James W. Flesher

Effect of various substituents in the 6-position on the relative carcinogenic activity of a series of benzo[a]pyrene derivatives☆

Abstract To test the hypothesis that polycyclic aromatic hydrocarbons capable of being converted to a reactive ester of the mesohydroxymethyl metabolite would be carcinogenic, a series of 6-substituted derivatives of benzo[ a ]pyrene (B[ a ]P) were tested for carcinogenicity in Sprague-Dawley rats by subcutaneous injection of the compound in sesame oil on alternate days for 30 doses. At the 0.2-μmol dose level B[ a ]P, 6-acetoxymethyl(6-AcOCH 2 )B[ a ]P, 6-hydroxymethyl(6-HOCH 2 )B[ a ]P, 6-methyl(6-CH 3 )B[ a ]P and 6-benzoyloxymethyl(6-BzOCH 2 )B[ a ]P were nearly equipotent, 6-formyl(6-OCH)-and 6-chloromethyl(6-ClCH 2 )B[ a ]P were less active, and 6-methoxymethyl (6-MeOCH 2 )B[ a ]P was inactive. At lower doses the order of potency was estimated to be: 6-AcOCH 2 - = 6-HOCH 2 - = or > B[ a ]P > 6-CH 2 - > 6-BzOCH 2 - > 6-ClCH 2 - > 6-OCH- > 6-BrCH 2 B[ a ]P. Incubation of these compounds in the presence of cofactors or cofactors plus a microsomal preparation of rat subcutis indicated that enzymic activation was necessary for metabolism to highly polar products and for conversion of 6-AcOCH 2 -, 6-BzOCH 2 - and 6-OCHB[ a ]P to 6-HOCH 2 B[ a ]P. The halomethyl compounds were converted to 6-HOCH 2 B[ a ]P in the absence of enzyme by hydrolysis. 6-MeOCH 2 B[ a ]P was unchanged in this system. These observations are consistent with the foregoing hypothesis with regard to derivatives of B[ a ]P and demonstrate that compounds of this series that are capable of conversion to the 6-HOCH 2 -derivatives are carcinogenic.

Bioalkylation and biooxidation of anthracene, invitro and invivo

Anthracene undergoes biomethylation in rat liver cytosol preparations in vitro and in rat subcutaneous tissue, in vivo. The in vitro reaction is dependent on cytosol preparations fortified by the addition of S-adenosyl-L-methionine. The products of the reaction are the meso-anthracenic or L-region derivatives 9-methylanthracene and 9,10-dimethylanthracene. The latter compound may be the simplest polynuclear aromatic hydrocarbon carcinogen known. These reactive methylated metabolites are readily oxidized in cytosol preparations and in subcutaneous tissue, in vivo, to hydroxymethyl and formyl derivatives. Oxidation takes place mainly on the methyl groups since ring oxidized products were not detected.

Biosynthesis of the potent carcinogen 7,12-dimethylbenz[a]anthracene☆

Earlier studies from this laboratory of the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and of benzo[a]pyrene as well as studies of mutagenic and carcinogenic activity of some of the metabolic products led to the concept that a necessary first step in carcinogenesis by most alkyl substituted polycyclic hydrocarbons is biotransformation to a meso-anthracenic hydroxyalkyl metabolite, whereas most hydrocarbons lacking alkyl substituents undergo a bio-alkylation substitution reaction in the mesoanthracenic position(s) or L-region as a necessary first step in carcinogenesis. According to this unified hypothesis, all strong polycyclic hydrocarbon carcinogens must either, themselves, bear a meso-anthracenic alkyl substituent or else undergo a bio-alkylation substitution reaction in vitro and in vivo. Here we report that the weak carcinogen benz[a]anthracene undergoes meso-anthracenic methylation by S-adenosyl-L-methionine (SAM), in the presence of a rat liver cytosol preparation, in vitro, to form DMBA and presumably the moderately active carcinogens 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene. These compounds are substrates for further L-region methylation to form the strong carcinogen, DMBA.

Benzylic carbonium ions as ultimate carcinogens of polynuclear aromatic hydrocarbons

Abstract Perturbational molecular orbital calculations were applied to a series of polynuclear aromatic hydrocarbons (PAH) to assess the energetics involved in generating electrophilic carbonium ions of two types, triol and benzylic carbonium ions. ΔE β , an index of carbonium ion formation and stability, was nearly always higher for benzylic carbonium ions attached to mesoanthracenic positions of PAHs than for terminal ring triol carbonium ions. Furthermore, the reactivity index N t for substitution reactions leading to benzylic carbonium ions was lowest in each molecule at the site corresponding to the most favorable ΔE β value. No similar relationship existed for ortholocalization energies of bonds at which addition reactions generate triol carbonium ions. Statistical analysis revealed that Iball indices for carcinogenicity showed higher correlation with both benzylic-relevant ΔE β and N t values than with triol carbonium ion ΔE β or ortholocalization energies. Various authors claim correlation between the energetics of triol carbonium ion generation and carcinogenicity, yet our observations along similar lines of reasoning demonstrate even stronger correlation and more favorable energetics for benzylic carbonium ions that require initial methylation at an exceptionally reactive center. Based on our calculations, we predict that animal tests, such as repeated application to mouse skin or repeated subcutaneous injection in mice or rats, should reveal benzylic carbonium ions arising from hydroxymethyl sulfate ester metabolites to be endowed with more complete carcinogenic potency than triol carbonium ions arising from dihydrodiol epoxide metabolites.

Bioalkylation of dibenz[a,b]anthracene in rat liver cytosol

Previous studies by other investigators have established that L-region methyl derivatives of dibenz[a,h]anthracene (DBA) were more carcinogenic than the parent hydrocarbon. The bioalkylation of DBA was investigated by incubating the hydrocarbon with rat liver cytosol fortified with S-adenosyl-L-methionine (SAM) in 0.1 M phosphate buffer (pH 7.4) for 1 h at 37 degrees C in air. The reaction was stopped by the addition of cold acetone and the mixture extracted with ethyl acetate and washed with water. The organic phase was evaporated and the residue dissolved in methylene chloride for analysis by reverse phase high performance liquid chromatography (HPLC) and gas chromatography/mass spectroscopy GC/MS. Products were found that were indistinguishable from 7-methyl-DBA and 7,14-dimethyl-DBA, 7-hydroxymethyl-DBA, 7-hydroxymethyl-14-methyl-DBA, and 7,14-dihydroxymethyl-DBA. The results suggest that unsubstituted carcinogenic hydrocarbons are preprocarcinogens that react with SAM in liver cytosol preparations, to form alkyl substituted procarcinogens, which are more potent than the corresponding preprocarcinogens.

Binding of 6-hydroxymethylbenzo[a]pyrene and 6-ace-toxymethylbenzo[a]pyrene to DNA

The carcinogenic hydrocarbons 6-hydroxymethylbenzo[a]pyrene (6-HOCH2-B[a]P) and 6-acetoxymethylbenzo[a]pyrene (6-AcOCH2-B[a]P) were examined for their ability to bind to rat and calf thymus DNA. The data indicate there are no appreciable differences in the amount of binding to the two types of DNA. Non-enzymatic binding of 6-HOCH2-B[a]P was low (5 mumol hydrocarbon/mol DNA P) but 6-AcOCH2-B[a]P was bound to a considerable extent (88.4--97.3 mumol hydrocarbon/mol DNA P). Non-enzymatic binding of 6-HOCH2-B[a]P was greatly increased in the presence of ATP. Binding of 6-HOCH2-B[a]P in the presence of liver microsomes from untreated rats or from rats pretreated with 3-methylcholanthrene (3-MC) never exceeded 5 mumol hydrocarbon/mol DNA P. Binding of 6-HOCH2-B[a]P in the presence of a PAPS generating system was less than non-enzymatic binding mediated by ATP and was dependent on the presence of ATP rather than ATP and sulfate. Binding was reduced by 50% when ADP was employed in the non-enzymatic reaction and was negligible in the presence of AMP or adenosine, indicating that a diphosphate group is necessary. Incubation of 6-HOCH2-B[a]P with DNA in the presence of ATP, CTP, GTP, or UTP showed that ATP was the most effective mediator of the binding reaction. These observations suggest that 6-HOCH2-B[a]P is converted to a phosphate ester which, like 6-AcOCH2-B[a]P, is much more reactive than 6-HOCH2-B[a]P itself.

MOLECULAR MODELING OF CARCINOGENIC POTENTIAL IN POLYCYCLIC HYDROCARBONS

Abstract The generality and validity of rules of molecular geometry for predicting the carcinogenic potential of unsubstituted polynuclear aromatic hydrocarbons (PAHs) were further tested. These rules are based on the number and location of sites for the electrophilic substitution in unsubstituted PAHs. When the rules of molecular geometry are applied in the absence of specific theoretical or experimental information, they have been shown to predict carcinogenic potential with a high degree of accuracy. However, the accuracy of the rules is even further improved when additional information about the number and location of sites for electrophilic substitution is examined. A theoretical method that probes for such exceptionally reactive centers has been provided by the molecular orbital calculations of Dewar and Dennington. Calculations of this sort have been applied with the rules in order to make predictions of carcinogenic potential in 90 alternant and nonalternant PAHs. These predictions, with very few exceptions, were in good agreement with currently available carcinogenicity test data. It is concluded that a relationship exists between carcinogenic potential, molecular geometry, and the relative energies for electrophilic substitution of the individual carbons in unsubstituted PAHs.

Oxidative metabolism of 7-methylbenz[a] anthracene, 12-methylbenz [a] anthracene and 7,12-dimethylbenz [a]-anthracene by rat liver cytosol

Earlier studies from this laboratory demonstrated that benz[a]anthracene (BA), 7-methylbenz[a]anthracene (7-MBA) and 12-methylbenz[a]anthracene (12-MBA) undergo a bio-alkylation substitution reaction in the meso-anthracenic position(s) or L-region leading to the biosynthesis of the potent carcinogen 7,12-dimethylbenz[a]anthracene (7,12-DMBA). These results support the hypothesis that for most, if not all, unsubstituted polycyclic aromatic hydrocarbon carcinogens, the chemical or biochemical introduction of an alkyl group in the meso-anthracenic position(s) or L-region is a structural requirement for strong carcinogenic activity. Here we report that the L-region methyl derivatives 7-MBA, 12-MBA and 7,12-DMBA are oxidized to hydroxymethyl derivatives by a rat liver cytosol preparation without any apparent oxidation of the ring positions.

Methyl-substitution of benzene and toluene in preparations of human bone marrow

The metabolism of benzene and toluene was investigated in preparations of human bone marrow incubated with S-adenosyl-L-methionine. Benzene undergoes a methyl-substitution reaction to yield toluene as a metabolite. Furthermore, toluene undergoes methyl-substitution in preparations of human bone marrow incubated with S-adenosyl-L-methionine to yield o-xylene, m-xylene, and p-xylene. Metabolites were detected by gas chromatography and mass spectroscopy. No metabolism of either benzene or toluene was detected when a boiled bone marrow preparation was used in the incubation, demonstrating the enzymatic nature of the S-adenosyl-L-methionine dependent methylation of both benzene and toluene.

Bioalkylation of benz[a]anthracene, 7-methylbenz[a]anthracene, and 12-methylbenz[a]anthracene in rat lung cytosol preparations.

Benz[a]anthracene (BA) and the monomethyl meso-anthracenic or L-region derivatives 7-methylbenz[a]anthracene (7-methylBA) and 12-methylbenz[a]anthracene (12-methylBA) underwent a bioalkylation substitution reaction in rat lung ctyosol preparations, fortified with S-adenosyl-L-methionine to form the more potent carcinogen 7,12-dimethylbenz[a]anthracene. The methyl groups of the highly reactive L-region methylated metabolites also underwent enzymatic hydroxylation in rat lung cytosol preparations to yield the corresponding hydroxymethyl derivatives, 7-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, and 7,12-dihydroxymethylbenz[a]anthracene. The biooxidation reaction took place enzymatically, and exclusively, or nearly so, at the reactive methyl groups attached to the meso positions or L-region of the hydrocarbon. Bioalkylation and biooxidation reactions did not occur when the hydrocarbons were incubated with a boiled cytosol preparation, indicating the need for enzymatic activation of the L-region methyl groups. Also, the bioalkylation reaction did not occur in the absence of S-adenosyl-L-methionine. Furthermore, the S-adenosyl-L-methionine-dependent reaction was inhibited by S-adenosyl-L-homocysteine, suggesting that the reaction is catalyzed by a cytosolic S-adenosyl-L-methionine-dependent methyltransferase.