It remains uncertain why non-genotoxic compounds that result in liver hypertrophy cause liver tumors. In an effort to resolve this issue, we examined whether liver post-mitochondrial fraction (S9) prepared from rats treated with non-genotoxic compounds affected the genotoxicity of pro-mutagens. Known hepatotoxic compounds, such as piperonyl butoxide (PBO), decabromodiphenyl ether (DBDE), beta-naphthoflavone (BNF), indole-3-carbinol (I3C) and acetaminophen (AA), were orally administered to male and female F344 rats at doses sufficient to cause liver hypertrophy. Rats received diets containing each test compound for 3 days, 4 weeks or 13 weeks, and were then kept for 4 weeks without the test chemical. S9 prepared from the livers of each group was used for the Ames test with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA). In both sexes, liver hypertrophy was observed following administration of all test compounds, and was then reversed to the control state when administration ceased. The mutagenicity of MeIQx, BaP and NDMA increased with the use of S9 derived from rats treated with non-genotoxic compounds other than AA. DBDE administration had a marked effect on the mutagenicity of BaP (over a 30-fold increase in females) and NDMA (about a 20-fold increase in males). To estimate the involvement of metabolic enzymes in the alteration of mutagenicity, we measured the activity of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) (phase I enzymes), and UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (phase II enzymes) in each S9 sample. The activity of phase I enzymes increased, even at the 3rd day following administration, and then decreased gradually, except in the case of AA, while the activity of phase II enzymes increased slightly. These results suggest that non-genotoxic hepato-hypertrophic compounds may be partly involved in carcinogenesis by modulating the metabolism of pre-carcinogens incorporated from the environment, in a manner that is dependent on sex and pre-incorporated chemicals.
Although nucleotide excision repair (NER) removes bulky adducts upon exposure to various chemicals, base excision repair (BER) removes altered bases that cause relatively minor disturbances to the helical DNA structure. We have shown that the action of BER can be distinguished from that of NER by the enhancement of a comet-positive response by 2′,3′-dideoxythymidine (ddT). Furthermore, in this study, the action of NER is shown to be distinguishable from that of BER by the reduction of a comet-positive response by α-amanitin (AMN). To study how bulky alkylated bases can be removed by NER or BER, we studied the reduction of a comet-positive response by AMN and its enhancement by ddT for n-alkyl methanesulfonates having an n-alkyl group with 1-7 carbons. For this purpose, n-alkyl methanesulfonates having an n-alkyl group with 3-7 carbons (n-propyl methanesulfonate, n-butyl methanesulfonate, n-pentyl methanesulfonate, n-hexyl methanesulfonate, and heptyl methanesulfonate) were synthesized. AMN reduced the comet-positive response for n-alkyl methanesulfonates having an n-alkyl group with ≧4 carbons and ddT enhanced it for n-alkyl methanesulfonates having an n-alkyl group with ≦5 carbons. Therefore, it is considered that NER acts to remove bases alkylated by n-alkyl groups with ≧4 carbons, BER acts to remove bases alkylated by n-alkyl groups with ≦5 carbons, and both NER and BER act to remove bases alkylated by n-alkyl groups with 4 and 5 carbons.
Histone H2AX phosphorylated at Ser139 (γ-H2AX) is a useful biomarker for DNA double-strand breaks. However, γ-H2AX detection has methodological disadvantages such as the requirement of expensive anti-γ-H2AX antibody and time-consuming handling for its staining. Mediator of DNA damage checkpoint 1 (MDC1) is a central adaptor protein which recruits various DNA damage response proteins to γ-H2AX and thus forms nuclear foci in the same location as γ-H2AX in response to DNA damage. Here, we describe an easy-to-use genotoxicity assay which combines enhanced green fluorescence protein (EGFP)-fused MDC1-expressing cells with a free R program for image-processing and quantification of foci area/nucleus. The workflow of this assay is simple: mutagen treatment, imaging, and R-processing. This assay does not need antibodies or staining handling and it detected the genotoxicity of a range of mutagens, including camptothecin (topoisomerase I inhibitior), cisplatin (crosslinker), and 4-nitroquinoline 1-oxide and benzo[a]pyrene (bulky DNA-adduct forming compounds), as increased fluorescence of EGFP-MDC1 foci. Furthermore, cotreatment with arabinofuranosyl cytosine/hydroxyurea and mutagens sensitized EGFP-MDC1 foci formation to bulky DNA adduct-type mutagens. Additionally, the established cells can be monitored in real-time using live cell imaging to obtain detailed dynamics of MDC1 in response to mutagens. The simple handling of this assay is expected to enable its full automation, thus making it useful for high-throughput genotoxicity screening of chemicals and monitoring of environmental mutagens.
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