Study On Ovary Oxidative Injury And Apoptosis In Freshwater Crab Sinopotamon Henanense Induced By Lead

Lead (Pb2+) is a heavy metal considered as one of the most toxic environmental and industrial pollutants, which causes gonadotoxic effect in animals.To explore the effects of lead (Pb2+) on the ovary, the freshwater crabs, Sinopotamon henanense, were exposed to0,3.675,7.35,14.70,29.40, and58.80mg-L-1Pb2+for5,10and15d respectively. The activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and the contents of malondialdehyde (MDA) in ovarian tissues were investigated to study the effect of Pb2+on oxidative stress. In order to study Pb2+-induced apoptosis after oxidative damage, the content of H2O2, the capabilities of anti-O2" and inhibit-OH were measured, meanwhile, the changes in ovarian cell apoptosis were measured by the H.E. staining and agarose gel electrophoresis of DNA. In order to preliminarily explore the mechanisms of apoptosis in the ovary of the crab after Pb2+administration, the activities of apoptosis-associated enzymes were investigated including Caspase-3, Caspase-9, constitutive nitric oxide synthase(cNOS), inducible nitric oxide synthase(iNOS) and total nitric oxide synthase(T-NOS). The results were taken as follows:(1) In the ovary, Pb2+significantly impacted the activities of SOD, CAT and GPx and altered the contents of MDA in the ovary. MDA content was found to increase persistently during Pb2+exposure. Low concentration of Pb2+can significantly increase antioxidant enzymes; however, for crabs exposed to high concentrations of Pb2+, the activities of the three antioxidant enzymes were all inhibited with prolongation of exposure time. The significant buildup of MDA in ovarian tissues following Pb2+exposure is indicative of Pb2+-induced oxidative damages to crab ovary.(2) The content of H2O2persistently increased following the increase in concentration of lead and prolongation of exposure time; on the contrary, the capabilities of anti-O2and inhibit-OH declined compared to the control. The ovary cells showed typical morphological characteristic of apoptosis:the karyotin became pyenosis and assembled at the edge after exposure to lead by H.E. staining. The Pb2+-induced ovarian apoptotic DNA fragmentation("ladder" pattern) was clearly revealed on the agarose gel.(3) At day10, compared with control, cNOS, iNOS, T-NOS, Caspase-3and Caspase-9activity gradually increased with increasing concentration of Pb2+. Lead induced a large amount of NO in the ovary of freshwater crab, and led to ovary cell apoptosis, possibly through Caspase-9-dependent mitochondrial pathway.Conclusions:(1) The activities of antioxidant enzyme could be strengthened initially and suppressed with the increase of concentration of lead and extension of exposure time. MDA content was found to increase persistently, indicating that the antioxidant system in ovary was damaged and the crab sufferd oxidative damage. Exposure to Pb2+could induce apoptosis of ovary cells of freshwater crab, via the mitochondria-dependent apoptosis pathway initiated by the activation of Caspase-9and Caspase-3.(2) The biochemical parameter of SOD, CAT, GPx and MDA, which could reflect cellular toxicity caused by lead, can be used as a biomarker for heavy metal-induced oxidative damages to aquatic animals.