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DNA-protein cross-links (DPCs) are generated by internal factors such as cellular aldehydes that are generated during normal metabolism and external factors such as environmental mutagens. A nucleoside analog, 5-aza-2'-deoxycytidine (5-azadC), is randomly incorporated into the genome during DNA replication and binds DNA methyltransferase 1 (DNMT1) covalently to form DNMT1-DPCs without inducing DNA strand breaks. Despite the recent progress in understanding the mechanisms of DPCs repair, how DNMT1-DPCs are repaired is unclear. The metalloprotease SPRTN has been considered as the primary enzyme to degrade protein components of DPCs to initiate the repair of DPCs. In this study, we showed that SPRTN-deficient (

Introduction: Endocrine-disrupting chemicals (EDCs) mimic or antagonize the actions of hormones, thus they exert potentially adverse health effects on organisms. Most EDCs act on nuclear receptors,...

application/pdf, Genes and Environment. 2020, 42,( Article number: 16), P.1-7

Background/Aim: Among colorectal cancer-associated intestinal microbiota, colibactin-producing (clb(+)) bacteria are attracting attention. We aimed to clarify the interaction between clb(+) Escherichia coli and normal colorectal epithelial cells in vivo and in vitro. Materials and Methods: Five-week-old female Balb/c mice were divided in an untreated group, a group treated with clb(+) E. coli isolated from a Japanese patient with colorectal cancer (E. coli-50), and a group treated with non colibactin-producing E. coli (E. coli-50/ΔclbP). Mice were sacrificed at 18 weeks of treatment. Results: Treatment with clb(+) E. coli increased positivity for H2A histone family member X phosphorylated at Ser-139 (γH2AX) in epithelial cells of the luminal surface of the mouse rectum but this did not occur in the E. coli-50/ΔclbP and untreated groups. In an in vitro setting, the ratio of apoptotic cells was increased and cell counts were reduced by treatment with clb(+) E. coli more than in untreated cells and normal rat colorectal epithelial cells. Conclusion: E. coli-50 induced DNA damage in the mouse rectum, possibly by direct interaction between clb(+) E. coli and normal colorectal epithelial cells. Our findings imply that regulation of clb(+) E. coli infection may be a useful strategy for colorectal cancer control.

Juvenile hormones (JHs) are sesquiterpenoids that play important roles in the regulation of growth, metamorphosis, and reproduction in insects. Synthetic JH agonists (JHAs) have been used as insecticides and are categorized as a class of insect growth regulators (IGRs). Natural JHs and synthetic JHAs bind to the JH receptor methoprene‐tolerant (Met), which forms a functional JH‐receptor complex with steroid receptor coactivators, such as Drosophila melanogaster Taiman (Tai). The ligand‐bound Met–Tai complex induces the transcription of JH response genes by binding to specific DNA elements referred to as JH response elements (JHREs). In the present study, we established a reporter gene assay (RGA) for detecting natural JHs and synthetic JHAs in a yeast strain expressing D. melanogaster Met and Tai. The yeast RGA system detected various juvenoid ligands in a dose‐dependent manner. The rank order of the ligand potencies of the juvenoids examined in the yeast RGA linearly correlated with those of RGAs for Met–Tai established in mammalian and insect cells. Our new yeast RGA is rapid, easy to handle, cost‐effective, and valuable for screening novel JHAs., Synthetic juvenile hormone agonists (JHAs) that have been used as insecticides mimic the action of insect JHs and disrupt signaling via the JH‐receptor methoprene‐tolerant (Met). In this study, we developed a novel reporter gene assay to measure ligand activity of natural JHs and synthetic JHAs by co‐expressing Drosophila melanogaster Met and steroid receptor coactivator Taiman in yeast Saccharomyces cerevisiae.

Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotoxic potency of the colibactin-producing (clb+) E. coli strains that carry the polyketide synthases "pks" gene cluster (pks+) and an isogenic clb- mutant in which the colibactin-producing ability is impaired. Measurement of phosphorylated histone H2AX indicated that DNA double strand breaks were induced in mammalian CHO AA8 cells infected with the clb+ E. coli strains. Induction of DNA damage response (SOS response) by crude extract of the clb+ strains was 1.7 times higher than that of the clb- E. coli in an umu assay with a Salmonella typhimurium TA1535/pSK1002 tester strain. Micronucleus test with CHO AA8 cells revealed that infection with the clb+ strains induced genotoxicity, i.e., the frequencies of micronucleated cells infected with clb+ strain were 4-6 times higher than with the clb- strain. Since the intestinal flora are affected by dietary habits that are strongly associated with ethnicity, these data may contribute to both risk evaluation and prevention of colorectal cancer in the Japanese population.

Worldwide, humans are exposed to a mean background radiation dose of about 2.4 mSv per year. Natural background radiation is a constant and ubiquitous environmental factor affecting all living things on this planet, and organisms have evolved in the presence of the natural radiation since the first forms of life appeared on Earth. However, its role in biology is still not completely understood. Here, we summarize the interesting biological responses in organism at below-background radiation and highlight recent development of the studies with molecular based analyses. To date, deprivation of natural background radiation has been carried out with shielding chambers or by conducting experiments in underground laboratories. In recent decades some research groups have reported changes to growth-related biological endpoints in microorganisms and mammalian cells under low-background radiation conditions. Moreover, new technologies, such as DNA microarray and next-generation sequencing, have begun to shed fresh light on the molecular mechanisms of these biological phenomena. Although there is no consistent explanation for the mechanisms so far, such studies might provide clues toward an understanding of the biological responses to natural background radiation.

Aryl hydrocarbons such as 3-nitrobenzanthrone (NBA), 4-aminobiphenyl (ABP), acetylaminofluorene (AAF), benzo(a)pyrene (BaP), and 1-nitropyrene (NP) form bulky DNA adducts when absorbed by mammalian cells. These chemicals are metabolically activated to reactive forms in mammalian cells and preferentially get attached covalently to the N2 or C8 positions of guanine or the N6 position of adenine. The proportion of N2 and C8 guanine adducts in DNA differs among chemicals. Although these adducts block DNA replication, cells have a mechanism allowing to continue replication by bypassing these adducts: translesion DNA synthesis (TLS). TLS is performed by translesion DNA polymerases—Pol η, κ, ι, and ζ and Rev1—in an error-free or error-prone manner. Regarding the NBA adducts, namely, 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone (dG-N2-ABA) and N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-ABA), dG-N2-ABA is produced more often than dG-C8-ABA, whereas dG-C8-ABA blocks DNA replication more strongly than dG-N2-ABA. dG-N2-ABA allows for a less error-prone bypass than dG-C8-ABA does. Pol η and κ are stronger contributors to TLS over dG-C8-ABA, and Pol κ bypasses dG-C8-ABA in an error-prone manner. TLS efficiency and error-proneness are affected by the sequences surrounding the adduct, as demonstrated in our previous study on an ABP adduct, N-(2′-deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP). Elucidation of the general mechanisms determining efficiency, error-proneness, and the polymerases involved in TLS over various adducts is the next step in the research on TLS. These TLS studies will clarify the mechanisms underlying aryl hydrocarbon mutagenesis and carcinogenesis in more detail.

Synthetic nonsteroidal ecdysone agonists, a class of insect growth regulators (IGRs), target the ecdysone receptor (EcR), which forms a heterodimer with ultraspiracle (USP) to transactivate ecdysone response genes. These compounds have high binding affinities to the EcR-USP complexes of certain insects and their toxicity is selective for certain taxonomic orders. In the present study, we developed reporter gene assay (RGA) systems to detect molting hormone (ecdysone) activity by introducing EcR-USP cDNA and a bacterial lacZ reporter gene into yeast. EcR and USP were derived from the insect species of three different taxonomic orders: Drosophila melanogaster (Diptera), Chilo suppressalis (Lepidoptera), and Leptinotarsa decemlineata (Coleoptera). Transcriptional coactivator taiman (Tai) cDNA cloned from D. melanogaster was also used in this RGA system. This yeast RGA system responded to various EcR ligands in a dose-dependent and ecdysteroid-specific manner. Furthermore, the insect order-selective ligand activities of synthetic nonsteroidal ecdysone agonists were linearly related to their binding activities, which were measured against in vitro translated EcR-USP complexes. Our newly established yeast RGA is useful for screening new molting hormone agonists that work selectively on target insects.

Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotoxic potency of the colibactin-producing (clb

Cadmium contamination still occurs in some parts of the world, and its concentrations in the environment are monitored in most countries due to its adverse effects on human health. We herein established yeast (Saccharomyces cerevisiae) reporter assay strains carrying plasmids with the yeast JLP1, SEO1, and CUP1 promoters connected to the bacterial lacZ reporter gene. The strain carrying the high-copy number pESC-JLP1-lacZ reporter plasmid was more responsive to cadmium than strains with other reporter plasmids. This JLP1-lacZ reporter assay strain will be useful for monitoring cadmium contamination in environmental water and soil as a first screening tool preceding official instrumental analyses, because the assay is rapid, easy to handle, and has the ability to process a large number of samples at a low cost.

Thyroid hormones (THs) are involved in the regulation of metabolic homeostasis during the development and differentiation of vertebrates, particularly amphibian metamorphosis, which is entirely controlled by internal TH levels. Some artificial chemicals have been shown to exhibit TH-disrupting activities. In order to detect TH disruptors for amphibians, we herein developed a reporter assay using yeast strains expressing the thyroid hormone receptors (TRs) α and β together with the transcriptional coactivator SRC-1, all of which were derived from the frog Xenopus tropicalis (XT). These yeast strains responded to endogenous THs (T2, T3, and T4) in a dose-dependent manner. They detected the TR ligand activities of some artificial chemicals suspected to exhibit TH-disrupting activities, as well as TR ligand activity in river water collected downstream of sewage plant discharges, which may have originated from human excrement. Moreover, the responses of XT TR strains to these endogenous and artificial ligands were stronger than those of yeast strains for human TRα and β assays, which had previously been established in our laboratory. These results indicate that the yeast reporter assay system for XT TRα and β is valuable for assessing TR ligand activities in environmental samples that may be particularly potent in amphibians.

Introduction The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are members of the nuclear receptor superfamily and ligand-dependent transcription factors, whose major ligands are glucocorticoid and mineralocorticoid, so-called corticosteroids. The corticosteroids are a class of substances that include steroid hormones naturally produced in the adrenal cortex of vertebrates and analogues of these hormones that are synthesized in industry. They are involved in a wide range of physiological processes including stress and immune responses, and the regulation of carbohydrate metabolism, protein catabolism, sodium homeostasis, and inflammation. These substances are potential environmental contaminants because they are clinically consumed in large amounts worldwide. To develop a simple and sensitive bioassay to detect corticosteroids, we newly established reporter assay yeasts expressing human GR and MR. Methods Ligand responses of the established assay yeasts were improved by forced expression of a human transcription coactivator SRC-1e. Further enhancement of the responses was achieved by inactivating the CWP and PDR genes that encode cell wall mannoproteins and plasma membrane efflux pumps, respectively, which may be attributable to an increased intracellular concentration of ligands. Results These new assay yeasts were more responsive to both natural and synthetic agonist ligands than the conventional assay yeasts. They detected both agonistic and antagonistic activities of mifepristone, spironolactone, and eplerenone in a receptor-selective manner. They also detected ligand activities contained in oral pharmaceutical tablets and human urine. Discussion This assay system will be a valuable tool to detect agonists as well as antagonists of corticosteroid receptors, in the fields of drug discovery and the assessment of environmental pollutants.

Introduction Retinoic acids are essential for embryonic development, tissue organization, and homeostasis and act via retinoic acid receptors (RARs) that form heterodimers with retinoid X receptors (RXRs). Human RARs and RXRs include the three subtypes α, β, and γ, which have varying distributions and physiological functions among human tissues. Recent reports show that subtype-specific binding of several chemicals to RARs or RXRs may lead to endocrine disruption. To evaluate these ligand-like chemicals, convenient assay systems for each receptor subtype are required. Methods We developed reporter assay yeasts to screen ligands for RXR subtype receptor homodimers. To screen RAR ligands, yeasts were engineered to express RAR subtypes with defective RXRα, which fails to bind to coactivators because of its shortened c-terminus. Results These assay yeasts were validated using known RXR- and RAR-specific ligands and subtype-specific responses were clearly shown. Subtype-specific ligand activities of the suspected chemical RAR or RXR ligands o - t -butylphenol, triphenyltin chloride, tributyltin chloride, and 4-nonylphenol were determined. Discussion The present assay yeasts may be valuable tools for subtype-specific assessments of unidentified environmental ligand chemicals and receptor-specific pharmaceuticals.

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a potent environmental mutagen that is found in diesel exhaust fumes and airborne particulates. It is known to produce several DNA adducts, including three major adducts N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-ABA), 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone (dA-N(6)-C2-ABA), and 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-C2-ABA) in mammalian cells. In the present study, we measured the quantity of the formation and subsequent reduction of these adducts in human hepatoma HepG2 cells that had been treated with 3-NBA using LC-MS/MS analysis. As a result, dG-C8-N-ABA and dG-N(2)-C2-ABA were identified as major adducts in the HepG2 cells, and dA-N(6)-C2-ABA was found to be a minor adduct. Treatment with 1μg/mL 3-NBA for 24h induced the formation of 2835±1509 dG-C8-N-ABA and 3373±1173 dG-N(2)-C2-ABA per 10(7) dG and 877±330 dA-N(6)-C2-ABA per 10(7) dA in the cells. The cellular DNA repair system removed the dG-C8-N-ABA and dA-N(6)-C2-ABA adducts more efficiently than the dG-N(2)-C2-ABA adducts. After a 24-h repair period, 86.4±11.1% of the dG-N(2)-C2-ABA adducts remained, whereas only 51.7±2.7% of the dG-C8-N-ABA adducts and 37.8±1.7% of the dA-N(6)-C2-ABA adducts were present in the cells. We also evaluated the efficiency of bypasses across these three adducts and their mutagenic potency by introducing site-specific mono-modified plasmids into human cells. This translesion DNA synthesis (TLS) assay showed that dG-C8-N-ABA blocked DNA replication markedly (its replication frequency was 16.9±2.7%), while the replication arrests induced by dG-N(2)-C2-ABA and dA-N(6)-C2-ABA were more moderate (their replication frequencies were 33.3±6.2% and 43.1±7.5%, respectively). Mutagenic TLS was observed more frequently in replication across dG-C8-N-ABA (30.6%) than in replication across dG-N(2)-C2-ABA (12.1%) or dA-N(6)-C2-ABA (12.1%). These findings provide important insights into the molecular mechanism of 3-NBA-mutagenesis.

Cadmium contamination still occurs in some parts of the world, and its concentrations in the environment are monitored in most countries due to its adverse effects on human health. We herein established yeast (Saccharomyces cerevisiae) reporter assay strains carrying plasmids with the yeast JLP1, SEO1, and CUP1 promoters connected to the bacterial lacZ reporter gene. The strain carrying the high-copy number pESC-JLP1-lacZ reporter plasmid was more responsive to cadmium than strains with other reporter plasmids. This JLP1-lacZ reporter assay strain will be useful for monitoring cadmium contamination in environmental water and soil as a first screening tool preceding official instrumental analyses, because the assay is rapid, easy to handle, and has the ability to process a large number of samples at a low cost.

We examined the genotoxicity of magnetite nanoparticles (primary particle size: 10 nm) on human A549 and Chinese hamster ovary (CHO) AA8 cells. Six hours' treatment with the particles dose-dependently increased the frequency of micronuclei (MN) in the A549 and CHO AA8 cells up to 5.2% and 5.0% at a dose of 200 µg/ml (34 µg/cm²), respectively. In A549 cells, treatment with the nano-particles (2 µg/ml) for 1 hr induced H2AX phosphorylation, which is suggestive of DNA double strand breaks (DSB). Treating CHO AA8 cells with 2 µg/ml (0.34 µg/cm²) magnetite for 1 hour resulted in a five times higher frequency of sister chromatid exchange (SCE) than the control level. We detected reactive oxygen species (ROS) in CHO cells treated with the particles. These findings indicate that magnetite nano-particles induce ROS in mammalian cells, leading to the direct or indirect induction of DSB, followed by clastogenic events including MN and SCE.

Benzo[a]pyrene (BaP) is a well-studied pro-carcinogen that is metabolically activated by cytochrome P450 enzymes. Cytochrome P4501A1 (CYP1A1) has been considered to play a central role in the activation step, which is essential for the formation of DNA adducts. This enzyme is strongly induced by many different chemical agents, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which binds to the aryl hydrocarbon receptor (AhR). Therefore, AhR activators are suspected to have the potential to aggravate the toxicity of BaP through the induction of CYP1A1. Besides, CYP1A1 inhibitors, including its substrates, are estimated to have preventive effects against BaP toxicity. However, strangely, increased hepatic BaP–DNA adduct levels have been reported in Cyp1a1 knockout mice. Moreover, numerous reports describe that concomitant treatment of AhR activators reduced BaP–DNA adduct formation. In an experiment using several human cell lines, TCDD had diverse modulatory effects on BaP–DNA adducts, both enhancing and inhibiting their formation. In this review, we focus on the factors that could influence the BaP–DNA adduct formation. To interpret these complicated outcomes, we propose a hypothesis that CYP1A1 is a key enzyme for both generation and reduction of (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), the major carcinogenic intermediate of BaP. Conversely, CYP1B1 is thought to contribute only to the metabolic activation of BaP related to carcinogenesis.

A protein with strong removal activity against the natural estrogen estriol was purified from a culture supernatant of Pleurotus eryngii var. tuoliensis C.J. Mou. The protein was characterized as a laccase and had a molecular mass of 60kDa on SDS-PAGE. The enzyme was most active at pH 7.0 and 50°C. The partial N-terminal amino acid sequence of the enzyme showed homology with laccases from mushrooms, such as Pleurotus ostreatus, Coriolus versicolor (current name: Trametes versicolor), Pycnoporus cinnabarinus, and P. eryngii. A recombinant yeast assay confirmed that laccase treatment was very efficient for removing the estrogenic activity of steroid estrogens. Our results suggest that the enzyme may be applicable as a potential factor for removing natural steroid hormones.

We have discovered that 3,3',5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC(50) of ~1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser(139))histone H2AX induction and cell growth inhibition was observed.

In the 1970s and 1980s, Planel et al. reported that the growth of paramecia was decreased by shielding them from background radiation. In the 1990s, Takizawa et al. found that mouse cells displayed a decreased growth rate under shielded conditions. The purpose of the present study was to confirm that growth is impaired in organisms that have been shielded from background radiation. Radioprotection was produced with a shielding chamber surrounded by a 15 cm thick iron wall and a 10 cm thick paraffin wall that reduced the γ ray and neutron levels in the chamber to 2% and 25% of the background levels, respectively. Although the growth of Paramecium tetraurelia was not impaired by short-term radioprotection (around 10 days), which disagreed with the findings of Planel et al., decreased growth was observed after long-term (40-50 days) radiation shielding. When mouse lymphoma L5178Y cells were incubated inside or outside of the shielding chamber for 7 days, the number of cells present on the 6th and 7th days under the shielding conditions was significantly lower than that present under the non-shielding conditions. These inhibitory effects on cell growth were abrogated by the addition of a ¹³⁷Cs γ-ray source disk to the chamber. Furthermore, no growth retardation was observed in XRCC4-deficient mouse M10 cells, which display impaired DNA double strand break repair.

The in vitro micronucleus (MN) test is widely used for screening genotoxic compounds, but it often produces false-positive results. To consider the significance of positive results, it is important to know whether DNA adducts are formed in the cells treated with the test compound. Recently, Matsuda et al. developed the DNA adductome approach to detect DNA adducts comprehensively ([4] Kanaly, et al., Antioxid. Redox Signal., 2006, 8, 993-1001). We applied this method to assess the DNA-damaging capability of in vitro MN test-positive compounds. CHL/IU cells were treated with compounds from three categories: (1) carcinogens causing DNA alkylation, ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine; (2) carcinogens producing DNA bulky adducts, 2-amino-6-phenyl-1-methylimidazo[4,5-b]pyrene, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and 4-nitroquinoline-1-oxide, and (3) non-carcinogens, caffeine, maltol, and sodium chloride, with or without metabolic activation. With the conditions in which all test compounds gave positive results in the MN tests, DNA was extracted from the cells and hydrolyzed to deoxyribonucleosides, which were subsequently subjected to LC/ESI-MS/MS analysis. All carcinogens (categories 1 and 2) produced various DNA adduct peaks, and some of the m/z peak values corresponded to known adducts. No non-carcinogens produced DNA adducts, indicating that these compounds produced MN through different mechanisms from the adduct formation. These results indicate that the adductome approach is useful to demonstrate DNA damage formation of MN test-positive compounds and to understand their mechanisms of action.

Endocrine disruptors are exogenous substances that act like hormones in the endocrine system and disrupt the physiologic function of endogenous hormones. In the present study, we established reporter yeast strains (Saccharomyces cerevisiae) expressing human estrogen receptors, ERalpha or ERbeta. These strains contain a reporter plasmid carrying an estrogen responsive element (ERE) upstream of the beta-galactosidase gene, and a plasmid expressing a steroid receptor coactivator, SRC-1e. Using these reporter strains, we demonstrated dose-dependent estrogenic activities of different categories of ligands, a natural hormone, 17beta-estradiol (E2); a synthetic drug, diethylstilbestrol (DES); phytoestrogens, genistein, daizein and emodin; and an environmental endocrine disrupter, bisphenol A. EC(50) values of E2 for ERalpha and ERbeta are 5.31 x 10(-10) and 5.85 x 10(-10) M, respectively. We also demonstrated that these yeasts were applicable for measuring estrogenic activities of environmental water samples. Most downstream sites of a river showed similar activity in both ERalpha and ERbeta assays. These yeast strains are useful and convenient for detecting and comparing the estrogenic ligand activities of environmental samples in response to ERalpha and ERbeta.

The mutagenic specificity of N-nitrosotaurocholic acid (NO-TCA) in human cells was investigated using supF shuttle vector plasmids. The plasmids pMY189 were treated with NO-TCA in vitro and introduced into normal fibroblasts (WI38-VA13) and nucleotide excision repair (NER)-deficient cells (XP2OS(SV)) for replication. The background mutation frequency of the supF gene was 4.1× 10-4 and 2.0×10-4 after replication in normal and NER-deficient cells, respectively. The mutation frequency increased 5 and 15 times in normal and NER-deficient cells, respectively, after the treatment of the plasmid with 50 mg/mL of NO-TCA. The higher mutation frequency in NER-deficient cells indicates that the DNA damage induced by NO-TCA is repaired by NER. Base sequence analysis of 101 and 94 plasmids with mutations in the supF gene propagated in normal and NER-deficient cells, respectively, revealed that the majority of the mutations were base substitutions (about 89 and 90%) and the rest were deletions and insertions (about 11 and 10%) in both cell lines. About half of the mutant plasmids contained a single base substitution. Of the single base substitutions, the most frequent mutations were G:C to A:T transitions (about 37 and 36%), followed by G:C to C:G transversions (about 31 and 28%) in both cell lines. The mutations were not distributed randomly but were located at several hot spots in the supF gene, and almost all hot spots were at G:C sites. These observations accord with previous findings that NO-TCA forms DNA adducts with dC and induces G:C to A:T base substitution in Salmonella typhimurium TA100.

Translesion DNA synthesis (TLS) is a tolerance pathway of replication block caused by DNA lesions. To measure the efficiency and fidelity of TLS in human cells, we established a shuttle vector assay by modifying of a bacterial TLS assay system. The assay consists of transfection of DNA repair-deficient human cells with a plasmid possessing a single DNA adduct, and transformation of indicator bacteria with plasmids extracted from the cells. We show that plasmid replication was suppressed to 1/9 by a single aminobiphenyl-dG adduct, and the mutant frequency of TLS-operated plasmids was 0.31, of which the major mutation (78%) was G to T transversion. The results demonstrate that this assay is applicable in practice for investigating TLS in human cells.

3-Nitrobenzanthrone (3-NBA) is a potent environmental mutagen and a potential human carcinogen present in diesel exhaust and airborne particulates. N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) has been shown to be a major reactive metabolite of 3-NBA, which mainly produces adducts with guanine and adenine in cellular DNA. Here we analyzed mutations induced by N-Aco-ABA using supF shuttle vector plasmids to elucidate the mutagenic specificity of 3-NBA in human cells. Base sequence analysis of more than 100 plasmids with supF mutations induced in wildtype and DNA repair-deficient XP cells revealed that the major mutation was base substitutions of which the majority (42 and 38%, respectively) were G:C to T:A transversions. The next major mutation was G:C to A:T and A:T to G:C base substitutions in wildtype and XP cells, respectively. The DNA polymerase stop assay using N-Aco-ABA-treated plasmids as a template showed that most stop signals, i.e., adducted sites, appeared at G:C sites. These results suggest that N-Aco-ABA binds preferably to guanine rather than adenine, and adducted adenine is repaired more efficiently by the nucleotide excision repair. Error-prone DNA polymerases could insert adenine at sites opposite to N-Aco-ABA-adducted guanine, which leads to G:C to T:A transversion. These findings could be very important to evaluate the human lung cancer risk of environmental 3-NBA.

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful nurtagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts fon-ned in 3-NBA-exposed human hepatoma HepG2 cells by a P-32-postlabeling/thin layer chromatography (TLC) method and a P-32-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36-36.4 mu M) for 3h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2 '-deoxyadenosin-N-6-yl)-3-aminobenzanthrone-3 '-phosphate (dA3 ' p-N-6-C2-ABA) and 2-(2 '-deoxyguanosin-N-2-yl)-3-aminobenzanthrone-3 '-phosphate (dG3 ' p-N-2-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3 ' p-N-6-C2-ABA and its relative adduct labeling (RAL) value at 36.4 mu M of 3-NBA was 200.8 +/- 86.1/10(8) nucleotide. (c) 2007 Elsevier B.V. All rights reserved.

3-Nitrobenzanthrone is a powerful bacterial mutagen and carcinogen to mammals. To obtain precise information on DNA-adduct formation by 3-nitrobenzanthrone, a number of DNA adducts, including N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (13 a), 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone (14 a), N-(2'-deoxyadenosin-8-yl)-3-aminobenzanthrone (15 a), 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone (16 a), and their N-acetylated counterparts 13 b, 14 b, 15 b, and 16 b were synthesized by palladium-catalyzed aryl amination of the corresponding nucleoside and bromobenzanthrone derivatives. Among these DNA adducts, DNA adducts 13 a, 13 b, 14 a, 14 b, and 16 a were identified in the reaction mixture of nucleosides (2'-deoxyguanosine, 2'-deoxyadenosine, or DNA) with N-acetoxy-3-aminobenzanthrone or N-acetyl-N-acetoxy-3-aminobenzanthrone, both of which are recognized as activated metabolites of 3-nitrobenzanthrone. The formation of these multiple DNA adducts may help explain the potent mutacarcinogenicity of 3-nitrobenzanthrone.

Polymorphism of the aryl hydrocarbon receptor (AhR) presumably induces genetic difference in the susceptibility of animals to aryl hydrocarbons. The activation of intracellular signaling following AhR binding to aryl hydrocarbons is highly correlated with the toxicity and carcinogenicity of these chemicals. Here we developed two reporter yeasts coexpressing AhR and AhR nuclear translocator (Arnt) proteins of a guinea pig and a Syrian hamster, known as the most sensitive and most resistant laboratory rodents to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. We previously constructed reporter yeasts expressing human and mouse AhR/Arnt. We conducted reporter assays to measure ligand activities of TCDD, 3-methylcholanthrene, β-naphthoflavone and indirubin in these yeasts. Ligand treatment induced a dose-dependent increase in β-galactosidase activity from a reporter plasmid in all 4 yeast strains. The assays showed that yeast expressing guinea pig AhR/Arnt is most sensitive and yeast expressing Syrian hamster AhR/Arnt is most insensitive to these ligands. The yeasts expressing human and mouse AhR/Arnt were in-between. These different ligand activities reflect the species specificity of AhR/Arnt, and may be related to the susceptibility of rodents to aryl hydrocarbons.

Particles present in urban air pollution are mainly derived from diesel- and gasoline-fueled vehicles. Exhaust emission is able to cause several health effects in humans including mutagenicity and ...

Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998). Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

Omeprazole (OME), a proton pump inhibitor used to treat gastritis, is also an aryl hydrocarbon receptor (AhR) activator. OME activates AhR in human hepatocytes and hepatoma cells, but not in mice in vivo or in vitro. We recently discovered that this species-specific difference results from a difference in a few amino acids in the ligand-binding domain of AhR. However, OME activates both mouse and human AhRs in the yeast reporter assay system. Nevertheless, the cause of this discrepancy in OME responses remains unknown. Here, we report that CYP1A1 mRNA expression in mouse cecum was elevated after OME administration, although the mouse is regarded as an OME-unresponsive animal. Using the yeast reporter assay system with human and murine AhRs, we found AhR agonist-like activity in the cecal extracts of OME-treated mice. We speculated that OME metabolites produced by cecal bacteria might activate murine AhRs in vivo. In high-performance liquid chromatography (HPLC) analysis, AhR agonist-like activity of cecal bacterial culture and cecal extracts were detected at the same retention time. AhR agonist-like activity was also detected in the HPLC fractions of yeast culture media containing OME. This unknown substance could induce reporter gene expression via mouse and human AhRs. The agonist-like activity of the OME metabolite was reduced by concomitant α-naphthoflavone exposure. These results indicate that a yeast-generated OME metabolite elicited the response of mouse AhR to OME in the yeast system, and that bacterial OME metabolites may act as AhR ligands in human and mouse intestines.

Cisplatin (cis-diamminedichloroplatinum(II)), a widely used anticancer drug, forms inter- and intra-strand DNA crosslinks. The major intra-strand crosslinks are Pt adducts at 1,2-d(GpG) and 1,3-d(GpNpG) (Pt-GG and Pt-GNG, respectively). Although most of the intra-strand crosslinks are removed by the nucleotide excision repair (NER), the remaining crosslinks can cause mutations through the translesion DNA synthesis (TLS) during chromosome replication. To understand the precise mechanism of cisplatin mutagenesis in human cells, the plasmid carrying a single Pt-GG or 1,3-d(GpTpG) crosslink (Pt-GTG) site-specifically in lacZ gene was constructed and propagated in NER-defective xeroderma pigmentosum cells. The plasmids retrieved from the cells were introduced into indicator bacterial cells to access frequencies of TLS and mutations. The experiments revealed that Pt-GTG blocked DNA replication more strongly and caused more mutations (29.1%) than Pt-GG (1.7%). Most mutations were G to A or T base changes at 5' G residue in the Pt-GTG crosslinks. These results indicate that the Pt-GTG crosslinks become effective obstacles for cancer cell division, and have an important role for cisplatin cancer therapy.

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.

3-Nitrobenzanthrone (NBA) is a powerful bacterial mutagen and a suspected human carcinogen present in diesel exhaust and airborne particulates [Enya, T., et al. (1997) Environ. Sci. Technol. 31, 2772-2776]. In the accompanying paper [Enya, T., et al. (1998) Chem. Res. Toxcol. 11, 1460-1467], N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA) was synthesized to yield the DNA adducts of NBA. In this work, to investigate the mutagenic specificity of NBA in human cells, we analyzed mutations induced by N-Aco-N-Ac-ABA using the supF shuttle vector plasmids. Base sequence analysis of 110 and 100 plasmids with mutations in the supF gene propagated in normal cells [WI38-VA13] and nucleotide excision repair deficient cells [XP2OS(SV)], respectively, revealed that the majority of the mutations were base substitutions (85 and 90%) and the rest were deletions and insertions (10 and 15%) in both cell lines. About half of the mutant plasmids had a single base substitution. Of the base substitutions, the most frequent mutation was G.C to T.A transversion (41 and 51%), followed by G.C to A.T transitions (18 and 24%) in either cell. The mutations were distributed not randomly but located at several hot spots, and almost all (nine of ten) hot spots were at the sites of G.C base pairs. The polymerase stop assay in the supF gene revealed that N-Aco-N-Ac-ABA preferentially bound to guanine residues, and mutation sites were generally consistent with the sites where the guanine adducts were formed.

Types of mutations induced by acrolein in the supF gene on the shuttle vector plasmid pMY189 replicated in normal human fibroblast cells were examined. Base sequence analysis of 92 plasmids with mutations in the supF gene revealed that the majority of the mutations were base substitutions (76%) and the others were deletions and insertions (24%). Single base substitutions were most frequently found (46%), while multiple base substitutions were 18% and tandem (two adjacent) base substitutions were 12% of the mutations. Of the base substitution mutations, G:C to T:A transversions were 44% and G:C to A:T transitions were 24%. The mutations were distributed not randomly but located at several hotspots. Acrolein produced DNA intra-strand cross-links between guanine residues, which might be responsible for rather high induction of the tandem base substitution mutations.

Acetaldehyde is present in tobacco smoke and automotive exhaust gases, is produced by the oxidation of ethanol, and causes respiratory organ cancers in animals. We show both the types and spectra of acetaldehyde-induced mutations in supF genes in double- and single-stranded shuttle vector plasmids replicated in human cells. Of the 101 mutants obtained from the double-stranded plasmids, 63% had tandem base substitutions, of which the predominant type is GG to TT transversions. Of the 44 mutants obtained from the single-stranded plasmids, 39% had tandem mutations that are of a different type than the double-stranded ones. The GG to TT tandem substitutions could arise from intra-strand crosslinks. Our data indicate that acetaldehyde forms intra- as well as inter-strand crosslinks between adjacent two-guanine bases. Based upon the following observations: XP-A protein binds to acetaldehyde-treated DNA, DNA excision repair-deficient xeroderma pigmentosum (XP) cells were more sensitive to acetaldehyde than the repair-proficient normal cells, and a higher frequency of acetaldehyde-induced mutations of the shuttle vectors was found in XP cells than in normal cells, we propose that the DNA damage caused by acetaldehyde is removed by the nucleotide excision repair pathway. Since treatment with acetaldehyde yields very specific GG to TT tandem base substitutions in DNA, such changes can be used as a probe to identify acetaldehyde as the causal agent in human tumors.