The primary motivation for conducting short-term tests for environmental mutagens and carcinogens has been to predict mutagens and/or carcinogens and to assess any associated risks. Organ-specific in vivo short-term tests in rodents are valuable because chemical carcinogenesis is generally organ-specific. I have attempted to develop various organ-specific in vivo short-term tests mainly in rodent liver and stomach. Recently, our collaborative study group, Toxicogenomics/Japanese Environmental Mutagen Society·Mammalian Mutagenicity Study Group (JEMS·MMS), attempted to use gene expression profiling in in vivo short-term tests, conducted DNA microarrays to extract candidate marker genes, and later shifted to quantitative real-time PCR (qPCR) to profile the expression of selected genes. We successfully discriminated 8 genotoxic hepatocarcinogens from 4 non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) based on the gene expression profiles for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2, and Tubb2c) in mouse liver at 4 and 48 h following a single intraperitoneal administration of chemicals as determined by qPCR. More recently, we successfully performed a similar study in rat liver. Previously, my collaborators and I developed various organ-specific in vivo short-term test methods, including UDS (unscheduled DNA synthesis); RDS (replicative DNA synthesis) using a liquid scintillation counter in rat glandular stomach, forestomach, colon, and liver and hairless mouse epidermis; DNA single-strand scission (DSS); and ornithine decarboxylase assay (ODC) in rat glandular stomach on 62 compounds. Developing short-term tests that are helpful for the risk assessment of human mutagens and carcinogens would contribute to the development of ideal prediction methods.
The comet assay can detect single-strand breaks (SSBs) as initial lesions and those that developed from alkali-labile sites under alkaline conditions (pH>12.6). While the incision of UV-induced pyrimidine dimers creates a long gap of about 30 bp during nucleotide excision repair (NER) that is re-synthesized and repaired by DNA polymerase δ/ε, base excision repair (BER) creates a short gap of 1-6 bp that is closed by DNA polymerase β. Some chemicals are known to affect the formation of comet tails by the inhibition of enzymes taking part in DNA repair pathways. In this study, we investigated how 2′,3′-dideoxythymidine (ddT) which is a precursor of DNA polymerase β inhibitor, 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP), affects comet-tail formation. The effects of three kinds of DNA re-synthesis inhibitors on the response of the comet assay were studied in TK+/− heterozygotes of TK6 human lymphoblastoid cells. Aphidicolin (APC) and the combination of cytosine-1-β-D-arabinofuranoside and hydroxyurea (araC/HU) enhanced comet-tail formation by UV, a UV-mimetic agent, 4-nitroquinoline-1-oxide (4NQO), and methylating agents, methyl methanesulfonate (MMS) and methyl nitrosourea (MNU). On the other hand, ddT enhanced comet-tail formation by MMS and MNU, but not UV and 4NQO. Since ddT did not affect comet-tail formation by MMS and MNU in TK−/− TK6 cells, ddTTP which is expected to be formed from ddT by thymidine kinase in TK+/− TK6 cells would inhibit the resealing of short gaps left by excision of damaged bases induced by MMS and MNU. Taking into consideration that 75% of BER was estimated to be due to DNA polymerase β in human cells, ddT can enhance a comet-positive response upon exposure to mutagens that produce damaged bases removed by BER, showing that BER depending on DNA polymerase β can be distinguished from NER by the enhancement of a comet-positive response by ddT.
We present here a mutation assay with little bias which incorporates high-throughput DNA sequencing technology. Our strategy is simple: 1) expose cells to a test compound, 2) isolate colonies, and 3) carry out whole-genome sequencing of the clones. In this pilot study, we used Salmonella typhimurium TA100 as a tester strain and successfully detected mutations induced by the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2). We believe that this new mutation assay will be a very useful tool in hazard assessment of chemicals.
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