In Vitro PIG-A Gene Mutation Assay in Human B-Lymphoblastoid TK6 Cells

Abstract

The X-linked PIG-A gene is involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD59 and CD55). In recent years, in vitro PIG-A assay has been established based on the high conservation of PIG-A/Pig-a loci among different species and the large data from the in vivo system. The purpose of this study was to extend the approach for PIG-A mutation assessment to in vitro human B-lymphoblastoid TK6 cells by detecting the loss of GPI-linked CD55 and CD59 proteins. TK6 cells were treated with three mutagens 7,12-dimethylbenz[a]anthracene (DMBA), N-ethyl-N-nitrosourea (ENU), etoposide (ETO), and two nonmutagens: cadmium chloride (CdCl₂) and sodium chloride (NaCl). The mutation rate of PIG-A gene within TK6 cells was determined on the 11th day with flow cytometry analysis for the negative frequencies of CD55 and CD59. The antibodies used in this production were APC mouse-anti-human CD19 antibody, PE mouse anti-human CD55 antibody, PE mouse anti-human CD59 antibody, and nucleic acid dye 7-AAD. An immunolabeling method was used to reduce the high spontaneous level of preexisting PIG-A mutant cells. Our data suggested that DMBA-, ENU-, and ETO-induced mutation frequency of PIG-A gene was increased by twofold compared with the negative control, and the effects were dose-dependent. However, CdCl₂ and NaCl did not significantly increase the mutation frequency of PIG-A gene, with a high cytotoxicity at a dose of 10 mmol/L. Our study suggested that the novel in vitro PIG-A gene mutation assay within TK6 cells may represent a complement of the present in vivo Pig-a assay, and may provide guidance for their potential use in genotoxicity even in cells with a significant deficiency of GPI anchor.

Keywords

PIG-A gene mutation assay - flow cytometry - TK6 cells - genotoxicity

^# These authors contributed equally to this work.

Publikationsverlauf

Eingereicht: 14. April 2021

Angenommen: 24. Juni 2021

Artikel online veröffentlicht:
02. September 2021

• References

• 1 Cimino MC. Comparative overview of current international strategies and guidelines for genetic toxicology testing for regulatory purposes. Environ Mol Mutagen 2006; 47 (05) 362-390
• 2 Miyata T, Takeda J, Iida Y. et al. The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis. Science 1993; 259 (5099): 1318-1320
• 3 Zurzolo C, Simons K. Glycosylphosphatidylinositol-anchored proteins: membrane organization and transport. Biochim Biophys Acta 2016; 1858 (04) 632-639
• 4 Kinoshita T. Biosynthesis and deficiencies of glycosylphosphatidylinositol. Proc Jpn Acad, Ser B, Phys Biol Sci 2014; 90 (04) 130-143
• 5 Krüger CT, Hofmann M, Hartwig A. The in vitro PIG-A gene mutation assay: mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol (GPI) status of TK6 cells. Arch Toxicol 2015; 89 (12) 2429-2443
• 6 Rees BJ, Tate M, Lynch AM. et al. Development of an in vitro PIG-A gene mutation assay in human cells. Mutagenesis 2017; 32 (02) 283-297
• 7 Bemis JC, Avlasevich SL, Labash C. et al. Glycosylphosphatidylinositol (GPI) anchored protein deficiency serves as a reliable reporter of Pig-a gene mutation: Support from an in vitro assay based on L5178Y/Tk^+/- cells and the CD90.2 antigen. Environ Mol Mutagen 2018; 59 (01) 18-29
• 8 Wang Y, Revollo J, McKinzie P. et al. Establishing a novel Pig-a gene mutation assay in L5178YTk^+/- mouse lymphoma cells. Environ Mol Mutagen 2018; 59 (01) 4-17
• 9 David R, Talbot E, Allen B, Wilson A, Arshad U, Doherty A. The development of an in vitro Pig-a assay in L5178Y cells. Arch Toxicol 2018; 92 (04) 1609-1623
• 10 Johnson GE, Slob W, Doak SH. et al. New approaches to advance the use of genetic toxicology analyses for human health risk assessment. Toxcol Res 2015; 4: 667-676
• 11 Bemis JC, Heflich RH. In vitro mammalian cell mutation assays based on the Pig-a gene: a report of the International Workshops on Genetic Toxicology (IWGT) workgroup. Mutat Res Genet Toxicol Environ Mutagen 2019; 847: 403039
• 12 Ellis P, Fowler P, Booth E. et al. Where will genetic toxicology testing be in 30 years' time? Summary report of the 25th Industrial Genotoxicity Group Meeting, Royal Society of Medicine, London, November 9, 2011. Mutagenesis 2014; 29 (01) 73-77
• 13 OECD iLibrary. OECD Guideline for the testing of chemicals, Section 4. In vitro mammalian cell gene mutation tests using the Hprt and xprt genes. Accessed July 2, 2021 at: https://www.oecd-ilibrary.org/docserver/9789264264809-en.pdf?expires=1624388874&id=id&accname=guest&checksum=48AA31AD17D5DC66BB8E902FEAC0F0D3.
• 14 OECD iLibrary. OECD Guideline for the testing of chemicals, secton 4. In vitro mammalian cell gene mutation tests using the Thymidine Kinase gene. Accessed July 2, 2021 at: https://www.oecd-ilibrary.org/docserver/9789264264908-en.pdf?expires=1624388821&id=id&accname=guest&checksum=655A70F3F7AB4229B6E026174C446532
• 15 Lorge E, Moore MM, Clements J. et al. Standardized cell sources and recommendations for good cell culture practices in genotoxicity testing. Mutat Res 2016; 809: 1-15
• 16 Kirkland D, Kasper P, Müller L, Corvi R, Speit G. Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop. Mutat Res 2008; 653 (1–2): 99-108
• 17 Kimoto T, Horibata K, Miura D. et al. The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals. Mutat Res 2016; 811: 3-15
• 18 Zielenska M, Beranek D, Guttenplan JB. Different mutational profiles induced by N-nitroso-N-ethylurea: effects of dose and error-prone DNA repair and correlations with DNA adducts. Environ Mol Mutagen 1988; 11 (04) 473-485
• 19 Ezoe S. Secondary leukemia associated with the anti-cancer agent, etoposide, a topoisomerase II inhibitor. Int J Environ Res Public Health 2012; 9 (07) 2444-2453
• 20 van Maanen JM, Lafleur MV, Mans DR. et al. Effects of the ortho-quinone and catechol of the antitumor drug VP-16-213 on the biological activity of single-stranded and double-stranded phi X174 DNA. Biochem Pharmacol 1988; 37 (19) 3579-3589
• 21 Ashby J, Tinwell H, Glover P. et al. Potent clastogenicity of the human carcinogen etoposide to the mouse bone marrow and mouse lymphoma L5178Y cells: comparison to Salmonella responses. Environ Mol Mutagen 1994; 24 (01) 51-60
• 22 Suzuki H, Nakane S. Differential induction of chromosomal aberrations by topoisomerase inhibitors in cultured Chinese hamster cells. Biol Pharm Bull 1994; 17 (02) 222-226
• 23 Kirkland D, Kasper P, Martus HJ. et al. Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests. Mutat Res Genet Toxicol Environ Mutagen 2016; 795: 7-30
• 24 Sakai A, Sasaki K, Muramatsu D. et al. A Bhas 42 cell transformation assay on 98 chemicals: the characteristics and performance for the prediction of chemical carcinogenicity. Mutat Res 2010; 702 (01) 100-122
• 25 Han EH, Hwang YP, Jeong TC, Lee SS, Shin JG, Jeong HG. Eugenol inhibit 7,12-dimethylbenz[a]anthracene-induced genotoxicity in MCF-7 cells: Bifunctional effects on CYP1 and NAD(P)H:quinone oxidoreductase. FEBS Lett 2007; 581 (04) 749-756
• 26 Honma M, Hayashi M, Sofuni T. Cytotoxic and mutagenic responses to X-rays and chemical mutagens in normal and p53-mutated human lymphoblastoid cells. Mutat Res 1997; 374 (01) 89-98
• 27 Honma M, Momose M, Tanabe H. et al. Requirement of wild-type p53 protein for maintenance of chromosomal integrity. Mol Carcinog 2000; 28 (04) 203-214
• 28 Nicklas JA, Carter EW, Albertini RJ. Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line. Environ Mol Mutagen 2015; 56 (08) 663-673