Metabolism Of 3-methylcholanthrene Research Articles

Substrate Enantioselective and Product Stereoselective Metabolism of Racemic 2-Hydroxy-3-methylcholanthrene by Rat Liver Microsomes

Abstract Racemic 2-hydroxy-3-methylcholanthrene (rac-2-OH-3MC) is a potent carcinogen and 2S-OH-3MC is the most abundant metabolite formed in the metabolism of 3-methylcholanthrene (3MC) by rat liver microsomes. Eighteen identifiable metabolites were formed in the metabolism of rac-2-OH-3MC by liver microsomes prepared from phenobarbital-treated rats. Metabolites were separated by sequential use of reversed-phase and normal-phase high performance liquid chromatography (HPLC) and characterized by ultraviolet-visible absorption, mass, and circular dichroism spectral, and chiral stationary phase (CSP) HPLC analyses. Identified metabolites were: 2-OH-3MC 7,8-dihydrodiol (enriched in 7R,8R enantiomer), 2-OH-3MC 9,10-dihydrodiol (2S,9R,10R:2R,9S,10S ≈ 27:73 and 2R,9R,10R:2S,9S,10S ≈ 81:19), 2-OH-3MC 11,12-dihydrodiol (2R,11R,12R:2S,11S,12S ≈ 5:95 and an essentially optically pure 2S,11R,12R enantiomer), 3MC trans-1,2-diol (1S,2S:1R,2R ≈ 68:32), 3MC cis-1,2-diol (1S,2R:1R,2S ≈ 18:82), 2-OH-3-hydroxymethyl-cholan...

Long-Term (Imprinting) Effects of Transplacental Treatment of Mice with 3-Methylcholanthrene or β-Naphthoflavone on Hepatic Metabolism of 3-Methylcholanthrene

Foetal mice of genotype AhbAhd (responsive to induction of metabolism of polycyclic aromatic hydrocarbons [PAH]) or AhdAhd (non-responsive) were exposed transplacentally on gestation day 17 to a single dose of 3-methylcholanthrene (MC, 5-175 mg/kg) with or without prior treatment on day 15 with beta-naphthoflavone (beta NF, 150 mg/kg). The mothers were themselves either induction-responsive [(C57BL/6 x DBA/2)F1] or non-responsive (DBA/2). Metabolism of [14C]MC by homogenates of livers from the transplacentally-exposed offspring was quantified at 9 months of age (first experiment) or 13 months (second experiment) with or without prior inducing treatment with MC. The foetal exposure to MC had a permanent effect on MC metabolism by the adult hepatic homogenates in both experiments. In most instances the effect was positive in direction and small in magnitude (15-30%). It was dose-dependent with regard to transplacental MC, occurred in both induced (AhbAhd) and non-induced (AhdAhd) individuals, and was significant only when the mother and/or the foetus was inducible. beta NF itself did not have a positive imprinting effect. In some cases it either reduced or potentiated the long-term imprinting effect of MC, depending on the MC dose and the phenotype of the mother. These results confirm that transplacental exposure to a carcinogenic PAH may permanently alter metabolism of the chemical in later life, and indicate that this imprinting action is dependent on induced metabolism of the chemical in the mother and/or foetus.

1-Hydroxy- and 2-hydroxy-3-methylcholanthrene: regioselective and stereoselective formations in the metabolism of 3-methylcholanthrene and enantioselective disposition in rat liver microsomes

Absolute configurations of enantiomeric 1-hydroxy-3-methylcholanthrene (1-OH-3MC) and 2-hydroxy-3-methyl-cholanthrene (2-OH-3MC) were determined by the exciton chirality circular dichroism (CD) method as their p-nitrobenzoate derivatives. Enantiomers of 1-OH-3MC were resolved by HPLC using a column packed with chiral stationary phase (CSP) (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to gamma-aminopropylsilanized silica. Enantiomers of 2-OH-3MC were resolved as diastereomeric (-)-methoxyacetates by normal-phase HPLC. 1-OH-3MC and 2-OH-3MC, formed in the metabolism of 3MC by liver microsomes from untreated, phenobarbital (PB)-treated and 3MC-treated male Sprague-Dawley rats, were first isolated as a mixture by reversed-phase HPLC and subsequently separated by normal-phase HPLC. Concentration ratios of [1-OH-3MC]:[2-OH-3MC] formed in the metabolism of 3MC by three rat liver microsomal preparations (at 0.5 mg protein per ml of incubation mixture and an incubation time of 10 min) were found to be: 30:70 (control), 21:79 (PB treated) and 10:90 (3MC treated) respectively. R/S enantiomer ratios of 1-OH-3MC formed in the metabolism of 3MC by three rat liver microsomal preparations were determined by CSP HPLC: 35:65 (control), 39:61 (PB treated) and 46:54 (3MC treated) respectively. R/S enantiomer ratios of 2-OH-3MC formed in the metabolism of 3MC by three rat liver microsomal preparations were determined by CD spectral data: 14:86 (control), 6:94 (PB treated) and 6:94 (3MC treated) respectively. Metabolism of racemic 1-OH-3MC and 2-OH-3MC by all three rat liver microsomal preparations was found to be substrate enantioselective; the rate of 1S-OH-3MC metabolism was faster than that of 1R-OH-3MC, whereas the rate of 2R-OH-3MC metabolism was faster than that of 2S-OH-3MC.

Metabolism of 3-methylcholanthrene in rat liver cytosol

The present study investigates the metabolism of the potent carcinogen 3-methylcholanthrene in rat liver cytosol preparations. Three metabolites of 3-methylcholanthrene were characterized by HPLC and GC/MS analysis. These metabolites were identified as 1-hydroxy-3-methylcholanthrene, 1-keto-3-methylcholanthrene and cholanthrene. The results of the present study, taken together with earlier studies, suggests that the first step in the metabolic activation of 3-methylcholanthrene is hydroxylation at the 1-position, the most easily oxidized reactive center in the molecule.

The metabolism of 3-methylcholanthrene by liver and lung microsomes: Effect of enzyme inducing agents

The metabolism of 3-methylcholanthrene (3-MC) by rat liver and lung microsomes was investigated. The animals were pretreated with various inducing agents to determine the effect which microsomal induction has on metabolic profile. Metabolites were separated by high pressure liquid chromatography. Major metabolism was associated with three peaks: 2-hydroxy-3-MC, 1-hydroxy-3-MC and a peak that co-chromatographs with 3-MC 11,12-oxide and probably contains phenols. Pretreatment of animals with various inducing agents caused an increase in total metabolism as well as in specific metabolites. However, lung microsomes from phenobarbital treated animals were less efficient at metabolizing 3-MC than control microsomes. Liver and lung microsomes converted 3-MC to qualitatively similar products. No metabolites were formed by lung microsomes that were not also formed by those from liver.

The formation of dihydrodiols by chemical or enzymic oxidation of 3-methylcholanthrene

The chemical oxidation of 3-methylcholanthrene in an ascorbic acidferrous sulphate-EDTA reaction mixture gave all five possible dihydrodiols. The structures and stereochemistry of the dihydrodiols were shown by UV, mass and NMR spectral studies and by chemical examination to be cis-2a,3-dihydroxy-3-methylcholanthrene, trans-4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene, cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. An examination by HPLC of the dihydrodiols formed in the metabolism of 3-methylcholanthrene by rat-liver microsomal preparations showed the presence of trans-4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, identified by comparison of their UV and chromatographic characteristics with those of authentic standards. Tentative identifications of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, cis-2a,3-dihydroxy-3-methylcholanthrene and cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene as metabolites were made from their mobilities using HPLC. A quantitative comparison of the dihydrodiols formed from 3H-labelled 3-methylcholanthrene by microsomal preparations from the livers of normal and 3-methylcholanthrene-treated rats was carried out. trans-9,10-Dihydro-9,10-dihydroxy-3-methylcholanthrene and cis- and trans-1,2-dihydroxy-3-methylcholanthrene were formed when 3-methylcholanthrene was incubated with mouse skin in organ culture.

The metabolism of 3-methylcholanthrene by rat liver microsomes — A reinvestigation

Metabolites of 3-methylcholanthrene (3-MC) formed by rat liver microsomes were analyzed by high pressure liquid chromatography. The metabolic profile is significantly different from previous studies using thin layer chromatography. The major metabolites include 1-and 2-hydroxy-3-MC. Use of the high pressure liquid chromatographic system allows for the separation of at least seven new metabolites. The amounts of three of these new metabolites are substantially decreased when the potent epoxide hydrase inhibitor 3,3,3-trichloropropene oxide is added to the incubation system. These results then suggest the formation of epoxides of 3-methylcholanthrene other than the K-region oxide.

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.