Mouse lymphoma thymidine kinase gene mutation assay: Follow‐up meeting of the international workshop on Genotoxicity testing—Aberdeen, Scotland, 2003—Assay acceptance criteria, positive controls, and data evaluation

Moore, Martha M.; Honma, Masamitsu; Clements, Julie; Bölcsföldi, George; Burlinson, Brian; Cifone, Maria A.; Clarke, Jane J.; Delongchamp, Robert R.; Durward, Robert; Fellows, Michael; Gollapudi, B. Bhaskar; Hou, Sai‐Mei; Jenkinson, P.; Lloyd, Melvin; Majeska, Jenness B.; Myhr, B.; O’Donovan, Michael R.; Omori, Takashi; Riach, Colin; San, Richard H.C.; Stankowski, Leon F.; Thakur, Ajit Kumar; Goethem, Freddy Van; Wakuri, Shinobu; Yoshimura, Isao

doi:10.1002/em.20159

Cited by 136 publications

(74 citation statements)

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“…AZT (0.8 and 1 mg/ml) (2,994 and 3,742 lM) produced clear increases in Tk MF over that of the vehicle control. The results were evaluated using the new criteria developed by the Mouse Lymphoma Expert Working Group of the International Workshop for Genotoxicity Testing (IWGT) [Moore et al, 2006]. For a response to be positive for the microwell version of the assay, these guidelines require an induced MF of at least 126 3 10 À6 [the Global Evaluation Factor (GEF)] in one or more treated cultures while showing cytotoxicity !10% RTG.…”

Section: Tk Mutation Assaymentioning

confidence: 99%

3′‐azido‐3′‐deoxythymidine induces deletions in L5178Y mouse lymphoma cells

Wang

Chen

Honma

et al. 2007

Environ and Mol Mutagen

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3'-Azido-3'-deoxythymidine (AZT), a nucleoside analogue used for the treatment of acquired immunodeficiency syndrome (AIDS), induced a significant dose-related increase in the thymidine kinase (Tk) mutant frequency (MF) in L5178Y/Tk(+/-) 3.7.2C mouse lymphoma cells. Treatment with 1 mg/ml (3,742 muM) AZT for 24 hr resulted in a MF of 407 x 10(-6) compared to a control MF of 84 x 10(-6). The MFs of the large and small colony mutants resulting from AZT exposure were 142 x 10(-6) and 265 x 10(-6), respectively. One hundred and fifty mutants from the 1 mg/ml (3,742 muM) AZT-treated culture and sixty-nine mutants from independent untreated cultures were isolated and analyzed. LOH analysis using a heteromorphic microsatellite locus located in the Tk gene was performed to determine the presence or absence of the Tk(+) allele. Eight other microsatellite markers spanning the entire mouse chromosome 11 also were examined for heterozygosity to determine the extent of LOH. In addition, Tk gene dosage analysis was conducted using Real-Time PCR in those mutants showing LOH at the Tk locus. The presence of only one Tk allele based on Real-Time PCR indicated that the mutant resulted from deletion while the presence of two alleles was consistent with a recombination event. More mutants from the AZT-treated culture showed Tk LOH than did independent mutants from the untreated cultures (91% vs. 64%) and the induced mutants also showed distinct chromosome 11 LOH patterns. The mutation spectrum of mutants from AZT-treated cells was also significantly different from that of spontaneous mutants. More deletions and fewer intragenic mutations were observed in the mutants from the AZT-treated culture than independent mutants from the untreated control. Our data indicate that AZT primarily induced LOH mutations in L5178Y mouse lymphoma cells and a large number of LOH mutations resulted from deletions.

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Section: Tk Mutation Assaymentioning

confidence: 99%

3′‐azido‐3′‐deoxythymidine induces deletions in L5178Y mouse lymphoma cells

Wang

Chen

Honma

et al. 2007

Environ and Mol Mutagen

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“…The data evaluation criteria developed by the mouse lymphoma assay expert workgroup of the international workgroup for genotoxicity tests (IWGT) were used to determine whether or not a response was positive or negative (Moore et al, 2006). Positive responses are defined as those where the induced MF in one or more treated cultures exceeds the global evaluation factor (GEF) of 126 mutants per 10 6 cells and there is also a dose related increase with MF.…”

Section: Data Analysesmentioning

confidence: 99%

Genotoxic effects of acrylamide and glycidamide in mouse lymphoma cells

Mei

Churchwell

et al. 2008

Food and Chemical Toxicology

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In addition to occupational exposures to acrylamide (AA), concerns about AA health risks for the general population have been recently raised due to the finding of AA in food. In this study, we evaluated the genotoxicity of AA and its metabolite glycidamide (GA) in L5178Y/ Tk +/− mouse lymphoma cells. The cells were treated with 2-18 mM of AA or 0.125-4 mM of GA for 4 h without metabolic activation. The DNA adducts, mutant frequencies and the types of mutations for the treated cells were examined. Within the dose range tested, GA induced DNA adducts of adenine and guanine [N3-(2-carbamoyl-2-hydroxyethyl)-adenine and N7-(2-carbamoyl-2-hydroxyethyl)-guanine] in a linear dose-dependent manner. The levels of guanine adducts were consistently about 60-fold higher across the dose range than those of adenine. In contrast, no GAderived DNA adducts were found in the cells treated with any concentrations of AA, consistent with a lack of metabolic conversion of AA to GA. However, the mutant frequency was significantly increased by AA at concentrations of 12 mM and higher. GA was mutagenic starting with the 2 mM dose, suggesting that GA is much more mutagenic than AA. The mutant frequencies were increased with increasing concentrations of AA and GA, mainly due to an increase of proportion of small colony mutants. To elucidate the underlying mutagenic mechanism, we examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for mutants induced by AA or GA. Compared to GA induced mutations, AA induced more mutants whose LOH extended to D11Mit22 and D11Mit74, an alteration of DNA larger than half of the chromosome. Statistical analysis of the mutational spectra revealed a significant difference between the types of mutations induced by AA and GA treatments (P = 0.018). These results suggest that although both AA and GA generate mutations through a clastogenic mode of action in mouse lymphoma cells, GA induces mutations via a DNA adduct mechanism whereas AA induces mutations by a mechanism not involving the formation of GA adducts.

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“…The evaluation and interpretation of results from the L5178Y mouse lymphoma assay has changed over the years and protocol updates have been recommended (Moore et al, 2000(Moore et al, , 2006(Moore et al, , 2007.…”

Section: In Vitro Mammalian Cell Gene Mutation Test (Oecd Tg 476)mentioning

confidence: 99%

Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment

2011

EFS2

296

136

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The Scientific Committee reviewed the current state-of-the-science on genotoxicity testing and provided a commentary and recommendations on genotoxicity testing strategies. A step-wise approach is recommended for the generation and evaluation of data on genotoxic potential, beginning with a basic battery of in vitro tests, comprising a bacterial reverse mutation assay and an in vitro micronucleus assay. Consideration should be given to whether specific features of the test substance might require substitution of one or more of the recommended in vitro tests by other in vitro or in vivo tests in the basic battery. In the event of negative in vitro results, it can be concluded that the substance has no genotoxic potential. In case of inconclusive, contradictory or equivocal results, it may be appropriate to conduct further testing in vitro. In case of positive in vitro results, review of the available relevant data on the test substance and, where necessary, an appropriate in vivo study to assess whether the genotoxic potential observed in vitro is expressed in vivo is recommended. Suitable in vivo tests are the mammalian erythrocyte micronucleus test, transgenic rodent assay, and Comet assay. The approach to in vivo testing should be step-wise. If the first in vivo test is positive, no further testing is necessary and the substance should be considered as an in vivo genotoxin. If the test is negative, it may be possible to conclude that the substance is not an in vivo genotoxin. However, in some cases, a second in vivo test may be necessary (e.g. if the first test is negative but more than one endpoint in the in vitro tests are positive, an in vivo test on a second endpoint may be necessary). The combination of assessing different endpoints in different tissues in the same animal in vivo should also be considered.

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Mouse lymphoma thymidine kinase gene mutation assay: Follow‐up meeting of the international workshop on Genotoxicity testing—Aberdeen, Scotland, 2003—Assay acceptance criteria, positive controls, and data evaluation

Cited by 136 publications

References 2 publications

3′‐azido‐3′‐deoxythymidine induces deletions in L5178Y mouse lymphoma cells

3′‐azido‐3′‐deoxythymidine induces deletions in L5178Y mouse lymphoma cells

Genotoxic effects of acrylamide and glycidamide in mouse lymphoma cells

Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment

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