Repeat treatment of organotypic airway cultures with ethyl methanesulfonate causes accumulation of somatic cell mutations without expansion of bronchial-carcinoma-specific cancer driver mutations.

The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.25-100 µg/mL ethyl methanesulfonate (EMS) for 28 days, followed by a 28-day recovery period. Mutagenesis was evaluated by Duplex Sequencing (DS), and clonal expansion of bronchial-cancer-specific cancer-driver mutations (CDMs) was investigated by CarcSeq to determine if both mutation-based endpoints can be assessed in the same system. Additionally, DNA damage and tissue-specific responses were analyzed during the treatment and following the recovery period. EMS exposure led to time-dependent increases in mutagenesis over the 28-day treatment period, without expansion of clones containing CDMs; the mutation frequencies remained elevated following the recovery. EMS also produced an increase in DNA damage measured by the CometChip and MultiFlow assays and the elevated levels of DNA damage were reduced (but not eliminated) following the recovery period. Cytotoxicity and most tissue-function changes induced by EMS treatment recovered to control levels, the exception being reduced proliferating cell frequency. Our results indicate that general, respiratory-tissue-specific and genotoxicity endpoints increased with repeat EMS dosing; expansion of CDM clones, however, was not detected using this repeat treatment protocol. DISCLAIMER: This article reflects the views of its authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.

Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

Erythrocyte PIG-A mutant frequencies in cancer patients receiving cisplatin.

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.

Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models.

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines.

Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays.

Propranolol is a widely used ß-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells. We found that NNP induced concentration-dependent mutations in the Ames test, both in two tester strains that detect base pair substitutions, TA1535 and TA100, as well as in the TA98 frameshift-detector strain. Although positive results were seen with rat liver S9, the hamster liver S9 fraction was more effective in bio-transforming NNP into a reactive mutagen. NNP also induced micronuclei and gene mutations in human lymphoblastoid TK6 cells in the presence of hamster liver S9. Using a panel of TK6 cell lines that each expresses a different human cytochrome P450 (CYP), CYP2C19 was identified as the most active enzyme in the bioactivation of NNP to a genotoxicant among those tested. NNP also induced concentration-dependent DNA strand breakage in metabolically competent 2-dimensional (2D) and 3D cultures of human HepaRG cells. This study indicates that NNP is genotoxic in a variety of bacterial and mammalian systems. Thus, NNP is a mutagenic and genotoxic nitrosamine and a potential human carcinogen.

Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Genotoxicity evaluation of nitrosamine impurities using human TK6 cells transduced with cytochrome P450s.

Many nitrosamines are recognized as mutagens and potent rodent carcinogens. Over the past few years, nitrosamine impurities have been detected in various drugs leading to drug recalls. Although nitrosamines are included in a 'cohort of concern' because of their potential human health risks, most of this concern is based on rodent cancer and bacterial mutagenicity data, and there are little data on their genotoxicity in human-based systems. In this study, we employed human lymphoblastoid TK6 cells transduced with human cytochrome P450 (CYP) 2A6 to evaluate the genotoxicity of six nitrosamines that have been identified as impurities in drug products: N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutanoic acid (NMBA), N-nitrosomethylphenylamine (NMPA), N-nitrosodiisopropylamine (NDIPA), and N-nitrosodibutylamine (NDBA). Using flow cytometry-based assays, we found that 24-h treatment with NDEA, NEIPA, NMBA, and NMPA caused concentration-dependent increases in the phosphorylation of histone H2A.X (γH2A.X) in CYP2A6-expressing TK6 cells. Metabolism of these four nitrosamines by CYP2A6 also caused significant increases in micronucleus frequency as well as G2/M phase cell-cycle arrest. In addition, nuclear P53 activation was found in CYP2A6-expressing TK6 cells exposed to NDEA, NEIPA, and NMPA. Overall, the genotoxic potency of the six nitrosamine impurities in our test system was NMPA > NDEA ≈ NEIPA > NMBA > NDBA ≈ NDIPA. This study provides new information on the genotoxic potential of nitrosamines in human cells, complementing test results generated from traditional assays and partially addressing the issue of the relevance of nitrosamine genotoxicity for humans. The metabolically competent human cell system reported here may be a useful model for risk assessment of nitrosamine impurities found in drugs.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

Effect of life stage and target tissue on dose-response assessment of ethyl methane sulfonate-induced genotoxicity.

In order to investigate the possibility that treatment age affects the genotoxic response to ethyl methane sulfonate (EMS) exposure, we dosed gpt-delta neonatal mice on postnatal days 1-28 with 5-100 mg/kg/day of EMS and measured micronucleus (MN) induction in peripheral blood and gpt gene mutation in liver, lung, bone marrow, small intestine, spleen, and kidney. The data were compared to measurements from similarly exposed adult gpt-delta mice. Our results indicate that the peripheral blood MN frequencies in mice treated as neonates are not substantially different from those measured in mice treated as adults. There were, however, differences in tissue-specific gpt mutation responses in mice treated with EMS as neonates and adults. Greater mutant frequencies were seen in DNA isolated from kidney of mice treated as neonates, whereas the mutant frequencies in bone marrow, liver, and spleen were greater in the animals treated as adults. Benchmark dose potency ranking indicated that the differences for kidney were significant. Our data indicate that there are differences in EMS-induced genotoxicity between mice treated as adults and neonates; the differences, however, are relatively small.

Appropriate in vivo follow-up assays to an in vitro bacterial reverse mutation (Ames) test positive investigational drug candidate (active pharmaceutical ingredient), drug-related metabolite, or drug-related impurity.

Kirkland et al. [Mutation Research/Genetic Toxicology and Environmental Mutagenesis 847 (2019) 403035, https://doi.org/10.1016/j.mrgentox.2019.03.008; Mutation Research/Genetic Toxicology and Environmental Mutagenesis 839 (2019): 21-35, https://doi.org/10.1016/j.mrgentox.2019.01.007] made recommendations on the use of the in vivo comet and transgenic rodent (TGR) gene mutation assays to screen for in vivo mutagenicity. Although it is not directly stated in either of these publications, we are concerned that the reports could potentially be used to support assertions that it is equally acceptable to follow up a positive bacterial reverse mutation (Ames) finding for an investigational drug with either the in vivo TGR mutation assay or an in vivo comet assay. For regulatory genotoxicity assessment, the in vivo follow-up for an in vitro bacterial mutation-positive drug, drug-related metabolite, or impurity should be based upon evaluating a similar endpoint (i.e., mutagenicity) as the intent is to determine if the findings of in vitro gene mutation correlate with findings of in vivo gene mutation (i.e., biologically relevant to the in vitro results). Thus, the most scientifically appropriate in vivo assays would be the TGR mutation assay or, in some circumstances, the in vivo Pig-a assay. An in vivo rodent comet assay or combination of the in vivo micronucleus and in vivo rodent comet assays would generally not be an appropriate follow-up test.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Genetic toxicity testing using human in vitro organotypic airway cultures: Assessing DNA damage with the CometChip and mutagenesis by Duplex Sequencing.

The organotypic human air-liquid-interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high-throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error-corrected next-generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 µg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time- and concentration-dependent increases in DNA damage and a concentration-dependent increase in mutant frequency. The mutations observed in the EMS-treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti-p63-positive basal cell frequency, but produced concentration-responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration-responsive decreases in culture viability, goblet cell and anti-Ki67-positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28-day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue-specific general toxicity.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

Invited review: human air-liquid-interface organotypic airway tissue models derived from primary tracheobronchial epithelial cells-overview and perspectives.

The lung is an organ that is directly exposed to the external environment. Given the large surface area and extensive ventilation of the lung, it is prone to exposure to airborne substances, such as pathogens, allergens, chemicals, and particulate matter. Highly elaborate and effective mechanisms have evolved to protect and maintain homeostasis in the lung. Despite these sophisticated defense mechanisms, the respiratory system remains highly susceptible to environmental challenges. Because of the impact of respiratory exposure on human health and disease, there has been considerable interest in developing reliable and predictive in vitro model systems for respiratory toxicology and basic research. Human air-liquid-interface (ALI) organotypic airway tissue models derived from primary tracheobronchial epithelial cells have in vivo-like structure and functions when they are fully differentiated. The presence of the air-facing surface allows conducting in vitro exposures that mimic human respiratory exposures. Exposures can be conducted using particulates, aerosols, gases, vapors generated from volatile and semi-volatile substances, and respiratory pathogens. Toxicity data have been generated using nanomaterials, cigarette smoke, e-cigarette vapors, environmental airborne chemicals, drugs given by inhalation, and respiratory viruses and bacteria. Although toxicity evaluations using human airway ALI models require further standardization and validation, this approach shows promise in supplementing or replacing in vivo animal models for conducting research on respiratory toxicants and pathogens.

Analysis of In Vivo Mutation in the Hprt and Tk Genes of Mouse Lymphocytes.

Determining mutant frequencies in endogenous reporter genes is a tool for identifying potentially genotoxic environmental agents, and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here, we describe a high-throughput method for identifying mouse spleen lymphocytes with mutations in the endogenous X-linked hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of HPRT- and TK-deficient cells. The same procedure can be utilized for quantifying Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), while mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutant clones can be further analyzed to classify the types of mutations in the Tk gene (small intragenic mutations vs. large chromosomal mutations) and to determine the nature of intragenic mutation at both the Hprt and Tk genes.

Mutation as a Toxicological Endpoint for Regulatory Decision-Making.

Mutations induced in somatic cells and germ cells are responsible for a variety of human diseases, and mutation per se has been considered an adverse health concern since the early part of the 20th Century. Although in vitro and in vivo somatic cell mutation data are most commonly used by regulatory agencies for hazard identification, that is, determining whether or not a substance is a potential mutagen and carcinogen, quantitative mutagenicity dose-response data are being used increasingly for risk assessments. Efforts are currently underway to both improve the measurement of mutations and to refine the computational methods used for evaluating mutation data. We recommend continuing the development of these approaches with the objective of establishing consensus regarding the value of including the quantitative analysis of mutation per se as a required endpoint for comprehensive assessments of toxicological risk. Environ. Mol. Mutagen. 61:34-41, 2020. © 2019 Wiley Periodicals, Inc.

Detection of In Vivo Mutation in the Hprt and Pig-a Genes of Rat Lymphocytes.

Assays for in vivo mutation are used to identify genotoxic hazards and phenotypes prone to genomic instability and cancer. The hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the phosphatidyl inositol glycan, class A (Pig-a) gene are endogenous X-linked genes that can be used as reporters of mutation in peripheral blood lymphocytes from most mammals. Here we describe methodology for measuring Hprt and Pig-a mutation in rat T-lymphocytes. The identification and selective expansion of mutant lymphocytes is based upon the phenotypic properties of Hprt- and Pig-a-deficient cells, that is, resistance to the purine analog, 6-thioguanine, or to the bacterial toxin, proaerolysin. Expanded mutants can be further analyzed by sequencing cDNA from the target transcripts for identification of small sequence alterations and by multiplex PCR analysis of genomic DNA for the detection of deletions.