The inducing and inhibiting effects of sodium valproate in vivo on the biotransformation systems of xenobiotics in isolated rat hepatocytes

1. The induction and inhibition of some biotransformation enzymes by valproate have been studied in hepatocytes isolated from rats treated with sodium valproate either i.p. or by subcutaneous implantation of osmotic pumps.

2. When valproate was given i.p., the cytochromes P-450 and b5, and aldrin epoxidase and glutathione S-transferase activities were significantly induced.

3. In contrast, valproate administered by osmotic pumps induced 7-ethoxycoumarin-O-deethylase activity, whereas aldrin expoxidase and glutathione S-transferase activities were significantly inhibited. At a valproate serum concentration of about 100 μg/ml for 2 weeks a significant induction of the cytochromes P-450 and b5 was observed.

4. Since there is a large difference between the half-lives of valproate in man and rodent, constant-rate delivery of valproate represents a better model for induction studies than i.p. injection.     

The inducing and inhibiting effects of sodium valproate in vivo on the biotransformation systems of xenobiotics in isolated rat hepatocytes

1. The induction and inhibition of some biotransformation enzymes by valproate have been studied in hepatocytes isolated from rats treated with sodium valproate either i.p. or by subcutaneous implantation of osmotic pumps. 2. When valproate was given i.p., the cytochromes P-450 and b5, and aldrin epoxidase and glutathione S-transferase activities were significantly induced. 3. In contrast, valproate administered by osmotic pumps induced 7-ethoxycoumarin-O-deethylase activity, whereas aldrin expoxidase and glutathione S-transferase activities were significantly inhibited. At a valproate serum concentration of about 100 micrograms/ml for 2 weeks a significant induction of the cytochromes P-450 and b5 was observed. 4. Since there is a large difference between the half-lives of valproate in man and rodent, constant-rate delivery of valproate represents a better model for induction studies than i.p. injection.     

Spectral and inhibitory interactions of methylenedioxyphenyl and related compounds with purified isozymes of cytochrome P-450

1. Spectral and inhibitory interactions of two methylenedioxyphenyl (MDP) compounds (dihydrosafrole (DHS) and 4,5-dichloro-1,2-methylenedioxybenzene (DCMB)) and 4-n-butyl dioxolane (BD) were studied in vitro in reconstituted systems incorporating cytochromes P-450b and P-450c, purified respectively from hepatic microsomes of phenobarbital (PB)- and β-naphthoflavone (βNF)-treated rats.

2. In NADPH-fortified reconstituted systems containing P-450b, DHS yielded a stable type III spectral complex with peaks at 428 and 458 nm; a complex with a single 456?nm peak was formed in systems containing cytochrome P-450c. DCMB formed unstable 456–458?nm spectral complexes with both isozymes, and BD generated an unstable complex with a single Soret peak near 428?nm with cytochrome P-450b; no spectral interaction occurred between BD and cytochrome P-450c. Carbon monoxide was formed in incubations of DCMB with both isozymes but was not observed with either DHS or BD.

3. Marked selectivity was observed in the ability of the test compounds to inhibit selected mono-oxygenase reactions in the reconstituted systems. Thus, while DHS was an effective inhibitor of cytochrome P-450b-mediated ethoxycoumarin O-deethylase (ECD), it failed to inhibit aldrin epoxidase (AE) in the same system; DCMB and BD inhibited both of these reactions. In reconstituted systems incorporating cytochrome P-450c, DHS and DCMB, but not BD, were effective inhibitors of ethoxyresorufin O-deethylase (ERD) activity but none of the compounds showed any inhibitory activity towards aryl hydrocarbon (benzo[a]pyrene)hydrolase (AHH) activity.

4. The results indicate that metabolite complex formation with cytochrome P-450 is not the sole criterion for inhibition of mono-oxygenase activity by MDF and related compounds, and that in some cases type I competitive interactions at the substrate binding sites may be the primary contributing factor.     

Differences in the biochemical properties of aldrin epoxidase, a cytochrome P-450-dependent monooxygenase, in various tissues

Aldrin epoxidase, a cytochrome P-450-dependent monooxygenase, was studied in the lung and kidney of male rats. The sensitivity of the liver enzyme activity to different chemicals in vitro was influenced by the treatment of the animals with phenobarbital or methylcholanthrene. These results confirm that more than one form of cytochrome P-450 supports aldrin epoxidase in the liver. The lung and kidney aldrin epoxidase activity was not modified by the administration of chemical inducers to the rats. In vitro, the lung and kidney aldrin epoxidase activities were activated by tetrahydrofurane and progesterone, respectively. The results obtained from the lung and kidney indicate that one single species of cytochrome P-450, associated with aldrin epoxidase, exists in these organs, but it may be a different type, or regulated in a different manner in these tissues.     

Effects of a hormone-supplemented medium on cytochrome P-450 content and mono-oxygenase activities of rat hepatocytes in primary culture

Cytochrome P-450 content and associated mono-oxygenation activities (7-ethoxycoumarin-O-deethylase, biphenyl-4-hydroxylase and 7-ethoxyresorufin-O-deethylase) of rat hepatocytes were found to decrease during the first 48 hr in primary culture in control (WOBA-M₂) medium. However, by culturing the hepatocytes in a hormone-supplemented medium (AB medium), all of these enzymes were maintained at higher levels after 12. 24 and 48 hr in culture. In particular. 7-ethoxyresorufin-O-deethylase activity was markedly enhanced after 12 and 24 hr in culture in AB medium to levels greater than that in isolated hepatocytes. Metabolic capacities of the cytochromes P-450 present in hepatocytes cultured in WOBA-M₂ medium vs AB medium were also quantitatively different at 12, 24 and 48 hr when specific activities/pmole of hemoprotein were compared. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments further suggested that a cytochrome P-450-related protein was maintained to a greater extent in AB medium than in WOBA-M₂ medium. It is proposed that AB medium may maintain a higher cytochrome P-450 concentration in cultured primary rat hepatocytes by increasing both the rate of hcme synthesis and the synthesis of a cytochrome P-450-related protein.     

Rat liver cytochrome P-450 isozymes as catalysts of aldrin epoxidation in reconstituted monooxygenase systems and microsomes

To explore which rat liver cytochrome P-450 species are involved in aldrin epoxidation, we have studied the catalytic activities of a series of cytochrome P-450 isozymes purified from untreated and inducer-treated Sprague-Dawley rats. Of ten cytochrome P-450 forms analyzed, seven isozymes, listed in order of decreasing activity, catalyzed aldrin epoxidation: P-450UT-A, P-450PB-C, P-450UT-H, P-450PB-B, P-450PCN-E, P-450UT-F, and P-450PB-D. P-450UT-I, P-450BNF-B, and P-450ISF-G were not very active at all. A novel aldrin metabolite, endo-dieldrin, was formed by cytochrome P-450UT-F in a 6-fold excess over dieldrin, which is the exo-isomer. The activity of aldrin epoxidase furthermore was assayed in liver microsomes from Sprague-Dawley rats of diverse physiological status and after pretreatment with various inducers resulting in a peculiar pattern of cytochrome P-450 isozymes. Untreated animals, at an age of 3 weeks, showed similar enzyme activities in both genders. During maturation, the activity of males increased by 3-fold, while the activity in females did not significantly change during this period. Pretreatment with pregnenolone-16-alpha-carbonitrile or dexamethasone strongly increased the activity in females. Pretreatment with dexamethasone did not increase the activity of males. A 50% depression of epoxidase activity was noted for males pretreated with 5,6-benzoflavone. Phenobarbital pretreatment increased the activity of females by 12-fold and of males by 2-fold. Males responded to pretreatment with polychlorinated biphenyls in a strain dependent fashion: enzyme activity was increased 2-fold in Sprague-Dawley rats but was not altered in Wistar rats. "Theoretical" values of microsomal epoxidase activity were calculated for weanling and adult Sprague-Dawley rats from turnover numbers and published data on the relative abundance of aldrin epoxidizing P-450 isozymes (Waxmann et al., Biochemistry 24, 4409, 1985). These values agreed with the activities determined. A similar statement can be made for male rats of both strains pretreated with inducers, when the ratio of enzyme activity of pretreated to control animals was used as a basis of comparison. The activity ratio of females pretreated with pregnenolone-16-alpha-carbonitrile, dexamethasone and phenobarbital, however, was much higher than the ratio calculated. Our results reveal that aldrin epoxidation is a reaction indicative of male specific and of phenobarbital-inducible cytochrome P-450 isozymes in rat liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of picloram on xenobiotic biotransformation in rat liver

1. The effect of picloram on model xenobiotic substrate biotransformation in vivo was studied in female and male rat liver.

2. Treatment with picloram had little effect on epoxide hydratase and glutathione S-transferase activity, but caused a dose-dependent increase in ethoxyresorufin-O-deethylase activity and a concomitant decrease in aldrin epoxidase activity in male rats.

3. Treatment of male rats with equivalent doses of 2-acetylaminofluorene, 2-amino-anthracene and picloram induced ethoxyresorufin-O-deethylase activity to the same degree.

4. Treatment of female rats with picloram resulted in dose-dependent increases in ethoxyresorufin and ethoxycoumarin-O-deethylation without decreasing aldrin epoxidase activity.

5. Picloram binds to liver microsomal preparations from rats pretreated with phenobarbitone and/or 3-methylcholanthrene, giving a type I spectrum.

6. The results indicate that picloram is a 3-methylcholanthrene-type inducer, and the implications are discussed.     

Low glutathione<Emphasis Type="Italic"> S</Emphasis>-transferase dogs

Liver and kidney glutathione S-transferase (GST) activities to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activities) were measured in 280 dogs from five different breeders, and significant individual differences in this activity were observed in both organs. Interestingly, 34 out of the 280 dogs (i.e. 12.1%) were those in which liver GST-D activities were less than 10 nmol/min per mg cytosolic protein, low GST dogs, and the other dogs were classified as middle and high GST dogs for which the liver GST-D activities were 10–80 and >80 nmol/min per mg protein, respectively, and occurred at similar percentages (41.4% for the middle GST dog and 46.4% for the high GST dog). Furthermore, the existence of the low GST dogs was not limited to one particular breeder. There was a good correlation (r=0.910) between the liver and kidney GST-D activities, showing low activity in not only the liver but also the kidney in the low GST dogs. Although liver GST activity to 1-chloro-2,4-dinitrobenzene as a substrate (GST-C activity), catalyzed by various GST isozymes in dogs, was significantly correlated with liver GST-D activity, GST-C activity showed more than 450 nmol/min per mg protein even in the low GST dogs. There was no significant difference in cytochrome P450 content, 7-ethoxycoumarin O-deethylase activity or UDP-glucuronosyltransferase activity to p-nitrophenol as a substrate between low GST dogs and the other dogs. Finally, remarkably high plasma concentrations of DCNB were observed in the low GST dogs after single doses of DCNB at 5 or 100 mg/kg. The individual differences in GST-D activity are probably attributable to the content and/or activity of the theta class GST isozyme Yd_fYd_f since it has been reported that glutathione conjugation of DCNB is specifically catalyzed by GSTYd_fYd_f in dogs. In conclusion, we identified a number of low GST dogs in which the GST-D activities were not observed either in vivo or in vitro. The feasibility of using a single low dose of DCNB to phenotype dogs based on GST-D activity was confirmed. It was also suggested that low GST dogs have high susceptibility, including unexpected toxicity or abnormal exposure, to chemicals metabolized by GSTYd_fYd_f.     

Maintenance of monooxygenase activities and detection of cytochrome P-450-mediated cytotoxicity in Mongolian gerbil hepatocyte cultures

1. Hepatocytes were isolated from untreated and phenobarbitone (PB)-treated Mongolian gerbils by lobe perfusion. Yields were approx. 20 ± 10⁶ cells/g liver and viability was 95 ± 1%.

2. PB treatment significantly increased the total cytochrome P-450 content, and the 7-ethoxycoumarin O-deethylase, p-nitrophenol hydroxylase and coumarin 7-hydroxylase activities, relative to those of untreated gerbils, measured in homogenates of freshly isolated hepatocytes.

3. After 24?h in culture the cytochrome P-450 content of hepatocyte homogenates from both untreated and PB-treated gerbils was 40–45% that of the corresponding values of freshly isolated hepatocytes. This decrease was accompanied by selective losses of cytochrome P-450-dependent enzyme activities.

4. Erythromycin and benzphetamine N-demethylase, and p-nitrophenol hydroxylase, activities were well maintained over 24?h in culture, whilst 7-ethoxycoumarin O-deethylase and coumarin 7-hydroxylase activities were poorly maintained. In general, the stability of the monooxygenase activities measured was improved by PB treatment of gerbils.

5. The toxicity of coumarin, precocene I and precocene II to gerbil hepatocyte cultures was dose-dependent. Precocene II was significantly more toxic to hepatocytes cultured from PB-treated, compared with untreated, gerbils.

6. Gerbil hepatocyte cultures would seem to be appropriate for investigating species differences in metabolism-mediated cytotoxicity.     

Phase I and phase II xenobiotic biotransformation in cultures and co-cultures of adult rat hepatocytes

The aim of this study was to measure the activity of phase I and II key enzymes in the biotransformation of xenobiotics and their inducibility by phenobarbital (2 mM) in two currently used in vitro models, namely adult rat hepatocytes, conventionally cultured or co-cultured with rat epithelial cells derived from primitive biliary duct cells. For phase I, the cytochrome P450 content and the enzymic activities of 7-ethoxycoumarin O-deethylase and aldrin epoxidase have been determined, for phase II glutathione S-transferase activity was measured. In conventional cultures, all phase I parameters investigated declined continuously as a function of culture time. Two mM phenobarbital had inducing effects on 7-ethoxycoumarin O-deethylase and glutathione S-transferases but not on aldrin epoxidase. In co-cultures, after an initial decrease, a steady state situation developed for all the parameters measured, lasting for at least 10 days. The cytochrome P450 content, the 7-ethoxycoumarin O-deethylase, aldrin epoxidase and glutathione S-transferase activities were maintained from 3 to 4 days on at 25, 100, 15 and 50%, respectively, of their corresponding value obtained for freshly isolated hepatocytes. After phenobarbital treatment, the parameters mentioned were significantly increased with the exception of the aldrin epoxidase activity of which the inducibility was nearly completely lost.     

Induction of xenobiotic biotransformation by the insecticide chlordimeform, a metabolite 4-chloro-o-toluidine and a structurally related chemical o-toluidine

Chlordimeform, 4-chloro-o-toluidine and o-toluidine have all been found to have carcinogenic properties. Due to an empirical link between such properties and alteration of some biotransformation enzymes, the abilities of these three chemicals to affect cytochrome P-450 mediated biotransformation, epoxide hydrolase and glutathione S-transferase have been examined. Chlordimeform had no effect on the cytochrome P-450 content, aniline p-hydroxylase or glutathione S-transferase activities, but induced ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase and epoxide hydrolase activities and decreased aldrin epoxidase and aminopyrine N-demethylase activities. The metabolite 4-chloro-o-toluidine increased cytochrome P-450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, glutathione S-transferase and epoxide hydrolase activities. o-Toluidine induced cytochrome P-450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, and aldrin epoxidase activities. Ethoxy-resorufin-O-deethylase activity was induced approximately eight times by chlordimeform and 18 times by 4-chloro-o-toluidine and o-toluidine. Induction was seen at 50 mg/kg with chlordimeform and at 10 mg/kg with the other treatments. Chlordimeform increased the 7 alpha and 16 alpha androstenedione hydroxylase pathways. 4-Chloro-o-toluidine increased the 7 alpha, 16 beta and 16 alpha hydroxylase pathways, while o-toluidine increased the 7 alpha, 6 beta, 16 beta and 16 alpha hydroxylase pathways. All three chemicals marginally decreased the testosterone pathways. SDS-PAGE of rat microsomes revealed an increase in a protein band of MW c54,000 for the chlordimeform and 4-chloro-o-toluidine treated groups. Taken together with the increase in ethoxyresorufin-O-deethylase activity these observations are consistent with the induction of hepatic isozyme P-450d. Thus each chemical has been shown to induce various pathways of biotransformation with increases in the P-450c and P-450d specific substrate ethoxyresorufin-O-deethylase being a consistent finding.     

Cytochromes P-450 in the intestinal mucosa of man

With monoclonal antibodies against cytochrome P-450(5) and P-450(4,5,6), 52 and 54 kDa bands are visualized in microsomes from proximal as well as distal human small intestine. These bands most probably correspond to cytochrome P-450(5) and P-450(4), respectively. This and several other cytochrome P-450 related proteins are present in hepatic microsomes from the same patient. In both hepatic and intestinal microsomes from this patient cytochrome P-450(8) is hardly detectable. In contrast to the small intestine and liver, large intestinal tissue from several other patients does not contain cytochrome P-450(5). Here the 54 kDa isoenzyme, possibly cytochrome P-450(4), is most prominent. Earlier we found a higher content of total cytochrome P-450 in the proximal as compared to the distal small intestine. A similar distribution is now found with regard to aldrin epoxidase activity.     

Attempts to phenotype human liver samples in vitro for debrisoquine 4-hydroxylase activity.

Twenty-eight samples of human liver have been characterised for cytochrome P-450 content, aldrin epoxidase, debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase activities. Evidence is presented here and elsewhere that bufuralol 1'-hydroxylase and debrisoquine 4-hydroxylase are activities catalysed by the same form of cytochrome P-450 in man, and that this form is different from that catalysing the epoxidation of aldrin. Attempts to phenotype liver samples in vitro, in the absence of any metabolic data in vivo for debrisoquine 4-hydroxylation status, met with limited success. A combination of enzyme assays will most probably be required in any such phenotyping of human liver samples.     

Rat hepatic microsomal enzyme induction by pretreatment with toxaphene and toxaphene fractions

The levels of hepatic microsomal induction caused by toxaphene were determined. Young Sprague-Dawley rats (70 g) were administered toxaphene (ip injection, daily for 5 d) at 0, 5, 25, and 100 mg/kg. All doses caused increases in liver/body weight ratio, cytochrome P-450 level, aminopyrine demethylation, and aldrin epoxidation. The aldrin epoxidase activity increased almost 700% at the 100-mg/kg dose. Toxaphene was separated into nonpolar (S-A) and polar (S-B) fractions and administered as before at 25 mg/kg. All treatments caused significant increases in cytochrome P-450, aminopyrine demethylation, and aldrin epoxidation. A comparison of the treatments, however, did not reveal any significant differences between the treatments.     

Effects of arsenite on hepatic mixed-function oxidase activity in rats

1. Injection of arsenite (As³⁺) to control rats results in losses of total hepatic cytochrome P-450 and significant decreases of ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD) activities. However, As³⁺ appears to decrease the activity of these enzymes differentially, with EROD showing greater sensitivity than ECOD.

2. Injection of As³⁺ to rats treated with phenobarbital and isosafrole significantly decreases the total content of hepatic cytochrome P-450 and various mixed function oxidase (MFO) activities, with the exception of ECOD which appears to be insensitive to As³⁺.

3. 3-Methylcholanthrene administration apparently protects against the effects of As³⁺ on the cytochrome P-450 system, since total content of the cytochrome P-450 and various MFO activities were all insensitive to this treatment.     

Inhibitors of cytochrome P-450 reduce cyclic GMP stimulation by glyceryl trinitrate in LLC-PK1 kidney epithelial cells

Summary The inhibitor of cytochrome P-450 cimetidine was used to asses the role of cytochrome P-450-dependent enzymes for cyclic GMP stimulation by glyceryl trinitrate in a kidney epithelial cell line (LLC-PK₁). Pretreatment of the cells with 0.1 mmol/1 cimetidine markedly decreased cyclic GMP stimulation by glyceryl trinitrate (0.03 –1 mol/l). In the presence of 0.1 mmol/1 cimetidine, the 14-fold cyclic GMP stimulation observed at 1 mol/l glyceryl trinitrate was reduced by 66%. Glyceryl trinitrate-induced cyclic GMP stimulation remained unaltered by ranitidine (0.1 mmol/1), which has a much lower affinity for the cytochrome P-450 enzyme system. Another inhibitor of cytochrome P-450, miconazole (0.1 mmol/1), also attenuated glyceryl trinitrate-induced cyclic GMP stimulation. In contrast, cimetidine and miconazole did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. These results suggest that in intact cells, glyceryl trinitrate-induced cyclic GMP stimulation is dependent on cytochrome P-450 enzymes which may be relevant for nitric oxide formation from organic nitrates.Send offprint requests to K. Schrör at the above address     

Oxidative biotransformation in primary cultures of chick embryo hepatocytes: induction of cytochrome P-450 and the metabolism of benzo(a)pyrene

Primary cultures of chick embryo hepatocytes are known to maintain their initial level of cytochrome P-450 for a number of days. To explore the possibilities of chick embryo hepatocyte cultures as a tool in drug metabolism, induction profiles of cytochrome P-450 were determined and the metabolism of benzo(a)pyrene as a model substrate was studied.Maximum induction by phenobarbitone and Aroclor 1254 is reached after 21 h and 18 h, respectively, both in the presence and absence of serum. For -naphthoflavone induction is maximal after 31 h in the presence and 43 h in the absence of serum. The levels of P-450 after induction are comparable to those found in vivo in rats: increases of 200% for phenobarbitone, 200% for -naphthoflavone and 210% for Aroclor 1254. Ethoxyresorufin-0-deethylase activities are induced by -naphthoflavone and Aroclor 1254, but as expected only slightly by phenobarbitone. In the absence of serum in the culture medium, for the control as well as the induced cells a plateau of activity is maintained for at least 24 h. In the presence of serum a decline in P-450 levels is observed. Especially in the case of Aroclor, an increase in porphyrin content of 320% of control values is seen at the same time.A number of representative metabolites of benzo(a)pyrene were quantitated during a 4-h incubation. Relative amounts are comparable to those observed with rat liver microsomes. As expected, -naphthoflavone and Aroclor induce the rate of metabolism (by 500% and 400%, respectively, in the absence of serum), but phenobarbitone has no or very little effect.Interestingly, when benzo(a)pyrene is incubated with control or phenobarbitone-induced cells an increase in rate of metabolite formation with time is observed: benzo (a)pyrene seems to induce its own metabolism. The chick embryo hepatocytes thus offer the possibility of studying induction and biotransformation in the same system at the same time, in vitro.     

The effect of ethanol administration on renal and hepatic mixed-function oxidases in the Fischer 344 rat

Administration of ethanol in the drinking water increases hepatic cytochrome P-450 content and xenobiotic metabolism. However, the effect on renal xenobiotic metabolism has not been investigated. Chronic consumption of ethanol (15% solution in the drinking water) increased hepatic cytochrome P-450 content as well as ethoxycoumarin-O-deethylase, aniline hydroxylase and benzphetamine-N-demethylase activities. No change in renal cytochrome P-450 was detected after chronic ethanol consumption whereas ethoxycoumarin-O-deethylase activity in renal microsomes was significantly increased.     

In vitro biotransformation of 2-methylpropene (isobutene) in rat lung tissue in comparison with liver tissue

 The epoxidation of the gaseous alkene 2-methylpropene or isobutene was studied in vitro in rat lung tissue in comparison with rat liver. Pulmonary tissue appears to be less exposed to the toxic epoxide metabolite than is the case for hepatic tissue. The results are correlated with the low capacity of the mixed function oxidase system, expressed by means of the cytochrome P-450 content and the 7-ethoxycoumarin O-deethylase activity, to form reactive intermediates. The activities of the principal epoxide detoxifying enzymes glutathione S-transferase and epoxide hydrolase represent only 5–10% of the values measured in rat liver. Received: 20 February 1995/Accepted: 20 June 1995     

Effects of polychlorinated terphenyls and paraffins on rat liver microsomal cytochrome P-450 and in vitro metabolic activities

The polychlorinated terphenyl Aroclor 5460 and the polychlorinated paraffins Witaclor 171 P and Witaclor 149 increased to different degrees the total microsomal concentration of cytochrome P-450 in the rat liver after intraperitoneal injection of 0.3, 1.0, and 1.0 g · kg^–1 body weight, respectively, each day for four days. The multiple forms of cytochrome P-450 were affected differently with an induction of RLvMc P-450₅₀ and RLvMc P-450₅₄ by all chemicals, and an additional induction of RLvMc P-450₅₅ by the polychlorinated terphenyl. The rat liver weights were extensively increased after treatment with the polychlorinated paraffins. Alterations were found in the in vitro metabolism of biphenyl, benzo(a)pyrene and the steroid hormones, 4-androstene-3,17-dione and 5-androstane-3,17-diol, after exposure to all chemicals. Changes in the in vitro formation of benzo(a)pyrene metabolites were found to correlate with changes in the multiple forms of cytochrome P-450. The present study demonstrate that only limited information can be obtained from alterations in the total concentration of cytochrome P-450 and show the importance of studying changes in the multiple forms and the metabolism of different substrates. Our results further indicate that exposure to any of the investigated polychlorinated chemicals may alter the biological effects of other environmental contaminants, drugs and endogenous substances which are metabolized by the cytochrome P-450 enzyme system.