Development of a yeast reporter gene assay to detect ligands of freshwater cladoceran Daphnia magna ultraspiracle, a homolog of vertebrate retinoid X receptors.

Endocrine-disrupting chemicals (EDCs) often affect homeostatic regulation in living organisms by directly acting on nuclear receptors (NRs). Retinoid X receptors (RXRs), the most highly conserved members of the NR superfamily during evolution, function as partners to form heterodimers with other NRs, such as retinoic acid, thyroid hormone, and vitamin D3 receptors. RXRs also homodimerize and induce the expression of target genes upon binding with their natural ligand, 9-cis-retinoic acid (9cRA), and typical EDCs organotin compounds, such as tributyltin and triphenyltin. In the present study, we established a new yeast reporter gene assay (RGA) to detect the ligands of freshwater cladoceran Daphnia magna ultraspiracle (Dapma-USP), a homolog of vertebrate RXRs. D. magna has been used as a representative crustacean species for aquatic EDC assessments in the Organization for Economic Corporation and Development test guidelines. Dapma-USP was expressed along with the Drosophila melanogaster steroid receptor coactivator Taiman in yeast cells carrying the lacZ reporter plasmid. The RGA for detecting agonist activity of organotins and o-butylphenol was improved by use of mutant yeast strains lacking genes encoding cell wall mannoproteins and/or plasma membrane drug efflux pumps as hosts. We also showed that a number of other human RXR ligands, phenol and bisphenol A derivatives, and terpenoid compounds such as 9c-RA exhibited antagonist activity on Dapma-USP. Our newly established yeast-based RGA system is valuable as the first screening tool to detect ligand substances for Dapma-USP and for evaluating the evolutionary divergence of the ligand responses of RXR homologs between humans and D. magna.

SPRTN and TDP1/TDP2 Independently Suppress 5-Aza-2'-deoxycytidine-Induced Genomic Instability in Human TK6 Cell Line.

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 (SPRTN-/-) human TK6 cells displayed high sensitivity to 5-azadC, and the removal of 5-azadC-induced DNMT1-DPCs was significantly slower in SPRTN-/- cells than that in wild-type cells. We also showed that the ubiquitination-dependent proteasomal degradation, which was independent of the SPRTN-mediated processing, was also involved in the repair of DNMT1-DPCs. Unexpectedly, we found that cells that are double deficient in tyrosyl DNA phosphodiesterase 1 and 2 (TDP1-/-TDP2-/-) were also sensitive to 5-azadC, although the removal of 5-azadC-induced DNMT1-DPCs was not compromised significantly. Furthermore, the 5-azadC treatment induced a marked accumulation of chromosomal breaks in SPRTN-/- as well as TDP1-/-TDP2-/- cells compared to wild-type cells, strongly suggesting that the 5-azadC-induced cell death was attributed to chromosomal DNMT1-DPCs. We conclude that SPRTN protects cells from 5-azadC-induced DNMT1-DPCs, and SPRTN may play a direct proteolytic role against DNMT1-DPCs and TDP1/TDP2 also contributes to suppress genome instability caused by 5-azadC in TK6 cells.

Identification of amino acid residues in the ligand-binding domain of the aryl hydrocarbon receptor causing the species-specific response to omeprazole: possible determinants for binding putative endogenous ligands.

Omeprazole (OME) induces the expression of genes encoding drug-metabolizing enzymes, such as CYP1A1, via activation of the aryl hydrocarbon receptor (AhR) both in vivo and in vitro. However, the precise mechanism of OME-mediated AhR activation is still under investigation. While elucidating species-specific susceptibility to dioxin, we found that OME-mediated AhR activation was mammalian species specific. Moreover, we previously reported that OME has inhibitory activity toward CYP1A1 enzymes. From these observations, we speculated that OME-mediated AhR target gene transcription is due to AhR activation by increasing amounts of putative AhR ligands in serum by inhibition of CYP1A1 activity. We compared the amino acid sequences of OME-sensitive rabbit AhR and nonsensitive mouse AhR to identify the residues responsible for the species-specific response. Chimeric AhRs were constructed by exchanging domains between mouse and rabbit AhRs to define the region required for the response to OME. OME-mediated transactivation was observed only with the chimeric AhR that included the ligand-binding domain (LBD) of the rabbit AhR. Site-directed mutagenesis revealed three amino acids (M328, T353, and F367) in the rabbit AhR that were responsible for OME-mediated transactivation. Replacing these residues with those of the mouse AhR abolished the response of the rabbit AhR. In contrast, substitutions of these amino acids with those of the rabbit AhR altered nonsensitive mouse AhR to become sensitive to OME. These results suggest that OME-mediated AhR activation requires a specific structure within LBD that is probably essential for binding with enigmatic endogenous ligands.

Microbial metabolites of omeprazole activate murine aryl hydrocarbon receptor in vitro and in vivo.

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.

Identification of small molecule proliferating cell nuclear antigen (PCNA) inhibitor that disrupts interactions with PIP-box proteins and inhibits DNA replication.

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.

Dioxin suppresses benzo[a]pyrene-induced mutations and DNA adduct formation through cytochrome P450 1A1 induction and (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide inactivation in human hepatoma cells.

Benzo[a]pyrene (BaP) is metabolically activated by cytochrome P450 enzymes, and forms DNA adduct leading to mutations. Cytochrome P450 1A1 plays a central role in this activation step, and this enzyme is strongly induced by chemical agents that bind to the aryl hydrocarbon receptor (AhR), which is also known as a dioxin receptor. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand has not been shown to form any DNA adduct, but has a possibility to aggravate the toxicity of precarcinogenic polycyclic hydrocarbons through the induction of metabolic enzymes. We treated human hepatoma cells (HepG2) with TCDD, and subsequently exposed them to BaP to elucidate the synergistic effects on mutations. Surprisingly, mutant frequency induced by BaP at the hypoxanthine-guanine phosphribosyltransferase (HPRT) locus was decreased by pretreatment with TCDD. In correlation with decrease in the mutant frequencies, BaP-DNA adduct formation was also decreased by TCDD pretreatment. This suppressive effect of TCDD was more potent when the cells were exposed to (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), a reactive metabolic intermediate of BaP. Among the enzymes catalyzing BaP oxidation and conjugation, cytochrome P450 1A1, 1A2, 3A4 and UDP-glucuronosyltransferase 1A1 mRNAs were induced by the exposure to TCDD. In cytochrome P450 1A1-deficient murine cells and cytochrome P450 1A1-uninducible human cells, TCDD could not suppress BPDE-DNA adduct formation. Further experiments using "Tet-On" cytochrome P450 1A1-overexpressing cells and a recombinant cytochrome P450 1A1 enzyme demonstrated that this is the key enzyme involved in the biotransformation of BaP, that is, both production and inactivation of BPDE. We conclude that TCDD-induced cytochrome P450 catalyzes the metabolism of BPDE to as yet-unidentified products that are not apparently DNA-reactive, thereby reducing mutations in hepatoma cells.

Translesion DNA synthesis across various DNA adducts produced by 3-nitrobenzanthrone in Escherichia coli.

To analyze translesion DNA synthesis (TLS) across lesions derived from the air pollutant 3-nitrobenzanthrone in Escherichia coli, we constructed site-specifically modified plasmids containing single molecule adducts derived from 3-nitrobenzanthrone. For this experiment, we adopted a modified version of the method developed by Fuchs et al. [29]. Each plasmid contained one of the following lesions in its LacZ' gene: N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-ABA); 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-C2-ABA); 2-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-C2-ABA); 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone (dA-N(6)-C2-ABA); N-(2'-deoxyguanosin-8-yl)-3-acetylaminobenzanthrone (dG-C8-N-AcABA); or 2-(2'-deoxyguanosin-8-yl)-3-acetylaminobenzanthrone (dG-C8-C2-AcABA). All of the adducts inhibited DNA synthesis by replicative DNA polymerases in E. coli; however, the extent of the inhibition varied among the adducts. All five dG-adducts strongly blocked replication by replicative DNA polymerases; however, the dA-adduct only weakly blocked DNA replication. The induction of the SOS response increased the frequency of TLS, which was higher for the dG-C8-C2-ABA, dG-C8-N-AcABA and dG-C8-C2-AcABA adducts than for the other adducts. In our previous study, dG-C8-N-ABA blocked DNA replication more strongly and induced mutations more frequently than dG-N(2)-C2-ABA in human cells. In contrast, in E. coli the frequency of TLS over dG-N(2)-C2-ABA was markedly reduced, even under the SOS(+) conditions, and dG-N(2)-C2-ABA induced G to T mutations. All of the other adducts were bypassed in a less mutagenic manner. In addition, using E. coli strains that lacked particular DNA polymerases we found that DNA polymerase V was responsible for TLS over dG-C8-N-AcABA and dG-C8-C2-AcABA adducts.

Adduct formation and repair, and translesion DNA synthesis across the adducts in human cells exposed to 3-nitrobenzanthrone.

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.

Induction of DNA Damage in Mouse Colorectum by Administration of Colibactin-producing Escherichia coli, Isolated from a Patient With Colorectal Cancer.

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.

Application of the DNA adductome approach to assess the DNA-damaging capability of in vitro micronucleus test-positive compounds.

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.

Detection of juvenile hormone agonists by a new reporter gene assay using yeast expressing Drosophila methoprene-tolerant.

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.

Genotoxicity of micro- and nano-particles of kaolin in human primary dermal keratinocytes and fibroblasts.

INTRODUCTION: Kaolin is a clay mineral with the chemical composition Al2Si2O5(OH)4. It is an important industrial material, and is also used as a white cosmetic pigment. We previously reported that fine particles of kaolin have genotoxic potency to Chinese hamster ovary CHO AA8 cells, and to the lungs of C57BL/6 J and ICR mice. In the present study, we evaluated the genotoxicity of different particle sizes of kaolin using primary normal human diploid epidermal keratinocytes and primary normal human diploid dermal fibroblasts, in addition to a CHO AA8 cell line. FINDINGS: After 6-h treatment with kaolin micro- and nano-particles of particle sizes 4.8 µm and 0.2 µm (200 nm), respectively, the frequencies of micronucleated cells increased in a dose-dependent manner. The frequency increased 3- to 4-fold by exposure to the particles at 200 µg/mL (i.e., 31.4 µg/cm2) in all cells tested. Two-way ANOVA revealed a significant main effect of particle size, and the nano-particles tended to have a higher potency of micronucleus (MN) induction. However, the cell type did not significantly affect the MN frequencies. In addition, one-hour treatment with the kaolin particles increased DNA damage in a dose-dependent manner in a comet assay. The %tail DNA was increased 8- to 20-fold by exposure to the particles at 200 µg/mL, for all cells tested. The kaolin nano-particles had higher DNA-damaging potency than the micro-particles. Furthermore, treatment with kaolin particles dose-dependently increased the production of reactive oxygen species (ROS) in all cells. Again, we observed that kaolin nano-particles induced more ROS than the micro-particles in all cells. CONCLUSION: Kaolin particles demonstrated genotoxicity in primary normal human diploid epidermal keratinocytes and fibroblasts as well as in CHO AA8 cells. Although no significant difference was observed among these three types of cells, fine particles of kaolin tended to have higher genotoxic potency than coarse particles. Since studies on its genotoxicity to skin have been scarce, the findings of the present study could contribute to safety evaluations of kaolin particles when used as a white cosmetic pigment.

Improvement of reporter gene assay for highly sensitive dioxin detection using protoplastic yeast with inactivation of CWP and PDR genes.

A yeast reporter gene assay system with improved performance for dioxin detection was established. Since yeast reporter gene assays are relatively simple, easy to handle, and inexpensive, they have been used for various assessments of environmental contaminants. We previously constructed a yeast assay strain expressing the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) carrying the lacZ reporter gene, for detection of dioxins. In the present study, genes encoding cell wall mannoproteins and ATP-binding cassette transporters in the yeast assay strains were deleted in order to increase the substance influx and prevent its efflux. We also established an assay procedure for protoplasts of these yeasts. These modifications improved the detection limit 40-fold and reduced the duration of the assay by 40%. By combining the yeast protoplast and a rapid sample preparation technique using disposal multilayer solid-phase extraction columns to remove unintended aryl hydrocarbons, this yeast reporter gene assay system detected the ligand activities of dioxins and related compounds in 1 g of forest soil containing dioxins at a concentration 10 times lower than the Japanese environmental standard for dioxins in soil.

Construction of reporter gene assays using CWP and PDR mutant yeasts for enhanced detection of various sex steroids.

BACKGROUND: Sex steroid hormone receptors are classified into three classes of receptors: estrogen receptors (ER) α and ß, androgen receptor (AR), and progesterone receptor (PR). They belong to the nuclear receptor superfamily and activate their downstream genes in a ligand-dependent manner. Since sex steroid hormones are involved in a wide variety of physiological processes and cancer development, synthetic chemical substances that exhibit sex steroid hormone activities have been applied as pharmaceuticals and consumed in large amounts worldwide. They are potentially hazardous contaminants as endocrine disruptors in the environment because they may induce inappropriate gene expression mediated by sex steroid hormone receptors in vivo. RESULTS: To develop simple reporter gene assays with enhanced sensitivity for the detection of sex steroid hormones, we newly established mutant yeast strains lacking the CWP and PDR genes encoding cell wall mannoproteins and plasma membrane drug efflux pumps, respectively, and expressing human ERα, ERß, AR, and PR. Reporter gene assays with mutant yeast strains responded to endogenous and synthetic ligands more strongly than those with wild-type strains. Sex steroid hormone activities in some pharmaceutical oral tablets and human urine were also detectable in these yeast assays. CONCLUSIONS: Yeast reporter gene assay systems for all six steroid hormone receptors, including previously established glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) assay yeasts, are now available. Environmental endocrine disrupters with steroid hormone activity will be qualitatively detectable by simple and easy procedures. The yeast-based reporter gene assay will be valuable as a primary screening tool to detect and evaluate steroid hormone activities in various test samples. Our assay system will strongly support the detection of agonists, antagonists, and inverse agonists of steroid hormone receptors in the field of novel drug discovery and assessments of environmental pollutants.

Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of Escherichia coli strains obtained from the Japan Collection of Microorganisms.

INTRODUCTION: Colibactin is a small genotoxic molecule produced by enteric bacteria, including certain Escherichia coli (E. coli) strains harbored in the human large intestine. This polyketide-peptide genotoxin is considered to contribute to the development of colorectal cancer. The colibactin-producing (clb +) microorganisms possess a 54-kilobase genomic island (clb gene cluster). In the present study, to assess the distribution of the clb gene cluster, genotyping analysis was carried out among E. coli strains randomly chosen from the Japan Collection of Microorganisms, RIKEN BRC, Japan. FINDINGS: The analysis revealed that two of six strains possessed a clb gene cluster. These clb + strains JCM5263 and JCM5491 induced genotoxicity in in vitro micronucleus (MN) tests using rodent CHO AA8 cells. Since the induction level of MN by JCM5263 was high, a bacterial umu test was carried out with a cell extract of the strain, revealing that the extract had SOS-inducing potency in the umu tester bacterium. CONCLUSION: These results support the observations that the clb gene cluster is widely distributed in nature and clb + E. coli having genotoxic potencies is not rare among microorganisms.

Genotoxicity of 3,6-dinitrobenzo[e]pyrene, a novel mutagen in ambient air and surface soil, in mammalian cells in vitro and in vivo.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP), newly identified in airborne particles and surface soil, is a potent mutagen in Salmonella typhimurium. The present study investigated the genotoxic potency of 3,6-DNBeP in vitro and in vivo using mammalian cell strains (Chinese hamster CHL/IU and human HepG2) and ICR mice, respectively. In the hprt gene mutation assay using HepG2 cells, the spontaneous mutant frequency was 61.1 per 10(5) clonable cells, which increased to 229 per 10(5) clonable cells after treatment with 1.0 microg/ml (3 microM) 3,6-DNBeP. Notably, in HepG2 cells with increased N-acetyltransferase 2 activity, the mutant frequency increased to 648 per 10(5) clonable cells by treatment of 1.0 microg/ml (3 microM) 3,6-DNBeP. The sister chromatid exchange frequency increased approximately three times the control level in HepG2 cells treated with 3,6-DNBeP at a concentration of 1.0 microg/ml (3 microM). In HepG2 and CHL/IU cells, the frequency of the cells with micronuclei was 0.9 and 1.2%, and the frequencies increased to 2.3 and 7.6% after 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment, respectively. The H2AX phosphorylation level increased 8-fold compared with the background level with 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment in HepG2 cells. Moreover, the comet assay showed that 3,6-DNBeP produced DNA damage in the cells of liver, kidney, lung and bone marrow in ICR mice 3 h after intraperitoneal injection at 40 mg/kg (0.12 mmol/kg) body weight. These data indicate that 3,6-DNBeP is genotoxic to mammalian cells in vitro and in vivo.

Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems.

BACKGROUND: Recently, manufactured nano/microparticles such as fullerenes (C60), carbon black (CB) and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN) test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles. RESULTS: In in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations. CONCLUSION: Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

Validation of a new yeast-based reporter assay consisting of human estrogen receptors alpha/beta and coactivator SRC-1: application for detection of estrogenic activity in environmental samples.

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 32nd summer school of the Research Community for Mechanisms of Mutations.

The 32nd summer school of the Research Community for Mechanisms of Mutations was held at Inter-University Seminar House in Hachioji city, Tokyo, from September 7 to 8, 2019. Thirty-eight people attended this annual event, and three eminent researchers were invited to discuss DNA damage induced by endogenous aldehydes, "action-at-a-distance mutagenesis" and a novel genome editing method, and DNA repair in fungi and plants. In addition to these plenary sessions, eleven participants presented their own research in oral sessions. More than half of the participants were young scientists such as graduate/undergraduate students, post-doctoral fellows and assistant professors. All members joined in enthusiastic discussions and acquired new scientific knowledge through these two days.

In vitro genotoxicity analyses of colibactin-producing E. coli isolated from a Japanese colorectal cancer patient.

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.