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.

Ionisierende Strahlung verursacht in Abhängigkeit von den strahlenphysikalischen Eigenschaften der Radionuklide Zellschäden unterschiedlicher Komplexität. An humanen Lymphozyten wurde untersucht, ob die biologische Wirksamkeit von Alpha- und Betastrahlung sowie der Einfluss von Doxorubicin der Qualität des Strahlenschadens zugewiesen werden kann. Die DNA-Schäden und deren Reparatur wurden mit zellbiologischen Methoden quantifiziert.

L´apoptosi, o mort cel.lular programada, és un procés actiu que mobilitza els recursos cel.lulars amb l´objectiu de mantenir l´homeostasi de l´organisme a expenses del suïcidi de cèl.lules individuals. Diferents estudis han mostrat un increment de l´activiat d´algunes cdk, especialment Cdk1 i Cdk2, en correlació amb la progressió dels primers estadis apoptòtics. En el nostre laboratori l´estudi de l´apoptosi en timòcits, els quals no tenen una activitat cdk significativa degut a l´aturada del cicle cel.lular en G1, demostren que la inducció de l´activitat de Cdk2 després del tractament amb radiació gamma o amb glucocorticoides és necessària per l´inici de l´apoptosi. Mentre cap de les ciclines conegudes sembla ser la proteïna activadora de Cdk2 en apoptosi, en el nostre laboratori hem identificat un nou membre de la família de les ciclines, denominada Ciclina O, capaç d´activar aquesta kinasa in vivo en línies cel.lulars. L´expressió d´aquesta nova ciclina en el timus, i altres teixits, s´indueix ràpidament després del tractament amb radiació gamma i coincideix amb l´aparició de l´apoptosi. Aquests resultats posicionen la Ciclina O com a millor candidat a ser l´activador de Cdk2 necessari per induïr la mort cel.lular programada en el timus, i probablement també en altres òrgans. / The apoptosis, also called programmed cell death, is an active process able to use the cellular mechanisms to kill individual cells in order to keep the functional homeostasis of the whole organism. Different studies had shown a correlation between the first apoptotic events and the induction of some cdk proteins, particularly Cdk1 and Cdk2. The studies of thymocytes in our laboratory, wich lacks the most amount of cdk activity related to the cell cycle because of its arrest in G1, had shown that the induction of Cdk2 activity after the treatment with gamma radiation or glucocorticoids is a necessary step for the apoptosis induction. While any of the cyclins described at the moment seems to be the Cdk2 activator for apoptosis a new member of the cyclin family able to activate the kinase Cdk2 in vivo in cell lines has been identified in our laboratory. The expresion of this cyclin, known as Cyclin O, is quickly induced in the thymus after the treatment with gamma radiation and correlates with the induction of apoptosis. These results position Cyclin O as the best candidate to activate Cdk2 and inuce the programmed cell death in the thymus, and probably other tissues.