Effect Of N-acetylcysteine On Mitochondrial Damage Induced By Mercury Chloride In Kidney

IntroductionMercury (Hg) is a toxic element of widespread environmental and clinical significance, but many of inorganic Hg-based products are still in use today. Kidney is the main target of the toxic action of the inorganic form of the metal-Mercury Chloride (HgCl2). While the mechanism of HgCl2 toxicity is not fully known, it has been hypothesized that it is associated with mitochondrial dysfunction. Mitochondria are essential for the production of ATP, and have important functions in regulation of different types of cell death, generation of reactive oxygen species (ROS) and calcium homeostasis. Previous studies have shown that HgCl2 decreases the contents of sulfhydryl groups, enhances the production of ROS and disturbs antioxidant defense mechanisms in kidney. However, the mechanism underlying mitochondrial damage in response to exposure to HgCl2 is still unclear. N-acetylcysteine (NAC) is the most common specific antidote used in emergency. NAC administration was found to protect age related stress-induced mitochondrial dysfunction in a rat model as well as in a different stress-induced model of mitochondrial dysfunction in mice. In the present study, we evaluated the effect of HgCl2 on rat renal cortical mitochondria. We investigated the effect of HgCl2 on the activities of Ca2+-ATPase, succinate dehydrogenase (SDH) and phospholipase A2 (PLA2), the contents of malondialdehyde (MDA) and cytochrome c (Cyt c), mitochondrial membrane potential (MMP) and mitochondrial membrane fluidity. Studies were also made on the assessment of the preventive effect of NAC on HgCl2. MethodsIn vitro, the mitochondrial pellets of every untreated rat were divided into five shares, and were incubated in the assay buffer containing different concentrations of HgCl2 (0,5,50,500μmol/L) at 37℃for 1 h. In NAC pre-treatment group, the mitochondrial fractions were incubated with NAC (100μmol/L) for 20min then the effect of the NAC was studied at a HgCl2 concentration of 50μmol/L. For the acute experimental studies,24 Wistar rats weighing approximately 200 g were randomly divided into control group, HgCl2 group, NAC pretreated group by body weight. They were injected intraperitoneally with saline (control group and HgCl2 group) or with 4mmol/kg NAC (NAC pretreated group). After 2 h, the rats were injected subcutaneouly with saline (control group) or with 18.4μmol/kg HgCl2 (HgCl2 group and NAC pretreated group). All administration was given at the dose of 5ml/kg. The animals in all groups were decapitated 24 h after the last dose application. For the subacute experimental studies,24 Wistar rats weighing approximately 180g were randomly divided into control group, HgCl2 group, NAC pretreated group by body weight. They were injected intraperitoneally with saline (control group and HgCl2 group) or with 0.4mmol/kg NAC (NAC pretreated group). After 2 h, the rats are injected subcutaneouly with saline (control group) or with 1.84μmol/kg HgCl2 (HgCl2 group and NAC pretreated group). All administration was given at the dose of 5ml/kg for 14 d. The animals in all groups were decapitated 24 h after the last dose application. Mitochondria were prepared from Wistar rats'renal cortices using differential centrifugation. The activities of Ca2+-ATPase, PLA2 and SDH, the contents of MDA and Cyt c, MMP and mitochondrial membrane fluidity were investigated.ResultsIn vitro experimental part:As compared to control group, at 50,500μmol/L HgCl2 concentration, PLA2 activity was increased significantly (P<0.05), SDH activities were reduced significantly (P<0.05), the contents of MDA and Cyt c were increased significantly (P<0.05), MMP was reduced significantly (P<0.05); at 5,50 500μmol/L HgCl2 concentration, Ca2+-ATPase activity was reduced significantly (P<0.05). In addition, dose-dependent decrease in the activities of Ca2+-ATPase and SDH, increase in PLA2 activity, increase in the contents of MDA and Cyt c, decrease in MMP were observed. As compared to 50μmol/L HgCl2 group, PLA2 activity was decreased significantly (P<0.05), the contents of MDA and Cyt c were reduced significantly (P<0.05), MMP was increased significantly (P<0.05) in NAC pre-treatment group. Pre-treatment with NAC protected against HgCl2 mediating increase in PLA2 activity, increase in the contents of MDA and Cyt c, reduction in MMP in vitro.In acute experimental part:As compared to control group, in HgCl2 group, the activities of Ca2+-ATPase and SDH were reduced significantly (P<0.05), PLA2 activity was increased significantly (P<0.01), the contents of MDA and Cyt c were increased significantly (P<0.05), MMP was reduced significantly (P<0.01), mitochondrial membrane fluidity was reduced significantly (P<0.05). As compared to HgCl2 exposed group, PLA2 activity was decreased significantly (P<0.01), SDH activity was increased significantly (P<0.05), the contents of MDA and Cyt c were reduced significantly (P<0.05), MMP was increased significantly (P<0.05), mitochondrial membrane fluidity was increased significantly (P<0.01) in NAC pre-treatment group. In morphological appearance, HgCl2 induced an extensive swelling response of mitochondria and NAC-pretreated improved mitochondria to a certain extent.In subacute experimental part:As compared to control group, in HgCl2 group, the activities of Ca2+-ATPase and SDH were reduced significantly (P<0.01, P<0.05), PLA2 activity was increased significantly (P<0.01), the contents of MDA and Cyt c were increased significantly (P<0.01), MMP was reduced significantly (P<0.05). As compared to HgCl2 exposed group, the activities of Ca2+-ATPase and SDH were increased significantly (P<0.05), PLA2 activity was decreased significantly (P<0.01), the contents of MDA and Cyt c were reduced significantly (P<0.05), MMP was increased significantly (P<0.05) in NAC pre-treatment group. Pre-treatment of NAC protected against the HgCl2 mediating reduction in the activities of Ca2+-ATPase and SDH, increase in PLA2 activity, increase in the contents of MDA and Cyt c, and reduction in MMP in vivo.Conclusions1. HgCl2 could decrease the activities of Ca2+-ATPase and SDH, increase the contents of MDA and Cyt c, increase PLA2 activity, reduce MMP and increase mitochondrial membrane fluidity in renal mitochondria. Moreover, in vitro experimental part, HgCl2 could dose-dependently decrease in the activities of Ca2+-ATPase and SDH, increase in the contents of MDA and Cyt c, increase in PLA2 activity and decrease in MMP in renal mitochondria.2. NAC pre-treatment could reverse the toxic effect of HgCl2 to a certain extent, although it could not completely avoid mitochondrial damage induced by mercury chloride in kidney.