M value of 10 umoles and an vmax of 15 nmoles/min/mg microsomal protein. These values were the same for all three organs within the margin of error. This result indicates that although different isozymes are present for L A metabolism they are indistinguishable by kinetic properties. In lung microsomes we tried to inhibit LA-metabolism by addition of anti-Cyp450IVB 1 (LA -concentration 20 umole). This addition showed doserelated inhibition of LA-hydroxylation; at 10 mg IgG/nmole eytochrome P450 only 40 - 60% of the control activity remained, with further reduction upon increased amounts of antibody. This effect could also be mimicked by addition of antibodies specific for other isozymes as Cyp4501A 1, IA2 andlIB4. Surprisingly the addition of preimmune serum and BSA in equivalent amounts also led to a 30 - 40% decrease in activity at protein concentrations of 20 mg/nmole cytochrome. We therefore conclude that in rabbit lung microsomes Cyp450IVB1 is only a minor contributor, if any, to LA metabolism. The apparent inhibition by antibodies in high concentration can be explained by distribution of laurie acid between the different pro-rein compartments, thus leading to a smaller substrate concentration available at the cytochrome P 450 system.

H~Is AG, Ps/Biologie-Texikologie, PB 12, P.O. Box 1320 D-4370 Marl, FRG

Supported by a grant from the Curt Bohnewand-fond. Walther Straub Institut filr Pharmakologie und Toxikologie, Nussbaumstrasse 26, 8000 M~nchen 2

R15 57

59

U P T A K E O F P O L Y C H L O R I N A T E D BIPHENYLS (PCB) IN CELL CULTURE SYSTEMS IS IMPROVED BY LIPOSOMES L. Potthoff*, J. Seedorf2, M. Dubowy s, K. Buff4, W. Leibold*

I N D U C T I O N A N D I S O E N Z Y M E P R O F I L E OF G L U T A T H I O N E T R A N S F E R A S E S IN H E P A T I C A N D E X T R A H E P A T I C C E L L LINES.

THE

S.F. R o e s c h Phospholipid-derived liposomes were reported to be suitable carriers for PCB. The transfer of lipophilic substances takes place by adsorption between liposomes and cells visualized by the model substance fluorophore 1,6-dlphenyl-1,3,5- hexatriene (DPH). Liposomes are able to distribute PCB homogenously in aqueous cell culture medium. Concentrations up to 78 m g PCB per kg liposome solution can be solved. Kinetic studies with a cell line (K 562) reveal a continous u p t a k e o f t h e PCB congener 2 , 2 ' , 4 , 4 " , 5 , 5 ' - h e x a e h l o r o b i p h e n y l (PCB No. 163) u p to 2 4 h o u r s w i t h l i p o s o m e s a s well a s w i t h m e t h a n o l a s s h u t t l e . While w i t h m e t h a n o l t h e maximum uptake of one million cells is 5 % o f 61 ~lg/kg in cell c u l t u r e medium, w i t h l i p o s o m e s m u c h h i g h e r c o n c e n t r a t i o n s a r e p o s s i b l e . A f t e r 24 h o u r s 18 % o f 5.6 m g / k g a r e i n c o r p o r a t e d in t h e cells. I n v e s t i g a t i o n s on t h e m e t a b o l i c a c t i v i t y o f l i n e s o f d i f f e r e n t c e l l - t y p e s ( J u r k a t , K 562, a n d P D e - B 1 ) m e a s u r e d b y the tetrazolium dye test documented that up to 4 mg/kg PCB No. 153 in l i p o s o m e s a d d e d f o r f o u r d a y s a r e t o l e r a ted within the acceptable range. Further investigations have to be done to determine the i n f l u e n c e o f PCB o n s e v e r a l f u n c t i o n s o f i m m u n e cells. *Immunology Unit, ZDept. of Chemistry, aDept, of Pharmacology and Toxicology, Veterinary School, 3000 Hannover, 4Research Centre for Environment and Health, 8042 Neuherberg, F R G

58 V79 CELLS GENETICALLY ENGINEERED FOR EXPRESSION OF CYTOCHROME P4501A2 AND M E T A B O L I S M OF A R O M A T I C AMINES AND C A F F E I N E C. J a n s s e n s and J. Doehmer A s p a r t o f o u r w o r k f o r the e s t a b l i s h m e n t o f a cell b a n k f o r p h a r m a c o l o g i c a l and t o x i c o l o g i c a l s t u d i e s V79 C h i n e s e h a m s t e r ceils were genetically engineered for stable expression of rat cytochrome P450IA2, which hydroxylates aromatic amines and demethylates c a f f e i n e . A c D N A l i b r a r y was m a d e f r o m A r o c l o r 1254 induced rat liver mRNA and was searched for cDNA e n c o d i n g C Y P 1 A 2 . Full l e n g t h c D N A w a s o b t a i n e d by j o i n i n g a small 5' c o d i n g c D N A f r a g m e n t and the rest of the c D N A . The c o n s t r u c t w a s v e r i f i e d by D N A s e q u e n c i n g . T h e r e c o n s t r u c t e d full l e n g t h c D N A w a s r e c o m b i n e d with a S V 4 0 e a r l y p r o m o t e r containing eukaryotic vector and transferred into V79 cells together with the selective marker gene neomycin phosphotransferase by the Ca/P coprecipitation technique. G418 resistant cell clones were checked for chromosomal i n t e g r a t i o n o f the c D N A , e x p r e s s i o n o f an a u t h e n t i c P 4 5 0 I A 2 mRNA and protein by Southern-, Northern-, Western-blotting, a n d i m m u n o f l u o r e s c e n c e s t a i n i n g o f f i x e d cells. E n z y m a t i c a c t i v i t y in P 4 5 0 I A 2 e x p r e s s i n g V79 d e r i v e d cell line X E M d - M Z was v a l i d a t e d by h y d r o x y l a t i o n o f 17B-estradiol and 2-aminofluorene. In addition, X E M d - M Z cells were s h o w n to be c a p a b l e o f specific d e m e t h y l a t i o n o f c a f f e i n e , w h i c h m a y b e u s e d as i n d i c a t o r d r u g in m e t a b o l i s m s t u d i e s (in c o l l a b o r a t i o n w i t h F u h r a n d S t a i b , K l i n i s c h e P h a r m a k o l o g i e , F r a n k f u r t a/M.). P 4 5 0 I A 2 a n d N - a c e t y l t r a n s f e r a s e e x p r e s s i n g V79 d e r i v e d c e l l line X E M d - N H was a p p l i e d f o r m u t a g e n i c i t y s t u d i e s o f a r o m a t i c a m i n e s s u c h as 2 - a m i n o f l u o r e n e and aminochrysene (in c o l l a b o r a t i o n with Glatt, Institut f. T o x i k o l o g i e , M a i n z ; W i e b e l , GSF, Neuherberg). I n s t i t u t ftir T o x i k o l o g i e , J o h a n n e s G u t e n b e r g - U n i v e r s i t ~ t Mainz, Obere Z a h l b a c h e r Str. 63, D-6500 Mainz, F R G

and S. H e s s e

H e p a t i c cell lines d e r i v e d f r o m rat H 4 I I E C 3 hep a t o m a c e l l s and the e x t r a - h e p a t i c cell lines V79, C 3 H I O T I / 2 , and WRC w e r e t e s t e d for t h e i r e x p r e s s i o n of g l u t a t h i o n e t r a n s f e r a s e (GST). G S T activities were measured using l-chloro-2,4-din i t r o b e n z e n e (CDNB) as s u b s t r a t e , m R N A e x p r e s s i o n of d i f f e r e n t GST forms, i.e. t h e i r subunits, was d e t e r m i n e d by N o r t h e r n blot a n a l y s i s e m p l o y i n g c D N A c l o n e s of i s o e n z y m e Y a ( ' l - l ' ) , Yc('2-2') and Y p ( ' 7 - 7 ' ) . All cell l i n e s t e s t e d s h o w G S T a c t i v i t i e s t o w a r d s C D N B r a n g i n g from 50-200 n m o l / m i n x mg p r o t e i n . E x p o s u r e of c e l l s to 20 ~ M b e n z ( a ) a n t h r a c e n e (BA) for 24 h does not s i g n i f i c a n t l y c h a n g e G S T a c t i v i t i e s , e x c e p t for a 1 . 5 - f o l d i n c r e a s e in the h e p a t i c lines 2 s F o u and H 4 I I E C 3 / G - . Y p - m R N A is f o u n d in all cell lines tested. U n d e r the same test c o n d i tions no s i g n a l for Y p - m R N A is s e e n in rat liver. BA d o e s not a f f e c t the e x p r e s s i o n of Ypm R N A in any of the cell lines. In c o n t r a s t to Y p - m R N A , Ya- and Y c - m R N A is d e t e c t e d o n l y in hep a t o m a c e l l s and rat l i v e r but not in the 3 ext r a h e p a t i c c e l l lines tested. BA i n d u c e s m R N A for Ya and Y c in h e p a t o m a lines 5L and H 4 I I E C 3 / T m o r e t h a n 2-fold. Cell lines e x p r e s s i n g s p e c i f i c G S T isoenzymes such as t h o s e i n v e s t i g a t e d h e r e s h o u l d be u s e f u l in a n a l y s i n g the m e t a b o l i s m of electrophilic compounds. GSF-Institut

fur T o x i k o l o g i e ,

D-8042 Neuherberg

60 1 , 6 - D I N I T R O P Y R E N E R E D U C T I O N IN P E R M A N E N T C E L L L I N E S IS M E D I A T E D BY S E V E R A L C Y T O S O L I C REDUCTASES U. H i l p e r - R e u t e r ,

O. C u m p e l i k and F.J. W i e b e l

We h a v e p r e v i o u s l y s h o w n that the h i g h l y m u t a genic diesel exhaust component 1,6-dinitropyrene (I,6-DNP) is m e t a b o l i z e d via o x i d a t i o n and red u c t i o n in m a m m a l i a n c e l l s (U. H i l p e r - R e u t e r et al. [1990] N a u n y n - S c h m i e d e b e r g ' s Arch. P h a r m a col. Suppl. 341, R23). - The p r e s e n t s t u d y was a i m e d at m e a s u r i n g the o v e r a l l c a p a c i t y of m a m m a l i a n cell lines for r e d u c t i o n of 1 , 6 - D N P and s e p e r a t i n g their c y t o s o l i c n i t r o r e d u c t a s e s by gel f i l t r a t i o n . - The r e s u l t s i n d i c a t e that the test cell lines h a v e s i m i l a r c a p a c i t i e s for r e d u c i n g 1,6-DNP. The e l u t i o n p r o f i l e s of the gel f i l t r a t i o n show s e v e r a l p e a k s c o n t a i n i n g 1 , 6 - D N P reductase activity. V79 and H4IIEC3/Gc e l l s h a v e a m a j o r p e a k of 1 , 6 - D N P r e d u c t a s e a c t i v i t y in the r e g i o n of 1 6 0 - 2 4 0 kDa. In c o n t r a s t , 5L and H e p G 2 c e l l s are d e v o i d of this p e a k but p o s s e s s a 1 , 6 - D N P r e d u c t a s e p e a k in the 46-96 kDa region. This p e a k is a l s o f o u n d in H 4 I I E C 3 / G - cells. X a n t h i n e o x i d a s e and N A D ( P ) H d e h y d r o g e n a s e ( D T - d i a p h o r a s e ) coelute at the r e g i o n s of 240 kDa and 68/78 kDa, r e s p e c t i v e l y . B e s i d e the two m a j o r 1 , 6 - D N P r e d u c t a s e peaks, a n u m b e r of m i n o r p e a k s are o b s e r v e d w h i c h h a v e not yet b e e n a s s o c i a t e d w i t h any k n o w n r e d u c t a s e form. - T h e r e s u l t s suggest, that a r e d u c t a s e f o r m of 1 6 0 - 2 4 0 kDa is r e s p o n s i b l e for the 1,6DNP t o x i c i t y w h i c h is o b s e r v e d in V79 and H 4 I I E C 3 / G - cells, but not in 5L or H e p G 2 cells. GSF-Inst.

f. T o x i k o l o g i e ,

D - 8 0 4 2 N e u h e r b e r g FRG

R16 61 RAM SEMINAL VESICLE CELL CULTURES, AN I N VITRO MODEL FOR STUDIES OF PROSTAGLANDIN H SYNTHASE (PHS) CATALYZED OXIDATION OF XENOBIOTICA. J. Foth and G.H, Degen P r o s t a g l a n d i n H s y n t h a s e ( P H S ) - p e r o x i d a s e has been s u g g e s t e d t o p r o v i d e a l t e r n a t i v e metabolic activation o f c e r t a i n x e n o b i o t i c s i n t i s s u e s low in monooxygenases (MFO), b u t so f a r i t has been difficult t o a s s e s s t h e r o l e o f PHS in m e d i a t i n g a d v e r s e e f f e c t s i n c e l l s o r in v~vo, SEMV c e l l s d e r i v e d f r o m ram s e m i n a l v e s i c l e s c o n v e r t a r a c h i d o n i c a c i d (ARA) e f f i c i e n t l y to prostaglandins but lack MFO-activity. They were used t o c o n d u c t s t u d i e s on t h e PHS-mediated m e t a b o l i s m o f e s t r o g e n s w i t h t h e goal o f r e l a t i n g t h i s t o an e n d p o i n t f o r g e n o t o x i c i t y inducible in t h i s in v i t r o model. D i e t h y l s t i l b e s t r o l (14C-DES, 2 NM) i s c o n v e r t e d t o s u l f a t e c o n j u g a t e s and t o the oxidative metabolite Z,Z-dienestrol (Z,ZDIES) by SEMV c e l l c u l t u r e s . A d d i t i o n o f ARA o r i n d o m e t h a c i n (5 #M) i n c r e a s e d and i n h i b i t e d Z,ZDIES f o r m a t i o n by 60 and 50 %, r e s p e c t i v e l y , indicating free fatty acid availability and h y d r o p e r o x i d e s as f a c t o r s i n f l u e n c i n g DES-oxidat i o n by PHS. A c o m p a r i s o n o f ARA c o n v e r s i o n t o p r o s t a g l a n d i n s and o f DES o x i d a t i o n r a t e s f u r t h e r r e v e a l s t h a t DES i s c o o x i d i z e d a l t h o u g h e n d o g e nous c o m p e t i n g c o s u b s t r a t e s f o r PHS a r e p r e s e n t , The r e s u l t s d e m o n s t r a t e t h a t SEMV c e l l s a r e a u s e f u l t o o l f o r s t u d i e s on t h e PHS-dependent o x i d a t i o n o f DES; s i n c e i n d u c t i o n o f m i c r o n u c l e i by DES has a l s o been o b s e r v e d in t h i s model i t c o u l d a l s o p r o v i d e c l u e s as t o t h e r o l e o f PHS in m e d i a t i n g g e n o t o x i c i t y o f DES and o t h e r xenobiotics. S u p p o r t e d by t h e Deutsche F o r s c h u n g s g e m e i n s c h a f t Institute o f T o x i c o l o g y and P h a r m a c o l o g y , SFB 172, V e r s b a c h e r S t r . 9, D-87OO WGrzburg

62

EXPRESSION A N D REGULATION OF PHENOL UDP-GLUC U R O N O S Y L T R A N S F E R A S E IN HEPATOCYTE CULTURES, HEPATOMA (H4IIE) CELLS A N D IN U N T R A N S FORMED A N D TRANSFORMED RAT LIVER EPITHELIAL CELLS E. R6hrdanz and P. Mfinzel UDP-Glucuronosyltransferases (UGTs) are involved in detoxication and elimination of endobiotics and of a vast array of xenobiotics including carcinogens. One isozyme of this supergene family, called phenol UGT or UGTI, is expressed at a low level in hepatocytes, but inducible by 3-methylcholanthrene (MC)-type inducers and by multiple other factors. The enzyme levels appear to be high in H41IE hepatoma cells and in rat liver epithelial cells (RLEs). In order to study mechanisms responsible for this constitutively higher expression of UGTI, a UGTI-selective DNA probe was used. Two oligonucleotide primers, prepared according to the cDNA of UGTI, and isolated rat liver DNA were used to synthesize a 2$0bp fragment (nucleotide 71350) by the polymerase chain reaction. In the presence of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) UGTI mRNA level was increased 3-fold in primary hepatocytes. Persistently increased expression (7-fold) was observed in H4IIE cells and expression was further inducible by TCDD. Regulation of UGTI was also studied in RLEs (McMahon et al. 1986, Cancer Res. ~fi, 4665). UGTI-expression depended on cell density and passage number. Expression of UGTI in RLEs of low passage number was highest at day 3 after plating whereas enzyme activity reached its maximum at day 6. Aflatoxin B~transformed RLEs and RLEs of higher passages showed even higher UGTI mRNA levels than RLEs of low passage number. The results suggest that (in contrast to primary hepatocytes) H411E cells, RLEs and transformed RLEs show high persistent expression of UGTI, a behaviour which is also found at cancer prestages (hepatocyte foci and bepatocyte nodules). The cell lines may be useful to elucidate mechanisms responsible for high constitutive expression of the isozyme.

Institute of Toxicology, University of Tiibingen, WilhelmstraBe 56 D-7400 Tiibingen, Germany

63 Biotransformation in carcinogen-induced diploid and polyploid hepatocytes separated by centrifugal elutriation J. B. Watkins III I, D. Thierau 2 and L.R. Schwarz 2 Recently it has been shown that sequential treatment of rats with partial hepatectomy, DEN and 2-acetylaminofluorene (2-AAF)('Seglen protocol') induces the emergence of diploid hepatocytes, which are thought to contain precursor cells of hepatocellular carcinomas (Saeter et al Carcinog. ~,939, 1988). It is not known whether these cells exhibit the 'toxin-resistance phenotyp' characterized by decreased activities of cytochrome P-450 dependent monooxygenaees and increased activities of several phase II enzymes. TO address the question of whether biotransformation is altered in diploid hepatocytes induced according to the 'Seglen protocol', diploid and polyploid hepatocytes were separated from hepatocytes suspensions isolated from treated rats by centrifugal elutriation. The purity of diploid and polyploid cell fractions amounted to 90 and 79 %, respectively. Benzo(a)pyrene hydroxylase, ethoxyresorufin O-deethylase (EROD) and methoxycoumarin O-demethylaee (MCOD) activities were 30-40% lower in diploid than in polyploid cells. Activities of glutathione S-transferases towards CDNB, UDP-glucuronosyltransferases towards 3-hydroxybenzo(a)pyrene(3-OHGT) or 4-hydroxybiphenyl and DT-diaphorase did not differ in the two elutriated fractions. Treatment with 2-AAF for 3 weeks induced EROD and 3-OH-GT 4-6 fold and 2-fold, respectively and administration of phenobarbital for 4 d increased EROD and MCOD activity 2- and 4-fold, 3-OH-GT activity and glutathione conjugation 2- and 1.5-fold, respectively, in both diploid and polyploid hepatocytes. In conclusion, although there is some decrease in monooxygenase activities, carcinogen-induced diploid hepatocytes do not show the typical 'toxin-resistance phenotype'. This finding is compatible with the hypothesis that proliferation and promotion of diploid hepatocytes by 2-AAF is caused by non-toxic mechanisms. 1 GSF-Institut fHr Toxikologie, D-8042 Neuberberg/MHnchen 2 Indiana Univ. School of Medicine, Bloomington, IN 47405

64 FRESHLY ISOLATED AS WELL AS CRYOPRESERVEDHEPATOCYTES ARE SUITABLE IN VITRO MODELS FOR THE BIOTRANSFORMATIONOF NEBRACETAM IN THE MOUSE, RAT, RABBIT, DOG, MONKEYAND MAN IN VIVO K.L. P l a t t , H. RuBm, E. Molitor, H. Maith, D. Utesch, B. Diener, W. Tr6ger ~, K. Mendla~, and W. Pollmann ~ The biotransformation and disposition of nebracetam (4aminomethyl-l-benzylpyrrolidin-2-one) was determined in the mouse, rat, rabbit, dog, monkey, and in man by oral administration of the carbon-14 labelled drug (2-5 mg/kg body weight) and measurement of the renal (82 - 96%) and the fecal (2 - 15%) excretion; nebracetam and i t s metabol i t e s excreted via the urine were separated by reversedphase HPLC which revealed a metabolic conversion of 35 88% and marked species differences in the metabolite profiles. Freshly isolated hepatocytes (mouse, r a t , r a b b i t ) as well as cryopreserved hepatocytes (rabbit, dog, monkey, man) were incubated with nebracetam (I0 ~M, 2 m i l l i o n c e l l s / m l ) For up to 5 h followed by HPLC separation of the metabol i t e s . The r e s u l t of t h i s in v i t r o study exhibited good q u a l i t a t i v e and s a t i s f a c t o r y q u a n t i t a t i v e agreement with the in vivo metabolism; the dog being the animal species which most closely resembles man in i t s biotransformation of nebracetam. This example demonstrates that freshly isolated as well as cryopreserved bepatocytes are suitable in v i t r o metabolism systems f o r the prediction of the hepatic metabolism of a drug in vivo. We, therefore, propose to select the r e l e vant animal species f o r chronic t o x i c i t y testing of a newly developed drug by in v i t r o metabolism studies with isolated hepatocytes. Supported by the Bundesministerium fur Forschung und Technologie I n s t i t u t f~r Toxikologie der Universit~t, Obere Zahlbacher StraBe 67, 0-6500 Mainz Abteilung Biochemie, Boehringer Ingelheim KG, D-6507 Ingelheim

R17 65 METABOLISM MEDIATED CYTOTOXICITY DETECTED BY COCULTURES OF MICROCARRIER-ATTACHEDRAT HEPATOCYTES AND BALB/C 3T3 CELLS J.U.Voss and H.Seibert The t o x i c i t y of numerous chemicals is modified by biotransformation reactions in the l i v e r . To assess the influence of xenobiotic-metabolizing pathways on the t o x i c i t y of chemicals in v i t r o , several approaches have been applied (e.g. S9-mix, freshly isolated or primary cultured hepatocytes in coculture with target cells). In an e f f o r t to improve the application of rat hepatocytes in cocultures, we developed a method to cultivate hepatocytes on microcarriers. Liver cells isolated from male rats by two-step collagenase perfusion were inoculated with collagen-coated microcarriers (Cytodex 3, Pharmacia) in Waymouth 752/I supplemented with I0% FCS, insulin, amino acids and HEPES. 24 hours after inoculation, about 34% of cells had attached to microcarriers corresponding to 0.14 mg protein/mg dry weight of microcarriers. Cells retained the a b i l i t y to metabolize xenobiotics as judged by the a c t i v i t y of 7-Ethoxycoumarindeethylase (EOD) f o r at least 48 hours. In the present study microcarrierattached hepatocytes were cocultured with BALB/c 3T3 fibroblasts as target cells. The cytotoxic effects of cyclophosphamide (CPA) were studied. Incubation conditions were examined with respect to incubation time and hepatocyte: 3T3-cell r a t i o . In the presence of hepatocytes CPA lead to a dose-dependent growth retardation of 3T3 cells as measured by protein content/ well, whereas no toxic effects on the hepatocytes were observed. Microcarrierattached hepatocytes can become a valuable tool to assess the influence of metabolism on t o x i c i t y of chemicals in cocultures. Abt. Toxikologie, Universit~t Kiel, Brunswiker Str. lO, 2300 Kiel, FRG

67 METABOHC FATE OF METHYLENE CHLORIDE IN HUMAN BLOOD R. Thier, R. Kreiling#,H. Ottenw~ilder*

An increased incidence of pancreatic tumors in humans following occupational exposure to methylene chloride has been reported by the European Community. In contrast, long-term inhalative studies with high concentrations of 2,000 and 4,000 ppm substance resulted in formation of liver and lung tumors in mice, but not in rats. This implies the existence of a species difference in either the metabolism of methylene chloride or its organ distribution. The aim of the study presented here was to investigate the distribution of radioactively labelled methylene chloride in the different components of human, since blood is the main transport vehicle for inhaled xenobiotics preceeding organ distribution. Ten human blood samples of healthy volunteers (9 ml each) were individually incubated in 22 ml head space vials with either 10 ~tl methylene chloride spiked with 14-C-labelled substance with a spec. act. of 1.3 MBq/mmol, or 0.2 ml saturated aqueous solution of the pure radiolabelled substance with a spec. act. of 925 MBq/mmol. Aliquots of 0.5 ml were drawn in 30 rain intervals and fractionated into plasma and cellular compartments by differential centrifugation. The lymphocytes were isolated by density centrifugation through a separation medium. The erythrocytes were separated from the remaining cellular components by filtration through microcristalline cellulose. The erythrocytes were lysed by addition of an equal volume of distilled water and subsequently divided into cell membranes and high and low molecular weight fractions of cytoplasm with a cutoff at 10,000 Da. Plasma differentiated likewise. Radioactivity was determined by liquid scintillation counting. The results show the existence of two distinct subpopulations among humans in terms of distribution of radioactivity between the high and low molecular weight fractions of blood plasma. In five samples, group A, radioactivity increased significantly over time in the low (< 10,000 Da) and high (> 10,000 Da) molecular weight fractions. The other samples, group B, showed no increase of radioactivity in either fraction. Radioactivity was not detectable in the erythrocytes of either group. Additional experiments showed a coincidence of the two groups with ,,conjugators" and ,,nonconjugators" for monohalogenated methanes in human erythrocytes. This suggests a common metabolic principle and an enzyme polymorphism for methylene chloride in human blood plasma. Institut ffir Arbeitsphysiologie, Ardeystr 67 4600 Dortmund 1 ~Bundesmln.f. Jugend,Frauen,Famiheund Gesundheit,Deulschherrenst~.2, 5300Bonn2 ~Fa. Hoechst, Gewerbetoxikologie, Postfach 800320, 6230 Frankfurt 80

66 ENZYME POLYMORPHISM FOR METHYL BROMIDE IN HUMAN ERYTHROCYTES MODULATES ITS GENOTOXIC RISK K.R. Schr/Sder, T. Langhof, H. Peter The genotoxic risk induced by xenobiotics can be modulated by enzyme polymorphism. Methyl bromide is conjugated in human erythrocytes with glutathione by approximately 60 % of the human population (conjugators), the rest lacking this ability (non-conjugators). The genotoxic potential of methyl bromide has been attributed to a direct alkylation of DNA; a detoxification of the substance would therefore reduce this genotoxic risk. Since blood is the first compartment and major transport vehicle for inhaled xenobiotics after passage of the lung, metabolism in erythrocytes as in the case of the ,,conjugators" could have a pronounced protective effect. In order to verify an interindividual difference in the genotoxic effect of methyl bromide, individual whole blood samples of 5 non-conjugators and 5 conjugators (3.5ml each) were incubated in 22ml head-space vials with an initial concentration of 3.6 Ixmol (5000 ppm) methyl bromide for one hour. Substrate disappearance in the gas phase of the vials was controlled by gas chromatographyon a Perkin Elmer Sigma 5 GC equipped with a 5 m 1/8" stainless steel column packed with Tenax TA 30-60 mesh, gas sample loop 0.25 ml. Following the incubation, lymphocytes were isolated by density centrifugation through a separation medium. After 24 hours of cultivation in a specific medium at 37°C, bromodesoxyuridine was added and the cells incubated for further 46 hours• Colcemide was then added for termination of mitosis; 2 hours later the cells were fixed and stained on glass slides for microscopic determination of sister cbromatid exchanges (SCE). The rate of SCE was found to be significantly higher in the case of the nonconjugators. Furthermore the rate of SCE in the lymphocytes of the conjugators was not enhanced following the exposure to methyl bromide compared to non-incubated controls. This indicates a protective effect of glutathione conjugation of methyl bromide in human blood, and the influence of the enzyme polymorphism for genotoxic risks. A further characterization of the enzymatic factor responsible for this phenomenonis therefore necessary. Preliminary experiments give evidence for the involvement of an enzyme which accepts both glutathione and cysteine as cofactor, indicating the existence of an enzyme with both glutathione transferase activity and glutathione peroxidase activity. Instimt ftir Arbeitsphysiologie, Ardeystr. 67, 4600 Dortmund 1

68 Chromium (VI) reducing capacity of human blood plasma in vitro M.Capellmann, H.M.Bolt Since chromates play a considerable role in industrial application, e.g. in welding or tanning, biological monitoring of exposed workers is important. In the metabolism of chromium(VI) the reducing capacity of human blood plasma is of importance, because reduction of Cr(VI) is regarded as a detoxification step. In the case of plasma, ascorbic acid accounts for the majority of its reducing capacity. To prove this, chromate solutions of different concentrations (20-80 ng/ml) were added to solutions containing ascorbic acid in the "physiological" concentration range (5-20 gl/ml), simulating the normal conditions in plasma (0.2 M HEPES-buffer, pH 7.4) and were also added to plasma separated from freshly drawn blood. The incubation was carried out at 37 °C. From both systems ascorbic acid and its oxidized form dehydroascorbic acid, were analyzed simultaneously by H P L C - s e p a r a t i o n and post c o l u m n d e r i v a t i z a t i o n with 1,2phenylendiamindihydrochloride. The decrease of chromate was measured by flow injection analysis using diphenylcarbazide as derivating reagent. The kinetics of the model system show a reaction of 2nd order at higher concentrations, while at lower concentrations the reaction is apparently autocatalysed. The results obtained by spiking human blood plasma are similar, as shown in a comparison between the reaction curves of the model solutions and those received after spiking human blood plasma. Institut ftir Arbeitsphysiologie an der Universit~it Dortmund Ardeystr. 67 W-4600 Dortmund 1

R18 69 METABOLISM OF MEBENDAZOLE IN A MODIFIED PERFUSED GUT SYSTEM H. Gottmanns, R. Kroker, and F.R, Ungemach*

71 ISOLATED

CHIRAL EFFECTS IN THE METABOLISM OF 1,3-BUTADIENE IN MALE B6C3F1 MICE U. Hindermeier, S. Deutschmann, D. Wistuba*

The benzimidazole anthelmintic mebendazole which is known for its inter- and intraindividual varying bioavailability had recently been investigated by an isolated perfused rat gut system for its absorption and biotransformation. Poor absorption and strongly varying metabolism under different conditions had been demonstrated (1}. However the validity of this perfusion system is limited due to the failure of a continuous flow at the serosal side for the removal of the absorbate. Thus there is no sufficient (gut wall/serosal absorbate) concentration gradient as driving force for the absorption process. Therefore in a modified system of the isolated perfused gut it has been tried to substitute the cut off of serosal blood flow. Into the flexible tube connection between the two chambers of a perfusator according to Fisher and Parsons (2l a cannula was inserted which allows to supply the serosal side of the isolated perfused gut segment with a continuous definite flow of an adequate medium. The modification of the system resulted in an increased absorption of metabolites followed by further metabolic steps with a rise of resecretion of the metabolites into the gut lumen and enhanced secretion of metabolites (especially glucurenidal into the absorbate while the absorption of the parent substance remained almost unchanged. The results indicate an increased absorption of mebendazole metabolites with different patterns under continuous serosal flow without markedly affecting the bioavailability of the active parent substance. 1) Gottmanns et al., N.S. Arch. Pharmacol. 337, 44, 1988 2l Fisher and Parsons, J. Physiol. 110, 36-46, 194-9

The plastic monomer 1,3-butadiene has been classified as a possible human carcinogen by the IARC (International Agency for Research on Cancer). Long-term inhalation studies showed a carcinogenic effect down to a concentration of 6 ppm in mice but not in rats. In the experiments presented here, male B6C3F1 mice were exposed in an open system to a constant inhalative concentration of 1,000 ppm 1,3butadiene for 8 hours. After the exposure, the animals were transferred to a closed desiccator. One hour after the transfer, 60 ml samples of the atmosphere in the desiccator were drawn with a syringe and flushed through closed 22 ml head space vials, which were subsequently frozen with dry ice. For further analysis; the vials were later thawed and subjected to complexation gas chromatography on a 25 m x 0.3 mm glss capillary colum coated with 0.125 mol nickel(1])bis[(3-heptafluorobutanoyl)-(1R)-camphorate)] on SE 30; 90 C, 1.5 bar N2. The animals were found to exhale the reactive intermediate 1,2-epoxy-3-hutene, the ratio between the S- and the Renantiomer of the epoxide beeing 74.1 to 25.9 %. This supports findings by Wistuba et al. (Chirality 1, 127-136, 1989) that the metabolism of 1,3butadiene by cytoehrome P-450 of mice and rats in vitro results in the formation of 70 % S- and 30 % R-enantiomers of 1,2-epoxy-3-butene. The results/n vitro and/n vivo suggest that chirality may play an essential role for the genotoxicity of the reactive metabolites of 1,3-butadiene and may be responsible for the observed species differences. Following 7 hour inhalative exposure to 1,3-butadiene concentrations of 10, 50, 100, 250, 500, 1,000 and 2,000 ppm, the mice were sacrificed and liver, lung and heart removed and shock frozen in liquid nitrogen. After thawing, the organs were homogenized and deproteinized with 4% aqueous sulfosalicylic acid. The glutathione content was determined with Ellman's reagent. Above concentrations of 500 ppm 1,3-butadiene, a dose-dependent depletion of glutathione was observed in all organs investigated. This indicates that in mice, glutathione conjugation of 1,2-epoxy-3-butene, the chiral reactive intermediate of butadiene, does not influence the ratio of both enantiomers of this epoxide in exhaled air.

Bundesgesundheitsamt, Postfach 330013, 1000 Berlin 33, FRG *lnstitut fur Pharmakologie u. Toxikolikologie, Freie Universitit Berlin, Koserstr.20, 1000 Berlin 33, FRG

Institut ffir Arbeitsphysiologle, Ardeystr. 67, 4600 Dortmund 1 *Inst. ffir Organ. Chemic der Univ. Tiibingen, Auf der Morgenstelle 18,. 7400 Ttibingen

7O COUMARINMETABOLISM IN ISOLATED PERFUSED RAT LIVER Th. Huwer, H.-J. Altmann, W. Grunow ........................................................ The biotransformation of coumarin (Cou) was studied using a recirculating isolated perfused rat liver system. Liver vitality was ensured by normal appearance, activity of lactate dehydrogenase in perfusate, oxygen-consumption, perfusion rate and bile flow over the experimental period of 4 hr. 14-C-Cou was added to the perfusate to yield a final concentration of 50, 150, 500 or i000 ~mol/l. Examination of the perfusate by means of HPLC revealed rapid metabolism. In the lower dose experiments (50 or 150 ~M), Cou was completely metabolized after 90 or 120 min. In the higher dose experiments the concentration of Cou at the end of experiment was 6% (500 ~M) or 15% (I000 ~M) of the applied concentration, o-Hydroxyphenylacetic acid (OHPAA) proved to be the most prominent metabolite, representing about 50% of all metabolites. Furthermore, 3-hydroxycoumarin (3OHC), o-coumaric acid (CA), ohydroxyphenyllactic acid and traces of 7-hydroxycoumarin (7OHC) were identified. In addition, several unknown metabolites were also detected. Biliary elimination of Cou-equivalents was dosedependent, ranging from 7% (I000 ~M) to 35% (50 ~M) of dose with a maximum between 30 and 60 min. The major part of radioactivity in bile was associated with unknown metabolites. Besides, we detected unchanged Cou and conjugates of OHPAA, 3OHC, CA and 70HC. Final total glutathione content of the liver ranged between 0.12 ~mol/g in the highest dose and 3 ~mol/g in the control group. 4-Glutathione and 4-mercapturic acid conjugates, as well as Cou-3-mercapturate recently isolated from rat urine, were absent in bile and perfusate. 5-7% of the applied dose remained in liver at the end of perfusion. A dose-dependent covalent binding of Coumetabolites to protein was observed. Max-von-Pettenkofer-Institut, BGA, Thielallee 88-92, i000 Berlin 33

72 SPECIES SPECIFIC PHARMACOKINETICS OF 2-NITROPROPANE IN RABBITS,ANDIN RATS P. Stei. G. Csan~v*. J.G. Fitser The solvent 2-Nitropropane (2-NP) produced liver necrosis and hepatocarciname in each of f0 rats exposed to 207 ppm for 6 months. However, in 5 rabbits exposed under identical conditions no alterations were detected (Lewis et al., J. Environ• Path. Toxicol. 2: 233, 1979). In pharmacokinetic studies with rats we could distinguish two different metabolic pathways for 2-NP, a saturable and a non-saturable one (Denk et al., Arch. ToxicoL SuppL 13: 330, 1969). Based on several further studies, we concluded that especially the non-saturable pathway would lead to hepatotoxicity and hepatocarcinogenicity in contrast to the saturable one (Denk et el., Arch. Toxicol. Suppl. 13: 330; Denk and Deml, this meeting). In order to test this conclusion we studied the pharmacokinetics of 2-NP in the rabbit strain that had proved not sensitive in the carcinogenicity study mentioned above. New Zealand White rabbits of both sexes were individually exposed in closed chambers (65 I) to atmospheric concentrations of 2-NP. Some animals were pretreated i.p. with pyrazole (320 rng/kg body weight) to inhibit cytochrome P450 mediated metabolism. Different amounts of 2-NP were administered by single injections into the systems leading to initial concentrations between 1000 and 6000 ppm. Concentrations in the atmosphere were measured by gas chromatography. Pharmacokinetic analysis of the experimental concentrationtime courses was done as previously described (Kessler et al., NATO ASI Series A: Life Sciences 159: 123,1989). The thermodynamic partition coefficient whole body/air was 170. This value was similar to that obtained in rats. Like in rats the uptake of gaseous 2-NP was 100%, its rate was limited by the alveolar ventilation which in this species is referred to be about 60000 mYh per animal of 2500 g (Guyton, Am. J. Physiol. 150: 70, 1947)• Furthermore also in rabbits two different metabolic pathways have been found but in contrast to rats no sex differences. The pathways were a saturable (Vmax = 126 [.umol/h/kg body weight]) and a non-saturable one• After pretreatment with pyrazole, only the latter pathway was still detectable. The capacity of this pathway was smaller than that of the saturable one below 300 ppm. In contrast, above 300 ppm it exceeded that of the saturable pathway• In rats however, the relative capacity of the saturable pathway was much smaller: Here, at 180 ppm (females) and 60 ppm (males) already the rates of both pathways were equal. At higher concentrations more 2-NP was metabolized via the non-saturable pathway. These findings support strongly the hypothesis formulated above concerning the different hepatotoxic and hepatocarcinogenic effectiveness of the two metabolic pathways of 2-NP. *Central Research Institute of Chemistry, Hungadan Academy of Sciences, Budapest, Hungary GSF - Institut f0r Toxikologie, Ingolst&dter Landstra6e 1, D-8042 Neuherberg

73

75

PHARMACOKINETICSOF STYRENE-7,8-OXIDEIN MOUSE AND RAT

TRACE ANALYSIS OF ETHYLENE OXIDE IN AMBIENT AND ALVEOLAR AIR M. Leutbecher, B. Marczynski*, U. FOst

X Jiang and 1~ Kessler

Styrene-7,8-oxide(SO) is a first metabolite of styrene (S). SO was mutageNc and induced tumors exclusively in the forestomach of mice and rats after oral administration. The body burden of SO generated from S depends on the

Ethylene oxide, one of the major products of the chemical industries, is an air p ollutan! emitted by various sources like exhaust fumes and cigarette

1990, Gentner Verlag, in press). Here, we present pharmacokinetic data of SO in B6C3F1 mice and in Sprague-Dawleyrats: 1. after inhalation el S and 2. after intraperitoneal (ip) and oral (po) administrationof SO. The concentration of SO in blood was directly determined (Kessler etal., J.Chromatogr. Biomed.Appl., 1990, in press). 1. In mice and rats, we found saturation kinetics for the metabolism of S w i t h Vmax=15 [p.mol/h]and atm.conc, at Vmax/2=270 [ppm],calculated tot I mouse, and with Vmax=56 [umol/hl and atm.conc, at Vmax/2=lgn [nnml c..~l~ill~t~dfor

environmental and/or endogenous sources, it is essential to detect extremely small amounts of the substance and its precursor in ambient and alveolar air. The carcinogenic potency of ethylene oxide has clearly been proven in animal experiments. In Germany, the biological monitoring concept (EKA) favors the determination of ethylene oxide in alveolar air and whole blood samples; the methods proposed by the regulatory organ have however a detection limit of 0.5 PPm.

conditions. In

rats,

SO

concentrations correlated

with

metabolic rates

of

S.

However, no such correlation was found in mice: The blood concentrations of SO increased linearly at concentrations of S between 250 and 800 [ppm]. The blood concentration of SO was 8 [ng/ml] in mice and 20 [ng/ml] in rats after exposures to 20 [ppm] of S. After exposures to 800 [ppm] of S the concentration of SO in blood of mice (4700/rig/roll) was much higher than that in rats (340 [ng/ml]). Fromthese results we expect that mice being exposed to high concentrations of S will be more sensitive than rats to effects evoked by the metabolite SO.

w,um me stomacn, investigationsm wrcosnowed mat me hair-,re or ;50 at 3/~{.; depended directly on the pH-value: In H2Obidest.(pH 6.2) half-life was 445 [min], in 1 mM HCI it was 0.4 [min]. The low amount of SO found in blood after po administration may explain the exclusive induction of forestomach tumors observed in long-term studies in both species.

GSF- Institut for roxikologie, IngolsNdter Landstr. 1, 8042 Neuherberg

v .....

3, . . . . . . . . . . .

,,~lu~

~L,,),~,,~.

~n~

a~my~

w~L~

p~lJ.utm~.,u

un

a z, m

capillary column packed with GS/Q, (J & W Sci., 0.5 mm i.d.). Two different methods were developed for these experiments. In the first case, ethylene oxide was reacted in aqueous solution with sodium pipefidinodithiocarbamate, a substance that selectively reacts with epoxides. The reaction products were extracted with methylene chloride and reacted with MSTFA(N-methyl-Ntrimethylsilyl-trifiuoroacetamide) togivethe volatileTMS derivatives. This solution was analyzed by GO/MS. Alternatively, air samples were condensed into a trap cooled with liquid nitrogen, reacted with hydrogen bromide and extracted with methylene chloride. The organic phase was analysed by GO/

--

-/-Jr- ............

j

v._

rE

........................................................

enables a quantification and differentiation of ethylene oxide resulting from either endogenous or exogenous sources or metabolized from endogenous or exogenous ethylene.

Institut fur Arbeitsphysiologie, Ardeystr. 67, 4600 Dortmund 1 * BG Krankenanstalt Bergmannsheil, Gilsingstr. 14, 4630 Bochum 1

74

76

SIMULTANEOUS DETERMINATION OF MERCAPTURIC ACIDS AND THE ENANT/OMERS OF MANDELIC ACE) IN THE URINE OF HUMANS EXPOSED TO STYRF.NF.

2-NITROPROPANE: RELATIONSHIP BETWEEN INDUCTION OF PRENEOPLASTIC FOOl IN RAT LIVER AND METABOLISM ~ n ~ t , ~,4 = n ~ t

Styrene is metabolized in humans to styrene oxide, which is genotoxic in various test systems. Detoxification of this epoxide follows two major pathways, the excretory products being mandelic acid/phenylglyoxylic acid and mercapturic acids. Since the epoxide hydrolase pathway quantitatively overweighs the GSH-transferase pathway in humans, mandelic acid has been chosen as a biomonitor for occupational exposure to styrene. Both styrene oxide and mandelic acid are chiral molecules. Therefore, the enantiomers S- and R-mandelic acid were analyzed selectively in the urine of 20 workers exposed to styrene. For this purpose, the compounds were isolated from acidified urine by solid phase extraction on Porapak Q and subsequently eluted from the adsorbent with acetonitrile. The S- and Renantiomers of mandelic acid were quantified by capillary gas chromatography on a chiral column. In a parallel analysis of the same urine samples, mercapturic acids and mandelic acid were separated on 20x20 cm silicagel tic plates with fluorescence indicator. The two metaboiites were differentiated by selective staining. The mercapturic acids used as markers were synthesized in incubations of N-acetylcysteine with the pure enantiomers of styrene oxide and further characterized by NMR spectroscopy. The ratio between the S- and R- forms was found to vary between 1:1 and 4:1. R-styrene oxide is a stronger mntagen than the S- form in the Ames test, indicating that enantioselective preference could lead to a difference in susceptibility to genotoxic effects of styrene oxide, In rats, the GSH transferase pathway has been found to play an important role in the metabolism of styrene oxide. In humans, an enzyme polymorphism hasbeen described for GSH transferase m in the liver; thiswas testedwith styrene oxide as substrate, About 40% of the population were found to be deficient in the enzyme. In our experiments, interindividual differences in the urinary excretion of the mercapturic acids by the exposed workers support the existence of the enzyme polymorphism. An individually different detoxification of styrene oxide via the GSH-transferase pathway could lead to an influence on the biological effective dose of styrene oxide and thus to a modulation of the genotoxic effects.

,'-r~uropropane ~z-t'~w) is an i n a u s m a l SOlvent. innalatlve e x p o s u r e of rats to high concentrations of about 200 ppm c a u s e d hepatotoxicity and liver carcinomas, 25 ppm h o w e v e r were ineffective (Lewis etal., J. Environ. Pathol. Toxicol. 2: 233, 1979; Griffin etal., Ecotoxicol, Environ. Safety, 4: 267,1980). For determining the concentration-response relationship we studied the metabolism of 2-NP in Sprague-Dawley rats of both s e x e s at concentrations between 0 and 250 ppm (Denk etal., Arch. Toxicol. Suppl. 13, 1989), and the induction of

Institut ffir Arbeitsphysiologie an der Universiffit Dortmund, Ardeystr. 67, 4600 Dortmund 1 * Inst. f. Arbeits- & Sozialmedizin, Uni Ttibingen, Wilhelmstr. 27, 7400 Tfibingen

p r e n e o p l a s t i c liver foci using the rat liver foci b i o a s s a y described by Oesterle and Deml (J. Cancer Res. Olin. Oncol. 105:141, 1983). In adult rats, 2-NP was metabolized via two different pathways: a saturable one of low capacity and high affinity according to Michaelis-Menten kinetics, and a non-saturable one following first-order kinetics. Striking sex differences were observed in the kinetics of metabolism: Vmax of the saturable pathway w a s higher in females, w h e r e a s first-order metabolism w a s faster in m a l e s than in f e m a l e s leading to higher rates of total metabolism. The n u m b e r s of loci were found to be related to the exposure co ncentrations of 2-NP resulting in upwards concave curves. The shapes of these curves may be explained by the interference of the saturable with the non-saturable pathway. This study indicates that no threshold d o s e in the hepatocarcinogenic effect of 2-NP can be expected. However, the risk is relatively lower at low concentrations of 2-NP. GSF - Institut for Toxikologie, Ingolstgtdter kandstr. 1 D-8042 Neuherberg

R 20 77 LIPID PEROXIDATION AS A INDUCED HEPATOTOXlCITY. M. Younes* and O. Strubelt

MECHANISM OF VANADATE-

The toxicity of sodium orthovanadate (2 mmol/I) towards isolated perfused rat livers was studied. In livers from fasted rats, vanadate led to a release of cytosolic and mitochondrial enzymes, an accumulation of calcium in the liver, a marked depletion of glutathione and an enhanced release of it into the perfusate, as well as to an augmented formation and release of thiobarbituric acid reaction material. Furthermore, a marked inhibition of oxygen consumption and a progressive decrease in perfusate flow rate due to vanadate-induced vasoconstriction were observed. Control experiments with similarly reduced perfusion rates in the absence of vanadate led to a release of cytosolic but not of mitochondrial enzymes. Also, calcium accumulation and lipid peroxidation (LPO) were not evident, indicating that reduced flow rate was only partly responsible for vanadate-induced hepatotoxicity. Feeding the animals resulting in an activation of anaerobic energy conservation reactions strongly attenuated vanadate-induced toxicity indicating that the energy status of the liver is the main target of venadate. LPO and liver damage were inhibited in parallel by allopurinol and deferrioxamine. Together with the strong cor~lation between LPO and hepatotoxicity, this suggests that LPO plays a causative role in vanadate-induced liver damage. Ommission of calcium from the perfusate did not prevent hepatotoxic responses to vanadate, indicating that calcium influx is not involved in vanadate-induced toxicity towards the perfused rat liver.

79 CELLULAR INTERACTION IN RAT HEPATOCYTE / KUPFFER CELL COCULTURE H.Desel, G.Latocha, M.MQIler, A.Steding, C.Guthardt, & C.SchrSder Damage of liver parenchymal cells (HC) in inflammatory liver disease is influenced by components of the cellular defense system. The role of liver macrophages (Kupffer cells) is not yet understood in detail. We have studied the interaction between these cell types in a hepatocyte / Kupffer cell (KC) coculture (1:1). Rat hepatocytes were cultured for 24 h. Rat KC were separated by elutriation centrifugation and added to the HC culture. Experiments were done after 48 h of coculture. Superoxide release after stimulation with zymosan or phorbol myristate acetate is strongly increased in coculture as compared to KC monoculture. - Production of malondialdehyde (MDA) in cell homogenates is enhanced in stimulated cocultures as compared to the unstimulated control experiment. Gross hepatocyte damage as determined by release of lactate dehydrogenase (LDH) is not observed with and without KC stimulation. - The toxicity of exogenous H202 to HC is' markedly enhanced by the presence of KC. MDA production in coculture is 3-10 times the production in HC monoculture. This effect can further be enhanced by KCstimulation. LDH release is also increased in coculture. H202 decomposition by HC via catalase and/or glutathione peroxidase is slowed down in the presence of KC.

-

-

Institute of Toxicology, Medical University of L~ibeck, Ratzeburger Allee 160, D-2400 L0beck, FRG. *Present address: Max von Pettenkofer-lnstitute, BGA, D-1000 Berlin 33, FRG.

These data demonstrate mutual communication between HC and KC in vitro: HC stimulate superoxide production and KC damage HC directly and sensitise HC for exogenous reactive H202. Abt. Klinische Pharmakologie, Universit&t G6ttingen, Robert-Koch-Stra8e 40, 3400 G6ttingen

78 INCREASE IN BILIARY PERMEABILITY BY ENDOTOXIN ENHANCED BY GALACTOSAMINE AND ESTRADIOLVALERATE H. Krell, R. Schuster and W. Schr6ero

8O CONCENTRATION OF 2,3,7,8-TCDD IN THE LIVER OF JUVENILE RATS AFTER CONTINUOUS EXPOSURE Elisabeth Koch, Martin Mayer, Jutta Hartmann

In order to study the mechanisms involved in the development of endotoxin-induced hepatitis and cholestasis, rats were treated with endotoxin (0.3 mg/kg), galactosamine (650 mg/kg) and estradiolvalerate (i m g / k g / w e e k for 5 weeks). In anesthetized animals, bile flow began to decrease within 2 hours when endotoxin was given in combination with galactosamine. In estradiolvalerate-pretreated rats, bile flow was lowered 5 hours after administration of endotoxin. In isolated perfused livers of treated rats, biliary clearance of 14C-sucrose or 14C-inulin was analyzed and p a r a c e l l u l a r and transcellular portions, resp., were quantitated by off-kinetics after withdrawal of the radioactive marker from the perfusate (Biochem.J. 241:635,1987). Analysis of the approximately biexponential off-kinetics of the markers in bile,C = C1e-klt+C2e -kat, revealed that the paracellular portion of the clearance (CI) accounted for the increase. Using a model to describe the p a r a c e l l u l a r access of inert solutes in terms of bile flow (F), diffusion and convection, diffusion p e r m e a b i l i t y coefficient K and reflection factor o were determined by fitting to the data the equation C~ = (I-o)F/I-O e -(~-~)F/K. An increase in p e r m e a b i l i t y by endotoxin was enhanced by pretreatment with estradiolvalerate and by combined administration of galactosamine. It is concluded that increased paracellular permeability of the bile tract is an early alteration induced by endotoxin which may be involved in pathogenesis of hepat±tis and cholestasis. Pharmakologisches Institut der Universit~t THbingen, Wilhelmstr. 56, D-7400 T~bingen.

Female Wistar rats were treated with a single loading-dose of 20, 50, 120 and 250 ng 14C-TCDD/kg s.c. and weekly maintenancedoses of 1/5 of the loading-dose. The controls were treated with the solvent. The weekly maintenance-doses were administered 21 days prior to mating and during pregnancy and the lactation period. After weaning (at 3 weeks o f age) the Fl-generation were treated with the same dose regime (loading-/maintenance-dose). Offspring from each ~oup, 4 males and 4 females, aged 4, 8 and 12 weeks were sacrificed and the hepatic TCDD concentrations (radioactivity) were measured. The table presents the 14C-TCDD concentrations as pg/g wet tissue: Age(weeks)

Dose Regime(loadiag-/mainteaance-dose) TCDD-20/4 TCDD-50/10 TCDD-120/24 TCDD-250/50

4 8 12

218-* 35 147-* 43 135-.115

544-*134 249-+ 45 243-+ 31

980_+296 534+108 514-+ 54

2171-+669 1088-+294 969-*248

The dose regime used was capable of maintaining the TCDD levels in adult rats and those older than 8 weeks. However, during the phase of rapid growth (4th to 8th week) this dosing schedule was insufficient to keep the tissue concentrations of TCDD constant. The maintenance-dose had to be increased to weekly doses of 2/5 of the loading-dose.

Supported by a grantfrom the Bundesministerium fi~rForschung und Technologie Institut ffir Toxikologie und Embryopharmakologie, Freie Universit/it Berlin, Garystr. 5, D-1000 Berlin 33

R 21

81

83

BIOLOGICAL ACTIVITIES OF MIXTURES OF POLYCHLORINATED DIBENZO-P-DIOXINS (PCDDs) AND THEIR CONSTITUENTS IN HUMAN HEPG2 CELLS H.-P. Litm and D. Scbxenk Polychlorinated dibenzo-p-dioxins (PCDDs) are environmental contaminants which cause a variety of toxic symptoms including body weight loss and thymic atrophy in rats. A good correlation has been reported between the toxic potency of various PCDDs in vivo and their potency to induce P450IA1 activities in the H4IIE rat hepatoma cell line (Safe S., Ann. Rev. Pharmacol. Toxicol. 26, 371-399, 1986). In a previous study it was found that inducing potencies of PCDDs in H4IIE ceils and in rat hepatocytes are similar. However, little is known about the inducing potency of PCDDs in human cells. Therefore, induction of P450IAl-dependent 7-ethoxyresorufin O-deethylase (EROD) was investigated in the human hepatoma cell line HepG2. The ECs0-value for the most potent PCDD, 2378-CI,DD (TCDD), determined in HepG2 cells (706 pg/plate) was much higher than those determined in rat H4IIE cells (89 pg/plate) and rat hepatocytes (37 pg/plate). The relative potency of other PCDDs was calculated from their respective EC5o values and is given as TCDD equivalents fiE). A similar rank order of TE for the 2378-substituted PCDDs was obtained in human and rat ceils, though in HepG2 cells 12378-C15DD (TE 0.75) and 123478-C16DD (TE 0.75) were nearly as potent as 2378-C1,DD. Furthermore, CIsDD did not lead to detectable EROD induction in the human cell line (TE < 0.001). For a complex PCDD mixture containing 49 congeners the experimentally obtained ECg0-value was in good agreement with that calculated from the sum of its 2378-substituted PCDDs, suggesting additive effects of the 6 most potent PCDDs. However, results obtained with mixtures containing > 50% CI~DD suggest partial antagonistic effects of ClsDD.

BIOLOGICAL ACTIVITY OF TODD, TBrDD AND THREE 2,3,7,8-MIXED HALOGENATED DIBENZO-P-DIOXINS

_

_

Institute of Toxicology, University of Tiibingen, WilhelmstraBe 56, D-7400 Tiibingen, Germany

82 BIOLOGICAL EFFECTS AND TISSUE CONCENTRATIONS OF H7CDD AND OCDD IN FEMALE WISTAR RATS Georg Golor, Maria Korte, Thomas Wiesmiiller., Wolfgang K6rner*, Hanspanl Hagenmaler* We investigated the liver enzyme induction in female Wistar rats after subcutaneous injection of 1,2,3,4,6,7,8-heptachlorodibenzo-pdioxin (H7CDD) and octachlorodibenzo-p-dioxin (OCDD) and correlated the effects with tissue concentrations of the substances (ng/g wet weight). The chemicals were disolved in a toluene/ DMSO (1+2) mixture (HTCDD) or toluene only (OCDD). A dose of 250/~g OCDD/kg body wt was injected once a week for 10 weeks. EROD (= Ethoxyresorufin O-deethylase) activity in liver microsomes and OCDD concentrations in liver and adipose tissue were determinated at the end of the treatment period and during 13 weeks after the last injection. In the second series rats were treated with a single dose of 30 ~zg HTCDD/kg body wt, EROD activity and tissue concentrations were determined three weeks after treatment. Congener Liver conc. EROD activity in liver (ng/g (pmolesx mg wet weight) prot-1x min "1) controls (n=6) HTCDD (n=4) OCDD (n=3)

0.007 (H7CDD)* 0.030 (OCDD)* 260 -+ 80 2500 + 190

77 -+ 20 990 -+ 440 320 _+ 90

* Liver concentrations in pooled material from six rats. We conclude that repeated applications of OCDD induced EROD activity, however, the inductive potency of H7CDD is clearly more than 10 times higher than that of OCDD. Studies supported by grants from the Bundesministeriumfiir Forschung und Technologie(07VDX01). Institut far Toxikologie und Embryopharmakologie, Freie UniversitiitBerlin, Garystr. 5, 1000Berlin 33 *Institutfar Organische Chemie, UniversitiitTabingen,Auf der Morgenstelle 18, 7400 Tiibingen

T h o m a s Schulz-Schalge, Karl-Heinz Schwind*, Otto Hutzinger* The combustion of brominated and chlorinated scavengers in leaded gasoline leads to the emission of polyhalogenated dibenzodioxins (PHDD) and dibenzofurans (PHDF). Considerable amounts of 2,3,7,8-PHDD were detected in the exhaust of vehicles. The toxic potency of these compounds is unkown, therefore we investigated the ethoxyresorufin-O-deethylase (I=ROD), as an indicator for Ah-receptor mediated reactions, in hepatic microsomes of adult male Wistar rats. The animals received a single subcutaneous injection of 2 nmole/kg body wt of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TODD) or 2,3,7,8tet rabromedibenzo-p-dioxin (TBrDD), 2-bromo-3,7,8-t richlorodibenzo-pdioxin (B1C3), 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin (B202), 2,3,7-tribromo-8-chlorodibenzo-p-dioxin (B301). The control rats received 100/~l/kg body wt of the vehicle (dimethylsulfoxide/toluene 2+1) only. The EROD induction was investigated 7, 14, 28, 42, 56, 70, 84, 98 days after administration. The following results were obtained.

Days

TODD

B1C3

B2C2

B3C1

TBrDD

7 5210 5850 4330 2730 6740 14 3740 3180 2910 2540 3840 28 1750 1600 n.d. 922 1800 42 903 874 696 653 1410 56 401 376 232 345 801 70 285 295 275 244. 546 84 169 130 131 226 371 98 162 169 105 132 410 control 31 (N = 35) , n.£1.= not determined values in pmoles resorufin x mg protein "'x min" (mean of three animals). The inductive potency of all investigated substances was similar, only B3C1 showed only about 50% of the EROD activity of TCDD seven days after application. 98 days after administration all substances, with the exception of TBrDD, showed almost the same EROD activity. TBrDD had at this time a 2.5times higher activity. These preliminary data suggest a comparable potency of EROD induction of all investigated 2,3,7,8-PHDDs and the necessity of consideration of these compounds for the calculation of toxic equivalency factors. These studies were supported by grant Nr. 07 VDX 019 from the Bundesministerium for Forschung und Technologie (BMFT). Institut for Toxikologie und Embtyopharmakologie, Freie Universit~t Berlin, GatystraBe 5, D- 1000 Berlin 33 *Lehrstuhl for 5kologische Chemie und Geochemie der Universit&t Bayreuth, Posffach 10 12 51, D-8580 Bayreuth

84 POLYCHLORINATED DIBENZO-p-DIOXINS (PCDDs) AND ETHINYLESTRADIOL AS GROWTH MODULATORS IN RAT HEPATOCYTE PRIMARY CULTURES A. Karger 2,3,7,8-C14DD (TCDD) has been shown to act as a liver tumor promotor in female rats (Pitot et al., Cancer Res. 40, 3616-3620, 1980). To elucidate mechanisms responsible for this sex specific tumor promoting activity, the influence of 2,3,7,8-C14DD, 1,2,3,4,6,7,8-C1~DD (HCDD) and C18DD (OCDD, containing 0.5% HCDD) on EGF-stimulated DNA synthesis was studied in primary hepatocyte cultures of male and female rats in the presence of 5 % fetal calf serum. Addition of TCDD (maximal effect at 10 ~2M), HCDD (maximal effect at 10-" M), and OCDD (maximal effect at 10.9 M) increased DNA synthesis 30-50% in a strictly EGF-dependent manner, the rank order of potency suggesting involvement of the Ah receptor. Induction of P4501Al-dependent 7-ethoxyresorufin O-deethylase activity occurred at PCDD concentrations which were higher than those leading to maximal stimulation of DNA synthesis. Addition of ethinylestradiol fiLrther increased TCDD-mediated stimulation of DNA synthesis. This effect was variable. However, the hepatocyte preparations responding strongly to TCDD also responded strongest to ethinylestradiol. In the presence of 3 x 10-~2 M TCDD a 2.5-fold stimulation of DNA synthesis was obtained at 20/zM ethinylestradiol. The results indicate that PCDDs enhance EGF-stimulated DNA synthesis in rat hepatocytes in the rank order of their binding affmity to the Ah receptor. Furthermore, synergistic effects of ethinylestradiol suggest that estrogens facilitate tumor promoting actions of PCDDs. Institute of Toxicology, University of Tiibingen, Wilhehnstral~e 56, D-7400 Tiibingen, Germany

R 22 85

87

EFFECTS OF UVER TUMOR PROMOTERS ON THE ACTION OF GROWTH

INDUCTION OF E T H O X Y R E S O R U F I N - O - D E E T H Y L A S E ACTIVITY BY VARIOUS AROMATIC AMINES DOES NOT CORRELATE WITH THEIR AH RECEPTOR AFFINITY P. Cikryt and W. Muster

FACTORS IN PRIMARY RAT HEPATOCYTES AND H411ECELLS

D.Wdlfle and K.Heidelberg The influence of liver tumor promoters on the regulation of DNA synthesis was studied in primary rat hepatocytes using 2,3,7,8-tetrachiorodibenzo-p-dioxin and 3,3',4,4'-tetrachlorobiphenyl

(TODD)

(TCB), known as inducers of drug metabolizing

enzymes. The plalotropic response of these agents includes the interaction w~th receptors of a variety of growth factors. We, therefore, investigated the effects of TODD and TCB on the regulation of growth-factor stimulated DNA synthesis in serum-free culturas of adult rat hepatocytes and H411Ehepstoma cells. The maximal stimulation of DNA synthesis of hepatocytes after 2 days in culture was achieved at 10"12M TODD and 10"7M TCB. These effects were dependent on the presence of insulin (10~8M) and dexamethasone (10"8M) in the culture media. The growth stimulation of hepatocytes by epidermal growth factor or alpha 1-adrenergic agents, e,g. phenylephrine, was further enhanced by TODD or TCB. On the other hand, no significant effect of these tumor promoters was observed on the growth of H411Ecells in hormone- or serum-supplemented media. Thus, the results suggest that TODD or TCB in stimulating the DNA synthesis of normal hepatocytes interact with growth factors and that very low concentrations of these tumor promoters, i.e. several orders of magnitude below those necessary for maximal enzyme induction, are active.

Dept of Toxicology, UniversityHamburgMedical School, Grindelallee 117, D-2000 Hamburg 13, F.R.G. Suppoded by a grant from Deutsche Forschungsgemalnschaft (DFG)

The induction of a number of drug-metabolizing enzymes by polycyclic aromatic hydrocarbons seems to be mediated by a cytosolic protein: the aromatic hydrocarbon (Ah) receptor. One of the most thoroughly studied Ah receptor-mediated responses is the induction of P4501A1 which can be easily monitored by the rate of ethoxyresorufinO-deethylation (EROD).The aromatic amines 2-acetylaminofluorene (2AAF) and 4-acetylaminofluorene (4AAF) differ in their toxic and carcinogenic properties. 2AAF is a wellknown inducer of drug-metabolizing enzymes, but 4AAF has not been characterized in this respect. Recently, we have shown that 2AAF binds to the Ah receptor and 4AAF does not. The objective of this study was to compare the inducing properties of 2AAF and 4AAF on two microsomal enzyme activites, namely EROD and pentoxyresorufin-O-deethylation (PROD) in male Wistar rats. 2AAF (0.02 % in the diet) and 4AAF (0.1% in the diet) were fed for 2, 7 and 21 days. The results demonstrate that both compounds are 'mixed-type' inducers of P450 enzymes both of the '3methylcholanthrene-type' and of the 'phenobarbital-type'. The inducing capacity of 2AAF was weak compared to 4AAF. 2AAF increased both EROD and PROD activity at most 2-fold whereas 4AAF induced EROD activity 11-fold (after 2 days of treatment) and PROD activity 56fold (7 days). The specificity of P4501A1 induction by 2AAF and 4AAF was proved by Western blot with enzyme-specific antibodies. We have also tested the Ah receptor affinity of a number of compounds which, according to the literature, are inducers of EROD activity in vivo. These compounds, like the heterocyclic aromatic amines 2amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminodipyrido(1,2a:3',2'-d)imidazole (Glu P-2) and 3-aminodipyrido(1,2-a:3',2'd)imidazole (Glu P-3) and the 3,3'-dichlorobenzidine homologue 4,4'methylenebis(2-chloroanilin) (MOCA), do not bind to the Ah receptor in vitro, but do induce EROD activity in vivo. The mechanism of P4501A1 induction by these chemicals is not clear. In conclusion, the induction of EROD activity in vivo, especially in the case of weak inducers, does not prove an interaction with the Ah receptor in vivo. Institute of Toxicology, University of W0rzburg, Versbacher Str. 9, W-8700 W0rzburg, Germany.

86

88

EFFECTS OF THE LIVER TUMOR PROMOTER 2,3,7,8-TETRACHLORODIBENZO-

THE ROLE

P-DIOXIN (TCDD) ON THE REGULATION OF PROTEIN KINASE C AND INOSITOLPHOSPHATE METABOLISM OF RAT HEPATOCYTES IN VITRO

C.Schmu~teand E.Becker To investigate the modulation of signal transfer pathways of hepatocytes by tumor promoters in vitrq, TODD was chosen as a prototyp of polychlorinated

aromatic

hydrocarbons which bind to a cytosolic receptor protein (Ah receptor). Furthermore, TODD activates protein kinase C (PKC) in the liver (Bombick st al., Biochem. Biophys. Res. Commun. 127:296, 1985). This enzyme is known to be involved in the regulation of agonist-stimulated inosito~trisphosphate (IP3) formation. The influence of low TODD concentrations (10"12M) on the PKC activity was studied in freshly isolated rat hepatocytes and in primary hepatocytes after 24 h in serum-free culture. TODD was found to be a poor activator of PKC compared to 12-O-tetradscanoylphorbol-13acetat (TPA,10"7M). In contrast to TPA, no inhibition of vasopressin-sfimulated IP3 formation was observed after a short-term treatment (15 rain) with TODD. Long-term treatment (18 h) of primary hepatocytes with TODD (10"12M) resulted only in a small stimulation of vasopressin-mediated IP3 formation (1,5 fold) compared to TPA (2 to 3 fold). Thus, the results indicste that the effects of TODD on PKC activities and IP3 formation are different from those of TPA and may be regarded as indirect actions of TODD.

DepL of Toxicology, UniversityHamburgMedical School, Grindelallee 117, D-2000 Hamburg 13, F.R.G. Supported by a grant from Deutsche Forschungsgemeinschaft (DFG)

OF ACUTE AND CHRONIC TOXICITY OF CARCINOGENIC AROMATIC AMINES IN RAT LIVER S. Ambs and H.-G. Neumann 2-Acetylaminofluorene (AAF, initiator and promotor) is a complete rat liver carcinogen. 2-Acetylaminophenanthrene (AAP) and 4-acetylaminostilbene (AAS) are incomplete rat liver carcinogens (initiatior). We studied the role of cytotoxicity for the promoting activity of AAF. Male Wistar rats were chronically fed 0.02% AAF for up to 16 weeks. The food intake of the animals decreased. Growth stopped. Various biochemical parameters were measured in blood, but the results could not explain the pathological changes seen in the liver after 8-12 weeks. Glucose-6-phosphatase activity in liver, decreased with a minimum after 5 weeks (histological diagnosis: no focal event). Serum levels of the thyroid hormons T3 and T4 declined with a minimum after 8 weeks. Experiments with isolated perfused livers taken from animals fed AAF showed that the efflux of glucose into the perfusate and that of glutathione into the bile were lower than in controls. Menadione caused a much higher glutathione efflux in livers from control animals than in dosed animals, although the glutathione content in treated animals was higher. Oxygen consumption in livers from dosed animals was ca. 20% higher than in control animals. Therefore~ we investigated the putative role of mitochondria for the acute and chronic toxicity of AAF. The uptake of ADP into mitochondria from control animals was inhibited by the N-acetoxy derivative of AAF, but not by those of AAS and AAp. The ADP-uptake was also reduced in AAFpretreated animals with a minimum after 3-4 weeks. The phosphate uptake was impaired by all hydroxamic acid esters and by N-hydroxy-AAF. The results are consistent with the hypothesis that AAF produces acute effects which do not lead to overt toxicity, but rather represent alterations in biochemical homeostasis. If this situation is maintained it may lead to chronic toxicity. Mitochondria might be targets for such primary effects. Present address: Institute of Toxicology, University of W~rzburg, Versbacherstr.9, D-8700 W~rzburg

R 23 89 DIFFERENCES IN INITIATING AND PROMOTING ACTIVITY OF AZO DYE ISOMERS IN RAT LIVER. G. Werle-Schneider, A. Wolf*, and W. Kunz. The azo dye 4-dimethylaminoazobenzene (4-DAB) is a very potent carcinogen in rodent liver with extremely short tumor induction times at high exposure levels. We have hypothesised that this effect is due to additional "intrinsic" promoting activity of the weakly initiating agent present at high dose levels. In this study we have extended these investigations using the azo dye isomers 3'-methyl-4-DAB (3'-MeDAB) and 2-methyl-4-DAB (2-Me-DAB) designated as "complete" and "incomplete" carcinogens based on their differential carcinogenic profile. As parameters of hepatocarcinogenic response, number and volume of enzyme-altered foci (EAF) in livers of rats treated with 3'Me-DAB and 2-Me-DAB were determined. The initiating potency of the two azo dyes was tested by brief exposure of rats to the test compounds followed by continuous treatment with the promotor phenobarbital. The promoting activity was analysed by continuous azo dye treatment of rats subsequent to a limited exposure to diethylnitrosamine (DEN) as an initiator. Our results demonstrate for both compounds similar initiating potencies but considerable differences in promoting activity. While 3'-Me-DAB led to an increase in the number and especially the size of EAF initiated by DEN, 2-Me-DAB did not exhibit such promoting effects. In different strains of Salmonella typhimurium, 3'-Me-DAB and 2-Me-DAB showed qualitatively and quantitatively similar mutagenicity profiles demonstrating a correlation between mutagenic effects in vitro and initiating activity in vivo, rather than between mutagenicity and overall carcinogenity of the two azo dyes. Treatment with 3'-Me-DAB led to elevated levels of reactive oxygen in liver microsomes in vitro and in rat liver in vivo indicating that oxygen radicals may be involved in the "promoting" activity of this azo dye. *Present adress: Department of Toxicology, Sandoz Pharma Ltd., Basel, Schweiz Deutsches Krebsforschungszentrum, Institut ffir Experimentelle Pathologie, Im Neuenheimer Feld 280, D-6900 Heidelberg.

9O

DIFFERENT SENSITIVITY OF THREE RAT TOWARDS INITIATING AGENTS D. Oesterle I and B. Schlatterer 2

LIVER

FOCI

8IOASSAYS

Three different protocols for rat liver loci bioassays have been compared with respect to their sensitivity in revealing the carcinogenic potential of chemicals: The initiation/promotion schedules according to Oesterle and Deml ( A ) , according to Pereira (B), and the initiation/ selection protocol according to Tatematsu et al. (C) (Oesterle et al., Carcinogenesis, i~0, 1891, 1989). As biological parameters preneoplastic foci were evaluated histochemically. They were identified by ATPase-deficiency and by reactivation of GGTase on serial sections. We compared six initiating agents of different carcinogenic potency in two doses: 4-aminobiphenyl (4-AB; 50 and 100 mg/kg body weight), dimethylbenzanthracene (DMBA; 25 and 50 mg/kg body weight), N-nitrosomorpholine (N-NM; 50 and 100 mg/kg body weight), diethylnitrosamine (DEN; i0 and 30 mg/kg body weight) all applied one time, and 2-acetylaminofluorene (2-AAF; 20 and 50mg/kg body weight), and benzidine (BZ; i0 and 20 mg/ kg body weight), applied five times. With all protocols the .strong initiator DEN caused the highest foci incidence, followed by N-NM, DMBA, 4-AB, 2-AAF and BZ. In (B) DMBA was more effective than N-NM. With ATPase as marker, protocol (A) was found to be most sensitive in view of number and size of the foci. With GGTase as marker, protocol (B) was most sensitive for the number, except of N-NM and DEN, here the higher numbers were found with (A). For foci area protocol (C) was most effective for half of the experiments, followed by (A) and (B). A dose~dependent effect was more often observed in protocols (B) and (C). In conclusion: From the three protocols used in this comparison, protocol (A) was found to be most suitable for the detection of the initiating potency of chemical carcinogens. IGSF - Forschungszentrum fur Umwelt und Gesundheit, Institut fur Toxikologie, D-8042 Neuherberg, 2Umweltbundesamt, D-1000 Berlin, FRG

92

ENZYMES OF THE CARBOHYDRATE M E T A B O L I S M AS M A R K E R S FOR FOCI TREATMENT WITH DEN AND CLOPHEN.A 1C. Einig, IE. Eigenbrodt, 3U. G e r b r a c h t

AND XENOBIOTIC INCIDENCE AFTER 50.

The rat l i v e r foci b i o a s s a y is w i d e s p r e a d l y u s e d as an in v i v o t e s t s y s t e m for the i d e n t i f i c a t i o n of chemically induced hepatocarcinogenesis. F o l l o w i n g the two s t a g e m o d e l of carcinogenesis w h i c h i m p l i c a t e the t r e a t m e n t w i t h an i n i t i a t o r and a p r o m o t o r , the rats r e c e i v e d o n c e i0 m g / k g b.wt of diethylnitrosamine (DEN) which is a strong liver-carcinogenic chemical. After 7 days the a n i m a l s w e r e t r e a t e d twice a w e e k w i t h the p r o m o t e r C l o p h e n A 50 (10 m g / k g b.wt) for the following i0 weeks. After the e x p e r i m e n t the rats w e r e k i l l e d a n d the l i v e r c r y o s t a t s l i c e s were stainded immunhistochemically or cytochemically. The enzymes lactate-dehydrogenase (LDH) and glucose-6-phosphate-dehydrogenase (G6PDH) of the carbohydrate metabolism and glutathion-S-transferase (GST) of the x e n o b i o t i c pathway were used for the endpoint determination. The m o s t of the foci w e r e r e c o g n i z e d in the animal group which had received the complete treatment schedule in c o n t r a s t to the groups which were treated only with DEN or Clophen A 50. A l s o the r e l a t i v e size distrib u t i o n of the foci i n d i c a t e that in the g r o u p w h i c h r e c e i v e d the c o m p l e t e s c h e d u l e the i s l a n d s were significantly i n c r e a s e d . T h e m o s t loci w e r e stained with GST suggesting t h a t this e n z y m e is e x p r e s s e d d u r i n g the e a r l y s t a g e of hepatocarcinogenesis. 1 Vet. Biochemie Universit~t Gie~en Frankfurterstr. i00 6300 G i e S e n 3

91

Pharmbiodyn

Denzlingen

EFFECT OF NAFENOPIN ON CELL TURNOVER AND THE EXPRESSION OF PEROXISOMAL ENZYMES, CYTOCHROME P-452, GLUTATHIONE-TRA~SFERASE-ISOENZYMES IN WE~I[L¥ B A S O P H I L I C F O C I O F R A T LIttER B. K r a u p p - G r a s l *

Peroxisome inducing agents, s u c h as n a f e n o p i n (NAF), are h e p a t o c a r c i n o g e n s in l i f e - t i m e r o d e n t e x p e r i m e n t s , In v a r i o u s a s s a y s p e r o x i s o m e p r o l i ferators are not genotoxic or tumor-initiators. In o u r r e c e n t experiments NAF enhanced tumor f o r m a t i o n t h r o u g h p r o m o t i o n of: i. A F B l - i n i t i a t e d l o c i in l i v e r s of y o u n g r a t s a n d 2. " s p o n taneous" f o c i w h i c h a p p e a r in l i v e r s of a g i n g rats. M o s t of t h e f o c i a n d t u m o r s s e e n in N A F t r e a t e d l i v e r s w e r e of w e a k c y t o p l a s m a t i c basophilia. Their phenotype was different from that of eosinophilic-clear cell and tigroid loci. Foci were further characterized by: i. d e t e r mination of their proliferative capacity as judged by DNA-synthesis and apoptotic activity, 2. e x p r e s s i o n of N A F - i n d u c i b l e peroxisomal Bo x i d a t i o n e n z y m e s a n d c y t o c h r o m e P-452, a n d G S H transferase-isoenzymes. Like tigroid loci but unlike eosinophilic-clear cell foci, weakly basophilic loci in N A F - t r e a t e d livers do not express placentar GSH-transferase. The express i o n of o t h e r G S H - t r a n s f e r a s e - i s o e n z y m e s in all f o c i s u b t y p e s is v a r i a b l e . T h e s a m e a p p l i e s for t h e e x p r e s s i o n of p e r o x i s o m a l e n z y m e s a n d c y t o c h r o m e P-452. H o w e v e r , t h e r a t e of D N A - s y n t h e s i s and apoptotic activity is highest in w e a k l y basophilic foci suggesting an increased cell t u r n o v e r in t h i s l i k e l y t u m o r p r e c u r s o r lesion. * Present address: Institut fur TumorbiologieKrebsforschung, Borschkegasse 8a, A-1090 Wien

R 24 93 ROLE OF PRO AND M A T U R E T G F B - I IN C E L L D E A T H (APOPTOSIS) OF HEPATOCYTES Fr. Oberhammer, R. Sedivy, R. Jirtle*, R. Schulte-Hermann . . . . . . . . . . . . . . . . . . . . . . . .

!

95 METALLOTHIONEIN, COPPER AND ZINC IN HUMAN LIVER DURING PRENATAL AND POSTNATAL DEVELOPMENT D. Klein a n d K. H. S u m m e r

. . . . . . . . . . . . . . . . . . . . . . .

Administration of non-genotoxic 'compounds stimulates DNA synthesis and liver growth due to both hyperplasia and hypertrophy. Upon withdrawal of this growth stimuli the increase in organ mass is found to be reversible. Thus in a study with cyproteroneacetate about 30-40% of liver enlargement disappeared within six days after cessation of treatment; this was accompanied by cell elimination through apoptosis. As the proteins of the TGF-B family are involved in organ involution (Mullerian Duct, prostate) we investigated the occurrence of TGF-BI (pro and mature part of the protein) during this regression by immunohistochemistry. The majority of apoptoses stained positive for the pro part of TGF-B suggesting a possible role for TGF-BI in this process. Further studies with isolated cultured hepatocytes revealed that apoptosis can be induced by TGFBI. This findings indicate that TGF-BI may function as one of the signal factors during apoptosis. Institut ffir Tumorbiologie-Krebsforschung Borschkegasse 8a, A-1090 Wien *Duke University, Medical Center, Durham, NC

94 G E N E X P R E E S S I O N A S S O C I A T E D W I T H APOPTOSIS ("PROGRAMMED" C E L L DEATH)

W. Bursch I, L. Fesus 2 and M. Tenniswood 3 Apoptosis is a type of cell death involved in the maintenance of cell number homeostasis in tissues. Apoptosis is regulated by a complex interaction between extrinsic (e.g. hormones, growth factors) and intrinsic (e.g. cell cycle related) factors; disturbance of its regulation appears to be involved in teratogenesis, carcinogenesis and immunosuppression. In the present study, the expression of tissue transglutaminase (TGase) and "testosterone repressed prostate message" (TRPM-2) during apoptosis was investigated. Apoptosis occurring during regression of hormonally induced rat liver hyperplasia and in mouse lymphoma cell cultures after glucocorticoid treatment were found to be associated with TGase and TRPM-2 expression. The possible role of the gene products in the apoptotic process will be discussed. IInstitut ffir Tumorbiologie-Krebsforschung Borschkegasse 8a, A-1090 Wien 2University of Debrecen, Hungary 3University of Ottawa, Canada

The fetus a n d n e o n a t a l offspring have high d e m a n d s for Cu a n d Zn. Since little information is available o n the role of metallothionein (MT) in the homeostatic control of t h e s e m e t a l s d u r i n g development, cytosolic MT, total a n d cytosolic Cu a n d Zn, a n d the portion of MT w h i c h b i n d s Cu (Cu-load of MT) were investigated in fetal a n d n e o n a t a l h u m a n liver. Liver samples were obtained from p r e t e r m a b o r t u s e s (22, 24 a n d 32 gestational weeks) a n d from children (2-15 months) whose d e a t h was due to s u d d e n i n f a n t d e a t h syndrome. Histological a n d pathological findings of the livers were normal. MT a n d m e t a l s were d e t e r m i n e d with the recently developed thiomolybdate a s s a y (1) a n d AAS, respectively. The MT-content was higher in fetal t h a n in n e o n a t a l liver. There w a s a linear correlation (r=0.996) between cytosolic MT a n d Zn in b o t h fetal a n d n e o n a t a l liver b u t n o t between MT a n d Cu. In c o n t r a s t to fetal liver, t h e Cu-load of MT in n e o n a t a l liver seems to be determined by the Z n / C u ratio in the cytosol. Whereas the fraction of cytosolic Z n r e m a i n e d c o n s t a n t at 66°/6 of the total, i n d e p e n d e n t of the stage of development, the fraction of cytosolic Cu increased from 26% in preterm liver to a b o u t 100% within 12 m o n t h s postnatally. These results suggest t h a t MT is involved in the regulation of Cu a n d Zn metabolism d u r i n g fetal a n d n e o n a t a l development. 1) Klein et al. (1990). Anal. Biochem. 189, 35-39 GSF-Institut ffir Toxikologie, D-8042 Neuherberg

96 NEPHROTOXICITY AND MYELOTOXICITY OF NEW ANTINEOPLASTIC PLATINUM (II) AND PLATINUM (IV) COMPLEXES IN RATS U. Horn, A. H~rtl, W. Neuhaus, U. St6ckel, H.-P. Schr6er, and H. Hoffmann In comparison with cis-DDP four new platinum (II) and platinum (IV) complexes (cis-diammineplatinum(II)-lactate; cis-diammine-platinum(II)lactate,L; cis-diammine-platinum(II)-dilactate; trans-dihydroxy-cis-dichlorodiammine-platinum(IV)) were evaluated for their acute nephrotoxic and myelotoxic ~otency in male rats (Shoe:WIST) following l.v. administration of maximum tolerated doses on 5 consecutive days. Parameters for nephrotoxicity determined on day 6, 13 and 22 after the first administration of the drug included blood urea nitrogen, serum creatinine, urine volume, urinary glucose and tubule cell excretion. Parameters for myelotoxicity determined on the same days included leucocytes, platelets, hemoglobin and hematocrit. Cis-DDP was found to be the most nephrotoxic compound. The myelotoxicity of the Pt(II) complexes appeared to be similar to that of cisDDP. In contrast the myelotoxic effect of the pt(IV) complex was of minor importance. Department of Pharmacology and Toxicology, Institute of Microbiology and Experimental Therapy, Beutenbergstrasse ii, 0-6900 Jena

R 25

97

99

E F F E C T OF ARSENICALS ON GLUCOSE U T I L I Z A T I O N IN MDCK-CELLS

HEAVY METAL IONS INFLUENCE THE HISTAMINE SECRETION FROM HUMAN ADENOIDAL MAST CELLS S,Bent, W.Schmutzler

B. LieN, H. MiJckter, E. Doldea, B. Fichfl Impaired carbohydrate utilization has been demonstrated in acute arsenic poisorting. The ensuing energy depletion has mainly been attributed to an inhibition of cellnlar pyruvate dehydrogenase although effects of arsenic on other metabolic pathways may be likewise important. Cultured ceils offer a suitable experimental system to trace the utilization of extracellular carbohydrates in the presence of arsenic. We have investigated the effect of organic (oxophenylarsine; PhAsO) and inorganic (As203) arsenicals on the availability of glucose to Madin-Darby canine kidney (MDCK) cells. Following a 3 h preincubation period with glucose-free Hanks' buffer MDCK cells (adherent or in suspension) were incubated (10 - 60 min, 37°C) with PhAsO (10-7 - 10-3 mniB) or As203 (10-6 - 10-2 tool/l) in the presence of D-glucose (0.01-25 mmolB) using D-[6-14C]-glucose as tracer. Cell-associated radioactivity, dye exclusion, extracellular LDH activity and potassium release were determined. A concentration-dependent inhibition of tracer accumulation was observed with both arsenicals suggesting an impaired uptake of glucose. With As 203 cytotoxic concentrations were required for half-maximum inhibition (IC50 0.5 mmolB). On the other hand, PhAsO inhibited glucose uptake in the micromolar range (IC50 10 p-molB). At these concentrations cellular viability as assessed by dye exclusion, LDH and potassium release, and cell morphology was not affected within the experimentaltimescale. We conclude that inhibition of glucose uptake may contribute to the increased acute toxicity of organic arsenicals by further aggravating the depletion of intracellular carbohydrates.

In peritoneal rat mast ceils histamine secretion is modulated by some heavy metal ions (Wieczorek et al. Allergologie 12, 158-160, 1989). Because of the known species specific differences we tested the effects of water soluble salts of mercury (HgC12), lead [Pb(CH~COO)~], cadmium (CdSO4) and bismuth [BiO(CIO4)] on histamine release from human adehoidal mast cells. The mast cells were isolated mechanically (Schmutzler et al. Int. Archs. Allergy appl. Immun. 77, 177-178, 1985). Histamine secretion was stimulated by Concanavalin A (Con A; 50mg/1) and was quantitated by the double isotope method. Mercury increased the histamine releas~e in stimulated and unstimulated cells at a concentration of 10- " M by at least 41% of the controll values. Cadmium exerted a biphasic reaction. At 10-6 M the spontaneous and Con A induced histamine release was inhibited by 13% and 24%. At the concentration of 1044 M cadmium resembled the effects of mercury with an increased release of 25% ~nd 16%. Lead showed a different pattern: At 10T M M it enhanced the histamine release by 30% in stimulated and by 55% in unstimul~tted mast ceils. It decreased the spontaneous mediator release at 10-" M but influenced the stimulated release only marginally. Bismuth decreased the histamine release from mast cells at 10-4 M by 18% and 32%. These data show that heavy metal ions influence allergic histamine release differently. Lead increases the mediator release at concentrations measurable in human blood. The bismuth induced inhibition might contribute to its therapeutical effectiveness in gastric ulcerations. lnstitut for Pharmakologie, Medizinische Fakult~it der Aachen, D-5100 Aachen, FRG

RWTH

Walther Straub-Institut f/Jr Pharmakologie und Toxikologie der LMU, Nugbaumstr. 26, D-8000 M~inchen 2

98 INFLUENCE OF C H E L A T I N G AGENTS ON BILIARY EXCREtION AND ON TOXIC EFFECTS OF D I A L K Y L T I N C O M P O U N D S IN RATS J. Merkord x, G. K r O n i n g and G , H e n n i g h a u s e n

100

D i a l k y l t i n compounds have b e e n used as biocides, catalysts and plast stabilizer. An important toxicological p r o p e r t y of the d i a l k y l t i n compounds may be the r e a c t i o n with dithiol groups. 2,3-dim e r c a p t o - l - p r o p a n o l (BAL, dimercaprol), meso-2,3d i m e r c a p t o s u c c i n i c acid (DMSA) and 2 , 3 - d i m e r c a p t o - l - p r o p a n s u l f o n i c acid {DMPS) were compared in their potency to d i m i n i s h the b i l i a r y e x c r e t i o n of o r g a n o t i n and the o r g a n o s p e c i f i c toxicity (lesions on liver, bile duct and pancreas) of dialkyltin d i c h l o r i d e s with alkyl chains of 4-6 Catoms in rats. D i m e r c a p r o l did not influence s i g n i f i c a n t l y the o r g a n o s p e c i f i c toxic effects of d i b u t y l t i n dichloride (DBTC), d i p e n t y l t i n d i c h l o r i d e (DPTC) and d i h e x y l t i n d i c h l o r i d e (DHexTC) on liver, bile d u c t and pancreas. The b i l i a r y excretion of o r g a n o t i n after treatment of a n a e s t h e s i z e d rats with DBTC, DPTC and DHexTC was r e d u c e d to 50 % of c o n t r o l by contemporary a d m i n i s t r a t i o n of DMSA and DMPS. The toxic effects of d i a l k y l t i n compounds on liver, bile duct and pancreas were s i g n i f i c a n t l y diminished by DMSA and DMPS. These results indicate an i n t e r a c t i o n of d i a l k y l tin compounds with alkyl chains of 4-6 C-atoms with d i t h i o l compounds in vivo. Compared with d i m e r c a p r o l DMSA and DMPS are more active and both compounds may be r e c o m m e n d e d for the treatment of acute poisonings with these organotins.

M. B6hme, W. R u m m e l

Xpresent adress: Institute of P h a r m a c o l o g y and Toxicology, U n i v e r s i t y of R o s t o e k , L e n i n a l l e e 70, 0-2500 Rostock, Germany

M e c h a n i s m s of methylmercury- and HgCl2-indueed C1secretion in the rat colon

T h e action of organic and inorganic mercury on colonic epithelium was studied with the whole-cell patch-clamp technique and the Ussing chamber. Methylmercury induced an increase of m e m b r a n e outward current in enterocytes of isolated crypts patched from the basolateral side. This action was inhibited by a CF channel blocker and a K + channel blocker indicating an increase of both the CI- and the K + conductance. In contrast, HgC12 did not affect m e m b r a n e current. In m u c o s a preparations, both compounds increased the shortcircuit current (Isc) and the tissue conductance (Gt) indicating an activated CI" secretion. The effect on Gt was m o r e pronounced, w h e n the H g compounds w e r e given to the mucosal side, whereas Isc response was m o r e sensitive to the H g compounds applied to the serosal side. Tetrodotoxin but not atropine suppressed the effect of serosally applied H g c o m p o u n d s on Isc indicating the m e d i a t i o n by non cholinergic s e c r e t o m o t o r neurons. Inhibition by i n d o m e t h a c i n gave evidence for the participation of neuronally acting prostaglandins. Addition of dithiothreitol reversed the actions of H g - c o m p o u n d s suggesting a reaction with SH-groups. Institut ftir P h a r m a k o l o g i e und Toxikologie, Universitiit des Saarlandes, D-6650 Homburg/Saar, F.R.G. Supported by SFB 246, project C2.

R 26 101 EFFECT OF HEAVY METAL IONS ON THE PHAGOCYTOSIS OF HUMAN BLOOD MONOCYTES G. Zwadlo-Klarwasser and C. G6ttsch Heavy metal ions are reported to decrease many macrophage functions e.g. antigenpresentation and killing of parasites and tumor cells. Their influence on the phagocytosis however is discussed controversially. We therefore studied the effect of the metals cadmium (CdS04), mercury (HgC12), lead (Pb(CHACOO)2) and bismuth (BiO (CIO4)) on the latex phagocytosis o f - h u m a n blood monoeytes. Ceils were isolated with Ficoll-Paque centrifugation and incubated for 90 min at 37" C with the latex particles and the metals in concentration ranges from 10 -4 to 10-8 M. Results were expressed as the relative number of monocytes (Mo) ingesting latex particles versus the total Mo population (% positive Mo) and as the particle number/positive Mo a~ parameter of phagocytic aetivty. Concentrations of 10-" M of bismuth, cadmium and lead and of 10 -5 M of mercury reduced the viability of the Mo from >95% to about 80% and decreased the phagocytic activity. Cadmium, lead and mercury at 10-~ M also diminuished the % positive Mo probably due to toxic effects. By contrast bismuth did not i n f l u e n c e l h e number of phagocytic Mo at 10-4 M and eve~ increased it at 10 - 6 M. Cadmium in concentrations lower than 10T M M had only weak effects while mercury strongly reduced the number and activity of phagocytic Mo up to 10 -8 M. Lea~l depressed the phagocytosis of Mo at 10 -6 M, but increased it at 1 0 - ° M . These results show that heavy metal ions affect the phagocytosis of blood monocytes differently. Mercury and lead inhibit the phagocytosis in concentrations only twofold higher than those found in human blood. Bismuth seems to activate Mo in terms of an increased phagocytosis. This activation may lead to an enhanced wound healing and may therefore contribute to the beneficial effects of bismuth on gastrointestinal lesions. Institute of Pharmacology, RWTH Aachen, Wendlingweg 2, 5100 Aachen, F.R.G.

103 RENAL TOLERANCE OF THE COMBINATION TREATMENT WITH FUROSEMIDE-CEFPIROME OR GENTAMICIN-CEFPIROME IN RATS C. Cojocel, H. H. Donaubauer, H. Fuchs, K. H. Langer and D. Mayer The present study was designed to investigate the morphological and functional effects on the rat kidney after i . v . treatment of female Wistar rats with cefpirome (CPO) (700 mg/kg/d) or with the combination of furosemide (FU)-cefpirome (20 + 700 mg/kg/d) or gentamicin (GNT)-cefpirome (20 + 700 mg/kg/d) for 30 days. CPO was administered i h after FU or GNT administration. In control groups, rats were given saline, CPO, FU or GNT alone. Morphological and functional changeswere monitored at the end of the treatment period by standard histology and measurements of the plasma concentrations of Na~, K÷, Ca++, C l , P042-, bilirubin, glucose, uric acid, creatinine, urea (BUN), GOT, GPT and alkaline phosphatase. The a b i l i t y of renal cortical slices from male rats to accumulate p-aminohippurate (PAH) and tetraethylammonium (TEA) or to synthesize glucose (gluconeogenesis) was also investigated after 5, 10 or 15 days of similar treatment. Except a marginal increase in BUN, there were no changes of the clinical chemistry parameters in rats treated with CPO alone or with the combinations FU-CPO or GNT-CPO. Treatment of rats with FU or GNT alone induced the expected calcification of tubular cells or tubule cell necrosis/atrophy, respectively. The combination treatments FU-CPOor GNT-CPOdid not potent i a t e the tubule damage induced by FU or GNT alone but rather showed a distinct protective effect. Furthermore, protective effects of the combination therapy were also observed on the a b i l i t y of the rat renal cortex to accumulate organic ions or to synthesize glucose. Hoechst AG, Postfach 800320, D-6230 Frankfurt/M 80, FRG

102 SMALL-SCALE EXPOSURE SYSTEM FOR MERCURY-(Hg-203) VAPOR. R. Fiohtner and S. Halbach.

104

The investigation of the toxicokinetics of mercury upon inhalation of the vapor (Hg °) requires the use of an exposure system for small animals with specific performances: rapid development and stability of preselected Hg ° concentrations, continuous operation over variable exposure times, easy monitoring of [Hg °] in air and ready determination of Hg in organs and carcass. These .~pecifications can be realized by generating Hg from the reduction of Hq ++ that had been labeled with radioactive Hg-203 ÷+. The commonly used reducing agents SnCI 2 or NaBH 4 gave unsatisfactory results, i.e. the time period for the buildup of the Hg ° concentration was too 10ng and stability could not be maintained. Therefore, the reducing properties of hypophosphoric acid (HPHo02) were tested. Continuous measurement with AAS ~howed that rise time of [Hg °] was below i0 min and the plateau was stable and higher than with the other reductants. The height of the plateau, i.e. [Hg °] in air was linearily correlated to the [Hg +÷] in solution. Estimation of Hg-203 in the wasted solution gave concentrations lower than 2% of that of the initial Hg ++ solution, i.e. vaporization of Hg was nearly complete. The time to attain 90% of the plateau [Hg °] in the exposure chamber can be calculated to be 3.7 min, which is in accordance with 4.6 min actually measured. Body burden and organ distribution of Hg were determined after exposure to 0.5, 1.0 and 2.0 mg Hg°/m 3 for i, 2 and 3 h. Under these conditions Hg uptake was linearily correlated to exposure time or concentration.

p-Aminophenol is nephrotoxic in rats and causes necrosis of the pars recta of the proximal tubules. Aminophenol is oxidized by hepatic and renal enzymes to a reactive quinone imine which is presumed to react with glutathione, y - G l u t amyltranspeptidase-dependent uptake of t h i s glutathione S-conjugate, which is structurally related to toxic S-conjugates formed from bromoquinone, into the kidney may explain nephrotoxicity. We have investigated the biosynthesis and in vitro nephrotoxicity of aminophenol-derived Sconjugates in rats and rat kidney cortex cells. After i.p. application of aminophenol, l-amino-3glutathione-S-yl-4-hydroxybenzene (AP-SG) was identified as a biliary metabolite by thermospray mass spectrometry and by its pH-dependent electronic spectra. AP-SG was cytotoxic in rat kidney cortex cells; cell killing by AP-SG was significantly reduced by inhibition of ~-glutamyltranspeptidase and by inhibition of cysteine conjugate 6-1yase. Moreover, inhibition of cytochrome P-450 by SKF-525A and prostaglandin synthase by indomethacin also protected the cells from AP-SG induced toxicity. These results suggest that biosynthesis and renal metabolism of AP-SG contributes to p-aminophenol nephrotoxicity. Institut f~r Toxikologie, Universit~t W~rzburg, Versbacher Str. 9, D-8700 W~rzburg, F.R.G.

GSF-Institut

f. Toxikologie,

8042 - Neuherberg

AMINOPHENOL GLUTATHIONE

M. Koob,

NEPHROTOXICITY: CONJUGATES

C. Klos,

BIOSYNTHESIS

OF T O X I C

C. Kramer and W. Dekant

R 27 105

107

I N V E S T I G A T I O N S ON POSSIBLE A C T I V A T I O N P A T H W A Y S OF THE B L A D D E R C A R C I N O G E N N - N I T R O S O BU T Y L - 3 - C A R B O X Y P R O P Y L A M I N E [BCPN] D. J a c o b u n d C. J a n z o w s k i

ARE REACTIVE RADICALS FORMED AS A CONSEQUENCE REPERFUSION OF THE ISCHEMIC HEART ?

Activation of N-nitrosobutyl-3-carboxypropylamine [BCPNI b y c~- or ~ - o x i d a t i o n m a y b e i n v o l v e d i n t h e mechanism of i t s o r g a n o t r o p i c e f f e c t to t h e u r i n a r y b l a d d e r . T h e r e f o r e i n - v i t r o m e t a b o l i s m of BCPN a n d i t s ~-oxidized metabolite N-nitrosobutyl-2-oxopropylamine [BOPN] w a s s t u d i e d . BOPN w a s d e b u t y l a t e d a t h i g h r a t e s w h e n i n c u b a t e d with rat liver microsomes, whereas pig urinary bladder m i c r o s o m e s w e r e m u c h l e s s a c t i v e . BCPN w a s d e a l k y l a t e d a t b o t h a l k y l g r o u p s to a l o w e x t e n t in t h e s e s y s t e m s . It a l s o w a s f o u n d to b e n o n g e n o t o x i c in N a m a l v a c e l l s , i r r e s p e c t i v e of t h e m o d e of a c t i v a t i o n . In c o n t r a s t , B O P N i n d u c e d D N A s i n g l e s t r a n d b r e a k s [SSB] w h e n i n c u b a t e d w i t h P B - i n d u c e d r a t l i v e r m i c r o s o m a l f r a c t i o n . N - N i t r o s o b u t y l u r e a [BNU], N - n i t r o s o - 3 ~ carboxypropylurea [CPNU] a n d N - n i t r o s o - 2 - o x o p r o p y l u r e a [OPNU] w e r e t e s t e d a s d i r e c t l y a c t i n g m o d e l compounds yielding the same ultimate electrophiles as BCPN a n d BOPN. O P N U i n d u c e d h i g h r a t e s of D N A SSB in N a m a l v a c e l l s a n d u r i n a r y b l a d d e r c e l l s w i t h n o i n d i c a t i o n for r e p a i r w i t h i n 4h. BNU s h o w e d t h e s a m e effect only at lO-fold higher concentrations. DNA damage w a s r e p a i r e d to a s i g n i f i c a n t e x t e n t [4hi, CPNU s h o w e d v e r y w e a k SSB i n d u c i n g a c t i v i t y . T h e r e s u l t s s u g g e s t t h a t ~ - o x i d a t i o n of BCPN m i g h t b e a p o t e n t i a l a c t i v a t i o n pathway. Department of F o o d Toxicology, University,

Chemistry and Environmental D-6750 Kaiserslautern

106 S T U D I E S ON THE M E C H A N I S M OF THE P A N C E E A T I C BC E L L T O X I C A C T I O N OF A L L O X A N S. L e n z e n A n e w i n d i c a t o r s y s t e m for e l u c i d a t i o n of a l l o x a n B - c e l l t o x i c a c t i o n was used: the i n h i b i t i o n of s p e r m i n e - i n d u c e d c a l c i u m u p t a k e by m i t o c h o n d r i a in p e r m e a b i l i z e d p a n c r e a t i c B - c e l l s f r o m o b / o b m i c e as m e a s u r e d w i t h a specially designed calcium minielectrode. The g l u c o k i n a s e is the s i g n a l r e c o g n i t i o n e n z y m e of the p a n c r e a t i c B - c e l l for i n i t i a t i o n of g l u c o s e - i n d u c e d i n s u l i n s e c r e t i o n . B o t h a l l o x a n and the s u g a r m a n n o h e p t u l o s e i n h i b i t glucose-induced insulin secretion through i n h i b i t i o n of this enzyme. However, o n l y a l l o x a n is B - c e l l t o x i c due to t o x i f i c a t i o n of a l l o x a n as a r e s u l t of the i n t e r a c t i o n w i t h the g l u c o k i n a s e . A l l o x a n not o n l y i n h i b i t s this e n z y m e but at the s a m e time y i e l d s d i a l u r i c a c i d t h r o u g h r e d u c t i o n of alloxan. T h r o u g h r e d o x c y c l i n g b e t w e e n a l l o x a n and d i a l u r i c a c i d c y t o t o x i c free r a d i c a l s are g e n e r a t e d , w h i c h are r e s p o n s i b l e for p a n c r e a t i c B - c e l l death. The half m a x i m a l i n h i b i t o r y c o n c e n t r a t i o n for i n h i b i t i o n of c a l c i u m u p t a k e in p e r m e a b i l i z e d o b / o b m o u s e B - c e l l s was 3.4 m M for a l l o x a n and 5.8 m M for d i a l u r i c acid. G l u c o s e p r o t e c t e d a g a i n s t the e f f e c t of a l l o x a n but not of d i a l u r i c a c i d a p p a r e n t l y t h r o u g h o c c u p y i n g the g l u c o k i n a s e g l u c o s e b i n d i n g site and t h e r e b y p r e v e n t i n g t o x i f i c a t i o n of alloxan. I n s t i t u t f~r P h a r m a k o l o g i e und T o x i k o l o g i e , Universit~t G6ttingen, D-3400 G6ttingen

H. N o h l

OF

and K. S t o l z e

R e p e r f u s i o n i n j u r y of i s c h e m i c o r g a n s is s u g g e s t e d to r e s u l t f r o m m e t e b o l i c d e r a n g e m e n t s initia t i n g an i m b a l a n c e d f o r m a t i o n of free o x y g e n radicals. M o s t i n v e s t i g a t o r s in this f i e l d have u s e d the s p i n t r a p 5 , 5 - d i m e t h y l p y r r o l i n e - l - o x i d e (DMPO) to s t a b i l i z e these short lived radicals and m a k e t h e m v i s i b l e b y m e a n s of E a R t e c h n i q u e . EaR-signals obtained from intravascular DMPO w e r e r e p o r t e d to i n d i c a t e the f o r m a t i o n of free O H ' - r a d i c a l s and in some c a s e s also c a r b o n cent e r e d r a d i c a l s . We w e r e u n a b l e to c o n f i r m these findings. C a r b o n c e n t e r e d r a d i c a l s w e r e not obtained irrespectively of conditions studied while oxygen centered DMPO-adducts could only be d e t e c t e d in r e a s o n a b l e a m o u n t s w h e n iron was add e d to the p e r f u s a t e . U n d e r t h e s e c o n d i t i o n s an ascorbyl-related EaR-signal c a m e hp w h i c h was also p r e s e n t w i t h o u t the a d d i t i o n of iron. However, the i n t e n s i t y of the latter varied with the a m o u n t of iron r e l e a s e d into the perfusate. Although our r e s u l t s do not exclude oxidative s t r e s s as the p a t h o g e n e t i c m e c h a n i s m of postisc h e m i c o r g a n injury, i n t r a v a s c u l a r f o r m a t i o n of o x y g e n - c e n t e r e d D M P O - a d d u c t s is u n l i k e l y to result f r o m free o x y g e n r a d i c a l s of the p e r f u s a t e . T h i s p o i n t w i l l b e d i s c u s s e d in t e r m s of b i o c h e mical and physicochemical considerations.

I n s t i t u t e for P h a r m a c o l o g y and T o x i c o l o g y , V e t e r i n a r y U n i v e r s i t y of V i e n n a , L i n k e B a h n g a s s e II, A-1030 Vienna

108 ALTERED OXYGEN-HEMOGLOBIN DISSOCIATION RATES IN SMOKERS S.Kunkel*, P.Ever *~ The importance of smoking as a possible risk factor in in coronary heart disease (CHD) may be related to the effect of carbon monoxide (C0) on oxygen exchange in hemoglobin (Hb). Therefore we have examined the kinetics of this process in vitro using a fast-reaction technique (stopped-flow). Blood from 14 smokers and 13 non-smokers was diluted (approx. 60umol/l Mb) with bis-tris buffer (pH 7.4; 0.2mol/]) containing 2 mmol 2,3-diphosphoglycerate and mixed with 20mmol/l sodium dithionite/bis-tris as oxygen acceptor in the reaction chamber of an Aminco-Dasar stopped-flow apparatus. The change in oxyhemoglobin absorption was followed at 576/593nm. The initial reaction (loss of the first oxygen molecules I) proceeded with a halflife of 14.2 ! 0.7 ms in non-smokers but in smokers this process was slower (ti/2 = 16.5 ± 0.7 ms; p < 0.05). In the later phase of the reaction (occuring over the period of 14-30 ms) the rate of oxygen release in smokers was also reduced (tl/2 = 9.1 t 0.7 ms versus 8.4 ~ 1.13 ms; p= 0.065). These changes are apparently due to the presence of CO (8.4% ~ 2.4%) is smokers since in subsidiary experiments the halflife of oxyhemog]0b~n in the late reaction phase increased from 7 to 21ms when the HbCO saturation was raised from 0.8% to 45%. These results support the view that CO modulates the release of oxygen from hemoglobin in smokers under normal conditions and that this phenomenon can impair oxgyen availability in CHD patients. i. Dalziel and O'Brien (1960) Biochem. J.,78,236-245. * Dept.Clin.Pharmacology, University Hospital Frankfurt **Inst. Pharmacology and Toxilogy, University of M~nchen

R 28 109 DINITROBENZENE DERIVATIVES INHIBIT PLATELET AGGREGATION BY INCREASING INTRACELLULAR CYCLIC GMP LEVELS F. v. Appen Preincubation of washed human platelets with 1-chloro-2,4dinitrobenzene (CDNB) or with 1,3-dinittobenzene leads to dose- and timedependent inhibition of agonist-induced platelet aggregatiom This inhibitory effect is correlated with an impaired agonist-induced intracellular calcium release. Using radioimmunoassays a steady increase of intracellular cyclic GMP level is observed during treatment of intact platelets with CDNB or dinitrobenzene. Compared with control cells a 6 to 10 fold increased activity of guanylate cyclase is detected in the eytosolic fraction of CDNB-pretreated platelets. However, CDNB and 1,3-dinitrobenzene are also potent activators of soluble guanylate cyclase activity in cell free assay systems. The presence or absence of low molecular weight thiols does not influence the activation of soluble guanylate cyclase by the dinitrobenzenes. With heme-free enzyme preparations, however, the stimulating effect of the dinitrobenzenes is abolished. The presented data provide evidence for an activation of soluble guanylate cyclase by dinitrobenzenes or their metabolites via interaction with its heine group.

111 UV-PHOTODEGRADATION OF QUINOLON~S - BINDING OF PHOTOPRODUCTS TO HUMANSKIN E.M. Tiefenbacher and H. Kurz Various 6- fluoroquinolones absorb radiation energy" from the UVrange of the electromagnetic spectrum. Phototoxic or photoallergic effects have been reported for some of these substances. Exact mechanisms of these reactions are unknown so far. We investigated the influence of UVA on aqueous solutions of ciprofloxacin, ofloxacin and fleroxacin, HPLC - chromatograms of the irradiated substances (UVA dose= 100 J / c m z ) revealed photodegradation and formation of several photoproducts. Binding of activated photoproducts to human cells is discussed as mechanism for phototoxic rea'ctions. We are interested in the binding of ciprofloxacin and its photoproducts to human skin. Skin was obtained from human thighs, reduced with N2 and l¥ophilisized to a homogenous powder. Binding was determined by equilibrium dialysis with ciprofloxacin in phosphate buffer (pH 7.4, 10-3 - 10-6 tool/l) or a similar, but irradiated solution (10 -4 tool/l, 100 J/cm 2) at 4° C for 20 h. In the range from 10-3mol/l to 10-6mol/l, ciprofloxacin binding is concentration dependent;10- 3tool/1 are bound to 14~3+1.8%, 10-6tool/1 to 82.0 + 3.0%. Irradiated ciprofloxacin is bound to the same extent as non-irradiated. After dialysis of irradiated 10-4 tool/1 ciprofloxacin solutions none of the photoproducts could be detected as free fraction. Using 10- 3 tool/1 solutions, one photoproduct, more polar than ciprofloxacin and binding to 47.3%, and two less polar photoproductm binding both to 75.0%, could be detected. Some photoproducts seem to bind to a higher degree to human skin than ciprofloxacin itself. It is not known so far whether binding of these substances to human skin is specific or non-specific~ _

Walther-Straub-Institute of Pharmacology and Toxicology, Ludwigs-Maximilians-University, NuSbaumstr. 26, D-8ooo Miinchen 2

Department of Biology, University of Konstanz, Universit~tsstr. 8-10, D-7750 Konstanz, F.R.G.

110 The long-term effect of the rodenticide brodifacoum on blood coagulation and on the hepatic vitamin K metabolism in rats J.J. Mosterd* and H.H.W. Thijssen Brodifacoum is a potent anticoagulant rodenticide which acts as a vitamin KI antagonist. An abnormal vitamin K1 metabolism was reported for factory workers for at least 18 months after accidental exposure to brodifacoum. However, clotting factor activity at that time was normal, suggesting a dissociation between the coumarin effect on vitamin K metabolism and clotting factor synthesis (Park et al, Br J Clin Pharmacol 21, 289, 1986). We investigated the long-term effects of brodifacoum on vitamin K metabolism in rats. Rats received a single dose of brodifacoum (0.2 mg/kg, p.o.). The anticoagulant effect, the hepatic vitamin K cycle and the microsomal warfarin binding were assayed during 30 days. Ex-vivo microsomal vitamin K epoxide reductase and microsomal warfarin binding remained inhibited for at least 30 days. Clotting factor synthesis, however, was restored from beyond day 7. The in-vivo vitamin K metabolism was also disturbed: accumulation of vitamin K epoxide in the liver following a pharmacological vitamin K dose was still manifest at day 30. Liver tissue and liver microsomal brodifacoum concentrations remained almost stable and no brodifacoum could be detected in the circulation. In conclusion, brodifacoum is a persistent rodenticide with high binding affiriity for the liver microsomal warfarin binding site, resulting in a long lasting suppression of the vitamin K cycle. Apparently, a partly operating vitamin K cycle suffices for normal clotting factor synthesis. In vivo, the vitamin K cycle can be uncoupled from the vitamin K dependent earboxylation reaction. Dept. of Pharmacology, University of Limburg, P.O. Box 616, 6200 MD maastricht, The Netherlands "Supported by the Dutch Heart Foundation

112 METALLOTHIONEIN IN HUMAN EPIDERMAL KERATINOCYTES IN CULTURE H. Kappus* and Ch. Reinhold'* Human skin is exposed to d r u g s , chemicals, irradiation etc. which induce toxicity. Some of the mechanisms involved include oxidative s t r e s s which leads to cellular damage of lipids, p r o t e i n s and DNA r e s u l t i n g in cell death, metabolic d i s t u r b a n c e s , mutagenicity and carcinogenicity. We studied the effects of organic peroxides in c u l t u r e d human k e r a t i n o c y t e s . In c o n t r a s t to other cells lipid peroxidation was h a r d l y inducible, although cellular toxicity o c c u r r e d (Kappus and A r t u c , Bioelectrochem. Bioe n e r g . 1 8 , 263, 1987; A r t u c et al., Arch. Dermatol. Res. 281, 49, 1989). Because metallothionein has been shown to t r a p h y d r o x y l radicals, we wondered w h e t h e r this molecule is protective a g a i n s t oxidative s t r e s s and w h e t h e r it is p r e s e n t in the keratinocytes u s e d . Keratinocytes were isolatted from various f r e s h human skin samples. Cell cult u r i n g was carried out according to s t a n d a r d methods. After removal of the cells from the dishes t h e y were homogenized and cytosol p r e p a r e d b y u l t r a c e n t r i f u g a t i o n . Metallothionein was determined b y the C d - s a t u r a t i o n method (Klein et a l . , Anal. Biochem. 189, 35, 1990). In f r e s h l y isolated keratinocytes 0.1 - 0.3 p.g metallothionein was measured p e r mg cytosolic p r o t e i n . In keratinocytes cult u r e d for 2-4 weeks the metallothionein content increased to 0.6 - 2.0 ~tg/mg p r o t e i n , p r o b a b l y due to culture condit_ions. When we c u l t u r e d keratinocy-tes in the p r e s e n c e of Zn ions the metallothlonein content increased u p to 15 fold compared to r e s p e c t i v e controls. These r e s u l t s indicate that metallothlonein is p r e s e n t in human epidermal cells (keratinocytes) and that it is inducible in culture b y a n u m b e r of f a c t o r s . T h u s , keratinocytes might be a suitable tool to s t u d y the protective role of metallothionein in oxidative s t r e s s and toxicity. * I n s t i t u t ffir Landstrafie 1, ** Hautklinik, D-1000 Berlin

Toxikologie, GSF, Ingolst~idter D-8042 N e u h e r b e r g FU, A u g u s t e n b u r g e r Platz 1, 65

R 29 113 INFLUENCE OF rHIRUDIN ON NICROTNROMBOSI$ INDUCED BY RUSSEL'$ VIPER VENOM (RVV) G. Nowak, E. Bucha and +J, N e i e r

The a d m i n i s t r a t i o n o f small amounts of RVV ( I 0 25 pg/kg x h) was followed by formation o f microthrombi in r a t lungs. Previous a p p l i c a t i o n o f 1 2 ~ I - f i b r i n o g e n (3.7 MBQ, I-2 h before the experiment) and 111-In-labelled platelets (I MSq, 2-24 h before the experiment) served to measure the microthrombosis continuously by means of a gamma-scintillation probe. Much lower venom doses were required for the induction of p l a t e ] e t deposition than for fibrin deposition in rat lungs, The a d m i n i s t r a t i o n of

rhirudin before the application of venom prevented the p l a t e l e t accumulation and the deposition o f f i b r i n o g e n , rasp. Already very low doses o f h i r u d i n w e r e able t o prevent f i b r i n d e p o s i t i o n whereas much higher doses were necessary to i n h i b i t p l a t e l e t d e p o s i t i o n . Preliminary LDso experiments with and without r h i r u d i n pretreatment resulted in the f o l l o w i n g f i n d i n g s : h i r u d i n proved t o have a b e n e f i c i a l effect on the animal blood coagulation system by reducing the l e t h a l i t y caused by RVV (4.3 f o l d lower LDs0 values a f t e r pretreatment with rhirudin). Present address: Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Nordh~user Str. 74, O-5010 Erfurt, FRG; ÷ P e n t a p h a r m AG Lid, Basel, Switzerland

115 CYTOSKELETAL CHANGES IN ENDOTHELIAL CELLS F O L L O W I N G A P P L I C A T I O N OF C.NOVYI A L P H A TOXIN A. Oksche, R. N a k o v and E- H a b e r m a n n Alpha toxin of C.novyi type A is a single-chain cytotoxin of about 200 kDa. The toxin (0.02 2pg/ml) induces retraction of the cell body and occasional membrane blebbing of cultured porcine p u l m o n a r y artery endothelial cells in a concentration dependent manner. Cells treated with the highest concentration for up to 6h remain adherent to their p r i m a r y support as shown by scanning electron microscopy. Cells pretreated with alpha toxin (125 ng/ml) for 24h and then passagad still adhere to their secondary support. However, such cells show neither spreading, nor sprouting, nor mitosis w h i c h indicates an effect of alpha toxih on their cytoskeleton. Changes in the arrangement of m i c r o f i l a m e n t s have been shown d i r e c t l y with fluorescent p h a l l o i d i n as a marker specific for F-actin. A l r e a d y in early stages of alpha toxin treatment (2pg/ml, 2h) stress fibers disintegrate an~ F-actin is enriched around the nucleus whereas microtubules and intermediate filaments are not altered. The amount of F-actin but not of total actin decreases when m e a s u r e d by DNAase inhibition and calculated as the fraction of total protein. It is concluded that alpha toxin alters the c y t o s k e l e t o n which then leads to retraction of endothelia and, finally, oedema formation. Since the morphological changes resemble those described for C.difficile cytotoxins A and 'B, the three toxins may share a common mode of action. However, the primary target of the toxins is still unknown.

R u d o l f - B u c h h e i m - I n s t i t u t f~r Pharmakologie der Universit~t Gie~en, Frankfurter Str. 107, D-6300 GieSen

114

116

THE CHROMAFFIN CELL: A SUITABLE MODEL FOR INVESTIGATING THE ACTIONS AND THE METABOLISM OF TETANUS AND BOTULINUM A NEUROTOXINS P. Marxen and H. Bigalke

RESTORATION OF NORADRENALINE RELEASE FROM TETANUS TOXIN-TREATED CHROMAFFIN CELLS F. Bartels

Tetanus (Tetx) and botulinum A neurotoxins (BoNtx) block the exocytotic release of catecholamines from bovine chromaffin cells, provided they are able to reach the cell interior, Chromaffin cells, being excellent models for the study of neurosecretion, are inherently deficient in polysialogangliosides, the binding sites for the toxins. Basically, there are three different ways of translocating the toxins into the cells: 1) Enrichment of cell membranes with polysialogangliosides enables the toxins to bind to the plasma membranes and hence to accumulate in the cells. 2) Poration of the cell membrane by cytolysins or digitonin allows the toxin molecules to diffuse into the cells. 3) Exposure to a strong electric field results in the formation of transient pores in the cell membrane through which the toxins may enter the cytosol.-To develop full inhibitory action in chemically permeabilized chromaffin cells, the toxins have to be applied in their reduced form. The use of radioiodinated toxins, introduced into chromaffin cells by electroporation, allowed the observation of the intracellular processing of these substances over a period of several days. The toxins and their derivatives were extracted, separated by SDSPAGE and detected by autoradiography. Increasing amounts of the toxins were split into two chains and further degraded to small fragments within a period of 5 days. The block of exocytosis, nonetheless, outlasts the intracellular survival of both toxins. This leaves room for speculations that a hitherto unidentified component in the exocytotic machinery, knocked out by the toxins, may have to be resynthesized to restore exocytosis, or that the capacity to block exocytosis resides in a small fagment of the toxin molecules. Furthermore, it cannot be excluded that an amount of toxin too small to be detected may suffice to maintain the block of exocytosis. Medizinische Hochschule Hannover, Abteilung Toxikologie, 3000 Hannover 61, FRG This work was supported by the DFG (Bi:274/4-1)

Tetanus toxin blocks carbachol-stimulated noradrenaline release from ganglioside-preloaded chromaffin cells. The block persists for several days. Specific anti-tetanus toxin antibodies, applied to digitonin-permeabilized chromaffin cells at a time when the block is fully evident, bind to intracellular tetanus toxin without being able to restore .exocytosis (Marxen et al., 1990). When tetanus toxin-treated cells are exposed to an electric field, pores open up in the plasma membrane. The pores are large enough for the toxin (and cytosolic substances) to move out of the ceils. In spite of this, the cells fail to resume the secretion of noradrenaline for at least 6 days, although in toxin-untreated cells, the exocytotic machinery has recovered from poration long ago. When specific anti-tetanus toxin antibodies are present during electropermeabilization, the intracellular interaction of tetanus toxin with its antibody restores exocytosis within 72 hours. It is concluded that tetanus toxin irreversibly alters a component essential for exocytosis and that, after the intracellular neutralization of tetanus toxin by its antibody, a slow recovery is achieved, probably by resynthesis of its as yet unidentified target. Marxen P., Ahnert-Hilger G., Wellh6ner H.H., and Bigalke H. (1990) Toxicon 28:1077-1082 Medizinische Hochschule Hannover, Abteilung Toxikologie, 3000 Hannover 61, FRG This work was supported by the DFG (Bi:274/4-1)

R 30 117

119

POINT HUTATIONS IN CYTOTOXIN GENE OF PSEUBOIqON~S

IN VITRO TOXICITY OF LITHIUM: EFFECTS, TISSUE

4ERUGII~A

CONCENTRATIONS - NO PROTECTIVE EFFECT OF MYO-INOSITOL Stephan Khig, Tetsuji Nagao, Mike Collins

G. Xiong and F. Lutz

Pseudomonas aerugino~a is a patho~nic organism causing l i f e threatening dise~ses by pr(w~ucing several toxic factors. One of the path~enic factors is an acidic protein of 28 kDa. The gene of that has ~)een cloned into pSN3, sequenced and expressed in E. c o l i in t h i s laboratory [ 1 ] . By research of deletion and point mutants, three dowains on the cytotoxin gene were found to c(~3nect with the cytotoxin toxic activities. Activity paramel~rs used were binding to rabbit ghosts and ~ r m e a b i l i t y increase of human granul~ytes. Severe1 point mutants on a domain of bp 700 - 858 were prel:kared. The r e s u l t s showed t h a t bp 745 - 778 a r e connected with biolc~ical functions. Both a c t i v i t i e s became very low when point mutants occured in that r e g i o n . Whereas the f u n c t i o n s were not changed when p o i n t mutations were c ~ t r u c t e d i n I~o 700 - 744 and 779 - 858. There are two cys t h e c y t o t o x i n p r o t e i n are two cys (c(~ons a t bp 68 - 70 and 64~ - 645) i n t h e c y t o t o x i n p r o t e i n . When bp 68 - 70 were d e l e t e d o r / a n d bp 645 - 645 were changed t o g l y c i n e , the c y t o t o x i n p r o t e i n sh(~ed low b i n d i n g a c t i v i t y and no g r a n u l o c y t e s w e l l i n g activity. I t seemed t h a t t h e $-S b r i ~ may be very i m p o r t a n t t o keep t o x i n conformation and biolcw3ical activities. [ 1 ] O r l i k - E i s e l G, Lutz F, E i s e l U, Struckmeier H, Kr~uter ~, Niemann H (1770) Arch Hicr(Y~iol 15~:5~1-5~8.

Lithium was tested at various concentrations using a rat wholeembryo culture system in order to establish a concentrationresponse relationship (c.f. table). CR Somites Score Abn (#g/ml) (ram) (n) (%) Control 5O 100 150 2OO

4.68 3.72* 3.48** -3.36** 2.28**

28 27* 27* 27* 24**

38 36* 34** 31"* 23**

60 100

CR = crown-rump length; Abn = frequency of abnormal embryos; • = 0.01 _ p _ A transversions at the first base and A - > T transversions or A - > G transitions at the second base of c-Ha-ras codon 61 were detected in 20 - 60% of spontaneous or carcinogen-induced liver tumors of the four sensitive mouse strains but not in liver tumors of the two insensitive mouse strains or the Wistar rat. Further analyses of c-Ha-ras codon 12 mutations in liver tumors from the insensitive rodent strains also failed to give any positive results. In early precancerous liver lesions, c-Ha-ras codon 61 mutations were found in 13 - 14% of lesions 6f the sensitive C3H/He and B6C3F1 mouse strains, whereas no such mutations could be detected in precancerous lesions of the insensitive C57BL/6J mouse. Taken together, our results indicate a close correlation between the mutational activation of the c-Ha-ras gene in liver tumors of the different rodent strains and their susceptibility to hepatocarcinogenesis, whereby the mutations appear to provide a selective growth advantage, leading to a clonal expansion of the mutated liver cell population, only in livers of sensitive but not of insensitive strains. ~Institute of Toxicology, University of Tiibingen, 7400 Tiibingen, Germany, 2Institute of Pathology, GSF, 8042 Neuherberg, Germany, 3McArdle Laboratory for Cancer Research, Madison, USA, 'Institute of Experimental Pathology, DKFZ, 6900 Heidelberg, Germany

134

136

INTERRELATIONSHIP OF MICRONUCLEUS FORMATION, GENOMIC

MUTATIONS IN THE HA-RAS PROTO-ONCOGENE IN LIVER TUMORS OF THE C3H MOUSE OCCURRING SPONTANEOUSLY OR AFI'ER TREATMENT WITH TUMOR PROMOTING AGENTS. R. Bauer-Hofmann, S. Kress, and J. Mahr

LOSS AND CELL TRANSFORMATION INDUCED BY DIETHYLSTILBESTROL (DES) D. Schiffmann, H. Stopper, P. Lenz, U. De Boni* and B. K. Vig** Certain nongenotoxic carcinogens (e.g.some estrogens like DES) cause mitotic disturbances resulting in displacement, nondisjunction and subsequent loss of chromatin elements. DES induces micronuclei (MN) and neoplastic transformation in Syrian hamster embryo (SHE) flbroblasts. However, the key events at the early onset of this multistage process are still under debate. Our previous results show that DES induced MN contain kinetochores (K+) suggesting the presence of whole chromosomes/chromatids. We now have analyzed the meta-anaphase ring arrangement of chromosomes in SHE cells. After DES treatment, a threefold increase in the number of chromosomes displaced from the ring was observed. In mouse LA 9 cells we found a similar increase, with a maiority of K+ chromosomes . Most likely, these displaced chromosomes can give rise to the observed K+-MN. Furthermore, a 10% subpopulation of these MN exhibits chromatin compaction (electron microscopy). Since such MN will not be reintegrated, genomic loss will occur, possibly depending on the type of chromosome / chromatid enclosed. In addition, the frequency of DES-mediated SHE cell transformation also is 10% of the one for MN formation. Further investigations will show, whether these phenomena are related to the loss of tumor suppressor genes. Institute of Toxicology,University of Wfirzburg,87 Wiirzburg, Germany *Dept. of Physiology, University of Toronto, Canada, **Dept. of Biology, University of Nevada Reno, USA.

In the present study we have analysed the frequency and pattern of point-mutations in codon 61 of the Ha-ras proto-oncogene in liver tumors of male C3H/He mice that occurred spontaneously or after treatment with 500 ppm phenobarbital or I0 ppm dieldrin in their diet. The liver tumor prevalence at 52 weeks after start of treatment was 41% (15/37) in control mice, and 63% (10/16) and 67% (10/15) in mice treated with phenobarbital or dieldrin, respectively, indicating that the two promoting agents led to an enhancement of the liver tumor frequency. Serial frozen sections were performed through the entire liver of each animal and stained for glucose-6-phosphatase activity. Liver lesions characterized by a loss in this enzyme activity ranging between 0.5 and 3.0 mm in diameter were punched out from the liver sections. For mutation analysis we used the method of in vitro amplification of DNA via the polymemse chain reaction combined with selective oligonucleotide hybridisation. Our preliminary results demonstrate the presence of codon 61 mutations both in spontaneous tumors and in tumors induced by prolonged treatment with phenobarbital and dieldrin. The mutation frequencies, however, showed striking differences between the three treatment groups examined. While approximately 60% (11 out of 17) of the spontaneous liver tumors contained point mutations in codon 61, the mutation frequency at this gene locus was lowered to a value of only 25 % in tumors occurring during treatment with either phenobarbital (4 out of 16 tumors) or dieldrin (6 out of 24 tumors). These results suggest that mutations in the Ha-ras gene in liver tumors of promotortreated mice represent background mutations not related to treatment and, secondly, that tumor promotors such as phenobarbital and dieldrin also confer a selective growth advantage on those initiated mouse hepatocytes that do not contain an activated Ha-ras gene. Deutsches Krebsforschungszentrum, Institut fiir Experimentelle Pathologie, Im Neuenheimer Feld 280, D-6900 Heidelberg.

R 35 137

139

DETECTION OF GENOMIC ALTERATIONS IN CARCINOGENINDUCED MOUSE LIVER TUMORS BY DNA FINGERPRINT ANALYSIS. O. Mtiller

COMPARISON OF PARTICULATE GUANYLYL CYCLASE FROM BOVINE ADRENAL CORTEX WITH A CLONED ANF-SENSITIVE GUANYLYL CYCLASE. J.-M. Helm, H.-J. Ftille, S. Singh , and R. Gerzer

According to the present understanding of the carcinogenic process, the transition of a normal cell to a tumor cell is mediated by sequential genetic alterations in critical cell regulatory genes such as protooncogenes or tumor suppressor genes that may lead to either the activation or the inactivation of these genes. Molecular correlates are structural changes of the genome ranging from specific point mutations to loss of entire chromosomes. Evidence for structural alterations in the genome of tumor cells has classically been accumulated from cytogenetic studies. In the present investigation DNA fingerprint analysis was used to study structural abnormalities in the genome of mouse liver tumor cells. Liver tumors were induced in three different strains of mice by single injection of 20 I.tg/g body wt. diethylnitrosamine on day 15 after birth. Liver tumors were removed 30 to 45 weeks later. Tumor DNA was isolated, digested with restriction enzymes and hybridized on Southern blots with wild-type bacteriophage M13 DNA as probe. The resulting fingerprints of tumor DNA were compared with those of D N A from normal liver tissue. Genomic aberrations were detected in two out of 68 tumors analyzed, one stemming from a C57BL/6J and the other from a C3H/He mouse. In spite of the fact that the frequency of genomic alterations observed in this study was relatively low, DNA fingerprint analysis has the advantage that, in contrast to classical cytogenetic analyses, frozen tissue can be used and analyses can be performed without selection of metaphase chromosomes.

Recently, an ANF-sensitive guanylyl cyclase (GC-A) has been cloned from a rat brain cDNA library (Nature 338:78-83 (1989)). A rat glioma cell line (C6) was permanently transfected with rat GC-A cDNA with the lipofectin method. Stable clones were selected with geneticin. The following characteristics of this cloned ANF-sensitive guanylyl cyclase were evaluated: stimulation of cyclic GMP accumulation as well as binding characteristics were measured in confluent monolayers, whereas guanylyl cyclase activity was measured with membranes from homogenized cells (100 000 x g; 30 rain). ANF, BNF, atriopeptin 1 (AP 1) as well as urodilatin, a recently described N-terminally elongated ANFanalog, were used. The characteristics of GC-A were compared with those of membrane guanylyl cyclase prepared from bovine adrenal cortex (BAC). In C6 cells expressing GC-A, ANF and BNF stimulated cyclic GMP accumulation to the same extent (ED5~'I nM), whereas AP 1 and urodilatin were less effective (EDs~,-10 nM). The analogs tested showed the same order of potency when g'uanylyl cyclase activity was evaluated with a membrane fraction from C6 cells expressing GC-A. In contrast, binding characteristics were comparable for ANF and the analogs BNF and urodilatin. In contrast to these results obtained with GC-A, AP 1 did not stimulate guanylyl cyclase activity in membranes from BAC, while urodilatin was as effective as ANF and BNF. In BAC membranes, binding of ANF and BNF was comparable. These results suggest that GC-A is different from the ANF-sensitive guanylyl cyclase in BAC membranes. Since the binding characteristics for the tested ANF analogs in C6 cells expressing GC-A were comparable with those on BAC membranes, while the order of potency for activation of guanylyl cyclase differed, signal transduction mechanisms in response to various natriuretic peptides may be distinct in BAC and GC-A.

Deutsches Krebsforschungszentrum, Institut fiir Experimentelle Pathologie, Im Neuenheimer Feld 280, D-6900 Heidelberg.

Supported by the Deutsche Forschungsgemeinschaft (Ge 399/3-3) Medizinische Klinik, Klinikum Innenstadt Ziemssenstr.1, D-8000 Miinchen 2, FRG

Universit~tt,

~o

138 I~AND EXHIBIT BINDING

der

I~ oGMP-DEPENDENT PROTEIN DIFFERENT PHOBPHOTRANSFERASE ACTIVITIES

W.Landgraf,

P.Ruth,

KINASE AND

B.May, A.Keilbach,

(oGK) oGMP

F.Hofmann

The enzymatic properties of recombinant type I~and I$ cGK were studied. Recombinant Iu- and I# cGK were transiently expressed by t r a n s f e c t i o n of the cloned cDNAs into COS-7 cells. Cytosolic extracts contained about 1 ~g of Iu- or I~ cGK per i0 u cells. The recombinant cGKs were indistInguishable in molecular weight from cGK purified either from bovine lung or tracheal s m o o t h muscle, The extracts were m e a s u r e d for phosphotransferase activit~ at 3 0 " C in the presence of an synthetic inhibltor p e p t i d e for cAMP-dependent protein kinase and the substrate peptide GRTGRRNSI. In the presence of cGMP or 8-Br-cGMP the catalytic rat~s w~re 5.0±.04 (3) and 4 9 ±.03 (3) ~mol.m~n'~.mg ~ for Iu- and I@ cGK, respectively. These catalytic rates are similiar to those obtained for the Iu- and I~ cGK purified from bovine tracheal smooth muscle. For the Iu cGK, half maximal stimulation of the phosphotransferase activity was obtained at 0.i ~M cGMP or 0.3 ~M 8-Br-cGMP. In contrast, the K_ values of the I~ cGK for cGMP and 8-Br-cGMP ~ere increased 13-fold and 41-fold, respectively. A similar shift in the activation curve was f o u n d between the purified bovine tracheal Iu- and I~ cGK. Equilibrium binding of cGMP at 4°C d e m o n that both isoforms bound 2 moles cGMP/ strated mol subunit. For the Iu cGK a high and low affinity cGMP binding site was detectable whereas for the IB cGK only a low affinity binding site was calculated. These results were confirmed by measuring the dissociation of cGMP at 4°C. For the Iu cGK a slowly and rapidly cGMP exchanging site was obtained, whereas two rapidly cGMP exfor I@ cGK. These changing sites were m e a s u r e d results show that the p h o s p h o t r a n s f e r a s e activity and the cGMP binding o f I ~ - and I~ cGK are regulated by its different aminoterminal domains. Inst.f. Pharmakologie und Toxikologie der Techn. Universit~t, Biedersteiner Str.29,W-8 M ~ n c h e n 40

EXPRESSION OF E . C O L I ~ F U N C• T I O N A L

THE HUMAN INTERACTION §

~z-ADRENOCEPTOR WITH 2 FORMS §

E.Selzer , S.Marullo" A.D.Strosberg

IN O F .. G ~

and W.Schutz

The coupling of the human B2-adrenoceptor expressed in E.coli was investigated using 2 splice variants of the ~-subunit of G s synthetized in E.coli (rG s ~ - ~ and rGs~ . " L ) and the . B~-subunit . . . .purified from bovlne braln. In competition blnd~ng experiments with (-) [125I]iodocyanopindolol (ICYP) and isoproterenol (ISO), rG s~- . ~ and. rGs~L.S~ recon. . o . • stltuted guanine nucleot1~e-sensltlve, hlgh-afflnity agonist binding. Titration of the B2-adrenoceptor at a fixed concentration ratio of ICYP/ISO (60pM/20nM) showed• that rG S ~ - ~ and. rG ~. were equi~L , , potent (EC~o-3nM) in reconstltut~ng ~ g h - a f f ~ n ~ t y binding. S~ecificity was also probed using rGs~PT , a mutant of rG.s -L with an altered carboxyterminus, a n d rG.~1 , w h l ~ were -20- and -200-fold less potent, ~ s p e c t ~ v e l y . A comparison of the S2-adrenoceptor expressed in E.coli with the receptor in $49 cyc-membranes revealed a similar affinity of rG s~-s and rG sa- for the recombinant and native . receptor. I n c ~ a t l o n of rGs~.s.B[ with partially solubilised E.coli membranes followed by dilution results in the stable incorporation of G s into the membranes as assessed by Western blots. Incorporated rGs¢-s is capable of coupling to the recom• • blnant receptor as determined not only by ICYP/ ISO competition curves but also by ISO-mediated acceleration of the rate for [35S]guanosine 5 ~-(3O-thio) triphosphate binding. These results show that (i) receptor-G protein coupling can be reconstituted in E. coli using recombinant components and that (ii) the ~2-adrenoceptor does not .discriminate between splice variants of Gs~. * I n s ¢ i t u t e of Phar~co[ogy, U n i v e r s i t y of V i e ~ a W~hcJnger SOt. 13a, A-lOgO Vienna § Ins~iout Cochin ~ G~n~ti~e ~ t 4 c u t a i r e , 22 rue M4chain, F-75014 Paris suppor¢~ ~ a grant f r ~ the "Fo~s zur F6rderung de~ wissenschafttichen Forschung" (P~22)

R 36

141 PERTUSSIS TOXIN ABOLISHES THE INHIBITORY ACTIVITY OF A SERUM FACTOR ON 8-ADREN~GICALLY IN-~JCED CYCLIC kMPPRODUCTION IN RAT GLIC~4A C6-BUrl CELLS A. Sarem-~slani and B.Hamprecht* A ser~n factor (SF) inhibits 8-adrenergically induced cyclic AMP-production in rat glioma cells C6-BU-I cells (v~n Calker et al., Hoppe Seyler's Z. Physiolog. chen. 1977;358:1188). Recent studies have shown a pertussis toxin (PTX) sensitive GTPase activity of fetal calf ser~n (FCS) on this cell line (Milligan, Biochem. J. 1987;245:501-505). We investigated the effect of PTX on the inhibitory activity of SF, SF was partially purified by extraction of lyophilized bovine serum with methanol followed by chrcr~tography on a silicagel-col~m~. The inkibitory activity was enhanced by addition of fatty acid free BSA to aqueous solutions of SF. C6-BU-I cells were cultured in the absence of FCS. Cyclic AMP levels are given in p~nol/r~ protein (means ± SD, n = 3). additions

without PTX 26

±

PTX (50 n g / m l )

1

27 ~

2

Isoproterenol (400 nM)

799 ± 90

832 ±

55

Isoproterenol (400 r~) + SF (i:i000)

402 ± 23

801 ± 119

These results indicate that SF inhibits the 8adrenergically induced cyclic AMP-preductic~ through a PTX-se~nsitive substrate, probably a C~-protein. Since Rireceptors have not been found in C6-BU-l-cells, SF could be a valuable tool to investigate Gi-protein-mediated signal transduction in this cell modell. The physiological role and the chemical structure of SF rerm~in to be clarified. Abteil~gf~rK]inischeCh~mie,Robert-Bosch-Kraken~,D-7000Stuttgart50 ~Physio[wisch-Ch~isch~l~itu~der~iversit~f,D-7400~bin~,~RG

142 C Y C L I C AMP M E D I A T E S U P - R E G U L A T I O N PROTEIN ALPHA-SUBUNITS I N CHICKEN

~

=

~

OF I N H I B I T O R Y G HEART MUSCLE C E L L S

..........................................

In a d d i t i o n to a d e c r e a s e i n t h e number o f s t i m u l a t o r y r e c e p t o r s , a d e n y l y l c y c l a s e s t i m u l a t o r y hormones have been shown to i n c r e a s e t h e l e v e l o f t h e a l p h a subunits of the inhibitory G-protein of adenylyl c y c l•a s e (G• i a ). The p u r p o s e o f t h e p r e s e n t s t u d y was t o l n v e s t l g a t e w h e t h e r hormonal u p - r e g u l a t l o n o f Gia i s a c y c l i c AMP-mediated p r o c e s s and w h e t h e r i t i s correlated with receptor down-regulation. Exposure of chicken heart muscle cells for 3 days w i t h t h e f i - a d r e n o c e p t o r (fi-AR) a g o n l s t i s o p r o t e r e n o l (lOuM) d e c r e a s e d t h e number o f fi.-AR a s d e t e r m i n e d by b i n d i n g s t u d i e s w i t h (3H)CGP ]2177 by a b o u t 60%. Q u a n t i t a t i o n of G. by p e r t u s s i s t o x i n - c a t a i y z e d ( 3 2 p ) A D P - r i b o s y l a ~ o n showed an i n c r e a s e i n t h e l e v e l o f Gi a by a b o u t a f a c t o r o f 2 i n membranes o f i s o p r o terenol-treated c e l l s . S i m i l a r to i s o p r o t e r e n o l ~ ~he receptor-dependent adenylyl cyclase stimulator p r o s t a g l a n d i n E 1 (lOuM) and t h e r e c e p t o r - i n d e p e n d e n t a d e n y l y l c y c l a s e s t i m u l a t e r f o r s k o l i n (lOuM) i n o f Gia by a b o u t a f a c t o r of t w o c r e a s e d the level and d e c r e a s e d t h e number of ~I-AR by 40 and 50%, respectively. In c o n t r a s t , t r e a t m e n t o f t h e h e a r t m u s c l e c e l l s i n t h e p r e s e n c e of t h e fi-AR a n t a g o n i s t w i t h weak p a r t i a l a g o n i s t i c a c t i v i t y c e l i p r o l o l (lOuM) d e c r e a s e d t h e number of 8I-AR by 20% b u t had no e f f e c t on t h e l e v e l o f G i a - p r o t e i n s . Conclusion: The d a t a s u g g e s t t h a t hormonal u p - r e g u ~-~-~. . i s m e d i a t e d b y. c y c l i c A~P. T h i s i n a c r e a s e in t ~ e l e v e l o f Gi a ~s n. o t s t r i c t l y correlat e d w i t h r e c e p t o r d o w n - r e g u l a t i o n . The ~-AR downr e g u l a t i o n i n d u c e d by p a r t i a l ~-AR a g o n i s t s seems to be m e d i a t e d . b y a c y c l i c A ~ P - i n d e p e n d e n t mechanism. M e d i z i n i s c h e K l i n i k I , U n i v e r s i t ~ t M~nchen, K l i n i k u m Grofihadern M a r c h i o n i n i s t r o 15~ D-8000 MHnchen 70~ F.RoGo

143 B-ARRESTIN AND 6-ADRENERGIC RECEPTOR KINASE MEDIATE HOMOLOGOUS DESENSITIZATION OF B-ADRENOCEPTORS M.J, Lohse Stimulation of B-adrenoceptors by agonists causes a specific loss of receptor responsiveness that is termed homologous desensitization. This process has been suggested to be mediated by a kinase that specifically phosphorylates agonist-oecupied B-receptors,termed B-adrenergic receptor kinase (BARK)• However, in a reconstituted system using the purified components B-receptor, Gs and BARK, BARK-mediated phosphorylation has only minor effects on B-receptor function, suggesting that additional factors we required for homologous desensitization• A protein that is proposed to represent such a factor was cloned and was termed B-arresfin. B-An'estin is similar to the retinal protein arresfin that has been implicated in inhibition of signal transmission from rhodopsin to Gt. Both proteins, B-arrestin and arrestin, were overexpressed in COS-cells and were purified from the cytosol to apparent homogeneity using sequential ammonium sulfate precipitation, gel filtration, and chromatography on hydroxylapatite and Mono-Q. In a. reconstituted system, both proteins were capable of inhibiting signal transduction from B-receptors to Gs, with B-arrestin being about 100-fold more potent. The reverse specificity was found in the rhodopsin-Gt-system: arrestin was much more potent than B-arrestin. Phosphorylafion of B-receptors by BARK resulted in, a 50-fold increase in the potency of B-arrestin. Under these conditions, half-maximal inhibition of B-receptor function was achieved at a ratio of B-arrestin to receptors of 1:1. B-Arrestin seems to compete with Gs for binding to the receptors. Thus, homologous desensitization appears to occur in the following steps: Agonist-occupied receptors are phosphorylated by BARK. This promotes the binding of g-arrestin, which prevents the interaction between receptor and Gs, and thus causes the desensitized state of the receptors. To determine the rate limiting step, stable cell lines were created overexpressing either BARK or B-arrestin, and different concentrations of B-receptors. Alterations of receptor function were observed only in cells expressing high levels of B-receptors: in these cells overexpression of BARK attenuated receptor signalling and caused increased homologous desensitization• This suggests that BARK-mediated phosphorylation is the rate-timiting step in homologous desensitization of B-receptors. Laboratory of Molecular Biology, University of Munich, Max-PlanckInstitute of Biochemistry, 8033 Martinsried, FRG•

144 CHOLERA TOXIN AND THE PHORBOL ESTER TPA SYNERGISTICALLY STIMULATE ADENYLYL CYCLASE IN PPJMARYASTROCYTE CULTURES P.J.Gebicke-Haerter, A Schobert, G. Hertting Cholera toxin (Ctx) ADP-ribosylates the a - s u b u n i t of G s , the stimulatory G protein of adenylyl cyclase (AC), which results in prolonged activation of the enzyme. It is shown here, that pretreatment of cultured rat astroglia with Ctx both markedly increased levels of intracellular cAMP and resulted in a change of astrocyte morphology into a stellate, ramified form. These effects were observed with as little as 1 ng Ctx/ml media. A significant rise of intracellular calcium by the action of the calcium ionophore A23187 had no influence on AC activities in both untreated or Ctx-treated cells. The phorbol ester TPA, however, synergistically enhanced cAMP levels in the presence of Ctx. This effect was abolished when cells were preincubated with T P A for 18 h to deplete protein kinase C (PKC). Pretreatment of cells with pertussis toxin (Ptx) and subsequent stimulations with A23187 or T P A had no effects on cAMP levels whatsoever. Simultaneous incubations with Ptx and Ctx and subsequent additions of stimuli revealed characteristics typical for Ctx alone. From these data it is concluded, that phorbol esterstimulated PKC either indirectly (via pp60 c'src) or directly phosphorylates the catalytic unit of AC. The described synergism arises from the concomitant activation of G s by Ctx. Institute of Pharmacology, University of Freiburg, HermarmHerder-Str. 5, D-7800 Freiburg i.Br.

R 37 145 RECONSTITUTION OF THE A~-ADENOSINE RECEPTOR WITH DEFINED G PROTEIN ~-SUBUNITS REVEALS A SELECTIVIT Y• F O R r G ~ - o ~

M~chael

Frelssmuth

and Maurine

L. Linder §

The ability of the bovine brain A~-adenosine receptor to discriminate between different G protein subtypes was tested using ~-subunits synthetized in E.coli (rG~-subunits). When combined with a three-fold molar excess of purified 5[-subunit and used at high concentrations, all three subtypes of rG~ and r G ~ were capable of reconstituting guanine nucleotide-sensitive, high-affinity binding of the agonist (-)~-3[~sI](iodo-4-hydroxyphenylisopropyl)adenosine (IHPIA) to the receptor. Titration of the receptor with increasing amounts of rGi~ I, rGi~2, rGi~3 and r G ~ revealed a -10-fold highe~ affinity fo~ rG~.~. This selectivity was also observed in the absence of the 6~subunit Other ~-subunits (rG sQ ' $ , rGs., rG S ~ PT, ~'L and rGz~ ) dld not interact with the receptor. In N-ethylmaleimide-treated bovine brain membranes, rG~=~ was the only rG. a -subunit capable of recon• , stltutlng high-affinity agonist binding. Similarly, r G ~ . competed potently with [~-~2P]GDP-prelabeled r ~ ~ for activation ,by the agonist-liganded At-adenosine receptor, whlle a ~50-fold molar excess of r G ~ was required to quench the receptormediated release of [~-32P]GDP from rG~.~. Hence, in spite of the extensive homology between o-subunits of the G~/Go-group, the At-adenosine receptor appears to discriminate between the subtypes. •

,

* Institute of Pharmacology, University of Vienna W~hringer Str. 13a, A-1090 Vienna § Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 supported by a grant from the "Fonds zur F6rderung der wissenschaftlichen Forschung" (P7622)

146 PERTUSSIS TOXIN-SENSITIVE AND -INSENSITIVE INCREASES IN CYTOSOLIC CALCIUM IN HUMAN ERYTHROLEUKEMIA (HEL) CELLS I. Schwaner The pluripotent human erythroleukemia cell line, HEL, possesses thrombocytic, erythrocytic and macrophage-like properties. ~-Adrenergic agonists increase cytosolic Ca2+ ([Ca2+]i) in HEL-cells via pertussis toxin (PT)-sensitive guanine nucleotide-binding proteins (G-proteins) of the Gi family (Michel MC et al. (1989) J Biol Chem264:4986). We studied the increases in [Ca2÷]i mediated by various receptor agonists and focussed on their PT-sensitivity. Thrombin, platelet-activating factor (PAF), prostaglandins E1 and E~ (PGEI and PGE~), the PGEz-analogue, sulprostone, the stable analogue of prostaglandin I2, iloprost, ATP and UTP increased [Ca2+]i through transient mobilization of Ca~+ f r o m intracellular stores and sustained influx from the extracellular space. ADP was less effective than ATP and UDP; UMP and AMP were ineffective. The kinetics of the Ca2+ signals induced by nucleotides and eicosanoids were (luite different. PT completely inhibited the rises in [CaZ÷]i stimulated by PGEz and sulprostone and p a r t i a l l y inhibited the one mediated by PGEI. Rises in [Ca2+]i induced by the other agonists were PT-insensitive. All increases in [Ca2*]i were desensitized in a homologous manner. PGE2 and sulprostone did not desensitize the effect of iloprost and vice versa. PGEI blunted the responses of the other eicosanoids but not vice versa. Our data suggest that (I) HEL cells possess PGEz- and PGI~-specific receptors and PGEI mimicks the effects of PGE2 and iloprost. ( I I ) HEL cells possess purinoand pyrimidinoceptors with properties similar to those of macrophages but not of thrombocytes. (III) PT-sensitive a n d - insensitive mechanisms play very different roles in the rises in [Ca2+]i mediated by various types of agonists. I n s t i t u t f(ir Pharmakologie, Freie Universit~t Berlin, Thielallee 69/73, D-IO00 Berlin 33, F.R.G.

147 ANTISENSE OLIGONUCLEOTIDES HYBRIDIZING WITH CODING SEQUENCESOF G-PROTEINe-SUBUNITS REDUCETHE HORMONALINHIBITION OF VOLTAGE-DEPENDENTCa2+ CURRENTSIN GH3 CELLS. C. Kleuss*, C. Ewel*, J. Hescheler, G. Schultz, and B. Wittig* While the number of known G-proteins is rapidly increasing, the identity of G-proteins involved in functional coupling between receptors and modulated effectors is not known for most systems. We tried to inhibit the expression of G-protein e-subunits by intranuclear injection of oligonucleotides into rat pituitary GH3 cells. Oligonucleotides were synthesized, which can either hybridize with a coding sequence common to known G-protein e-subunits or a coding sequence found in the two forms of the GO ~-subunit (~oi and eo2). Intranuclear injection of either oligonucleotide markedly reduced the inhibition of voltage-dependent Ca2÷ currents by somatostatin or carbachol. The reduction of Ca2+ current inhibition was observed 12 hours after injection; a maximum was reached between 24 and 48 hours. Antisense ol.igonucleotides hybridizing with coding sequences for either the eol- or the :o2-subunit selectively reduced the Ca2+ current inhibition induced by carbachol or somatostatin, respect i v e l y . Oligonucleotides complementary to coding sequences of e-subunits of Gs and the Gi family and sense oligonucleotides were ineffective. These results are consistent with the involvement of Go-subtypes in the inhibitory Ca2+ current modulation and establish intranuclear injection of antisense oligonucleotides as a tool for the identification of G-protein functions. * I n s t i t u t fur Molekularbiologie und Biochemie, Freie Universit~t Berlin, Arnimallee 22, D-tO00 Berlin 33 Institut fur Pharmakologie, Freie Universit~t Berlin, Thielallee 69-73, D-tO00 Berlin 33

148 PEPTIDE ANTIBODIES DISTINGUISH BETWEEN SUBTYPES OF GO ~-SUBUNITS K. Spicher, F.-J. K]inz, B. N~rnberg, I. Tychowiecka, W. Rosenthal Go is a highly abundant pertussis toxin-sensitive G-protein in neuronal, neuroendocrine and pituitary cells, in which i t may be involved in the inhibitory modulation of voltage-dependent Ca2+ channels. Its e-subunit (eo) exists in multiple forms. This diversity is at least in part explained by alternative splicing of a single transscript, yielding the two products, eol and eo2 (Hsu et a l . , J. Biol. Chem. 265, 11220-11226, 1990; Strathmann et a l . , Proc. Natl. Acad. Sci. USA 87, 6477-6481, 1990). The proteins encoded by eol and ~o2 mRNAdiffer in their Nterminal thirds. Antibodies against synthetic peptides corresponding to confined regions of Go e-subunits were raised in rabbits. Recombinant proteins (obtained by expression of ~oI and eo2 cDNA in E. coli) and membranes of insulin-secreting cell lines (SV 40-transformed pancreat i c B-cells from hamster, HIT; rat insulinoma cells, RINm5F) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. An antibody (AS 6) raised against a peptide common to both eol and Co2 (one-letter code C-NLKEDGISAAKDVK) recognized eol and eo2 fusion proteins and 39 and 40 kDa proteins in membranes of HIT and RINmSF cells. An antibody (AS 244) raised against a peptide specific for eol (one-letter code SKNRSPNKEIYCHM) recognized the %1 but not the eo2 fusion protein and 40 but not 39 kDa proteins in membranes of HIT and RINm5F cells. An antibody (AS 201) raised against a peptide specific for eo2 (one-letter code C-GPSAFTEAVAHIQGQY) recognized the mo2 but not the eol fusion protein and 39 but not 40 kDa proteins in membranes of HIT and RINm5F cells. The data indicate that 39 and 40 kDa GO e-subunits are encoded by %2 and %1 mRNA, respectively. Supported by the Deutsche Forschungsgemeinschaft. I n s t i t u t fur Pharmakologie, Freie T h i e l a l l e e 69/73, D-IO00 Berlin 33

Universit~t

Berlin,

R 38

149

151

C O M P L E M E N T A T I O N OF FORMYL PEPTIDE RECEPTORM E D I A T E D SIGNAL T R A N S D U C T I O N IN XENOPUS OOCYTES P. Gierschik, P. Stannek, S0 Gierschik, M. Voigt*, and P. Schultz

Formylmethionine-containing peptides (e.g. fMetLeu-Phe) stimulate a v a r i e t y of neutrophil functions by interacting with specific cell surface receptors which are coupled via a G-protein(s) to stimulation of p h o s p h o l i p a s e C (PLC). To characterize the formyl peptide r e c e p t o r - m e d i a t e d signal transduction at the molecular level, we have cloned the receptor cDNA and expressed the receptor protein in ~enopus laevis oocytes. R e c e p t o r a c t i v i t y was determined e l e c t r o p h y s i o l o ~ i c a l l y by measuring f M e t - L e u - P h e - e l i c i t e d opening of C a ~ ÷ - d e p e n d e n t C]- channels. Injection of pure formyl peptide receptor mRNA did not lead to p e p t i d e - d e p e n d e n t changes in m e m b r a n e current. In contrast, m a r k e d alterations of membrane currents were o b s e r v e d in response to fMet-Leu-Phe when the receptor mRNA was s u p p l e m e n t e d with poly(A) ÷ RNA isolated from undifferentiated human leukemia (HL-60) cells. Injection of the latter RNA alone did not lead to e l e c t r o p h y s i o l o g i c a l responses to fMetLeu-Phe. These results d e m o n s t r a t e that Xenopus oocytes injected with pure formyl peptide receptor mRNA lack a specific component which is essential for formy] peptide receptor-mediated signal t r a n s d u o t i o n and is present in immature granulocytes. Identification of this factor most likely a G-protein subunit or a PLC isoenzyme - will provide important information about the molecular mechanisms by which G-protein-coupled receptors stimulate phospholipase C. Pharmakologisches Institut and *Zentrum f6r Molekulare Bio]ogle, U n i v e r s l t ~ t Heidelberg, Im Neuenheimer Feld 366 and *280, D-6900 H e i d e l b e r g

A D P - R I B O S Y L A T I O N OF THE G E L S O L I N ACTIN C O M P L E X BY CLOSTRIDIAL TOXINS M. Wi!lel t A. Weqner ~ and K. Aktories *,~ C. botulinum C2 toxin and C__.perfrinqens iota toxin A D P - r i b o s y l a t e monomeric but not polymsrized actin. The actin-binding protein gelsolin which forms dimeric (GA) and trimeric (GAA) complexes with actin, severs, nucleates and caps filamentous actin thereby regulating the dynamic transition between monomeric and p o l y m e r i z e d actin. Here we studied the ADPr i b o s y l a t i o n of actin in the gelsolin actin complex. GA and GAA were isolated from the lysate of human platelets or leukocytes by DNAse I-affinity c h r o m a t o g r a p h y or by using anti-gelsolin antibody and were characterized by gel filtration on HPLC. Iota toxin ADP-ribosylated GA and GAA as effectively as G-actin. The time course and the NAD dependence of iotatoxin induced A D P - r i b o s y l a t i o n of actin and GA were identical. In contrast, C2'toxin ADP-ribosylated GA less effectively than actin. GA bound A D P - r i b o s y l a t e d actin (AN) to form the GAAr complex with an about 3-fold higher affinity than unmodified actin. Also A D P - r i b o s y l a t e d GA~ formed the GArA complex with actin. The nucleation activities of GA and of GAr were identical. In contrast, the nucleation activity of A D P - r i b o s y l a t e d GAAr was largely decreased compared with unmodified GAA. The data show that GA and GAA are substrates of ADPribosylating toxins and indicate that the two actin molecules of the GAA complex are functionally different. I R u d o l f - B u c h h e i m - I n s t i t u t f~r Pharmakologie der U n i v e r s i t ~ t Gieaen, D-6300 Gie~en, ~Institut f~r Physiologische Chemic, RuhrUniversit~t, D-4630 Bochum, SPharmakologisches Institut des Universit~tsklinikums Essen, D-4300 Essen

150

152

COUPLING TO ACTIN

OF fMLP-RECEFTORS FILAMENTS

Karl-Norbert Jesaitis

Klotz,

Dan

FROM HUMAN NEUTROPHILS

Siemsen,

Algirdas

J.

Desensitization of formyl peptide induced responses of human neutrophils like superoxlde p r o d u c t i o n has been shown to be a c c o m p a n i e d by a simultaneous segregation of f M L P - r e c e p t o r s into an actin- and fodrin-rich domain of the plasma m e m b r a n e w h i c h is d e p l e t e d in guanyl n u c l e o t i d e b i n d i n g proteins (G proteins). It is not known, however, w h e t h e r the receptors only co-localize w i t h or also bind to actin. Receptors solubilized from neutrophil membranes with o c t y l g l u c o s i d e as a d e t e r g e n t sediment as a 7 S form in a 5-20% sucrose gradient. In the presence of GTP, w h i c h u n c o u p l e s the receptors from G proteins, they sediment as a 4 S form. If receptors were s o l u b i l i z e d with Triton X--100, however, the f M L P - r e c e p t o r s sediment to the pellet along w i t h unsolubilized material in the presence and absence of GTP. This d e m o n s t r a t e s that the receptors were linked to complexes w h i c h are d i s r u p t e d by o c t y l g l u c o s l d e but p r e s e r v e d in Triton X--100. Since Triton X--100 ~oes not solubilize the c y t o s k e l e t o n release of receptors from the pellet was attempted in the presence of 600 mM KCl, a condition which specifically d i s r u p t s actin filaments. S o l u b i l i z a t i o n of the neutrophil membranes in Triton X--100 under this condition released the receptors from the pellet. This result suggests that fMLP-receptors are linked to actin filaments which may play a direct role in the regulation of receptor function. D e p a r t m e n t of Chemistry, M o n t a n a State University, Bozeman, MT 59717, USA

A NOVEL C3-LIKE A D P - R I B O S Y L T R A N S F E R A S E PRODUCED BY CLOSTRIDIUM LIMOSUM I. Just and G. Schallehn* Clostridium botulinum exoenzyme C3 ADPribosylates the small G T P - b i n d i n g proteins rho and rac. Here we report on the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a C3-1ike A D P - r i b o s y l t r a n s ferase (ADP-r) p r o d u c e d by a C. limosum (C.1.) strain isolated from a human lung abscess. The C.1. ADP-r (Mr 25,000) was p u r i f i e d from the culture supernatant by ammonium sulfate precipitation, gel filtration, anion exchange c h r o m a t o g r a p h y and, finally, by elution from SDS-PAGE. As known for C3 exoenzyme, the C.I. ADP-r m o d i f i e d the small G T P - b i n d i n g proteins rho and rac w i t h a K. value of about 0,4 ~M for NAD. rho/rac p r e v i o u s l y A D P - r i b o s y l a t e d by C3 was d e - A D P - r i b o s y l a t e d by C.I. Thus, the exoenzymes apparently A D P - r i b o s y l a t e d the same amino acid. Both enzymes showed similar pI (C3: 10.6 and C.l: 10.3) and proteolytic peptide analysis. Furthermore, they were immunologically related. In contrast to C3, the C.I. ADP-r was auto-ADP-ribosylated. These data indicate that C.I. produces an exoenzyme which belongs to the novel class of C3-1ike ADP-ribosyltransferases. Pharmakologisches Institut des Universit~tsklinikums Essen, Hufelandstr. 55, D-4300 Essen I, FRG *Institut f~r Mad. M i k r o b i o l o g i e und Immunologic der U n i v e r s i t ~ t Bonn, Sigmund-Freud-Str. 25, D-5300 Bonn, FRG

R 39

153

155

I N H I B I T I O N OF THE C L O S T R I D I U M B O T U L I N U M EXOENZYME C3-INDUCED A D P - R I B O S Y L A T I O N OF r h o / r a c PROTEINS BY M A S T O P A R A N A N D COMPOUND 48/80 G. Koch and B. Habermann* The small G T P - b i n d i n g p r o t e i n s r h o and r a c are A D P - r i b o s y l a t e d by Clostridium botulinum C3 A D P - r i b o s y l t r a n s f e r a s e . Mastoparan, a peptide toxin from wasp venom, and other histamine liberators have been reported to activate h e t e r o t r i m e r i c G proteins (G~, Ge) d i r e c t l y w i t h o u t receptor interaction (Higashijima T., J. Biol. Chem. 265, 14176, 1990). Here we inv e s t i g a t e d the effects of m a s t o p a r a n and other histamine liberators on the low molecular weight GTP-binding proteins rho and rac (Mr 21 kDa). C 3 - - i n d u c e d A D P - r i b o s y l a t i o n of 21 kDa human p l a t e l e t membrane proteins was inhibited by c o m p o u n d 48/80, m a s t o p a r a n and melittin half-maximally at 15, 20 and 22 DM concentrations, respectively and maximally (>90%) at 100, 100 and 50 DM concentrations, respectively. I n h i b i t i o n of A D P - r i b o s y l a t i o n was e n h a n c e d by GTP[S]. In contrast, apamin, a bee venom without histamine-liberating activity, had no effect. M a s t o p a r a n inhibited C3-catalyzed ADP-ribosylation of a porcine brain r h o p r o t e i n p r e p a r a t i o n but not ADPr i b o s y l a t i o n of r e c o m b i n a n t r h o expressed in E. coll. In control experiments no influence of c o m p o u n d 48/80 on C3 N A D - g l y c o h y d r o l a s e a c t i v i t y was found. The data suggest that histamine liberators not only activate h e t e r o t r i m e r i c G proteins but also interact with small G T P - b i n d i n g proteins.

CHARACTERIZATION OF GUANINE NUCLEOTIDE-BINDING PROTEINS IN INTRACELLULAR, GOLGI-ASSOCIATED VESICLES FROM RAT ADIPOCYTES A. Schiirmann-Bartsch and H.G. Joost Glucose transporters in insulin-sensitive cells exhibit a characteristic subcellular distribution between plasma membranes and an intracellular, vesicular compartment from which they are translocated to the plasma membrane in response to insulin. We have previously proposed that guanine nucleotide-binding proteins participate in the regulation of glucose transport activity through a direct interaction with the carrier protein. In the present study we investigated the subcellular localization of guanine nudeotide-binding proteins in relation to glucose transporters. Isolated adipocytes were incubated in the presence or absence of insulin, homogenized, and fractionated by dkfferential centrifugation. Total [~sS]GTPrS-binding, [Z~S]GTPrS-binding to 23-28 kDa G-proteins, and immunoreactivity of G-protein c~ and fl-subunits were determined in plasma membranes and a Golgi-enriched fraction of intracellular microsomes (lowdensity microsomes). Total GTPrS-binding and 23-28 kDa G-proteins were evenly distributed between plasma membranes and low-density microsomes. In contrast, levels of ~-subunits of Gs and G± were ten-fold lower in the low-density microsomes than in plasma membranes;/3-subunits were not detectable in the low-density microsomes. Insulin gave rise to the characteristic translocation of glucose transporters but failed to alter the subcellular distribution of any of the G-proteins. Further separation of the low-density microsomes on a discontinuous sucrose gradient revealed that none of the GTP-binding proteins copurified with glucose transporters. Moreover, the ~-subuults of G~ and G~ as detected with antiserum against a common peptide sequence (c%omon) could be separated by this procedure. These data indicate that adipocytes contain considerable amounts of 23-28 kDa G-proteins, and moderate amounts of Gz and G~ in intracellalar, Golgi-associated vesicles other than those containing the glucose transporter. Thus, the presumed regulatory interaction of glucose transporters and G-proteins does not involve a dose spatial relationship. The functional significance of the presence of G-protein a-subunits in distinct, intracellular vesicles remains to be elucidated. Abteilung Pharmakologle und Toxikologie I, Institut fiir Pharmakologie und Toxikologie der Universit~it G/Sttingen, Robert-Koch-Str. 40, D-3400 G6ttingen, FRG.

* R u d o l f - B u c h h e i m Institut f~r Pharmakologie, J u s t u s - L i e b i g U n i v e r s i t ~ t Gie~en, Frankfurter Str. 107, D-6300 Gie~en. P h a r m a k o l o g i s c h e s Institut, U n i v e r s i t ~ t Essen, Hufelandstr. 55, D-4300 Essen.

154

156

ACTIVATION OF HUMAN N E U T R O P H I L S BY M A S T O P A R A N J. Norqauer, M. Eberle and H.D. Lemke*

C R O S S - T A L K BETWEEN P H O P H O L I P A S E A2 AND PHOSPHOLIPASE C IN HUMAN GRANULOCYTES M. Camps and E. Strohmaier

Mastoparan (MP), a peptide toxin from wasp venom has been r e p o r t e d to activate regulatory G-proteins without receptor interaction. Here we studied the effects of MP on human neutrophils. MP induced biphasic changes in the o r g a n i z a t i o n of the cytoskeleton with rapid Factin p o l y m e r i z a t i o n followed by a slow and sustained d e p o l y m e r i z a t i o n below the initial baselevel F-actin content. Incubation of neutrophils with pertussis toxin (PT) inhibited M P - s t i m u l a t e d F-actin polymerization, however did not prevent sustained depolymerization of F-actin. Analysis of phospholipids performed in parallel revealed that MP stimulated rapid formation of p h o s p h o i n o s i t o l 3,4,5-trisphosphate (PIP3) and consumption of phosphoinositol-4,5-bisphosphate. PT treatment blocked MP-induced formation of PIP3. Furthermore, MP stimulated release of p r i m a r y granules in a manner sensitive to intracellular Ca z÷ . Cytochalasin B (CB) enhanced M P - s t i m u l a t e d secretion. PT treatment of neutrophils r e d u c e d M P - s t i m u l a t e d secretion in the presence of CB but not in its absence. MP also triggered superoxide anion production and induced complement type 3 receptor up-regulation. The M P - i n d u c e d superoxide anion production was enhanced by CB. The data indicate that beside PT-sensitive Gproteins other mechanisms are involved in the activation of human neutrophils by MP. ~udolf-Buchheim-Institut f~r Pharmakologie, Frankfurter Str. 107, D-6300 Gie~en and *Enka AG, D-8753 Obernburg.

Phospholipase C (PLC)-mediated hydrolysis of phosphatidyl inositol 4 , 5 - b i s p h o s p h a t e (PIP2) is a major m e c h a n i s m by which extracellular mediators regulate the functions of their target cells. Human granulocytes contain a soluble PLC that is stimulated b y a soluble OTP-binding protein(s). We have p r e v i o u s l y d e m o n s t r a t e d that this soluble PLC is also a c t i v a t e d by exogeneous G-protein 8~-subunits and that this stimulation is additive to stimulation of PLC by the endogeneous GTP-bindlng protein. Here, we present evidence that soluble granulocyte PLC is m a r k e d l y s t i m u l a t e d by products of phospholipase A2 (PLA~) and identify the s t i m u l a t o r y principle as arachidonic acid (AA). S t i m u l a t i o n of PLC by AA was not due to formation of AA metabolites as the effect of AA was not p r e v e n t e d by several cyelooxygenase or lipoxygenase inhibitors. Stimulation of PLC was also seen with other long (n~]8) chain cJs-unsaturated fatty acids, but was not observed with short chain (n~16) cischain unsaturated, trans-unsaturated, or saturated fatty acids. Most importantly, however, the abllity of AA to stimulate inosito] phosphate formation did not appear to be related to its amphiphilic nature, since its effect was fully additive to the effect of deoxycholate, a well known amphiphilic stimulator of PLC. These results suggest that AA g e n e r a t e d by PLA2 may be d i r e c t l y involved in stimulating p h o s p h o l i p a s e C, which would represent a novel m e c h a n i s m for cross-talk between these important transmembrane signalling systems. Pharmakologisches Institut, Univers~t~t Heidelberg, INF 366, D-6900 Heidelberg

R 40 157

159

THE SOLUBLE P H O S P H A T I D Y L I N O S I T O L 4,5-BISPHOSPHATE P H O S P H O M O N O E S T E R A S E OF HL-60 G R A N U L O G Y T E S ~. Hou Cytosolic fractions of human HL-60 granulocytes contain two enzymatic activities that hydrolyze phosphatidylinositol 4,5-bisphosphate (PIPs) : phospholipase C (a phosphodiesterase) and a p h o s p h o m o n o e s t e r a s e (PME) that hydrolyzes PIP2 to PIP and Pi • The hydrolysis of PIP~ to PIP and Pi was investigated using HL-60 cytoso] and sonicated phospholipid vesicles containing [~H]PIPz as substrate. PIPs hydrolysis was markedly reduced in the presence of 2,3-diphosphog]ycerate (2,3-DPG). Half-maximal and maximal inhibition were o b s e r v e d at ~ 5 and 30 mM, respectively. Neither 2- nor 3-monophosphoglycerate affected PIP~ hydrolysis. Control experiments performed under the same conditions but using [zH]PIP as substrata did not reveal PIP p h o s p h o r y l a t ~ o n or PIP hydrolysis. Thus, HL-60 cytosol contains a PIP~-speciflc PME that is specifically inhibited by 2,3-DPG. We suggest that 2,3-DPG acts by mimicking the two vicinal phosphates present in the 4,5-posltlon of PIPs. In the presence of 2,3-DPG, the n o n h y d r o l y z a b l e GTP-analogue GTP[S] (I00 ~M) m a r k e d l y stimulated the formation of PIP from PiPs. This effect was not m i m i c k e d by ATP[S], and was also seen in the presence of 3 mM ATP. As PIP p h o s p h o r y l a t i o n is not evident under the conditions used in th5s study, GTP[S] is likely to stimulate PIPs PME rather than inhlb~ting PIP kinase. Taken together, the results show the existence in HL-60 cytosol of a P I P ~ - s p e c i f i c PME which is m a r k e d l y stimulated by GTP[S], p r e s u m a b l y via a c t i v a t i o n of a soluble GTP-binding protein present in HL-60 cytosol. Identification of this protein is subject to further investigation. Pharmakologisches Institut, Universit~t Heidelbergi INF 366, D-8900 Heidelberg

STIMULATION AND

158 A SYNTHETIC LIPOPEPTIDE INDUCES EXPRESSION OF NADPHOXIDASE IN HL-60 LEUKEMIC CELLS THROUGHPERTUSSIS TOXINSENSITIVE AND -INSENSITIVE MECHANISMS R. Seifert

INHIBITION OF HUMAN

PLATELET ADENYLYL

CYCLASE BY TRANSDUCIN B~-SUBUNYIS M. Ronzani, C. Stannek and K.H. Jakobs The influence of 13~- GTP > GppNHp), while with other nucleotides only small (GDP[fiS]) or no effects (GMP, ATP) of fMet-Leu-Phe were observed. Finally, the fMet-Leu-Phe-induced release was not seen in membranes of pertassis toxin-treated HL 60 cells and was also not seen at high ( > 5 nM) GTP[rS] concentrations present in the prelabelling period. The data indicate (1) that binding of GTP['rS} to membrane G proteins is not irreversible and (2) that agonistactivated receptors can even interact, either directly or indirectly, with GTP[rS]-liganded G proteins leading to release of bound guanine nucleotide. Pharmakologisches Insfitut tier Universitat Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, FRG

5'-N-Ethylcarboxamido[3H]adenosine ([3H]NECA) has been shown to bind to A t and A x adenosine receptors and also to non-receptor binding sites. In this study, we describe a novel binding site for adenosine that can be detected in solubilized extracts from bovine brain membranes. Bovine striatal membranes were solubilized with CHAPS (3-[3-(cholamidopropyl)dimethylammonio]-lpropanesulfonate), and the soluble extract was subjected to gel filtration on a Sepharose CL-6B-cohimn. [3H]NECA-binding activity was eluted in three separate peaks. The first peak was eluted with the void volume and contained appro]dmately 10% of the total [3H]NECA binding sites. According to its pharmacological profile it was identified as the A 2 adenosine receptor. The second peak contained 40% of the [3H]NECA binding sites with pharmacological properties typical of the non-receptor NECA-binding protein adenotin. In the third peak a [3H]NECA-binding site was eluted that could clearly be distinguished from A 2 adenosine receptors and from adenotin by a characteristic pharmacological profile. The radiofigand [3H]NECA showed a saturable binding to these fractions with a Braax value of 12,900 fmol/mg protein and a K d value of 22 riM. In competition experiments, adenosine, NECA, NAD, inosine, 5'-AMP, and SAH (S-adenosyl-L-homocysteine) were the most potent ligands (K i values 15, 23, 57, 71, 82 and 82 nM, respectively). In contrast to adenosine A 2 receptors, this site did not bind the A2-selective agonist CGS 21,680 (2-[p-(2-carboxyethyl)- phenethylamino]-5'-N-ethylcarboxamido adenosine). R-N 6-phenylisopropyladenosine (R-PIA) and S-N 6-phenylisopropytadenosine (S-PIA) were almost equipotent (K. values 3.6 and 2.2/~M respectively). A f f i .. 1. ' . nttles of adenosine receptor antagomsts were 100 to 1,oo0fold lower at th~s new binding site compared to A 2 adenosine receptors. A binding site with an identical pharmacological profile could be detected in soluble extracts from bovine cortical membranes. These results suggest the existence of a novel high affinity binding site for adenosine of unknown function in bovine brain membranes. Pharmakologisches Institut dar Universit~t, Im Neuenheimer Feld 366, D-6900 Heidelberg, FRG.

R 42

165

167

EXTRACELLULAR ADENOSINE 5'-TRIPHOSPHATE (ATP) ANTAGONIZES THE EFFECTS OF POTASSIUM CHANNEL OPENERS IN GUINEA-PIG HEART PAPILLARY MUSCLE *,zp. Illes a n d *A. B e r t

EXCITATORY EFFECTS OF THE ADENOSINE ANALOGUES, PIA AND NECA, MEDIATED BY A NON-A~ ADENOSINE RECEPTOR

P a p i l l a r y m u s c l e s w e r e i s o l a t e d from t h e r i g h t v e n t r i c l e of guinea-pig heart and stimulated with electrical pulses. The e v o k e d a c t i o n p o t e n t i a l s (AP) w e r e r e c o r d e d b y i n t r a c e l l u l a r microelectrodes; the simultaneously elicited contractions w e r e m e a s u r e d i s o m e t r i c a l l y . We u s e d c r o m a k a l i m a n d Re 31-6930 [2- (6-eyano-2,2-dimethyl-2H1-benzopyran- 4-yl)pyridine 1-oxide]; these compounds open potassium chann e l s , w h i c h a r e s e n s i t i v e to i n t r a c e l l u l a r ATP (KAte c h a n n e l s ) . T h e a p p l i e d c o n c e n t r a t i o n s of c r o m a k a l i m a n d Re 3 1 6 9 3 0 m a r k e d l y s h o r t e n e d t h e AP d u r a t i o n a n d d e p r e s s e d t h e c o n t r a c t i l e f o r c e , b u t did n o t c h a n g e e i t h e r t h e r e s t i n g m e m b r a n e p o t e n t i a l , or t h e a m p l i t u d e a n d t h e m a x i m u m r i s e v e l o c i t y of t h e AP. G l i b e n c l a m i d e c l o s e s K a ~ c h a n n e l s ; t h i s s u b s t a n c e w a s i n a c t i v e on i t s own, b u t r e d u c e d t h e e f f e c t s of c r o m a k a l i m a n d Re 3 1 - 6 9 3 0 . ATP a n d i t s e n z y m a t i c a l l y s t a b l e a n a l o g u e a , l ~ - m e t h y l e n e ATP ( a d ~ - m e A T P ) a c t e d in a s i m i l a r m a n n e r a s g l i b e n c l a m i d e . G l i b e n c l a m i d e a n d a,13meATP s h i f t e d t h e c o n c e n t r a t i o n - r e s p o n s e c u r v e of c r o m a k a l i m in a p a r a l l e l w a y to t h e r i g h t . N e i t h e r t h e P~p u r i n o c e p t o r a n t a g o n i s t s u r a m i n n o r t h e P , - (A~-) p u r i n o ceptor antagonist 8-cyclopentyl-l,3-dipropylxanthine (DPCPX) c o u n t e r a c t e d a,l~-meATP. T h e c a l c i u m c h a n n e l b l o c k e r n i t r e n d i p i n e a l s o s h o r t e n e d t h e AP d u r a t i o n a n d depressed the contractile force. However, both glibenclamide a n d a,l~-meATP f a i l e d to i n t e r f e r e w i t h n i t r e n d i p i n e . In c o n c l u s i o n , ATP m a y a l t e r t h e e f f e c t s o f p o t a s s i u m c h a n n e l openers both at lntra- and extracellular sites. However, e x t r a c e l l u l a r ATP d o e s n o t a c t v i a t h e k n o w n s u b t y p e s of P~-purinoceptor.

Angela Ameri In a previous study it was shown that the adenosine analogue, PIA, at a concentration of 2.5 ~mol/l either reduced or increased the excitability of hippocampal CA1 neurones. The inhibitory effect was abolished by the selective A1 receptor antagonist, DPCPX, and thus was mediated by the adenosine A1 receptor. To further characterize the receptor involved in producing excitatation, intracellular recordings were made from CA1 neurones in slices of the rat hippocampus. Drugs were applied by superinsion. The slices were pretreated with DPCPX (0.5 tzmol/l) to block A~ receptor-mediated responses. PIA was then applied at different concentrations in solutions containing DPCPX (0.5 ~nol/l). DPCPX alone produced a depolarization, an increase of membrane resistance, and spontaneous discharges of action potentials. PIA (1 - 5 #tool/l) caused a concentration-dependent excitation of all neurones tested. It further depolarized the neurones, increased their membrane resistance and spontaneous impulse discharges. Also NECA (5 ~mol/l), which inhibited neuronal excitability when applied alone, produced an excitation during block of the A1 receptors by DPCPX. The excitatory effects of PIA and NECA disappeared during superfusion of a medium containing theophylline in combination with DPCPX and the agonists. Forskolin failed to change the membrane properties of the CA1 neurones so that an involvement of cAMP in the excitatory effects of the adenosine agonists described could be excluded. The results provide evidence ~ a t PIA and NECA increase neuronal excitability by binding to an adenosine receptor other than that of the A1 type. This receptor is probably of the A2 type, because the excitatory effects were abolished theophylline. This study was supported by the SFB 246. Institut fiir Pharmakologie und Toxikologie der Universitat des Saarlandes, D-6650 Homburg/Saar.

~ D e p a r t m e n t o f P h a r m a c o l o g y , Byk G u l d e n P h a r m a c e u t i c a l s , B y k - G u l d e n - S t r a s s e 2, D - 7 7 5 0 K o n s t a n z a n d Z D e p a r t m e n t of P h a r m a c o l o g y , U n i v e r s i t y of F r e i b u r g , H e r m a n n - H e r d e r S t r a s s e 5, D - 7 8 0 0 F r e i b u r g , F.R.G.

166

168

ADENOSINE RECEPTOR ACTIVATION REDUCES ISOPROTERENOLSTIMULATED ACTIVITY OF PHOSPHATASE INHIBITOR-I IN INTACT GUINEA PIG VENTRICLES J. Neumann ], R. C, G u p ~ , A. M~ Watanab~o The adenosine agonist (-)-N -phenylisopropyladenosine (PIA) attenuates the isoproterenol (Iso)-induced increase in force of contraction and protein phosphorylation in perfused hearts. These dephosphorylations can only in part be explained by a reduction in cAMP. However, it is conceivable that an activation of protein phosphatases is involved. The activity of phosphatases could be regulated by the protein phosphatase ir~hibitor-I which is only inhibitory after phosphorylation by protein kinsse A. Therefore, we measured the activity of inhlbitor-i (I-l) from isolated pe~fused electrically stimulated (3Hz) guinea-pig ventricles. The inhibitory effect of I-I was measured against the catalytic subunit of rabbit skeletal muscle type 1 phosphatase and expressed as % of maximal inhibitory activity. Under these conditions Iso (I0 ruM, 1 min) stimulated I-I activity from 30.5 ± 3.8 % to 48.6 ± 4.5 %. Additionally applied PIA (I ~M) reduced I-I activity to 36.2 ± 4.3 % (p I- (4.74 > 2,50 > 0.70 pmol/mg protein at lnM total radioligand concentration; potassium salts). This rank order very closely resembled Eisenman's binding selectivity sequence VII for monovalent anions (Eisenman, Biophys. J. 2(2):259, 1962) and clearly differed from the diffusion coefficient rank order for hydrated monovalent anions (i.e. I" > CI" > F'). The rank order for monovalent cations was K+ > Na+ > Li + > Cs+ (2.50 > 2.26 > 1.44 > 0.88 pmol/mg; chloride salts) which is also clearly distinct from their diffusion coefficient rank order (Le, Cs + > K+ > Na + > Li+). The relevance of these rank orders for the estimation of the anion and cation binding domain size of the mitochondrial DHP receptor complex is discussed. Supported by FWF grants P7492-MED and K0042-MED and Dr.Legerlotz foundation. Institut for Biochemische Pharmakologie, P.Mayr-Str.1,A-6020 Innsbruck

195 COS CELLS AS A MODEL FOR STUDYING DRUG EXPORT BY PGLYCOPROTEIN (PGP): STEREOISOMERS OF CALCIUM ANTAGONISTS BLOCK CELLULAR VINBLASTINE EFFLUX WITH EQUAL EFFICACY. E. Schwarz, M. Kouba, G.Vogt and V. H611t

PGP is a multidrug transporter encoded by the MDRI gene. Enhanced expression of PGP is responsible for the c e l l u l a r efflux of several drugs used in cancer therapy leading to multidrug resistance. In addition, other drugs, such as calcium channel antagonists , have been shown to be transported by PGP and to overcome multidrug resistance in cancer patients. We now report that COS-7 monkey kidney tumor cells express high levels of both MDR1 mRNA and PGP as revealed oby Northern b l o t and Western b l o t a n a l y s i s . Moreover, ~ H - v i n b l a s t i n e (VBL) is effectively export#d by COS-7 c e l l s . The c e l l u l a r accumulation of ~H-VBL was inhibited by several a n t i c a n c e r drugs and by o t h e r drugs known to be s u b s t r a t e s o f PGP. Moreover, t r a n s f e c t i o n of COS c e l l s w i t h a v e c t o r expressing MDR1 ant~sense RNA r e s u l t e d in an increased accumulation o f ~H-VBL. A s e r i e s o f stereoisomers w i t h phenylalkylamine s t r u c t u r e (verapamil, devapamil, emopamil) and dihydropydine structure (nimodipine~ f e l o d i p i n ~ i s r a d i p i n e , n i t r e n d i p i n e and n i g u l d i p i n e ) increased ~H-VBL accumulation in COS c e l l s at micromolar concentration. The rank order of potency in COS c e l l s was s i m i l a r t o t h a t in PGP overexpressing doxorubicin-resistant mouse leukemia c e l l s (D89-065). There was no e s s e n t i a l d i f f e r e n c e between the v a r i o u s steroisomers in increasing VBL accumulation in both c e l l t y p e s . The n i g u l d i p i n e enantiomers belong to the most potent drugs. Since ( - ) - n i g u l d i p i n e displays an about 40f o l d lower a f f i n i t y f o r calcium channel binding s i t e s than ( + ) - n i g u l d i p i n e , but is about e q u a l l y p o t e n t in i n h i b i t i n g c e l l u l a r e f f l u x of VBL, i t may be a useful drug f o r reversing multidrug resistance in cancer patients. Department of Physiology, University of Munich, P e t t e n k o f e r s t r . 12, D-8000 MQnchen 2, FRG;

194

196

SIALIC ACID MAY PLAY A FUNCTIONAL ROLE WITHIN THE CALCIUM CekHA~NEL G. Werner, K. Addicks , and M. Sarram

PRE3/ENTION OF VERATRIDINE INDUCED INTI~J~SYNAPTOSOMAL [Ca2+]i INCREASEBY R 56B65 B.A. Osikowska-Evers, F. Tegtmeier, T. Peters Calcium overload occurring during ischemialhypoxia influences functional and structural changes in neurons which f i n a l l y lead to cell injury or death. Calcium entry blockers, by lowering neuronal calcium increase, protect brain from ischemiclhypoxic injury. Synaptosomes depolarized by veratridine are a useful tool to mimic hypoxiclischemic insults on the nerve terminal i~n vitro. ~_~ V~vo brain studies show that R 56B65 (N-[l-[4-(4-fluorophenoxy)butyl]-4-piperidinyl] -N-methyl-2-benzothiazolamine) is protective in cerebral Ischemia (1). Therefore, the effect of R 56865 on veratridine induced [Ca2+li increase was studied in rat cor~ical synaptosomes, using fluorimetric measurement of [Ca~+]1 with Fura-2.

Cellular calcium metabolism may be influenced by the glycocalyx component sialic acid (Langer etal., Pharmacol Ther 16: 331-376, 1982). We here report on the influence of this anionic sugar on the myocardial and vascular activity of calcium antagonists. Sialie acid removal from the external matrix of isolated gmnea pig left atrial strips supramaximall}, stimulated at 2 Hz (resting force 4,9 mN) or rabbit thoracic aortic ring segments (resting tension 2,0 g) was performed by incubation with neuraminidase (2,0 units/nil; 2h; Type V, Sigma) in carbogen-saturated Tyrode (30°C) or KrebsHenseleit solution (37o C) following an equilibration period of lh and was controlled electron microscopically. After frequently washing out the enzyme, aortic rings were precontracted by 130 mM KC1. The action of different calcium antagonists was evaluated in cumulative dose-response-curves (0,1 nM-100 #M) by measuring myocardial contractile force isometrically and vascular tension isotonieally using a strain gauge. Control experiments with untreated organs were conducted in parallel. Neuraminidase treatment resulted in a sialie acid release of 62,0---2,2% (S.E.M.; atria; n = 3) and 57,3 +-1,3% (aorta; n = 5), as determined according to the thiobarbituric acid method of Warren (J Biol Chem 234: 1971-1975, 1959). Total sialic acid content following 0,1 M H2SO 4 was (nmol/mg wet weight): 1,30--,0,03 (atria), 1,70--.0,05 (aorta). [:Ialf-maximal negative inotropic effects (ICsI/, in /~M+-S.E.M.; p < 0 , 0 5 ; n=6-9) by the calcium antagonistff~studied in control experiments and neuraminidase pretreated guinea.pig atria, were obtained with nisoldipi.n,e at 0,5--0,1 and 0,2 ± 0,04 , gallopamil ~t 0,5-+0,1 and 0,3±0,02, diltiazem at 8,7-+1,2 and 5,4---0,8, fendiline at 24,3+-3,0 and 24,3±2,7. Similar results, however, ICs0 being in the nanomolar concentration range, were obtained In rabb~t aorta. Thus, sialic acid may play a functional role in calcium channels, possibly facilitating calcium influx. Institut ftir Pharmakologie der Universit~it zu K61n, Gleueler Str. ~4, D-5000 K61n 41, FRG }Zentrum der Anatomie der Universit~it zu K61n, Joseph-Stelzmann Str. 9, D-5000 K61n 41, FRG

Veratridine (l x lO-5 M) increased [Ca2+]i in control synaptosomes from If3 18 (SD) to 368M± BO (~2~,~ nM (n - lO) within 2 min. mR 56B65 (l x lO-B - l x 5 M; n = B) inhibited strongly (9 ± 9 % (SD) and B7 ± 3 % (SD~; respectlvely) veratridine induced intrasynaptosomal [Ca~+]i increase in a dose dependent manner with an IC 50 of l x lO-6 M. Possibly, Ca2+ entry into synaptosomes occurs through voltage-gated Ca2+ channels opened by veratridine induced membrane depolarization. However, Ca2+ influx directly through the veratridine sensitive Na+ channel, and/or Na+ICa~+ exchanger working in a reverse mode, can not be excluded. I t remains to be elucidated which of these pathways is influenced by R 56865. Nevertheless, above data f u l l y support _ i _Bn vivo findlngs indicating that cerebroprotective effects of R 56865 (1) are partly due to its calcium-antagonistic properties (2). (1) Scheller et a l . , Naunyn-Schmied. Arch. Pharmacol., Suppl 339, R I07 (19BY) (2) Koch et a i . , Cardiovasc. Drug Rev. B, 238-254 (1990) JANSSEN RESEARCHFOUNDATION, D-4040 NEUSS21

R 50 197 THE RELATIONSHIP OF INOSITOL 1,4,5-TRISPHOSPHATEINDUCED CALCIUM RELEASE TO VARIOUS CALCIUM TRANSPORT MECHANISMS IN SUBCELLULARFRACTIONS FROM RAT LIVER H.-P. Bode and M. Trautmann* In contrast to the endoplasmic reticulum calcium pump, the calcium transporters present in some organelles of the vacuolar apparatus (chromaffin granules and the inositol 1,4,5-trisphosphate (IP3)-sensitive calcium store of exocrine pancreas c e l l s ) as well as the plasma membrane calcium pump appear to be calcium proton exchanging ATPases, with obligatory coupling of calcium and proton transport. Examining vesicular calcium uptake in subcellular f r a c t i o n s , the l a t t e r mechanisms of calcium transport therefore can be i d e n t i f i e d using agents that affect the i n t r a v e s i c u l a r proton concentration. To investigate whether vacuolar organelles or the plasma membrane are targets of IP 3 in rat l i v e r c e l l s , we have examined the effect of the vacuolar proton pump i n h i b i t o r bafilomycin and the potassium proton exchanging ionophore nigericin on vesicular calcium transport in subcellular fractions from r a t l i v e r . Bafilomycin had no effect on calcium uptake by any of the f r a c t i o n s . Purified Golgi and endosomal fractions showed no calcium pumping a c t i v i t y . Nigericin p a r t l y inhibited calcium uptake in an early sedimenting microsomal f r a c t i o n , but did so only in the presence of an outside to inside potassium concent r a t i o n gradient across the vesicular membranes, suggesting that this effect was due to a l k a l i n i z a t i o n of the vesicular lumen. Nigericin did not a l t e r the amount of calcium releasable by IP 3 from the same f r a c t i o n . We conclude that n i g e r i c i n acts on calcium pumping inside-out plasma membrane vesicles, which are devoid of IP3-receptors. In summary, no evidence favoring association of the IP3-receptor with an organelle d i s t i n c t from endoplasmic reticulum in subcellular fractions from rat l i v e r was found.

199 T H E DIIq,,Y,D R O P Y R I D I N E ( _ + ) - N I G U L D I P I N E I N H I B I T S TT Y P E C a z'" C U R R E N T S IN A T R I A L M Y O C Y T E S . K. Seydl The whole cell tight seal recording technique v~as used to investigate the effects of the ~ovel drug niguldipine on Ca 2+ currents in guineapig atrial cells. C£ z+ currents were separated into T-type and L-type components by an appropriate stimulus protocol, i. e. first, T-type C~z~" currents were evoked by depolarizing voltage pulses from_ a holding potential of -90 mV to -40 mV and then, L-type Cg z+ currents were activated by a subsequent depolarization to 0 mV. Fast sodium currents were eliminated by substituting choline for Na and outward currents were suppressed by the use of Cs instead of K in both extracellular and intracellular solution. T- and L-type current characteristics were further confirmed by a selective inhibition by 40 /~M Ni and by 1 #M_ ~-+)-PN 200-110, respectively. Based on this characterization of C~z ÷ current components we tested for the effects (-+)-niguldipine (N/G), Extracellular application of 1 ~M NIG resulted in a potent blockade of T-type C£z + current~ ,(to 7-+4 % of control, n=7) and in a weaker inhibition of L-t3)pe Ca z ÷ currents (to 22-+9 % of control, n=6). Current to voltage relationships clearly showed that both Ca 2+ currents are blocked at all voltages examined (-60 to -40 mV). Inhibition of T- and L-type currents occurred in a slow fashion. At a concentration of 1 # M NIG half maximal reduction in T-type current a,lnplitude was observed within tl/,, = 52 sec. The ICCn for T-type CUz ÷ current inhibition was estimate8 to be 0.13 ~M. TH%~IC50 for NIG on L-type Ca z+ currents could not be clearly resolved~tue to the slow ~.~set of NIG action, which is obscured by a concomitant L-type Ca 2 current run-down. This study clearly indicates a T-type Ca 2+ current blocking activity of NIG with a high relative affinity, NIG appears, however, not to be selective among Tand L-type Cd 2+ currents. (Supported by Austrian Research Funds, project S 45-03) Institute for Biophysics, University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria

Inst.f.Pharmakol., K.v.Frisch S t r . , Lahnberge und *Zentr.f.lnnere Med., Klinikum d. Univ., D-3550 Marburg

198 R E V E R S A L OF A N T H R A C Y C L I N E A C C U M U L A T I O N DEFICITS IN M U L T I D R U G R E S I S T A N T F R I E N D L E U K E M I A CELLS B Y T H E DIHYDROPYRIDINE B859-35, THE R-ENANTIOMER OF NIGULDIPINE. A. Reymann*, C. Woermann*, and M. Dietel+

M u l t i d r u g r e s i s t a n c e (HI)R) i s a ma3or o b s t a c l e i n c a n c e r c h e m o f h e r a p y . The use o f t h e e x p e r i m e n t a l c h e m o s e n s i t i z e r v e r a p a m i l , known t o b l o c k a n t i c a n c e r e x t r u s i o n from MOl~-ma]ignancies i n v i t r o , i s h a m p e r e d b y i n vivo cardiovascular toxicity observed at concentrations r e q u i r e d £or t h e r e v e r s a l of MDR ( D i c k s o n R.B. a n d G o t t e s m a n n ~.N., T r e n d s P h a r m a c o l Sci 11, 305, 1990). The d i h y d r o p y r i d i n e B859-35 ( R - e n a n t i o m e r of n i g u l d i pine), a n a g e n t w i t h m i n o r c a r d i o v a s c u l a r e f f e c t s , was studied in an a n t h r a c y c l l n e accumulation assay in routine leukemia cells strain F4-6RADR e x p r e s s i n g p g l y c o p r o k e i n . Cells w e r e p r e i n c u b a t e d w i t h B859-35 (i0 rain, d i m e t h y l s u l f o x i d e i% v / v a s s o l v e n t ) f o l l o w e d b y 60 min i n c u b a t i o n i n t h e p r e s e n c e of r a d i o l a b e l l e d c y t o s t a t i c s (i ~mol/l), and c e n t r i f u g e d t h r o u g h a s i l i cone o i l l a y e r i n t o t r i c h l o r o a c e t i c a c i d (10% w/v). I~859-35 e l e v a t e d low a c c u m u l a t i o n o f d o x o r u b i c i n , d a u norubicin (DNR) or mitoxantrone in resistant F 4 - 6 R A D R cells to levels measured in drug-sensitive F4-6P precursor cells. Concentration-response studies on D N R accumulation revealed that B859-35 was m o r e potent than verapami] (ECs0 0.73 //mol/l vs. 5.4 //tool/l). In parallel to increased D N R contents (pmol/mg protein, F4-6RADR, solvent: 303+27; B859-35 3,3 ~mol/l: 1067-+174; sensitive F4-6P, solvent: 948+110; n=8-9, SEM), the amount of D N R tightly b o u n d after washing of the pellet obtained from F 4 - 6 R A D R (i.e. protein, DNA, RNA), was increased 3.9-fold to levels observed in F4-6P. The results indicate that B859-35 is a potent chemosensitizer, with regard to in-vitro accumulation of cytostatics and improved d r u g access to binding sites in F 4 - 6 R A D R leukemia cells of the M D R phenotype. (Supported b y the DFG, SFB 232,C3 and Di 276/i-3.) *Abteilung Allgemeine Pharmakologie, Unlversit~tsKrankenhaus Eppendorf, Universitat Hamburg, Martin~stra~e 52, D-2000 H a m b u r g 20, FRG. (+) Institut fur PathologJe, Universik~t Kiel, Michaelisstra~e Ii, D2300 Kiel I, F E G

2O0 EFFECTS OF A CARDIODEPRESSANT FACTOR ON ACTION POTENTIALS AND IONIC CURRENTS iN ISOLATED GUINEAPIG CARDIOMYOCYTES. B.Koidl*, S.Hallstr6m**, U.M~ller***, K.Werdan***, and G.Schlag** A cardiodepressant factor (CDF) was isolated from the plasma of dogs post hypovolemic-traumatic shock by column chromatography. The CDF-fraction contains a hydrophilic peptide ( M W < I O 0 0 ) . By means of photometric measurement of cell pulsation and patch-electrode recording of membrane potential and whole cell ionic current its influence on excitation and contraction was studied in isolated guinea pig cardiomyocytes. CDF reduced dose dependently down to a pathophysiologically relevant concentration of 1.84 ml shock plasma aliquot/ml the amplitude of contraction in electrically stimulated cells. This effect could be rapidly reversed by wash-out and could be abolished within seconds by elevation of the calcium concentration within the bath from 1.8 to 10 mmol/I. Action potential measurements showed a distinct reduction of the plateau-phase and an increase in the duration at 50 and 90 percent of repolarization. Voltage-clamp measurements revealed an inhibition of the calcium current and a change in the N-shaped current voltage relationship of the potassium current suggesting an increase in IK1 and a decrease in the delayed rectifier component IK. The negative inotropic effect as well as its rapid reversibility by elevation of calcium in the the bathing solution indicate a similar point of attack of CDF and calcium antagonists. The effects on IK are not necessarily in contradiction with this interpretation. The rapid reversibilty of the effect however suggests a very different access of the substance to the receptors of the calcium channel. *lnst.f.Med.Physik u. Biophysik d. Univ.Graz, A-8010 Graz * * L u d w i g Boltzmann-lnstRut f. Exp. u. Klin.Traumatologie, Wien, * * *Med.Klinik I d.Univ.M(Jnchen, Klinikum GrolShadern

R51 201

203

Increasing and d e c r e a s i n g effects of extracellular m a g n e s i u m on inward currents in heart ventricular cells W. Vierling, A. Dichtl

DILTIAZEM AND (+)-TETRANDRINE INCREASE THE CA2+ AFFINITY OF THE L-TYPE ~Az + CHANNEL: STEREOSELECTIVE REVERSAL B Y A NOVEL CA2+ - ANTAGONIST R. Staudinger and H. Glossmann

The influence of an increase in the extracellular m a g n e s i u m c o n c e n t r a t i o n on inward currents of isolated m y o c y t e s from g u i n e a - p i g hearts was investigated in whole cell v o l t a g e - c l a m p experiments. From a h o l d i n g potential of -80 mY in a c o n d i t i o n i n g clamp step to -40 mV the sodium current was inactivated. The slow calcium inward current was e l i c i t e d in a second step to 0 mV. M a g n e s i u m c o n c e n t r a t i o n - d e p e n d e n t l y and reversibly decreased this current (by about 40% after an increase from 1.2 to 9.6 mmol/l, taking the spontaneous run down into account). C o n s i d e r i n g the v o l t a g e - c u r r e n t r e l a t i o n s h i p of the calcium inward current by changing the second clamp step, m a g n e s i u m decreased the maxim a l l y available current, but a d d i t i o n a l l y shifted the r e l a t i o n s h i p by about 10 mY to more positive potentials. A l t e r i n g the c o n d i t i o n i n g dep o l a r i z a t i o n to -55 mV increased an early current component at the second d e p o l a r i z a t i o n (probably an incompletely inactivated sodium current) which was further strongly augmented after addition of magnesium. This can be explained by a shift of the dependence of the inward current on the potential of the first depolarization (by ca. +5 mV). It is concluded that magnesium in g u i n e a - p i g ventricular heart exerts an inhibiting effect on calcium inward current, but a d d i t i o n a l l y influences voltage-current relationships by inc r e a s i n g the "effective" m e m b r a n e potential.

Positive heterotropic allosteric regulators of dihydropyridine (DHP) blndlng2+(( + )-cis-diltiazem, ( + )-tetrandrme," trans-diclofurime) to L- tv_oe Ca channels increase the affinity of the channel for C£Z+ [ Ca"z+ site I] (Vitamins and Hormones 44, 155-328). BM 20.1140 (BM, ethgl.2,2-diphenyl-4-(1-pyrrolidino)-5-(2-picolyl)oxy-valerate)) is a novel Ca ~+- antagonist (pA 2 for (-)-BM= 8.64; for (+)-BM= 7.29; taenia caeci). (-)-BM but noi (+)-BM effects were antagonized by DHPchannel activators. (-)-BM is one of the most potent negative heteroropic allosteric regulators of (+)-cis-diltiazem binding to skeletal muscle ~l-SUbunits (K05 = 1.12 4aM). (-+)-BM, (-)-BM and (+)-BM accelerat4 association 'of (+)-[aH]-isradipine, (+)-BM accelerates isradipine dissociation from 0.024 min "1 to 0.093 min "1. (+)-BM and ()-BM are noncompetitive inhibitors for the phenylalkylamine site. (-)BM is a potent stimulator of equilibrium DHP binding, depending on channel subtype; (+)-BM is without effect, but reverses the positive allosteric effect of (-)-BM with a Ki= 3.3 nM. Likewise (+)-BM reverses the effects of (+)-tetrandrine, ~' + )-cis-diltiazem, fostedil and trans-diclofurime. The high- affinity Ca 2"+ site i on the el- subunit is formed •upon binding . of DHP's (K05 . = 315 . . nM [Ca2+),tree ) • (+)tetrandrme (10 #M) increases the channel affimty 8- fold (Kn ~ = 40 nM [Ca2+]lf.r~^) perhaps by increasing the number of Ca 2~: c~rdinating carbonyl oxygens. (+)-BM (10 #M) prevents the colfformatmnal change induced by (+)-tetrandrine (-K , ~ = 236 nM [Ca2+]fr^~ , , ( + ) - c.i s • . ~J.D it~c dlltmzem and the DHP's act from the outer face of the elJSUbumt (J. Gen. Physiol. 95, 1-27), whereas the phenylalkylamine binding site is at the intracellular fac9 (Striessnig et al., Proc. Natl. Acad. Sci. U.S.A., in press) close to Caz÷ site II (Nature 346, 321-322). The location of Ca z÷ 'site I is not yet known. Supported by FWF (S-4501) Institut fiir Biochemische Pharmakologie, Peter Mayrstr.1, A-6020 Innsbruck, Austria

Institut f~r P h a r m a k o l o g i e und Toxikologie der T e c h n i s c h e n Universit~t M~nchen, Biedersteiner Str. 29, D-8000 M ~ n c h e n 40

202

2O4

FENDILINE ACTS AS A DIRECT BLOCKER OF L-TYPE CALCIUM CHANNELS IN GUINEA-PIG VENTRICULAR CELLS W. Schreibmayer and O. Tripathi*

SYNTHESIS AND SAR STUDY OF THE C a - A N T A G O N I S T I C PROPERTIES OF A SERIES OF N E W D I P H E N Y L A L K Y L ~ I N E S P. Caldirola, P. Zandberg and H. Timmerman

The modulation of the L-type calcium currents of guinea-pig ventricular myocytes by fendlline ( ),-[.-Phenyl-N-(1-phenylethyl)benzenepropanamine) was investigated with the patch-clamp method. The Ltype calcium current is Necked directly by fendiline with an IC50 of 17.0 -+ 2.4~M. Its kinetics is modulated in a specific way: the availability curve of c'alcium channels is shifted to negative membrane potentials and inactivation time constants are shortend. Additional administration of the agonistic dihydropyridine compound (4R,4S)-Bay K 8644 (racemic Methyl-l,4-dihydro-2,6-dimethyl-3-nitro4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate) at a concentration of 1/~M in the presence of 30~uMfendiline resulted in a further block of calcium channels instead of the expected increase in the cardiac calcium current. Neither nifedipine, diltiazem nor verapamil resulted in a similar paradoxic effect in combination with (4R,4S)-Bay K 8644. Instead in all cases the expected agonistic behaviour of (4R,4S)-Bay K 8644 was observed. The most likely explanation for diphenylalkylamine action on the voltage sensitive calcium channel is, thus, binding to a receptor site that is physically different from the sites for dihydropyridines, phenylalkylamines and benzothiazepines.

A c c o r d i n g t ~ binding studies the following subgroups of Ca ++ can be differentiated: Ia: dihydropyridines; Ib: diphenylalkylamines; II: verapamil; III: benzothiazepines. SAR studies for dip h e n y l a l k y l a m i n e s are currently missing. Thus we have synthesized and tested the C a - a n t a g o n i s t i c p o t e n c y of d i p h e n y l a l k y l a m i n e s of the following structure: R1~

This work was supported by the Austrian Research Fund ($4504B) *:Physiology Division, Central Drug Research Institute, Lucknow 226001, India; Department of Medical Physics and Biophysics, University of Graz, Harrachgasse 21/IV, A-8010 Graz, Austria

c--x R2

/ R4

The compounds are rather simple m o l e c u l e s of ~,~ich the skeleton is fit for rational manipulation. By considering: the length of the chain b e t w e e n the benzhydryl m o i e t y and the nitrogen; including an ether function (X= O;S), saturation and unsaturation of the chain (X= CH2; CH) ; the substitution of the n i t r o g e n (R3; R4) , a series of amines is obtained. The compounds have been tested in a 3Hnitrendipine binding assay and in a number of in vitro systems such as smooth m u s c l e r e l a x a t i o n using d i f f e r e n t contractants and an a n t i p l a t e l e t a g g r e g a t i o n test. These compounds are not strong C a + + - e n t r y blockers (Ki is 10-6); their pharmacological properties can be explained by interference w i t h the Ca ++ homoeostasis via other ways. The synthesis will be presented together w i t h b i o l o g i c a l data and SAR investigations. D e p a r t m e n t of P h a r m a c o c h e m i s t r y , Vrije U n i v e r s i teit, De Boelelaan 1083, NL-I081 HV A m s t e r d a m / The N e t h e r l a n d s

R 52 205 MYOCARDIAL AND VASCULAR ACTIVITY OF SOME AGONISTIC 1,4-DIHYDROPYRIDINES IN VITRO. R. Ratke, W. Klaus and U. Fricke Calcium agonists of the 1,4-dihydropyridine (DHP) type are positive inotropic and cause vasoconstriction by facilitating the calcium inward current into cardiomyocytes and smooth muscle cells through voltage-dependent channels (Schramm et. al. Arzneim.-Forsch./Drug Res. 33 (1983) 1286; Alonso et. al., Gen. Pharmac. 20, 827 1989). Because of their pronounced vasoconstrictor activity, currently known calcium agonists cannot be used clinically as cardiostimulants. We here describe the action of (-)-S-Bay K 8644 [(-)-S-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2trifluoromethylphenyl)-pyridine-5-carboxylate] and some new related compounds on porcine right ventricular trabeculae and coronary arteries: Bay W 5035 [2-aminoethyl ester derivative], Bay P1974 [6-hydroxyhexyl ester derivative]. Porcine ventricular trabeculae were stimulated with 1 Hz in an oxygenated KrebsHenseleit-solution (resting tension 5raN; 30°C). Porcine right coronary artery segments were examined in the same solution after a testcontraction with 60 mmol/l KC1, washout and moderate depolarization with 15 retool/1 KCI (resting tension 20 raN; 37°C). Alldrugs were added cumulatively. At porcine trabeculae Bay W 5035 (IC50 1,46_+0,16 xl0 "° mol/1) displays a bell shaped dose response curve with omuch lower affinity than (@S-Bay K 8644 (ICso 1,35- 0,11 xlo -~ mol/1) but comparable activity. In contrast, Bay W 5035 showed no detectible_constriction of the coronary arteries at a concentration up to 10-0 mol/l. Bay P 1974 is slightly positive inotropic and vasoconstrictive. Interestingly, Bay K 8644 is able to enhance the 60 mmol/1 KCl-tone of the artery while Bay W 5035 does not affect the state of contraction and Bay P 1974 acts like an antagonist. This behaviour may be explained by a different affinity of the agonistic resp. antagonistic enantiomers of these compounds. From our experiments ~t seems likely that the cardioselectivity of DHP calcium agonists can be improved by molecular variations. Institut ffir Pharmakologie der Universit~it zu K61n, Gleueler Strage 24, W-5000 K61n 41

207 C H A R A C T E R I Z A T I O N OF C A L C I U M A N T A G O N I S T I C E F F E C T S O F T H R E E M E T A B O L I T E S OF T H E N E W A N T I H Y P E R T E N S I V E AGENT NAFTOPIDIL: (NAPHTHYL)HYDROXY-NAFTOPIDIL, (PHENYL)HYDROXY-NAFTOPIDIL, AND O-DESMETHYLN A F T O P I D I L . M. G r u n d k e and H.M. H i m m e l T h e a n t i h y p e r t e n s i v e d r u g n a f t o p i d i l b l o c k s ~la d r e n o c e p t o r s and i n h i b i t s Ca2 + e n t r y v i a p o t e n t i a l - d e p e n d e n t c h a n n e l s in v a s c u l a r and cardiac m u s c l e (Himmel et al. 1991, J C a r d i o v a s c P h a r m a col, in press). N a f t o p i d i l u n d e r g o e s e x t e n s i v e b i o t r a n s f o r m a t i o n in vivo. W e t h e r e f o r e h a v e s t u d i e d t h e p o s s i b l e p h a r m a c o l o g i c a l e f f e c t s of its m e t a b o l i t e s ( n a p h t h y l ) h y d r o x y - , (phenyl)hydroxy-, o - d e s m e t h y l - n a f t o p i d i l (NHN, PHN, DMN) in p a p i l l a r y m u s c l e s and i s o l a t e d m y o c y t e s of t h e g u i n e a - p i g heart. In p a p i l l a r y muscles, 30 ~ M of all c o m p o u n d s r e d u c e d the f o r c e of cont r a c t i o n (PD2-values = 5.5) and decreased the A P D 2 0 and APDs0. A P D g 0 w a s s l i g h t i y i n c r e a s e d by PHN, h a r d l y a f f e c t e d by DMN, and s h o r t e n e d by NHN. The A P a m p l i t u d e and V m a x w e r e s l i g h t l y r e d u c e d b y PHN, but little i n f l u e n c e d b y DMN and NHN. In v o l t a g e - c l a m p e d v e n t r i c u l a r cells, the c a l c i u m c u r r e n t Ica w a s c o n c e n t r a t i o n - d e p e n d e n t ly depressed b y the t h r e e c o m p o u n d s in t h e r a n g e of i-I00 ~M. M e m b r a n e c u r r e n t s m e a s u r e d w i t h s l o w r a m p p u l s e s w e r e m a r k e d l y r e d u c e d by i00 # M of e i t h e r N H N or P H N i n d i c a t i n g an i n h i b i t o r y e f f e c t on K + - c o n d u c t a n c e . Our d a t a s h o w t h a t NHN, PHN, a n d DMN are e f f e c t i v e as C a 2 + - a n t a g o n i s t s in vitro. Thus, the m e t a b o l i t e s are exp e c t e d t o c o n t r i b u t e to the a n t i h y p e r t e n s i v e a c t i o n of t h e i r p a r e n t c o m p o u n d n a f t o p i d i l . P h a r m a k o l o g i s c h e s Institut, U n i v e r s i t ~ t - G H S Essen, H u f e l a n d s t r . 55, 4300 E s s e n i, G e r m a n y

206 THE CONTRIBUTION OF a- AND CA++-CHANNEL BLOCKING PROPERTIES TO THE VASODILATING EFFECT OF NAFTOPIDIL

208

W. Bartsch, A. Schbffel, M. Kohler, B. Eberwein, R.G. Hooper and L. Kling Previous experiments have provided evidence that the antihypertensive effect of naftopidii is attributable not only to its s-blocking property but also 1o Ca~-antagonism. To evaluate the importance of these properties investigations were performed to calculate the pA~.values of different vasodilating compounds against noradrenaline (NA) and CaCI~ as contracting agents. METHOD: Rabbit aortic spiral strips were used for the experiments. Norodrenaline and Ca ~ were used as agonists. The vasoconstricting effect of the stimulants was determined by adding incremental concentrations of the compounds to the bath. Various concentrations of the antagonists were used and the shift of the concentration-response curves of the agonists were calculated. The method of Aruhnlakshana and Schild has been used for the calculation of the

Increase in cytosolic Ca ~+ is a crucial step for the stimulation of insulin secretion. Depolarising initiators of hormone release are known to elevate Ca s÷ influx via opening of voltage dependent Ca 2÷ channels. We have studied the characteristics of Ca z÷ antagonist binding to a high affinity binding site in the particulate fraction of insulin secretory RINmSF cells. In intact RINmSF cells the dihydropyridine (DHP)-type Ca z÷ antagonist [3H]-(+)-PN 200-110: (+)-[5-methyl-aH]-[isopropyl-4-(2,1,3-benzoxa diazol-4-yl)1,4-dihydro- 2,6-dimethyl-5-methoxy-carbonyl-pyridine-3-carboxylate] concentration dependently inhibited 25 m M KCl-induced insulin release with an IC~o of 0.01 laM. In the particulate fraction [aH]-(+)-PN 200110 bound in a stereoselective manner to a high affinity site, with a I~ of 7.0 9:4.8 n M and a Bmax of 858 9:556 fmo]/mg protein (means 9: SD, n=6). Benzothiazepine-type Ca z÷ antagonist d-cis-diltiazem stimulated binding of [aH]-(+)-PN 200-110 in a temperature dependent manner, whereas the calmodulin antagonists (amiodarone and CGS 9343B: 1,3-dihydro- 1-[1- ((4-methyl-4H,6H-pyrrolo[1,2-a] -[4,1] benzoxazepin-4-yl)~methyl)- 4-piperidinyl]- 2H-benz imidazol-2-one maleate) inhibited binding of [~H]-(+)-PN 200-110. CGS 9343B was roughly 50 times less potent than amiodarone. These results demonstrate the presence of a Ca 2~ entry blocking site in the insulin secretory cells (RINm5F) with analogous properties to the DHP binding subunit of the L-type Ca ~÷ channel from skeletal muscle.

INTRODUCTION:

PA2-vaines. RESULTS: Substance

Prazosln

Nicardipine

Urapidil

Naftopidil

(mS)

(R)

(S)

pA 2 NA

8.85

> 5.00

6.25

7.10

6.92

6.48

pA 2 Ca ~

> 5.00

10.11

> 4.0

5.90

6.12

5.92

Ratio > 7080 < 0.000008 > 178 16 6.3 3.6 The drugs investigated are presented in the table. Obviously, from the pA 2values and the ratios of calcium channel to nocadrenaline antagonistic activity, prazosin may be characterized as a typical c~-blocking agent and nicardipine as a typical Ca~-channel blocking agent. Urapidil showed a c~-blockadeo Interestingly, naftapidil and its enantiomers do not show any marked discrimination between its activity on the response to noradrenaliue or Ca b. ions. The ratio of 16 should not be interpreted as a marked predominance of c~blockade. Both the enantiomers show a narrower ratio between ct- and Ca ~blocking properties, which may be explained by biological variation. CONCLUSION: These results suggest that the hypotensive effect of naftopidil is not only the result of the ct-blockade but also of the Ca++-channel blocking activity. l)ep. of Pharrnacol.,BoehringerMannhehnGmbH, P.O. Box 31 01 20, D-6800 Mannheim

Ca z÷ ANTAGONIST BINDING TO TI~ FRACTION O F INSULIN SECRETORY CELLS.

PARTICULATE M. R@rd~e]dt

Pharmazeutisches Institut, Lehrstuhl f~ir PharmakologJe, Auf der Morgenstelle 8, D-7400 Tl~bingen, FRG

R 53 211

209 CALMODULIN INHIBITION BY DIPHENYLALKYLAMINES R . M a n n h o l d a n d W. V o i g t

HIGH-AFFINITY INTERACTION OF POLYMYXIN B,WITH CALMODULIN B.H. Schmidt, J. Traber, and L. Hegemann

P o t e n c y and s e l e c t i v i t y of c u r r e n t l y a v a i l a b l e c a l m o d u l i n ( C a M ) - a n t a g o n i s t s is r a t h e r l i m i t e d . We a t t e m p t e d to i m p r o v e t h e s e p r o p e r t i e s b y o p t i m i z i n g the C a M - a n t a g o n i s t p r e n y l a m i n e . T e s t system used was the CaM-stimulated phosphodiesterase. I n s e r t i o n of a d o u b l e b o n d or s u l p h u r in t h e benzh y d r y l p a r t of t h e m o l e c u l e i n c r e a s e s , i n s e r t i o n of o x y g e n d e c r e a s e s p o t e n c y . C h a i n l e n g t h is o p timal w i t h 4 a t o m s - n i t r o g e n - 3 atoms. R e g a r d i n g benzhydryl substitution halogenation surmounts m e t h y l a t i o n ; p a r a - s u b s t i t u t i o n is s u p e r i o r to os u b s t i t u t i o n . S e c o n d a r y n i t r o g e n s e e m s to be adv a n t a g e o u s as c o m p a r e d to t e r t i a r y a m i n e . L i p o p h i l i c i t y d e p e n d e n c e of C a M - a n t a g o n i s t i c p o t e n c y d e c r e a s e s in t h e f o l l o w i n g m a n n e r : saturated chain >double bond-> sulphur-> oxygen derivatives. A m o n g s t the c o m p o u n d s d e s c r i b e d n e w C a M - a n t a g o nists with remarkably high potency have been detected.

The cyclic peptide antibiotic polymyxin B is considered a rather selective inhibitor of protein kinasn C and is thnrefore widely used as a pharmacological tool to assess the involvement of this enzyme in physiological processes. We however found that polymyxin B inhibits calmodulin-dependent cyclic 3':5'-nucleotide phosphodiesterase from bovine heart in vitro at nanomolar concentrations 2+ (IC -values 80 nM at 500 ~M Ca ), that is at concentra50 tions about 200 fold lower than those required for inhibition of protein kinase C. This effect appears to be mediated by a direct interaction of polymyxin B with the z+ Ca -receptor protein calmodulin. Inhibition of phosphodiesterase was selective for the calmodu~in-dependent isoenzyme and competitive with respect to Ca z+ and calmodulin. The electrophoretic migration velocity of calmodulin in non-denaturating polyacrylamide gels was slowed down in the presence of the antibiotic. Finally, polymyxin B, covalently bound to an agarose matrix, could be used as an affinity label for calmodulin. Thus, polymyxin B may be regarded as one of the most poten~ and selective calmodulin antagonists presently available. Institute for Neurobiology, Troponwerke GmbH & Co. KG, Berliner Straee 156, D-5000 K61n 80, FRG. * Institute of Molecular Biology and Medical Biotechnofogy, University of Utrecht, The Netherlands.

C--X

R~

/ R4

I n s t i t u t e of L a s e r m e d i c i n e , H e i n r i c h - H e i n e - U n i v e r s i t y of D N s s e l d o r f , U n i v e r s i t ~ t s s t r a s s e 7, D-4OOO D~sseldorf / FRG

210

212

INFLUENCE OF DOPANINE RECEPTOR AGON~STB AND ANTAGONISTS ON CALNODULIN T~ANSLOCATION IN DIFFERENT BRAIN REGIONS. N.S. Popov

MUSCARINIC ACETYLCHOLINE RECEPTORS REGULA'VE THE RELEASE OF GTP[rS] FROM G PROTEINS IN PORCINE ATILIAL MEMBRANES G. Hilf

Acute and c h r o n i c e x p e r i m e n t s were p e r f o r m e d to s t u d y the e f f e c t s of i n t r a p e r i t o n e a l l y adm i n i s t e r e d dopamine a g o n i s t s and a n t a g o n i s t s on the t r a n s l o c a t i o n of c y t o s o l i c and membrane-bound c a l m o d u l i n i n the s t r i a t u m and h±ppocampus of the r a t b r e ± n . S i n g l e doses o f apomorphine and a l o w - d o s e amphetamine (1.25 mg/kg) r e s u l t e d i n a t r a n s l o c a t i o n of c a l m o d u l i n , as measured by a decrease i n membrane-bound and i n c r e a s e i n c y t o s o l i c calmod u l i n i n the s t r i a t u m , whereas b r o m o c r y p t i n e was i n e f f e c t i v e . A~phetamine e x e r t e d a s i m i l a r e f f e c t i n the h±ppocampus as w e l l . Howe v e r , a h i g h - d o s e amphetamine CSmg/kg) had an o p p o s i t e e f f e c t on t r a n s l o c a t i o n , £n t h a t t h e r e was an i n c r e a s e i n membrane-bound c a l m o d u l i n . C h r o n i c a l l y a p p l i e d amphetamine (5 mg/kg) and h a l o p e r i d o l (1 m g / k g ) , i . e . under c o n d i t i o n s of dopamine r e c e p t o r s u p e r sensitivity, tended to decrease c y t o s o l i c c a l m o d u l i n i n the s t r ± a t u m and hippocampus, and to i n c r e a s e membrane-bound c a l m o d u l i n . The f i n d i n g e and, e s p e c i a l l y , the c h r o n i c e f f e c t s o f h a l o p e r ± d o l and the h i g h dose o f amphetam±ne are i n t e r p r e t e d i n the l i g h t of the c u r r e n t c o n c e p t which s u g g e s t s t h a t t r a n s m i t t e r s and i n t r a n e u r o n e l s i g n a l l i n g systems c o n v e r g e , t h e r e b y i n f l u e n c i n g the more comp l e x processes of neuronal p l a s t i c i t y , rather than r e c e p t o r s e n s ± t i v ± t y o n l y . Institute o f Pharmacology and T o x i c o l o g y , N e d i c a l Academy Nagdeburg, L e i p z i g e r s t r . 44 0-3090 Nagdeburg, S a c h s e n - A n h a l t , F~G

Under appropriate conditions, i.e. in the presence of GDP, muscarinic acetylcboline receptors (mAcChR) stimulate the binding of the GTP analog GTP[rS] to G proteins in porcine atrial membranes. On the other hand, mAcChR can also regulate the dissociation of bound [35S]GTP[rS]

from prelabelled

atrial membranes.

The release of bound

[35S]GTP[rS] ffom the membranes induced by carbacbol (CCh) was rapid and was halfm~imal at about 0.3,~M CCh. Atropine competitively antagonized the stimulatory effect of CCh. In the absence of CCh, the mAcChR antagonists atropine and pirenzepine reduced the basal release of [35S]GTP[rS] by maximally about 25 %, with half-maximal effecs at 5 and 500 nM, respectively. The regulation of [35S]GTP[rS] release by mAcCbR agonists and antagonists was strictly dependent on the presence of a second guanine nucleotide in the release assay. At maximally effective concentrations, GTP[~-S], GTP, GppNHp, GDP and G.DP[I~S] promoted the stimulatory effect of CCh on [35S]GTP[~-S] release to a similar extent. GMP and ATP were ineffective up to 1 raM. In addition to guanine nucleotides, the CCh-stimulated release of bound GTP[rS] was dependent on Mg2+, being maximally effective at about 10 ,~M. Prelabelling of the membranes with increasing concentrations of GTP[~-S] largely decreased the CCh-stimulated release of bound GTP[~-S]. The data indicate that mAcChR can interact with GTP[~'S]-liganded G proteins in porcine atrial membranes, resulting in release of bound nucleoside triphosphate. This release may be caused by a direct recoptor-G protein interaction or by a receptor- and guanine nucleotide-dependent G protein-G protein interaction. Finally, the antagonist data support the hypothesis that even agonist-free mAcChR can productively interact with G proteins. Pharmakologisehes Institut tier Universi~t Heidelberg, Im Neuenbeimer Feld 366, D.-6900 Heidelberg, FRG

R 54

213

215

TIME C O U R S E OF T H E E F F E C T OF ISOPRENALINE ON T H E INHIBITORY G - P R O T E I N ~ - S U B U N I T IN T H E HEART. U. Mende, D. Geertz, H. Scholz, J. Schulte am Esch and R. Sempell

QUANTIFICATION OF GIC ( _ BY A NOVEL RADIOIMMUNOASSAY IN CARDIOMYOPATHIC HUMAN HEARTS M.B0hm, K.Larisch and E.Erdmann

In h u m a n end-stage heart failure due to idiopathic dilated cardiomyopathy the increase of the inhibitory G-protein ~-subunit (¢Gi) is thought to be due to elevated catecholamine levels. In order to clarify in vivo the pathophysiological role of catecholamines we investigated in a rat model the time course of the effect of chronic isoprenaline (ISO)-infusion on myocardial ~ G i content determined b y pertussis toxincatalyzed ADP-ribosy]ation. In addition we studied in left papillary muscles the influence of a 4 d a y ISOinfusion on the effect of carbachol (Carb) on force of contraction. Rats (n=7-8) were treated by a subcutaneous infusion with osmotic mlnipumps of ISO (2.4 m g / k g - d), propranolol (PROP, 9.9 m g / k g • d), ISO+ P R O P or 0.9% NaC] (CTR) for i, 2, 3, 4 or 8 days. ISOtreated rats revealed a constant increase in ventricular/body weight ratio of about 35% (p 9 0 % of t h a t of Ca ++. pD2 v a l u e s w e r e for D E N (n=6): 5 . 8 5 ± 0 . 1 3 ; +30 n M ICI: 5 . 9 2 ± 0 . 2 2 ; +i0 n M CGP: 5 . 3 3 ± 0 . 1 0 ; + I C I + C G P : 5 . 3 0 ± 0 . 1 5 ; for DOB (n=5): 6 . 2 6 ± 0 . 1 3 ; +30 n M ICI: 5 . 7 6 ± 0 . 1 3 ; + 3 0 0 n M CGP: 4 . 9 4 ± 0 . 0 7 ; +ICI+CGP: 4.63±0.12. For both a g o n i s t s a n e a r l y i:i r a t i o b e t w e e n B A R o c c u p a n c y and contractile response was observed indicating t h e l a c k of s p a r e r e c e p t o r s for the PIE of DEN a n d D O B in h u m a n r i g h t atrium. W e c o n c l u d e t h a t D E N is a B ~ A R - s e l e c t i v e a g o n i s t ; D O B c a u s e s its P I E m a i n l y v i a B~AR w i t h a s m a l l b u t s i g n i f i c a n t c o n t r i b u t i o n b y B~AR; X A M h a s no P I E in h u m a n r i g h t atrium.

phenoxyethylamino)-3-(4-(l-me±hyl-4-±rifluorome%hyl-2imidazolyl)phenoxy)-2-propanol methanesulfonate). Results: PM: Bmax ~*/~2 FW: Bmax ~I/~2 VA: Bmax ~i/~2 IS: Bmax

NF IDC ICM 70.~±6.2 25.8±2.8* 26.2±3.4* 78.9/21.1 60.2/39.8+ 77.8/22.2 68.9±7.2 24.2±3.2* 25.1±2.9" 83.1/16.9 62.8/37.2+ 80.2/19.8 63.1±6.9 23.8±3.4* 24.4±2.8* 82.2/17.8 64.3/35.7 76.6/23.2 66.7±6.2 22.1±3.1" 25.0±2.7* ~1/~2 76.8/23.2 62.3/37.7+ 76.6/22.1 *p 2 y e a r s post g a s t r e c t o m y ) a n d in a group of 5 h e a l t h y m a l e v o l u n t e e r s (group II, 24-27 years, 68-84 kg) a f t e r oral a d m i n i s t r a tion as a s o l i d f o r m and a solution (pH: 8-8,5) in a d o s e of 225 mg. group I g r o u p II preparation solid liquid solid liquid cm~ (~g/ml) 0 , 2 6 " x 3,1 2,5 3,4 t ..... (min) 132"x 17~ 74 61 A U C = ~ ( ~ g * m i n * m l -:L) 5 8 " x 360 460 487 t - T e s t (p3000] after treatment for 3-4 weeks, 10502100 mg BSN/day, average bismuth levels of 3-4 ~g/l were found. Ground level: 0-2.6 ~g/l before application. The overall maximum level in any trial was 21.3 ~g/l. Thus all values range below the alert level of 50 ~g/l. Serum levels: Nitrate. Methaemoqlobin NO significant increase was found neither in nitrate nor in methaemoglobin concentrations in any trial. Surprising methaemoglobin levels were found to be often decreased after therapy with BSN. Average nitrate concentrations were 3.8±1.3 mg/l before and 4.0±1.9 mg/l after 4 weeks treatment [2100 mg/day]. Cimetidine patients showed comparable values: 3.4±1.6 mg/l before and 4.6±1.9 mg/l after therapy. *Medical Department, Marburg, FRG

Temmler Werke, Temmlerstr.

2, D-3550

R 120 477

479

COMPARATIVE BIOAVAILABILITY OF THREE ORAL FORMULATIONS

ENHANCED BIOAVAILABILITY OF CICLOSPORINE (CYA) AFTER ORAL APPLICATION OF SANDIMMUN R i.v. TO PATIENTS ON CHRONIC CYA TREATMENT. H.iven 1L, R.Preuss2L, R.Wacke 1R, R.Bast2R

OF 8-METHOXYPSORALEN *

8. Quednow and T. Walther In the phototherapy o f p s o r i a s i s , t h r e e i n t e r n a t i o n a l l y introduced 8-methoxypsoralen p r e p a r a t i o n s were t e s t e d f o r their bioavailability. A f t e r s i n g l e doses o f 30 mg 9-methoxypsoralen each in s o f t - g e l a t i n capsules (1), h a r d - g e l a t i n capsules (2) and t a b l e t s (3), the serum c o n c e n t r a t i o n p a t t e r n s were determined in nine p a t i e n t s over 8 hours by means o f an HPLC method. The b i o a v a i l a b i l i t y parameters determined did not suggest t h a t bioequivalence e x i s t e d . Hence, dosage as a f u n c t i o n o f the s p e c i f i c p r e p a r a t i o n i s d e s i r a b l e t o obtain s i m i l a r serum l e v e l s and, thus, a comparable p h o t o t h e r a p e u t i c e f f e c t , 8ioavailability

parameters (Median) o f G e r o x a l e n ® ( 1 ) ,

Oxsoralen@)(2) and

Mopsora~en(~(3) 1

2

3

AUC [rig. m1-1. h - i ]

293,0

i61,8

135,8

C max

~47,0

~04,0

98,0

1,5

1,5

1

bg.ml- 1

t

max

1

[hi *

Department o f Dermatology, U n i v e r s i t y o f Leipzig L i e b i g s t r . 21, 0-7010 Leipzig

The intestinal absorption of CyA is incomplete, ranging between 10 and 60% in man. Experiments in rats showed that the bioavailability of CyA from Sandimmun i.v. when given orally was nearly twice that from the oral application form. The ratio of emulsifier to CyA (g/g) which is 3.2 for the oral and 13 for the i.v. preparation was considered responsible for the increased bioavailability (Iven,Harries, Naunyn-Schmiedeberg's Arch. Pharmacol. 341; R7, 1990). We now extended these studies to patients with kidney grafts. So far, 6 patients with a stable kidney graft function receiving CyA orally on a twice daily schedule took part in the study. During three consecutive days after the morning dose, 9 blood samples were collected at appropriate time intervals from an indwelling catheter. On day 1 and 2 the patients took their usual Sandimmun solution, on day 3 Sandimmun i.v. was given orally once, taking into account the difference in concentration between Sandimmun oral and i.v. solution. Whole blood CyA concentrations were determined with the specific Sandoz 3H-RIA and AUC was calculated using the trapezoidal rule. AUC 0-12h (ng*h*m1-1 ) Pat. Sandimmun oral Sandimmun i.v. A B C increase* I(L) 915 1163 +27.1% II(L) 2774 2080 3571 +28.7% Ill(L) 2298 2183 2508 + 9.1% IV(L) 2235 2671 3064 + 14.7% V(R) 1805 2018 3123 +54.8% VI(R) 3953 3506 4588 + 16.1% * as compared to the higher of the two control AUCs Under control conditions AUCs may differ considerably (A;B), however, in all 6 patients AUC was highest after oral dosing of Sandimmun i.v.(C) with a mean increase of 25.1 ± 6.7%. These results so far confirm our observations in rats.

I n s t i t u t e o f C l i n i c a l Pharmacology, Medical Academy o f Magdeburg, L e i p z i g e r Str. 44, 0-3090 Magdeburg, FRG

Institute of Pharmacology (1) and Clinics for Internal Medicine (2) of LObeck (L) and Rostock (R) University, D-2400 LObeck

478

_48O

PHARMACOKINETIC OF NICOTINE AFTER TOPICAL APPLICATION S.Caspary, BoKeller-Stanislawski, T.Huber, P.-G.Merz

INTERACTIONS WITH CYCLOSPORIN A: INDUCERS AND INHIBITORS OF ITS METABOLISM EVALUATED WITH LIVER MICROSOMAL INCUBATIONS. S. Bauer1'2, S. Olaizola-Horn2, M. Reisinger~, H. HOller1

Transdermal nicotine systems (Nicotinell R 30) were applied daily to the skin of healthy nicotine-dependent smokers for 4 days. Blood samples were taken for the measurement of nicotine and cotinine in plasma using capillary gas chromatography with nitrogen detection. The plasma concentration-time profiles of nicotine and cotinine and the pharmacokinetic parameters Cmax, tmax, AUC and the elimination-half life were determined under steady-state conditions (results see table below). Nicotine levels after application of the 30 cm 2 patch were in accord with published values for I0, 20 and 2x20 cm 2 patches. The elimination half-life of nicotine after removal of the patch was longer than reported values obtained after i.v. application.

Table I: Pharmacokinetic parameters of nicotine after NicotinellR 30 in the 4th dosage period Parameter Nicotine Cmax tma x AUC tl/2 Cotinine AUC tl/2

Mean

±

SD

16.0 8.5 254 4.2

± ± ± ±

2.8 2.4 74 2.2

ng/ml h ng*h/ml h

4028 22.7

± ±

1852 6.4

ng*h/ml h

Dep. Clin. Pharmacoloqy, University Hospital Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt/Main 79

Clinically relevant drug-interactions with the immunosuppressant cyclosporin (CyA) are mainly due to interactions with its metabolism. Cytochrome P-450111A isoenzymes have been described as CyA metabolizing enzymes (Kronbach et al., Clin Pharm Ther 43: 630). As shown here, also compounds like 8-naphthoflavone (Nf) result in the induction of specific reactions of CyA metabolism. Antihypertensives are often nessessary comedication for CyA treated patients, therefore, their influence on CyA elimination is of interest. Methods: Induction was evaluated in liver microsomes from male SpragueDawley rats after pretreatment (50 mg/kg i.p.; 4 days). Inhibition of CyA metabolism was tested with human liver microsomes. The 1 ml microsomal incubations were adjustet to microsomal protein concentration of 2.2 mg/ml and substrate concentrations between 4 and 160 nmol/ml were used. Inhibitor concentrations were between 0.01 and 1.5 ,umol/ml. Results: While the predominantly CyA metabolizing enzymes belong to the cytochrome P-450111Afamily, also isoenzymes belonging to family I contribute to CyA metabolism, as shown in the table by the respective Vmax values (means of 5 different preparations, pmol/mg protein/min). Pretreatment Metab. 1 Metab.17 Metab,18 Untreated 4 2 2 Dexamethasone (Dx) 9 5.5 2 B-Naphthoflavone (Nf) 3.5 2 5.5 Dx and Nf 17.5 9.5 7 The inhibitory effects of the following calcium channel blockers were evaluated: Diltiazem, Verapamil, Nicardipin, Nitrendipin, Nifedipin, Nimodipin, Nis01dipinand Diperdipin. Significant inhibition was determined for all these substances, with Ki values between 5 and 80 ~uM. Significant clinical interactions have been observed for Diltiazem, Verapamil and Nicardipin. Institute l~r Klinische Pharmakologie: 1 Klinikum Steglitz, Freie Universit&t Berlin, ~ Charitd, Humboldt-Universit~t, Berlin.

R 121 481

488

CICLOSPORIN(Cs) AND ITS METABOLITES(CsM) IN A COMBINATION THERAPY WITH AZATHIOPRINE(Aza) AND PREDNISOLONE(P) IN KIDNEY ALLOGRAFT RECIPIENTS J.$. Bleck ~, V.Kliemz, U.Christians 3, H.Repp4, U.Frei z

GENOI~fPIC DETERMINATION OF CYTOCHROME P-450IID6, GLUTATHIONE S-TRANSFERASE ~, AND N-ACETYLTRANSFERASE IN LUNG CANCER PATIENTS I. Roots, J. BrockrnSller, N. Drakoulis, M. Beland, K. Grof~, D. Grot~

Triple drug therapy (Aza,Cs,P) may be a successful regime in patients with clinical signs of Cs-toxicity. It was the aim of this investigation to study metabolic pattern of Cs and its CsM in patients treated with Aza/Cs/P (strived trough level I00 ng) in comparison to Cs/P treated kidney allograft recipients(strived trough level 130 ng). In 40 patients, 19 treated with Aza/Cs/P (Aza:l.74±0.37 mg/kg/d, Cs:4.0±1.2 mg/kg/d, P:7.5mg/d) and and 21 treated with Cs/P (Cs:4.3±1.5 mg/kg/d, P:7.5 mg/d) Cs and 17 CsM in blood(trough level) and 24h-urine were measured by HPLC. The groups were comparable in age, sex and time after transplantation. Results: There was no difference in liver function, but serum creatinine concentrations were significantly elevated in the Aza/Cs/P-group (2~8±99 vs. 168±77 ~mol/l, p(0.013). Although both groups reached the different Cs trough level as desired(Aza/Cs/P:99±10 vs. 128±6 ng/ml,p

German Society for Pharmacology and Toxicology. Abstracts of the 32nd spring meeting, 12-15 March 1991, Mainz.

Naunyn-Schmiedeberg's Arch Pharmacol (1991) 343 (Suppl.): R1-R132 002812989100017F 1 CHARACTERIZATION OF THE MOUSE-MAS ONCOGENE R. Metzger, B. Bunnema...
17MB Sizes 0 Downloads 0 Views