Highly selective bioactivation of 1- and 2-hydroxy-3-methylcholanthrene to mutagens by individual human and other mammalian sulphotransferases expressed in Salmonella typhimurium.

The benzylic alcohols 1- and 2-hydroxy-3-methylcholanthrene (OH-MC) are major primary metabolites of the carcinogen 3-methylcholanthrene (MC). We investigated them for mutagenicity in TA1538-derived Salmonella typhimurium strains expressing mammalian sulphotransferases (SULTs). 1-OH-MC was efficiently activated by human (h) SULT1B1 (2400 revertants/nmol), weakly activated by hSULT1C3 and hSULT2A1 (2-9 revertants/nmol), but not activated by the other hSULTs studied (1A2, 1A3, 1C2 and 1E1). Mouse, rat and dog SULT1B1 activated 1-OH-MC (8-100 revertants/nmol) with much lower efficiency than their human orthologue. The other isomer, 2-OH-MC, was activated to a potent mutagen by hSULT1A1 (4000-5400 revertants/nmol), weakly activated by hSULT1A2 or hSULT2A1 (1-12 revertants/nmol), but not activated by the other hSULTs. In contrast to their human orthologue, mouse, rat and dog SULT1A1 did not appreciably activate 2-OH-MC (<1 to 6 revertants/nmol), either. Instead, mouse and rat SULT1B1, unlike their human and canine orthologues, demonstrated some activation of 2-OH-MC (15-100 revertants/nmol). Docking analyses indicated that 1- and 2-OH-MC might bind to the active site of hSULT1A1 and hSULT1B1, but only for (S)-2-OH-MC/hSULT1A1 and (R)-1-OH-MC/hSULT1B1 with an orientation suitable for catalysis. Indeed, 1- and 2-OH-MC were potent inhibitors of the hSULT1A1-mediated sulphation of acetaminophen [concentration inhibiting the enzyme activity by 50% (IC50) 15 and 13nM, respectively]. This inhibition was weak with mouse, rat and dog SULT1A1 (IC50 ≥ 4 µM). Inhibition of the SULT1B1 enzymes was moderate, strongest for 1-OH-MC/hSULT1B1. In conclusion, this study provides examples for high selectivity of bioactivation of promutagens by an individual form of human SULT and for pronounced differences in activation capacity between orthologous SULTs from different mammalian species. These characteristics make the detection and evaluation of such mutagens extremely difficult, in particular as the critical form may even differ for positional isomers, such as 1- and 2-OH-MC. Moreover, the species-dependent differences will complicate the verification of in vitro results in animal studies.

Alveolar hypoxia promotes murine lung tumor growth through a VEGFR-2/EGFR-dependent mechanism.

Patients with chronic obstructive pulmonary disease (COPD) are at an increased risk for the development of lung cancer, the mechanisms for which are incompletely understood. We hypothesized that the hypoxic pulmonary microenvironment present in COPD would augment lung carcinogenesis. Mice were subjected to chemical carcinogenesis protocols and placed in either hypoxia or normoxia. Mice exposed to chronic hypoxia developed tumors with increased volume compared with normoxic controls. Both lungs and tumors from hypoxic mice showed a preferential stabilization of HIF-2α and increased expression of VEGF-A, FGF2, and their receptors as well as other survival, proliferation, and angiogenic signaling pathways regulated by HIF-2α. We showed that tumors arising in hypoxic animals have increased sensitivity to VEGFR-2/EGFR inhibition, as chemoprevention with vandetanib showed markedly increased activity in hypoxic mice. These studies showed that lung tumors arising in a hypoxic microenvironment express increased growth, angiogenic, and survival signaling that could contribute to the increased lung cancer risk in COPD. Furthermore, the differential sensitivity of tumors arising in hypoxia to VEGFR-2/EGFR inhibition suggests that the altered signaling present in tumors arising in hypoxic lung might be therapeutically exploited in patients with underlying COPD.

UVA photoirradiation of oxygenated benz[a]anthracene and 3-methylcholanthene--generation of singlet oxygen and induction of lipid peroxidation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread genotoxic environmental pollutants and potentially pose a health risk to humans. Although the biological and toxicological activities, including metabolism, mutagenicity, and carcinogenicity, of PAHs have been thoroughly studied, their phototoxicity and photo-induced biological activity have not been well examined. We have long been interested in phototoxicity of PAHs and their derivatives induced by irradiation with UV light. In this paper we report the photoirradiation of a series of oxygenated benz[a]anthracene (BA) and 3-methylcholanthene (3-MC) by UVA light in the presence of a lipid, methyl linoleate. The studied PAHs include 2-hydroxy-BA (2-OH-BA), 3-hydroxy-BA (3-OH-BA), 5-hydroxymethyl-BA (5- CH2OH-BA), 7-hydroxymethyl-BA (7-CH2OH-BA), 12-hydroxymethyl-BA (12-CH2OH-BA), 7-hydroxymethyl-12- methyl-BA (7-CH2OH-12-MBA), 5-formyl-BA (5-CHO-BA), BA 5,6-cis-dihydrodiol (BA 5,6-cis-diol), 1-hydroxy-3- methylcholanthene (1-OH-3-MC), 1-keto-3-methylcholanthene (1-keto-3-MC), and 3-MC 1,2-diol. The results indicate that upon photoirradiation by UVA at 7 and 21 J/cm2, respectively all these compounds induced lipid peroxidation and exhibited a relationship between the dose of the light and the level of lipid peroxidation induced. To determine whether or not photoirradiation of these compounds by UVA light produces ROS, an ESR spin-trap technique was employed to provide direct evidence. Photoirradiation of 3-keto-3-MC by UVA (at 389 nm) in the presence of 2,2,6,6-tetramethylpiperidine (TEMP), a specific probe for singlet oxygen, resulted in the formation of TEMPO, indicating that singlet oxygen was generated. These overall results suggest that UVA photoirradiation of oxygenated BA and 3-methylcholanthrene generates singlet oxygen, one of the reactive oxygen species (ROS), which induce lipid peroxidation.

Characterization of metabolites of benz(j)aceanthrylene in faeces, urine and bile from rat.

1. The excretion of benz[j]aceanthrylene (B[j]A) and the biotransformation products found in faeces, urine and bile of rat exposed to [3H]-labelled B[j]A have been studied. 2. About 95% of the administered radioactivity was excreted within 7 days, 79% via faeces and 16% via urine, and most of the radioactivity in urine and faeces was excreted within 2 days. 3. The B[j]A metabolites excreted between days 1 and 2, including those excreted in bile during the first 5.5 h in a separate experiment, were further characterized by HPLC, UV and electrospray/atmospheric pressure chemical ionization mass spectrometry. 4. In faeces, bile and urine, hydroxylated B[j]A metabolites predominated. The major metabolites in faeces were B[j]A-1,2-dihydrodiol-8-hydroxy and B[j]A-1,2-dihydrodiol-10-hydroxy. These metabolites were found as conjugated metabolites in the bile. The glucuronide conjugate of B[j]A-1,2-dihydrodiol-10-hydroxy was also a major metabolite in urine. Two sulphate conjugates of oxidized B[j]A were detected in bile, a sulphate conjugate of a B[j]A-dihydrodiol-phenol and B[j]A-1,2-dihydrodiol-10-sulphate. Trans-B[j]A-1,2-dihydrodiol was detected in urine, faeces and bile. 5. These findings support the hypothesis that epoxidation at the cyclopenta ring is an important biotransformation pathway for B[j]A in vivo. In addition to the characterized metabolites, a large fraction of polar compounds, possibly glutathione conjugates, was also excreted in urine and bile.

Metabolism and activation of cyclopenta polycyclic aromatic hydrocarbons in isolated human lymphocytes, HL-60 cells and exposed rats.

The metabolism of radiolabelled benz(j)aceanthrylene (B(j)A) was studied in suspensions of isolated human peripheral mononuclear blood cells (lymphocytes), using high performance liquid chromatography (HPLC). The only known metabolite found after 24 h exposure to 30 microg/ml (120 microM) B(j)A, was B(j)A-1,2-dihydrodiol, representing approximately 35% of the total metabolites formed. B(j)A, benz(l)aceanthrylene (B(l)A) and benzo(a)pyrene (B(a)P) all caused DNA adducts in human lymphocytes, as well as in the human promyelocytic cell line HL-60 cells, as measured by the 32P-postlabelling technique (30 microg/ml, 24 h). The total DNA adduct levels in human lymphocytes exposed to B(j)A, B(l)A or B(a)P were 0.13 +/- 0.03, 1.10 +/- 0.62 and 0.37 +/- 0.10 fmol/microg DNA, respectively, and similar levels were obtained in HL-60 cells (0.18 +/- 0.14, 0.97 +/- 0.35 and 0.29 +/- 0.17 fmol/microg DNA, respectively). For each compound, the human lymphocytes and HL-60 cells in addition showed similar DNA adduct patterns. Cells exposed to B(j)A revealed only one DNA adduct, which, judged by its TLC properties, resulted from B(j)A-1,2-epoxide. As measured by the alkaline filter elution technique, only low levels of single strand DNA breaks (SSB) were observed in both human lymphocytes and HL-60 cells after exposure to B(j)A, B(l)A or B(a)P (24 h, 30 microg/ml). By adding cytosine-1-beta-D-arabinofuranoside (Are C) and hydroxyurea (HU) 1 h prior to analysis to prevent strand break rejoining, some increase in SSB (2-3 times) was observed in the lymphocytes. Co-incubation of human lymphocytes with liver microsomes from PCB-treated rats for 1 h and exposure to B(j)A or B(l)A, increased the DNA adduct levels in the lymphocytes to 12.3 and 37.8 fmol/microg DNA, respectively. A large increase in SSB was also observed, whereas no such increase was observed after co-incubation with human liver microsomes. In vivo exposure of rats to 30 mg/kg B(j)A (i.p.) for 24 h revealed one major DNA adduct in lymphocytes and lung tissue (only one of three rats showed an adduct in liver tissue), apparently resulting from B(j)A-1,2-epoxide. The total DNA adduct level in the lymphocytes was 0.09 fmol/microg DNA, and in lung tissue between 0.10 and 0.62 fmol/microg DNA. Overall, the present data suggests that oxidation at the cyclopenta-ring is an important activation pathway for B(j)A in rats as well as in humans.

Carcinogenicity of 1-hydroxy-3-methylcholanthrene and its electrophilic sulfate ester 1-sulfooxy-3-methylcholanthrene in Sprague-Dawley rats.

Previous experiments have demonstrated that the carcinogen 1-hydroxy-3-methylcholanthrene is a metabolite of 3-methylcholanthrene. 1-Sulfooxy-3-methylcholanthrene, prepared by chemical synthesis from 1-hydroxy-3-methylcholanthrene, was shown to be a direct acting electrophilic mutagen and DNA damaging agent. These results imply that 1-hydroxy-3-methylcholanthrene could be metabolically activated to an ultimate electrophilic and carcinogenic form of 1-hydroxy-3-methylcholanthrene and 3-methylcholanthrene in a reaction catalyzed by 3'-phosphoadenosine-5'-phosphosulfate-dependent sulfotransferase activity. 1-Hydroxy-3-methylcholanthrene and its aralkylating reactive ester, 1-sulfooxy-3-methylcholanthrene, were individually administered to groups of 12 female Sprague-Dawley rats at a 0.2 mumol dose, three times weekly, for 20 doses. 1-Sulfooxy-3-methylcholanthrene induced sarcomas at the site of injection in 8 of 12 rats (66%) by 52 weeks, whereas 1-hydroxy-3-methylcholanthrene induced sarcomas at the site of injection in 5 of 12 rats (42%) by 52 weeks. These results, taken together with the results of previous experiments, strongly support the hypothesis that the activated electrophilic mutagen 1-sulfooxy-3-methylcholanthrene plays a major role as an ultimate electrophilic and carcinogenic form of 1-hydroxy-3-methylcholanthrene, a major metabolite of 3-methylcholanthrene.

Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in different types of isolated rat lung cells.

The genotoxic effects of the environmental contaminants benz[j]aceanthrylene (B[j]A), benz[l]aceanthrylene (B[l]A) and benzo[a]pyrene (B[a]P), and the metabolism of radiolabelled B[j]A, were studied using rat lung microsomes and various types of isolated rat lung cells from control and Aroclor 1254 (PCB) treated animals. All three compounds (10 or 20 microg/plate) resulted in low, but detectable, levels of His+ revertants in the Salmonella assay when plated with control lung microsomes. The two cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) B[j]A and B[l]A, gave increased levels of revertants when plated with microsomes from PCB-treated animals. Clara cells, type 2 cells and alveolar macrophages isolated from control rats were exposed to B[j]A, B[l]A or B[a]P (30 microg/ml, 1 h), but neither of the cell types showed any DNA damage when measured by alkaline filter elution. However, both B[j]A and B[l]A (30 microg/ml, 2 h) caused DNA adducts in all three cell types, measured by the 32P-post-labelling technique, whereas no B[a]P adducts were detected (30 microg/ml, 2 h). The total DNA adduct levels in Clara cells, type 2 cells and macrophages exposed to B[j]A were 0.085 +/- 0.033, 0.053 +/- 0.001 and 0.170 +/- 0.030 fmol/microg DNA, respectively, whereas the total levels in cells exposed to B[l]A were 0.140 +/- 0.070, 0.140 +/- 0.030 and 0.220 +/- 0.080 fmol/microg DNA, respectively. Cells exposed to B[j]A revealed only one adduct which corresponds with the B[j]A-1,2-oxide DNA adduct. Judged from high performance liquid chromatography (HPLC) analysis using radiolabelled B[j]A (30 microg/ml, 30 min), the major metabolite formed in control microsomes was B[j]A-1,2-diol. Thus, oxidation at the cyclopenta ring appears to be the most important activation pathway for B[j]A with control rat lung cells. Exposure of lung cells to CP-PAH (30 microg/ml, 2 h) isolated from PCB pretreated rats resulted in slightly increased DNA adduct levels in Clara cells and macrophages when compared to cells isolated from control rats. Furthermore, the adduct pattern had shifted, and no apparent B[j]A-1,2-oxide adduct could be detected on the thin layer chromatography (TLC) plate. In contrast, the major metabolite formed with microsomes from PCB-treated animals was still the B[j]A-1,2-diol.

3-Hydroxymethylcholanthrene: metabolic formation from 3-methylcholanthrene and stereoselective metabolism by rat liver microsomes.

3-Hydroxymethylcholanthrene (3-OHMC) was identified as one of the three initial hydroxylation products formed in the metabolism at the aliphatic carbons of 3-methylcholanthrene (3MC) by rat liver microsomes. The 3-OHMC formed in 3MC metabolism by liver microsomes prepared from untreated (control) rats, and rats treated with phenobarbital, 3MC, and polychlorinated biphenyls (Aroclor 1254) was determined by HPLC analysis and the effect of enzyme inducers on its formation was phenobarbital > polychlorinated biphenyls > 3MC > control. Incubation of 3-OHMC with rat liver microsomes produced the following identifiable products: 1-hydroxy-3-hydroxymethylcholanthrene (enriched in 1S-enantiomer, enantiomer excess 14-50%), 2-hydroxy-3-hydroxymethylcholanthrene (enriched in 2S-enantiomer, enantiomer excess 30-92%), 3-hydroxymethylcholanthrene-1-one, 3-hydroxymethylcholanthrene-2-one, 8-hydroxy-3-hydroxymethylcholanthrene, 3-hydroxymethylcholanthrene trans-9,10-dihydrodiol (enriched in 9R,10R-enantiomer, enantiomer excess 64-86%), 3-hydroxymethylcholanthrene trans-7,8-dihydrodiol (enriched in 7R,8R-enantiomer), and 3-hydroxymethylcholanthrene trans-11,12-dihydrodiol (enriched in 11R,12R-enantiomer). The enantiomer compositions were determined by circular dichroism spectral analysis and/or chiral stationary phase HPLC analysis.

Biotransformation of the cyclopenta-fused polycyclic aromatic hydrocarbon benz[j]aceanthrylene in isolated rat liver cell: identification of nine new metabolites.

The biotransformation of benz[j]aceanthrylene (B[j]A) was studied in suspensions of hepatocytes isolated from Aroclor 1254-treated or untreated rats. Using radiolabeled cofactors and metabolic inhibitors combined with UV, mass and 1H-NMR spectroscopy, we have detected five known metabolites and characterized nine new metabolites: metabolite 1 was tentatively assigned as B[j]A-1,2-dihydrodiol-8-sulfate; metabolite 2, B[j]A-1,2,9,10-tetrahydrotetrol; metabolite 3, B[j]A-1,2-dihydrodiol-10-O-glucuronide; metabolite 4, B[j]A-1-one-8-sulfate; metabolite 5, B[j]A-1,2-dihydrodiol-10-sulfate; metabolite 6, the sulfate conjugate of B[j]A-dihydrodiol-phenol; peak 7 in the chromatogram is a mixture of one glutathione conjugate and two sulfate conjugates of a B[j]A-metabolite; metabolite 8, B[j]A-10-O-glucuronide; metabolite 8', B[j]A-1,2-dihydrodiol; metabolite 9, B[j]A-10-sulfate; metabolite 9', B[j]A-9,10-dihydrodiol and metabolite 10, B[j]A-9,10-dihydro-9-hydroxy-10-sulfate. The metabolites identified support the notion that epoxidation at the cyclopenta region is an important activation step of B[j]A. Furthermore, sulfation appears to play a very important role in the conversion of hydroxylated B[j]A metabolites into more polar excretable products.

Intrinsic mutagenicity and electrophilicity of 1-sulfooxy-3-methylcholanthrene: implications for metabolic activation of the carcinogen 3-methylcholanthrene.

Hydroxylation of a meso-anthracenic carbon atom with subsequent formation of a reactive ester bearing a good leaving group (e.g., sulfate) has been proposed as a possible biochemical mechanism responsible for DNA binding, mutagenicity and tumorigenicity of 3-methylcholanthrene, one of the most potent carcinogenic polycyclic aromatic hydrocarbons in experimental animals. In support of this supposition, the chemically synthesized sulfuric acid ester, 1-sulfooxy-3-methylcholanthrene (1-SMC) was directly mutagenic in bacteria and covalently bound to DNA without metabolic activation. The intrinsic mutagenicity of this reactive ester was significantly potentiated by addition of extra acetate or chloride anions to the media. Reduced glutathione and ascorbic acid protected against 1-SMC-induced mutagenesis. These findings suggest 1-SMC as a potential ultimate electrophilic and tumorigenic metabolite of 3-methylcholanthrene.

Morphological transformation of C3H10T1/2CL8 cells by cyclopenta-fused derivatives of benzo[a]pyrene and benzo[e]pyrene.

Cyclopenta-fused homologs of polycyclic aromatic hydrocarbons (PAH) have proven to be more genotoxic and tumorigenic than their parent PAHs. In an effort to uncover their mechanisms of metabolic activation, the morphological transforming activities of dibenzo[k,mno]acephenanthrylene (CP(3,4)B[a]P), dibenzo[j,mno]acephenanthrylene (CP(1,12)B[a]P) and naphtho[1,2,3,4-mno]acephenanthrylene (CPB[e]P) were studied in C3H10T1/2CL8 mouse embryo fibroblasts. CP(3,4)B[a]P, a PAH with a blocked K region and unblocked bay region, was highly active inducing an average of 1.1 Type II and III foci/dish at 5 micrograms/ml with an average of 67% of the dishes containing foci. This activity was similar to that of benzo[a]pyrene. CP(1,12)B[a]P and CPB[e]P were inactive. The relative positions of the cyclopenta-ring and bay region may play an essential role in the metabolic activation of these PAHs and their biological activities.

Mouse skin tumor-initiating activity of benz[j]aceanthrylene in SENCAR mice.

Benz[j]aceanthrylene (B[j]A), a cyclopenta-fused derivative of benz[a]anthracene, has been reported to be an active bacterial cell and mammalian cell gene mutagen, a morphological transforming agent in C3H10T1/2CL8 mouse embryo fibroblasts and a mouse lung tumorigen in strain A/J mice. B[j]A was evaluated as a skin tumor initiator in female SENCAR mice and was found to induce papilloma formation in the range of 40-400 micrograms/mouse. B[j]A was found to be extremely active, inducing 8.7 papillomas/mouse after an initiating dose of 40 micrograms/mouse. At this dose, 100% of the mice bore tumors. Comparison with four other cyclopenta-fused polycyclic aromatic hydrocarbons suggests that B[j]A is extremely potent.

Activation and metabolism of benz[j]aceanthrylene-9,10-dihydrodiol, the precursor to bay-region metabolism of the genotoxic cyclopenta-PAH benz[j]aceanthrylene.

Benz[j]aceanthrylene, a cyclopentafused polycylic aromatic hydrocarbon produced in combustion emissions, possesses a bay region and an etheno bridge which may both contribute to the overall genotoxicity of the compound. In order to assess the role of activation at the bay region, the precursor epoxide benz[j]aceanthrylene 9,10-oxide, its dehydration product 10-hydroxybenz[j]aceanthrylene, the key dihydrodiol 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene and the bay-region diol-epoxide 7,8-epoxy-9,10-dihydroxy-7,8,9,10- tetrahydrobenz[j]aceanthrylene were evaluated in the bacterial histidine-reversion plate incorporation assay (Ames assay) with Salmonella typhimurium strain TA98. The diol-epoxide alone showed direct-acting mutagenicity (10 revertants per nmole), which was decreased by addition of exogenous metabolic activation (Aroclor 1254-treated rat-liver S9), whereas all the other compounds tested were activated by increasing concentrations of S9. The potency of the diol-epoxide was not sufficient to account for the activity of the parent compound. Identification by proton nuclear magnetic resonance and mass spectrometry of the major products of further metabolism by Aroclor 1254-treated rat-liver S9 of the bay region precursor dihydrodiol 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene indicated that oxidation occurred predominantly at the etheno bridge, to give 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene-2(1H)-one, arising by (non-enzymic) rearrangement of the etheno bridge epoxide and the tetrol 1,2,9,10-tetrahydroxy-1,2,9,10- tetrahydrobenz[j]aceanthrylene. The bay region tetrol 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenz[j] aceanthrylene was observed, implying further bay-region metabolism; re-aromatization of the benzo ring to benz[j]aceanthrylene-9,10-diol also occurred. Thus oxidation at the etheno bridge accounts for the majority of the activity of benz[j]aceanthrylene and its derivatives when Aroclor 1254-treated rat-liver S9 is used for exogenous metabolic activation.

Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in isolated rat hepatocytes and rabbit lung cells.

Benz[j]aceanthrylene (B[j]A) and benz[l]aceanthrylene (B[l]A), two isomeric cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) structurally related to 3-methylcholanthrene, were studied with respect to their genotoxic effects in isolated liver and lung cells. Both compounds were found to cause DNA adducts measured by the 32P-postlabelling technique. The level of DNA-adducts in rat hepatocytes exposed to 30 micrograms/ml B[l]A and B[j]A for 4 h were 46.5 +/- 22.0 and 8.3 +/- 5.1 fmol/micrograms DNA respectively. Using butanol extractions, the major and one of the minor B[j]A adducts co-chromatographed with B[j]A-1,2-oxide adducts of 2'-deoxyadenosine and 2'-deoxyguanosine. Thus, oxidation at the cyclopenta-ring of B[j]A appears to be an important activation pathway. In hepatocytes, 3-30 micrograms/ml of B[j]A and B[l]A induced DNA damage and repair measured both as increased alkaline elution of DNA and as increased incorporation of [3H]TdR in the DNA. B[l]A was somewhat more potent than B[j]A in inducing DNA repair. Reactive CP-PAH intermediates formed in the hepatocytes caused mutations in Salmonella typhimurium TA98 upon co-incubation. DNA adducts were also observed in isolated rabbit lung cells exposed to 30 micrograms/ml B[l]A or B[j]A for 2 h. A total of 14.5 +/- 6.9, 2.9 +/- 2.1 and 0.2 +/- 0.6 fmol B[l]A adducts/micrograms DNA were observed in Clara cells, type II pneumocytes and alveolar macrophages respectively. The main B[l]A adduct observed in the liver cells was not found in the lung cells. On the other hand, the levels of B[j]A adducts in the lung cells were in the range 4-14% of that found in liver cells, and no major differences between the various lung cells were observed. Neither B[l]A nor B[j]A induced DNA damage measured by alkaline elution in the lung cells, indicating that these adducts are not alkali labile.

32P-postlabelling in studies of polycyclic aromatic hydrocarbon activation.

Polycyclic aromatic hydrocarbons (PAHs) undergo metabolic activation reactions to yield intermediates that react with DNA to form covalent adducts. PAH administration leads to the formation of various types of DNA adducts that may differ between species, strains and tissues due to differences in metabolic activation and repair. The structures of PAH-DNA adducts can be identified by three approaches: co-chromatography with synthetic mononucleotide adduct standards; examining the adducts resulting from metabolism of pathway intermediates; or chemically blocking metabolic activation at specific sites on the PAH. Administration of putative metabolic intermediates of a PAH leads to enhanced formation of DNA adducts resulting from further activation along that pathway. Conversely, chemically blocking a bond or position on a PAH prevents adducts arising from activation at that site. By comparing the DNA adduct spectra generated by metabolites, blocked forms, and parent PAH administration, the pathways important in the metabolic activation of the PAH in each tissue may be deduced. Partial identification of these adducts may also be made by co-chromatography with the products of reactions between synthetic reactive intermediates and defined polydeoxynucleotides, and more thorough identification by using synthetic DNA adduct standards. These approaches have all been successfully applied to studies of PAH activation, and are reviewed here.

Ki-ras oncogene mutations in tumors and DNA adducts formed by benz[j]aceanthrylene and benzo[a]pyrene in the lungs of strain A/J mice.

Strain A/J mice received intraperitoneal injections of benz[j]aceanthrylene (B[j]A) or benzo[a]pyrene (B[a]P). At 24, 48, and 72 h, lung tissues were removed for analysis of B[a]P- or B[j]A-derived DNA adduct formation during the first 3 d of exposure. One group of mice exposed to these hydrocarbons was kept for 8 mo to determine lung tumor multiplicity, the occurrence of mutations in codons 12 and 61 of the Ki-ras gene in the tumors that arose, the relationship between Ki-ras oncogene mutations in tumors, and the presence and quantity of genomic DNA adducts. The major DNA adduct in the lungs of mice exposed to B[a]P was N2-(10 beta-[+B, 7 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl)-deoxyguanosine (BPDE-I-dGuo) arising from bay-region diolepoxide activation of B[a]P and was consistent with the occurrence of tumors with mutations GGT-->TGT (56%), GGT-->GTT (25%), and GGT-->GAT (19%) in codon 12, all involving mutations of a guanine. B[j]A, a demethylated analogue of 3-methylcholanthrene (3-MCA) with an unsaturated cyclopenta ring, produced 16-to 60-fold more tumors at equivalent doses than did B[a]P; the mutations in tumors were GGT-->TGT (4%), GGT-->GTT (30%), and GGT-->CGT (65%). Analysis of adduction patterns in DNA suggested that B[j]A was activated to form DNA-binding derivatives in A/J mouse lungs primarily at the cyclopenta ring even though B[j]A contains a bay region. As reported in the published literature, the mutation spectrum induced by 3-MCA in Ki-ras codon 12 of mouse cells is similar to that of B[a]P but not to that of its close relative B[j]A. In contrast to B[j]A, 3-MCA is activated mostly via a bay-region diol-epoxide since its cyclopenta ring is saturated and not easily epoxidates. Therefore, we propose that the GGT-->CGT mutations produced by B[j]A in Ki-ras codon 12 were mostly the result of cyclopenta-ring-derived adducts.

Morphological transformation and DNA adduct formation by benz[j]aceanthrylene and its metabolites in C3H10T1/2CL8 cells: evidence for both cyclopenta-ring and bay-region metabolic activation pathways.

Benz[j]aceanthrylene (B[j]A), a cyclopenta-fused polycyclic aromatic hydrocarbon related to 3-methylcholanthrene, has been studied to identify the major routes of metabolic activation in transformable C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts in culture. Previous studies have reported that the major (55% of total) B[j]A metabolite formed by C3H10T1/2 cells was (+/-)-trans-9,10-dihydro-9,10-dihydroxy-B[j]A (B[j]A-9,10-diol), the dihydrodiol in the bay-region ring, with moderate amounts (14% of total) of (+/-)-trans-1,2-dihydro-1,2-dihydroxy-B[j]A (B[j]A-1,2-diol), the cyclopenta-ring dihydrodiol. The morphological transforming activities of three potential intermediates formed by metabolism of B[j]A by C3H10T1/2 cells, (+/-)-anti-trans-9,10-dihydro-9,10-dihydroxy-B[j]A-7,8-oxide (B[j]A-diol-epoxide), B[j]A-9,10-oxide, and B[j]A-1,2-oxide as well as the two B[j]A-dihydrodiols were examined. B[j]A, B[j]A-diol-epoxide, B[j]A-1,2-oxide, and B[j]A-9,10-diol were found to have moderate to strong activities with B[j]A-diol-epoxide the most active compared to B[j]A, while B[j]A-1,2-diol was inactive. B[j]A-9,10-oxide was found to be a weak transforming agent. At 0.5 microgram/ml, the following percentage of dishes with type II or III foci were observed: B[j]A, 59%; B[j]A-diol-epoxide, 75%; B[j]A-1,2-oxide, 25%; and B[j]A-9,10-diol, 17%. DNA adducts of B[j]A, B[j]A-9,10-diol, B[j]A-diol-epoxide, B[j]A-9,10-oxide, and B[j]A-1,2-oxide in C3H10T1/2 cells were isolated, separated, identified, and quantitated using the 32P-postlabeling method. B[j]A forms two major groups of adducts: one group of adducts is the result of the interaction of B[j]A-1,2-oxide with 2'-deoxyguanosine and 2'-deoxyadenosine; the second group of adducts is a result of the interaction of B[j]A-diol-epoxide with 2'-deoxyguanosine and 2'-deoxyadenosine. Qualitative and quantitative analysis of the postlabeling data suggests that B[j]A is metabolically activated by two distinct routes, the bay-region diol-epoxide route and the cyclopenta-ring oxide route, the former being the most significant.

Direct labeling of DNA-adducts formed from carcinogenic diol-epoxides with a fluorescent reporter compound specific for the cis vic-diol group.

The fluorescent reporter group, N-(5-FLUORESCEINYL),N'-(3-BORONATOPHENYL)THIOUREA (FABA) has been synthesized. This boronate group makes the reporter specific for cis vic-diols. The reporter group is bound to DNA-adducts formed from the reaction of calf-thymus DNA and benzanthracene trans-10,11-dihydrodiol,8,9-epoxide (anti), benzo(a)anthracene-trans-3,4-dihydrodiol-1,2-epoxide (anti) but is not bound to 3-methylcholanthrene-11,12 dihydro-epoxide. Femtomole quantities of adduct may be detected.

Bacterial mutagenicity of new cyclopenta-fused cata-annelated polycyclic aromatic hydrocarbons, and identification of the major metabolites of benz[j]acephenanthrylene formed by Aroclor-treated rat liver microsomes.

Three novel cyclopenta-fused polycyclic aromatic hydrocarbons were synthesized, benz[d]aceanthrylene, benz[k]aceanthrylene, and benz[j]acephenanthrylene, and evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. The two benzaceanthrylene derivatives were active at low S9 concentrations in strain TA98 (4 and 27 rev/nmole respectively), as had been predicted from the calculated delta Edeloc/beta values of the carbocations derived from opening of the cyclopenta-fused epoxide rings, but the majority of this mutagenicity appeared to be due to free-radical decomposition products of spontaneous endo-peroxide formation. These compounds were therefore not further investigated. Benz[j]acephenanthrylene was also an indirect-acting frameshift mutagen (8-12 rev/nmole in strain TA98), but unlike most of the previously assayed cyclopenta-fused polycyclic aromatic hydrocarbons exhibited no peak of activity at low S9 protein concentration. The principal metabolites formed from this compound by microsomes from Aroclor-treated rat liver were benz[j]acephenanthrylene-4,5-dihydro-4,5-diol (necessarily derived from hydration of benz[j]acephenanthrylene 4,5-oxide) and benz[j]acephenanthrylene-9,10-dihydro-9,10-diol (precursor to benz[j]acephenanthrylene-9,10-dihydrodiol 7,8-oxide, the bay-region diol-epoxide). Consideration of the reduced activity of this compound compared to the related structure chrysene, the S9 dependence curves, and the predicted delta Edeloc/beta values of the postulate active species, suggests that in contrast to most other cyclopenta-fused polycyclic aromatic hydrocarbons, bay-region diol-epoxide formation plays a greater role than epoxidation of the cyclopenta-fused ring in the metabolic activation of benz[j]acephenanthrylene.

Metabolism of benz[j]aceanthrylene (cholanthrylene) and benz[l]aceanthrylene by induced rat liver S9.

The metabolites of benz[j]aceanthrylene (B[j]A) produced by incubation with liver S9 proteins from rats induced with Aroclor-1254 and phenobarbital have been identified as: trans-B[j]A-1,2-dihydrodiol, B[j]A-9,10-dihydrodiol, B[j]A-11,12-dihydrodiol, and 10-hydroxy-B[j]A. The major metabolite formed (58-60%) by both induced S9 preparations was trans-B[j]A-1,2-dihydrodiol, the cyclopenta-ring dihydrodiol while oxidation at the k-region or the proximal-bay region was minor. There were no statistical differences in individual or total B[j]A metabolite rates between the 2 induced S9 preparations. B[l]A was metabolized by Aroclor-1254 and phenobarbital induced rat liver S9 preparations to trans-B[l]A-1,2-dihydrodiol, B[l]A-7,8-dihydrodiol, and B[l]A-4,5-dihydrodiol. The major B[l]A metabolite formed (28-40%) by both induced S9 preparations was B[l]A-7,8-dihydrodiol, the k-region dihydrodiol. Cyclopenta-ring oxidation to trans-B[l]A-1,2-dihydrodiol was approximately 50% of that observed for k-region oxidation. Both induced S9s produced similar rates of B[l]A metabolites except for B[l]A-7,8-dihydrodiol formation which was higher for Aroclor-1254-induced S9.