Search

The role of acetylation in the genotoxicity of the heterocyclic amine, batracylin, was evaluated in Salmonella typhimurium strains expressing various levels of N- and O-acetyltransferase activity. A significant correlation was observed between batracylin-induced mutagenicity and bacterial N-acetyltransferase activity. Strains with the greatest capacity for N-acetylating batracylin (YG 1012 and YG 1024) were the most sensitive to the mutagenic effects of the drug. The number of revertants/nmol batracylin and the formation of acetylbatracylin were approximately 50-fold greater in YG 1024 compared to TA 98 which expresses endogenous levels of N-acetyltransferase. A similar response was observed with strains YG 1012 and TA 1538. Strains (TA 98/1,8-DNP6 or TA 1538/1,8-DNP6) which lack the ability to N-acetylate batracylin were the least sensitive to the mutagenic effects of the drug. At 1 microgram/plate of batracylin, the number of revertants in TA 1538 and TA 98 was 4-fold higher than that observed in TA 1538/1,8-DNP6 and TA 98/1,8-DNP6. To determine if batracylin was a substrate for human N-acetyltransferases, assays were performed in bacteria expressing NAT1 or NAT2. Both strains were capable of N-acetylating batracylin. The strain expressing NAT2 (DJ 460) formed a significantly greater amount of acetylbatracylin, as well as batracylin-induced revertants, compared to the strain expressing NAT1 (DJ 400). These results demonstrate that the mutagenicity of batracylin is directly related to N-acetyltransferase activity. Data obtained in bacteria expressing either human NAT1 and NAT2 show that batracylin is capable of being bioactivated by both human enzymes. In addition, the higher enzyme activity and mutagenicity in bacteria expressing NAT2 suggests that batracylin is a substrate of this enzyme in humans.

Fluoranthene (FA) is frequently among the more abundant components detected in environmental mixtures of polycyclic aromatic hydrocarbons. Several methylated fluoranthenes, although less prevalent than FA, have also been detected as environmental pollutants. While FA is inactive as a tumorigenic agent on mouse skin, it does induce lung and liver tumors in newborn mice. Among the five isomers of methylfluoranthene, only 2-methylfluoranthene (2-MeFA) and 3-methylfluoranthene (3-MeFA) are active as tumor initiators on mouse skin. A comparative bioassay was performed to determine the relative tumorigenic activity of FA, 2-MeFA and 3-MeFA in newborn CD-1 mice. All three compounds were assayed at doses of 3.46 and 17.3 mumol. The bioassay was terminated when mice were 1 year old. At a dose of 17.3 mumol, FA and 2-MeFA induced a similar incidence of lung tumors (65-96%) in both male and female mice. However, tumor multiplicity in the lung was different between FA and 2-MeFA. At a dose of 17.3 mumol, the multiplicity of lung tumors observed for mice administered 2-MeFA ranged from 3.04 to 3.94 tumors per mouse. In contrast, animals treated with FA developed only an average of 1.12-2.45 tumors per mouse. 3-MeFA did not induce a statistically significant incidence of lung tumors in either male or female mice. All three compounds when administered to newborn mice did induce a significant incidence of liver tumors among male mice. The relative tumorigenic potency observed was FA > or = 2-MeFa >> 3-MeFA. Only 2-MeFA at a dose of 17.3 mumol was tumorigenic in the liver of female mice. These bioassay results are contrasted with prior studies on the in vitro genotoxic activity and metabolic activation pathways of FA, 2-MeFA and 3-MeFA.

Quinoline in the presence of microsomal activation exhibits mutagenic activity in Salmonella typhimurium TA100 and induces unscheduled DNA synthesis (UDS) in rat hepatocytes. Structure-activity studies were performed to determine those positions on quinoline critically associated with its genotoxicity. In assays performed to determine the ability of 2-, 4-, 6- and 8-methylquinoline to induce UDS, only 4- and 8-methylquinoline produced a positive response. This represents an improved correlation for these methylquinolines with tumorigenic activity as compared to that previously observed with mutagenic activity in S. typhimurium TA100. The complete isomeric series of fluoroquinolines were evaluated as mutagens in S. typhimurium TA100 and for their potential to induce UDS in rat hepatocytes. The only isomers that did not exhibit significant mutagenic activity were 2- and 3-fluoroquinoline. Among these isomeric fluoroquinolines 5-, 6-, 7- and 8-fluoroquinoline are capable of inducing UDS. No significant effect on UDS was observed for 2-, 3- or 4-fluoroquinoline. These data indicate that positions 1-3 in quinoline are critical sites associated with its genotoxicity.

Indeno[1,2,3-cd]pyrene (IP) is a non-alternant polycyclic aromatic hydrocarbon that has tumor-initiating activity on mouse skin and is carcinogenic in newborn mice and in rat lungs. Previous studies have shown that 8- and 9-hydroxyIP and IP-1,2-diol are major metabolites formed in vivo in mouse skin. 8-HydroxyIP-1,2-diol and 9-hydroxyIP-1,2-diol are also observed as in vivo metabolites of IP. Although 8-hydroxyIP had marginal tumor-initiating activity on mouse skin, IP-1,2-diol and its epoxide precursor, IP-1,2-oxide, had similar tumorigenic activity as IP. In the present study fluorine probes have been employed to investigate the contribution of metabolic activation at the 1,2 and 7-10 positions of IP. At a total initiating dose of 4.0 mumol, 2-fluoroIP induced skin tumors in 76% of the treated animals with an average of 3.9 tumors/mouse. At the same dose, IP induced a 72% incidence of tumor-bearing mice with 2.1 tumors/mouse. In contrast, 8,9-difluoroIP elicited a tumorigenic response in 40% of the treated animals with 0.6 tumors/animal. Five mice from each experimental group were killed at the conclusion of the initiation phase of the bioassay and DNA was isolated from the treated areas of skin. 32P-Postlabeling analysis of the hydrolyzed DNA indicated that IP forms one major detectable DNA adduct that migrates close to the origin. This adduct is absent in mice treated with 8,9-difluoroIP. In contrast, 2-fluoroIP forms one major adduct spot with different retention behavior as compared with the adduct formed from IP. DNA from mice treated topically with IP-1,2-diol and IP-1,2-oxide was subjected to 32P-postlabeling analysis. IP-1,2-diol forms one major DNA adduct spot with mobility similar to that observed for the IP-DNA adduct. IP-1,2-oxide displayed an intense pattern of DNA adducts centered around the location of the IP-DNA adduct. No adducts were detected which had mobility similar to that formed from 2-fluoroIP. These results are consistent with IP undergoing metabolic activation at positions 7-10 either alone or in conjunction with dihydrodiol formation at the 1,2 position.

The tumor initiating activities on mouse skin of benzo[b]fluoranthene, (B[b]F), benzo[j]fluoranthene (B[j]F), benzo[k]fluoranthene (B[k]F] and three of their dihydrodiols, 9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene (B[b]F-9,10-diol), 9,10-dihydro-9,10-dihydroxybenzo[j]fluoranthene (B[j]F-9,10-diol), and 8,9-dihydro-8,9-dihydroxybenzo[k]fluoranthene (B[k]F-8,9-diol) were evaluated. Among the parent hydrocarbons, B[b]F was the most potent tumor initiator, with activity greater than that of B[j]F but less than that of benzo[a]pyrene. B[k]F also showed tumor initiating activity, in contrast to its lack of complete carcinogenic activity on mouse skin. B[b]F-9,10-diol, which can form a bay region dihydrodiol epoxide, was as active as B[b]F. B[j]F-9,10-diol, which would form its dihydrodiol epoxide in a four sided pseudo-bay region, was less active than B[j]F. B[k]F-8,9-diol was inactive. These results, together with parallel metabolic studies, suggest that the formation of bay region dihydrodiol epoxides may not be the major activation mechanism in benzofluoranthene tumorigenesis.

In this study we assayed the effects of snuff and nicotine on the DNA methylation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a powerful tobacco-specific N-nitrosamine. Male F344 rats were pretreated for 2 weeks with either a solution of a snuff extract or 0.002% nicotine in the drinking water. Subsequently, the rats were given a single dose of NNK and the effects of snuff and nicotine on the methylation of guanine by NNK in the DNA of target organs of this carcinogenic nitrosamine were determined. Formation of 7-methylguanine in the liver, nasal mucosae and oral cavity and of O6-methylguanine in the liver and oral cavity was much lower in the rats pretreated with snuff extract than in those not pretreated. On the other hand, pretreatment of the rats with nicotine had no significant effect on the methylation of DNA by NNK nor on the elimination constants of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridly)-1-butanol.

The metabolism of 3-, 8- and 9-fluorobenzo[k]fluoranthene (B[k]F) relative to B[k]F was investigated. The major metabolites of B[k]F formed in vitro using rat liver S-9 metabolism systems were 8,9-dihydro-8,9-dihydroxyB[k]F, the 2,3-quinone of B[k]F and 3-, 8- and 9-hydroxyB[k]F. Fluorine substitution within the structure of B[k]F substantially altered the types of metabolites formed in vitro. The most pronounced effect was observed with 9-fluoroB[k]F. In contrast to B[k]F, the 8,9-dihydro-8,9-dihydroxy-, 9-hydroxy- and 10,11-dihydro-10,11-dihydroxy derivatives were not detected as metabolites of 9-fluoroB[k]F. However, either the 2,3- or 4,5-dihydrodiol of 9-fluoroB[k]F was detected. In the case of 8-fluoroB[k]F, neither the 8- nor 11-hydroxy- derivatives were detected. The principle dihydrodiols formed from 8-fluoroB[k]F were the 10,11-dihydrodiol and either the 2,3-or 4,5-dihydrodiol. The pattern of metabolites formed with 3-fluoroB[k]F was similar to that observed with B[k]F with the exception that neither the 3- nor 4-hydroxy derivatives were formed. Mass spectral data indicated that fluoro substitution is not lost to any appreciable extent during the metabolism of 3-, 8- and 9-fluoroB[k]F. The mutagenic activity of these B[k]F fluoro derivatives along with B[k]F, 2,3-dihydro-2,3-dihydroxyB[k]F, the 2,3-quinone of B[k]F and 8,9-dihydro-8,9-dihydroxyB[k]F were evaluated in Salmonella typhimurium TA100 in the presence of rat liver S-9 metabolism systems. 3-FluoroB[k]F was more mutagenic than B[k]F, while both 8- and 9-fluoroB[k]F were less active. While the 2,3-dihydrodiol and 2,3-quinone were weakly active, the 8,9-dihydrodiol had similar mutagenic potency to B[k]F.

The metabolites of fluoranthene, 2-methylfluoranthene, and 3-methylfluoranthene obtained upon incubation with liver homogenate from Aroclor pretreated rats were assayed for mutagenicity in Salmonella typhimurium TA100. The mutagenic metabolites of fluoranthene and 2-methylfluoranthene were identified as 2,3-dihydro-2,3-dihydroxyfluoranthene and 4,5-dihydro-4,5-dihydroxy-2-methylfluoranthene, respectively. In contrast to these results, the major proximate mutagen detected among the in vitro metabolites of 3-methylfluoranthene was 3-hydroxymethylfluoranthene. Comparison of the mutagenic potential of 2-hydroxymethylfluoranthene demonstrated that the latter was a more powerful mutagen. Quantitative analyses of the metabolites of fluoranthene with that of 3-hydroxymethylfluoranthene demonstrated that the latter was a more powerful mutagen. Quantitative analyses of the metabolites of fluoranthene, 2-methylfluoranthene, and 3-methylfluoranthene indicated that similar amounts of dihydrodiols were formed. 4,5-Dihydro-4,5-dihydoxy-3-methylfluoranthene, however, was not found to be a potent mutagenic metabolite. These data suggest that the activation pathway to ultimate mutagens may differ for 2- and 3-methylfluoranthene.

5-Methylchrysene (5-MeC) can form two bay region dihydrodiol epoxides: 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I) which has the methyl group and the epoxide ring in the same bay region, and 7,8-dihydroxy-9,10-epoxy-7,8,10-tetrahydro-5-methylchrysene (DE-II). In a previous study, we observed that the ratio of DE-I:DNA adducts to DE-II:DNA adducts in mouse epidermis, 24 h after application of [3H]5-MeC metabolites was 2.7 to 1. To investigate the basis for this observation we have now studies: (i) the formation of [3H]5-MeC in mouse epidermis in vivo at various time intervals from 0.33 to 24 h; (ii) the persistence of DE-I:DNA adducts and DE-II:DNA adducts in mouse epidermis at 4-48 h after application of [3H]5-MeC; and (iii) the reactions of DE-I and DE-II with calf thymus DNA in vitro. In contrast to results obtained with mouse liver 9000 grams supernatant, the dihydrodiol precursors of DE-I and DE-II were present in equivalent quantities in mouse epidermis in vivo at every time point studied. The ratio of DE-I:DNA adducts to DE-II:DNA adducts in mouse epidermis was constant throughout the time period studied. However, the extent of formation of DE-I:DNA adducts was greater than that of DE-II:DNA adducts upon reaction of DE-I or DE-II with calf thymus DNA in vitro. These results suggest that differences in reactivity with DNA of DE-I and DE-II may bw responsible for the higher levels in mouse epidermis of DE-I:DNA adducts compared with DE-II:DNA adducts and provide a possible basis for the observed enhancing effect of a bay region methyl group on the carcinogenicity of polynuclear aromatic hydrocarbons.

Methods were developed to determine the biological half-lives and rates of distribution and elimination of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in the F344 rat. The formation and persistence of an in vivo equilibrium between NNK and NNAL were also studied. The method consists of extraction and elution of the nitrosamines through a Clin-Elut column with ethyl acetate, followed by concentration and analysis by a gas chromatography-thermal energy analyzer. The biological half-lives for NNN and NAT were 184 and 540 min, those for NNK and NNAL ranged from 25 to 37 and 184 to 298 min, respectively. A relatively short biological half-life for the TSNA suggests a correlation with carcinogenic potency.

Benzo[f]quinoline and benzo[h]quinoline are widespread environmental pollutants which have been found to be mutagenic. The metabolism of benzo[f]quinoline and benzo[h]quinoline was investigated using a liver homogenate from Aroclor-pretreated rats. The metabolites of benzo[f]quinoline which were identified were 7,8-dihydroxy-7,8-dihydrobenzo[f]quinoline, 9,10-dihydroxy-9,10-dihydrobenzo[f]quinoline, 7-hydroxybenzo[f]quinoline, and benzo[f]quinoline-N-oxide. Metabolism studies on benzo[f]quinoline performed in the presence of the epoxide hydratase inhibitor, 3,3,3-trichloropropylene oxide, demonstrated that the formation of both of these dihydrodiols can be inhibited. The major metabolites of benzo[h]quinoline were identified as 5,6-dihydroxy-5,6-dihydrobenzo[h]quinoline and 7,8-dihydroxy-7,8-dihydrobenzo[h]quinoline. Benzo[h]quinoline-N-oxide was not detected as a metabolite. In the presence of an epoxide hydratase inhibitor, the major metabolites of benzo[h]quinoline were 5,6-epoxybenzo[h]quinoline and 7-hydroxybenzo[h]quinoline. The difference in the metabolism to N-oxides observed between benzo[h]quinoline and benzo[f]quinoline is consistent with previous observations in which sterically hindered aromatic ring nitrogen compounds such as benzo[h]quinoline are more resistant to N-oxide formation. The nitrogen atom of these aza-arenes with its lone pair of electrons has a significant influence on sites at which dihydrodiols are formed. The data suggest that the aromatic ring nitrogen of these azaphenanthrenes has an effect similar to that of a methyl substituent in directing their metabolic oxidation.

The extent to which some of the more prevalent and potent carcinogens in cigarette smoke could be transferred from circulating blood into the milk of lactating rats was determined. One hour after i.v. administration of benzo[a]pyrene (BaP), N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to the dams, the levels of these carcinogens were determined in both blood and milk specimens. The average amount of radioactivity detected 1 h after administration of 14C-labeled BaP was 0.21% of the administered dose per ml of milk as compared with 0.17% per ml of blood. The amount of NNN in milk ranged from 0.20 to 0.36% of the administered dose per ml which closely paralleled the levels detected in blood. NNK is readily converted in vivo to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The sum of NNK and NNAL was similar in the blood and milk of treated dams. There was, however, a major difference in the ratio of NNAL/NNK as detected in milk and blood. The ratio of NNAL/NNK in blood ranged from 1.3:1 to 1.9:1 while the ratio in milk ranged from 2.4:1 to 3.3:1. In a comparative study of the levels of NNN in the blood and milk of lactating rats at less than 1.0, 20, 60, 120 and 240 min after administration, it was confirmed that similar concentrations of NNN are present in blood and milk 1 h after administration. These data indicate that these carcinogens, which are present in both cigarette smoke and tobacco, can be transferred into the milk of lactating rats.

The metabolism by rat liver 9000 x g supernatant of the environmental carcinogen benzo[b]fluoranthene was investigated. The major metabolites were identified, by comparison to synthetic samples, as 5- and 6-hydroxybenzo[b]fluoranthene and 4- and 7-hydroxybenzo[b]fluoranthene. The principal dihydrodiol metabolite formed under these conditions was trans-11,12-dihydro-11,12-dihydroxybenzo[b]fluoranthene, which was identified by comparison to the synthetic compound. 1,2-Dihydro-1,2-dihydroxybenzo[b]fluoranthene was identified, by its mass spectrum and by comparison if its u.v. spectrum to that of a synthetic model compound, 11-ethylidene-11H-benzo[b]fluorene. No evidence was obtained for the formation of 7b,8-dihydro-7,8-dihydroxybenzo[b]fluoranthene or trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene. The latter would be the precursor to a bay region dihydrodiol epoxide of benzo[b]fluoranthene.

Indeno[1,2,3-cd]pyrene is a ubiquitous environmental pollutant which is active as a tumor initiator and complete carcinogen on mouse skin and is carcinogenic in rat lung. The major metabolites of indeno[1,2,3-cd]pyrene as formed in vivo in mouse skin have been identified. 8-Hydroxyindeno[1,2,3-cd]pyrene is the most abundant metabolite identified. 9-Hydroxyindeno[1,2,3-cd]pyrene and trans-1,2-dihydro-1,2-dihydroxyindeno[1,2,3-cd]pyrene are also major in vivo metabolites in mouse skin. Several minor metabolites were also identified. Among these are trans-1,2-dihydro-1,2,8-trihydroxyindeno[1,2,3-cd]pyrene, trans-1,2-dihydro-1,2,9-trihydroxyindeno[1,2,3-cd]pyrene, indeno[1,2,3-cd]pyrene-1,2-dione, and 10-hydroxyindeno[1,2,3-cd]pyrene. The tumor-initiating activity of several of the major in vivo metabolites of indeno[1,2,3-cd]pyrene has been investigated on mouse skin. Trans-1,2-dihydro-1,2-dihydroxyindeno[1,2,3-cd]pyrene and 1,2-dihydro-1,2-epoxyindeno[1,2,3-cd]pyrene both produced an 80% incidence of tumor-bearing mice at a total initiating dose of 1.0 mg. The activity of this K-region dihydrodiol and K-region oxide was, however, less than that of the parent hydrocarbon. These data suggest that 1,2-dihydro-1,2-epoxyindeno[1,2,3-cd]pyrene, which is an ultimate mutagenic metabolite of indeno[1,2,3-cd]pyrene, is not the ultimate tumorigenic metabolite on mouse skin. 8-Hydroxyindeno[1,2,3-cd]pyrene, which is mutagenic when assayed in the presence of a microsomal activation system, exhibited only weak tumor-initiating activity. These results indicate that the principal metabolic activation pathways associated with the mutagenic activity of indeno[1,2,3-cd]pyrene are not related to its tumor-initiating activity on mouse skin.