Dimethyl sulfoxide (DMSO) is generally believed to be non-cytotoxic at the concentrations used in the routine Ames test. In the present study, we found that DMSO reduced the number of bacterial survivors at a concentration of 14% in the treatment mixture (0.1 mL of DMSO in 0.7 mL of treatment mixture) under some assay conditions. The cytotoxicity emerged even before preincubation, and it was largely dependent on the bacterial strains used and the cell density of the treated cells; a low toxicity was detected at a low cell density. The most potent cytotoxicity was observed in the TA100 and TA1535 strains with S9 mix, where the reduction in survivors was about 60-90%, before or after preincubation. This reduction was, however, not accompanied by a significant diminution in the bacterial background lawn. The reduction in survivors does not seem to affect the sensitivity of mutagen detection, because no significant decrease in the number of induced revertants was observed in the preincubation assay using a direct mutagen, methyl methanesulfonate.

抄録全体を表示

Heterocyclic amines (HCAs) are a family of mutagenic and carcinogenic compounds produced during cooking or other burning processes, and exist in the environment. HCAs are metabolically activated by cytochrome P450, conjugated by phase II enzymes, to react with guanine bases. The aim of this study is to establish a chemical model for cytochrome P450 as an alternative to S-9 mix for detecting HCA mutagenicity in Salmonella strain. A chemical model was developed by comparing the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in the presence of an iron porphyrin and an oxidant. The iron porphyrin derivatives, water-soluble 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrinatoiron (III) chloride (4-MPy) or water-insoluble 5,10,15,20-tetrakis (pentafluorophenyl)porphyrinatoiron (III) chloride (F₅P), and the oxidant tert-butyl hydroperoxide (t-BuOOH), magnesium monoperoxyphthalate or iodosylbenzene were used. 4-MPy or F₅P with t-BuOOH activated Trp-P-2, and the activity was similar with either porphyrin. Water-soluble model has a better chance to detect unstable compound, since the tester strain was exposed in the whole incubation period in the mutation procedure with 4-MPy. The effectiveness of 4-MPy/t-BuOOH was evaluated with other HCAs; IQ, MeIQ, MeIQx, Glu-P-1, Glu-P-2, PhIP, Trp-P-1, MeAαC and AαC. All HCAs except for MeAαC and AαC were mutagenic in Salmonella typhimurium TA1538. MeAαC and AαC were not mutagenic in TA1538, but they were mutagenic in S. typhimurium TA1538/pYG219, which overexpresses O-acetyltranferase on the TA1538 genetic background. Although the HCAs mutagenicity with the chemical model was weaker than that with S-9 mix, the chemical models activated HCAs without S-9 mix in the Ames assay.

抄録全体を表示

It has been shown that cells having mutant p53 are more sensitive to genotoxic effects than cells having wild-type p53. To determine whether p53 status affects the sensitivity of the comet assay, we compared the results of the comet assay in WTK1 cells having mutant p53 and TK6 cells having wild-type p53 in the presence and absence of DNA repair inhibitors. Cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and bleomycin (BLM) for 2 h in the presence or absence of araC and hydroxyurea (araC/HU) or irradiated to UVC. UVC irradiated cells were cultured for 2 h with or without araC/HU. Immediately after the exposure to chemical mutagens or 2 h after UVC irradiation, slides for the comet assay were prepared. In the absence of araC/HU, the lowest genotoxic doses (LGDs) were higher in TK6 than in WTK1 cells, showing that wild-type p53 reduces comet assay sensitivity. On the other hand, in the presence of araC/HU, the LGDs were almost the same in WTK1 and TK6 cells. In the presence of araC/HU, positive responses to UVC irradiation were observed earlier in TK6 than in WTK1 cells. Therefore, SSB formation followed by SSB rejoining during the nucleotide excision repair process is considered to occur earlier in TK6 than in WTK1 cells, which results in a lower comet assay sensitivity in cells having wild-type p53. Comet assay response to mutagens inducing DNA damages that are repaired by the excision repair system tended to be affected by p53 status.

抄録全体を表示