Abstract
DNA double-strand breaks (DSBs), one of the most severe lesions of DNA damage triggered by various genotoxic insults, can lead to chromosome change, genomic instability, and even tumorigenesis if not repaired efficiently. In response to DNA damage, histone H2AX molecules are rapidly phosphorylated at serine 139 near the site of DNA DSBs and form γ-H2AX foci. As an early important cellular event linked to DNA damage and repair, γ-H2AX is a highly sensitive biomarker for “monitoring” DNA damage and consequently is a useful tool in genetic toxicology screen. We and other researchers have used γ-H2AX as a marker to assess the potential genotoxic effects of some nanoparticles in vitro and in vivo. In this chapter, we describe several useful methods for γ-H2AX detection, which can be used to evaluate the potential genotoxic effects of nanoparticles.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Moore JD, Krebs JE (2004) Histone modifications and DNA double-strand break repair. Biochem Cell Biol 82(4):446–452
Khanna KK, Jackson SP (2001) DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet 27(3):247–254
Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273(10):5858–5868
Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM (2000) A critical role for Histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10:886–895
Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ (2001) ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 276(45):42462–42467
Ward IM, Chen J (2001) Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem 276:47759–47762
Bassing CH, Chua KF, Sekiguchi J, Suh H, Whitlow SR, Fleming JC, Monroe BC, Ciccone DN, Yan C, Vlasakova K, Livingston DM, Ferguson DO, Scully R, Alt FW (2002) Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc Natl Acad Sci U S A 99(12):8173–8178
Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O et al (2002) Genomic instability in mice lacking histone H2AX. Science 296(5569):922–927
Sedelnikova OA, Rogakou EP, Panyutin IG, Bonner WM (2002) Quantitative detection of (125)IdU-induced DNA double strand breaks with γ-H2AX antibody. Radiat Res 158(4):486–492
Nikolova T, Dvorak M, Jung F, Adam I, Krämer E, Gerhold-Ay A, Kaina B (2014) The γH2AX assay for genotoxic and nongenotoxic agents: comparison of H2AX phosphorylation with cell death response. Toxicol Sci 140(1):103–117
Watters GP, Smart DJ, Harvey JS, Austin CA (2009) H2AX phosphorylation as a genotoxicity endpoint. Mutat Res 679(1–2):50–58
Gonzaleza L, Lisonb D, Krisch-Volders M (2008) Genotoxicity of engineered nanomaterials: a critical review. Nanotoxicology 2(4):252–273
Landsiedel R, Kapp MD, Schulz M, Wiench K, Oesch F (2009) Genotoxicity investigation on nanomaterials: methods, preparation and characterization of test material, potential artifacts and limitations-many questions, some answers. Mutat Res 681(2–3):241–258
Yousuke T, Tatsushi T, Yuko I (2012) Flow cytometric evaluation of nanoparticles using side-scattered light and reactive oxygen species-mediated fluorescence-correlation with genotoxicity. Environ Sci Technol 46(14):7629–7636
Zhao X, Takabayashi F, Yuko I (2016) Coexposure to silver nanoparticles and ultraviolet A synergistically enhances the phosphorylation of histone H2AX. J Photochem Photobiol B 162:213–222
Åkerlund E, Cappellini F, Di Bucchianico S, Islam S, Skoglund S, Derr R, Odnevall Wallinder I, Hendriks G, Karlsson HL (2018) Genotoxic and mutagenic properties of Ni and NiO nanoparticles investigated by comet assay, γ-H2AX staining, Hprt mutation assay and ToxTracker reporter cell lines. Environ Mol Mutagen 59(3):211–222. https://doi.org/10.1002/em.22163
Wan R, Mo Y, Feng L, Chien S, Tollerud DJ, Zhang Q (2012) DNA damage caused by metal nanoparticles: involvement of oxidative stress and activation of ATM. Chem Res Toxicol 25(7):1402–1411
Wan R, Mo Y, Zhang Z, Jiang M, Tang S, Zhang Q (2017) Cobalt nanoparticles induce lung injury, DNA damage and mutations in mice. Part Fibre Toxicol 14(1):1–15. https://doi.org/10.1186/s12989-017-0219-z
Kim SW, Roh J, Park CS (2016) Immunohistochemistry for pathologists: protocols, pitfalls, and tips. J Pathol Transl Med 50:411–418
Cynthia S, Gerald L, Jeffrey W, Carlin O, Ronald H (2002) Qualitative and quantitative analysis of nonneoplastic lesions in toxicology studies. Toxicol Pathol 30(1):93–96
Acknowledgment
This work was partly supported by the National Natural Science Foundation of China (81473009), and an Intramural Research Development Grant from Fujian Medical University (JS14001) to Dr. Rong Wan; and NIESH/NIH (ES023693 and ES028911), KSEF-148-502-16-381, Kentucky Lung Cancer Research Program, and an Intramural Research Incentive Grants (50992) from UofL to Dr. Qunwei Zhang.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wan, R., Mo, Y., Tong, R., Gao, M., Zhang, Q. (2019). Determination of Phosphorylated Histone H2AX in Nanoparticle-Induced Genotoxic Studies. In: Zhang, Q. (eds) Nanotoxicity. Methods in Molecular Biology, vol 1894. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8916-4_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8916-4_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8915-7
Online ISBN: 978-1-4939-8916-4
eBook Packages: Springer Protocols