Alan D. MacNicoll

The formation of dihydrodiols by the chemical or enzymic oxidation of benz[a]anthracene and 7,12-dimethylbenz[a]anthracene

When benz[a] anthracene was oxidised in a reaction mixture containing ascorbic acid, ferrous sulphate and EDTA, the non-K-region dihydrodiols, trans-1,2-dihydro-1,2-dihydroxybenz[a] anthracene and trans-3,4-dihydro-3,4-dihydroxybenz[a] anthracene together with small amounts of the 8,9- and 10,11-dihydrodiols were formed. When oxidised in a similar system, 7,12-dimethylbenz[a] anthracene yielded the K-region dihydrodiol, trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and the non-K-region dihydrodiols, trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-10,11-dihydro-10,11-dihydroxy-7,12-dimethylbenz[a] anthracene and a trace of the 1,2-dihydrodiol. The structures and sterochemistry of the dihydrodiols were established by comparisons of their UV spectra and chromatographic characteristics using HPLC with those of authentic compounds or, when no authentic compounds were available, by UV, NMR and mass spectral analysis. An examination by HPLC of the dihydrodiols formed in the metabolism, by rat-liver microsomal fractions, of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene was carried out. The metabolic dihydriols were identified by comparisons of their chromatographic and UV or fluorescence spectral characteristics with compounds of known structures. The principle metabolic dihydriols formed from both benz[a] anthracene and 7,12-dimethylbenz[a] anthracene were the trans-5,6- and trans-8,9-dihydrodiols. The 1,2- and 10,11-dihydrodiols were identified as minor products of the metabolism of benz [a] anthracene and the tentative identification of the trans-3,4-dihydriol as a metabolite was made from fluorescence and chromatographic data. The minor metabolic dihydriols formed from 7,12-dimethylbenz[a] anthracene were the trans-3,4-dihydrodiol and the trans-10,11-dihydriol but the trans-1,2-dihydrodiol was not detected in the present study.

The metabolism of a series of polycyclic hydrocarbons by mouse skin maintained in short-term organ culture

Investigations on the metabolism of 3H-labelled chrysene, benz[a]anthracene, 7-methylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, dibenz[a,c]anthracene and dibenz[a,h]anthracene by mouse skin maintained in short-term organ culture were carried out. Estimations of the distribution of the metabolites of each hydrocarbon present after 24 h showed that there were wide variations both in the rates at which the hydrocarbons were metabolised and in the amounts of metabolites covalently bound to skin macromolecules. All the hydrocarbons were metabolised to dihydrodiols, which were identified by comparison on high pressure liquid chromatography (HPLC) with the authentic compounds, and these were the same diols as those that were formed in previous experiments with rat-liver microsomal fractions. However, free dihydrodiols represented only relatively small proportions of the total amounts of metabolites formed. All the hydrocarbons yielded dihydrodiols of the type that could give rise to bay-region diol-epoxides, when further metabolised, some of which are thought to be involved in hydrocarbon carcinogenesis.

A comparison of warfarin resistance and liver microsomal vitamin K epoxide reductase activity in rats

Vitamin K-1 epoxide reductase activity was investigated in liver microsomal preparations from warfarin-resistant and -susceptible rats. One rat strain (TAS) is susceptible to the anticoagulant and lethal effects of warfarin and the other two strains are homozygous for warfarin resistance genes from either wild Welsh (HW) or Scottish (HS) rats. The enzyme in microsomal preparations from HW rat livers apparently has a reduced affinity for both warfarin and vitamin K-1 2,3-epoxide. The kinetic parameters for the enzyme activity in HS microsomal preparations indicated, however, that vitamin K-1 epoxide reductase in this warfarin-resistant strain was very similar, in respect of substrate and inhibitor affinities, to that prepared from susceptible (TAS) animals. Analysis of vitamin K-1 epoxide reductase activity in the livers of animals that had been orally treated with sodium warfarin (20 mg/kg body wt.) indicated that enzyme activity was inhibited in all three strains, although this dose is lethal only to animals of the TAS strain.

The development of a blood clotting response test for discriminating between difenacoum-resistant and susceptible Norway rats (Rattus norvegicus, berk.)

1. A new test for identifying levels of difenacoum resistance in the Norway rat is described, based upon the differential physiological response to difenacoum administration. 2. This test is based on changes in blood clotting activity over 4 days, following administration of the rodenticide difenacoum in conjunction with menadione (vitamin K3). 3. The anticoagulant effect is reduced only in rats that are resistant or tolerant to difenacoum. 4. This test procedure is quicker than traditional feeding tests, and identifies the degree of resistance in both laboratory and wild rats that have difenacoum resistance genes.

The involvement of a non-‘bay-region’ diol-epoxide in the metabolic activation of benz[a]anthracene in hamster embryo cells

The major hydrocarbon-nucleoside adduct present in hydrolysates of DNA from hamster embryo cells that had been treated with 3H-labelled benz[a]anthracene in culture has been examined by chromatography on Sephadex LH-20 columns and by high-pressure liquid chromatography. The results show that this adduct most probably arises from r-8,t-9-hydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[a]anthracene (anti-BA-8,9.-diol 10,11-oxide). On the basis of this and other evidence, this non-bay-region diol-epoxide appears to be a reactive intermediate involved in the metabolic activation of benz[a]anthracene.

The formation of dihydrodiols in the chemical or enzymic oxidation of dibenz[a,c]anthracene, dibenz[a,h]-anthracene and chrysene.

The formation of trans-dihydrodiols from dibenz[a,c]anthracene, dibenz[a,h]anthracene and chrysene by chemical oxidation in an ascorbic acid-ferrous sulphate-EDTA system and by rat-liver microsomal fractions has been studied using a combination of thin-layer (TLC) and high pressure liquid chromatography (HPLC) to separate the mixtures of isomeric dihydrodiols. The 1,2- and 3,4-dihydrodiols of dibenz[a,c]anthracene, the 1,2-,3,4- and 5,6-dihydrodiols of dibenz[a,h]anthracene and the 1,2-, 3,4- and 5,6-dihydrodiols of chrysene were formed in chemical oxidations. These dihydrodiols were also formed when the three parent hydrocarbons were metabolized by rat-liver microsomal fractions and, in addition, dibenz[a,c]anthracene yielded the 10,11-dihydrodiol. The 1,2- and 3,4-dihydrodiols of dibenz[a,c]anthracene have not been reported previously either as metabolites of the hydrocarbon or as products of chemical syntheses and the 5,6-dihydrodiol of chrysene was not detected in earlier metabolic studies.

The formation of dihydrodiols by the chemical or enzymic oxidation of 7-hydroxymethyl-12-methylbenz[a] anthracene and the possible role of hydroxymethyl dihydrodiols in the metabolic activation of 7,12-dimethylbenz[a]anthracene

The formation of dihydrodiols from 7-hydroxymethyl-12-methylbenz[alpha]anthracene by rat-liver microsomal fractions, by mouse skin in short-term organ culture and by chemical oxidation in an ascorbic acid/ferrous sulphate/EDTA system has been studied using a combination of thin-layer chromatography and high pressure liquie chromatography. The 3,4-, 8,9- and 10,11-dihydrodiols were formed in all three systems. The 5,6-dihydrodiol was formed in rat-liver microsomal fractions and in chemical oxidation but was not detected as a metabolite of [7-3H]hydroxymethyl-12-methylbenz[alpha]anthracene when this compound was incubated with mouse skin in short-term organ culture. The possible role of hydroxymethyl dihydrodiols in the in vivo metabolic activation of 7,12-dimethylbenz[alpha]anthracene in mouse skin has been studied using Sephadex LH-20 column chromatography. The results show that the hydrocarbon-nucleic acid products formed following the treatment of mouse skin in vivo with [7,12-3H]dimethylbenz[alpha]anthracene are not the same as those that are formed following the treatment of mouse skin under the same conditions with either 7-hydroxymethyl-12-methylbenz[alpha]anthracene or 7-methyl-12-hydroxymethylbenz[alpha]anthracene.

The metabolic activation of benz[a]anthracene in three biological systems

The 3,4- and 8,9-dihydrodiols of benz[alpha]anthracene (BA) are formed as metabolites of the parent hydrocarbon by rat-liver microsomes, by mouse skin and by hamster embryo cells. In incubations with rat-liver microsomal fractions, only small amounts of the 3,4-dihydrodiol of BA were detected relative to other dihydrodiol metabolites and only small amounts of BA-deoxyribonucleoside adducts derived from the related diol-epoxide, t-3, r-4-dihydroxy-t-1,2-oxy-1,2,3,4-tetrahydrobenz[alpha]anthracene (anti-BA-3,4-diol 1,2-oxide), were detected relative to adducts derived from r-8,t-9-dihydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[alpha]anthracene (anti-BA-8,9-diol 10,11-oxide). However, in studies with mouse skin and hamster embryo cells, larger amounts of free 3,4-dihydrodiol were detected and a larger proportion of the hydrocarbon-deoxyribonucleoside adducts resulted from the reaction of anti-BA-3,4-diol 1,2-oxide with DNA.

Comparison of the half-lives and regeneration rates of blood clotting factors II, VII, and X in anticoagulant-resistant and susceptible Norway rats (Rattus norvegicus Berk.)

The half-lives and regeneration rates of clotting factors II, VII, and X in the plasma of anticoagulant-resistant and susceptible rats were determined. There is little or no difference in the half-lives of factors II and X in anticoagulant-resistant rats compared to susceptible rats, but the half-life of factor VII is longer in anticoagulant-resistant rats. In anticoagulant-resistant rats critical clotting factors appear to be carboxylated in preference to factor II, whereas the opposite occurs in susceptible rats; this may contribute to an animals resistance status.

Inhibition by warfarin of liver microsomal vitamin K-reductase in warfarin-resistant and susceptible rats

The NADH-dependent vitamin K-reductase activity of liver microsomes from three closely related rat strains has been studied. One strain (TAS) is susceptible and two strains (HW and HS) resistant to the anticoagulant and lethal effects of warfarin. The effects of cofactors, temperature, detergent and dithiothreitol on vitamin K1 reduction and solvent extraction of substrate and product have been investigated. Vitamin K-reductase activity was inhibited by approximately 13 and 8% respectively when microsomal preparations from TAS and HW animals were incubated with 50 microM vitamin K1 and 10 microM warfarin. In HS rat liver microsomes the enzyme was highly resistant to inhibition by warfarin. Evidence is presented and discussed that suggests that NADH-dependent vitamin K-reductase may be inhibited in the anticoagulant effect of warfarin and may be altered as a result of expression of the warfarin-resistance gene in HS rats. The enzyme activity studied was probably not a DT-diaphorase although both NADH and NADPH acted as cofactors for the reaction.