The aim of the first part of this project is to determine if manganese [Mn(II)] can protect HeLa cells against oxidative stress caused by hydrogen peroxide. Indeed, when HeLa cells were treated with different concentrations of hydrogen peroxide for 24h, we observed an increase in lactic dehydrogenase (LDH) release, protease activity including caspase-3 like activity, a decrease in intracellular thiol level, and a dramatic diminution of poly(ADP ribose) polymerase, an enzyme involved in DNA repair. However, all of these changes were prevented if 100 micromolar Mn(II) was present during the hydrogen peroxide treatment. The amount of poly(ADP ribose) polymerase, determined by Western blot, increased more in cells exposed to both Mn(II) and hydrogen peroxide than in untreated cells or cells treated with hydrogen peroxide alone. The protective effect of Mn(II) against hydrogen peroxide toxicity can be explained by its ability to completely remove hydrogen peroxide (1 mM) within 5 minutes in contrast to 20 minutes in the absence of Mn(II). This result is in line with other results previously published by our laboratory showing the catalase activity of Mn(II). Despite its protective effect against hydrogen peroxide toxicity at low concentration, Mn(II) at high concentration produces toxic effects. In the second part of the project, we demonstrated that manganese chloride (1-2 mM) is able to induce classical markers of apoptotic death in HeLa cells, including cell morphology, caspase activation, cleavage of poly(ADP ribose) polymerase (a known endogenous substrate for caspase-3 protease), and DNA condensation. No significant differences in LDH release, as an indicator for cell necrosis, between control and Mn(II)- treated cells were observed, suggesting that the integrity of the plasma membrane may be preserved. In addition, a remarkable elevation of the intracellular level of reactive oxygen species was observed in Mn(II)-treated cells, monitored by staining cells with two different oxidation-sensitive fluorescent probes, 2,7-dichlorofluorescein diacetate and 123 dihydrorhodamine. Production of reactive oxygen species is accompanied both by an increase in high molecular weight glutathionylated protein as assessed by Western blot analysis and HNE- modified proteins as judged by immunostaining of cells. How reactive oxygen species were produced, their nature, and whether they act down or upstream of caspase is still unknown. The involvement of mitochondria in apoptosis caused by Mn(II) is more probable since their shape and probably their number are affected in Mn(II)-treated cells compared to control cells, as determined by cytochrome c immunostaining and mitochondria-specific fluorescent probes. - Apoptosis, HeLa cells, Manganese(II)
