Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2′-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and 13C- and 15N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.