Further investigations into the effect of non-benzo ring bay-region methyl substituents on tumor-initiating activity of polycyclic hydrocarbons

The effect of a methyl substituent at the non-benzo ring bay-region position of benzo[e]pyrene (B[e]P), cholanthrene (CA) and dibenz[a,j]anthracene (DB[a,j]A) on skin tumor-initiating activity was examined. A methyl substituent at the 1-carbon of B[e]P enhanced tumor-initiating activity of the parent compound (0.18 vs. 2.4 papillomas/mouse for B[a]P vs. 1-methyl-B[e]P, respectively, with an 800 nmol initiating dose). A methyl substituent at the 7- and 14-carbons of DB[a,j]A increased the activity of this weak initiator more than 13 times. The introduction of a methyl substituent at the non-benzo ring bay-region position of CA (i.e. carbon atom 6) dramatically increased tumor-initiating activity in SENCAR mice. 6-Methyl-CA was found to be a more potent skin tumor-initiator than 3-methyl-CA, and nearly as potent as 7,12-dimethylbenz[a]anthracene, one of the most potent initiators yet tested in the 2-stage initiation-promotion mouse skin model. When taken together with previous results from our laboratories, the data further support the generality of the enhancing effect of a non-benzo ring bay-region methyl substituent on polycyclic hydrocarbon tumor-initiation.     

Covalent binding of polycyclic aromatic hydrocarbons to adenine correlates with tumorigenesis in mouse skin.

Mouse epidermal homogenates were utilized to convert various polycyclic aromatic hydrocarbons to metabolites capable of binding covalently with nucleic acids. Poly(G) showed the highest capacity to bind covalently with the hydrocarbons; however, there was no correlation between binding to poly(G) and mouse skin tumorigenicity. On the other hand, covalent binding to poly(A) correlated well with values obtained for binding to DNA and mouse skin tumorigenicity. The order of binding to poly(A) was; 7,12-dimethylbenza[a]anthracene greater than benzo[a]pyrene greater than dibenz[a,h]anthracene greater than dibenz[a,c]anthracene.     

Toxic chemicals in sediments and biota from a creosote-polluted harbor: relationships with hepatic neoplasms and other hepatic lesions in English sole (Parophrys vetulus)

High prevalences of idiopathic hepatic lesions, including neoplasms(e.g., hepatocellular carcinomas, cholangiocellular carcinomas)(27%, 20 of 75 fish) and foci of cellular alteration (putative‘preneoplastic’ lesions) (44%, 33 of 75 fish), werefound in English sole (Parophrys vetulus) exposed to creosote-contaminatedsediments in Eagle Harbor, Puget Sound, WA. Sediments from thecontaminated region of the harbor contained particularly highconcentrations of aromatic hydrocarbons (e.g., benzo[a]pyreneand benz[a]anthracene), and a variety of nitrogen-containingaromatic compounds (e.g., carbazole and acridine). The compositionof the aromatic compounds was characteristic of creosote. Dramaticallylower concentrations of aromatic compounds were found in sedimentsfrom a reference site in which the bottom-dwelling fish examinedwere free of detectable neoplastic or ‘preneoplastic’hepatic lesions. Food organisms in the stomachs of the Englishsole from Eagle Harbor contained substantially higher concentrationsof aromatic hydrocarbons than comparable organisms from thereference site. The concentrations of individual aromatic hydrocarbonsin muscle and liver from the Eagle Harbor fish were low; however,high concentrations of metabolites of aromatic compounds werepresent in the bile. The findings strongly suggest an associationbetween exposure to creosote and the prevalence of hepatic lesions,including neoplasms, in the bottom-dwelling fish, and furthermoresupport the putative role of aromatic hydrocarbons in livercarcinogenesis in fish.     

Liver microsome-mediated mutagenicity of dihydrodiols derived from dibenz(a,c)anthracene in S. typhimurium TA100

The mutagenic activities of the trans-1,2- and 3,4-diols ofdibenz(a,c)anthracene, both of which could yield ‘bay-region’diol-epoxides on further metabolism, were lower than those ofthe parent hydrocarbon and of the related 10,11-diol.     

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.     

Fluorescence spectra of nucleoside-hydrocarbon adducts formed in mouse skin treated with 7,12-dimethylbenz[a]anthracene

Hydrolysates of DNA that had been isolated from mouse skin treatedwith ³H-labelled 7,12-dimethyl-benz[a]anthracene (DMBA) weresubjected to chromatography on Sephadex LH20 columns and ³H-labelledproducts that eluted in the region expected for nucleoside-hydrocarbonadducts were purified further by high pressure liquid chromatography(h.p.l.c). The fluorescence spectra of three major productsthat were resolved by this method were determined using photoncountingspectrophotofluorimetry. The fluorescence spectra of all threeproducts were anthracene-like and similar to the spectra ofnucleoside-hydrocarbon adducts obtained from DNA that was incubatedwith 3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a]anthracene1,2-oxide (DMBA-3,4-diol 1,2-oxide). This is consistent withthe idea that the metabolic activation of DMBA in mouse skinoccurs through the formation of ‘bay-region’ diol-epoxidesin the 1,2,3,4-ring.     

Polycyclic hydrocarbons induction of diphtheria toxin-resistant mutants in human cells

Stable spontaneous mutants resistant to diphtheria toxin are present in the human cell line (EUE) at a frequency of 0-8 x 10(-6). Mutation increases by a number of polycyclic hydrocarbons have been used as an estimate of their carcinogenic potency. Eight polycyclic hydrocarbons of decreasing carcinogenic potency were assayed: 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, benz[a]anthracene, dibenz[a,c]anthracene, dibenz[a,h]anthracene, chrysene, anthracene, and a well known mutagenic substance, ethyl methanesulfonate. In our system, which does not require an exogenous source for metabolic activation, the most potent hydrocarbons, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benzo[a]pyrene revealed a strong mutagenic effect, whereas three non-carcinogenic hydrocarbons, anthracene, benz[a]anthracene and chrysene were not mutagenic. Our results indicate that there is a relationship between mutagenesis and carcinogenic potency for the tested polycyclic hydrocarbons. The maximum recovery of diphtheria toxin mutants was observed after an expression time of three weeks, corresponding to 10 cell generations.     

Monooxygenase induction by various xenobiotics and its influence on the rat liver microsomal metabolite profile of benz[a]anthracene

Several pesticides (lindane, carbaryl, pentachlorophenol, DDT), polycyclic aromatic hydrocarbons (PAH) and heterocyclic analogues (fluoranthene, dibenz[a,h]anthracene, dibenz[a,h]acridine, indeno[1,2,3-cd]pyrene, 10-azabenzo[a]pyrene) and pharmaceuticals (diphenylhydantoin, ethinylestradiol, levonorgestrel) were tested for their potencies to induce monooxygenase activities in the rat liver by means of recording the metabolite profile of benz[a]anthracene in rat liver microsomal incubations. Some of them were found to be weak or moderate inducers, but even less efficient ones altered the benz[a]anthracene metabolite profile significantly. Only indeno [1,2,3-cd]pyrene stimulated the bay-region oxidation of benz[a]anthracene. A sex-dependent metabolism was observed in both untreated and contraceptive-pretreated Wistar rats.     

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.     

Microsome-mediated reactions of phenols of polycyclic hydrocarbons with DNA

Phenolic metabolites of 7 polycyclic hydrocarbons were incubated with rat-liver microsomal fractions in the presence of DNA. Hydrocarbon-nucleoside adducts with chromatographic properties similar to those of diol-epoxide-deoxyribonucleoside adducts, were detected in hydrolysates of DNA that had been incubated with phenols of benzo[a]pyrene, benz[a]anthracene and chrysene. Adducts were not detected when phenols of phenanthrene, pyrene, dibenz[a,c]anthracene or dibenz[a,h]anthracene were further metabolised. The possible contribution of phenolic metabolites to the carcinogenic activity and DNA binding of polycyclic hydrocarbons is discussed.     

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.     

Anticarcinogenic and cocarcinogenic effects of benzo[e]pyrene and dibenz[a,c]anthracene on skin tumor initiation by polycyclic hydrocarbons

In the present study, we have examined the effects of benzo[e]pyrene(B[e]P) and dibenz[a,c]anthracene (DB[a,c]A) on the skin tumor-initiatingactivities of methylated and nonmethylated polycyclic aromatichydrocarbons (PAH). B[e]P, when applied 5 min prior to initiationwith seven different PAH skin carcinogens, effectively inhibitedthe tumorinitiating activities of 7,12-dimethylbenz[a]anthracene(DMBA) and dibenz[a,h]anthracene (DB[a,h]A) but had little orno effect on the tumor-initiating activities of 3-methylcholanthrene(MCA), 7-methylbenz[a]anthracene (7-MBA), 12-methylbenz[a]anthracene(12-MBA), and 5-methylchrysene (5-MeC). B[e]P potentiated thetumor-initiating activity of benzo[a]pyrene (B[a]P) by 30%.DB[a,c]A, when applied 5 min prior to initiation, inhibitedthe tumor-initiating activities of DMBA, MCA, and DB[a,h]A buthad little or no effect on the tumor-initiating activities ofB[a]P, 7-MBA, 12-MBA, and 5-MeC. DB[a,c]A, when applied 12,24, or 36 h prior to initiation with B[a]P, which allowed timefor induction of epidermal monooxygenase enzymes, inhibitedtumor initiation. The covalent binding of DMBA and B[a]P toepidermal DNA was examined under the influence of B[e]P. Dosesof 20 and 200 nmol B[e]P given 5 min prior to 10 nmol [³H]DMBAreduced binding to 47 and 22%, respectively, of the controlvalue. In contrast, doses of 200 or 2000 nmol B[e]P given 5min prior to 200 nmol [³H]B[a]P had little or no effect on totalbinding. The data indicate that one cannot predict anti andcocarcinogenic effects of B[e]P and DB[a,c]A on the basis ofa presence or absence of a methyl substituent. In addition,fundamental differences exist in the processing and metabolismof DMBA and B[a]P by mouse epidermal cells.     

Metabolic activation of 7-ethyl- and 7-methylbenz[a]anthracene in mouse skin

The carcinogen 7-methylbenz[a]anthracene (7-MBA) is consideredto be metabolically activated via its bay-region dihydrodiol-epoxide,trans-3,4-dihydro-3,4-dihydroxy-7- methyl-benz[a]anthracene1,2-oxide (7-MBA-3,4-diol 1,2-oxide). When tested on mouse skin,a target tissue for polycyclic aromatic hydrocarbon carcinogenesis,7-ethylbenz[a]anthracene (7-EBA) was much less active than 7-MBA,and this difference may be due to differences in the pathwaysby which the two compounds are metabolized and activated. Inthe present work, the metabolism by mouse-skin microsomes ofboth hydrocarbons to dihydrodiols has been examined. Both weremetabolized to a similar extent with the 8,9-dihydrodiols beingdetected as the predominant metabolites. The 3,4-, 5,6- and10,11-dihydrodiols of 7-MBA and the 3,4- and 10,11-di-hydrodiolsof 7-EBA, were also detected. 7-MBA was found to bind covalentlyto microsomal protein at 10 times the level of 7-EBA. The covalentbinding of benz[a]anthracene (BA), 7-EBA and 7-MBA to DNA inmouse skin following topical application was determined usingthe ³²P-postlabelling assay. The results correlated with therelative carcinogenic activities of the compounds with 7-MBAbinding at five and nine times the level of 7-EBA and BA respectively.For all three hydrocarbons, the major hydrocarbon: ³²P-labellednucleoside bisphosphate, eluted in the same area of the TLCmaps, suggesting the involvement of a common type of bay-regiondihydrodiol-epoxide intermediate.     

Tumor-initiating activity of the bay-region dihydrodiols and diolepoxides of dibenz[a,j]anthracene and cholanthrene on mouse skin

The tumor-initiating activity of dibenz[a,j]anthracene, (±)dibenz[a,j]anthracene-trans-3,4-dioland (±)dibenz[a,j]anthracene-anti-3,4-diol-1,2-epoxidein mouse skin was examined and compared to that of cholanthrene,(±)cholanthrene-trans-9, 10-diol and (± )cholanthrene-anti-9,10-diol-7,8-epoxide.The tumor-initiating activity of these compounds dissolved inacetone or in tetrahydrofuran (THF) was also compared. In acetone,dibenz[a,j]anthracene was a weak tumor initiator with maximaltumor yields of 1.27 and 3.00 per mouse at 400 and 800 nmoldoses, respectively. At the 400 nmol dose, the diol of thiscompound was slightly more active than the parent compound whilethe tumorigenic activity of the diol-epoxide was significantlyhigher. The diol-epoxide was almost three times more activethan the parent compound as a tumor-initiator. Cholanthrenewas a moderate tumor-initiator with maximal tumor yields of6.90 and 8.86 tumors per mouse at 200 and 600 nmol doses, respectively,after 20 weeks of promotion. At comparable doses, (±)cholanthrene-trans-9,10-diolwas 50% as potent as cholanthrene as a tumor initiator whereasthe diol-epoxide was only minimally active. Replacing the acetonewith THF as solvent vehicle increased the tumor-initiating activityof cholanthrene-diol-epoxide; however, the parent compound stillretained higher tumor-initiating activity than its bay-regiondiol-epoxide. The low tumorigenic activity of cholanthrene-diol-epoxideis thought to reflect the high chemical reactivity and low stabilityof this derivative, which may prevent it from penetrating toepidermal targets. In contrast, the bay-region diol-epoxidederivative of dibenz-[a,j]anthracene appears to be stable enoughto exert greater biologic activity when applied to mouse skin.     

Dibenz[a,h]anthracene-induced subcutaneous tumors in mice. Strain sensitivity and the role of carcinogen metabolism

Four inbred strains of mice (C3H/HeJ, C57BL/6J, AKR/J, and DBA/2J)were injected s.c. with 150 µg of dibenz[a,h]-anthraceneand followed for 9 months. Strains C3H/HeJ and C57BL/6J weremost susceptible to dibenz[a,h]anthracene-induced carcinogenesiswith 80% and 53% incidence of tumors, respectively. StrainsAKR/J and DBA/2J were much less sensitive with only one tumorobserved out of a total of 60 treated mice. Dibenz[a,h]anthracenewas shown to induce hepatic aryl hydrocarbon hydroxylase activityin the two sensitive strains, C3H/HeJ and C57BL/6J, but notin the two resistant strains, AKR/J and DBA/2J. When 3-methyl-cholanthrene-treatedliver microsomes from the four strains were studied for dibenz[a,h]anthracenemetabolism in vitro, the two sensitive strains not only demonstrateda 3- to 4-fold greater overall rate of metabolism than the tworesistant strains, but also showed a quantitative shift witha greater percentage of the total metabolites being the 3,4-diolof dibenz[a,h]anthracene. This diol is the presumed precursorto the apparent ultimate carcinogen, dibenz[a,h]anthracene 3,4-diol-1,2-epoxide.     

The influence of 18 environmentally relevant polycyclic aromatic hydrocarbons and Clophen A50, as liver monooxygenase inducers, on the mutagenic activity of benz[a]anthracene in the Ames test

In the presence of various liver S9 preparations induced bydifferent polycyclic aromatic hydrocarbons, the weak carcinogen,benz[a]anthracene, exhibited mutagenic activities comparableto those of benzo[a]pyrene in the Ames test. A series of positivecorrelations were found between mutagenic activity and the hydroxylationproducts of benz(a)anthracene at the 3, 4-, 5, 6- and 8, 9-positions,of which 5, 6-position was the most pronounced one (p < 0.01).Our results suggest that the observed mutagenic activity isdue mainly to the generation of K-region epoxides.     

The metabolic activation of chrysene by hamster embryo cells

The major deoxyribonudeoside—hydrocarbon adducts presentin hydrolysates of DNA isolated from hamster embryo cells treatedwith chrysene were examined by chromatography on Sephadex LH20and by h.p.l.c. on Zorbax ODS. The results show that both majoradducts have chromatographic properties identical to those ofadducts formed when r-1, t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysenereacts with DNA and provide evidence that metabolic activationof chrysene occurs via the formation of this ‘bay-region’diolepoxide.     

The carcinogenic activities in mice of compounds related to benz[a]anthracene

The carcinogenic activities of a number of aromatic hydrocarbons have been compared with some metabolic intermediates by subcutaneous injection into C57 black mice. The monohydroxymethyl derivatives of 7,12-dimethylbenz[a]anthracene and some related compounds were found to be active carcinogens, but were much less so than the parent hydrocarbon. Epoxides formed at the K-region ofchrysene, benz[a]anthracene, 7-methylbenz[a]antnracene and dibenz[a,h]anthracene produced tumours when given at highdose levels, but were not as active as the parent hydrocarbons. The epoxide derived from phenanthrene was inactive.     

Preparation of microgram quantities of BaP-DNA adducts using isolated rat hepatocytes in vitro

The analysis of carcinogen-DNA adducts generally requires thepreparation (by chemical or biological means) of DNA adductstandards, in amounts sufficient for chemical characterization.We have established conditions for the in vitro biological preparationof microgram quantities of DNA adducts derived from benzo[a]pyrene(BaP), fluoranthene and 7,12-dimethylbenzanthracene, using isolatedrat hepatocytes. The metabolic activation of 180 µM (BaP)by isolated rat hepatocytes in a calf-thymus-DNA (CT-DNA)-supplementedmedium resulted in the formation of 2.9 µg of BaP adductedto 56.7 mg of DNA. The average level of binding in this experimentwas 148 ± 8 pmol BaP bound/1 mg DNA, which compares favorablyto the 10–30 pmol BAP/1 mg DNA which is typical of mouseskin adducts in vivo. In another experiment, BaP-DNA adductformation in calf-thymus DNA added to hepatocyte incubationswas further increased to 327 ± 27 pmol/mg DNA, by physicalshearing of the DNA prior to the incubation. The HPLC profileof the BaP adducts produced using hepatocytes plus CT-DNA isvirtually indistinguishable from that produced by tumor-initiatingdoses of BaP applied to mouse skin in vivo, and the major DNAadduct formed by the hepatocytes co-elutes with the (+)-anti-diol-epoxideadduct of deoxyguanosine. Similar experiments using fluorantheneand 7,12-dimethylbenz-anthracene also resulted in substantialDNA adduct formation; however, incubations using dibenz[a,h]anthracenedid not. These results indicate that isolated rat hepatocytesin vitro can be useful for the preparation of DNA adducts ofa number of polycyclic aromatic hydrocarbons, in quantitiessufficient for chemical characterization.     

Comparative tumor-initiating activity of methylene-bridged and bay-region methylated derivatives of benz[a]anthracene and chrysene

Polycyclic aromatic hydrocarbons (PAHs) with a methylenebridgespanning the bay region are often detected in higher concentrationsin the environment than PAHs which have a bay-region methylsubstituent. The tumor-initiating activity of methylene-bridgedbay-region derivatives of chrysene and benz[a]anthracene wasevaluated on the skin of female CD-1 mice. 4,5-Methylenechryseneelicited twice as many tumors as chrysene at each of the dosestested. At total initiation doses of 1.5, 0.5 and 0.15 µmol/mouse4,5-methylenechrysene gave rise to 8.5, 6.8 and 1.1 tumors/animalrespectively. There was a 100% incidence of tumor-bearing miceat the two higher doses and a 65% incidence at the lowest dose.This methylenebridged hydrocarbon was less tumorigenic than5-methylchrysene at each dose tested. Animals treated with 1,l2-methylenebenz[a]anthracenedeveloped 7.6, 4.9 and 1.6 tumors/animal attotal initiatingdoses of 4.0, 2.0 and 0.5 µmol/mouse. The incidence oftumor-bearing mice was 90% atthe higher doses and 75% at thelowest dose tested. 12-Methylbenz[a]anthracene was more tumorigenicthan the methylene-bridged derivative at each dose. The keto-derivativeof 1,12-methylenebenz[a]anthracene did not display significant(P> 0.05) tumor-initiating activity at these dose levels.Results from these bioassays indicate that methylenebridgedbay-region derivatives of chrysene and benz[a]anthracene contributetothe overall genotoxicity of environmentally occurring PAHs.