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Integration of liver and blood micronucleus and Pig-a gene mutation endpoints into rat 28-day repeat-treatment studies: Proof-of-principle with diethylnitrosamine.

ABSTRACT: Regulatory guidance documents stress the value of assessing multiple tissues and the most appropriate endpoints when evaluating chemicals for in vivo genotoxic potential. However, conducting several independent studies to consider multiple endpoints and/or tissue compartments is resource intensive. Furthermore, conventional approaches for scoring genotoxicity endpoints are slow, tedious, and less objective than what would be considered ideal. In an effort to address these issues with current practices, we attempted to i) employ flow cytometry-based methods to score liver micronuclei, blood micronuclei, and blood Pig-a gene mutation, and ii) integrate the endpoints into a common general toxicology study design-the rat 28-day repeat dose study. A proof-of-principle experiment was performed with 6-week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) for 28 consecutive days. One day later blood was collected for micronucleated reticulocyte (MN-RET) and Pig-a mutation assays, and liver tissue was obtained for micronucleated hepatocyte (MNHEP) scoring. MN-RET frequencies were not affected by DEN exposure, and mean Pig-a mutant cell frequencies were only slightly elevated. On the other hand, % MNHEP showed marked, dose-related increases (2.2, 7.2, and 9.1 mean fold-increase for 5, 10, 15?mg DEN/kg/day, respectively). Concurrent with MNHEP analyses, assessments of Ki-67-positive events and the proportion of 8n nuclei provided evidence for treatment-related changes to hepatocyte proliferation. Collectively, these results reinforce the importance of evaluating chemicals' genotoxic potential in liver in addition to hematopoietic cells, and suggest that several automated measurements can be successfully integrated into repeat-dose studies for higher efficiencies and better utilization of fewer animals.

SUBMITTER: Khanal S

PROVIDER: S-EPMC5896020 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Integration of liver and blood micronucleus and Pig-a gene mutation endpoints into rat 28-day repeat-treatment studies: Proof-of-principle with diethylnitrosamine.

Khanal Sumee S Singh Priyanka P Avlasevich Svetlana L SL Torous Dorothea K DK Bemis Jeffrey C JC Dertinger Stephen D SD

Mutation research. Genetic toxicology and environmental mutagenesis 20180223

Regulatory guidance documents stress the value of assessing multiple tissues and the most appropriate endpoints when evaluating chemicals for in vivo genotoxic potential. However, conducting several independent studies to consider multiple endpoints and/or tissue compartments is resource intensive. Furthermore, conventional approaches for scoring genotoxicity endpoints are slow, tedious, and less objective than what would be considered ideal. In an effort to address these issues with current pra ...[more]

PMID: 29555062

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Project description:In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methylcytosine to detect the presence of 5 methylcytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used. A method that detects DNA methylation that is not restricted to specific sequences would provide a useful platform to analyse methylation distribution among the genes in any given phenotypic condition.Availability of additional approaches to examine DNA methylation would lead to increase in our understanding of the methylome and would help define the intrinsic and extrinsic factors which govern it. Microarray based high throughput methods have been extensively used for gene expression analysis. The method described uses a microarray to analyse DNA methylation levels in a gene/cDNA/oligo specific manner. Here, genomic DNA is fragmented and used for hybridization to microarray slides. The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC( 5-methyl cytidine) antibody (monoclonal). The resolution provided by this method is dependent upon the microarray platform used. This method can be adapted to any platform thus enabling seamless integration with existing technology of gene expression analysis

Project description:Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.

Project description:BackgroundThe question whether lymphocyte radiosensitivity is representative of patients' response to radiotherapy (RT) remains unsolved. We analyzed lymphocyte cytogenetic damage in patients who were homogeneously treated with preoperative radiochemotherapy (RCT) for rectal cancer within clinical trials. We tested for interindividual variation and consistent radiosensitivity after in-vivo and in-vitro irradiation, analyzed the effect of patients' and RCT characteristics on cytogenetic damage, and tested for correlations with patients' outcome in terms of tumor response, survival and treatment-related toxicity.MethodsThe cytokinesis-block micronucleus cytome (CBMNcyt) assay was performed on the peripheral blood lymphocytes (PBLCs) of 134 patients obtained before, during, at the end of RCT, and during the 2-year follow-up. A subset of PBLCs obtained before RCT was irradiated in-vitro with 3 Gy. RCT included 50.4 Gy of pelvic RT with 5-fluorouracil (5-FU) alone (n = 78) or 5-FU plus oxaliplatin (n = 56). The analyzed variables included patients' age, gender, RT characteristics (planning target volume size [PTV size], RT technique), and chemotherapy characteristics (5-FU plasma levels, addition of oxaliplatin). Outcome was analyzed as tumor regression, patient survival, and acute and late toxicity.ResultsCytogenetic damage increased significantly with the radiation dose and varied substantially between individuals. Women were more sensitive than men; no significant age-dependent differences were observed. There was a significant correlation between the cytogenetic damage after in-vitro irradiation and in-vivo RCT. We found a significant effect of the PTV size on the yields of cytogenetic damage after RCT, while the RT technique had no effect. Neither the addition of oxaliplatin nor the 5-FU levels influenced cytogenetic damage. We found no correlation between patient outcome and the cytogenetic damage.ConclusionsWe found consistent cytogenetic damage in lymphocytes after in-vivo RCT and in-vitro irradiation. Gender was confirmed as a well-known, and the PTV size was identified as a less well-known influencing variable on lymphocyte cytogenetic damage after partial-body irradiation. A consistent level of cytogenetic damage after in-vivo and in-vitro irradiation may indicate the importance of genetic factors for individual radiosensitivity. However, we found no evidence that in-vivo or in-vitro irradiation-induced cytogenetic damage is an adequate biomarker for the response to RCT in rectal cancer patients.

Dataset Information

Integration of liver and blood micronucleus and Pig-a gene mutation endpoints into rat 28-day repeat-treatment studies: Proof-of-principle with diethylnitrosamine.

Publications

Integration of liver and blood micronucleus and Pig-a gene mutation endpoints into rat 28-day repeat-treatment studies: Proof-of-principle with diethylnitrosamine.

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