Study On The Toxicity And Combined Effects Of Cadmium And Pentachlorophenol On Primary Cultures Of Carassius Auratus Hepatocytes

With the rapid development of industrial and the improvement of people’s living standard, a large number of wastewater containing PBT pollutants discharged into water bodies directly or indirectly and water environment were polluted seriously. As the PBT pollutants have the "environmental sustainability" property, and it can be accumulated in the environment, accumulation of more than one type of PBT pollutants in the environment system becomes increasingly common. Thus, the study of the compound toxicological effects of multiple PBT pollutants in the environment system and its mechanism were paid more attention by the people. Although the cell toxicity and apoptosis induced by PBT pollutant were researched, there is no research about combined effects of PBT pollutants on cell toxicity is reported. And the mechanism of cell apoptosis induced by PBT pollutants remains unclear. Therefore, Carassius auratus hepatocytes were isolated by trypsin separation in this study, and the individual and combined effects of different concentration of Cd (0,0.01,0.1,1,10,100μmol/L) and PCP (0,0.01,0.1,1,10,100μmol/L) on Carassius auratus hepatocytes were researched.(1) The separation, training, identification of primary cultured Carassius auratus hepatocytes were discussed, and the influence of cell viability of the individual and combined effects of Cd and PCP were researched. Also, the morphological changes of primary cultured Carassius auratus hepatocytes were determined. It was showed that parenchymal cells of Carassius auratus can be separated by Trypsin digestion method, but it contains a lot of non-parenchymal cells. The purity of the Carassius auratus hepatocytes can be reached to more than92%by differential centrifugation for several times. With the increasing of Cd and PCP concentrations, cell viability was decreased, and it showed a dose-response relationship. Compared with the control, it showed no significant difference of low concentration of Cd or PCP treatment, and the high concentration of Cd or PCP treatment reached significant level. It exhibited interactions of combined group. Factorial analysis showed that it was Synergism between Cd and PCP. Apoptotic features of Carassius auratus hepatocytes can be detect by transmission electron microscopy, such as the apparent cytoplasmic concentration, nuclear chromatin condensation, nuclear membrane disappears, mitochondrial swelling and vacuolization, mitochondrial cristae broken or disappeared, apoptosis emergence.(2) The toxic effects of Cd and PCP along and combined with primary cultured Carassius auratus hepatocytes were studied by microcalorimetry. Results showed that, the metabolic thermogenic curve of primary cultured Carassius auratus hepatocytes was divided into stagnation phase, active recovery phase, stability phase and activity declining phase. The results showed that Cd and PCP bring out relevant changes of the value the rate constant (k) in the activity recovery phase, the maximum heat production (Pmax), total heat production (Q), generation time (tG). The rate constant and total heat production declines gradually while the concentration of Cd or PCP increase. But the generation time and the inhibition increased with the concentration of Cd or PCP increase. This indicated that the metabolic activity of Carassius auratus hepatocytes were inhibited by Cd or PCP, and the toxic effect of Cd was higher than PCP, the joint toxicity of them was synergism.(3) The influence of the individual and combined effects of Cd and PCP on the activities of CAT、POD、SOD and isozymes of primary cultured Carassius auratus hepatocytes were researched, and the content of MDA、GSH、GSSG were determined. CAT activity was activated at low concentrations of Cd and PCP, and with the concentration of Cd and PCP increase, CAT activity gradually decreased until inhibited. SOD and GST activity decreased, with the concentration of Cd and PCP increase. Low concentration of Cd can induce GSH-Px activity. When the concentration is more than1μmol/L, GSH-Px activity was inhibited, and the difference was significant. But GSH-Px activity changed little, when the concentration of PCP is lower than10μmol/L, high concentration of PCP significantly inhibit GSH-Px activity. GR activity decreased with the concentration of Cd increase. At low concentration of PCP, GR activity was activated. When the concentration of PCP is greater than1μmol/L, the activity of GR is decreased significantly. MDA content increased with the concentration of Cd and PCP increase. Whereas, GSH content decreased with the concentration of Cd and PCP increase, and it showed a good dose-response relationship. With the concentration of Cd increase, GSSG content was increased and then decreased gradually, when the concentration of Cd was100μmol/L, the difference reached significant level (P<0.05). The combined effects of Cd and PCP showed some interaction. GSSG content increased gradually with the concentration of PCP increase, and it showd a good dose-respones relationship. The joint toxicity of them was synergism.(4) The influence of the individual and combined effects of Cd and PCP on Na+,K+-ATPase、Ca2+,Mg2+-ATPase and the content of intracellular calcium of primary cultured Carassius auratus hepatocytes were studied. Under the effect of Cd, the activity of Na+,k+-ATPase decreased gradually. Under the effect of PCP, the activity of Na+,k+-ATPase was first increased and then decreased. It exhibited same trend of Cd and PCP on Ca2+,Mg2+-ATPase activity, that is to say, Ca2+,Mg2+-ATPase activity increased at low concentration of Cd and PCP, while the enzyme activity decreased at high concentration of Cd and PCP. The content of [Ca2+]i changed little under low concentrations of Cd and PCP, and it increased significantly under high concentration of Cd and PCP. The Na+,K+-ATPase and Ca2+,Mg2+-ATPase activity of Carassius auratus hepatocytes were all lower than the control, under the the combined effects of Cd and PCP. Factorial analysis showed that there is interaction between Cd and PCP, it showed synergistic effects. The content of [Ca2+]i of Cd and PCP combined groups were higher than the control. Also, the factorial analysis showed that there are synergistic effects between Cd and PCP.(5) The influence of the individual and combined effects of Cd and PCP on ΔΨm、 DNA Ladde and apoptosis rate of primary cultured Carassius auratus hepatocytes were observed. Also the molecular mechanism of apoptosis of primary cultured Carassius auratus hepatocytes induced by Cd and PCP were discussided. Mitochondrial membrane potential of hepatocytes decreased with the concentration of Cd and PCP increase, and the difference reached significant level. The change of apoptosis rate was not obvious under low concentration of Cd and PCP. When the concentration of Cd and PCP reached10μmol/L, apoptosis rate increased significantly. The change trend of DNA Ladder was consistent with apoptosis, it showed typical "ladder" at the high concentration of Cd and PCP. Mitochondrial membrane potential decreased under the combined effects of Cd and PCP. Factorial analysis showed that the interaction is not obvious at low concentration of Cd and PCP. When the concentration reached10μmol/L, interaction becomes clear, it showed synergistic effect. Apoptosis rate of the combined groups increased. Factorial analysis showed that the interaction is not obvious at low concentration of Cd and PCP. When the concentration reached10μmol/L, it showed synergistic effect.