title: Expression of human oxoguanine glycosylase 1 or formamidopyrimidine glycosylase in human embryonic kidney 293 cells exacerbates methylmercury toxicity in vitro.
abstract: Exposure to methylmercury (MeHg) acutely at high levels, or via chronic low-level dietary exposure from daily fish consumption, can lead to adverse neurological effects in both the adult and developing conceptus. To determine the impact of variable DNA repair capacity, and the role of reactive oxygen species (ROS) and oxidatively damaged DNA in the mechanism of toxicity, transgenic human embryonic kidney (HEK) 293 cells that stably express either human oxoguanine glycosylase 1 (hOgg1) or its bacterial homolog, formamidopyrimidine glycosylase (Fpg), which primarily repair the oxidative lesion 8-oxo-2'-deoxyguanosine (8-oxodG), were used to assess the in vitro effects of MeHg. Western blotting confirmed the expression of hOgg1 or Fpg in both the nuclear and mitochondrial compartments of their respective cell lines. Following acute (1-2h) incubations with 0-10μM MeHg, concentration-dependent decreases in clonogenic survival and cell growth accompanied concentration-dependent increases in lactate dehydrogenase (LDH) release, ROS formation, 8-oxodG levels and apurinic/apyrimidinic (AP) sites, consistent with the onset of cytotoxicity. Paradoxically, hOgg1- and Fpg-expressing HEK 293 cells were more sensitive than wild-type cells stably transfected with the empty vector control to MeHg across all cellular and biochemical parameters, exhibiting reduced clonogenic survival and cell growth, and increased LDH release and DNA damage. Accordingly, upregulation of specific components of the base excision repair (BER) pathway may prove deleterious potentially due to the absence of compensatory enhancement of downstream processes to repair toxic intermediary abasic sites. Thus, interindividual variability in DNA repair activity may constitute an important risk factor for environmentally-initiated, oxidatively damaged DNA and its pathological consequences.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 3M2, Canada.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Ondovcik
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Preston
authors.forename: Gordon P
authors.initials: GP
authors.lastname: McCallum
authors.forename: Peter G
authors.initials: PG
authors.lastname: Wells
mesh_terms: D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects*; D045647:DNA Glycosylases / Q000235:genetics*; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D043382:DNA-Formamidopyrimidine Glycosylase / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008767:Methylmercury Compounds / Q000008:administration & dosage / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012307:Risk Factors; D015854:Up-Regulation
chemical_list: D008767:Methylmercury Compounds; D017382:Reactive Oxygen Species; D007770:L-Lactate Dehydrogenase; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; D043382:DNA-Formamidopyrimidine Glycosylase; C004925:methylmercuric chloride
keywords: 0.1% Tween-20 in 1X Tris-buffered saline; 1,3-N,N′-bis(2-chloroethyl)-N-nitrosourea; 2,6-diamino-4-hydroxy-5-formamidopyromidine; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester; 8-oxo-2′-deoxyguanosine; 8-oxodG; AP; AP endonuclease 1; APE1; ARP; BCNU; BER; CM-H(2)DCFDA; DCF; DNA ligase 3α; DNA polymerase β; DNA repair; FapyG; Formamidopyrimidine glycosylase; Fpg; HEK; HPLC-UV; LDH; LIG3α; MTT; MYH; MeHg; Methylmercury; MutY homolog; Ogg1; Ogg1(−/−); Ogg1-null; Oxoguanine glycosylase 1; POLβ; ROS; Reactive oxygen species; TBST; X-ray cross-complementing 1; XRCC1; aldehyde reactive probe; apurinic/apyrimidinic; base excision repair; dG; deoxyguanosine; dichlorodihydrofluorescein; formamidopyrimidine glycosylase; hOgg1; high-performance liquid chromatography with detection by ultraviolet absorbance; human embryonic kidney; human oxoguanine glycosylase 1; lactate dehydrogenase; methylmercury; oxoguanine glycosylase 1; reactive oxygen species