Bay-region distortions in a methanol adduct of a bay-region diol epoxide of the carcinogen 5-methylchrysene

The three-dimensional structure of the product of the reactionof a diol epoxide of the carcinogen 5-methyl-chrysene with methanolhas been determined by an X-ray diffraction analysis. The diolepoxide used to obtain this compound contains a stereochemicallyhindered bay region because of the location of the 5-methylgroup, and this might be expected to affect the type of chemicalreaction that occurs. The crystal structure analysis of thisadduct of a polycyclic aromatic hydrocarbon (PAH) showed thata methoxy group has been added at the carbon atom of the epoxygroup that is nearest to the aromatic system. The bond thatis formed is axial to the ring system so that the carbon andhydrogen atoms of the methoxy group are considerably displacedfrom the PAH ring plane. The bay-region methyl group at position5 is displaced out of the ring plane in the opposite direction.The major steric distortion in this methanol adduct is shown,by a comparison with crystal structures of related non-methylatedcompounds, to be in the area of the 5-methyl group and not inthe tetrol-bearing ring. The steric effects that caused theaxial conformation of the newly formed bond would also be expectedto pertain in the DNA adduct of a PAH with a bay-region methylgroup. Since the presence of the bay-region methyl group in5-methylchrysene has been shown to enhance the carcinogenicityof this PAH over the parent compound or compounds with methylgroups in other positions of the molecule, it might be anticipatedthat this axial bond is found in carcinogenic lesions in DNA,and that any factor that ensures this axial conformation mayaccentuate the carcinogenic potential of a PAH of the appropriatesize.     

Characteristics of benzo[a]pyrene and A-ring reduced 7,12-dimethyl benz[a]anthracene induced neoplastic transformation of human cells in vitro

The polynuclear aromatic hydrocarbons (PAH) benzo[a]pyrene (BP) and the A-ring reduced analogue of 7,12-dimethylbenz[a]anthracene (DMBA), 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene (TH-DMBA) are carcinogenic to human cells. The unsaturated PAH, DMBA exhibits no carcinogenic activity on human cells as measured by growth in soft agar. The TH-DMBA and BP treated cells exhibit a colony frequency in soft agar of 84 and 86, respectively. These anchorage independent cells, when seeded on the chick embryonic skin (CES) organ cultures, are invasive and form a fibrosarcoma. It is highly unlikely that TH-DMBA, which does not contain an aromatic A-ring, can undergo metabolism in human cells in culture to form a bay region 3,4-dihydrodiol-1,2-epoxide. These results suggest that an alternate mechanism for the induction of carcinogenesis is appropriate to explain the absence of bay region diol-epoxide metabolite as the ultimate form of the carcinogen in TH-DMBA induced carcinogenesis in human diploid cells.     

DNA adducts and personal air monitoring of carcinogenic polycyclic aromatic hydrocarbons in an environmentally exposed population

The effect of personal exposure to air pollution on DNA adductsin humans was analyzed in a group (n = 30) of women workingoutdoors uo to 8 h/day) as postal workers or gardeners in thecity of Teplice, Czech Republic (Northern Bohemia), where winterinversions may result in high levels of air pollution from coalcombustion. Ten of these women were followed up during the nextwinter season by repeated personal exposure monitoring and analysisof the DNA adducts in their white blood cells (in four samplingperiods). Personal exposure monitoring for respirable particles(<2.5 µm) was conducted for the 24 h period prior tocollection of blood and urine. Particle extracts were analyzedfor carcinogenic polycyclic aromatic hydrocatbons (PAH). Urinesamples were collected for cotinine analysis to control forexposure to tobacco smoke. DNA isolated from white blood cellswas analyzed by ³²P-postlabeling with the butanol enrichmentprocedure. There were 21 non-smokers and nine light smokersin the pilot study (November 1992) and only non-smokers in thefollow-up study (winter season 1993/94). In both studies highpersonal exposure variability between the individuals sampledon the same day was observed. In the pilot study we found asignificantly higher (P < 0.05), level of DNA adducts inthe 14 non-smoking women sampled on November 24, when theirexposure to carcinogenic PAH was also significantly higher (P< 0.05), compared with seven non-smoking women sampled onNovember 26. We also found a significant correlation (r = 0.541,P < 0.016) between individual exposure to carcinogenic PAHand DNA adducts for the group of non-smoking (n = 21). No significantdifference in DNA adduct levels was found between non-smokersand smokers. In the follow-up study, during one sampling periodthe ambient and personal air monitors exhibited a significantlyelevated exposure to respirable particles and carcinogenic PAH.Analyzing data from the follow-up study, a significant effectof personal exposure on DNA adduct levels and their relationshipwith short-term exposure to carcinogenic PAH was found. Theresults suggest that DNA adduct levels in white blood cellsreflect a short-term environmental exposure.     

Association between polycyclic aromatic hydrocarbon-DNA adduct levels in maternal and newborn white blood cells and glutathione S-transferase P1 and CYP1A1 polymorphisms.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants; a number are carcinogenic. Metabolic polymorphisms may modulate susceptibility to PAH-induced DNA damage and carcinogenesis. This study investigates the relationship between PAH-DNA adduct levels (in maternal and newborn WBCs) and two polymorphisms: (a) an MspI RFLP in the 3' noncoding region of cytochrome P4501A1 (CYP1A1); and (b) an A-->G transition in nucleotide 313 of glutathione S-transferase P1 (GSTP1), resulting in an ile105val substitution. CYP1A1 catalyzes the bioactivation of PAH; the CYP1A1 MspI RFLP has been associated with cancer of the lung. GSTP1 catalyzes the detoxification of PAH; the val allele has greater catalytic efficiency toward PAH diol epoxides. The study involves 160 mothers and their newborns from Poland. Regression models controlled for maternal smoking and other confounders. No association was seen between maternal adduct levels and either polymorphism, separately or combined. However, adduct levels were higher among newborns with the CYP1A1 MspI restriction site (heterozygotes and homozygotes combined) compared with newborns lacking the restriction site (P = 0.06). Adducts were higher among GSTP1 ile/val and ile/ile newborns compared with GSTP1 val/val newborns (P = 0.08). Adduct levels were 4-fold higher among GSTP1 ile/ile newborns having the CYP1A1 restriction site compared with GSTP1 val/val newborns who lacked the CYP1A1 restriction site (P = 0.04). This study demonstrates a significant combined effect of phase I and phase II polymorphisms on DNA damage from PAHs in fetal tissues. It illustrates the importance of considering interindividual variation in assessing risks of transplacental exposure to PAHs.     

Selective interactions of cytochromes P-450 with the hydroxymethyl derivatives of 7, 12-dimethylbenz[a]anthracene

Competition between a hydroxylated metabolite and the parentpolycyclic aromatic hydrocarbon (PAH) for metabolism at cytochromesP-450 may result in the generation of hydroxylated dihydrodiolepoxides. The effectiveness of the competition between 7-hydroxymethyI-12-methylbenz[a]-anthracene(7HOMMBA) or 12-hydroxymethyl-1–7-methyl-benz[a]anthracene(12HOMMBA) and 7, 12-dimethylbenz[a]-anthracene (DMBA) is highlydependent on the form(s) of cytochrome P-450 in the microsomes.The inhibitory effects of exogenously added 7HOMMBA or 12HOMMBAon DMBA metabolism were 30- to 50-fold greater in 3-methyl-cholanthrene(MC-induced rat liver microsomes (K_i = 0.4 µM) comparedto either uninduced or phenobarbital (PB-induced liver microsomes(K_i = 14 and 11 µM, respectively). Similarly, productinhibition of total DMBA metabolism by metabolites generatedin situ was significant only in MC-induced liver microsomes(K'_i = 2.5 µM). Metabolism of 7HOMMBA in these microsomeswas strongly restricted by an unusual substrate inhibition derivedfrom the inhibitory binding of a second molecule of 7HOMMBA.This same phenomenon was observed with reconstituted cytochromeP-450c but not with PB-induced or uninduced microsomes. Complexformation by binding of DMBA, 7HOMMBA, and 12HOMMBA to purifiedP-450c reconstituted in phospholipid micelles was determinedby optical spectroscopy and fluorescence quenching. Bindingaffinities of both the 7HOMMBA and 12HOMMBA (K_d = 95 and 110nM, respectively), were 2.5-fold higher compared to that ofDMBA (K_d = 265 nM). These results provide a first demonstrationthat hydroxylation of a PAH can lead to preferential metabolismthrough an increased affinity for cytochrome P-450.     

Mutagenic specificity of syn-benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide in the dihydrofolate reductase gene of Chinese hamster ovary cells

Polycyclic aromatic hydrocarbons (PAHs) with sterically hinderedfjord region diol epoxides are interesting with respect to theirpotency as carcinogens, interactions with DNA and mutagenicspecificities. Unlike the bay region PAH derivative, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene(BPDE), reactive metabolites of two fjord region PAH, trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-Phenanthrene[(±)-anti-BcPHDE] and trans-11,12-dihydroxy-syn-13,14-epoxy-11,12,13,14-tetrabenzo[g]chrysene[(±)-syn-BgCDE], react with DNA to yield high levelsof adenine adducts. We previously found that forward mutationsinduced by (±)-anti-BcPHDE in the dihydrofolate reductase(dhfr) gene of Chinese hamster ovary (CHO) cells preferentiallytargeted mRNA splice acceptor sites. (±)-anti-BcPHDEand (±)-syn-BgCDE are structurally similar; they differonly by the presence of an additional benzene ring. Thus weused (±)-syn-BgCDE to learn if the mutational targetbias reflects aspects of the mutagen structure or its capacityto efficiently modify deoxyadenosine (dA) in vivo. dhfr^–mutants were induced after treatment of hemizygous UA21 cellswith a 0.75 µM dose of (±)-syn-BgCDE. Cell survivalafter carcinogen exposure was 40%. The induced mutation frequencywas 9x10^–6, nearly 10-fold higher than the spontaneousone, but     

Multi-step metabolism of the carcinogen dibenzo[a,e]fluoranthene. II. Metabolic pathways

The structural identification of nineteen metabolites of dibenzo[a,e]fluoranthene(DBF) obtained by incubation in rat and mouse liver microsomes,allows one to establish a qualitative and semi-quantitativemetabolic chart, involving up to three distinct oxidative attacks.The primary steps lead to dihydrodiols on rings A and D andphenols on rings A and E. Secondary vicinal epoxidation of dihydrodiolsis a minor route as compared to attack at a second peripheralring. Even after a third oxidation, one of the peripheral ringsA, D and E remains unsubstituted. A model for cytochrome P-450enzymatic activity which takes into account most of the observalionsis proposed. It requires that the catalytic site for monooxygenationis 0.6 nm apart from the center of an hydrophobic protein siteaccommodating one of the unsubstituted peripheral beuzenoidrings. Both trans diequatorial dihydrodiols of ring A and Dcorresponding to the ‘bay’ and ‘pseudo bayregion’ of DBF appear in the activation pathways for thein vivo carcinogenesis. The ultimate metabolite reacting withDNA is thus, most probably, a vicinal dihydrodiol epoxide ofring A or D. The great complexity of the metabolic chart ofDBF as compared to other carcinogenic polycyclic aromatic hydrocarbonsleaves also the possibility of sequential reactions at thesetwo distinct sites of the molecule.     

Effect of arsenic on benzo[a]pyrene DNA adduct levels in mouse skin and lung

Concomitant exposures to arsenic and polycyclic aromatic hydrocarbons(PAHs) such as benzo[a]pyrene (BaP) are widespread. While BaPacts by binding to and inducing mutations in critical siteson DNA, the mechanism(s) of arsenic carcinogenesis remains unknown.Data from epidemiological studies of arsenic copper smelterworkers and arsenic ingestion in drinking water suggest a positiveinteraction for arsenic exposure and smoking and lung cancer.A previous in vitro study showed that arsenic potentiated theformation of DNA adducts at low doses of BaP and arsenic. Thepresent study was conducted to test the effect of arsenic onBaP–DNA adduct formation in vivo. We hypothesized thatarsenic co-treatment would significantly increase BaP adductlevels in C57BL/6 mouse target organs: skin and lung. Treatmentgroups were: five mice, -BaP/-arsenic; five mice, -BaP/+arsenic;15 mice, +BaP/-arsenic; 15 mice, +BaP/+arsenic. Mice in theappropriate groups were provided sodium arsenite in drinkingwater (2.1 mg/l), ad libitum, for 13 days (starting 9 days beforeBaP treatment), and 200 nmol BaP/25 ml acetone (or acetone alone)was applied topically, once per day for 4 days. DNA was extractedfrom skin and lung and assayed by ³²P-postlabeling. Statisticalcomparisons were made using independent t-tests (unequal variancesassumed). BaP–DNA adduct levels in the +BaP groups weresignificantly higher than -BaP controls. Arsenic co-treatmentincreased average BaP adduct levels in both lung and skin; theincrease was statistically significant in the lung (P = 0.038).BaP adduct levels in the skin of individual animals were positivelyrelated to skin arsenic concentrations. These results corroborateour in vitro findings and provide a tentative explanation forarsenic and PAH interactions in lung carcinogenesis.     

Polycyclic aromatic hydrocarbon-DNA adduct formation in prostate carcinogenesis

The evidence for polycyclic aromatic hydrocarbons (PAH) playing a role in prostate carcinogenesis comes mainly from associations between reported PAH exposures and prostate cancer in epidemiologic studies. Associations between prostate cancer and DNA repair genotypes and phenotypes have also been reported, lending further credence to a PAH-induced carcinogenesis pathway in prostate cancer. Recent work that demonstrates the human prostate has metabolic enzyme activity necessary for PAH activation and will form DNA adducts upon exposure to PAH further supports PAH carcinogenesis. We have demonstrated the presence of PAH-DNA adducts in prostate cancer cases, but further validation of this biomarker as a carcinogenic agent in human prostate is needed.     

Covalent DNA adducts formed in mouse epidermis by benzo(g)chrysene

The metabolic activation in mouse skin of benzo(g)chrysene (B(g)C),a moderately carcinogenic polycyclic aromatic hydrocarbon (PAH)present in coal tar, was investigated. Male Parkes mice weretreated topically with 0.5 µmol B(g)C and DNA was isolatedfrom the treated areas of skin at various times after treatmentand analysed by 32P-post-labelling. Seven major adduct spotswere detected, at a maximum level of 6.55 fmol adducts/µgDNA. Mouse skin treated with the PAH benzo(c)phenanthrene (B(c)Ph)gave a total of 0.24 fmol adducts/µg DNA. B(g)C-DNA adductspersisted in skin for at least 3 weeks. Treatment of mice with0.5 µmol of the optically pure putative proximate carcinogens,the (+)- and (–)-trans benzo(g)chrysene-11, 12-dihydrodiols,led to the formation of adducts which co-migrated on TLC andHPLC with those formed in B(g)C-treatedmice, which suggestedthat the detected adducts were formed by the fjord region B(g)C-11,12-dihydrodiol-13, 14-epoxides (B(g)CDEs). To test this, thefour optically pure synthetic B(g)CDEs were reacted in vitrowith DNA and the heteroco-polymers poly(dA.dT) and poly(dG-dC)and these samples 32P-postlabelled. Co-chromatography, onbothTLC and HPLC, of in vitro and in vivo adducts indicated thatB(g)Cis activated in mouse skin through formation of the (–)-anti-(11R,12S,13S,14R) and (+)-syn-(11S, 12R,13S,14R) B(g)CDEs. (–)-anti-B(g)CDEformed five adducts with DNA, two of them with adenine and threewith guanine bases. (+)-syn-B(g)CDE formed one adduct with eachof these bases in DNA. The adenine adducts accounted for 64%of the total major adducts formed in B(g)C-treated mouse skin.The route of metabolic activation of B(g)C is similar to thatreported for B(c)Ph, but the extent of activation to the fjordregion diol-epoxides is significantly greater in the case ofB(g)C, as demonstrated by the higher levels of adduct formationin vivo.     

Arteriosclerotic plaque development is 'promoted' by polynuclear aromatic hydrocarbons

In previous work we found that weekly injections of the polynucleararomatic hydrocarbon (PAH) carcinogen 7,12-dimethylbenz[a]anthracene(DMBA) induced spontaneous aortic plaques in cockerels to growto a larger size and at a faster rate than plaques in controlanimals. To determine whether plaque-stimulating ability isrelated to carcinogenic potency or mutagenicity we have nowtested a variety of agents, including PAH carcinogens, non-PAHcarcinogens and weakly carcinogenic PAHs. Cockerels were injectedweekly (from 4–20 weeks of age) with one of the followingcompounds: benzo[a]pyrene (B[a]P), benzo[e]pyrene (B[e]P), dibenz[a,h]anthracene (AH), dibenz [a,c]anthracene (AC), 3-methylcholanthrene(MCA), acetylaminofluorene (AAF), N-methyl-N,N'-nitro-nitrosoguanidine(MNNG) or anthracene (ANT). Plaques were present in the abdominalaortas of all animals. Plaque volumes were 8–14 timesgreater in AC-, B[a]P-, B[e]P-, MCA- and AH-treated cockerelsthan in controls. Plaques were slightly larger in the AAF-treatedgroup than in control animals, and in the ANT- and MNNG treatedgroups were indistinguishable in size from plaques in controlanimals. The largest plaque volumes were in AH treated cockerelsand were comparable in size to those elicited by DMBA treatment.The accelerated development of plaques is consistent with a‘promotional’role for these agents. There was a poor correlation betweenmutagenicity or carcinogenicity and plaque ‘promotion’,which may reflect a role for different metabolites in theseprocesses.     

Tumor-initiating activity on mouse skin of bay region diol-epoxides of 5,6-dimethylchrysene and benzo[c]phenanthrene

Previous metabolism and DNA-binding studies indicated that 5,6-dimethylchrysene(5,6-diMeC) is metabolically activated in mouse skin throughformation of its 1,2-dihydrodiol (5,6-diMeC-1,2-diol) and bayregion diol-epoxide (anti-5,6-diMeC-1,2-diol-3,4-epoxide). Thesemetabolites were tested as tumor initiators on mouse skin. Includedfor comparison were syn-5,6-diMeC-1,2-diol-3,4-epoxide and anti-4,3-di-hydroxy-2,1-epoxy-4,3,2,1-tetrahydrobenzo[c]phenanthrene(anti-B[c]Ph-4,3-diol-2,1-epoxide). At an initiating dose of100 nmol/mouse, 5,6-diMeC-1,2-diol and anti-5,6-diMeC-1,2-diol-3,4-epoxidewere significantly more tumorigenic than 5,6-diMeC, inducing7.1 and 3.9 skin tumors per mouse respectively compared to 1.1induced by 5,6-diMeC. Similar results were obtained at an initiatingdose of 33 nmol/mouse. This is the first example of a methylatedpolynuclear aromatic hydrocarbon bay region diol-epoxide whichis more tumorigenic than its parent hydrocarbon on mouse skin.syn-5,6-diMeC-1,2-diol-3,4-epoxide was only weakly tumorigenic.Comparisons of anti-5,6-diMeC-1,2-diol-3,4-epoxide and anti-B[c]Ph-4,3-diol-2,1-epoxidedemonstrated that the latter was a stronger tumor initiator.The results of this study confirm the bay region diol-epoxidemetabolic activation pathway of 5,6-diMeC but do not providean explanation for the relatively weak tumorigenicity of thishydrocarbon on mouse skin.     

32P-Postlabelling analysis of the covalent binding of benzo[ghi]perylene to DNA in vivo and in vitro

Benzo[ghi]perylene (B[ghi]P) is a polycyclic aromatic hydrocarbon(PAH), present in complex combustion products, and evidencefor its carcinogenic activity in experimental animals is equivocal,and yet it has demonstrable mutagenic activity in vitro. Inorder to investigate the possible DNA binding properties ofB[ghi]P, groups of male Parkes mice were treated topically with1.0 µmol of B[ghi]P. Mice were killed up to 3 months aftertreatment, DNA was isolated from the treated areas of skin andanalysed for adducts by ³²P-postlabelling. Maximum levels ofbinding (0.57 fmol/µg DNA) were detected 2 days aftertreatment and adducts were found to persist for at least 12weeks after treatment, a property of many PAHs whh known tumor-initiatingactivity. B[ghi]P also became bound to DNA in vitro in the presenceof 3-methylcholanthrene-induced rat liver microsomal preparations.When chromatographed on PEI–cellulose, the major adductsformed by B[ghi]P in vivo and in vitro appeared to be identical.However, they were found to be different when compared by reversed-phaseHPLC. These differences might explain, in part, the differencesin the biological activity of B[ghi]P in vivo and in vitro,.The behaviour of B[ghi]P when present in a mixture was alsoexamined. B[ghi]P was applied topically to mouse skin with sixother PAHs at a dose level of 0.25 µmol/PAH/mouse. DNAisolated 24 h after treatment was analysed for adducts by ³²P-postlabelllng.Whilst the total level of binding was 30% lower than expectedfrom the sum of the binding levels that resulted when the hydrocarbonswere applied singly, the formation of B[ghi]P–DNA adductsdid not appear to be inhibited. The results have demonstratedthat B[ghi]P has significant DNA binding ability in vivo andin vitro and on the basis of its DNA binding ability in mouseskin it would be predicted to be at least a weak tumour initiator.The formation of DNA adducts by B[ghi]P when present in an artificialmixture of PAHs suggests that B[ghi]P may contribute to theDNA binding activity of more complex carcinogenic mixtures.     

Bay- and fjord-region distortions in dibenz[a,j]anthracene and tetrabenzo[de,hi,mn,qr]naphthacene

The crystal structure of 7,14-dimethyldibenz[a,j]anthracene(DMDBA) has been determined, and the crystal structure of tetrabenzo[de,hi,mn,gr]naphthacene(TBNC) has been redetermined at higher precision than previouslyreported. These molecules are polycyclic aromatic hydrocarbons(PAHs) that have, respectively, two hindered bay regions andtwo fjord regions; the former PAH is a known carcinogen. Theextensive out-of-plane bending as a result of steric overcrowdingin the bay and fjord regions in these PAHs is shown by thesestudies. For DMDBA, the angle between the 14-methyl group andthe outer rings is 32.6°. For TBNC, the angle between theouter rings of the molecule is 31.9°. These structures arecompared with those of related structures of 7,12-dimethylbenz[a]anthraceneand dibenzo[g,p]chrysene. It appears that steric overcrowdingin such PAHs can cause distortions of up to 33°C. Such stericovercrowding will affect the conformations of bay- and fjord-regiondiolepoxides, which are the presumed activated metabolites inthe carcinogenic process.     

r-1,t-2,3,c-4-Tetrahydroxy-1,2,3,4-tetrahydrophenanthrene in human urine: a potential biomarker for assessing polycyclic aromatic hydrocarbon metabolic activation.

Individual differences in the metabolic activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons (PAHs) may influence cancer risk. This has been investigated in many studies using genotyping approaches, but the results to date have been inconsistent. We propose that carcinogen metabolite phenotyping would be a more reliable way to determine the role of host metabolism in PAH-related cancer. Many PAHs are metabolically activated by formation of bay-region diol epoxides. Phenanthrene, generally considered to be noncarcinogenic, is the simplest PAH with a bay region and is metabolized to diol epoxides by the same enzymes and with the same stereochemistry as the prototypic carcinogenic PAH, benzo[a]pyrene. The major end product of this metabolic activation pathway is r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (trans, anti-PheT). We have developed a method for the analysis of trans, anti-PheT in human urine. r-1,t-2,4,c-3-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (trans, syn-PheT) was used as internal standard. After hydrolysis by beta-glucuronidase and sulfatase, solid phase extraction, and high-performance liquid chromatography collection, the sample was silylated and analyzed by gas chromatography-negative ion chemical ionization-mass spectrometry-selected ion monitoring at m/z 372. The resulting chromatograms were remarkably clean and trans, anti-PheT was readily detected in all human urine samples. Levels of trans, anti-PheT were 791 +/- 363 pmol/mg creatinine (n = 20) in psoriasis patients treated with a PAH-containing ointment, 25.7 +/- 16.8 pmol/mg creatinine (n = 32) in coke oven workers exposed to PAH, 4.58 +/- 2.95 pmol/mg creatinine (n = 31) in smokers, and 1.51 +/- 1.15 pmol/mg creatinine (n = 30) in nonsmokers. Levels of trans, anti-PheT correlated with levels of 1-hydroxypyrene in the urine of coke oven workers, smokers, and nonsmokers. Thus, trans, anti-PheT appears to be an excellent biomarker of PAH uptake. Levels of trans, anti-PheT were 8,000-19,000 times higher than those of the corresponding metabolite of benzo[a]pyrene. The results of this study demonstrate that trans, anti-PheT can be detected in human urine. We propose that measurement of this metabolite of phenanthrene may be important as part of a carcinogen metabolite-phenotyping approach to determine individual response to PAH exposure.     

Anti-benzo[a]pyrene diolepoxide--DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking

The level of (±)-r-7, t-8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene(anti-BPDE) bound to DNA of lymphocytes plus monocytes in 39coke oven workers exposed to polycyclic aromatic hydrocarbons(PAH) and 39 non-exposed persons (controls) were investigated,each of the groups consisting of smokers and non-smokers. Theadduct level was measured by an improved HPLC/fluorescence method(Rojas, M., Alexandrov, K., van Schooten, F.J., Hillebrand,M., Kriek, E. and Bartsch, H., Carcinogenesis, 15, 557–560,1994) through the release of the corresponding benzo[a]pyrene(B[a]P) tetrols. The anti-BPDE-DNA adduct was detected in 51%of coke oven workers exposed to PAH and in 18% of the non-exposed(control) subjects. The mean level of anti-BPDE-DNA adducts/10⁸nucleotides in coke oven workers (15.7 ± 37.8) was     

Dibenzo[A,L]pyrene-induced genotoxic and carcinogenic responses are dramatically suppressed in aryl hydrocarbon receptor-deficient mice

Dibenzo[a,l]pyrene (DB[a,l]P), a notorious air pollutant, is the most powerful carcinogenic polycyclic aromatic hydrocarbon (PAH) ever tested. Although the carcinogenicity of PAH may be primarily mediated by the aryl hydrocarbon receptor (AhR), the in vivo role of AhR in skin carcinogenesis remains to be defined. In this context, we investigated the genotoxic and carcinogenic responses of the AhR-deficient mouse skin to DB[a,l]P. A single painting resulted in a striking epidermal hyperplasia in AhR+/+ mice but not in AhR-/- mice. Bromodeoxyuridine-labeling index and accumulation of p53 protein in epidermal cells of AhR+/+ mice were 8- and 33-fold higher than those of AhR-/- mice, respectively. 32P-Postlabeling assay for DB[a,l]P-DNA adducts displayed a 2-fold increase in the AhR+/+ mouse skin. After DB[a,l]P exposure, AhR-/- mice arranged a nearly 60% reduction in the induction of epidermal cytochrome P450 (CYP)1A1, but CYP1B1 was constitutively expressed in both genotypes of mice, irrespective of DB[a,l]P treatment. As compared with AhR+/+ mice, AhR-/- mice had both significantly lower incidence (100% vs. 33%) and multiplicity (2.7 vs. 0.46) of skin tumors by the complete carcinogenesis study. These observations indicate that a reduced tumor yield in AhR-/- mice may be secondary to reduction of inducible CYP1A1 activation and subsequent DNA adduction. It is evident from our continuous work that although AhR is likely to play a central role in epidermal proliferation and possibly neoplastic transformation, the relative importance of AhR for carcinogenesis may be different among PAH examined.     

Comparison of 7,12-dimethylbenz[a]anthracene metabolism and DNA binding in mammary epithelial cells from three rat strains with differing susceptibilities to mammary carcinogenesis

The capacity of polycyclic aromatic hydrocarbons such as 7,12-dimethylbenz[a]anthracene(DMBA) to induce mammary carcinomas has been studied in threerat strains. Wistar/Furth (WF) rats are highly susceptible toDMBA-induced mammary carcinogenesis, Copenhagen (Cop) rats arecompletely resistant, and Fischer 344 (F344) rats have an intermediatesusceptibility. We have previously shown that WF rats possess‘enhancer genes’, which enhance susceptibility toinduced mammary cancer. Cop rats, however, possess a single‘suppressor’ gene which confers complete resistanceto mammary cancer. Both gene types are apparently absent inF344 rats. In order to determine possible mechanisms of actionof these enhancer and suppressor genes, we have examined DMBAmetabolism and DNA binding in mammary epithelial cells isolatedfrom each rat strain. Quantitative analyses of both metabolismand DNA binding indicate no significant differences among thestrains. In addition, HPLC analyses of DMBA metabolites andDMBA-DNA adducts were essentially identical. These data suggestthat the genes controlling susceptibility and resistance tomammary carcinogenesis in these rat strains are likely to beactive at later stages of the carcinogenic process.     

Differential ability of Ambystoma tigrinum hepatic microsomes to produce mutagenic metabolites from polycyclic aromatic hydrocarbons and aromatic amines

A number of carcinogenic aromatic amines when activated by liver microsomes from a salamander, Ambystoma tigrinum, are mutagenic for Salmonella tester strains sensitive to frameshift mutagens. However, 2 polycyclic aromatic hydrocarbons (PAH) (benzo[a]pyrene (BaP) and perylene) that are rendered mutagenic by mammalian microsomes are not activated by Ambystoma mixed-function oxidases. BaP was chosen for study because it is a well-known environmental carcinogen; perylene, an isomer of BaP, has been implicated as an etiological agent in cutaneous neoplasia in Ambystoma. These results support the observation that amphibians are quite resistant to PAH carcinogenesis and suggest that aromatic amines may be more appropriate model carcinogens.     

Metabolism of l-nitrobenzo[a]pyrene by rat liver microsomes to potent mutagenic metabolites

1-, 3- and 6-Nitrobenzo[a]pyrene (nitro-BaP), which are prototypesof nitro polycyclic aromatic hydrocarbons (nitro-PAHs) derivedfrom a carcinogenic parent PAH, benzo[a]-pyrene, are environmentalcontaminants and potent bacterial mutagens. In this study, theaerobic and hypoxic metabolism of 1-nitro-BaP by rat liver microsomeswas studied. Aerobic metabolism of 1-nitro-BaP yielded 1-nitro-BaPtrans-7, 8- and 9, 10-dihydrodtol, while metabolism under hypoxicconditions yielded 1-amino-BaP. The metabolites formed fromaerobic metabolism of 1-nitro-BaP and 1-nitro-BaP trans-9, 10-dihy-drodiolby liver microsomes of untreated rats and rats pretreated with3-methylcholanthrene and phenobarbital were quantified. Comparisonof these results with those obtained with BaP and BaP trans-9,10-dihydrodiol indicates that nitro substitution at the 1-positionof BaP markedly affects the regioselectivity of the P-450-containingenzymes. 1-Nitro-BaP and the three metabolites were potent mutagensin Salmonella typhimurium TA98, both in the absence and in thepresence of an exogenous metabolic activation system (S9). Thedirect and S9-mediated mutagenkities of 1-nitro-BaP and thetwo dihydrodiols were decreased in the nitroreductase-deficientstrain TA98NR, while TA98/1, 8-DNP₆, an 0-acetylase-defidentstrain, was less sensitive to the two dihydrodiols, both withand without S9, and 1-nitro-BaP with S9. 1-Amino-BaP was equallymutagenic in all three tester strains. These observations indicatethat: (i) the metabolism of 1-nitro-BaP involves several pathwaysleading to mutagenic activation; (ii) the major activation pathwaysof 1-nitro-BaP involve nitroreduction; (iii) nitroreductionfollowed by O-acetylation is the major activation pathway of1-nitro-BaP trans-7, S- and 9, 10-dihydrodiol; and (iv) 1-amino-BaPis a potent direct-acting mutagen.