Terue Kasai

High-performance liquid chromatography-fluorescence determination of dinitropyrenes in soil after column chromatographic clean-up and on-line reduction

In order to quantify 1,3-dinitropyrene (DNP), 1,6-DNP and 1,8-DNP in soil, we developed an efficient clean-up procedure and a sensitive determination method using fluorescence detection. DNP isomers were efficiently cleaned by three stages of fractionation, i.e., a silica gel open column chromatography using stepwise elution and two further purification steps by high-performance liquid chromatography (PPLC) using a monomeric-type octadecylsilyl (ODS) column and a polymeric-type ODS column. The recoveries of DNPs during the whole clean-up process were 94% or more. The fraction corresponding to DNPs was injected into an analytical polymeric-type ODS column for HPLC to separate DNP isomers. The effluent from the analytical ODS column was directly introduced to a catalyst column, which was packed with 5 microns alumina coated with platinum and rhodium (Pt-Rh), in order to reduce DNPs to diamino compounds, and then the fluorescence of diaminopyrenes was detected. The immediate detection of diaminopyrene isomers after on-line reduction afforded a sensitive detection of DNP isomers. The detection limits for DNPs were in the range of 0.7 to 4 pg. These developed methods were applied to four soil samples collected at parks in residential areas.

Comparison of the mutational specificity induced by environmental genotoxin nitrated polycyclic aromatic hydrocarbons in Salmonella typhimurium his genes

Mutagenicity of 15 nitrated polycyclic aromatic hydrocarbons (nitro PAHs), which were detected in ambient air particles and/or combustion source emissions, were examined using a set of six Salmonella typhimurium tester strains (TA7001 to TA7006), and the mutational specificity was characterized by the comparison of the mutagenic potencies of nitro-PAHs in the tester strains. Each strain carries a unique missense mutation in the histidine operon and is reverted by only one specific base-substitution out of six possible changes. All nitro-PAHs tested were mutagenic in multiple strains, and were classified into four categories based on the strains predominantly reverted. 1-Nitropyrene (1-NPy), 2,7-dinitrofluoren-9-one and 1,3-, 1,6- and 1,8-dinitropyrene isomers exerted the highest mutagenicity in strain TA7005 (C.G-->A.T transversion) followed by strain TA7006 (C.G-->G.C transversion). 2- And 3-nitrofluoren-9-one isomers, 2-NPy and 2,7-dinitrophenanthrene were also markedly mutagenic in strain TA7005 but not in strain TA7006. For 2-, 3- and 9-nitrophenanthrene isomers, 2-nitrofluoranthene (2-NFT) and 4-NPy, TA7004 (G.C-->A.T transition) was the most responsive strain. 3-NFT was unique, showing the highest mutagenicity in strain TA7002 (T.A-->A.T transversion). All nitro-PAHs tested induced C.G-->A.T transversion, which is observed as the most frequent base-substitution mutation of p53 tumor suppressor gene in human lung cancer.

Genotixicity in vivo of phenazine and aminophenazines assayed in the wing spot test and the DNA-repair test with Drosophila melanogaster

The genotoxicity and DNA-damaging activity of 6 phenazine and aminophenazine derivatives were assayed in the wing spot and DNA-repair tests in Drosophila melanogaster. Phenazine (Pz), and all aminophenazines tested, namely, 1-aminophenazine (APz), 2-APz, 2,3-diaminophenazine (DAPz), 2,7-DAPz and 2,7-diamino-3,8-dimethylphenazine (DADMPz), exhibited mutagenicity significantly in the wing spot test. The activities in the wing spot test were ranked in a sequence DADMPz > (2,7-DAPz, 2,3-DAPz) > (2-APz, 1-APz, Pz). In the DNA-repair test, 2,3-DAPz, 2,7-DAPz, and DADMPz clearly showed DNA-damaging activity, but Pz, 1-APz and 2-APz were inactive. Based on these results, we predict that DADMPz, 2,3-DAPz and 2,7-DAPz are likely to be more carcinogenic than 2-APz, 1-APz or Pz.

Metabolic activation of 2- and 3-nitrodibenzopyranone isomers and related compounds by rat liver S9 and the effect of S9 on the mutational specificity of nitrodibenzopyranones

The effect of rat liver S9 on the mutagenicity of 10 nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) was evaluated with Salmonella typhimurium TA98NR using S9 from phenobarbital-, 3-methylcholanthrene (MC)-, beta-naphthoflavone- and polychlorobiphenyl-treated and untreated rats. 2-Nitrofluorene (2-NFI), 2-nitrofluoren-9-one (2-NFlone), 2-nitrocarbazole (2-NCz), 3-NCz, 2-nitrodibenzothiophene (2-NDBT), 2-nitro-6H-dibenzo[b,d]pyran-6-one (2-NDBP) and 3-NDBP were metabolically activated by one or more of 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 of S9, regardless of S9 induction. 2-NDBP 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. NADH and NADPH were the most effective electron donors on the activation of 2-NDBP by S9, 2-NDBP was also metabolically activated by NADH plus commercial preparations of xanthine oxidase. These activations of 2-NDBP were inhibited by allopurinol, indicating that cytosolic xanthine oxidase in rat liver S9 participates in the activation of 2-NDBP. The potency of 2- and 3-NDBP isomers as base-substitution mutagens was also enhanced by S9. In the presence of S9, both compounds showed the highest mutagenicity in strain TA7005 (C.G-->A.T) followed by strains TA7004 (G.C-->A.T), TA7006 (C.G-->G.C) and TA7002 (T.A-->A.T), and this mutation specificity was similar to that without S9, indicating that the mechanism of mutagenesis caused by NDBP isomers with S9 is similar to that without S9.

Metabolic activation of nitrodibenzofurans by rat liver in Salmonella/mutagenicity test

The effect of metabolic activation on the mutagenicity of nitrodibenzofurans (NDF) by rat liver S9 was evaluated with S. typhimurium tester strains. Except for 1-nitrodibenzofuran (NDF), five tested NDFs were mutagenic in strains TA98 and TA98/1,8-DNP6 without S9 mix but were not mutagenic in strain TA98NR. NDFs mutagenized strain TA98NR with S9 mix, and the NAD(P)H system plus 3-methylcholanthrene-induced S9 (3-MC-S9) was the most effective. The specificity of S9 enzyme(s) participating in the activation of NDFs was different from that of endogenous enzyme(s) in strain TA98, i.e., the order of mutagenic potency of NDFs in strain TA98 without S9 mix was 2,8- = 2,7-->3-->2-->4-->1-nitrated dibenzofuran and 2-NDF and 2,8-dinitrodibenzofuran (DNDF) were more mutagenic than 3-NDF and 2,7-DNDF, respectively, in strain TA98NR with S9 mix. The mutagenic potency of 2-NDF, 4-NDF, 2,7-DNDF and 2,8-DNDF in strain TA98NR with S9 mix was stronger than those in strain TA98 without S9 mix and the cytosolic fraction of the 3-MC-S9 accounted for more of the activation than the microsomal fraction. Studies with electron donors and inhibitors indicated that xanthine oxidase and/or NAD(P)H-quinone oxido-reductase (NQOR) participated in the activation of NDFs. The mutagenic potency of NDFs in strain TA98NR with S9 mix (3-MC-S9) was reflected in the induction of NQOR by pretreatment of rats with 3-MC.

Genotoxicity of 2-Amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) in Drosophila melanogaster and Enhancement of DNA Damaging Activity by Pretreatment with β-Naphthoflavone and Ethanol

The genotoxicity of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) was assayed by the wing spot test for detecting somatic cell mutations and the DNA-repair test for detecting DNA damage with Drosophila melanogaster. Although PhIP increased the frequency of small single spots in a dose-dependent manner in the wing spot test, no large single spots and twin spots were induced to a significant extent. In the DNA-repair test PhIP showed clear DNA-damaging activity. The DNA-damaging potency of PhIP was significantly increased when larvae were pretreated with β-naphthoflavone (BNF) 24-48 h after oviposition. Excessive reduction of the Rec- male/Rec+ female ratio was also observed in ethanol-pretreated stock compared with stock not pretreated. These results indicate that alcohol and BNF, both enzyme inducers, enhance the in vivo DNA-damaging activity of PhIP.