Monitoring Lead Toxicity by Huh7-1x-ARE-luc Cell Line Lucifersae Biosensor

Kavoosi, S.; Shirzad, H.; Jalili, Sh.; Sadeghizadeh, M.; Motahari, P.

Monitoring Lead Toxicity by Huh7-1x-ARE-luc Cell Line Lucifersae Biosensor

Authors

S. Kavoosi ¹

H. Shirzad ²

Sh. Jalili ²

M. Sadeghizadeh ¹

P. Motahari ¹

¹ Genetics Department, Biologic Sciences Faculty, Tarbiat Modares University, Tehran, Iran

² Research Institute of Police Science and Social Studies, Tehran, Iran

Abstract

Aims: The accumulation of free radicals in the body leads to damages to cellular biopolymers through oxidative stress. Due to the increasing proliferation of heavy metals in soil and water environments, finding efficient methods for diagnostic detection and measurement of heavy metals in contaminated environments is very important. Cell-based biosensors can produce a measurable signal in response to specific chemical or physical agent in their environment. In this study, stable hepatoma Huh7 ARE-reporter cell line was developed containing luciferase gene with the aim of monitoring lead toxicity. This biosensor is reported to be able to detect lead by expressed signal which is measurable. The luciferase assay and Real time-PCR were performed.

Materials and Methods: In this experimental research, the Huh7-1x-ARE-luc was stably transferred in to the Huh7 cells and transfected cells were selected. After 5 passages, stable clones were isolated to confirm plasmid entrance. Luciferase activity of the Huh7-1x-ARE-luc cell line was performed with 0-80μM lead concentration to induce oxidative stress response. Cell viability was assessed by MTT. With Real time PCR, AREKEAP1 pathway gene expression were detected. Statistical analysis was performed by ΔCt method, using graphpad prism 6 software.

Findings: The gene expression of the reporter gene increased with increasing oxidative stress. Reducing the expression of the reporter gene was observed after 30 μM. 35 μM lead inhibited 50% cell metabolism. Expression of antioxidant pathway genes was significantly increased in 30 μM leaded cells compared to control gene.

Conclusion: The biosensor prepared from Huh7-1x-ARE-luc cell line of the reporter gene can be a convenient and efficient means for measuring oxidative compounds such as heavy metals such as lead.

Keywords

Reporter Gene

Biosensor

Oxidative stress

Antioxidant Response Element

Subjects

Agricultural Biotechnology

Radi R. Peroxynitrite, a stealthy biological oxidant. J Biol Chem. 2013;288(37):26464-72. [Link] [DOI:10.1074/jbc.R113.472936]

Bauer CE, Elsen S, Bird TH. Mechanisms for redox control of gene expression. Annu Rev Microbiol. 1999;53(1):495-523. [Link] [DOI:10.1146/annurev.micro.53.1.495]

Bermingham-McDonogh O, Gralla EB, Valentine JS. The copper, zinc-superoxide dismutase gene of Saccharomyces cerevisiae: Cloning, sequencing, and biological activity. Proc Natl Acad Sci U S A. 1988;85(13):4789-93. [Link] [DOI:10.1073/pnas.85.13.4789]

Aslund F, Zheng M, Beckwith J, Storz G. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. Proc Natl Acad Sci U S A. 1999;96(11):6161-5. [Link] [DOI:10.1073/pnas.96.11.6161]

Bai X, Chen Y, Hou X, Huang M, Jin J. Emerging role of NRF2 in chemoresistance by regulating drug-metabolizing enzymes and efflux transporters. Drug Metab Rev. 2016;48(4):541-67. [Link] [DOI:10.1080/03602532.2016.1197239]

Inoue F, Ahituv N. Decoding enhancers using massively parallel reporter assays. Genomics. 2015;106(3):159-64. [Link] [DOI:10.1016/j.ygeno.2015.06.005]

Simmons SO, Fan CY, Yeoman K, Wakefield J, Ramabhadran R. NRF2 oxidative stress induced by heavy metals is cell type dependent. Curr Chem Genomics. 2011;5(1):1-12. [Link] [DOI:10.2174/1875397301105010001]

Floyd RA. Neuroinflammatory processes are important in neurodegenerative diseases: An hypothesis to explain the increased formation of reactive oxygen and nitrogen species as major factors involved in neurodegenerative disease development. Free Radic Biol Med. 1999;26(9-10):1346-55. [Link] [DOI:10.1016/S0891-5849(98)00293-7]

Vasiliou V, Ross D, Nebert DW. Update of the NAD(P)H: Quinone oxidoreductase (NQO) gene family. Hum Genomics. 2006;2(5):329-35. [Link] [DOI:10.1186/1479-7364-2-5-329]

Gilfillan SC. Lead poisoning and the fall of Rome. J Occup Environ Med. 1965;7(2):53-60. [Link]

Patterson CC. Contaminated and natural lead environments of man. Arch Environ Health. 1965;11(3):344-60. [Link] [DOI:10.1080/00039896.1965.10664229]

Warniment C, Tsang K, Galazka SS. Lead poisoning in children. Am Fam Physician. 2010;81(6):751-7. [Link]

Hayes JD, McMahon M. NRF2 and KEAP1 mutations: Permanent activation of an adaptive response in cancer. Trends Biochem Sci. 2009;34(4):176-88. [Link] [DOI:10.1016/j.tibs.2008.12.008]