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Background: Cisplatin is a primary chemotherapy choice for various solid tumors. DNA damage caused by cisplatin results in apoptosis of tumor cells. Cisplatin‐induced DNA damage, however, may also result in mutations in normal cells and the initiation of secondary malignancies. In the current study, we have used the erythrocyte PIG‐A assay to evaluate mutagenesis in non‐tumor hematopoietic tissue of cancer patients receiving cisplatin chemotherapy. Methods: Twenty‐one head and neck cancer patients undergoing treatment with cisplatin were monitored for the presence of PIG‐A mutant total erythrocytes and the young erythrocytes, reticulocytes (RETs), in peripheral blood for up to five and a half months from the initiation of the anti‐neoplastic chemotherapy. Results: PIG‐A mutant frequency (MF) in RETs increased at least two‐fold in 15 patients at some point of the monitoring, while the frequency of total mutant RBCs increased at least two‐fold in 6 patients. A general trend for an increase in the frequency of mutant RETs and total mutant RBCs was observed in 19 and 18 patients, respectively. Only in one patient did both RET and total RBC PIG‐A MFs did not increase at any time‐point over the monitoring period. Conclusion: Cisplatin chemotherapy induces moderate increases in the frequency of PIG‐A mutant erythrocytes in head and neck cancer patients. Mutagenicity measured with the flow cytometric PIG‐A assay may serve as a tool for predicting adverse outcomes of genotoxic antineoplastic therapy. Cisplatin chemotherapy induces moderate increases in the frequency of PIG‐A mutant erythrocytes in head and neck cancer patients. Mutagenicity measured with the flow cytometric PIG‐A assay may serve as a tool for predicting adverse outcomes of genotoxic antineoplastic therapy. [ABSTRACT FROM AUTHOR]

Procarbazine (PCZ) and N‐propyl‐N‐nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI‐anchored marker‐deficient mutant‐phenotype red blood cells (RBCs) in the flow cytometry‐based rat RBC Pig‐a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig‐a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59‐deficient mutant‐phenotype BMEs from PCZ‐ and PNU‐treated rats and examined their endogenous X‐linked Pig‐a gene by next generation sequencing. Pig‐a mutations consistent with the properties of PCZ and PNU were found in sorted mutant‐phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig‐a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment‐induced mutations were distributed across the protein coding sequence of the Pig‐a gene. The causal relationship between BMEs and RBCs and the agent‐specific mutational spectra in CD59‐deicient BMEs indicate that the rat RBC Pig‐a assay, scoring CD59‐deficient mutant‐phenotype RBCs in peripheral blood, detects Pig‐a gene mutation. [ABSTRACT FROM AUTHOR]

Somatic mutations accumulate in the human genome and are correlated with increased cancer incidence as humans age. The standard model for studying the carcinogenic effects of exposures for human risk assessment is the rodent 2‐year carcinogenicity assay. However, there is little information regarding the effect of age on cancer‐driver gene mutations in these models. The mutant fraction (MF) of Kras codon 12 GGT to GAT and GGT to GTT mutations, oncogenic mutations orthologous between humans and rodents, was quantified over the lifespan of B6C3F1 mice. MFs were measured in lung and liver tissue, organs that frequently develop tumors following carcinogenic exposures. The MFs were evaluated at 4, 6, 8, 12, 21, and 85 weeks, with the 12‐week and 21‐week time points being coincident with the conclusion of 28‐day and 90‐day exposure durations used in short‐term toxicity testing. The highly sensitive and quantitative Allele‐specific Competitive Blocker PCR (ACB‐PCR) assay was used to quantify the number of mutant Kras codon 12 alleles. The mouse lung showed a slight, but significant trend increase in the Kras codon 12 GAT mutation over the 85‐week period. The trend with age can be equally well‐fit by several non‐linear functions, but not by a linear function. In contrast, the liver GAT mutation did not increase, and the GTT mutation did not increase for either organ. Even with the slight increase in the lung GAT MFs, our results indicate that the future use of Kras mutation as a biomarker of carcinogenic effect will not be confounded by animal age. Environ. Mol. Mutagen. 59:715–721, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

We have established a flow cytometry‐based Pig‐a assay for rat bone marrow erythroid cells (BMEs). The BME Pig‐a assay uses a DNA‐specific stain and two antibodies: one against the transmembrane transferrin receptor (CD71 marker) and the other against the GPI‐anchored complement inhibitory protein (CD59 marker). In F344 male rats treated acutely with a total of 120 mg/kg of N‐ethyl‐N‐nitrosourea (ENU) the frequency of CD59‐deficient phenotypically mutant BMEs increased approximately 24‐fold compared to the rats concurrently treated with the vehicle. Such an increase of mutant BMEs coincides with increases of CD59‐deficient reticulocytes measured in rats treated with similar doses of ENU. Sequence analysis of the endogenous X‐linked Pig‐a gene of CD59‐deficient BMEs revealed that they are Pig‐a mutants. The spectrum of ENU‐induced Pig‐a mutations in these BMEs was consistent with the in vivo mutagenic signature of ENU: 73% of mutations occurred at A:T basepairs, with the mutated T on the nontranscribed strand of the gene. T→A transversion was the most frequent mutation followed by T→C transition; no deletion or insertion mutations were present in the spectrum. Since BMEs are precursors of peripheral red blood cells, our findings suggest that CD59‐deficient erythrocytes measured in the flow cytometric erythrocyte Pig‐a assay develop from BMEs containing mutations in the Pig‐a gene. Thus, the erythrocyte Pig‐a assay detects mutation in the Pig‐a gene. Environ. Mol. Mutagen. 59:722–732, 2018. © 2018 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The X‐linked Pig‐a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig‐a mutant cells fail to synthesize GPI and to express GPI‐anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig‐a mutation in various in vivo assays. Here, we describe an in vitro Pig‐a mutation assay in L5178YTk+/– mouse lymphoma cells, in which mutant‐phenotype cells are measured by flow cytometry using a fluorescent anti‐CD90 antibody. Increased frequencies of CD90‐deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N‐ethyl‐N‐nitrosourea (ENU), ethyl methanesulphonate, and 7,12‐dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting‐dilution cloning assay using proaerolysin selection. Individual CD90‐deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig‐a gene. Pig‐a mutations with agent‐specific signatures were found in nearly all clones that developed from sorted CD90‐deficient cells. These results indicate that a Pig‐a mutation assay can be successfully conducted in L5178YTk+/− cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig‐a assays. Environ. Mol. Mutagen. 59:4–17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA. [ABSTRACT FROM AUTHOR]

Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a 'clone-by-clone' approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand. Environ. Mol. Mutagen. 57:114-124, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNPs) are among the most commercially used nanomaterials and their toxicity and genotoxicity are controversial. Although many in vitro studies have been conducted to evaluate the genotoxicity of AgNPs, in vivo genotoxicity studies on the nanomaterials are limited. Given the unique physicochemical properties and complex pharmacokinetics behavior of nanoparticles (NPs), in vivo genotoxicity assessment of AgNPs is badly needed. In this study, the clastogenicity and mutagenicity of AgNPs with different sizes and coatings were evaluated using mouse micronucleus (MN) assay, Pig-a assay and Comet assay. Five 7-week-old male B6C3F1 mice per group were treated with 5 nm polyvinylpyrrolidone (PVP)-coated AgNPs at a single dose of 0.5, 1.0, 2.5, 5.0, 10.0 or 20.0 mg/kg body weight (bw) via intravenous injection for both the MN and Pig-a assays; or with 15-100 nm PVP- or 10-80 nm silicon-coated AgNPs at a single or 3-day repeated dose of 25.0 mg/kg bw for the MN assay and Comet assay in mouse liver. Inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) analyses indicated that AgNPs reached the testing tissues (bone marrow for the MN and Pig-a assays and liver for the Comet assay). Although there was a reduction of reticulocytes in the PVP-coated AgNPs-treated animals, indicating cytotoxicity of the AgNPs, none of the treatments resulted in a significant increase of either mutant frequencies in the Pig-a gene or the percent of micronucleated reticulocyte over the concurrent controls. However, both the PVP- and silicon-coated AgNPs induced oxidative DNA damage in mouse liver. These results demonstrate that the AgNPs can reach mouse bone marrow and liver, and generate cytotoxicity to the reticulocytes and oxidative DNA damage to the liver. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7-week-old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes-modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue ( P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage ( P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non-neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species. Environ. Mol. Mutagen. 55:24-34, 2014. © 2013 Wiley Periodicals, Inc.† [ABSTRACT FROM AUTHOR]

Somatic mutations accumulate in the human genome and are correlated with increased cancer incidence as humans age. The standard model for studying the carcinogenic effects of exposures for human risk assessment is the rodent 2-year carcinogenicity assay. However, there is little information regarding the effect of age on cancer-driver gene mutations in these models. The mutant fraction (MF) of Kras codon 12 GGT to GAT and GGT to GTT mutations, oncogenic mutations orthologous between humans and rodents, was quantified over the lifespan of B6C3F1 mice. MFs were measured in lung and liver tissue, organs that frequently develop tumors following carcinogenic exposures. The MFs were evaluated at 4, 6, 8, 12, 21, and 85 weeks, with the 12-week and 21-week time points being coincident with the conclusion of 28-day and 90-day exposure durations used in short-term toxicity testing. The highly sensitive and quantitative Allele-specific Competitive Blocker PCR (ACB-PCR) assay was used to quantify the number of mutant Kras codon 12 alleles. The mouse lung showed a slight, but significant trend increase in the Kras codon 12 GAT mutation over the 85-week period. The trend with age can be equally well-fit by several non-linear functions, but not by a linear function. In contrast, the liver GAT mutation did not increase, and the GTT mutation did not increase for either organ. Even with the slight increase in the lung GAT MFs, our results indicate that the future use of Kras mutation as a biomarker of carcinogenic effect will not be confounded by animal age. Environ. Mol. Mutagen. 59:715-721, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

The X-linked Pig-a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig-a mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig-a mutation in various in vivo assays. Here, we describe an in vitro Pig-a mutation assay in L5178YTk +/- mouse lymphoma cells, in which mutant-phenotype cells are measured by flow cytometry using a fluorescent anti-CD90 antibody. Increased frequencies of CD90-deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate, and 7,12-dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting-dilution cloning assay using proaerolysin selection. Individual CD90-deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig-a gene. Pig-a mutations with agent-specific signatures were found in nearly all clones that developed from sorted CD90-deficient cells. These results indicate that a Pig-a mutation assay can be successfully conducted in L5178YTk +/- cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig-a assays. Environ. Mol. Mutagen. 59:4-17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA., (Published 2017. This article is a US Government work and is in the public domain in the USA.)