title: Arsenic induces VL30 retrotransposition: the involvement of oxidative stress and heat-shock protein 70.
abstract: Arsenic is an environmental contaminant with known cytotoxic and carcinogenic properties, but the cellular mechanisms of its action are not fully known. As retrotransposition consists a potent mutagenic factor affecting genome stability, we investigated the effect of arsenic on retrotransposition of an enhanced green fluorescent protein (EGFP)-tagged nonautonomous long terminal repeat (LTR)-retrotransposon viral-like 30 (VL30) in a mouse NIH3T3 cell culture-retrotransposition assay. Flow cytometry analysis of assay cells treated with 2.5-20μM sodium arsenite revealed induction of retrotransposition events in a dose- and time-dependent manner, which was further confirmed as genomic integrations by PCR analysis and appearance of EGFP-positive cells by UV microscopy. Specifically, 20μM sodium arsenite strongly induced the VL30 retrotransposition frequency, which was ~90,000-fold higher than the natural one and also VL30 RNA expression was ~6.6-fold. Inhibition of the activity of endogenous reverse transcriptases by efavirenz at 15μM or nevirapine at 375μM suppressed the arsenite-induced VL30 retrotransposition by 71.16 or 79.88%, respectively. In addition, the antioxidant N-acetyl-cysteine reduced the level of arsenite-induced retrotransposition, which correlated with the rescue of arsenite-induced G2/M cell cycle arrest and cell toxicity. Treatment of assay cells ectopically overexpressing the human heat-shock protein 70 (Hsp70) with 15μM sodium arsenite resulted in an additional ~4.5-fold induction of retrotransposition compared with normal assay cells, whereas treatment with 20μM produced a massive cell death. Our results show for the first time that arsenic both as an oxidative and heat-shock mimicking agent is a potent inducer of VL30 retrotransposition in mouse cells. The impact of arsenic-induced retrotransposition, as a cellular response, on contribution to or explanation of the arsenic-associated toxicity and carcinogenicity is discussed.
authors.affiliation: Laboratory of General Biology, Medical School, University of Ioannina, 45110 Ioannina, Greece.
authors.forename: Georgios
authors.initials: G
authors.lastname: Markopoulos
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Noutsopoulos
authors.forename: Stefania
authors.initials: S
authors.lastname: Mantziou
authors.forename: Georgios
authors.initials: G
authors.lastname: Vartholomatos
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Monokrousos
authors.forename: Charalampos
authors.initials: C
authors.lastname: Angelidis
authors.forename: Theodore
authors.initials: T
authors.lastname: Tzavaras
mesh_terms: D000818:Animals; D001151:Arsenic / Q000633:toxicity*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction / Q000379:methods; D018626:Retroelements; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018626:Retroelements; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001151:Arsenic
keywords: Hsp70.; VL30; arsenic; cytotoxicity; retrotransposition; retrotransposon