Chem.-Biol. Interactions, 79 (1991) 79--89 Elsevier ScientificPublishers Ireland Ltd.
79
EFFECT OF VARIOUS CHEMICALS ON THE ALDEHYDE DEHYDROGENASE ACTIVITY OF THE RAT LIVER CYTOSOL
MARIOS MARSELOSand VASILIS VASILIOU Department of Pharmacology, Medical School. University of loannin~l. P.O. Box 1186. GR-45110 loannina (Greece)
(Received October 27th, 1990) (Revision received March 8th, 1991) (Accepted March 15th, 1991)
SUMMARY
The cytosolic activity of aldehyde dehydrogenase (ALDH) was studied in the rat liver, after acute administration of various carcinogenic and chemically related compounds. Male Wistar rats were treated with 27 different chemicals, including polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, azo dyes, as well as with some known direct-acting carcinogens. The cytosolic ALDH activity of the liver was determined either with propionaldehyde and NAD (P/NAD), or with benzaldehyde and NADP (B/NADP). The activity of ALDH remained unaffected after treatment with 1-naphthylamine, nitrosamines and also with the direct-acting chemical carcinogens tested. On the contrary, polycyclic aromatic hydrocarbons, polychlorinated biphenyls (Arochlor 1254) and 2-naphthylamine produced a remarkable increase of ALDH. In general, the response to the effectors was disproportionate between the two types of enzyme activity, being much in favour for the B/NADP activity. This fact resulted to an inversion of the ratio B/NADP vs. P/NAD, which under constitutive conditions is lower than 1. In this respect, the most potent compounds were found to be polychlorinated biphenyls, 3-methylcholanthrene, benzo(a)pyrene and 1,2,5,6-dibenzoanthracene. Our results suggest that the B/NADP activity of the soluble ALDH is greatly induced after treatment with compounds possessing aromatic ring(s) in their molecule. It is not known, if this response of the hepatocytes is related with the process of chemical carcinogenesis.
K e y w o r d s : Aldehyde dehydrogenase -- Induction -- Carcinogens -- Liver -- Rat Correspondence to: M. Marselos, Professorof Pharmacology,Departmentof Pharmacology.,Medical
School, GR-451 10 Ioannina, Greece. 0009-2797/91/$03.50 © 1991 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
80 INTRODUCTION
The enzyme aldehyde dehydrogenase (ALDH, EC 1.2.1.3) is detected in the mitochondrial, the microsomal and the cytosolic fraction of the rat liver [1,21. Despite the fact that only a small part of the total ALDH activity is confined to the cytosol, there are at least three cytosolic isozymes which can be induced by a variety of xenobiotics [3--11]. Phenobarbital and also other type I inducers of the microsomal drug metabolism can increase the activity of two different ALDH isozymes, which have distinct kinetic properties [7,81. One of these two isozymes (~-isozyme) is under genetic control and can be induced only in selected strains and substrains of rats, designated as RR animals [1,3,6]. In addition to these two isozymes, another cytosolic isozyme (r-ALDH, ALDH 3) [9] has been found to get induced in all rat strains tested, after treatment with polycyclic aromatic hydrocarbons, but not with phenobarbital [10,11]. All inducible isozymes are different proteins, in terms of substrate preference, use of coenzyme, sensitivity to inhibitors, and several other physicochemical or immunochemical properties [5,11--13]. Conventionally, the measurement of the ALDH activity induced with phenobarbital is carried out with propionaldehyde and NAD (P/NAD activity), whereas benzaldehyde and NADP are used for ALDH 3 (B/NADP activity). Induction of ALDH 3 by 3-methylcholanthrene has been demonstrated also in cultures of a human hepatoma cell line (HepG2), and in primary cultures of human and rat hepatocytes [14,15]. Relatively high levels of ALDH 3 activity could be detected in some chemically initiated hepatomas, a fact that explains the term 'tumour-ALDH' (r-ALDH) earlier applied for this isozyme [16--181. The aim of the present study was to examine the possible expression of ALDH 3 activity in the rat liver, after treatment with various known chemical carcinogens. Other substances with comparable structure were also tested, although they do not possess carcinogenic properties. The animals used in the present series of experiments were Wistar rats of the rr-substrain, which has been shown to lack any activity of the possibly interfering ~-isozyme [111. MATERIALS AND METHODS
Chemicals All chemicals used were reagent grade. Pyrazole was obtained from Fluka (W. Germany), propionaldehyde and benzaldehyde from Ferak (W. Germany), butadiene epoxide, ethylene dibromide, 4-dimethyl-aminoazobenzene and 2-propionolactone from Aldrich (U.S.A.). The nitroso-compounds were obtained from Serva (France). All other chemicals were obtained from Sigma Chemical Co. (U.S.A.). Treatment of the animals Male albino rats (weighing 200--250 g) of the Wistar/Mol/Io/rr substrain were used, originating from the Animal Center of the University of Kuopio (Finland).
81
These inbred animals have been separated, according to their genetically determined ability to respond to phenobarbital treatment, as far as the induction of ALDH is concerned [6]. In the present study, we used only animals of the nonresponsive group. The rats were kept in plastic cages (Makrolon) with bedding of wood (Populus sp.) and they had free access to tap water and pelleted chow (Elviz, Greece). All xenobiotics were administered by intraperitoneal injection, and the animals were killed 24 h after the last injection. The chemicals were dissolved in olive oil or saline, and they were injected in volumes ranging from 0.5 to 4 ml, depending on their solubility. Experimental and control animals were divided in groups of six. The same volume of the vehicle was injected in the respective controls (for details see Table I). This study comprised a large number of experiments over a long period of time, and therefore it was necessary to minimize all possibilities of variation in the experimental procedures. The rats were acclimatized in the animal house at least 1 month before the initiation of the treatment, and they were killed at the same time in the morning. Fluorescent light was on for 12 h, and the temperature was 20--250C. The preparation of the hepatic samples and the measurement of the enzyme activities, were performed by identical techniques.
Preparation of the cytosolic fraction After killing the rats by decapitation, the livers were homogenized in 3 vols. of ice-cold 0.25 M sucrose solution. The homogenate was first spun at 10 000 x g for 30 min. An equal volume of 0.024 M CaC12 in 0.25 M sucrose was added to the supernatant. The diluted supernatant was stirred and left to stand on ice for 10 min. The microsomal fraction was sedimented by centifugation at 10 000 x g for 30 rain [19]. The soluble fraction was used for the assays. Aldehyde dehydrogenase was measured at 37°C, by following the reduction of NAD(P) to NAD(P)H at 340 nm in a Hitachi 100--80A spectrophotometer. The reaction cuvette contained sodium pyrophosphate buffer (0.075 M, pH 8.0) i mM pyrazole (to inhibit alcohol dehydrogenase), 1 mM NAD and 5 mM propionaldehyde (P/NAD activity). For measuring the B/NADP activity, the substrate was benzaldehyde (5 raM) and the coenzyme was NADP (2.5 mM). In either enzyme assay, the reaction was started by adding the substrate after a 5-rain preincubation. A blank was run without the substrate [15]. Protein determination was carried out with the biuret method [20] using bovine serum albumin as the standard. Statistical analysis was performed by Student's t-test. RESULTS
No gross signs of toxicity, such as unwashed fur, haemorrhage, decreased mobility or decreased body temperature, were observed in any of the animal groups. The compounds used have been classified into four groups, according to their chemical structure or their possible implication in the process of chemical carcinogenesis (Table I) [21,22].
82 TABLE I TREATMENT OF THE ANIMALS Compound
Dose (mg/kg)
Administration (vehicle)
Duration (days)
Benzantbracene 1,2,3,4,-Dibenzanthracene 1,2,5,6,-Dibenzanthracene Benzo[a]pyrene Dimetylbenzanthracene 3-Methylcholanthrene Chrysene
(BA) (DBA1) (DBA2) (BP) (DMB) (MC) (CHR)
80 80 80 80 80 80 80
i.p. i.p. i.p. i.p. i.p. i.p. i.p.
(oil) (oil) (oil) (oil) (oil) (oil) (oil)
4 4 4 4 4 4 4
2-Acetylaminofluorene 1-Naphthylamine 2-Naphthylamine Dimethylnitrosamine Diethylnitrosamine Carbon tetrachloride Dimethylaminoazobenzene
(AAF) (1-NAF) (2-NAF) (DMN) (DEN) (CLTL) (DAMB)
80 80 80 10 10 5 100 10
i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p.
(oil) (oil) (oil) (NaCI) (NaCl) (oil) (oil) (oil)
4 4 4 3 3 3 4 4
Aflatoxin B1 Uretbane D-Ethionine Dietylstilboestrol Cyclophosphamide Chlorophenothane Arochlor 1254
(AFB) (UR) (D-ETH) (DES) (CPA) (DDT) (AR 1254)
80 100 100 100 30 100 250
i.p. i.p. i.p. i.p. i.p. i.p. i.p.
(oil) (NaCI) (NaCI) (oil) (NaCI) (oil) (oil)
4 4 4 4 4 4 1
/3-Propionolactone Butadiene epoxide Ethylene dibromide N-Methyl-N'-nitronitrosoguanidine N-Methylnitrosourea N-Methylnitrosourethane
(~-PRL) (BUEP) (ETHDB),
50 l0 20
i.p. (NaCI) i.p. (NaCI) !.p. (oil)
4 4 7
(NMNGU) (NMNU) (NMNUT)
20 20 20
i.p. (NaCl) i.p. (NaC1) i.p. (NaC1)
7 7 7
Polycyclic aromatic hydrocarbons (PAHs) With propionaldehyde as the s u b s t r a t e and N A D as the coenzyme (P/NAD), a 2-, 6- and 8-fold increase in the ALDH activity was detected in the animals treated with BA, DMBA and DBA1, respectively (Table II). Treatment with DBA2, BP and MC produced a 20-, 30- and 40-fold increase in the P/NAD ALDH activity. When ALDH was measured with benzaldehyde and NADP (B/NADP), the elfects were considerably more pronounced, especially with DBA2, BP and MC which produced an induction of 300-, 400- and 500-fold, respectively.
83
Control animals had only traces of the B/NADP activity, so that the ratio of the two constitutive activities was found to be lower than 1 (B/NADP versus P/NAD). However, after administration of PAHs, the B/NADP activity showed a dramatic increase which led to the inversion of this ratio (Table II). T A B L E II E F F E C T OF V A R I O U S C H E M I C A L S ON T H E CYTOSOL
A L D H A C T I V I T Y OF T H E
RAT L I V E R
*Significantly d i f f e r e n t controls (P < 0.001). The symbols a re in Table I. A L D H a c t i v i t y a (nmol NAD(P)H/min pe r m g protein) P/NAD
B/NADP
Control BA DBA1 DBA2 DMB BP MC CHR
7,2 29.4 47.3 150.3 14.8 239.0 280.7 18.9
± -~ ± ± ± ± ± ±
1.2 2.5" 4.1" 20.4* 1.0" 30.4* 40.2* 2.5*
Control AAF 1-NAF 2-NAF DMN DEN
7.4 11.8 6.9 15.7 7.5 6.4
± ± ± ± ~±
Control CTCL DAMB (100 m g / k g ) (10 m g / k g )
7.8 5.8 6.9 10.0
Control AFB UR D-ETH DES CPA DDT AR-1254 Control /3-PRL BUEP ETHDB NMNGU NMNU NMNUT
R
1.2 74.0 78.0 423.0 33.2 453.7 552.5 74.0
+ ± :e ± :~ :~ :e ±
0.3 14.7" 9.6* 27.9* 5.9* 50.0* 55.4* 14.7"
0.17 3.91 1.66 2.82 2.24 1.75 1.95 3.91
1.8 2.3 2.2 1.6" 0.8 0.8
1.1 8.2 1.0 36.4 0.8 1.3
:e + :e ± :e ±
0.4 0.5* 0.2 6.1" 0.2 0.4
0.15 0.60 0.14 2.32 0.11 0.20
± ± ± ±
0.2 0.6* 0.6* 0.7
1.2 1.2 1.6 19.0
± ± ± ±
0.1 0.4 0.5 1.9"
0.15 0.21 0.23 1.90
8.6 6.8 7.6 6.8 9.7 6.9 8.3 155.0
+ ± ± :e ± ± ± ~:
1.3 2.1 0.4 1.2 1.4 2.6 2.1 19.1"
2.0 1.5 1.3 0.8 2.4 1.3 7.5 709.0
± ± ~± ± ± ± ±
0.4 0.4 0.5 0.4 0.8 0.6 1.2" 132.0"
0.23 0.22 0.17 0.12 0.22 0.19 0.90 4.57
8.2 7.6 10.2 9.0 7.6 10.4 7.8
± ± ± ± ± + +
1.5 0.3 0.3 1.0 0.8 0.2 1.3
+ ± + ± ± ± +
0.5 0.4 0.7* 0.2 0.3 0.3 0.6
0.19 0.12 0.79 0.12 0.18 0.13 0.12
1.6 0.9 8.5 1.1 1.4 1.4 0.9
aEach value r e p r e s e n t s the m e a n ± S.D. of 6 rats. R, t he ratio of B / N A D P v e r s u s P / N A D activity.
84
Aromatic amines and nitrosamines The P/NAD enzyme activity was slightly increased ( x 2) in the animals treated with 2-NAF. More significant was the induction of ALDH, when measured as B/NADP activity, after treatment with A A F ( x 7) or 2-NAF ( x 30). However, inversion of the ratio of the two activities (P/NAD versus B/NADP), like the one observed after PAHs-treatment, was detected only in the animals treated with 2-NAF (Table II). Direct-acting carcinogens From the direct-acting carcinogenic compounds tested in this study (Table II), only BUEP produced an increase ( x 7) of ALDH when measured as B/NADP activity. Nevertheless, in absolute values of enzyme activity this increase was not high enough to bring about an inversion of the ratio B/NADP versus P/NAD, which thus remained lower than 1. TABLE iII A C T I V I T I E S OF T H E L I V E R C Y T O S O L I C A L D H IN U N T R E A T E D AND P A H - T R E A T E D RATS A S S A Y E D W I T H D I F F E R E N T S U B S T R A T E S a
Substrate
A L D H activity ~, (nmol NAD(P)H/min per mg protein)
Concentration
(mM) Control
Experimental
Formaldehyde
1
NAD NADP
Propionaldehyde
0.05
NAD NADP NAD NADP
5.3 3.0 10.2 7.4
± ± ± ±
1.0 0.8 1.0 1.8
5.2 6.8 220.5 172.0
± ± ± ±
0.3 0.5* 5.4* 16.0"
NAD NADP NAD NADP
4.6 1.3 6.6 2.8
± ± ± ±
0.3 0.5 0.8 1.0
483.3 609.3 539.0 819.5
± ± ± ±
25.4* 83.6* 33.7* 96.5*
5
Benzaldehyde
1 5
9.2 ± 2.2 1.3 ± 0.7
11.6 ± 1.0 2.5 ± 0.7
D-Glucuronolactone
50
NAD NADP
1.8 ± 0.3 0.9 ± 0.1
1.8 ± 0.6 1.2 ± 0.4
Furaldehyde
50
NAD NADP
3.4 ± 0.5 2.6 ± 0.6
172.0 ± 22.5* 284.3 ± 45.5*
4-Carboxybenzaldehyde
1
NAD NADP
6.9 ± 2.1 5.9 ± 1.3
10.9 ± 0.2 9.8 ± 0.9*
Phenylaeetaldehyde
1
NAD NADP
5.4 + 0.8 3.8 ± 0.4
11.7 + 0,2" 7.8 + 1.0"
a3-Methyleholanthrene was given to rats (100 m g / k g , i.p. x 4) as a typical P A H inducer of the A L D H activity, bEach value represents the mean ± S.D. of 6 rats. *Significantly different from controls (P < 0.001).
85
Miscellaneous agents Treatment with a mixture of polychlorinated biphenyls (Arochlor 1254) caused a significant induction of the ALDH activity, measured as P/NAD ( x 15) or as B/NADP (x 350). On the contrary, the hepatotoxic agents CTCL and DAMB caused a slight but statistically significant inhibition of the P/NAD activity (20%, P < 0.001). When a 10-fold lower dose of DAMB was used, an increase of the B/NADP activity ( x 15) was obtained.
Oxidizing properties of the ALDH, after treatment with PAHs Different substrates were used for investigating the oxidizing capacity of ALDH, under normal conditions and also after treatment with methylcholanthrene (Table III). With propionaldehyde, a 25-fold induction could be detected with either NAD or NADP, at a final substrate concentration of 5 raM. When a micromolar concentration of propionaldehyde was used, only a 2-fold increase in the activity of ALDH was found in the treated animals. The induction with methylcholanthrene could not be detected at all, when the substrates were formaldehyde (1 raM) or D-glucuronolactone (50 mM). A rather poor measurement of the induced ALDH can be achieved with 1 mM concentration of the substrates phenyl acetaldehyde and 4-carboxybenzaldehyde. The highest enzyme activities could be found with benzaldehyde as the substrate ( x 100 with NAD and x 450 with NADP). Also furaldehyde proved to be a rather satisfactory substrate, at the concentration used (50 raM), with either NAD ( x 50) or NADP (x 100) as the coenzyme. DISCUSSION
Induction of the activity of ALDH in the hepatic cytosol was first shown by Deitrich [3] after administration of phenobarbital. Soon after this initial observation, it was found that also TCDD and the PAHs can greatly increase the activity of another hepatic isozyme of ALDH in the rat cytosol (r-isozyme or ALDH 3), which is almost undetectable under normal conditions [4,5,23,24]. This ALDH activity is different from the one induced by phenobarbital and it can be best measured with benzaldehyde as the substrate and NADP as the coenzyme [8,24]. The results presented here confirm these earlier findings and show that the inducible ALDH 3 can be easily detected also by using furaldehyde as the substrate, whereas carboxybenzaldehyde and phenylacetaldehyde seem to be very poor substrates. In addition, the B/NADP activity was found to be similarly increased by the polycyclic aromatic hydrocarbons DBA1, DBA2 and DMB, by the aromatic amine 2-NAF, the aromatic azo dye DMAB and a mixture of polychlorinated biphenyls (Arochlor 1254). An overall enhancement of the ALDH activity may be the response of hepatocytes to several xenobiotics, which act either like phenobarbital or like methylcholanthrene and produce a rather preferential effect on the P/NAD or the B/NADP activity. Therefore, it is feasible to classify various xenobiotics with inducing properties by measuring both ALDH activities and then examining the ratio B/NADP versus P/NAD. Although for the phenobarbital type of inducers this ratio is lower than 1, an inversion is produced with methylcholanthrene and
86
other aromatic compounds. This phenomenon ('ALDH-inversion') has been observed also in vitro, in cultures of HepG2 cells or in primary cultures of human hepatocytes [14]. A slight increase of the hepatic B/NADP activity can be detected in the rat also after treatment with phenobarbital (15,25), which is negligible compared to the effect of PAHs. A similar result was obtained in the present series of experiments, after administration of AAF, DDT and BUEP. In comparison with MC and other PAHs, however, all these agents are causing only a minor increase of the B/NADP activity, which remains much lower than the respective increase of the P/NAD activity. As a result of this, the ratio B/NADP versus P/NAD remains lower than 1, when type I inducers are used. It is not possible to rule out the putative presence of additional inducible isoforms of ALDH. After treatment with phenobarbital, there is at least one more isozyme that exhibits an almost 3-fold increase of activity in all animals tested, particularly at micromolar substrate concentrations [7,8]. This issue was not further addressed in this series of experiments, because such minor activity changes were not expected to affect significantly the dramatic induction achieved by PAHs. A closer examination of Table Ill, however, may suggest an overlapping of induced activities, especially when measured with benzaldehyde or furaldehyde as the substrates. A similar multiple expression of inducible isozymes has been observed with UDP-glucuronosyltransferase, and the overlapping enzyme activity could be assessed with a simple mathematical model [26]. High doses of DAMD, as well as CTCL, led to a slight but significant decrease in the P/NAD activity, most probably as a result of a diffuse hepatotoxic action [21]. However, this response is rather unspecific, since it could not be detected after administration of AFB, DEN or DMN, which are also known to exert toxic effects on the hepatocytes [27]. Despite the rather large number of chemicals which can increase or decrease the activity of ALDH, the expression of this enzyme in the rat is not changed in the proliferating hepatic tissue, in cholestatic liver injury, or after surgical sympathectomy of the liver [28]. A strong correlation has been demonstrated between the degree of ALDH 3 induction and the mutagenic potency of PAHs, as determined by the Ames~Salmonella test [29]. The role of this isozyme in the normal metabolic pathways of the liver is generally unclear, since none of the known endogenous aldehydic compounds has an affinity for ALDH 3 high enough to furnish satisfactory evidence for a possible participation in the physiology of the hepatocyte [30]. However, it may be involved in certain pathophysiological processes, as suggested by the relationship between an increase in this type of ALDH activity and some experimental protocols of chemically initiated hepatocarcinogenesis [16--18]. The foreign compounds inducing the cytosolic enzyme ALDH 3 have at least one aromatic ring in their molecule. Since they do not possess any aldehydic group, they cannot be considered as direct substrates for ALDH. A single chemical property can be the common denominator in the inducing capability of a large and seemingly heterogeneous group of substances, as has been shown in the induction of quinone reductase [31]. It is not clear whether there is also a common
87
factor in the action of the inducing compounds detected by us in the present study. Triggering the production of endogenous mono- and dialdehydes, through lipid peroxidation, cannot be excluded as a possible mechanism. However, these aldehydes are rather poor substrates for ALDH 3, as has been found in relevant kinetic studies [13,30]. It has been suggested that benzopyrenes form 4,5-dihydrodiols and 4,5-dialdehydes as intermediate products during their epoxidation [32]. If this happens also with other PAHs, the increase of the ALDH activity can be considered as a result of substrate induction. It is worth noticing that MC, given to rats in vivo [8,24] or applied to hepatocyte cultures in vitro [14], induces the B/NADP activity after a lag time of about 3 days. Also, the effect of MC is blocked by inhibitors and enhanced by inducers of the mixed-function oxidase [14,33]. These observations support the hypothesis that the eventual inducer of ALDH 3 is a metabolite of MC. The exact chemical structure and the possible biological properties of this metabolite remain unknown. A similar substrate-induction has been detected in the case of UDP-glucuronosyltransferase, by using eugenol [34]. We have already proposed that ALDH can be used as a sensitive bioassay for the exposure to certain types of xenobiotics [23]. The present results show that this is true for substances with aromatic rings, and especially for the polycyclic aromatic hydrocarbons. On the other hand, the interrelationship of the B/NADP and the P/NAD activity provides a simple and quick method for discriminating between type I and type II inducers of the microsomal drug metabolizing enzymes. There is also some evidence that a decrease of the P/NAD activity may reflect a hepatotoxic effect depending on the chemical used, or the dose schedule applied. ACKNOWLEDGMENTS
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31
32 33 34
V. Vasiliou, K. Athanasiou and M. Marselos, The use of ALDH induction as a carcinogenic risk marker in comparison with a typical in vitro mutagenicity system, Proc. Advanced Research Workshop on the "Biologically Based Methods for Cancer Risk Assessment", pp. 231--240, C.C. Travis (Ed.), Plenum Press, New York and London, 1988. M. Marselos and R. Lindahl, Substrate preference of a cytosolic aldehyde dehydrogenase inducible in rat liver by treatment with 3-methylcholanthrene, Toxicol. Appl. Pharmacol., 95 (1988) 339--345. P. Talalay, M.J. de Long and H.J. Prochaska, Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis, Proc. Natl. Aead. Sci. U.S.A., 85 (1988) 8261--8265. M.R. Osborne and N.T. Crosby, Benzopyrenes, Cambridge University Press, Cambridge, 1987, pp. 52--72. V. Vasiliou ana M. Marselos, Changes in the inducibility of a hepatic aldehyde dehydrogenase by various effectors, Arch. Toxicol., 63 (1989) 221--225. J.A. Boutin, A.M. Batt and G. Siest, Effect of pretreatment with hydroxylated xenobiotics on the activities of rat liver UDP-glueuronosyltransferases, Xenobiotica, 13 (1983) 755--761.
79
EFFECT OF VARIOUS CHEMICALS ON THE ALDEHYDE DEHYDROGENASE ACTIVITY OF THE RAT LIVER CYTOSOL
MARIOS MARSELOSand VASILIS VASILIOU Department of Pharmacology, Medical School. University of loannin~l. P.O. Box 1186. GR-45110 loannina (Greece)
(Received October 27th, 1990) (Revision received March 8th, 1991) (Accepted March 15th, 1991)
SUMMARY
The cytosolic activity of aldehyde dehydrogenase (ALDH) was studied in the rat liver, after acute administration of various carcinogenic and chemically related compounds. Male Wistar rats were treated with 27 different chemicals, including polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, azo dyes, as well as with some known direct-acting carcinogens. The cytosolic ALDH activity of the liver was determined either with propionaldehyde and NAD (P/NAD), or with benzaldehyde and NADP (B/NADP). The activity of ALDH remained unaffected after treatment with 1-naphthylamine, nitrosamines and also with the direct-acting chemical carcinogens tested. On the contrary, polycyclic aromatic hydrocarbons, polychlorinated biphenyls (Arochlor 1254) and 2-naphthylamine produced a remarkable increase of ALDH. In general, the response to the effectors was disproportionate between the two types of enzyme activity, being much in favour for the B/NADP activity. This fact resulted to an inversion of the ratio B/NADP vs. P/NAD, which under constitutive conditions is lower than 1. In this respect, the most potent compounds were found to be polychlorinated biphenyls, 3-methylcholanthrene, benzo(a)pyrene and 1,2,5,6-dibenzoanthracene. Our results suggest that the B/NADP activity of the soluble ALDH is greatly induced after treatment with compounds possessing aromatic ring(s) in their molecule. It is not known, if this response of the hepatocytes is related with the process of chemical carcinogenesis.
K e y w o r d s : Aldehyde dehydrogenase -- Induction -- Carcinogens -- Liver -- Rat Correspondence to: M. Marselos, Professorof Pharmacology,Departmentof Pharmacology.,Medical
School, GR-451 10 Ioannina, Greece. 0009-2797/91/$03.50 © 1991 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
80 INTRODUCTION
The enzyme aldehyde dehydrogenase (ALDH, EC 1.2.1.3) is detected in the mitochondrial, the microsomal and the cytosolic fraction of the rat liver [1,21. Despite the fact that only a small part of the total ALDH activity is confined to the cytosol, there are at least three cytosolic isozymes which can be induced by a variety of xenobiotics [3--11]. Phenobarbital and also other type I inducers of the microsomal drug metabolism can increase the activity of two different ALDH isozymes, which have distinct kinetic properties [7,81. One of these two isozymes (~-isozyme) is under genetic control and can be induced only in selected strains and substrains of rats, designated as RR animals [1,3,6]. In addition to these two isozymes, another cytosolic isozyme (r-ALDH, ALDH 3) [9] has been found to get induced in all rat strains tested, after treatment with polycyclic aromatic hydrocarbons, but not with phenobarbital [10,11]. All inducible isozymes are different proteins, in terms of substrate preference, use of coenzyme, sensitivity to inhibitors, and several other physicochemical or immunochemical properties [5,11--13]. Conventionally, the measurement of the ALDH activity induced with phenobarbital is carried out with propionaldehyde and NAD (P/NAD activity), whereas benzaldehyde and NADP are used for ALDH 3 (B/NADP activity). Induction of ALDH 3 by 3-methylcholanthrene has been demonstrated also in cultures of a human hepatoma cell line (HepG2), and in primary cultures of human and rat hepatocytes [14,15]. Relatively high levels of ALDH 3 activity could be detected in some chemically initiated hepatomas, a fact that explains the term 'tumour-ALDH' (r-ALDH) earlier applied for this isozyme [16--181. The aim of the present study was to examine the possible expression of ALDH 3 activity in the rat liver, after treatment with various known chemical carcinogens. Other substances with comparable structure were also tested, although they do not possess carcinogenic properties. The animals used in the present series of experiments were Wistar rats of the rr-substrain, which has been shown to lack any activity of the possibly interfering ~-isozyme [111. MATERIALS AND METHODS
Chemicals All chemicals used were reagent grade. Pyrazole was obtained from Fluka (W. Germany), propionaldehyde and benzaldehyde from Ferak (W. Germany), butadiene epoxide, ethylene dibromide, 4-dimethyl-aminoazobenzene and 2-propionolactone from Aldrich (U.S.A.). The nitroso-compounds were obtained from Serva (France). All other chemicals were obtained from Sigma Chemical Co. (U.S.A.). Treatment of the animals Male albino rats (weighing 200--250 g) of the Wistar/Mol/Io/rr substrain were used, originating from the Animal Center of the University of Kuopio (Finland).
81
These inbred animals have been separated, according to their genetically determined ability to respond to phenobarbital treatment, as far as the induction of ALDH is concerned [6]. In the present study, we used only animals of the nonresponsive group. The rats were kept in plastic cages (Makrolon) with bedding of wood (Populus sp.) and they had free access to tap water and pelleted chow (Elviz, Greece). All xenobiotics were administered by intraperitoneal injection, and the animals were killed 24 h after the last injection. The chemicals were dissolved in olive oil or saline, and they were injected in volumes ranging from 0.5 to 4 ml, depending on their solubility. Experimental and control animals were divided in groups of six. The same volume of the vehicle was injected in the respective controls (for details see Table I). This study comprised a large number of experiments over a long period of time, and therefore it was necessary to minimize all possibilities of variation in the experimental procedures. The rats were acclimatized in the animal house at least 1 month before the initiation of the treatment, and they were killed at the same time in the morning. Fluorescent light was on for 12 h, and the temperature was 20--250C. The preparation of the hepatic samples and the measurement of the enzyme activities, were performed by identical techniques.
Preparation of the cytosolic fraction After killing the rats by decapitation, the livers were homogenized in 3 vols. of ice-cold 0.25 M sucrose solution. The homogenate was first spun at 10 000 x g for 30 min. An equal volume of 0.024 M CaC12 in 0.25 M sucrose was added to the supernatant. The diluted supernatant was stirred and left to stand on ice for 10 min. The microsomal fraction was sedimented by centifugation at 10 000 x g for 30 rain [19]. The soluble fraction was used for the assays. Aldehyde dehydrogenase was measured at 37°C, by following the reduction of NAD(P) to NAD(P)H at 340 nm in a Hitachi 100--80A spectrophotometer. The reaction cuvette contained sodium pyrophosphate buffer (0.075 M, pH 8.0) i mM pyrazole (to inhibit alcohol dehydrogenase), 1 mM NAD and 5 mM propionaldehyde (P/NAD activity). For measuring the B/NADP activity, the substrate was benzaldehyde (5 raM) and the coenzyme was NADP (2.5 mM). In either enzyme assay, the reaction was started by adding the substrate after a 5-rain preincubation. A blank was run without the substrate [15]. Protein determination was carried out with the biuret method [20] using bovine serum albumin as the standard. Statistical analysis was performed by Student's t-test. RESULTS
No gross signs of toxicity, such as unwashed fur, haemorrhage, decreased mobility or decreased body temperature, were observed in any of the animal groups. The compounds used have been classified into four groups, according to their chemical structure or their possible implication in the process of chemical carcinogenesis (Table I) [21,22].
82 TABLE I TREATMENT OF THE ANIMALS Compound
Dose (mg/kg)
Administration (vehicle)
Duration (days)
Benzantbracene 1,2,3,4,-Dibenzanthracene 1,2,5,6,-Dibenzanthracene Benzo[a]pyrene Dimetylbenzanthracene 3-Methylcholanthrene Chrysene
(BA) (DBA1) (DBA2) (BP) (DMB) (MC) (CHR)
80 80 80 80 80 80 80
i.p. i.p. i.p. i.p. i.p. i.p. i.p.
(oil) (oil) (oil) (oil) (oil) (oil) (oil)
4 4 4 4 4 4 4
2-Acetylaminofluorene 1-Naphthylamine 2-Naphthylamine Dimethylnitrosamine Diethylnitrosamine Carbon tetrachloride Dimethylaminoazobenzene
(AAF) (1-NAF) (2-NAF) (DMN) (DEN) (CLTL) (DAMB)
80 80 80 10 10 5 100 10
i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p.
(oil) (oil) (oil) (NaCI) (NaCl) (oil) (oil) (oil)
4 4 4 3 3 3 4 4
Aflatoxin B1 Uretbane D-Ethionine Dietylstilboestrol Cyclophosphamide Chlorophenothane Arochlor 1254
(AFB) (UR) (D-ETH) (DES) (CPA) (DDT) (AR 1254)
80 100 100 100 30 100 250
i.p. i.p. i.p. i.p. i.p. i.p. i.p.
(oil) (NaCI) (NaCI) (oil) (NaCI) (oil) (oil)
4 4 4 4 4 4 1
/3-Propionolactone Butadiene epoxide Ethylene dibromide N-Methyl-N'-nitronitrosoguanidine N-Methylnitrosourea N-Methylnitrosourethane
(~-PRL) (BUEP) (ETHDB),
50 l0 20
i.p. (NaCI) i.p. (NaCI) !.p. (oil)
4 4 7
(NMNGU) (NMNU) (NMNUT)
20 20 20
i.p. (NaCl) i.p. (NaC1) i.p. (NaC1)
7 7 7
Polycyclic aromatic hydrocarbons (PAHs) With propionaldehyde as the s u b s t r a t e and N A D as the coenzyme (P/NAD), a 2-, 6- and 8-fold increase in the ALDH activity was detected in the animals treated with BA, DMBA and DBA1, respectively (Table II). Treatment with DBA2, BP and MC produced a 20-, 30- and 40-fold increase in the P/NAD ALDH activity. When ALDH was measured with benzaldehyde and NADP (B/NADP), the elfects were considerably more pronounced, especially with DBA2, BP and MC which produced an induction of 300-, 400- and 500-fold, respectively.
83
Control animals had only traces of the B/NADP activity, so that the ratio of the two constitutive activities was found to be lower than 1 (B/NADP versus P/NAD). However, after administration of PAHs, the B/NADP activity showed a dramatic increase which led to the inversion of this ratio (Table II). T A B L E II E F F E C T OF V A R I O U S C H E M I C A L S ON T H E CYTOSOL
A L D H A C T I V I T Y OF T H E
RAT L I V E R
*Significantly d i f f e r e n t controls (P < 0.001). The symbols a re in Table I. A L D H a c t i v i t y a (nmol NAD(P)H/min pe r m g protein) P/NAD
B/NADP
Control BA DBA1 DBA2 DMB BP MC CHR
7,2 29.4 47.3 150.3 14.8 239.0 280.7 18.9
± -~ ± ± ± ± ± ±
1.2 2.5" 4.1" 20.4* 1.0" 30.4* 40.2* 2.5*
Control AAF 1-NAF 2-NAF DMN DEN
7.4 11.8 6.9 15.7 7.5 6.4
± ± ± ± ~±
Control CTCL DAMB (100 m g / k g ) (10 m g / k g )
7.8 5.8 6.9 10.0
Control AFB UR D-ETH DES CPA DDT AR-1254 Control /3-PRL BUEP ETHDB NMNGU NMNU NMNUT
R
1.2 74.0 78.0 423.0 33.2 453.7 552.5 74.0
+ ± :e ± :~ :~ :e ±
0.3 14.7" 9.6* 27.9* 5.9* 50.0* 55.4* 14.7"
0.17 3.91 1.66 2.82 2.24 1.75 1.95 3.91
1.8 2.3 2.2 1.6" 0.8 0.8
1.1 8.2 1.0 36.4 0.8 1.3
:e + :e ± :e ±
0.4 0.5* 0.2 6.1" 0.2 0.4
0.15 0.60 0.14 2.32 0.11 0.20
± ± ± ±
0.2 0.6* 0.6* 0.7
1.2 1.2 1.6 19.0
± ± ± ±
0.1 0.4 0.5 1.9"
0.15 0.21 0.23 1.90
8.6 6.8 7.6 6.8 9.7 6.9 8.3 155.0
+ ± ± :e ± ± ± ~:
1.3 2.1 0.4 1.2 1.4 2.6 2.1 19.1"
2.0 1.5 1.3 0.8 2.4 1.3 7.5 709.0
± ± ~± ± ± ± ±
0.4 0.4 0.5 0.4 0.8 0.6 1.2" 132.0"
0.23 0.22 0.17 0.12 0.22 0.19 0.90 4.57
8.2 7.6 10.2 9.0 7.6 10.4 7.8
± ± ± ± ± + +
1.5 0.3 0.3 1.0 0.8 0.2 1.3
+ ± + ± ± ± +
0.5 0.4 0.7* 0.2 0.3 0.3 0.6
0.19 0.12 0.79 0.12 0.18 0.13 0.12
1.6 0.9 8.5 1.1 1.4 1.4 0.9
aEach value r e p r e s e n t s the m e a n ± S.D. of 6 rats. R, t he ratio of B / N A D P v e r s u s P / N A D activity.
84
Aromatic amines and nitrosamines The P/NAD enzyme activity was slightly increased ( x 2) in the animals treated with 2-NAF. More significant was the induction of ALDH, when measured as B/NADP activity, after treatment with A A F ( x 7) or 2-NAF ( x 30). However, inversion of the ratio of the two activities (P/NAD versus B/NADP), like the one observed after PAHs-treatment, was detected only in the animals treated with 2-NAF (Table II). Direct-acting carcinogens From the direct-acting carcinogenic compounds tested in this study (Table II), only BUEP produced an increase ( x 7) of ALDH when measured as B/NADP activity. Nevertheless, in absolute values of enzyme activity this increase was not high enough to bring about an inversion of the ratio B/NADP versus P/NAD, which thus remained lower than 1. TABLE iII A C T I V I T I E S OF T H E L I V E R C Y T O S O L I C A L D H IN U N T R E A T E D AND P A H - T R E A T E D RATS A S S A Y E D W I T H D I F F E R E N T S U B S T R A T E S a
Substrate
A L D H activity ~, (nmol NAD(P)H/min per mg protein)
Concentration
(mM) Control
Experimental
Formaldehyde
1
NAD NADP
Propionaldehyde
0.05
NAD NADP NAD NADP
5.3 3.0 10.2 7.4
± ± ± ±
1.0 0.8 1.0 1.8
5.2 6.8 220.5 172.0
± ± ± ±
0.3 0.5* 5.4* 16.0"
NAD NADP NAD NADP
4.6 1.3 6.6 2.8
± ± ± ±
0.3 0.5 0.8 1.0
483.3 609.3 539.0 819.5
± ± ± ±
25.4* 83.6* 33.7* 96.5*
5
Benzaldehyde
1 5
9.2 ± 2.2 1.3 ± 0.7
11.6 ± 1.0 2.5 ± 0.7
D-Glucuronolactone
50
NAD NADP
1.8 ± 0.3 0.9 ± 0.1
1.8 ± 0.6 1.2 ± 0.4
Furaldehyde
50
NAD NADP
3.4 ± 0.5 2.6 ± 0.6
172.0 ± 22.5* 284.3 ± 45.5*
4-Carboxybenzaldehyde
1
NAD NADP
6.9 ± 2.1 5.9 ± 1.3
10.9 ± 0.2 9.8 ± 0.9*
Phenylaeetaldehyde
1
NAD NADP
5.4 + 0.8 3.8 ± 0.4
11.7 + 0,2" 7.8 + 1.0"
a3-Methyleholanthrene was given to rats (100 m g / k g , i.p. x 4) as a typical P A H inducer of the A L D H activity, bEach value represents the mean ± S.D. of 6 rats. *Significantly different from controls (P < 0.001).
85
Miscellaneous agents Treatment with a mixture of polychlorinated biphenyls (Arochlor 1254) caused a significant induction of the ALDH activity, measured as P/NAD ( x 15) or as B/NADP (x 350). On the contrary, the hepatotoxic agents CTCL and DAMB caused a slight but statistically significant inhibition of the P/NAD activity (20%, P < 0.001). When a 10-fold lower dose of DAMB was used, an increase of the B/NADP activity ( x 15) was obtained.
Oxidizing properties of the ALDH, after treatment with PAHs Different substrates were used for investigating the oxidizing capacity of ALDH, under normal conditions and also after treatment with methylcholanthrene (Table III). With propionaldehyde, a 25-fold induction could be detected with either NAD or NADP, at a final substrate concentration of 5 raM. When a micromolar concentration of propionaldehyde was used, only a 2-fold increase in the activity of ALDH was found in the treated animals. The induction with methylcholanthrene could not be detected at all, when the substrates were formaldehyde (1 raM) or D-glucuronolactone (50 mM). A rather poor measurement of the induced ALDH can be achieved with 1 mM concentration of the substrates phenyl acetaldehyde and 4-carboxybenzaldehyde. The highest enzyme activities could be found with benzaldehyde as the substrate ( x 100 with NAD and x 450 with NADP). Also furaldehyde proved to be a rather satisfactory substrate, at the concentration used (50 raM), with either NAD ( x 50) or NADP (x 100) as the coenzyme. DISCUSSION
Induction of the activity of ALDH in the hepatic cytosol was first shown by Deitrich [3] after administration of phenobarbital. Soon after this initial observation, it was found that also TCDD and the PAHs can greatly increase the activity of another hepatic isozyme of ALDH in the rat cytosol (r-isozyme or ALDH 3), which is almost undetectable under normal conditions [4,5,23,24]. This ALDH activity is different from the one induced by phenobarbital and it can be best measured with benzaldehyde as the substrate and NADP as the coenzyme [8,24]. The results presented here confirm these earlier findings and show that the inducible ALDH 3 can be easily detected also by using furaldehyde as the substrate, whereas carboxybenzaldehyde and phenylacetaldehyde seem to be very poor substrates. In addition, the B/NADP activity was found to be similarly increased by the polycyclic aromatic hydrocarbons DBA1, DBA2 and DMB, by the aromatic amine 2-NAF, the aromatic azo dye DMAB and a mixture of polychlorinated biphenyls (Arochlor 1254). An overall enhancement of the ALDH activity may be the response of hepatocytes to several xenobiotics, which act either like phenobarbital or like methylcholanthrene and produce a rather preferential effect on the P/NAD or the B/NADP activity. Therefore, it is feasible to classify various xenobiotics with inducing properties by measuring both ALDH activities and then examining the ratio B/NADP versus P/NAD. Although for the phenobarbital type of inducers this ratio is lower than 1, an inversion is produced with methylcholanthrene and
86
other aromatic compounds. This phenomenon ('ALDH-inversion') has been observed also in vitro, in cultures of HepG2 cells or in primary cultures of human hepatocytes [14]. A slight increase of the hepatic B/NADP activity can be detected in the rat also after treatment with phenobarbital (15,25), which is negligible compared to the effect of PAHs. A similar result was obtained in the present series of experiments, after administration of AAF, DDT and BUEP. In comparison with MC and other PAHs, however, all these agents are causing only a minor increase of the B/NADP activity, which remains much lower than the respective increase of the P/NAD activity. As a result of this, the ratio B/NADP versus P/NAD remains lower than 1, when type I inducers are used. It is not possible to rule out the putative presence of additional inducible isoforms of ALDH. After treatment with phenobarbital, there is at least one more isozyme that exhibits an almost 3-fold increase of activity in all animals tested, particularly at micromolar substrate concentrations [7,8]. This issue was not further addressed in this series of experiments, because such minor activity changes were not expected to affect significantly the dramatic induction achieved by PAHs. A closer examination of Table Ill, however, may suggest an overlapping of induced activities, especially when measured with benzaldehyde or furaldehyde as the substrates. A similar multiple expression of inducible isozymes has been observed with UDP-glucuronosyltransferase, and the overlapping enzyme activity could be assessed with a simple mathematical model [26]. High doses of DAMD, as well as CTCL, led to a slight but significant decrease in the P/NAD activity, most probably as a result of a diffuse hepatotoxic action [21]. However, this response is rather unspecific, since it could not be detected after administration of AFB, DEN or DMN, which are also known to exert toxic effects on the hepatocytes [27]. Despite the rather large number of chemicals which can increase or decrease the activity of ALDH, the expression of this enzyme in the rat is not changed in the proliferating hepatic tissue, in cholestatic liver injury, or after surgical sympathectomy of the liver [28]. A strong correlation has been demonstrated between the degree of ALDH 3 induction and the mutagenic potency of PAHs, as determined by the Ames~Salmonella test [29]. The role of this isozyme in the normal metabolic pathways of the liver is generally unclear, since none of the known endogenous aldehydic compounds has an affinity for ALDH 3 high enough to furnish satisfactory evidence for a possible participation in the physiology of the hepatocyte [30]. However, it may be involved in certain pathophysiological processes, as suggested by the relationship between an increase in this type of ALDH activity and some experimental protocols of chemically initiated hepatocarcinogenesis [16--18]. The foreign compounds inducing the cytosolic enzyme ALDH 3 have at least one aromatic ring in their molecule. Since they do not possess any aldehydic group, they cannot be considered as direct substrates for ALDH. A single chemical property can be the common denominator in the inducing capability of a large and seemingly heterogeneous group of substances, as has been shown in the induction of quinone reductase [31]. It is not clear whether there is also a common
87
factor in the action of the inducing compounds detected by us in the present study. Triggering the production of endogenous mono- and dialdehydes, through lipid peroxidation, cannot be excluded as a possible mechanism. However, these aldehydes are rather poor substrates for ALDH 3, as has been found in relevant kinetic studies [13,30]. It has been suggested that benzopyrenes form 4,5-dihydrodiols and 4,5-dialdehydes as intermediate products during their epoxidation [32]. If this happens also with other PAHs, the increase of the ALDH activity can be considered as a result of substrate induction. It is worth noticing that MC, given to rats in vivo [8,24] or applied to hepatocyte cultures in vitro [14], induces the B/NADP activity after a lag time of about 3 days. Also, the effect of MC is blocked by inhibitors and enhanced by inducers of the mixed-function oxidase [14,33]. These observations support the hypothesis that the eventual inducer of ALDH 3 is a metabolite of MC. The exact chemical structure and the possible biological properties of this metabolite remain unknown. A similar substrate-induction has been detected in the case of UDP-glucuronosyltransferase, by using eugenol [34]. We have already proposed that ALDH can be used as a sensitive bioassay for the exposure to certain types of xenobiotics [23]. The present results show that this is true for substances with aromatic rings, and especially for the polycyclic aromatic hydrocarbons. On the other hand, the interrelationship of the B/NADP and the P/NAD activity provides a simple and quick method for discriminating between type I and type II inducers of the microsomal drug metabolizing enzymes. There is also some evidence that a decrease of the P/NAD activity may reflect a hepatotoxic effect depending on the chemical used, or the dose schedule applied. ACKNOWLEDGMENTS
This work was supported by a grant from the Greek Ministry of Research and Technology. REFERENCES 1 R.A. Deitrich, Tissue and subcellular distribution of mammalian aldehyde-oxidizing capacity, Biochem. Pharmacol., 15 (1966) 1911--1922. 2 S.O.C. Tottmar, H. Pettersson and K.-H. Kiessling, The subcellular distribution and properties of aldehyde dehydrogenase in rat liver, Biochem. J., 135 (1973) 577--586. 3 R.A. Deitrich, Genetic aspects of increase in rat liver aldehyde dehydrogenase induced by phenobarbital, Science, 173 {1971) 334--336. 4 M. Marselos and O. H~nninen, Enhancement of D-glucuronolactone and aldehyde dehydrogenase activity in the rat liver by inducers of drug metabolism, Biochem. Pharmacol., 24 (1974) 1457--1466. 5 R.A. Deitrich, P. Bludeau, T. Stock and M. Roper, Induction of different rat liver supernatant aldehyde dehydrogenase by phenobarbital and tetrachlorodibenzo-p-dioxin, J. Biol. Chem., 252 (1977) 6169--6176. 6 M. Marselos, Genetic variation of drug metabolizing enzymes in the Wistar rat, Acta Pharmacol. Toxicol., 39 (1977) 186--197.
88 7 8
9
10 ll
12 13 14
15 16 17
18 19 20 21 22
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