Alan Hewer

Formation of K-region epoxides as microsomal metabolites of pyrene and benzo[a]pyrene

Abstract Epoxides of pyrene and benzo[a]pyrene have been detected for the first time as microsomal metabolites of these polycyclic hydrocarbons. The epoxides, which have been identified as the K-region derivatives, pyrene 4,5-oxide and benzo[a]pyrene 4, 5-oxide respectively, were formed by the NADPH-dependent mixed function oxidase of a rat-liver microsomal incubation system where the epoxide hydrase was inhibited. In the absence of hydrase inhibition, benzo[a]pyrene was converted into a metabolite with the Chromatographic properties of a K-region dihydrodiol, trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, which has not previously been described as a metabolite of this hydrocarbon. Pyrene 4,5-oxide and benzo[a]pyrene 4,5-oxide rearrange in acid to compounds with the properties of 4-pyrenol and 4-benzo[a]pyrenol respectively, are converted by microsomal epoxide hydrase into compounds indistinguishable from the corresponding 4, 5-dihydro-4,5-dihydroxy derivatives and react with glutathione to yield conjugates. Both epoxides are reactive towards polyguanylic acid. The significance of these results is discussed in relation to our hypothesis that polycyclie hydrocarbon carcinogenesis results from somatic mutations caused by epoxides that are formed from the hydrocarbons by metabolism.

Tumour necrosis factor-alpha mediates tumour promotion via a PKC alpha- and AP-1-dependent pathway.

Tumour necrosis factor-α (TNF-α) deficient mice (TNF-α−/− mice) are resistant to skin carcinogenesis. Cellular signalling via the transcription factor complex AP-1 is thought to play a key role in tumour promotion. The induction of a specific subset of AP-1 responsive genes thought to be important for tumour development, namely GM–CSF, MMP-9 and MMP-3, was suppressed in TNF-α−/− compared to wild-type mouse skin in response to the tumour promotor TPA. The differential induction of these genes correlated with a temporal shift in AP-1 activation and c-Jun expression in TNF-α−/− compared to wild-type epidermis. The major receptor for TPA-induced signalling in basal keratinocytes, PKCα, was also differentially regulated in wild-type compared with TNF-α−/− epidermis. A marked delay in TPA-induced intracellular translocation and downregulation of PKCα was observed in TNF-α−/− epidermis, which correlated with the deregulated TPA-induced AP-1 activation and c-Jun expression. The frequency of DNA adduct formation and c-Ha-ras mutations was the same in wild-type and TNF-α−/− epidermis after DMBA treatment, suggesting that TNF-α was not involved in tumour initiation. These data suggest that the pro-inflammatory cytokine TNF-α is a critical mediator of tumour promotion, acting via a PKCα- and AP-1-dependent pathway. This may be one mechanism by which chronic inflammation increases susceptibility to cancer.

Monitoring occupational exposure to carcinogens: detection by 32P-postlabelling of aromatic DNA adducts in white blood cells from iron foundry workers

Blood samples were volunteered by workers in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. DNA was isolated from peripheral white blood cells and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1. The DNA digest was then incubated with [gamma-32P]ATP and polynucleotide kinase. Aromatic adducts present in the digest that were resistant to nuclease P1 were thus 32P-labelled while unmodified nucleotides were not. The 32P-labelled adducts were resolved by t.l.c. and detected by autoradiography. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene (high greater than 0.2, medium 0.05-0.2, low less than 0.05 microgram BP/m3 air). Aromatic adducts were found to be present in DNA from 3/4 samples from the high exposure group, 8/10 samples from the medium exposure group. 4/18 samples from the low exposure group and 1/9 samples from the unexposed controls. The levels of adducts found in the high and medium group samples ranged up to 1 adduct in 10(7) nucleotides but the levels formed in the low exposure group samples were not significantly different from those in unexposed controls. No differences related to the smoking habits of the subjects were observed. Most of the DNA adducts detected had chromatographic mobilities distinct from those formed when the 7,8-diol 9,10-oxide of BP reacted with DNA. The results indicate that highly-exposed individuals are more likely to contain aromatic DNA adducts in their white blood cells, but large interindividual variations were evident. In addition, multiple samples from the same subjects indicate that qualitative and quantitative changes in adduct patterns occur with time. This pilot study suggests that 32P-postlabelling may be useful in monitoring human exposure to known and to previously unidentified environmental genotoxic agents.

Sex differences in risk of lung cancer: Expression of genes in the PAH bioactivation pathway in relation to smoking and bulky DNA adducts

It is controversial whether women have a higher lung cancer susceptibility compared to men. We previously reported higher levels of smoking‐related bulky DNA adducts in female lungs. In a pilot study (27 cases), we also found a higher level of female lung cytochrome P4501A1 (CYP1A1) gene expression. In the present extended study we report on the pulmonary expression of several genes involved in polycyclic aromatic hydrocarbon bioactivation in relation to sex, smoking and DNA adducts. CYP1A1, CYP1B1, aryl hydrocarbon receptor and microsomal epoxide hydrolase gene expression was measured by quantitative real‐time reverse transcriptase‐PCR in 121 normal lung tissue samples. The expression of CYP1A1 and CYP1B1 was significantly higher among current smokers compared to ex‐smokers and never‐smokers. Among current smokers, females had a 3.9‐fold higher median level of CYP1A1 compared to males (p = 0.011). CYP1B1 expression was not related to sex. Lung DNA adducts (measured by 32P‐postlabeling) were highly significantly related to CYP1A1 (p < 0.0001) irrespective of smoking‐status. Our results are consistent with the hypothesis that CYP1A1 plays a significant role in lung DNA adduct formation and support a higher susceptibility to lung cancer among females.

The metabolic activation of 7-methylbenz(a)anthracene in mouse skin

The metabolism of 7-methylbenz(a)anthracene by rat-liver preparations and by mouse skin has been studied using a combination of thin-layer and high pressure liquid chromatography and all five possible trans-dihydrodiols have been detected as metabolites but in different proportions. The roles of these dihydrodiols and of the related vicinal diol-epoxides in the metabolic activation of 7-methylbenz(a)anthracene in mouse skin has been studied using Sephadex LH-20 column chromatography. The results show that the hydrocarbon-nucleic acid products formed in mouse skin in vivo most probably arise from 3,4-dihydro-3,4-dihydroxy-7-methylbenz(a)anthracene 1,2-oxide which, on the basis of this and other evidence, appears to be the reactive intermediate involved in the metabolic activation of 7-methylbenz(a)anthracene in this tissue.

32P-postlabeling analysis of DNA adducts.

(32)P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of the DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of (32)P-orthophosphate from [γ-(32)P]ATP; chromatographic separation of labeled adducts; and detection and quantification by means of their radioactive decay. Using 10 μg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 10(9)-10(10) normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair.

Detection of bulky DNA lesions in the liver of patients with Wilson's disease and primary haemochromatosis

In the human metal storage disorders of Wilsons disease and primary haemochromatosis, ion transport and excretion dysfunctions result in the intracellular deposition of copper and iron, respectively. These aberrant accumulations of transition metal ions lead to extensive tissue damage, especially in the liver. In order to investigate the possible role of metal ion-mediated oxygen free radical-generated DNA damage in these processes, DNA was isolated from liver of eight Wilsons disease patients and six haemochromatosis patients. Significant levels of bulky DNA damage were detected in these samples by 32P-postlabelling analysis, but were not found in liver DNA from age-matched controls. This form of novel DNA damage was detected in six out of eight Wilsons patients, varying between approximately 1 and 100 base modifications per 10(8) nucleotides, and in all of the haemochromatosis samples examined; the levels of modified species per 10(8) nucleotides varying from approximately 2 to 50. HPLC analysis of these bulky DNA lesions demonstrated that the species formed in Wilsons disease and in haemochromatosis were chromatographically identical but were not the same as putative purine dimers that can be generated in DNA by in vitro incubation with Cu+/Fe2+ and H2O2 (although the possibility that the adducts detected are closely related has not been ruled out). Analysis of the oxidative base lesion 8-hydroxydeoxyguanosine showed that levels were not elevated in liver DNA from either Wilsons disease or haemochromatosis sufferers. In fact, a statistically significantly lower level of this lesion was found in Wilsons disease patients than in controls. These data suggest that bulky DNA damage present in the liver of both wilsons disease and primary haemochromatosis patients may play a more important role in the induction of tissue damage than 8-hydroxydeoxyguanosine. The novel DNA damage detected by 32P-poslabelling may also be a significant factor in the initiation of neoplasia leading to malignant hepatoma in haemochromatosis patients.

DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats.

Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust, 3-nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspected human carcinogen forming multiple DNA adducts in vitro. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA), and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) were identified as 3-NBA metabolites. In order to gain insight into the pathways of metabolic activation leading to 3-NBA-derived DNA adducts we treated Wistar rats intraperitoneally with 2mg/kg body weight of 3-NBA, 3-ABA, 3-Ac-ABA, or N-Ac-N-OH-ABA and compared DNA adducts present in different organs. With each compound either four or five DNA adduct spots were detected by TLC in all tissues examined (lung, liver, kidney, heart, pancreas, and colon) using the nuclease P1 or butanol enrichment version of the 32P-postlabelling method, respectively. Using HPLC co-chromatographic analysis we showed that all major 3-NBA-DNA adducts produced in vivo in rats are derived from reductive metabolites bound to purine bases and lack an N-acetyl group. Our results indicate that 3-NBA metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) undergo several biotransformations and that N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be the common intermediate in 3-NBA-derived DNA adduct formation. Therefore, 3-NBA-DNA adducts are useful biomarkers for exposure to 3-NBA and its metabolites and may help to identify enzymes involved in their metabolic activation.

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 formation of "K-region" epoxides as hepatic microsomal metabolites of 7-methylbenz(a)anthracene and 7,12-dimethylbenz(a)anthracene and their 7-hydroxymethyl derivatives.

Abstract Epoxides have been detected as metabolites of 7-methylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene and their 7-hydroxymethyl derivatives using rat-liver niicrosomal systems. The metabolites, which are formed by the microsomal NADPH-dependent mixed-function oxidase, were detected in extracts of incubation mixtures that contained an inhibitor of the enzyme “epoxide hydrase”. The epoxide metabolites were converted (a) by “epoxide hydrase” into products that were chromatographically identical with the related “K-region” trans -dihydrodiols, (b) by acid into products with the chromatographic properties of the related “K-region” phenols and (c) by reaction with GSH into products with the Chromatographic properties of GSH conjugates. The identification of three metabolites as “K-region” epoxides was confirmed by recrystallization of the radioactive metabolites to constant specific activity in the presence of the appropriate unlabelled “K-region” epoxide. In common with other “K-region” epoxides, the epoxide metabolites were found to react with polyguanylic acid. When tested in Sprague-Dawley rats, the “K-region” epoxide of 7-hydroxymethyl-12-methylbenz[a] anthracene did not induce adrenal necrosis. cis -5,6-Dihydro-5,6-dihydroxy-7-methylbenz [a]anthracene and cis -5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene were each metabolized by rat liver homogenates to the related 7-hydroxymethyl derivative.