Development of an<i>in vitro PIG-A</i>gene mutation assay in human cells

Rees, Benjamin; Tate, Matthew; Lynch, Anthony M.; Thornton, Catherine A.; Jenkins, Gareth; Walmsley, Richard M.; Johnson, George E.

doi:10.1093/mutage/gew059

Cited by 13 publications

(15 citation statements)

References 56 publications

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“…However, these assays are time‐consuming and therefore not suitable for high throughput screening. What is more, they are performed with cell lines derived from mice (L5178Y) or hamster (CHO, AS52 and V79), which are mainly p53 defective and of little relevance for humans as well as being devoid of biotransformation properties (Whitwell et al, ; Rees et al, ). For this reason, we chose the HepG2 cells, the same cell model we used for the γH2AX assay, to perform the in vitro PIG‐A gene mutation assay originally developed with the TK6 cell line (Krüger et al, ; ; Nicklas et al, ; Rees et al, ) and more recently on the L5178Y cell line (Bemis et al, ; David et al, ; Wang et al, ).…”

Section: Discussionmentioning

confidence: 99%

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Genotoxicity and mutagenicity assessment of food contaminant mixtures present in the French diet

Kopp

Vignard

Mirey

et al. 2018

Environ and Mol Mutagen

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Through diet, people are exposed simultaneously to a variety of contaminants (e.g. heavy metals, mycotoxins, pesticides) that could have combined adverse effects on human health. A previous study identified six main mixtures of food contaminants to which French adult consumers are exposed. These complex mixtures are comprised of 11 to 19 chemicals that have numerous toxic properties. In the present study, we investigated the genotoxic effects of these food contaminants, as single molecules and in mixtures that reflect their occurrence in the French diet, using the γH2AX assay in two human cell lines (HepG2, LS-174 T). Results of detailed analysis of the 49 individual contaminants (including 21 tested in this study) demonstrated a positive genotoxic response to 14 contaminants in HepG2 and 12 in LS-174 T cells. Next, our results indicated that two mixtures out of six triggered significant γH2AX induction after 24 hr of treatment, at concentrations for which individual compounds did not induce any DNA damage, suggesting more than additive interactions between chemicals. γH2AX positive mixtures were then tested for mutagenicity with the innovative in vitro PIG-A assay in HepG2 cells coupled with the soft agar colony formation assay. The two γH2AX positive mixtures led to a significant increase in the frequency of PIG-A GPI-deficient cells and in the number of colonies formed in soft agar. In conclusion, our study demonstrates that two mixtures of contaminants present in the French diet induce genotoxicity and mutagenicity, and that the combined effects of single molecules present in these mixtures are likely not additive, highlighting potential problems for hazard assessment of mixtures. Environ. Mol. Mutagen. 59:742-754, 2018. © 2018 Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

“…Based on the results of recent studies, the mutagenicity of mixtures was assessed with the in vitro PIG‐A assay (Krüger et al, ; ; Rees et al, ). 1.10 6 HepG2 cells were seeded in 25 cm 2 culture flasks.…”

Section: Methodsmentioning

confidence: 99%

Genotoxicity and mutagenicity assessment of food contaminant mixtures present in the French diet

Kopp

Vignard

Mirey

et al. 2018

Environ and Mol Mutagen

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“…Some of the MLA-positive cases were from positive results of high concentration (> 500 μg/mL) of test solution and possibly due to the high sensitivity of the assay (Morita et al, 2014;Parry et al, 2010), while these positive results had eventually been negated by other assays. Thus, the new assays, for example, in vitro PIG-A gene mutation assay (Rees et al, 2017), may be needed for better understanding mutagenicity of antibiotics as well as other pharmaceuticals.…”

Section: Resultsmentioning

confidence: 99%

<i>In vitro</i> genotoxicity test package of antibiotics for human use submitted to the Japanese regulatory agency during 2004–2015

Sekizawa

Hoshino

Takasu

2017

Fundam. Toxicol. Sci.

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-The Ames test is used for the mutagenic assessment of drugs; however, it may not provide an accurate genotoxic profile for bactericidal compounds. This study was performed to clarify 1) whether the total number of genotoxicity assays performed (#Assays) was greater during antibiotic development than during the development of other drugs, particularly antivirals, possibly due to the requirement for additional assessments, 2) whether the maximum doses of the Ames test were less when an alternative assay had been performed for antibiotics, and 3) whether some particular alternative assay had an advantage to minimize #Assays in the last decade. Genotoxicity data submitted to the Pharmaceuticals and Medical Devices Agency in Japan during 2004 -2015 were used. The #Assays was greater and the maximum doses of the Ames tests were lower for antibiotics, which was more obvious when alternative mutagenic assays had been performed. The mouse lymphoma assay or hypoxanthine-guanine phosphoribosyl transferase gene mutation assay was performed preferentially as an alternative. For antibiotic development, preferred genotoxicity test packages should be discussed in the future for a better understanding of the genotoxic potential of antibiotics.

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“…In mammals, the X‐linked phosphatidyl inositolglycan class A ( Pig‐a ) gene is involved in GPI synthesis and present in a single functional copy, i.e., in males there is one X chromosome, and in females, while there are two X chromosomes, one is permanently inactivated early in embryogenesis. Recently, multiple models utilizing the endogenous Pig‐a gene as a reporter of mutation have been developed for well‐characterized mammalian cell lines, for several mammalian species commonly used in biomedical research, and for humans [Miura et al, ,b; Phonethepswath et al, ; Miura et al, ; Dertinger et al, ; Dobrovolsky et al, ; Kimoto et al, ; Dertinger et al, ; Krüger et al, ; Krüger et al, ; Rees et al, ; Wang et al, ]. There are models that detect mutant–phenotype cells in limited dilution culture (LDC) assays using aerolysin as a selecting agent, and there are models that detect GPI‐ and GPI‐anchored marker‐deficient phenotype cells using high throughput flow cytometry [Miura et al, ].…”

Section: Introductionmentioning

confidence: 99%

Analysis of mutation in the rat Pig‐a assay: I) studies with bone marrow erythroid cells

Revollo

Pearce

Dad

et al. 2018

Environ and Mol Mutagen

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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.

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Development of anin vitro PIG-Agene mutation assay in human cells

Cited by 13 publications

References 56 publications

Genotoxicity and mutagenicity assessment of food contaminant mixtures present in the French diet

Genotoxicity and mutagenicity assessment of food contaminant mixtures present in the French diet

<i>In vitro</i> genotoxicity test package of antibiotics for human use submitted to the Japanese regulatory agency during 2004–2015

Analysis of mutation in the rat Pig‐a assay: I) studies with bone marrow erythroid cells

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