Correlation between cytochrome P-448 content and benzopyrene hydroxylase activity during the induction of microsomal monooxygenases using methylcholanthrene-type xenobiotics

Using antibodies against electrophoretically homogeneous cytochrome P-448 from rat liver microsomes induced by 3-methylcholanthrene, the changes in the immunologic identity and contents by cytochrome P-448 induced by 3-methylcholanthrene, 3.4-benzpyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were studied. No cytochrome P-448 was detected in the liver microsomes of control or phenobarbital-induced rats. This form of the cytochrome makes up to about 35% of the total content of the CO-binding hemoprotein during TCDD induction and up to 90% during 3-methylcholanthrene and 3,4-benzpyrene induction. On the other hand, 3-methylcholanthrene, 3,4-benzpyrene and TCDD significantly and equally activates the cytochrome P-448-dependent benzpyrene hydroxylase, since the antibodies against cytochrome P-448 inhibit benzpyrene metabolism in the microsomes by 85-90%. The possible reasons for the TCDD-induced increase in the catalytic activity of cytochrome P-448 as compared to the immunologically identical cytochrome P-448 induced by 3-methylcholanthrene and 3,4-benzpyrene, are discussed.     

Cytochrome P-448 induction in liver microsomes of mice of inbred strains after administration of xenobiotics of MC-type

Using immunochemical methods, the identity of cytochrome P-448 from liver microsomes of mice of "inducible" and "non-inducible" lines during induction by xenobiotics of MX-type (3-methylcholanthrene, 3,4-benzpyrene, 2,3,7,8-tetrachlorodibenzodioxin) was established. This hemoprotein form was shown to play a role in 3,4-benzpyrene metabolism. Monospecific antibodies to purified cytochromes P-448 and P-450 were obtained; the cytochrome P-448 content in microsomes was measured by rocket immunoelectrophoresis. The content of cytochrome P-448 in control and phenobarbital-induced microsomes makes up to 10-15% of the total hemoprotein content determinable from the CO-spectra. 3-Methylcholanthrene and 3,4-benzpyrene injected into "non-inducible" mice cause no increase in the content of this hemprotein form, whereas in mice induced with 2,3,7,8-tetrachlorodibenzodioxin it rises to 50%. Under these conditions, an almost 100% inhibition of 3,4-benzpyrene metabolism by antibodies to cytochrome P-448 is observed. Antibodies against cytochrome P-448 obtained from liver microsomes of 3-methylcholanthrene-induced mice cause a 90% inhibition of 3,4-benzpyrene in microsomes induced with 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin.     

NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

Effects of 3-methylcholanthrene pretreatment on microsomal hydroxylation of 2-acetamidofluorene by various rat hepatomas

1. The effects of 3-methylcholanthrene pretreatment on both N- and ring hydroxylation of 2-acetamidofluorene by microsomal preparations from various well-differentiated and poorly differentiated hepatomas, primary tumours and their host livers and kidneys were studied. 2. Well-differentiated Morris hepatomas 5123C, 5123D, 5123CTC and 7800 and their host livers had low hydroxylating activity. Pretreatment with 3-methylcholanthrene caused a several-fold increase in both N- and ring hydroxylation in the host livers whereas in all tumours except 5123CTC it caused a many-fold increase only in ring hydroxylation. 5123CTC tumour in addition showed a fourfold increase in N-hydroxylating activity. 3. Hydroxylating activities of poorly differentiated Morris hepatoma 7288CTC and Novikoff hepatoma were low and they could not be altered by 3-methylcholanthrene pretreatment. 4. Primary hepatomas produced by administration of 4-dimethylamino-3'-methylazobenzene could be stimulated to some extent on 3-methylcholanthrene pretreatment; however, primary mammary tumour produced by administration of 3-methylcholanthrene was not responsive to 3-methycholanthrene pretreatment. 5. Like host livers, kidneys of tumour-bearing animals could also be stimulated to some extent by 3-methylcholanthrene pretreatment.     

Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens in rat liver

The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a) pyrene and β-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers (phenobarbital, 1,1,1-trichloro 2,2-bis (ρ-chlorophenyl) ethane (DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons.     

Effect of phenobarbital and 3-methylcholanthrene treatment on NADPH- and NADH-dependent production of reactive oxygen intermediates by rat liver nuclei.

The effect of inducing the rat liver nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene on NADPH- and NADH-dependent production of reactive oxygen intermediates was evaluated. The inducing agents produced a 2-fold increase in cytochrome P-450, a 50 to 70% increase in NADPH-cytochrome c reductase activity, and a 20 to 30% increase in NADH-cytochrome c reductase activity. Associated with these increases was a corresponding increase in NADPH- and NADH-dependent production of hydroxyl radical (.OH)-like species and of H2O2. Rates of .OH production were inhibited by catalase and partially sensitive to superoxide dismutase. The increase in nuclear production of .OH-like species after drug treatment appears to be due a corresponding increase in H2O2 generation. In contrast to H2O2 and .OH generation, production of thiobarbituric acid-reactive material by nuclei was not increased by the phenobarbital or 3-methylcholanthrene treatment. Redox cycling agents such as menadione and paraquat increased oxygen radical generation to similar extents in the control and the induced nuclei. These results indicate that induction of the nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene can result in a subsequent increase in production of reactive oxygen intermediates in the presence of either NADPH or NADH.     

Comparison of phenobarbital-and carcinogen-induced aldehyde dehydrogenases in the rat

There is a genetically determined variation in the inducibility of a high-K_m cytoplasmic aldehyde dehydrogenase activity in the rat liver by treatment with phenobarbital. In the present experiments this activity increased after phenobarbital administration in the phenobarbital-responsive rats also in the intestinal postmitochondrial supernatant fraction. Phenobarbital-nonresponsive rats did not exhibit such an increase after drug treatment. Intraperitoneal administration of 2,3,7,8,-tetrachlorodibenzo-pdioxin, strongly enchanced the cytoplasmic enzyme activity in the liver of both responsive and nonresponsive rats. This effect was also seen in the serum but not in the intestinal or hte kidney. Intragastric administration of 3-methylcholanthrene, 3,4,-benzpyrene or chrysene induced the activity in liver and intestine but not in serum or kidney. The activity in liver was also induced by long-term feeding with 2-acetamido-fluorene. The activities induced by tetrachlorodibenzodioxin or the carcinogens had similar behaviour in isoelectric focusing in gel slabs and in gel chromatography, suggesting a possible common identity of these induced enzymes. The activity induced by these agents could be clearly differentiated both from the activity induced by phenobarbital and from the normal cytoplasmic activities.     

Drug induction of hepatic glutathione S-transferases in male and female rats*.

The induction of the glutathione S-transferases by phenobarbital and polycyclic hydrocarbons was studied in male and female rats. Administration of phenobarbital resulted in 60-80% increase in S-aryl and S-aralkyl enzyme specific activities, whereas the S-epoxide and S-alkyl activities were increased by 30-40%. In following the sequence of induction, the former two activities were noted to reach peak activities before an increase in the latter two activities was observed. Both 3-methylcholanthrene and 3,4-benzopyrene were shown toi nduce these four enzymic activities, although without the discrimination between pairs of activities noted with phenobarbital. No change in Km accompanied the increase in Vmax. after induction by drugs, and no change occurred in Ki for sulphobromophthalein inhibition. Significantly lower enzyme specific activities were found for three of the activities studied in female rats but no difference was observed in the S-alkyltransferase activity. However, the proportional increase in the enzymic activities in response to phenobarbital was the same in males and females. These studies demonstrate the drug induction of a group of cytosolic drug-metabolizing enzymes as well as the identification of sex differences in these activities.     

Use of inhibition by metyrapone in a "mutual depletion system" for evaluation of active centres of various arylhydrocarbon hydroxylases]

It was shown that metyrapone, the inhibitor of arylhydrocarbonhydroxylase, taken at concentrations equimolar to that of cytochrome P-450 non-competitively inhibits the hydroxylation of 3,4-benzpyrene in the liver microsomes of inbred mice of the C57BL/6 and AKR strains. In a given "mutual depletion inhibition system" the concentration of the catalytically active centres of microsomal cytochrome (Ecac), their turnover number (TNcac) and "true" dissociation constant of the enzyme-inhibitor complex were determined in the control and 3-methylcholanthrenetreated mice of both strains. The increased rate of 3,4-benzpyrene hydroxylation in the liver of 3-methylcholanthrene-induced "sensitive" C57BL/6 mice is determined by the increase of Ecac (and, in a lesser degree, of TNcac) per molecule of cytochrome P-448. In the liver microsomes of "induction-resistant" AKR mice an injection of 3-methylcholanthrene results in a slight increase of Ecac and a simultaneous decrease of TNcac. It was assumed that contrary to the present-day concepts, an aberrant microsomal hemoprotein with a genetically determined low molecular activity is synthesized in mice of "resistant" AKR strain.     

BENZPYRENE HYDROXYLASE ACTIVITY IN ISOLATED PARENCHYMAL AND NONPARENCHYMAL CELLS OF RAT LIVER

Previous studies have implicated the reticuloendothelial cells of the liver in certain aspects of steroid metabolism. The similarity in the metabolism of steroids and polycyclic hydrocarbons suggested that the nonparenchymal cells possibly play a role in these areas. The present study presents evidence that at least one of the microsomal NADPH-requirig enzymes, benzpyrene hydroxylase, is present in nonparenchymal cells and, furthermore, is "inducible." In adult rats treated with 3-methylcholanthrene or β-naphthoflavone, the nonparenchymal cells exhibited increases in benzpyrene hydroxylase activity of 17-fold and five-fold, respectively. Treatment with phenobarbital resulted in only a slight increase in enzyme activity. Enzyme activity in parenchymal cells under similar conditions was increased sixfold and fivefold by 3-methylcholanthrene and β-naphthoflavone, respectively, but not by phenobarbital.     

The effects of 3-methylcholanthrene on macrophage respiratory burst and biotransformation activities in the common carp (Cyprinus carpio L.)

The sensitivity of phagocytic cell function as a bioindicator of pollution stress by polycyclic aromatic hydrocarbons was evaluated in the common carp (Cyprinus carpio L). The time course response of the head-kidney macrophage respiratory burst was measured 1, 2, 3, 5 and 7 days after intraperitoneal injection of a prototypical Cyp 1A inducer (3-methylcholanthrene). This immune activity was compared to the rate of induction of total cytochrome P450, ethoxyresorufin O-deethylase activity (EROD) and glutathione S-transferase activity (GST) in the liver and head-kidney. 3-methylcholanthrene (40 mg kg(-1)) caused a rapid increase in the macrophage respiratory burst. This response was maximal at day 3 post exposure and coincided with maximum induction of cytochrome P450 and EROD activity in liver and head-kidney. Moreover, alpha-naphtoflavone, which functions as both an Ah receptor antagonist and an inhibitor of cytochrome P450 1A activity, reversed the 3-methylcholanthrene induction of immune and enzymatic parameters measured, suggesting metabolic processes. Taken together these results suggest that the induction of macrophage oxidative function may be an equally sensitive marker of exposure to polycyclic aromatic hydrocarbon as the induction of biotransformation activities and confirm that responses mediated by the Ah receptor are similar, if not identical, to those of mammals.     

Benzpyrene hydroxylase activity in hepatic microsomal and solubilized systems containing rabbit or rat cytochrome P-448 or P-450

Treatment of adult, male rabbits and rats with 3-methylcholanthrene results in the formation of hepatic microsomal cytochrome P-448. In the rat, this occurs coincidently with an increase in hepatic microsomal benzpyrene hydroxylase activity. In the rabbit, benzpyrene hydroxylase activity is decreased following treatment with 3-methylcholanthrene. Benzpyrene hydroxylase activity in solubilized, reconstituted mixed-function oxidase systems containing rat cytochrome P-448 is about seven times higher than in systems containing rabbit cytochrome P-448. Evidence obtained by spectral analysis suggests that rabbit P-448 is combined with a type I compound. Residual ¹⁴C-3-methylcholanthrene does not appear to be responsible for the differences observed between rat and rabbit cytochrome P-448.     

Liver microsomal cytochromes P-450 and azoreductase activity.

Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is proportional to the levels of microsomal cytochrome P-450 from control or phenobarbital-pretreated rats and mice or cytochrome P-448 from 3-methylchol-anthrene-pretreated animals. In the "inducible" C57B/6J strain of mice, 3-methylcholanthrene and phenobarbital pretreatment cause an increase in cytochrome P-448 and P-450 levels, respectively, which is directly proportional to the increase of azoreductase activity. However, in the "noninducible" DBA/2J strain of mice, only phenobarbital treatment causes the increase both in cytochrome P-450 levels and azoreductase activity, while 3-methylcholanthrene has no effect. These experiments suggest that the P-450 type cytochromes are responsible for azoreductase activity in liver microsomes.     

Altered activation/detoxication enzymology following neonatal diethylstilbestrol treatment

The effects of neonatal exposure to diethylstilbestrol (DES) on hepatic activation/detoxication enzyme levels in the adult rat were investigated. Neonatal exposure of male rats to DES (DES males) decreased the endogenous levels of UDP-glucuronyltransferase as compared to control males. Female rats exposed neonatally to DES (DES females) had higher endogenous epoxide hydrolase and glutathione transferase activity levels than control females. Adult animals treated neonatally with DES also had altered metabolic potential following exposure to 3-methylcholanthrene and phenobarbital. The DES males treated in adulthood with 3-methylcholanthrene had higher benzo(a)pyrene hydroxylase activities and lower UDP-glucuronyltransferase activity levels than did control males treated in adulthood with 3-methylcholanthrene. The DES males exposed in adulthood to phenobarbital had reduced cytochrome P-450 and glutathione transferase activity levels as compared with respective controls. The DES females treated in adulthood with 3-methylcholanthrene had lower benzo(a)pyrene hydroxylase and epoxide hydrolase activity levels than control females receiving 3-methylcholanthrene. The DES females challenged in adulthood with phenobarbital also had decreased benzo(a)pyrene hydroxylase, epoxide hydrolase, UDP- glucuronyltransferase, and glutathione transferase activity levels as compared with respective controls. Our results demonstrated that neonatal exposure to DES changed the endogenous levels of specific hepatic enzymes and altered the metabolic response of these adult animals to a carcinogen and a drug.     

Kinetic characteristics of 3,4-benzpyrene hydroxylation in the liver microsomes of mice

The kinetic parameters of binding and hydroxylation of hydrophobic substrate 3,4-benzpyrene have been studied in liver microsomes of untreated and 3-methylcholanthrene treated mice. The reaction of benzpyrene-hydroxylase has been established to be described by hyperbolic curve, which characterizes the dependence of [ES] and d(P)/dt on [E0] for reactions in biphasic system. A key role of microsomal membraneous phospholipids has been revealed in competitive inhibition of 3,4-benzpyrene hydroxylation. For the adequate application of Michaelis--Menten theory for benzpyrene-hydroxylation reaction a modified method of 3,4-benzpyrene-hydroxylation in the samples with low content of protein in microsomal fraction is suggested.     

Age-dependent in situ hepatic and gill CYP1A activity in the see-through medaka (Oryzias latipes)

We used a recently introduced strain of medaka, the see-through medaka, whose internal organs can be seen through the skin, to develop an in situ toxicity assay of ethoxyresorufin-O-deethylase (EROD) activity that detected fluorescence from resorufin, a metabolite of ethoxyresorufin and thus an indicator of CYP1A activity. EROD activity in the liver and gills of 2-week post-hatch see-through medaka exposed simultaneously to various concentrations of 3-methylcholanthrene and 200 microg/L ethoxyresorufin for 24 h was proportional to the 3-methylcholanthrene dose. Activities in the liver and gills peaked at 40 microg/L of 3-methylcholanthrene and then decreased at higher doses, possibly because of 3-methylcholanthrene toxicity. At 1-week post-hatch stage, however, constant high EROD activity was observed in controls and at all 3-methylcholanthrene doses. Four-week post-hatch see-through medaka exhibited less EROD activity than 2-week post-hatch see-through medaka, and activity in the liver peaked at 100 microg/L of 3-methylcholanthrene. Adult see-through medaka were not suitable for fluorescence detection owing to their thick skin, muscle and/or tissue. In tests of oxidative activity response to ethoxyresorufin, 1-day and 1-week post-hatch see-through medaka exhibited high intrinsic EROD activity in the liver, gills, and other organs in the absence of 3-methylcholanthrene. This intrinsic activity declined with growth and explained the high constant EROD activity at 1-week post-hatch stage.     

Genetic regulation of UDP-glucuronosyltransferase induction by polycyclic aromatic compounds in mice. Co-segregation with aryl hydrocarbon (benzo(alpha)pyrene) hydroxylase induction.

Induction of hepatic 4-methylumbelliferone UDP-glucuronosyltransferase (EC 2.4.1.17) by polycyclic aromatic compounds, such as 3-methylcholanthrene or beta-naphthoflavone, occurs in C57BL/6N, A/J, PL/J, C3HeB/FeJ, and BALB/cJ but not in DBA/2N, AU/SsJ, AKR/J, or RF/J inbred strains of mice. This pattern of five responsive and five nonresponsive mouse strains parallels that of the Ah locus, which controls the induction of aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (EC 1.14.14.2). Induction of the transferase is maximal in C57BL/6N mice with 200 mg of 3-methylcholanthrene/kg body weight; no induction occurs in nonresponsive DBA/2N mice even at a dose of 400 mg/kg. The rise of inducible transferase activity lags 1 or more days behind the rise of inducible hydroxylase activity and peaks 5 days after a single dose of 3-methylcholanthrene. In offspring from the appropriate backcrosses and intercross between C57BL/6N and DBA/2N parent strains, the genetic expression of 3-methylcholanthrene-inducible transferase activity is inherited as an additive (co-dominant) trait. This expression differs distinctly from that of the inducible hydroxylase activity, which is inherited almost exclusively as a single autosomal dominant trait in these same animals. The more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the transferase more than 3-fold in C57BL/6N mice and less than 2-fold in DBA/2N mice, whereas the hydroxylase is induced equally (about 8-fold) in both strains. A dose of 3-methylcholanthrene given 3 days after 2,3,7,8-tetrachlorodibenzo-p-dioxin, at a time when hydroxylase induction in both strains is very high, does not enhance the rise in inducible transferase activity seen in C57BL/6N or DBA/2N mice which have received 2,3,7,8-tetrachlorodibenzo-p-dioxin alone. These data indicate that (a) the inducibility of two metabolically coordinated membrane-bound enzyme activities may be regulated by a single genetic locus, and (b) although the hydroxylase can be fully induced in the nonresponsive DBA/2N strain by 2,3,7,8-tetrachlorodibenzo-p-dioxin prior to 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene, presumably present in the liver, are incapable of inducing further the transferase activity. The difference in sensitivity between 3-methylcholanthrene and the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin for both the hydroxylase and the transferase activities suggests the possibility of a common receptor in regulating both enzyme induction processes.     

Induction of choline kinase by polycyclic aromatic hydrocarbons in rat liver. II. Its relation to net phosphatidylcholine biosynthesis

The effect of a single dose (50 mg/kg body weight) of 3-methylcholanthrene on de novo phosphatidylcholine biosynthetic activities in rat liver was studied both in a cell-free system and with slice experiments. 3-Methylcholanthrene caused a significant depression of either [methyl-14C]choline or [2-(3)H]glycerol incorporation into phosphatidylcholine when the precursor was incubated with liver slices. At the same time, there occurred a significant accumulation of radioactivity in either cholinephosphate or diacylglycerol molecule from [14C]choline or [3H]glycerol, respectively, suggesting that 3-methylcholanthrene could cause an inhibitory effect on hepatic phosphatidylcholine synthesis at the cholinephosphotransferase or/and cholinephosphate cytidylyltransferase step. Subsequent studies, where the activities of the three enzymes involved in de novo phosphatidylcholine synthesis were compared between control and 3-methylcholanthrene-pretreated rat liver subcellular fractions, demonstrated that the cholinephosphotransferase step could be the site of inhibition by 3-methylcholanthrene. On the other hand, 3-methylcholanthrene caused a significant induction of choline kinase activity in a time-dependent manner and, at the same time, the cholinephosphate pool size in liver cytosol was enlarged 2-3-fold when compared to the respective control. The overall results suggested strongly that 3-methylcholanthrene causes the counteractive effects on the de novo phosphatidylcholine biosynthesis, induction of choline kinase activity and inhibition of cholinephosphotransferase activity, both of which could participate in a concomitant increase in cholinephosphate pool size in rat liver.     

The metabolism of 3-methylcholanthrene and some related compounds by rat-liver homogenates

1. A chromatographic investigation of the products of the metabolism of 3-methylcholanthrene by rat-liver homogenates showed the formation of compounds with the properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene and 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. A glutathione conjugate that is probably S-(11,12-dihydro-12-hydroxy-3-methyl-11-cholanthrenyl)glutathione was also detected. 3-Methylcholanthrene-1- and -2-one and -1,2-quinone were also present, but these products may have arisen by the chemical oxidation of the corresponding hydroxy compounds. 2. Other metabolic products were tentatively identified as 9- and 10-hydroxy-3-methylcholanthrene, 4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene and 3-hydroxymethylcholanthrene. 3. 1- and 2-Hydroxy-3-methylcholanthrene were converted by homogenates into the related ketones and into products with the properties of cis- and trans-1,2-dihydroxy-3-methylcholanthrene: 3-methylcholanthren-1- and -2-one were converted into their related hydroxy compounds and into the isomeric 1,2-dihydroxy compounds. The isomeric 1,2-dihydroxy compounds were each partly converted into the other isomer by these homogenates. All the above substrates also yielded products that appeared to be derivatives of 3-hydroxymethylcholanthrene. 4. 3-Methylcholanthrylene was converted by rat-liver homogenates into products with the properties of trans-1,2-dihydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene and 3-methylcholanthren-2-one. A small amount of the cis-1,2-dihydroxy compound was also formed, together with a glutathione conjugate that is possibly S-(2-hydroxy-3-methyl-1-cholanthrenyl)glutathione or its positional isomer. 5. An unidentified product was detected in the metabolism of 3-methylcholanthrene, the monohydroxy compounds, the ketones and the dihydroxy compounds, the formation of which appeared to involve metabolism at the 1,2-bond. 6. 11,12-Epoxy-11,12-dihydro-3-methylcholanthrene was converted by rat-liver homogenates into products with the properties of 11-hydroxy-3-methylcholanthrene (or, less likely, the 12-isomer), 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and the glutathione conjugate described above. Products with the properties of these compounds were formed when the epoxide was allowed to react with glutathione in an aqueous medium. 7. Mouse-liver homogenate converted 3-methylcholanthrene into products with the chromatographic properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, 3-methylcholanthrene-1- and -2-one and -1,2-quinone and the unidentified hydroxy-3-methylcholanthrenes. 8. The syntheses of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 3-methylcholanthren-2-one, 2-hydroxy-3-methylcholanthrene, 3-methylcholanthrylene, 11,12-epoxy-11,12-dihydro-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene are described.