Cytoprotective Effects Of Selenium On Cadmium-induced Growth Inhibition And Apoptosis In Llc-pk 1 Cells

Cadmium is a widespread environmental and industrial pollutant, which is classified by IARC as Group I carcinogen to humans. It has been well established that chronic exposure to cadmium causes irreversible kidney damage and renal tubular dysfunction. During the last decade, a number of studies have shown that Cd induces apoptosis of the proximal tubular cells. At present, oxidative stress has been considered an important possible mechanism of cadmium toxicity. Nevertheless, the exact mechanism in Cd-induced apoptosis is still unclear.Selenium (Se), an essential trace element,is involved in functions of several enzymes and proteins such as glutathione peroxidases (GPx), selenoprotein P and thioredoxin reductases. GPx is an antioxidant enzyme which protects membrane lipids and macromolecules against the oxidative damage generated by peroxides. Most of the selenoproteins have ROS scavenging activities, so that the action of selenium has been known as an antioxidant system in cell survival. However, the underlying mechanisms involved in the cytoprotective effects of selenium against Cd-induced apoptosis remains to be elucidated. The present study was conducted to explore the protection mechanism of selenium on Cd-induced apoptosis in LLC-PK1 cells via ROS and mitochondria function. Objective: To determine the effect of selenium on cadmium-induced cell growth inhibition and apoptosis in LLC-PK1 cells.Methods: MTT assay was used to observe the effects on growth inhibition induced by cadmium in LLC-PK1 cells. Annexin V-FITC / PI was used to analyze apoptosis by flow cytometry.Results: Our study clearly revealed the protective effects of selenium were observed to inhibit cadmium-induced LLC-PK1 cells growth in a dose-dependent manner, and at 18 h, the total (early + late) apoptotic cells drastically enhanced from 2.05% (control) to 82.33% at 20μM Cd, selenium or NAC pretreatment before Cd exposure to the cells resulted in protection against Cd-induced apoptosis, as compared to that observed in the cells treated with Cd alone.Conclutions: Selenium plays a protective role on cadmium-induced cell growth inhibition and apoptosis in LLC-PK1 cells. Objective: The aim of our study was to explore the mechanism of selenium on Cd-induced apoptosis in LLC-PK1 cells via reactive oxygen species (ROS) and mitochondria linked signal pathway.Methods: We used an oxidant-sensitive fluorescent probe, DCFH-DA to examine the production of reactive oxygen species, the mitochondrial membrane potential was investigated with Rh123. Cytochrome c was determined using a quantitative immunoassay. Caspase-3 and -9 activities were measured through cleavage of a colorless substrate specific for caspase-3 (Ac-DEVD-pNA) or caspase-9 (Ac-LEHD-pNA) releasing the chromophore, p-nitroaniline (pNA). The levels of bax, bcl-2 proteins were detected by western blot.Results: (1) ROS was peaked within 12 h in the LLC-PK1 cells after Cd treatment, and selenium pretreatment before Cd exposure to the cells resulted in the recovery of ROS generation levels to basal levels. (2) A decrease inΔψm occurred as early as 3 h, and the loss inΔψm became significant at 6 h and 12 h . Pretreatment with selenium reversed the reduction ofΔψm resulted from Cd as indicated by a decrease in Rh-123 fluorescence. And cyt c in cytosolic markedly increased by Cd was restored by selenium or NAC treatment. (3) all the doses of selenium showed a significant decrease in caspase-9 activity as compared to Cd alone treated cells in a dose-dependent manner. (4) The level of bax protein increased and the Bcl-2 protein decreased in LLC-PK1 cells treated with 20μM Cd for 12 h, LLC-PK1 cells which were pretreated with selenium (5, 10, 20μM) showed down-regulation for the level of bax protein and up-regulation for Bcl-2 protein in a dose-dependent manner.Conclutions: Cd-induced apoptosis was mediated by oxidative stress and selenium produced a significant protection against Cd–induced apoptosis in LLC-PK1 via ameliorating the mitochondrial dysfunction.