RESUMO
Error-corrected next-generation sequencing (ecNGS) is an emerging technology for accurately measuring somatic mutations. Here, we report paired-end and complementary consensus sequencing (PECC-Seq), a high-accuracy ecNGS approach for genome-wide somatic mutation detection. We characterize a novel 2-aminoimidazolone lesion besides 7,8-dihydro-8-oxoguanine and the resulting end-repair artifacts originating from NGS library preparation that obscure the sequencing accuracy of NGS. We modify library preparation protocol for the enzymatic removal of end-repair artifacts and improve the accuracy of our previously developed duplex consensus sequencing method. Optimized PECC-Seq shows an error rate of <5 × 10-8 with consensus bases compressed from approximately 25 Gb of raw sequencing data, enabling the accurate detection of low-abundance somatic mutations. We apply PECC-Seq to the quantification of in vivo mutagenesis. Compared with the classic gpt gene mutation assay using gpt delta transgenic mice, PECC-Seq exhibits high sensitivity in quantitatively measuring dose-dependent mutagenesis induced by Aristolochic acid I (AAI). Moreover, PECC-Seq specifically characterizes the distinct genome-wide mutational signatures of AAI, Benzo[a]pyrene, N-Nitroso-N-ethylurea and N-nitrosodiethylamine and reveals the mutational signature of Quinoline in common mouse models. Overall, our findings demonstrate that high-accuracy PECC-Seq is a promising tool for genome-wide somatic mutagenesis quantification and for in vivo mutagenicity testing.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Animais , Camundongos , Consenso , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos Transgênicos , Mutagênese/genética , Mutação , Análise de Sequência de DNA/métodos , MasculinoRESUMO
DNA polymerase zeta (Polζ) is a heterotetramer composed of the catalytic subunit Rev3l, Rev7 and two subunits of Polδ (PolD2/Pol31 and PolD3/Pol32), and this polymerase exerts translesion DNA synthesis (TLS) in yeast. Because Rev3l knockout results in embryonic lethality in mice, the functions of Polζ need further investigation in vivo. Then, we noted the two facts that substitution of leucine 979 of yeast Rev3l with methionine reduces Polζ replication fidelity and that reporter gene transgenic rodents are able to provide the detailed mutation status. Here, we established gpt delta mouse knocked in the constructed gene encoding methionine instead of leucine at residue 2610 of Rev3l (Rev3l L2610M gpt delta mice), to clarify the role of Polζ in TLS of chemical-induced bulky DNA adducts in vivo. Eight-week-old gpt delta mice and Rev3l L2610M gpt delta mice were treated with benzo[a]pyrene (BaP) at 0, 40, 80, or 160 mg/kg via single intraperitoneal injection. At necropsy 31 days after treatment, lungs were collected for reporter gene mutation assays. Although the gpt mutant frequency was significantly increased by BaP in both mouse genotypes, it was three times higher in Rev3l L2610M gpt delta than gpt delta mice after treatment with 160 mg/kg BaP. The frequencies of G:C base substitutions and characteristic complex mutations were significantly increased in Rev3l L2610M gpt delta mice compared with gpt delta mice. The BaP dose-response relationship suggested that Polζ plays a central role in TLS when protective mechanisms against BaP mutagenesis, such as error-free TLS, are saturated. Overall, Polζ may incorporate incorrect nucleotides at the sites opposite to BaP-modified guanines and extend short DNA sequences from the resultant terminal mismatches only when DNA is heavily damaged.
Assuntos
Benzo(a)pireno/toxicidade , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA/metabolismo , Mutagênese , Alanina Transaminase/genética , Animais , Domínio Catalítico , Adutos de DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Feminino , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
As the carcinogenic risk of herbs containing aristolochic acids (AAs) is a global health issue, quantitative evaluation of toxicity is needed for the regulatory decision-making and risk assessment of AAs. In this study, we selected AA I (AAI), the most abundant and representative compound in AAs, to treat transgenic gpt delta mice at six gradient doses ranging from 0.125 to 4 mg/kg/day for 28 days. AAI-DNA adduct frequencies and gpt gene mutation frequencies (MFs) in the kidney, as well as Pig-a gene MFs and micronucleated reticulocytes (MN-RETs) frequencies in peripheral blood, were monitored. The dose-response (DR) relationship data for these in vivo genotoxicity endpoints were quantitatively evaluated using an advanced benchmark dose (BMD) approach with different critical effect sizes (CESs; i.e., BMD5, BMD10, BMD50 and BMD100). The results showed that the AAI-DNA adduct frequencies, gpt MFs and the MN-RETs presented good DR relationship to the administrated doses, and the corresponding BMDL100 (the lower 90% confidence interval of the BMD100) values were 0.017, 0.509 and 3.9 mg/kg/day, respectively. No positive responses were observed in the Pig-a MFs due to bone marrow suppression caused by AAI. Overall, we quantitatively evaluated the genotoxicity of AAI at low doses for multiple endpoints for the first time. Comparisons of BMD100 values across different endpoints provide a basis for the risk assessment and regulatory decision-making of AAs and are also valuable for understanding the genotoxicity mechanism of AAs.
Assuntos
Ácidos Aristolóquicos/toxicidade , Adutos de DNA , Dano ao DNA , Proteínas de Escherichia coli/metabolismo , Taxa de Mutação , Pentosiltransferases/metabolismo , Animais , Benchmarking , Proteínas de Escherichia coli/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes para Micronúcleos , Testes de Mutagenicidade , Mutagênicos/toxicidade , Pentosiltransferases/genéticaRESUMO
Quantitative analysis of the mutagenicity and carcinogenicity of the low doses of genotoxic carcinogens present in food is of pressing concern. The purpose of the present study was to determine the mutagenicity and carcinogenicity of low doses of the dietary genotoxic carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Male F344 gpt delta transgenic rats were fed diets supplemented with 0, 0.1, 1, 10 or 100 ppm IQ for 4 weeks. The frequencies of gpt transgene mutations in the liver were significantly increased in the 10 and 100 ppm groups. In addition, the mutation spectra was altered in the 1, 10 and 100 ppm groups: frequencies of G:C to T:A transversion were significantly increased in groups administered 1, 10 and 100 ppm IQ in a dose-dependent manner, and the frequencies of G:C to A:T transitions, A:T to T:A transversions and A:T to C:G transversions were significantly increased in the 100 ppm group. Increased frequencies of single base pair deletions and Spi- mutants in the liver, and an increase in glutathione S-transferase placental form (GST-P)-positive foci, a preneoplastic lesion of the liver in rats, was also observed in the 100 ppm group. In contrast, neither mutations nor mutation spectra or GST-P-positive foci were statistically altered by administration of IQ at 0.1 ppm. We estimated the point of departure for the mutagenicity and carcinogenicity of IQ using the no-observed-effect level approach and the Benchmark dose approach to characterise the dose-response relationship of low doses of IQ. Our findings demonstrate the existence of no effect levels of IQ for both in vivo mutagenicity and hepatocarcinogenicity. The findings of the present study will facilitate an understanding of the carcinogenic effects of low doses of IQ and help to determine a margin of exposure that may be useful for practical human risk assessment.
Assuntos
Testes de Carcinogenicidade , Carcinógenos/toxicidade , Testes de Mutagenicidade , Mutagênicos/toxicidade , Quinolinas/toxicidade , Animais , Testes de Carcinogenicidade/métodos , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Mutagênese/efeitos dos fármacos , Testes de Mutagenicidade/métodos , Quinolinas/química , Ratos , Ratos Endogâmicos F344 , Ratos TransgênicosRESUMO
As part of the hazard and risk assessment of chemicals in man, it is important to assess the ability of a chemical to induce mutations in vivo. Because of the commonalities in the molecular initiating event, mutagenicity in vitro can correlate well to the in vivo endpoint for certain compound classes; however, the difficulty lies in identifying when this correlation holds true. In silico alerts for in vitro mutagenicity may therefore be used as the basis for alerts for mutagenicity in vivo where an expert assessment is carried out to establish the relevance of the correlation. Taking this into account, a data set of publicly available transgenic rodent gene mutation assay data, provided by the National Institute of Health Sciences of Japan, was processed in the expert system Derek Nexus against the in vitro mutagenicity endpoint. The resulting predictivity was expertly reviewed to assess the validity of the observed correlations in activity and mechanism of action between the two endpoints to identify suitable in vitro alerts for extension to the in vivo endpoint. In total, 20 alerts were extended to predict in vivo mutagenicity, which has significantly improved the coverage of this endpoint in Derek Nexus against the data set provided. Updating the Derek Nexus knowledge base in this way led to an increase in sensitivity for this data set against this endpoint from 9% to 66% while maintaining a good specificity of 89%.
Assuntos
Simulação por Computador , Mutagênese/efeitos dos fármacos , Testes de Mutagenicidade , Mutagênicos/química , Animais , Humanos , Mutagênicos/toxicidade , Projetos de Pesquisa , Sensibilidade e EspecificidadeRESUMO
Flame retardant polybrominated diphenyl ethers (PBDEs) are a class of persistent organic pollutants (POPs). 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a representative PBDE congener with widespread distribution and relatively high toxicity potential. Although it has been reported that BDE-47 can cause DNA damage in various in vitro systems, few studies have provided in vivo genotoxicity information. The aim of the present study was to investigate the genotoxicity of BDE-47 in mice. Male gpt delta mice were administered BDE-47 by gavage at 0, 0.0015, 1.5, 10 and 30 mg/kg/day, and 6 days per week for six consecutive weeks. Before the first treatment, and at 2.5 and 5 weeks after the first treatment, peripheral blood was collected from tails and the micronucleus assay and the Pig-a gene mutation assay were performed. After the last treatment, the mutant frequencies of the gpt gene in the liver and the germ cells from seminiferous tubules were determined. All these assays failed to produce positive results, suggesting that BDE-47 was neither clastogenic nor mutagenic in both target and non-target tissues in gpt delta mice.
Assuntos
Proteínas de Escherichia coli/genética , Éteres Difenil Halogenados/toxicidade , Proteínas de Membrana/genética , Mutagênicos/toxicidade , Pentosiltransferases/genética , Animais , Dano ao DNA/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Fígado/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Testes para Micronúcleos , Testes de Mutagenicidade , MutaçãoRESUMO
1,4-Dioxane is a widely used synthetic industrial chemical and its contamination of drinking water and food is a potential health concern. It induces liver tumors when administered in the drinking water to rats and mice. However, the mode of action (MOA) of the hepatocarcinogenicity of 1,4-dioxane remains unclear. Importantly, it is unknown if 1,4-dioxane is genotoxic, a key consideration for risk assessment. To determine the in vivo mutagenicity of 1,4-dioxane, gpt delta transgenic F344 rats were administered 1,4-dioxane at various doses in the drinking water for 16 weeks. The overall mutation frequency (MF) and A:T- to -G:C transitions and A:T- to -T:A transversions in the gpt transgene were significantly increased by administration of 5000 ppm 1,4-dioxane. A:T- to -T:A transversions were also significantly increased by administration of 1000 ppm 1,4-dioxane. Furthermore, the DNA repair enzyme MGMT was significantly induced at 5000 ppm 1,4-dioxane, implying that extensive genetic damage exceeded the repair capacity of the cells in the liver and consequently led to liver carcinogenesis. No evidence supporting other MOAs, including induction of oxidative stress, cytotoxicity, or nuclear receptor activation, that could contribute to the carcinogenic effects of 1,4-dioxane were found. These findings demonstrate that 1,4-dioxane is a genotoxic hepatocarcinogen and induces hepatocarcinogenesis through a mutagenic MOA in rats. Because our data indicate that 1,4-dioxane is a genotoxic carcinogen, we estimated the point of departure of the mutagenicity and carcinogenicity of 1,4-dioxane using the no-observed effect-level approach and the Benchmark dose approach to characterize its dose-response relationship at low doses.
Assuntos
Carcinógenos/toxicidade , Dioxanos/toxicidade , Fígado/efeitos dos fármacos , Mutagênicos/toxicidade , 8-Hidroxi-2'-Desoxiguanosina , Animais , Testes de Carcinogenicidade , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Dioxanos/administração & dosagem , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Fígado/patologia , Masculino , Testes de Mutagenicidade , Mutagênicos/administração & dosagem , Nível de Efeito Adverso não Observado , Ratos Endogâmicos F344 , Ratos TransgênicosRESUMO
Ochratoxin A (OTA) is a carcinogen targeting proximal tubules at the renal outer medulla (ROM) in rodents. We previously reported that OTA increased mutant frequencies of the red/gam gene (Spi(-)), primarily deletion mutations. In the present study, Spi(-) assays and mutation spectrum analyses in the Spi(-) mutants were performed using additional samples collected in our previous study. Spi(-) assay results were similar to those in our previous study, revealing large (>1kb) deletion mutations in the red/gam gene. To clarify the molecular progression from DNA damage to gene mutations, in vivo comet assays and analysis of DNA damage/repair-related mRNA and/or protein expression was performed using the ROM of gpt delta rats treated with OTA at 70, 210 or 630 µg/kg/day by gavage for 4 weeks. Western blotting and immunohistochemical staining demonstrated that OTA increased γ-H2AX expression specifically at the carcinogenic target site. In view of the results of comet assays, we suspected that OTA was capable of inducing double-strand breaks (DSBs) at the target sites. mRNA and/or protein expression levels of homologous recombination (HR) repair-related genes (Rad51, Rad18 and Brip1), but not nonhomologous end joining-related genes, were increased in response to OTA in a dose-dependent manner. Moreover, dramatic increases in the expression of genes involved in G2/M arrest (Chek1 and Wee1) and S/G2 phase (Ccna2 and Cdk1) were observed, suggesting that DSBs induced by OTA were repaired predominantly by HR repair, possibly due to OTA-specific cell cycle regulation, consequently producing large deletion mutations at the carcinogenic target site.
Assuntos
Carcinógenos/toxicidade , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Ocratoxinas/toxicidade , Deleção de Sequência/efeitos dos fármacos , Animais , Sequência de Bases , Peso Corporal/efeitos dos fármacos , Carcinógenos/administração & dosagem , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Ensaio Cometa , Proteínas de Escherichia coli/genética , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Testes de Mutagenicidade/métodos , Ocratoxinas/administração & dosagem , Tamanho do Órgão , Pentosiltransferases/genética , Ratos , Ratos TransgênicosRESUMO
8-Hydroxydeoxyguanosine (8-OHdG) is well known not only as an effective biomarker of oxidative stress but also as a mutagenic DNA modification. Incorporation of dAMP at the opposite site of 8-OHdG induces G>T or A>C transversions. However, in vivo analyses of gene mutations caused by potassium bromate (KBrO3), which can induce 8-OHdG at carcinogenic target sites, showed that G>T was prominent in the small intestines of mice, but not in the kidneys of rats. Because KBrO3 was a much clearer carcinogen in the kidneys of rats, detailed analyses of gene mutations in the kidney DNA of rats treated with KBrO3 could improve our understanding of oxidative stress-mediated carcinogenesis. In the current study, site-specific reporter gene mutation assays were performed in the kidneys of gpt delta rats treated with KBrO3. Groups of 5 gpt delta rats were treated with KBrO3 at concentrations of 0, 125, 250, or 500 ppm in the drinking water for 9 weeks. At necropsy, the kidneys were macroscopically divided into the cortex and medulla. 8-OHdG levels in DNA extracted from the cortex were dramatically elevated at concentrations of 250 ppm and higher compared with those from the medulla. Cortex-specific increases in mutant frequencies in gpt and red/gam genes were found at 500 ppm. Mutation spectrum and sequence analyses of their mutants demonstrated significant elevations in A>T transversions in the gpt gene and single base deletions at guanine or adenine in the gpt or red/gam genes. While A>T transversions and single base deletions of adenine may result from the oxidized modification of adenine, the contribution of 8-OHdG to gene mutations was limited despite possible participation of the 8-OHdG repair process in guanine deletion.
Assuntos
Bromatos , DNA , Rim , Ratos , Camundongos , Animais , 8-Hidroxi-2'-Desoxiguanosina , Mutação , Adenina , Carcinogênese , Carcinógenos , GuaninaRESUMO
BACKGROUND: Carbendazim (methyl 2-benzimidazolecarbamate, CASRN: 10605-21-7) exhibits spindle poisoning effects and is widely used as a fungicide. With respect to genotoxicity, carbendazim is deemed to be non-mutagenic in vitro, but it causes indicative DNA damage in vivo and chromosome aberrations in vitro and in vivo. In this study, we examined the mutagenicity of carbendazim in vivo. RESULTS: MutaMice were treated with carbendazim orally at doses of 0 (corn oil), 250, 500, and 1,000 mg/kg/day once a day for 28 days. A lacZ assay was used to determine the mutant frequency (MF) in the liver and glandular stomach of mice. MutaMice were administered up to the maximum dose recommended by the Organization for Economic Co-operation and Development Test Guidelines for Chemicals No. 488 (OECD TG488). The lacZ MFs in the liver and glandular stomach of carbendazim-treated animals were not significantly different from those in the negative control animals. In contrast, positive control animals exhibited a significant increase in MFs in both the liver and glandular stomach. CONCLUSIONS: Carbendazim is non-mutagenic in the liver and glandular stomach of MutaMice following oral treatment.
RESUMO
Helicobacter pylori is a human carcinogen, but the mechanisms evoked in carcinogenesis during this chronic inflammatory disease remain incompletely characterized. We determined whether chronic H. pylori infection induced mutations in the gastric mucosa of male and female gpt delta C57BL/6 mice infected for 6 or 12 mo. Point mutations were increased in females infected for 12 mo. The mutation frequency in this group was 1.6-fold higher than in uninfected mice of both sexes (P < 0.05). A:T-to-G:C transitions and G:C-to-T:A transversions were 3.8 and 2.0 times, respectively, more frequent in this group than in controls. Both mutations are consistent with DNA damage induced by oxidative stress. No increase in the frequency of deletions was observed. Females had more severe gastric lesions than males at 6 mo postinfection (MPI; P < 0.05), but this difference was absent at 12 MPI. In all mice, infection significantly increased expression of IFNgamma, IL-17, TNFalpha, and iNOS at 6 and 12 mo, as well as H. pylori-specific IgG1 levels at 12 MPI (P < 0.05) and IgG2c levels at 6 and 12 MPI (P < 0.01 and P < 0.001). At 12 MPI, IgG2c levels in infected females were higher than at 6 MPI (P < 0.05) and also than those in infected males at 12 MPI (P < 0.05). Intensity of responses was mediated by sex and duration of infection. Lower H. pylori colonization indicated a more robust host response in females than in males. Earlier onset of severe gastric lesions and proinflammatory, Th1-biased responses in female C57BL/6 mice may have promoted mutagenesis by exposing the stomach to prolonged oxidative stress.
Assuntos
Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/genética , Helicobacter pylori/patogenicidade , Mutação , Animais , Anticorpos Antibacterianos/sangue , Citocinas/genética , Proteínas de Escherichia coli/genética , Feminino , Mucosa Gástrica/imunologia , Gastrite/genética , Gastrite/metabolismo , Gastrite/microbiologia , Gastrite/patologia , Expressão Gênica , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/imunologia , Humanos , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese , Óxido Nítrico Sintase Tipo II/genética , Estresse Oxidativo , Pentosiltransferases/genética , Caracteres Sexuais , Fatores de TempoRESUMO
BACKGROUND: Styrene (CAS 100-42-5) is widely used as polystyrene and acrylonitrile-butadiene-styrene resin such as plastic, rubber, and paint. One of the primary uses of styrene is food utensils and containers, but a small amount of styrene transferred into food can be ingested by eating. Styrene is metabolized into styrene 7,8-oxide (SO). SO is mutagenic in bacteria and mouse lymphoma assays. It is clastogenic in cultured mammalian cells. However, styrene and SO are not clastogenic/aneugenic in rodents, and no rodent in vivo gene mutation studies were identified. METHODS: To investigate the mutagenicity of orally administered styrene, we used the transgenic rodent gene mutation assay to perform an in vivo mutagenicity test (OECD TG488). The transgenic MutaMouse was given styrene orally at doses of 0 (corn oil; negative control), 75, 150, and 300 mg/kg/day for 28 days, and mutant frequencies (MFs) were determined using the lacZ assay in the liver and lung (five male mice/group). RESULTS: There were no significant differences in the MFs of the liver and lung up to 300 mg/kg/day (close to maximum tolerable dose (MTD)), when one animal with extremely high MFs that were attributed to an incidental clonal mutation was omitted. Positive and negative controls produced the expected results. CONCLUSIONS: These findings show that styrene is not mutagenic in the liver and lung of MutaMouse under this experimental condition.
RESUMO
BACKGROUND: tert-Butyl hydroperoxide (TBHP; CAS 75-91-2), a hydroperoxide, is mainly used as a polymerization initiator to produce polyethylene, polyvinyl chloride, and unsaturated polyester. It is a high-production chemical, widely used in industrial countries, including Japan. TBHP is also used as an additive for the manufacturing of food utensils, containers, and packaging (UCP). Therefore, there could be consumer exposure through oral intake of TBHP eluted from UCPs. TBHP was investigated in various in vitro and in vivo genotoxicity assays. In Ames tests, some positive results were reported with and/or without metabolic activation. As for the mouse lymphoma assay, the positive result was reported, regardless of the presence or absence of metabolic activation enzymes. The results of some chromosomal aberrations test and comet assay in vitro also demonstrated the genotoxic positive results. On the other hand, in in vivo tests, there are negative results in the bone marrow micronucleus test of TBHP-administered mice by single intravenous injection and the bone marrow chromosomal aberration test using rats exposed to TBHP for 5 days by inhalation. Also, about dominant lethal tests, the genotoxic positive results appeared. In contrast, there is little information about in vivo mutagenicity and no information about carcinogenicity by oral exposure. RESULTS: We conducted in vivo gene mutation assay using MutaMice according to the OECD Guidelines for the Testing of Chemicals No. 488 to investigate in vivo mutagenicity of TBHP through oral exposure. After repeated dosing for 28 days, there were no significant differences in the mutant frequencies (MFs) of the liver and glandular stomach up to 300 mg/kg/day (close to the maximum tolerable dose (MTD)). The positive and negative controls produced the expected responses. CONCLUSIONS: These findings show that orally administrated TBHP is not mutagenic in the mouse liver and glandular stomach under these experimental conditions.
RESUMO
The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.
RESUMO
Since mutagenicity which can induce permanent transmissible changes in the structure of the genetic material is one of the major causes of cancer, research for genotoxicity including mutagenicity has focused on cancer hazard identification. Thus, it has been assumed that there was no threshold in mutagenesis. On the other hand, tumor development induced by not only non-genotoxic carcinogen but also genotoxic carcinogens will likely show a practical threshold. Therefore, statistical evaluation can provide value of the benchmark dose lower confidence limit (BMDL) calculated by approaches for the determination of genetic toxicity point of departure (PoD). In addition, disruption of epigenetic regulation which affect transcription through alteration of chromatin structure is considered to be important in future genotoxicity research. Taking into account benchmark dose or epigenetics will help improve assessment of genotoxicity, which offer promising insight into understanding genomic instability. Overall, this review presents current trends for future assessments of genotoxicity.
Assuntos
Epigênese Genética , Neoplasias , Instabilidade Genômica , Humanos , Testes de Mutagenicidade , Medição de RiscoRESUMO
We assessed the genotoxicity of 30 food-flavoring chemicals used in Japan that have not been investigated before. These 30 food-flavoring chemicals have representative chemical structures belonging to 18 chemical classes. The Ames and chromosomal aberration (CA) tests (in vitro tests) were first conducted in accordance with the "Food Additive Risk Assessment Guidelines" of the Japan Food Safety Commission. If the in vitro test yielded a positive result, an in vivo micronucleus test or a transgenic mouse gene mutation assay was performed to verify the in vitro test results. Of the 30 food-flavoring chemicals, 3 yielded a positive result in both Ames and CA tests. Another 11 chemicals yielded positive results in the CA test. However, none of the chemicals yielding positive in vitro test results yielded positive results in the in vivo tests. These findings indicate no genotoxicity concerns of the food-flavoring chemicals belonging to the abovementioned 18 chemical classes used in Japan unless there are other structural modifications.
RESUMO
Silymarin, a natural flavonoid from the seeds of milk thistle, is used for chemoprevention against various cancers in clinical settings and in experimental models. To examine the chemopreventive mechanisms of silymarin against colon cancer, we investigated suppressive effects of silymarin against carcinogenicity and genotoxicity induced by 1,2-dimethylhydrazine (DMH) plus dextran sodium sulfate (DSS) in the colon of F344 gpt delta transgenic rats. Male gpt delta rats were given a single subcutaneous injection of 40 mg/kg DMH and followed by 1.5% DSS in drinking water for a week. They were fed diets containing silymarin for 4 weeks, starting 1 week before DMH injection and samples were collected at 4, 20 and 32 weeks after the DMH treatment. Silymarin at doses of 100 and 500 p.p.m. suppressed the tumor formation in a dose-dependent manner and the reduction was statistically significant. In the mutation assays, DMH plus DSS enhanced the gpt mutant frequency (MF) in the colon, and the silymarin treatments reduced the MFs by 20%. Silymarin also reduced the genotoxicity of DMH in a dose-dependent manner in bacterial mutation assay with Salmonella typhimurium YG7108, a sensitive strain to alkylating agents, and the maximum reduction was >80%. These results suggest that silymarin is chemopreventive against DMH/DSS-induced inflammation-associated colon carcinogenesis and silymarin might act as an antigenotoxic agent, in part.
Assuntos
1,2-Dimetilidrazina/toxicidade , Neoplasias do Colo/prevenção & controle , Dano ao DNA , Sulfato de Dextrana/toxicidade , Inflamação/etiologia , Silimarina/uso terapêutico , Transferases (Outros Grupos de Fosfato Substituídos)/fisiologia , Animais , Antioxidantes/uso terapêutico , Carcinógenos/toxicidade , Colo/efeitos dos fármacos , Colo/metabolismo , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/imunologia , Masculino , Mutação/genética , Ratos , Ratos Endogâmicos F344 , Ratos TransgênicosRESUMO
The recent discovery that the potent carcinogen acrylamide (AA) is present in a variety of fried and baked foods raises health concerns, particularly for children, because AA is relatively high in child-favoured foods such as potato chips and French fries. To compare the susceptibility to AA-induced genotoxicity of young versus adult animals, we treated 3- and 11-week-old male gpt delta transgenic F344 rats with 0, 20, 40 or 80 p.p.m. AA via drinking water for 4 weeks and then examined genotoxicity in the bone marrow, liver and testis. We also analysed the level of N7-(2-carbamoyl-2-hydroxyethyl)-guanine (N7-GA-Gua), the major DNA adduct induced by AA, in the liver, testis and mammary gland. At 40 and 80 p.p.m., both age groups yield similar results in the comet assay in liver; but at 80 p.p.m., the bone marrow micronucleus frequency and the gpt-mutant frequency in testis increased significantly only in the young rats, and N7-GA-Gua adducts in the testis was significantly higher in the young rats. These results imply that young rats are more susceptible than adult rats to AA-induced testicular genotoxicity.
Assuntos
Acrilamida/toxicidade , Envelhecimento/efeitos dos fármacos , Mutagênicos/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Ensaio Cometa , Adutos de DNA/metabolismo , Feminino , Masculino , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Mutação/genética , Especificidade de Órgãos/efeitos dos fármacos , Ratos , Ratos TransgênicosRESUMO
As humans are exposed to a variety of chemical agents as well as radiation, health effects of radiation should be evaluated in combination with chemicals. To explore combined genotoxic effects of radiation and chemicals, we examined modulating effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting methylating agent, against genotoxicity of γ-radiation. Human lymphoblastoid TK6 cells and its mismatch-deficient derivative, i.e., MT1 cells, were treated with MNNG for 24h before they were exposed to γ-irradiation at a dose of 1.0 Gy, and the resulting genotoxicity was examined. In TK6 cells, the pretreatments with MNNG at low doses suppressed frequencies of the thymidine kinase (TK) gene mutation and micronucleus (MN) formation induced by γ-irradiation and thus the dose responses of TK and MN assays were U-shaped along with the pretreatment doses of MNNG. In contrast, the genotoxic effects of MNNG and γ-irradiation were additive in MT1 cells and the frequencies of TK mutations and MN induction increased along with the doses of MNNG. Apoptosis induced by γ-radiation was suppressed by the pretreatments in TK6 cells, but not in MT1 cells. The expression of p53 was induced and cell cycle was delayed at G2/M phase in TK6, but not in MT1 cells, by the treatments with MNNG. These results suggest that pretreatments of MNNG at low doses suppress genotoxicity of γ-radiation in human cells and also that mismatch repair proteins are involved in the apparent adaptive responses.
Assuntos
Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Metilnitronitrosoguanidina/farmacologia , Adaptação Biológica , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular , Raios gama , Humanos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Mutação/efeitos dos fármacos , Timidina Quinase/metabolismoRESUMO
BACKGROUND: Base pair mismatches in genomic DNA can result in mutagenesis, and consequently in tumorigenesis. To investigate how mismatch repair deficiency increases mutagenicity under oxidative stress, we examined the type and frequency of mutations arising in the mucosa of the small intestine of mice carrying a reporter gene encoding guanine phosphoribosyltransferase (gpt) and in which the Msh2 gene, which encodes a component of the mismatch repair system, was either intact (Msh2+/+::gpt/0; Msh2-bearing) or homozygously knockout (KO) (Msh2-/-::gpt/0; Msh2-KO). RESULTS: Gpt mutant frequency in the small intestine of Msh2-KO mice was about 10 times that in Msh2-bearing mice. Mutant frequency in the Msh2-KO mice was not further enhanced by administration of potassium bromate, an oxidative stress inducer, in the drinking water at a dose of 1.5 g/L for 28 days. Mutation analysis showed that the characteristic mutation in the small intestine of the Msh2-KO mice was G-to-A transition, irrespective of whether potassium bromate was administered. Furthermore, administration of potassium bromate induced mutations at specific sites in gpt in the Msh2-KO mice: G-to-A transition was frequently induced at two known sites of spontaneous mutation (nucleotides 110 and 115, CpG sites) and at nucleotides 92 and 113 (3'-side of 5'-GpG-3'), and these sites were confirmed to be mutation hotspots in potassium bromate-administered Msh2-KO mice. Administration of potassium bromate also induced characteristic mutations, mainly single-base deletion and insertion of an adenine residue, in sequences of three to five adenine nucleotides (A-runs) in Msh2-KO mice, and elevated the overall proportion of single-base deletions plus insertions in Msh2-KO mice. CONCLUSIONS: Our previous study revealed that administration of potassium bromate enhanced tumorigenesis in the small intestine of Msh2-KO mice and induced G-to-A transition in the Ctnnb1 gene. Based on our present and previous observations, we propose that oxidative stress under conditions of mismatch repair deficiency accelerates the induction of single-adenine deletions at specific sites in oncogenes, which enhances tumorigenesis in a synergistic manner with G-to-A transition in other oncogenes (e.g., Ctnnb1).