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P I.16 Effect of exogenous metabolic activation on the mutagenicity of 2- and 3-nitrodibenzopyranone isomers and related compounds in Salmonella typhimurium
SIO
S-I: Metabolism of mutagens and carcinogens
06-alkylguaoloe-DNA alkyltraoferasel promote dlbromoalkaoe lethality and mutagenldty In Escherichia coli K11 Nieves Abril, Francisco L. Luque-Romero, Maria-Jose Prieto-Alamo, Carmen Pueyo. Departamento de Bioquimica y Biologic Molecular, Unioersidad de Cordoba, E-14071 Cordoba. Espana We have previously reported that the DNA alkyltransferase (ATase) encoded by the E. coli ogt gene enhances markedly the mutagenic potency of 1,2dibromoethane (DBE) and dibromomethane (DBM). Here we demonstrate that Ogt sensitises also the bacteria to the lethal effects of these carcinogens and that the sensitisation to both lethality and mutagenesis by DBE and DBM is a property shared by other ATases. In order of effectiveness the ATases ranked: murine> human> Ogt > rat. A truncated Ada version that retains the 06-methylguanine binding domain of the protein resembled Ogt ATase in its capacity to sensinse E. coli to the genotoxic action of both dibromoalkanes, while the full length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by these compounds. The ability to sensitise E. coli to the lethal and mutagenic effects ofdibromoalkanes seems restricted to ATases, since overexpression of thioredoxin (Trx) or glutaredoxin (Grxl) in ogt- ada- cells showed no effect, in Spite of the reported potential of dibromoalkanes to alkylate the cysteine-residue in the active site ofTrx. These results suggest the possible relevance of this ATasemediated mechanism to cause genotoxic damage in mammals. Our current working hypothesis is that the ATase might form a covalent complex between its own active-site cysteine residue and the compound. Such a reaction might activate the dibromoalkanes, and mediate its binding to DNA as postulated for the glutathione-dependent activation pathway.
The present study was undertaken to establish whether liver and kidney enzyme systems, from rat and mouse, have the potential to metabolise and bioactivate agaritine, !l-N-(y-L(+)glutamyl)-4_ (hydroxymethyl)phenylhydrazine, the most abundant hydrazine present in the edible mushroom Agaricus bisporus. Agaritine was weakly mutagenic, in the absence of an activation system, in Salmonella typhimurium strain TAI04. Rat kidney homogenates, in contrast to hepatic microsomes, enhanced the mutagenic response; moreover, hepatic microsomes could further potentiate the mutagenic response induced by the kidney. Agaritine was a good substrate for purified y-glutarnyl transpeptidase, being converted to a major metabolite 4-(hydroxymethyl)phenylhydrazine, formed as a result of the loss of the glutamyl moiety. Kidney homogenates from the rat and mouse also catalysed this reaction, the former being the more effective. Metabolism of agaritine was suppressed by serine-borate, an inhibitor of y-glutamyl transpeptidase. Kidney homogenates from rat and mouse could metabolise agaritine to intermediate(s) that bound covalently to proteins, with the rat preparations being the more effective; covalent binding was inhibited by glutathione. In contrast, hepatic preparations alone were ineffective in producing such covalent binding but did further increase the covalent binding mediated by the kidney preparations. The above observations collectively demonstrate that rat and mouse kidney homogenates can convert agaritine metabolically to electrophilic products that are genotoxic and bind covalently to proteins. Furthermore, hepatic microsomes can further rnetabolise the kidney-generated metabolites to even more genotoxic products. Keyword(s): Agariline; Agaricus bisporus; Mushroom
Ip 1.181
Keyword(s): Mutagenesis-promotion; Toxicity-promotion; Dibromoalkanes; DNA.alkyltransferase
The role or glutalhlone S-transferase genes In the suscepti_ bility to genetlc damage In humans and rodents
Andrew K1igerman, David DeMarini, Carolyn Doerr, Nancy Hanley, Velva Milholland, Alan Tennant. U.S. EPA. Research 'Iriangle Parle, NC. USA
IP 1.161
Effect of exogenous metabolic activation on the mutagenicIty of 2- and 3-nltrodlbenzopyranone Isomers and related compounds In Salmonella typhlmurlum
Tetsushi Watanabe, Terue Kasai, Teruhisa Hirayama. Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607, Japan The effect of rat liver S9 on the mutagenicity of 2· and 3-nitro-6H· dibenzolb.djpyran-e-one (nitrodibenzopyranone, NDBP) and 8 nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) was evaluated with Salmonella Iyphimurium TA98NR. S9 was prepared from the rats treated with phenobarbital, 3-methylcholanthrene (Me), b-naphthoflavone and polychlorobiphenyl and untreated rats. 2-Nitroftuorene, 2-nitroftuoren-9-one (2-NFlone), 2nitrocarbazole (2·NCz), 3-NCz. 2-nitrodlbenzothiophene. 2-NDBP and 3NDBP were metabolically activated by the S9 fractions, and the highest enhancement of the mutagenic potency of nitro- PAHs was observed with 3-MC-induced S9. Only in the case of 3-NFlone was the mutagenicity in strain TA98NR decreased by the addition ofS9, regardless ofS9 induction. 2NDBP was most efficiently activated among nitro-PAHs tested by all S9 fractions used. The cytosolic fraction of S9 accounted for more of the activation of 2-NDBP than the microsomal fraction. 2-NDBP was also metabolically activated by NADH plus commercial preparations of xanthine oxidase. These activations of2-NDBP were inhibited by allopurinol, indicating that cytosolic xanthine oxidase in rat liver S9 participates in the activation of 2-NDBP.
To understand better the species differences in carcinogenicity induced by 1,3-butadiene (BD), we exposed splenic and peripheral blood lymphocytes from rats, mice, and humans to 3,4-cpoxy-I-bulene (EB) or 1,2:3,4diepoxybutane (DEB). These studies indicate that EB does not induce measurable cytogenetic damage in either rat or mouse Go splenocytes as measured by either sister chromatid exchange (SCE) or chromosome aberration (CA) analyses. However, DEB was a potent inducer of both SCEs and CAs in Go splenic and peripheral blood lymphocytes. A comparison of the responses among species showed that the rat and mouse were approx~ma~ly equisensitive to the cytogenetic damaging effects of DEB, but the SituatIon for the human subjects was more complex. The presence or absence. ~f the GSTTI gene (expressed in erythrocytes) determined the relative sensittvity of the PBLs to the cytogenetic damaging effects ~f DE~ ': E~ was a mod.erately potent SeE-inducer when administered to actively dividing cells dunng the last 24 h of culture in all three species. However, DEB was > IOx more potent than EB for SCE induction. Similar to what was found with DEB, GSTMI had no effect on the senstivity to EB, whereas the lack of GSTTI markedly increased the cells' sensitivity to EB. Studies underway are examining rodents for increased susceptibility to EB and DEB due to variations in the rodent homologs of GSTTI and GSTMI genes. (This abstract does not necessarily reflect EPA policy). Keyword(s): glutathione S-transferase; butadiene; SCEs
Keyword(s). Metabolic activation; Nitro-6H-dibenzo[b,d]pyran-6-0ne; xanthine oxidase
Ip 1.191
Ip 1.171
1. Pluth, M. Ramsey, 1. Tucker. Lawrence Livermore Laboratory. Livermore. CA, USA
Bloactlvatlon or the mushroom hydrazlne. al:arltlne
Kim Walton l , Maurice M. Coombs', Fenton S. Catterall", Ron WalkerI, Costas lcannides'. 1 School of Biological Sciences, University of Surrey, Guildford. Surrey, GU} 5XH. UK; 2 Department of Chemistry, University of Surrey. Guildford, Surrey, GU} 5XH, UK
Relationship between genotypes and cbromosomal aberration frequencies In a normal population
Individual differences in cancer susceptibility may be attributed in part to genetic differences in the genes which code for enzymes involved in metabolic activation and detoxification of environmental procarcinogens. Polymorph isms of certain genes functioning in this manner (CYP2D6,
S-I: Metabolism of mutagens and carcinogens
06-alkylguaoloe-DNA alkyltraoferasel promote dlbromoalkaoe lethality and mutagenldty In Escherichia coli K11 Nieves Abril, Francisco L. Luque-Romero, Maria-Jose Prieto-Alamo, Carmen Pueyo. Departamento de Bioquimica y Biologic Molecular, Unioersidad de Cordoba, E-14071 Cordoba. Espana We have previously reported that the DNA alkyltransferase (ATase) encoded by the E. coli ogt gene enhances markedly the mutagenic potency of 1,2dibromoethane (DBE) and dibromomethane (DBM). Here we demonstrate that Ogt sensitises also the bacteria to the lethal effects of these carcinogens and that the sensitisation to both lethality and mutagenesis by DBE and DBM is a property shared by other ATases. In order of effectiveness the ATases ranked: murine> human> Ogt > rat. A truncated Ada version that retains the 06-methylguanine binding domain of the protein resembled Ogt ATase in its capacity to sensinse E. coli to the genotoxic action of both dibromoalkanes, while the full length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by these compounds. The ability to sensitise E. coli to the lethal and mutagenic effects ofdibromoalkanes seems restricted to ATases, since overexpression of thioredoxin (Trx) or glutaredoxin (Grxl) in ogt- ada- cells showed no effect, in Spite of the reported potential of dibromoalkanes to alkylate the cysteine-residue in the active site ofTrx. These results suggest the possible relevance of this ATasemediated mechanism to cause genotoxic damage in mammals. Our current working hypothesis is that the ATase might form a covalent complex between its own active-site cysteine residue and the compound. Such a reaction might activate the dibromoalkanes, and mediate its binding to DNA as postulated for the glutathione-dependent activation pathway.
The present study was undertaken to establish whether liver and kidney enzyme systems, from rat and mouse, have the potential to metabolise and bioactivate agaritine, !l-N-(y-L(+)glutamyl)-4_ (hydroxymethyl)phenylhydrazine, the most abundant hydrazine present in the edible mushroom Agaricus bisporus. Agaritine was weakly mutagenic, in the absence of an activation system, in Salmonella typhimurium strain TAI04. Rat kidney homogenates, in contrast to hepatic microsomes, enhanced the mutagenic response; moreover, hepatic microsomes could further potentiate the mutagenic response induced by the kidney. Agaritine was a good substrate for purified y-glutarnyl transpeptidase, being converted to a major metabolite 4-(hydroxymethyl)phenylhydrazine, formed as a result of the loss of the glutamyl moiety. Kidney homogenates from the rat and mouse also catalysed this reaction, the former being the more effective. Metabolism of agaritine was suppressed by serine-borate, an inhibitor of y-glutamyl transpeptidase. Kidney homogenates from rat and mouse could metabolise agaritine to intermediate(s) that bound covalently to proteins, with the rat preparations being the more effective; covalent binding was inhibited by glutathione. In contrast, hepatic preparations alone were ineffective in producing such covalent binding but did further increase the covalent binding mediated by the kidney preparations. The above observations collectively demonstrate that rat and mouse kidney homogenates can convert agaritine metabolically to electrophilic products that are genotoxic and bind covalently to proteins. Furthermore, hepatic microsomes can further rnetabolise the kidney-generated metabolites to even more genotoxic products. Keyword(s): Agariline; Agaricus bisporus; Mushroom
Ip 1.181
Keyword(s): Mutagenesis-promotion; Toxicity-promotion; Dibromoalkanes; DNA.alkyltransferase
The role or glutalhlone S-transferase genes In the suscepti_ bility to genetlc damage In humans and rodents
Andrew K1igerman, David DeMarini, Carolyn Doerr, Nancy Hanley, Velva Milholland, Alan Tennant. U.S. EPA. Research 'Iriangle Parle, NC. USA
IP 1.161
Effect of exogenous metabolic activation on the mutagenicIty of 2- and 3-nltrodlbenzopyranone Isomers and related compounds In Salmonella typhlmurlum
Tetsushi Watanabe, Terue Kasai, Teruhisa Hirayama. Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607, Japan The effect of rat liver S9 on the mutagenicity of 2· and 3-nitro-6H· dibenzolb.djpyran-e-one (nitrodibenzopyranone, NDBP) and 8 nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) was evaluated with Salmonella Iyphimurium TA98NR. S9 was prepared from the rats treated with phenobarbital, 3-methylcholanthrene (Me), b-naphthoflavone and polychlorobiphenyl and untreated rats. 2-Nitroftuorene, 2-nitroftuoren-9-one (2-NFlone), 2nitrocarbazole (2·NCz), 3-NCz. 2-nitrodlbenzothiophene. 2-NDBP and 3NDBP were metabolically activated by the S9 fractions, and the highest enhancement of the mutagenic potency of nitro- PAHs was observed with 3-MC-induced S9. Only in the case of 3-NFlone was the mutagenicity in strain TA98NR decreased by the addition ofS9, regardless ofS9 induction. 2NDBP was most efficiently activated among nitro-PAHs tested by all S9 fractions used. The cytosolic fraction of S9 accounted for more of the activation of 2-NDBP than the microsomal fraction. 2-NDBP was also metabolically activated by NADH plus commercial preparations of xanthine oxidase. These activations of2-NDBP were inhibited by allopurinol, indicating that cytosolic xanthine oxidase in rat liver S9 participates in the activation of 2-NDBP.
To understand better the species differences in carcinogenicity induced by 1,3-butadiene (BD), we exposed splenic and peripheral blood lymphocytes from rats, mice, and humans to 3,4-cpoxy-I-bulene (EB) or 1,2:3,4diepoxybutane (DEB). These studies indicate that EB does not induce measurable cytogenetic damage in either rat or mouse Go splenocytes as measured by either sister chromatid exchange (SCE) or chromosome aberration (CA) analyses. However, DEB was a potent inducer of both SCEs and CAs in Go splenic and peripheral blood lymphocytes. A comparison of the responses among species showed that the rat and mouse were approx~ma~ly equisensitive to the cytogenetic damaging effects of DEB, but the SituatIon for the human subjects was more complex. The presence or absence. ~f the GSTTI gene (expressed in erythrocytes) determined the relative sensittvity of the PBLs to the cytogenetic damaging effects ~f DE~ ': E~ was a mod.erately potent SeE-inducer when administered to actively dividing cells dunng the last 24 h of culture in all three species. However, DEB was > IOx more potent than EB for SCE induction. Similar to what was found with DEB, GSTMI had no effect on the senstivity to EB, whereas the lack of GSTTI markedly increased the cells' sensitivity to EB. Studies underway are examining rodents for increased susceptibility to EB and DEB due to variations in the rodent homologs of GSTTI and GSTMI genes. (This abstract does not necessarily reflect EPA policy). Keyword(s): glutathione S-transferase; butadiene; SCEs
Keyword(s). Metabolic activation; Nitro-6H-dibenzo[b,d]pyran-6-0ne; xanthine oxidase
Ip 1.191
Ip 1.171
1. Pluth, M. Ramsey, 1. Tucker. Lawrence Livermore Laboratory. Livermore. CA, USA
Bloactlvatlon or the mushroom hydrazlne. al:arltlne
Kim Walton l , Maurice M. Coombs', Fenton S. Catterall", Ron WalkerI, Costas lcannides'. 1 School of Biological Sciences, University of Surrey, Guildford. Surrey, GU} 5XH. UK; 2 Department of Chemistry, University of Surrey. Guildford, Surrey, GU} 5XH, UK
Relationship between genotypes and cbromosomal aberration frequencies In a normal population
Individual differences in cancer susceptibility may be attributed in part to genetic differences in the genes which code for enzymes involved in metabolic activation and detoxification of environmental procarcinogens. Polymorph isms of certain genes functioning in this manner (CYP2D6,