Rat bladder cell-mediated mutagenesis od Chinese hamster V79 cells and metabolism of benzo[a]pyrene.

Primary rat bladder epithelial cells were cocultivated with Chinese hamster V79 cells in the presence of carcinogens, and the induction of 6-thioguanine resistance in the V79 cells was used as a marker of cell-mediated mutagenesis. The carcinogens dimethylnitrosamine, 7,12-dimethylbenz[a]anthracene, and benzo[a]pyrene (BP) were mutagenic to V79 cells in the presence of bladder cells but not in their absence. Analysis of BP metabolites formed by bladder cells indicated that 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, and 9-hydroxybenzo[a]pyrene were the major organic-soluble metabolites formed. Glucuronide and sulfate conjugates of BP metabolites were also produced by bladder cells. Mutagenesis data from the rat bladder system and previous data from rat liver and lung cell-mediated mutagenesis systems indicate that the cell-mediated mutagenesis approach may provide a useful approach for studying the organotropic effect of chemical carcinogens. Furthermore, the finding that rat bladder epithelium can metabolize some carcinogens offers new possibilities for the mechanism of initiation of bladder cancer.

Comparative tumor-initiating activity of complex mixtures from environmental particulate emissions on SENCAR mouse skin.

The value of the SENCAR mouse for testing tumorigenic properties of complex mixtures on mouse skin was studied. Seven complex mixtures were obtained as dichloromethane extracts of collected particulate emissions from three diesel-fueled automobiles, a heavy-duty diesel engine, a nonleaded gasoline-fueled automobile, a coke oven battery, and a roofing tar pot. These emissions were applied topically at multiple doses to both male and female SENCAR mice that were subsequently promoted with 12-O-tetradecanoylphorbol 13-acetate. Two statistical analyses were applied to the data to rank the samples and to provide 95% confidence intervals. One analysis used tumor multiplicity data, applied them to a nonlinear Poisson model, and the second analysis used tumor incidence data and applied them to a log-probit model. Both analyses ranked the complex mixtures in similar order. Benzo[a]pyrene content alone could not account for all the tumorigenic activity in each complex mixture, indicating that other components also contribute to the overall tumorigenic activity.

The effect of modifiers of microsomal enzymes on chemical oncogenesis in cultures of C3H mouse cell lines.

Two cell lines, both derived from the C3H mouse and each having different responses (oncogenic and cytotoxic) to polycyclic aromatic hydrocarbon oncogens, were studied with respect to their drug-metabolizing enzymes. The 10T1/2CL8 cells (a C3H mouse embryo fibroblastic cell line) were much more effective in converting 3-methylcholanthrene (3-MC) to 3-MC water-soluble metabolites, 3-MC phenols, and 3-MC-bound cellular macromolecules than were CVP3SC6 cells (a new line of C3H mouse adult ventral prostate fibroblasts). Basal aryl hydrocarbon hydroxylase activity was higher in 10T1/2CL8 cells than in CVP3SC6 cells, while the reverse was found for epoxide hydrase activity (using 3-methylcholanthrene-11, 12-oxide as substrate. 3-MC or benz(a)anthracene induced epoxide hydrase activity in both cell lines to about the same extent. 3-MC did not induce aryl hydrocarbon hydroxylase activity in CVP3SC6 cells. Aryl hydrocarbon hydroxylase activity was markedly induced in both cell lines by benz(a)anthracene and was slightly induced in 10T1/2CL8 cells by 3-MC. In a chemical oncogenesis cell culture system, transformation of 10T1/2CL8 cells mediated by 3-MC could be increased two- to threefold by treating the cell cultures with: either benz(a)anthracene, styrene oxide, cyclohexene oxide, or 1,2,3,4-tetrahydrona=phthalene-1,2-oxide; or with cyclohexene or 1,2-dihydrona-phthalene, alkene precursors of cyclohexene oxide and 1,2,3,4-tetrahydronaphthalene-1,2-oxide, respectively. When 10T1/2CL8 cells were treated with a combination of benz(a)anthracene and cyclohexene, 3-MC-mediated transformation was increased 7.8-fold. CVP3SC6 cells that were not transformed by 3-MC or other hydrocarbon oncogens were transformed by a combined treatment with benz(a)anthracene, 1,2-dihydronaphthalene, and 3-MC.

Metabolism of alpha-naphthoflavone by rat liver microsomes.

alpha-Naphthoflavone (ANF) or 7,8-benzoflavone, a synthetic flavonoid, has been widely used in biochemical and biological studies concerning the mechanisms of action of chemical carcinogens. It has been shown previously that ANF inhibits benzo(a)pyrene metabolism by beta-naphthoflavone (BNF)-induced rat liver microsomes but has no inhibitory effects on benzo(a)pyrene metabolism in phenobarbital (PB)-induced rat liver microsomes. This study shows that ANF gives type 1 binding spectra with and is metabolized by both BNF- and PB-induced rat liver microsomes. Specific metabolites identified by ultraviolet and mass spectra and in some cases by cochromatography with authentic standards were: 6-hydroxy-alpha-naphthoflavone, 9-hydroxy-alpha-naphthoflavone, alpha-naphthoflavone-5,6-oxide, and 5,6-dihydro-5,6-dihydroxy-alpha-naphthoflavone. Metabolism at the 5,6 bond of ANF accounted for 73 and 86% of the total organic soluble metabolites produced by PB- and BNF-induced microsomes, respectively. This result is in concert with previous observations on the role of 6 substitution and the loss of inhibitory activity of ANF in BNF-induced rat liver microsomes. Metabolism of ANF is mediated by the cytochrome P-450 mixed-function oxidases, because it is dependent on NADPH and inhibited by carbon monoxide and other cytochrome P-450 inhibitors. BNF-induced microsomes metabolize ANF to 5,6-dihydro-5,6-dihydroxy-alpha-naphthoflavone to a much greater extent than do PB-induced microsomes.

Inhibition of benzo(a)pyrene-induced transformation of C3H/10T1/2 cells by allylisopropylacetamide and isopropylvaleramide.

Allylisopropylacetamide (AIA) and isopropylvaleramide (IVA) have been demonstrated previously to protect in vivo against the acute toxicity and adrenal necrotic effect of 7,12-dimethylbenz(a)anthracene. In the present study, the influence of these two amides on the in vitro transforming ability of two potent carcinogens, benzo(a)pyrene [B(a)P] and 7,12-dimethylbenz(a)anthracene, on C3H10T1/2 cells was investigated. Both AIA and IVA showed a dose-dependent inhibition of B(a)P-induced transformation of C3H10T1/2 cells when added simultaneously for 24 hr with the carcinogen. While pretreatment, simultaneous treatment, and posttreatment of the cells with AIA or IVA inhibited transformation, the 24-hr posttreatment was somewhat more effective. The protective effect did not appear to results from alterations in B(a)P metabolism inasmuch as aryl hydrocarbon hydroxylase activity and the metabolic products of B(a)P detected by high-pressure liquid chromatography were not changed by AIA or IVA treatment. Furthermore, AIA and IVA did not selectively kill chemically transformed C3H10T1/2 cells, as indicated by the absence of their effect on an established, chemically transformed cell line. AIA and IVA also inhibited 7,12-dimethylbenz(a)anthracene-induced transformation of C3H10T1/2 cells. These data suggest that AIA and IVA may be useful protective agents and that they presumably exert their protective effect at some stage between the activation of the carcinogen and the expression of the transformed phenotype.

Separation of glutathione S-transferase activities with epoxides from the mouse liver h-protein, a major polycyclic hydrocarbon-binding protein.

The C3H mouse liver h-protein is a cytoplasmic protein to which metabolites of carcinogenic polycyclic hydrocarbons bind covalently following i.p. injection. It has a number of physical properties similar to those of the glutathione S-transferases (EC 2.5.1.18). These properties include molecular weight (40,000), number of subunits (2), basic isoelectric point around 8.0, sedimentation coefficient (3.5S), and subcellular localization. In this communication, we have shown that glutathione S-transferase activities with 1,2-epoxy(3-p-nitrophenoxy)propane and benz[a]anthracene 5,6-oxide as substrates were separated from the h-protein on carboxymethylcellulose and isoelectrofocusing columns. The purification of the mouse h-protein as a [3H]-7,12-dimethylbenz[a]anthracene conjugate or as the free form is also described.

Formation and persistence of novel benzo(a)pyrene adducts in rat lung, liver, and peripheral blood lymphocyte DNA.

Male CD rats were injected with single i.p. doses of benzo(a)pyrene (B(a)P), and peripheral blood lymphocytes (PBLs), livers, and lungs were removed at various times after administration. DNA adducts were analyzed in each tissue by 32P postlabeling with nuclease P1 enhancement. Sister chromatid exchange frequencies were concomitantly measured in cultured whole blood. B(a)P-DNA adducts were observed in all three tissues from animals sacrificed between 1 and 56 days after injection. Maximal adduction levels occurred at about 4 days after administration, followed by a gradual loss of adducts over the period examined. The apparent half-lives of total DNA adducts were 15 days in liver, 17 days in PBLs, and 22 days in lung. Induced sister chromatid exchanges were linearly related to the amount of DNA adducts remaining in the PBLs at the time of harvest up to 56 days and were significantly elevated above concurrent controls up to 14 days. One of the major adducts found in each tissue was N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a) pyrene]yl)deoxyguanosine. An additional novel major adduct was found in the liver DNA and is derived from the further metabolism of B(a)P-trans-7,8-dihydrodiol. A second major novel B(a)P adduct was found in the DNA of lung tissues and accounts for about 40% of the total adducts present. Experimental evidence suggests that this adduct is derived from a metabolic pathway that includes the formation of 9-hydroxy-B(a)P.

Metabolism of benzo(a)pyrene in monolayer cultures of human bronchial epithelial cells from a series of donors.

Benzo(a)pyrene [B(a)P] metabolism was measured in monolayer cultures of human bronchial epithelial cells derived from 18 specimens of explanted tissue. Bronchial epithelial cells converted B(a)P to dihydrodiols, phenols, quinone derivatives, and polyhydroxylated forms. Sulfate and glucuronide conjugates of B(a)P metabolites were also detected. Both total metabolism and distribution of metabolites showed a 10-fold or greater variation in cultures from different specimens. When the data were divided according to smoking status, however, no differences in total metabolism, extent of conjugation, or distribution of metabolites could be demonstrated between the two groups. Wide variation (over 1000-fold) in the cytotoxicity of B(a)P towards cells derived from different specimens was demonstrated but could not be directly correlated to the extent of metabolic activation. The results suggest that human bronchial epithelial cells which are newly grown from explanted tissue of smokers in culture do not demonstrate enzymatic induction. Variation among individuals observed in these studies probably represents basal differences in metabolic capability.

DNA adducts of the antitumor agent diaziquone.

We have studied adduct formation of the antineoplastic agent diaziquone (AZQ; NSC 182986) with DNA and nucleotides in vitro. The aziridine moieties of AZQ can be expected to interact covalently with DNA which, in turn, presumably elicits the antitumor activity. We analyzed AZQ-DNA adducts by a modified 32P-postlabeling assay involving purification of the nuclease P1-enriched labeled adducts by high-salt C18 reversed-phase thin-layer chromatography and separation of the eluted adducts on a polyethyleneimine-cellulose layer using non-urea salt solutions. Modification of calf thymus DNA with AZQ produced two major (22% and 40%) and at least eight minor adducts. At equal concentrations of AZQ and DNA (1 micrograms/microliters each), peak binding was observed in about 2 h [1926 +/- 378 (SD) fmol/micrograms of DNA] with the binding levels remaining practically unchanged through 4 h. However, incubation for 24 h resulted in over 40% decline, indicating adduct instability. AZQ was found to be highly reactive in vitro as evidenced by its substantial binding (49 +/- 14 fmol/micrograms of DNA) even at a DNA:AZQ ratio of 100:1. When incubated with mononucleotides, AZQ reacted extensively with adenine, guanine, and cytosine but only slightly with thymine. Cochromatography of the modified DNA and nucleotides revealed that one of the major adducts and several minor adducts were guanine derived. The aziridine rings of AZQ were found to be the main reactive sites as its monoaminoalcohol derivative showed as much DNA reactivity as did the parent compound, but no activity was observed when both aziridine groups were hydrolyzed to diaminoalcohols. The improved 32P-postlabeling assay seems capable of detecting relatively polar adducts such as those formed with AZQ at a level of one adduct/10(9) nucleotides.

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.

Chemical enhancement of viral transformation in Syrian hamster embryo cells by gaseous and volatile chlorinated methanes and ethanes.

Methods were developed for exposing cells in vitro to gases or vapors of volatilized organic liquids. Compounds were selected for their industrial importance, environmental impact, and suspected role in the etiology of some human cancers. Exposure chambers were designed for easy insertion of dishes of cultured cells and were equipped with inlet and outlet ports for introduction and purging of test gases. A gas delivery system utilizing a mass flow meter was used for the quantitative distribution of test gases into exposure chambers. For volatile compounds, appropriate volumes of cold (4 degrees) liquids in glass Petri dishes were quickly placed into chambers, the system sealed, and the compounds rapidly volatilized at 37 degrees. For exposure, the cells and chambers were placed in an incubator and rocked at a constant rate so that a portion of the cells was always in direct contact with the test gases or vapors. Known sample volumes were removed after various treatment times and test gas concentrations determined by standard gas chromatographic techniques. After exposure, the cells were removed and assayed for viability and increased sensitivity to viral transformation. Under these experimental conditions, the volatile liquids 1,1,1-trichloroethane, dichloromethane, chloroform, 1,2-dichloroethane, and 1,1-dichloroethane significantly enhanced transformation of Syrian hamster embryo cells by SA7 adenovirus, while acetone exerted no effect. The gases chloromethane and vinyl chloride were also active in this test system, while bromomethane, methane, and ethane were inactive. Incorporation of some of these compounds into liquid cell culture medium for cell treatment was either unsuccessful or produced only a weak enhancement response. Methodology is now available to evaluate volatile and gaseous carcinogens or mutagens and can be used to identify their mechanisms of action and the relative hazards of these agents to human health.

Formation of benzo(a)pyrene/DNA adducts and their relationship to tumor initiation in mouse epidermis.

The tumorigenicity of benzo(a)pyrene [B(a)P] applied topically as a skin tumor initiator in Sencar mice and the formation of epidermal B(a)P/deoxyribonucleoside adducts were compared over a similar range of doses (50 to 1600 nmol). The tumor-initiating activity of B(a)P, its covalent binding to mouse epidermal DNA, and the formation of the major hydrocarbon/deoxyribonucleoside adduct showed approximately parallel dose-response curves. The major hydrocarbon/deoxyribonucleoside adduct formed cochromatographed with marker adducts of (N2-(10S-[7R,8S,9R-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]y) deoxyguanosine while other minor adducts also were observed. The disappearance of DNA-bound products in the epidermis was followed for 21 days after an initiating dose of B(a)P (100 nmol) was applied topically to the mice. The half-lives of the B(a)P/deoxyribonucleoside adducts and the total radioactivity bound to the DNA were 4.5 and 5.5 days, respectively. However, in spite of the loss of measurable DNA-bound material, the tumor yield was unchanged regardless of whether promotion was begun 7 or 21 days after initiation. The results suggest a possible causal relationship between B(a)P/deoxyribonucleoside adduct formation and papilloma formation in mouse skin.

Mutagenesis and morphological transformation of mammalian cells by a non-bay-region polycyclic cyclopenta(cd)pyrene and its 3,4-oxide.

Cyclopenta(cd)pyrene, a constituent of environmental emissions, has been found to mutate and transform mammalian cells in culture. Cyclopenta(cd)pyrene 3,4-oxide, a presumed metabolite, was found to be a direct-acting mutagen and to transform mammalian cells. These results suggest that cyclopenta(cd)pyrene 3,4-oxide may be an ultimate mutagenic form of the parent hydrocarbon.

Identification of cocarcinogens and their potential mechanisms of action using C3H10T 1/2 CL8 mouse embryo fibroblasts.

The cocarcinogenic action of five agents which increase microsomal mixed-function oxidase activity in vivo was examined in the C3H10T 1/2 CL8 transformation assay. The compounds studied were benz(a)anthracene, 5,6-benzoflavone, phenobarbital, pregnenolone-16 alpha-carbonitrile, and Aroclor 1254. After a 48-hr pretreatment with the agent, the cells were then treated with benzo(a)pyrene [B(a)P] and the agent for an additional 24 hr. All agents except for Aroclor 1254 increased B(a)P-mediated transformation in C3H10T 1/2 CL8 cells. Benz(a)anthracene, 5,6-benzoflavone, phenobarbital, and pregnenolone-16 alpha-carbonitrile also increased the overall metabolism of B(a)P in C3H10T 1/2 CL8 cells to 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene; 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 9-hydroxybenzo(a)pyrene, and 3-hydroxybenzo(a)pyrene. Growth studies indicated that all four agents had no stimulatory effect which might have explained the increases in transformation frequency. This suggests that these agents exert their cocarcinogenic action via increases in the enzyme-mediated pathways of B(a)P metabolism.

Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells.

The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct basis. However, the relationships between frequency of genetic effect or morphological transformation and B(a)P-DNA binding or BPDE I-dGuo were linear within a given assay. In order to compare biological end points of diverse frequencies in diverse biological systems, a doubling adduct level, expressed as the number of BPDE I-dGuo adducts per unit of DNA required to double the induced frequency of biological response, was applied to the data.

Tumorigenesis and genotoxicity of ethyl carbamate and vinyl carbamate in rodent cells.

Vinyl carbamate (VC) is a suspect metabolic intermediate in ethyl carbamate (EC) carcinogenesis. In the present studies, EC and VC were evaluated for their relative abilities to induce adenomas and sister chromatid exchanges (SCEs) in lung cells of A/J, C3HeB/FeJ, and C57BL/6J strain mice. For both end points, animals were administered a single i.p. injection of the test chemical. Percentage of mice with adenomas and number of adenomas per mouse were compared among the three strains 24 weeks following exposure to EC or VC. Although the relative order of strain sensitivity was the same for both chemicals: A/J greater than C57BL/6J greater than C3HeB/FeJ, VC was much more potent than EC. For SCE analysis of primary lung cells cultured from treated animals, EC and VC showed potency differences similar to those observed for tumorigenesis. All three mouse strains revealed significant dose-dependent increases in SCE frequency. However, there was no strain specificity for this effect. SCE persistence over time was also compared in treated A/J and C57BL/6J mice. Although EC- and VC-induced SCE frequencies declined over a 2-week observation period, again, there was no strain specificity for this effect. VC was also tested for enhancement of SA7 virus transformation of Syrian hamster embryo cells. Significant concentration-dependent increases in cell transformation frequency were observed.

Adenomas induced by polycyclic aromatic hydrocarbons in strain A/J mouse lung correlate with time-integrated DNA adduct levels.

The induction of DNA adducts and adenomas in the lungs of strain A/J mice has been investigated following the single i.p. administration of each of the following polycyclic aromatic hydrocarbons (PAH): pyrene, dibenz[a,h]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, 5-methylchrysene, and cyclopenta[c,d]pyrene. DNA adducts were measured by 32P-postlabeling at times between 1 and 21 days following injection, while adenomas were counted at 240 days after treatment. Pyrene did not induce either DNA adducts or lung adenomas at any of the doses examined. Each of the remaining PAH induced both adenomas and DNA adducts in a dose-dependent manner, with dibenz[a,h]anthracene > 5-methylchrysene > cyclopenta[c,d]pyrene > benzo[a]pyrene > benzo[b]fluoranthene. DNA adducts reached maximal levels between 3 and 9 days after injection, followed by a gradual decrease. The time-integrated DNA adduct level (TIDAL) was calculated by numerically integrating the areas under the adduct persistence curves extrapolated to 240 days for each PAH at each dose level. This value represents the effective total molecular dose of PAH that was delivered to the lung DNA over the entire course of tumorigenesis. A strong correlation of lung adenoma induction with the TIDAL values was observed for each PAH. The slopes of the tumors versus TIDAL value relationships were essentially identical for 5-methylchrysene, cyclopenta[cd]pyrene, benzo[a]pyrene, and benzo[b]fluoranthene. The slope of this relationship for dibenz[a,h]anthracene was markedly greater. The essentially identical induction of adenomas as a function of TIDAL values for these PAH suggests that the formation and persistence of DNA adducts determines their carcinogenic potency.

Mutagenicity of cyclopenta-fused isomers of benz(a)anthracene in bacterial and rodent cells and identification of the major rat liver microsomal metabolites.

The microsomal metabolites and mutagenic activity of four cyclopenta-fused benz(a)anthracenes, benz(j)aceanthrylene [B(j)A], benz(e)aceanthrylene [B(e)A], benz(l)aceanthrylene [B(l)A], and benz(k)acephenanthrylene [B(k)A], have been studied. Aroclor 1254-induced rat liver microsomes metabolized B(j)A to B(j)A-1,2-dihydrodiol, B(j)A-9,10-dihydrodiol, B(j)A-11,12-dihydrodiol, and 10-hydroxy-B(j)A; B(e)A-1,2-dihydrodiol, B(e)A-3,4-dihydrodiol, and B(e)A-5,6-dihydrodiol; B(l)A to B(l)A-1,2-dihydrodiol, B(l)A-4,5-dihydrodiol, and B(l)A-7,8-dihydrodiol; and B(k)A to B(k)A-4,5-dihydrodiol and B(k)A-8,9-dihydrodiol. With each polycyclic aromatic hydrocarbon, metabolism occurred on the cyclopenta ring. All four isomers were active as gene mutagens in Salmonella typhimurium and in Chinese hamster V79 cells. In the S. typhimurium mutation studies, using Aroclor 1254-induced rat liver S9, B(j)A, B(e)A, and B(l)A required significantly less microsomal protein for maximal mutation response than B(k)A and B(a)P, suggesting a one-step activation mechanism, presumably on the cyclopenta-fused ring. B(j)A, B(e)A, and B(l)A were significantly more mutagenic than B(k)A and B(a)P in S. typhimurium. In the Aroclor 1254-induced rat liver S9-mediated V79 mutagenesis system, all four isomers were active, with B(l)A the most active. When Syrian hamster embryo cells were used as the metabolic activation component for V79 cells, only B(l)A produced a significant response and was equivalent in activity to B(a)P. A helical configuration for B(l)A is inferred from the identification of two trans-B(l)A-1,2-dihydrodiols, syn and anti, which have been synthesized, separated, and characterized. The metabolically formed dihydrodiol is anti-trans-B(l)A-1,2-dihydrodiol, and experimental evidence suggests that the metabolically formed B(l)A-1,2-oxide is the anti-isomer. Synthetic B(l)A-1,2-oxide was found to be a direct-acting mutagen in S. typhimurium and Chinese hamster V79 cells and is estimated to account for up to 40% of the mutagenic activity of the parent hydrocarbon. Therefore, certain cyclopenta-ring fusions on benz(a)anthracene appear to markedly increase its genotoxic and carcinogenic activities.

A preliminary structure-activity study of the mixed-function oxidase inhibitor 7,8-benzoflavone.

A series of substituted and structural analogues of 7,8-benzoflavone were examined for their ability to inhibit benzo[a]pyrene oxidation by the mixed-function oxidases found in hepatic microsomes prepared from 3-methylcholanthrene- and phenobarbital-induced rats. Of all the benzoflavones tested, only 6-amino-7,8-benzoflavone possessed significant inhibitory activity toward both classes of induced mixed-function oxidases. Parameters which were found to be necessary for maximal inhibitory activity were the maintenance of an unsubstituted or specifically substituted exocyclic phenyl group on position 2, the preservation of the pyran-4-one ring, and a 6 position which is either unsubstituted or substituted with an oxidizable moiety.

Synthesis and biological studies of 3-(beta-D-ribofuranosyl)-2,3,-dihydro-6H-1,3-oxazine-2,6-dione, a new pyrimidine nucleoside analog related to uridine.

Reaction of the trimethylsilyl derivative of 2,3-dihydro-6H-1,3-oxazine-2,6-dione (2, "uracil anhydride") with protected 1-O-acetylribofuranoses in the presence of stannic chloride gave the corresponding block nucleosides. 3-(2,3-5-Tri-O-2',2',2'-trichloroethoxycarbonyl-beta-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione (4c) thus prepared from the protected sugar 3c, 1-O-acetyl-2,3,5-tri-O-(2,2,2-trichloroethoxycarbonyl)ribofuranose, gave, on removal of the protecting groups with zinc dust,3-(beta-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione (1). The structure of 1 was confirmed by uv, ir, NMR, and CD spectral data and was shown to be an N nucleoside. Uracil anhydride, 2, and, to a lesser extent, its ribonucleoside 1 exert a moderate growth inhibition of mouse leukemia L5178Y, HeLa, and Novikoff hepatoma cells i- culture. Both compounds produce weak inhibition of vaccinia viral replication in HeLa cells.