In Vitro Mouse Lymphoma (L5178Y Tk +/− -3.7.2C) Forward Mutation Assay

Schisler, Melissa R.; Moore, Martha M.; Gollapudi, B. Bhaskar

doi:10.1007/978-1-62703-529-3_2

Cited by 16 publications

(22 citation statements)

References 22 publications

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“…DNA adducts were determined using the nuclease P 1 enrichment version of the 32 P-postlabelling assay as previously described (Arlt et al 2017; Schmeiser et al 2013). DNA samples (4 μg) were digested with micrococcal nuclease (240 mU, Sigma-Aldrich) and calf spleen phosphodiesterase (60 mU, Calbiochem) for 3 h at 37 °C; and enriched and labelled as reported.…”

Section: Methodsmentioning

confidence: 99%

The impact of p53 on aristolochic acid I-induced nephrotoxicity and DNA damage in vivo and in vitro

et al. 2019

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Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(−/−) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by 32P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography–mass spectrometry (GC–MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice and exposed them to AAI in vitro (50 μM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(−/−) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(−/−) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.

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Section: Methodsmentioning

confidence: 99%

The impact of p53 on aristolochic acid I-induced nephrotoxicity and DNA damage in vivo and in vitro

et al. 2019

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“…L5178Y/TK +/− mouse lymphoma cells ATCC clone 3.7.2C was prepared for the assay as previously described [18], [19], [25]–[27]. The mutagenic potential of the test articles (PA and 2-BP) was measured by its ability to induce TK +/− → TK −/− (trifluorothymidine (TFT)-resistant phenotype) mutations.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Potential Genotoxicity of HIV Entry Inhibitors Derived from Natural Sources

et al. 2014

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AIDS is a global pandemic that has seen the development of novel and effective treatments to improve the quality of life of those infected and reduction of spread of the disease. Palmitic Acid (PA), which we identified and isolated from Sargassum fusiforme, is a naturally occurring fatty acid that specifically inhibits HIV entry by binding to a novel pocket on the CD4 receptor. We also identified a structural analogue, 2-bromopalmitate (2-BP), as a more effective HIV entry inhibitor with a 20-fold increase in efficacy. We have used the structure-activity relationship (SAR) of 2-BP as a platform to identify new small chemical molecules that fit into the various identified active sites in an effort to identify more potent CD4 entry inhibitors. To validate further drug development, we tested the PA and 2-BP scaffold molecules for genotoxic potential. The FDA and International Conference on Harmonisation (ICH) recommends using a standardized 3-test battery for testing compound genotoxicity consisting of the bacterial reverse mutation assay, mouse lymphoma assay, and rat micronucleus assay. PA and 2-BP and their metabolites tested negative in all three genotoxicty tests. 2-BP is the first derivative of PA to undergo pre-clinical screening, which will enable us to now test multiple simultaneous small chemical structures based on activity in scaffold modeling across the dimension of pre-clinical testing to enable transition to human testing.

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“…The MLA is an in vitro mammalian cell forward gene mutation assay that can detect thymidine kinase ( Tk ) gene mutations induced by test substances. In this assay, mutant colonies can be classified as small (slow growing) or large (normal growing) and mutagenic chemicals induce different frequencies of these small and large colonies (Clive et al ; Moore et al , ; Schisler et al ). Generally, chemicals acting primarily as clastogens induce a relatively higher proportion of small colony Tk mutants, whereas chemicals that primarily induce point mutations induce a relatively higher proportion of large colony mutants (Applegate et al ).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of U. S. National Toxicology Program (NTP) mouse lymphoma assay data using International Workshop on Genotoxicity Tests (IWGT) and the Organization for Economic Co‐Operation and Development (OECD) criteria

Schisler

Gollapudi

Moore

2018

Environ and Mol Mutagen

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The forward gene mutation mouse lymphoma assay (MLA) is widely used, as part of a regulatory test battery, to identify the genotoxic potential of chemicals. It identifies mutagens capable of inducing a variety of genetic events. During the 1980s and early 1990s, the U.S. National Toxicology Program (NTP) developed a publicly available database (https://tools.niehs.nih.gov/cebs3/ui/) of MLA results. This database is used to define the mutagenic potential of chemicals, to develop structure–activity relationships (SAR), and to draw correlations to animal carcinogenicity findings. New criteria for MLA conduct and data interpretation were subsequently developed by the International Workshop for Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development (OECD). These recommendations are included in a new OECD Test Guideline (TG490). It is essential that early experimental data be re‐examined and classified according to the current criteria to build a curated database to better inform chemical‐specific evaluations and SAR models. We re‐evaluated more than 1900 experiments representing 342 chemicals against the newly defined acceptance criteria for background mutant frequency (MF), cloning efficiency (CE), positive control values (modified for this evaluation due to lack of colony sizing), appropriate dose selection, and data consistency. Only 17% of the evaluated experiments met all acceptance criteria used in this re‐evaluation. Results from 211 chemicals were determined to be uninterpretable, 92 were positive, and 39 equivocal. The authors could not classify any responses as negative because colony sizing was not performed for any of these experiments and it is clear, based on many experiment with unacceptably low background and positive control MFs, that mutant colony recovery was often suboptimal. This re‐evaluation provides a curated database for the MLA. A similar curation should be done for other widely used genetic toxicology assays, but will be more difficult for certain assays (e.g., in vitro chromosomal aberrations) because important parameters such as level of cytotoxicity were often not evaluated/reported. Environ. Mol. Mutagen. 59:829–841, 2018. © 2018 Wiley Periodicals, Inc.

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In Vitro Mouse Lymphoma (L5178Y Tk +/− -3.7.2C) Forward Mutation Assay

Cited by 16 publications

References 22 publications

The impact of p53 on aristolochic acid I-induced nephrotoxicity and DNA damage in vivo and in vitro

The impact of p53 on aristolochic acid I-induced nephrotoxicity and DNA damage in vivo and in vitro

Evaluation of Potential Genotoxicity of HIV Entry Inhibitors Derived from Natural Sources

Evaluation of U. S. National Toxicology Program (NTP) mouse lymphoma assay data using International Workshop on Genotoxicity Tests (IWGT) and the Organization for Economic Co‐Operation and Development (OECD) criteria

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