f / r m m u o / c ~ g i& T ~ \ K o / u ~1992. I 71. 420425.
Enrichment of Metabolites in the Cerebrospinal Fluid of Cod (Gadus morhua) Following Oral Administration of Hexachlorobenzene and 2,4’,5=Trichlorobiphenyl K. Ingebrigtsen’, H. Hektoen*, T. Anderson3, E. Klasson Wehler4, A. Bergman‘ and 1. Brandt‘ ‘Departnient of Pharmacology and Toxicology, The Norwegian College of Veterinary Medicine, P.O. Box 8 I46 Dep.. N-0033 Oslo I. /Department of Laboratory Animal Services, National Institute of Public Health, Geitmyrsveien 75. N-0462 Oslo 2, Norway. ’Norwegian Institute for Water Research, N-0808 Oslo, Norway. ’Department of Zoophysiology. University of Goteborg. S-400 3 I Goteborg, ‘Environmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 9 I Stockholm. and ’Department of Pharmacology and Toxicology. The Swedish University of Agricultural Sciences, Biomedicum, S-751 23 Uppsala, Sweden (Received May 21. 1992; Accepted July 8. 1992) Ah.~/rtrct:The disposition of “C-labelled hexachlorobenzene (HCB) and 2,4’,5-trichlorobiphenyl (triCB) was studied in ./i~s For both compounds tape section autorddiogrdphy cod (Gridrr.v niorlrucr) and rainbow trout ( O ~ i ~ . o r / i j ~ i cniykiss). revealed substantial amounts of radiolabelled material in the central nervous system (CNS) of cod, whereas only traces of radioactivity were observed in the CNS of rainbow trout. Furthermore. an enrichment of radiolabelled compound in the cerebrospinal fluid (CSF) was observed in the cod, whereas no radioactivity could be detected in the CSF of rainbow trout. According to autoradiography. the CNS of cod dosed with HCB contained the parent compound, whereas the major part of radioactivity in CSF was due to HCB metabolites. Thin-layer chromatography of extracts from cod dosed with triCB showed the presence of parent compound in the CNS, whereas part of the radioactivity in the CSF was due to triCB metabolites. The activities of cytochrome P-450 and UDP-glucuronosyltransferase in the CNS of cod and rainbow trout were determined in microsomal and mitochondria1 fractions. Both species expressed activities which were i n the same order of magnitude as those reported for the corresponding fractions from rat brain. Incubation of triCB with cod brain mitochondria and microsomes resulted in the formation of two polar metabolites. it is suggested that cod may be more vulnerable than raindow trout regarding neurotoxicological effects of HCB. triCB and related environmental pollutants.
Comparative studies have revealed a profound interspecies difference in the disposition of various persistent chlorinated hydrocarbons between cod and rainbow trout. Thus, ordl administration of “C-labelled hexachlorobenzene (HCB) (Ingebrigtsen & Solbakken 1985), octachlorostyrene (OCS) (Ingebrigtsen ct a/. 1988). 2,3,3’,4,4’-pentachlorobiphenyl (pentaCB) (Ingebrigtsen P I a/. 1990) and 2.3.7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Hektoen et ( I / . 1992) resulted in a substantial accumulation of radiolabelled compound in the central nervous system (CNS) of cod. None of these compounds were found to give a corresponding accumulation in the CNS of rainbow trout (Oncorhync*hu.srnykiss) (Ingebrigtsen & Skaare 1983; Ingebrigtsen r t a/. 1988; Ingebrigtsen 1’1 ( I / . 1990; Hektoen rt d.1992). Whether this species difference in CNS-accumulation of chlorinated hydrocarbons reflects species differences in the total amount of body fat, or if it is due to the presence of a binding mechanism in the cod brain is not known at present. Nevertheless, in comparison to rainbow trout, the high residues attained in the CNS of cod following oral administration of persistent chlorinated hydrocarbons may be of toxicological significance. A reevaluation of data revealed that there was also a species-specific enrichment of radioactivity in the cerebrospinal fluid (CSF) of cod dosed with the “C-labelled xenobi-
otics previously studied. HCB and 2,4’,5-trichlorobiphenyl (triCB) were chosen as model compounds in order to further study this specific labelling of the CNS and CSF in cod.
Materials and Methods Tripe section ciuturudiugrupky. Uniformly labelled “C-hexachlorobenzene (HCB) (specific activity 35.3 Ciimol) was obtained from New England Nuclear Corporation (Boston, MA. U.S.A.). The radiochemical purity was > 99”/”. 2,4’,5-tri~hIoro-(’~C)biphenyl (triCB) (specific activity 18.0 Ciimol) was synthesized from “Clabelled 4-chloroaniline and 1.4-dichlorobenzene as described by Bergman & Wachtmeister (1977). The radiochemical purity was > 99%. Cod and rainbow trout weighing 150-210 g were kept in 250 I glass tanks supplied with running sea water (33% salinity, 8-C) and fed commercial fish pellets (15% fat) once a week. The fish were adapted to the test conditions for three weeks before the start of the experiment. The test compounds were dissolved in peanut oil and administered intragastrically in gelatin capsules at a dose of 33.3 pCiikg body weight. One month after administration the fish were euthanized in a solution of benzocaine in water. frozen in liquid nitrogen and mounted in a gel of carboxymethylcellulose. Sagital whole-body sections (30 pm) from different levels of the body were collected at -20 on tape (no. 821. 3M Co.. St. Paul, Minn., U.S.A.) in a PMV cryomicrotome (PMV 450 MP, Palmstierna Mekaniska Verkstad, Stockholm, Sweden). After freeze-drying at -20 for 24 hr the sections were submitted to the autoradiographic
42 I
HEXACHLOROBENZENE METABOLITES IN C O D CSF procedure described by Ullberg (1954 & 1977). In order to avoid artifacts due to melting and diffusion of fat in the dehydrated sections, all handling took place below -20 (Appelgren 1967). Exposure took place at -80 . Adjacent sections of cod administered ‘“C-HCB were heated to 50 for 24 hr after freeze-drying. Further processing of the sections was performed as described above. The X-ray films (Structurix D7R. Agfa-Gevaert. Antwerp. Belgium) were developed and fixed after 4 months of exposure. Siyurtition und churc~ctcri~citio~~ q / i?irtriholiies. Samples of CNS. CSF and bile were taken from cod administered triCB. Thin-layer chromatography (TLC) was performed on straight phase silicagel (Merck. F254. 0.25 nim thick). Two solvent systems were used for the separations: A: hexane:ethyl acetate (4:l) and B: butano1:acetic acid:water ( 12:3:5).The profile of radioactive compounds was determined by a radio TLC-Analyzer (RITA-3200, Raytest) and by autoradiography, using D7pDW x-ray film (Agfa-Gevaert. Antwerp, Belgium). Unlabelled triCB and 4-hydroxy-3.3’.4’-trichlorobiphenyl were used as reference-compounds. Extraction and enrichment of bile and CSF were performed by using SepPak C,,-cartridges (Waters). The cartridges were rinsed with methanol (20 ml) followed hy a phosphate buffer (pH 6, 0.1 M, 10 rnl) before they were used. The samples, CSF ( < 0 . 5 ml) and bile (0.5 ml), were diluted with the phosphate buffer to 2 ml and transferred to the SepPak cartridge. After rinsing the cartridge with pH-buffer ( 5 ml). the triCB-derived compounds were eluted with methanol ( 3 + 3 ml). The content of radioactivity in the fractions was determined. The volume of the CSF-sample was concentrated to 10 pI and the bile was concentrated to have 10000 d p m i l 0 pl. All of the CSF sample and 10 p1 or the bile were used for TLCanalysis. The brain tissue was homogenized in dichloromethane (three times) with an Ultra-Turrax homogenizer. The tissue-residue was allowed to precipitate and the solvent was taken off. The total radioactivity in the pooled extracts was determined before the solvent was evaporated and the lipid weight determined. Lipids were removcd by gel permeation chromatography and fractions of 10 in1 were collected as described by Klasson-Wehler ci 01. (1987). The radioactivity in all fractions was determined. Fractions were combined to give a lipid fraction, an intermediate fraction and a fraction containing triCB and conventional triCB metabolites. The latter fraction was analyzed by TLC.
Mi,/trhrdism cmd enzj~~nolog.): Whole brains from untreated cod and rainbow lrout were homogenized by a glass-teflon Potter-Elvehjeni homogenizer and sonicated for 10 sec. in a 0.1 M sodium phosphate bufl‘er containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 rnM butylated hydroxytoluene. I niM dithiothreit and 0.1 mM EDTA. Nuclei and cell debris were pelleted by centrifugation for 10 min. at 1.240 x g. The resulting supernatant was centrifuged at 15.000 x g for 20 min. The resulting supernatant was centrifuted at I80.000 x g for 90 inin. The 15,000 x g pellet and 180,000 x g pellets were found to enrich mitochondria and microsomes, respectively (Andersson L? Goks~iyr.unpublished results). The activity of the cytochrome P-450 dependent 7-ethoxycoumarin-0-deethylase (ECOD) was assayed as described by Forlin & Hansson (1982). The formation of 7-hydroxycoumarin was measured according to Greenlee & Poland ( I 978). UDP-glucuronosyltransferase activity toward5 p-nitrophenol was assayed as described by Andersson ct u / (19x5) in digitonin activated samples. Incubation of liver microsomes o r brain mitochondria1 and microsomal fractions with triCB was performed in a total volume of I nil 0.05 M sodium phosphate buffer (pH 7.4) containing 40 nmol of the radiolabelled triCB (0.7 pCi). The incubations contained 2.3 and 6.0 rng protein for the rainbow trout brain mitochondria and rnicrosomal fraction. respectively. The cod brain mitochondria and rnicrosomal incubations contained 10.5 and 5.6 mg protein respectively. The liver microsomal incubations contained 3.5 and
Fig. 1. Autoradiograms of tissue sections from the brain of ( A ) cod (Garfus morhua) and (B) rainbow trout (Oncor/iynchusni.vkis.7) 30 White days after oral administration of “‘C-2,4’.5-trichlorobiphenyl. areas correspond to high concentrations of radioactivity. CNS = central nervous system; CSF =cerebrospinal fluid. Note the low degree of radioactivity in the CNS and the lack of radioactivity in the CSF of rainbow trout.
2.1 mg protein for the rainbow trout and cod. respectively. The reaction was started by adding 0.8 mg NADPH and terminated after 2 hr incubation by the addition of cold aceton ( I ml). The reaction mixture was extracted three times with ethyl acetate (2 ml). The ethyl acetate was evaporated under a stream of nitrogen and the residue was dissolved in ethyl acetate and applied to a silica gel plate and developed in hexanexthy1 acetate (9:1 ).
Results
Tupe section uutorudiogrupky. Autoradiograms of sections from cod brain and rainbow trout brain one month after oral administration of I4C-2,4’,5-TriCB are shown in fig. l a and lb, respectively. The amount of radioactivity in the cod brain by far exceeded that of the rainbow trout. Furthermore. a substantial radiolabelling was present in the CSF of cod, while n o radioactivity could be detected in the CSF of rainbow trout. Fig. 2a, b and 3a, b are autoradiograms of sections from cod brain and cod liver, respectively, one month after oral administration of I4C-HCB. Fig. 2a and 3a represent sections which have been handled below - 20 and exposed at -80 , whereas fig. 2b and 3b represent
472
K. INGEBRIGTSEN ET A L .
adjacent sections which have been heated to 50- for 24 hr prior to exposure. I n the sections handled below - 20’ , high levels of radioactivity were present in the CNS and the C S F as well as in the liver and bile. In the sections heated to 50 , thc radioactivity in the C N S and the liver had evaporated completely. whereas radiolabelling in the C S F and the bile remained unchanged. Sq)urutioii mid ( . I i ~ ~ . ~ ( . t ~ ~ . i o- -/ ~nieruholites. tion The total content of radioactivity in the pooled bile from cod dosed with “C-triCB was approximately 9 x lo6 dpm. The methanol-fraction, containing 96% of the radioactivity, was concentrated and subsequently analyzed by TLC. According to TLC analysis, the bile mainly contained polar compounds (fig. 4) but also a minor compound with a polarity similar t o the unmetabolized triCB, as well as traces of a compound with an intermediate polarity. The latter compound had a R,-value similar to that of the hydroxylated triCB used as reference compound and this may indicate the presence of an unconjugated OH-triCB in the bile.
Fig. 3. Autoradiograms of tissue sections from the liver of cod (Gudirs morhutr) 30 days after oral administration of ‘T-hexachlorobenzene. A: Tissue section handled below -20-C; B: Adjacent section heated to +SO C prior to exposure. White areas correspond to high concentrations of radioactivity. Note that all radioactivity in the liver has evaporated during heat treatment, whereas radioactivity in the bile remains unchanged.
The C S F isolated for analysis contained a total of 7000 dpm, and all of the radioactivity eluted in the methanolfraction from the SepPak cartridge. According to fig. 4, the radioactivity consisted of both unmetabolized triCB and one or more polar metabolites with similar chromatographic properties as the polar metabolites in the bile. The radioactivity in the brain was quantitatively extractable and 95% of the radioactivity was isolated in the triCBfraction from the G P C fractionation. TLC-analysis showed that the radioactivity consisted of unmetabolized triCB only (fig. 4).
Fig, 2 , Autoradiogra111s of tissue sections from the brain of cod (Gtrdirs rlrorhrrcr) 30 days after oral administration of “C-hexachloro-
benzene. A: Tissue section handled below -20 C; B: Adjacent section heated to + S O C prior to exposure. White areas correspond to high concentrations of radioactivity. CNS=central nervous system; CSF=cerebrospinal fluid, Note that a , l radioctivity in the CNS has evaporated during heat treatment, whereas radioactivity in the CSF remains unchanged.
Metubolism und en:yrnologj~. About equal levels of cytochrome p-450 dependent ECoD activities could be measured in microsomal and mitochondrial fraction of the cod and rainbow trout brain (table 1). The localization ofp-450 dependent enzyme activities in the mitochondrial fraction should not be due to contamination with microsonies since subfractionation studies have shown that the mitochondrial fraction of cod and rainbow trout brain was contaminated
423
HEXACHLOROBENZENE METABOLITES IN C O D CSF
with less than 3% of microsomes as estimated by marker enzyme measurements (Anderson & Gokseyr, unpublished results). Furthermore, an UDP-glucuronosyltransferase active towards p-nitrophenol could be measured in both the mitochondrial and microsomal fractions from cod and rainbow trout (table I ) . Finally, incubation of cod liver microsomes with triCB resulted in formation of one main polar metabolite which was separated by thin layer chromatography (fig. 5). A peak with the same retention could be seen in cod brain mitochondria and microsomes. However, another more polar peak was also recorded in the cod brain. Incubation with rainbow trout liver microsomes gave the same pattern as for the cod liver microsomes whereas only weak signals were obtained for rainbow trout brain microsomes and mitochondria (data not shown).
Table I. ECOD (pmol/min./mg) and UDPGT (nmol/min./mg) activities in microsomal and mitochondria1 fractions of brain and liver from cod (Gadus morhuo) and rainbow trout (Oncur/i.ync/ius n7ykis.s).
Roinbou truut Brain mitochondria Brain microsomes Liver microsomes
UDPGT (nmol/min./mg)
0.460* (0.240)** 0.340 (0.340) 26.800 (1 1.500)
0.027 (0.01 5 ) 0.024 (0.027) 0.420 (0.077)
Cod
Brain mitochondria Brain microsomes Liver microsomes
Discussion
ECOD (pmol imin. img)
0.760 (0.760) 0.600 (0.300) 21.600 (7.010)
The present study confirms and extends previous observations that there is a species specific uptake of chlorinated hydrocarbons in the CNS of cod. Thus, substantial amounts of radiolabelled material are accumulated in the CNS following oral administration of ‘‘C-labelled triCB, pentaCB (Ingebrigtsen ot ul. 1990), HCB (Ingebrigtsen & Solbakken 1985), OCS (Ingebrigtsen et al. 1988) and TCDD (Hektoen er (11. 1992) to cod, whereas only traces of radioactivity are observed in the CNS of rainbow trout following oral exposure to these compounds. I n the present study, HCB and triCB were selected as model compounds for further investigations on the disposition ofchlorinated hydrocarbons in the brain of cod. HCB has a high vapor pressure, a property that thas been utilized in “high temperature autoradiography” to separate HCB from its nonvolatilc metabolites (lngebrigtsen & Nafstad 1983; Ingebrigtsen & Solbakken 1985). The choice of triCB
CI
FRONT+
ORIGIN-,
Fig. 4. Autoradiogram of a TLC-plate developed in hexane:ethyl acelate (4: I ). Samples of cerebrospinal lluid (CSF). central nervous system (CNS) and bile from cod administered “C-2.4’,5-trichlorobiphenyl (triCB) are shown. The upper spots correspond to (triCB).
* Each value ** S.D.
0.029 (0.049) 0.015
(0.01 I ) 0.320 (0.140)
represents the mean of 4 individuals
as a model compound was based on previous studies in mice (Brandt et al. 1982) and in rat (Bakke et LII. 1983) which showed that triCB is readily metabolized. Although the rate of metabolism of HCB and triCB in cod is unknown, previous studies with HCB (Ingebrigtsen & Solbakken 1985) and the present study with triCB indicate that polar metabolites of both compounds are excreted in the bile of this species. The radioactivity in the C N S and liver of “C-HCR-exposed cods was most likely due to parent compound, since the radiolabelled material almost completely evaporated following heating of the sections. However, the observation that the radioactivity in the C S F and bile was non-volatile implies the presence of HCB metabolites in these fluids. The radiaoctivity in the C N S of triCB exposed cods was due to unmetabolized triCB, whereas part of the radioactivity in the C S F was due to triCB metabolites (fig. 4). In addition. also triCB was present in the CSF. The CSF represents an hitherto unknown site of enrichment for xenobiotics in cod. The radioactivity in the CSF of HCB or triCB-exposed cod may be due to hepatic metabolites that have passed the blood brain barrier or to metabolites formed in the CNS and subsequently translocated into the CSF. In order to investigate the latter possibility, the activity of xenobiotic metabolizing enzymes in the CNS of cod and rainbow trout was compared to the metabolic activity of their livers. The results showed that the CNS of both species contained cytochrome P-450dependent ECOD activities. Compared with the microsomal liver values measured in the two fish species the activities in brain mitochondria and microsomes were low. The presence of cytochrome P-450dependent xenobiotic metabolism in the brain mitochondrial fraction has previously been shown in mammalian studies (Iscan et al. 1990), and the cod and rainbow trout brain mitochondrial and microsomal ECOD activities were
K. INGEBRIGTSEN ET A L
424
A
counts 10
10 -
B
5
5-
application zone 1
2
4
I
substrate
0
6
8
10
,
1
1
12
14
cm
D
counts 10 -
L
counts
substrate
amlication zone
1
5-
application zone
substrate
1I -
application zone
substrate
Fig. 5. Profiles of thin layer chromotograms from extracts of incubations with ''C-2,4',5-trichlorobiphenyl.A: Blank. B: Cod liver microsomes. C: Cod brain mitochondria. D:Cod brain microsomes. Note corresponding peaks in B. C and D.
on the same level as those reported for corresponding rat brain fractions (Naslund et a/. 1988). Furthermore, an UDP-glucuronosyltransferase activity was recorded in the brain of both species. Although the activities were low compared to the microsomal liver values, they were in the same order of magnitude as those previously reported for rat cerebral microsomal UDP-glucuronosyltransferase activities (Ghersi-Egea 1987). Finally, the incubation experiments revealed that brain homogenates from both species were able to transform triCB to polar metabolites. It cannot be excluded that HCB and triCB are metabolized in the CNS of cod, and that the metabolites are secreted into the CSF. Noteworthy, the levels of both HCB and triCB in the CNS of rainbow trout were low, and the lack of radioactivity in the CSF of rainbow trout may therefore be due to a low level of Substrate for the xenobiotic metabolizing enzymes in this tissue. The observation that part of the radioactivity in the CSF of triCB dosed cods was due to unmetabolized triCB is difficult to interprete, since the lipophilic triCB is not expected to be present in a water phase unless it was bound
to a watersoluble carrier molecule (Brandt et d.1982). An intriguing possibility is that the enrichment of unmetabolized triCB as well as triCB- and HCB-derived metabolites was due to the presence of a transport protein in the CSF. It has recently been reported that several xenobiotics including HCB and its hydroxylated metabolite pentachlorophenol together with certain PCBs and PCB-derived metabolites bind to the thyroid hormone transport protein, transthyretin, in rodents (Van den Berg et a/. 1991). Furthermore, epithelial cells in the choroid plexus of rodents and man are highly specialized for the synthesis of transthyretin which is subsequently released into the C S F (Dickson et al. 1987). Whether thyroid hormone transport proteins are present in the C S F of fish, however, is not known. In conclusion, the present study supports previous observations that chlorinated hydrocarbons are extensively accumulated in the CNS of cod, whereas only traces are taken up in the CNS of rainbow trout. The results also imply that, in the cod, HCB and triCB are accumulated as unmetabolized compound in the C N S and that there is an enrichment of HCB- and triCB-derived metabolites in the CSF. These
HEXACHLOROBENZENE METABOLITES IN C O D CSF
interspecies differences in the disposition of chlorinated hydrocarbons implicate that cod may be more vulnerable than rainbow trout as concerns neurotoxicological effects of such compounds.
References Andersson. T.. M. Pesonen & C. Johansson: Differential induction of cytochrome P-450-dependent monooxygenase, epoxide hydrolase, glutathione transferase and UDP-glucuronosyltransferase activities in the liver of rainbow trout by b-naphthoflavone or Clophen A50. Biochem. Phurmucol. 1985, 34, 3309-3314. Appelgren. L.-E.: Sites of steroid hormone formation. Autoradiographic studies using labelled precursors. Aera Phjsiol. Srand 1967, Suppl. 301, I 108. Bakke. J. E.. V. J. Feil & A. Bergman: Metabolites of 2.4',5-trichlorobiphenyl in rats. Xenohiofiea 1983. 13, 555-564. Bergman. A. & C. A. Wachtmeister: Synthesis of ('4C)biphenyl and of some chlorinated ( I4C)biphenylscontaining 4-chloro-, 2,4dichloro- and 2,3,6-trichlorophenyl nuclei from the corresponding labelled anilines. Chernospherr 1977, 11, 759-767. Brandt. I., P. 0. Darnerud, A. Bergman & Y. Larsson: Metabolism of ?.4'.5-trichlorobiphenyl: Enrichment of hydroxylated and methyl sulphone metabolites in the uterine luminal fluid of pregnant mice. Chem. Biol. Interactions 1982. 40, 45 56. Dickson. P. W.. A. R. Aldred, J. G. T. Menting, P. D. Marley, W. H.Sawyer & G . Schreiber: Thyroxine transport in choroid plexus. J. Biol. Cheni. 1987, 262. 13907-1 39 15. F6rlin. L. & T. Hansson: Effects of treated municipal wastewater on the hepatic xenobiotic and steroid metabolism in trout. Eeotosieol. Environm. Sufetv 1982. 6. 41 4 8 . Ghersi-Egea, J. F., B. Walther, D. Decolin, A. Minn & G. Siest: The activity of I-naphthol-UDP-glucuronosyltransferase in the brain. Neurophtrrmticolog~1987, 26, 367-372. Greenlee, W. E & A. Poland: An improved assay of7-cthoxycoumarin-0-deethylase activity: Induction of hepatic enzyme activity in C57BLi6J and DBA/2A mice by phenobarbital, 3-methylcholJ . Pharmacol. antrene and 2,3.7.8-tetrachlorodibenzo-p-dioxin. E.rp. Therap. 1978, 205. 596 605. Hektoen, H., K. Ingebrigtsen, E. M. Brevik & M. Oehme: Interspecies differences in tissue distribution of 2,3,7,8-tetrachlorodibenz-
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o-p-dioxin between cod (Gadus morhua) and rainbow trout (Oncorhynchus mykiss). Chemosphere 1992, 24, 58 1-587. Ingebrigtsen, K., H. Hektoen, T. Andersson. A. Bergman & 1. Brandt: Species-specific accumulation of the polychlorinated biphenyl (PCB) 2,3,3',4,4'-pentachlorobiphenyl in fish brain: A comparison between cod (Gadus morhua) and rainbow trout (Oncorhynchus mykiss). Phnrtnarology & Tosicolog~, 1990, 67, 344345. Ingebrigtsen, K. & I. Nafstad: Distribution and elimination of "Chexachlorobenzene after single oral exposure in the male rat. Acta pharmacol. et toxicol. 1983, 52, 254260. Ingebrigtsen, K. & J. U. Skaare: Distribution and elimination of ''C-hexachlorobenzene after single oral exposure in the rainbow trout (Sulmo gairdneri). J . Toxicol. Environ. Hculth 1983, 12, 309-3 16. Ingebrigtsen, K. & J. E. Solbakken: Distribution and elimination of ''C-hexachlorobezene after single oral exposure in cod (Cadus morhua) and flounder (Platichthys flesus). J. Touicol. Environ. Health 1985, 16, 179-205. Ingebrigtsen, K., J. E. Solbakken, G . Norheim & 1. Nafstad: Distribution and elimination of ''C-octachlorostyrene i n cod (Gadus morhua), rainbow trout (Salmo gairdneri) and blue mussel (Myti/us edulis). J. Toxieol. Environ. Health. 1988. 25, 361-372. Iscan, M., K. Reuhl, B. Weiss & M. D. Maines: Regional and subcellular distribution of cytochrome P-450-dependent drug metabolism in monkey brain: The olfactory bulb and the mitochondrial fraction have high levels of activity. Biochemical. Biophysical. Res. Commun. 1990, 169. 858-863. Klasson-Wehler, E., B. Kowalski. A. Bergman & I. Brandt: Metabolism of 2,3,4',6-tetrachlorobiphenyl:Formation and tissue localization of mercapturic acid pathway metabolites in mice. Xenohiotica 1987, 17, 471486. Naslund, B. M. A,, H. Galumann, M . Warner, J.-A. Gustafsson & T. Hansson: Cytochrome P450 b and c in the rat brain and pituitary gland. Mol. Pharmacol. 1988, 33, 31-37. Ullberg, S.. Studies on the distribution and fate of "S-labelled benzylpenicillin in the body. Acta Radiol. 1954. Suppl. 118, 1-1 10. Ullberg, S.:The technique of whole body autoradiography. Cryosectioning of large specimens. Science Tools, Special issue on whole body autoradiography. The LKB Instrument Journal. Stockholm, Sweden 1977, 1-29. van den Berg, K. J., J. A. G . M. van Raaij, P. C. Bragt & W. R. F. Notten: Interactions of halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in vivo. Arch. Toxicol. 1991, 65, 15-19.
Enrichment of Metabolites in the Cerebrospinal Fluid of Cod (Gadus morhua) Following Oral Administration of Hexachlorobenzene and 2,4’,5=Trichlorobiphenyl K. Ingebrigtsen’, H. Hektoen*, T. Anderson3, E. Klasson Wehler4, A. Bergman‘ and 1. Brandt‘ ‘Departnient of Pharmacology and Toxicology, The Norwegian College of Veterinary Medicine, P.O. Box 8 I46 Dep.. N-0033 Oslo I. /Department of Laboratory Animal Services, National Institute of Public Health, Geitmyrsveien 75. N-0462 Oslo 2, Norway. ’Norwegian Institute for Water Research, N-0808 Oslo, Norway. ’Department of Zoophysiology. University of Goteborg. S-400 3 I Goteborg, ‘Environmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 9 I Stockholm. and ’Department of Pharmacology and Toxicology. The Swedish University of Agricultural Sciences, Biomedicum, S-751 23 Uppsala, Sweden (Received May 21. 1992; Accepted July 8. 1992) Ah.~/rtrct:The disposition of “C-labelled hexachlorobenzene (HCB) and 2,4’,5-trichlorobiphenyl (triCB) was studied in ./i~s For both compounds tape section autorddiogrdphy cod (Gridrr.v niorlrucr) and rainbow trout ( O ~ i ~ . o r / i j ~ i cniykiss). revealed substantial amounts of radiolabelled material in the central nervous system (CNS) of cod, whereas only traces of radioactivity were observed in the CNS of rainbow trout. Furthermore. an enrichment of radiolabelled compound in the cerebrospinal fluid (CSF) was observed in the cod, whereas no radioactivity could be detected in the CSF of rainbow trout. According to autoradiography. the CNS of cod dosed with HCB contained the parent compound, whereas the major part of radioactivity in CSF was due to HCB metabolites. Thin-layer chromatography of extracts from cod dosed with triCB showed the presence of parent compound in the CNS, whereas part of the radioactivity in the CSF was due to triCB metabolites. The activities of cytochrome P-450 and UDP-glucuronosyltransferase in the CNS of cod and rainbow trout were determined in microsomal and mitochondria1 fractions. Both species expressed activities which were i n the same order of magnitude as those reported for the corresponding fractions from rat brain. Incubation of triCB with cod brain mitochondria and microsomes resulted in the formation of two polar metabolites. it is suggested that cod may be more vulnerable than raindow trout regarding neurotoxicological effects of HCB. triCB and related environmental pollutants.
Comparative studies have revealed a profound interspecies difference in the disposition of various persistent chlorinated hydrocarbons between cod and rainbow trout. Thus, ordl administration of “C-labelled hexachlorobenzene (HCB) (Ingebrigtsen & Solbakken 1985), octachlorostyrene (OCS) (Ingebrigtsen ct a/. 1988). 2,3,3’,4,4’-pentachlorobiphenyl (pentaCB) (Ingebrigtsen P I a/. 1990) and 2.3.7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Hektoen et ( I / . 1992) resulted in a substantial accumulation of radiolabelled compound in the central nervous system (CNS) of cod. None of these compounds were found to give a corresponding accumulation in the CNS of rainbow trout (Oncorhync*hu.srnykiss) (Ingebrigtsen & Skaare 1983; Ingebrigtsen r t a/. 1988; Ingebrigtsen 1’1 ( I / . 1990; Hektoen rt d.1992). Whether this species difference in CNS-accumulation of chlorinated hydrocarbons reflects species differences in the total amount of body fat, or if it is due to the presence of a binding mechanism in the cod brain is not known at present. Nevertheless, in comparison to rainbow trout, the high residues attained in the CNS of cod following oral administration of persistent chlorinated hydrocarbons may be of toxicological significance. A reevaluation of data revealed that there was also a species-specific enrichment of radioactivity in the cerebrospinal fluid (CSF) of cod dosed with the “C-labelled xenobi-
otics previously studied. HCB and 2,4’,5-trichlorobiphenyl (triCB) were chosen as model compounds in order to further study this specific labelling of the CNS and CSF in cod.
Materials and Methods Tripe section ciuturudiugrupky. Uniformly labelled “C-hexachlorobenzene (HCB) (specific activity 35.3 Ciimol) was obtained from New England Nuclear Corporation (Boston, MA. U.S.A.). The radiochemical purity was > 99”/”. 2,4’,5-tri~hIoro-(’~C)biphenyl (triCB) (specific activity 18.0 Ciimol) was synthesized from “Clabelled 4-chloroaniline and 1.4-dichlorobenzene as described by Bergman & Wachtmeister (1977). The radiochemical purity was > 99%. Cod and rainbow trout weighing 150-210 g were kept in 250 I glass tanks supplied with running sea water (33% salinity, 8-C) and fed commercial fish pellets (15% fat) once a week. The fish were adapted to the test conditions for three weeks before the start of the experiment. The test compounds were dissolved in peanut oil and administered intragastrically in gelatin capsules at a dose of 33.3 pCiikg body weight. One month after administration the fish were euthanized in a solution of benzocaine in water. frozen in liquid nitrogen and mounted in a gel of carboxymethylcellulose. Sagital whole-body sections (30 pm) from different levels of the body were collected at -20 on tape (no. 821. 3M Co.. St. Paul, Minn., U.S.A.) in a PMV cryomicrotome (PMV 450 MP, Palmstierna Mekaniska Verkstad, Stockholm, Sweden). After freeze-drying at -20 for 24 hr the sections were submitted to the autoradiographic
42 I
HEXACHLOROBENZENE METABOLITES IN C O D CSF procedure described by Ullberg (1954 & 1977). In order to avoid artifacts due to melting and diffusion of fat in the dehydrated sections, all handling took place below -20 (Appelgren 1967). Exposure took place at -80 . Adjacent sections of cod administered ‘“C-HCB were heated to 50 for 24 hr after freeze-drying. Further processing of the sections was performed as described above. The X-ray films (Structurix D7R. Agfa-Gevaert. Antwerp. Belgium) were developed and fixed after 4 months of exposure. Siyurtition und churc~ctcri~citio~~ q / i?irtriholiies. Samples of CNS. CSF and bile were taken from cod administered triCB. Thin-layer chromatography (TLC) was performed on straight phase silicagel (Merck. F254. 0.25 nim thick). Two solvent systems were used for the separations: A: hexane:ethyl acetate (4:l) and B: butano1:acetic acid:water ( 12:3:5).The profile of radioactive compounds was determined by a radio TLC-Analyzer (RITA-3200, Raytest) and by autoradiography, using D7pDW x-ray film (Agfa-Gevaert. Antwerp, Belgium). Unlabelled triCB and 4-hydroxy-3.3’.4’-trichlorobiphenyl were used as reference-compounds. Extraction and enrichment of bile and CSF were performed by using SepPak C,,-cartridges (Waters). The cartridges were rinsed with methanol (20 ml) followed hy a phosphate buffer (pH 6, 0.1 M, 10 rnl) before they were used. The samples, CSF ( < 0 . 5 ml) and bile (0.5 ml), were diluted with the phosphate buffer to 2 ml and transferred to the SepPak cartridge. After rinsing the cartridge with pH-buffer ( 5 ml). the triCB-derived compounds were eluted with methanol ( 3 + 3 ml). The content of radioactivity in the fractions was determined. The volume of the CSF-sample was concentrated to 10 pI and the bile was concentrated to have 10000 d p m i l 0 pl. All of the CSF sample and 10 p1 or the bile were used for TLCanalysis. The brain tissue was homogenized in dichloromethane (three times) with an Ultra-Turrax homogenizer. The tissue-residue was allowed to precipitate and the solvent was taken off. The total radioactivity in the pooled extracts was determined before the solvent was evaporated and the lipid weight determined. Lipids were removcd by gel permeation chromatography and fractions of 10 in1 were collected as described by Klasson-Wehler ci 01. (1987). The radioactivity in all fractions was determined. Fractions were combined to give a lipid fraction, an intermediate fraction and a fraction containing triCB and conventional triCB metabolites. The latter fraction was analyzed by TLC.
Mi,/trhrdism cmd enzj~~nolog.): Whole brains from untreated cod and rainbow lrout were homogenized by a glass-teflon Potter-Elvehjeni homogenizer and sonicated for 10 sec. in a 0.1 M sodium phosphate bufl‘er containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 rnM butylated hydroxytoluene. I niM dithiothreit and 0.1 mM EDTA. Nuclei and cell debris were pelleted by centrifugation for 10 min. at 1.240 x g. The resulting supernatant was centrifuged at 15.000 x g for 20 min. The resulting supernatant was centrifuted at I80.000 x g for 90 inin. The 15,000 x g pellet and 180,000 x g pellets were found to enrich mitochondria and microsomes, respectively (Andersson L? Goks~iyr.unpublished results). The activity of the cytochrome P-450 dependent 7-ethoxycoumarin-0-deethylase (ECOD) was assayed as described by Forlin & Hansson (1982). The formation of 7-hydroxycoumarin was measured according to Greenlee & Poland ( I 978). UDP-glucuronosyltransferase activity toward5 p-nitrophenol was assayed as described by Andersson ct u / (19x5) in digitonin activated samples. Incubation of liver microsomes o r brain mitochondria1 and microsomal fractions with triCB was performed in a total volume of I nil 0.05 M sodium phosphate buffer (pH 7.4) containing 40 nmol of the radiolabelled triCB (0.7 pCi). The incubations contained 2.3 and 6.0 rng protein for the rainbow trout brain mitochondria and rnicrosomal fraction. respectively. The cod brain mitochondria and rnicrosomal incubations contained 10.5 and 5.6 mg protein respectively. The liver microsomal incubations contained 3.5 and
Fig. 1. Autoradiograms of tissue sections from the brain of ( A ) cod (Garfus morhua) and (B) rainbow trout (Oncor/iynchusni.vkis.7) 30 White days after oral administration of “‘C-2,4’.5-trichlorobiphenyl. areas correspond to high concentrations of radioactivity. CNS = central nervous system; CSF =cerebrospinal fluid. Note the low degree of radioactivity in the CNS and the lack of radioactivity in the CSF of rainbow trout.
2.1 mg protein for the rainbow trout and cod. respectively. The reaction was started by adding 0.8 mg NADPH and terminated after 2 hr incubation by the addition of cold aceton ( I ml). The reaction mixture was extracted three times with ethyl acetate (2 ml). The ethyl acetate was evaporated under a stream of nitrogen and the residue was dissolved in ethyl acetate and applied to a silica gel plate and developed in hexanexthy1 acetate (9:1 ).
Results
Tupe section uutorudiogrupky. Autoradiograms of sections from cod brain and rainbow trout brain one month after oral administration of I4C-2,4’,5-TriCB are shown in fig. l a and lb, respectively. The amount of radioactivity in the cod brain by far exceeded that of the rainbow trout. Furthermore. a substantial radiolabelling was present in the CSF of cod, while n o radioactivity could be detected in the CSF of rainbow trout. Fig. 2a, b and 3a, b are autoradiograms of sections from cod brain and cod liver, respectively, one month after oral administration of I4C-HCB. Fig. 2a and 3a represent sections which have been handled below - 20 and exposed at -80 , whereas fig. 2b and 3b represent
472
K. INGEBRIGTSEN ET A L .
adjacent sections which have been heated to 50- for 24 hr prior to exposure. I n the sections handled below - 20’ , high levels of radioactivity were present in the CNS and the C S F as well as in the liver and bile. In the sections heated to 50 , thc radioactivity in the C N S and the liver had evaporated completely. whereas radiolabelling in the C S F and the bile remained unchanged. Sq)urutioii mid ( . I i ~ ~ . ~ ( . t ~ ~ . i o- -/ ~nieruholites. tion The total content of radioactivity in the pooled bile from cod dosed with “C-triCB was approximately 9 x lo6 dpm. The methanol-fraction, containing 96% of the radioactivity, was concentrated and subsequently analyzed by TLC. According to TLC analysis, the bile mainly contained polar compounds (fig. 4) but also a minor compound with a polarity similar t o the unmetabolized triCB, as well as traces of a compound with an intermediate polarity. The latter compound had a R,-value similar to that of the hydroxylated triCB used as reference compound and this may indicate the presence of an unconjugated OH-triCB in the bile.
Fig. 3. Autoradiograms of tissue sections from the liver of cod (Gudirs morhutr) 30 days after oral administration of ‘T-hexachlorobenzene. A: Tissue section handled below -20-C; B: Adjacent section heated to +SO C prior to exposure. White areas correspond to high concentrations of radioactivity. Note that all radioactivity in the liver has evaporated during heat treatment, whereas radioactivity in the bile remains unchanged.
The C S F isolated for analysis contained a total of 7000 dpm, and all of the radioactivity eluted in the methanolfraction from the SepPak cartridge. According to fig. 4, the radioactivity consisted of both unmetabolized triCB and one or more polar metabolites with similar chromatographic properties as the polar metabolites in the bile. The radioactivity in the brain was quantitatively extractable and 95% of the radioactivity was isolated in the triCBfraction from the G P C fractionation. TLC-analysis showed that the radioactivity consisted of unmetabolized triCB only (fig. 4).
Fig, 2 , Autoradiogra111s of tissue sections from the brain of cod (Gtrdirs rlrorhrrcr) 30 days after oral administration of “C-hexachloro-
benzene. A: Tissue section handled below -20 C; B: Adjacent section heated to + S O C prior to exposure. White areas correspond to high concentrations of radioactivity. CNS=central nervous system; CSF=cerebrospinal fluid, Note that a , l radioctivity in the CNS has evaporated during heat treatment, whereas radioactivity in the CSF remains unchanged.
Metubolism und en:yrnologj~. About equal levels of cytochrome p-450 dependent ECoD activities could be measured in microsomal and mitochondrial fraction of the cod and rainbow trout brain (table 1). The localization ofp-450 dependent enzyme activities in the mitochondrial fraction should not be due to contamination with microsonies since subfractionation studies have shown that the mitochondrial fraction of cod and rainbow trout brain was contaminated
423
HEXACHLOROBENZENE METABOLITES IN C O D CSF
with less than 3% of microsomes as estimated by marker enzyme measurements (Anderson & Gokseyr, unpublished results). Furthermore, an UDP-glucuronosyltransferase active towards p-nitrophenol could be measured in both the mitochondrial and microsomal fractions from cod and rainbow trout (table I ) . Finally, incubation of cod liver microsomes with triCB resulted in formation of one main polar metabolite which was separated by thin layer chromatography (fig. 5). A peak with the same retention could be seen in cod brain mitochondria and microsomes. However, another more polar peak was also recorded in the cod brain. Incubation with rainbow trout liver microsomes gave the same pattern as for the cod liver microsomes whereas only weak signals were obtained for rainbow trout brain microsomes and mitochondria (data not shown).
Table I. ECOD (pmol/min./mg) and UDPGT (nmol/min./mg) activities in microsomal and mitochondria1 fractions of brain and liver from cod (Gadus morhuo) and rainbow trout (Oncur/i.ync/ius n7ykis.s).
Roinbou truut Brain mitochondria Brain microsomes Liver microsomes
UDPGT (nmol/min./mg)
0.460* (0.240)** 0.340 (0.340) 26.800 (1 1.500)
0.027 (0.01 5 ) 0.024 (0.027) 0.420 (0.077)
Cod
Brain mitochondria Brain microsomes Liver microsomes
Discussion
ECOD (pmol imin. img)
0.760 (0.760) 0.600 (0.300) 21.600 (7.010)
The present study confirms and extends previous observations that there is a species specific uptake of chlorinated hydrocarbons in the CNS of cod. Thus, substantial amounts of radiolabelled material are accumulated in the CNS following oral administration of ‘‘C-labelled triCB, pentaCB (Ingebrigtsen ot ul. 1990), HCB (Ingebrigtsen & Solbakken 1985), OCS (Ingebrigtsen et al. 1988) and TCDD (Hektoen er (11. 1992) to cod, whereas only traces of radioactivity are observed in the CNS of rainbow trout following oral exposure to these compounds. I n the present study, HCB and triCB were selected as model compounds for further investigations on the disposition ofchlorinated hydrocarbons in the brain of cod. HCB has a high vapor pressure, a property that thas been utilized in “high temperature autoradiography” to separate HCB from its nonvolatilc metabolites (lngebrigtsen & Nafstad 1983; Ingebrigtsen & Solbakken 1985). The choice of triCB
CI
FRONT+
ORIGIN-,
Fig. 4. Autoradiogram of a TLC-plate developed in hexane:ethyl acelate (4: I ). Samples of cerebrospinal lluid (CSF). central nervous system (CNS) and bile from cod administered “C-2.4’,5-trichlorobiphenyl (triCB) are shown. The upper spots correspond to (triCB).
* Each value ** S.D.
0.029 (0.049) 0.015
(0.01 I ) 0.320 (0.140)
represents the mean of 4 individuals
as a model compound was based on previous studies in mice (Brandt et al. 1982) and in rat (Bakke et LII. 1983) which showed that triCB is readily metabolized. Although the rate of metabolism of HCB and triCB in cod is unknown, previous studies with HCB (Ingebrigtsen & Solbakken 1985) and the present study with triCB indicate that polar metabolites of both compounds are excreted in the bile of this species. The radioactivity in the C N S and liver of “C-HCR-exposed cods was most likely due to parent compound, since the radiolabelled material almost completely evaporated following heating of the sections. However, the observation that the radioactivity in the C S F and bile was non-volatile implies the presence of HCB metabolites in these fluids. The radiaoctivity in the C N S of triCB exposed cods was due to unmetabolized triCB, whereas part of the radioactivity in the C S F was due to triCB metabolites (fig. 4). In addition. also triCB was present in the CSF. The CSF represents an hitherto unknown site of enrichment for xenobiotics in cod. The radioactivity in the CSF of HCB or triCB-exposed cod may be due to hepatic metabolites that have passed the blood brain barrier or to metabolites formed in the CNS and subsequently translocated into the CSF. In order to investigate the latter possibility, the activity of xenobiotic metabolizing enzymes in the CNS of cod and rainbow trout was compared to the metabolic activity of their livers. The results showed that the CNS of both species contained cytochrome P-450dependent ECOD activities. Compared with the microsomal liver values measured in the two fish species the activities in brain mitochondria and microsomes were low. The presence of cytochrome P-450dependent xenobiotic metabolism in the brain mitochondrial fraction has previously been shown in mammalian studies (Iscan et al. 1990), and the cod and rainbow trout brain mitochondrial and microsomal ECOD activities were
K. INGEBRIGTSEN ET A L
424
A
counts 10
10 -
B
5
5-
application zone 1
2
4
I
substrate
0
6
8
10
,
1
1
12
14
cm
D
counts 10 -
L
counts
substrate
amlication zone
1
5-
application zone
substrate
1I -
application zone
substrate
Fig. 5. Profiles of thin layer chromotograms from extracts of incubations with ''C-2,4',5-trichlorobiphenyl.A: Blank. B: Cod liver microsomes. C: Cod brain mitochondria. D:Cod brain microsomes. Note corresponding peaks in B. C and D.
on the same level as those reported for corresponding rat brain fractions (Naslund et a/. 1988). Furthermore, an UDP-glucuronosyltransferase activity was recorded in the brain of both species. Although the activities were low compared to the microsomal liver values, they were in the same order of magnitude as those previously reported for rat cerebral microsomal UDP-glucuronosyltransferase activities (Ghersi-Egea 1987). Finally, the incubation experiments revealed that brain homogenates from both species were able to transform triCB to polar metabolites. It cannot be excluded that HCB and triCB are metabolized in the CNS of cod, and that the metabolites are secreted into the CSF. Noteworthy, the levels of both HCB and triCB in the CNS of rainbow trout were low, and the lack of radioactivity in the CSF of rainbow trout may therefore be due to a low level of Substrate for the xenobiotic metabolizing enzymes in this tissue. The observation that part of the radioactivity in the CSF of triCB dosed cods was due to unmetabolized triCB is difficult to interprete, since the lipophilic triCB is not expected to be present in a water phase unless it was bound
to a watersoluble carrier molecule (Brandt et d.1982). An intriguing possibility is that the enrichment of unmetabolized triCB as well as triCB- and HCB-derived metabolites was due to the presence of a transport protein in the CSF. It has recently been reported that several xenobiotics including HCB and its hydroxylated metabolite pentachlorophenol together with certain PCBs and PCB-derived metabolites bind to the thyroid hormone transport protein, transthyretin, in rodents (Van den Berg et a/. 1991). Furthermore, epithelial cells in the choroid plexus of rodents and man are highly specialized for the synthesis of transthyretin which is subsequently released into the C S F (Dickson et al. 1987). Whether thyroid hormone transport proteins are present in the C S F of fish, however, is not known. In conclusion, the present study supports previous observations that chlorinated hydrocarbons are extensively accumulated in the CNS of cod, whereas only traces are taken up in the CNS of rainbow trout. The results also imply that, in the cod, HCB and triCB are accumulated as unmetabolized compound in the C N S and that there is an enrichment of HCB- and triCB-derived metabolites in the CSF. These
HEXACHLOROBENZENE METABOLITES IN C O D CSF
interspecies differences in the disposition of chlorinated hydrocarbons implicate that cod may be more vulnerable than rainbow trout as concerns neurotoxicological effects of such compounds.
References Andersson. T.. M. Pesonen & C. Johansson: Differential induction of cytochrome P-450-dependent monooxygenase, epoxide hydrolase, glutathione transferase and UDP-glucuronosyltransferase activities in the liver of rainbow trout by b-naphthoflavone or Clophen A50. Biochem. Phurmucol. 1985, 34, 3309-3314. Appelgren. L.-E.: Sites of steroid hormone formation. Autoradiographic studies using labelled precursors. Aera Phjsiol. Srand 1967, Suppl. 301, I 108. Bakke. J. E.. V. J. Feil & A. Bergman: Metabolites of 2.4',5-trichlorobiphenyl in rats. Xenohiofiea 1983. 13, 555-564. Bergman. A. & C. A. Wachtmeister: Synthesis of ('4C)biphenyl and of some chlorinated ( I4C)biphenylscontaining 4-chloro-, 2,4dichloro- and 2,3,6-trichlorophenyl nuclei from the corresponding labelled anilines. Chernospherr 1977, 11, 759-767. Brandt. I., P. 0. Darnerud, A. Bergman & Y. Larsson: Metabolism of ?.4'.5-trichlorobiphenyl: Enrichment of hydroxylated and methyl sulphone metabolites in the uterine luminal fluid of pregnant mice. Chem. Biol. Interactions 1982. 40, 45 56. Dickson. P. W.. A. R. Aldred, J. G. T. Menting, P. D. Marley, W. H.Sawyer & G . Schreiber: Thyroxine transport in choroid plexus. J. Biol. Cheni. 1987, 262. 13907-1 39 15. F6rlin. L. & T. Hansson: Effects of treated municipal wastewater on the hepatic xenobiotic and steroid metabolism in trout. Eeotosieol. Environm. Sufetv 1982. 6. 41 4 8 . Ghersi-Egea, J. F., B. Walther, D. Decolin, A. Minn & G. Siest: The activity of I-naphthol-UDP-glucuronosyltransferase in the brain. Neurophtrrmticolog~1987, 26, 367-372. Greenlee, W. E & A. Poland: An improved assay of7-cthoxycoumarin-0-deethylase activity: Induction of hepatic enzyme activity in C57BLi6J and DBA/2A mice by phenobarbital, 3-methylcholJ . Pharmacol. antrene and 2,3.7.8-tetrachlorodibenzo-p-dioxin. E.rp. Therap. 1978, 205. 596 605. Hektoen, H., K. Ingebrigtsen, E. M. Brevik & M. Oehme: Interspecies differences in tissue distribution of 2,3,7,8-tetrachlorodibenz-
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o-p-dioxin between cod (Gadus morhua) and rainbow trout (Oncorhynchus mykiss). Chemosphere 1992, 24, 58 1-587. Ingebrigtsen, K., H. Hektoen, T. Andersson. A. Bergman & 1. Brandt: Species-specific accumulation of the polychlorinated biphenyl (PCB) 2,3,3',4,4'-pentachlorobiphenyl in fish brain: A comparison between cod (Gadus morhua) and rainbow trout (Oncorhynchus mykiss). Phnrtnarology & Tosicolog~, 1990, 67, 344345. Ingebrigtsen, K. & I. Nafstad: Distribution and elimination of "Chexachlorobenzene after single oral exposure in the male rat. Acta pharmacol. et toxicol. 1983, 52, 254260. Ingebrigtsen, K. & J. U. Skaare: Distribution and elimination of ''C-hexachlorobenzene after single oral exposure in the rainbow trout (Sulmo gairdneri). J . Toxicol. Environ. Hculth 1983, 12, 309-3 16. Ingebrigtsen, K. & J. E. Solbakken: Distribution and elimination of ''C-hexachlorobezene after single oral exposure in cod (Cadus morhua) and flounder (Platichthys flesus). J. Touicol. Environ. Health 1985, 16, 179-205. Ingebrigtsen, K., J. E. Solbakken, G . Norheim & 1. Nafstad: Distribution and elimination of ''C-octachlorostyrene i n cod (Gadus morhua), rainbow trout (Salmo gairdneri) and blue mussel (Myti/us edulis). J. Toxieol. Environ. Health. 1988. 25, 361-372. Iscan, M., K. Reuhl, B. Weiss & M. D. Maines: Regional and subcellular distribution of cytochrome P-450-dependent drug metabolism in monkey brain: The olfactory bulb and the mitochondrial fraction have high levels of activity. Biochemical. Biophysical. Res. Commun. 1990, 169. 858-863. Klasson-Wehler, E., B. Kowalski. A. Bergman & I. Brandt: Metabolism of 2,3,4',6-tetrachlorobiphenyl:Formation and tissue localization of mercapturic acid pathway metabolites in mice. Xenohiotica 1987, 17, 471486. Naslund, B. M. A,, H. Galumann, M . Warner, J.-A. Gustafsson & T. Hansson: Cytochrome P450 b and c in the rat brain and pituitary gland. Mol. Pharmacol. 1988, 33, 31-37. Ullberg, S.. Studies on the distribution and fate of "S-labelled benzylpenicillin in the body. Acta Radiol. 1954. Suppl. 118, 1-1 10. Ullberg, S.:The technique of whole body autoradiography. Cryosectioning of large specimens. Science Tools, Special issue on whole body autoradiography. The LKB Instrument Journal. Stockholm, Sweden 1977, 1-29. van den Berg, K. J., J. A. G . M. van Raaij, P. C. Bragt & W. R. F. Notten: Interactions of halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in vivo. Arch. Toxicol. 1991, 65, 15-19.
