The Toxicity Of Cadmium And Nonylphenol In Hepatocytes Of Carassius Auratus And Assessment Of Combined Toxicity

Cadmium(Cd)and Nonylphenol(NP)are now recognized to be two of most important environmental contaminants. Due to their widely used in industrial , the amount of them and other relative toxicological chemicals such as nonylphenol ethoxylates(precursor substances of nonylphenol)are increasing rapidly in the environment in recent years. Since Cd and NP are released simultaneously into environment from abundant of natural and man-made sources,the adverse health effects caused by combination Cd and NP has resulted in a significant public health concern. The liver is the target organ and the primary accumulation site of chronic Cd of NP exposure.The nephrotoxicity induced by Cd and/or NP have been extensively studied and widely reported in environmentally and occupationally exposed human subjects,as well as in various experimental models.Most studies were implicated in exposure of Cd/NP singlely on the liver. However, systemic studies of toxic damage on the combination of Cd and NP were little referred.The first part:Isolating hepatocytes from hepatic tissue in Carassius auratus with trypsin and culturing hepatocytes with M199 medium and NBS. The primary hepatocytes of Carassius auratus were confirmed with morphologic observation and trypan blue dye. The survival hepatofcytes of intact and spheric-shape motionlessly attached to the bottom of the culture plates, and survival rate of hepatocytes was above 90%. The methods of isolation and culture of Carassius auratus hepatocytes were successful obtained. Besides, we did other three tests : sterilizing tissue with chlorhexidine gluconate , purifying hepatocytes with percoll and culturing cells with NBS or FBS. We found that the combination of chlorhexidine gluconate and NBS are good for hepatocytes.The second part: Cadmium chloride toxicity on the hepatocytes. We studied the performance of the free Ca²⁺ ,the Ca²⁺-ATPase and metallothionein in primary cultured hepatocytes of Carassius auratus after treated with Cadmium chloride. Hepatocytes were incubated with cadmium chloride at 5,10,15 and 20μmol/L for 24 h. Fluo-2/AM fluorescent labeling is used to show the Ca²⁺ level. The activity of Ca²⁺-ATPase was measured by spectrophotometry graphite , cadmium by atomic adsorption spectroscopy and metallothionein(MT) by ELISA. Fluorescence intensity of Ca²⁺ was the higher in the treatment groups than in control group; the activity of Ca²⁺-ATPase significantly increased (P<0.01) Ca²⁺-ATPase activity in treatment group were 6.73, 6.68, 7.19, 6.18 times higher than that in the control group respectively. MT expression level significantly increased in each treatment groups(P <0.01), and MT content of 5μmol / L group increased by 17.15 % (P<0.01). The results indicated that cadmium increased levels of Ca²⁺ and MT in hepatocytes of Carassius auratus, and MT chelated cadmium in hepatocytes, which may be one of the mechanisms that low the cadmium toxicological influence.The third part: Nonylphenol toxicity on the hepatocytes. We studied the performance of the free Ca²⁺ ,Ca²⁺-ATPase and metallothionein in primary cultured hepatocytes of Carassius auratus after treated with nonylphenol. Hepatocytes were incubated with nonylphenol at 10^-7,10^-6,10^-5,10^-4,10^-3mol/L for 24 h. The Ca²⁺ level, activity of Ca²⁺-ATPase , MT were measured by the same methods as in Cd. test. The results showed that the cell growth rate was inhibited by nonylphenol at all treatment groups, and the growth rate was significantly inhibited by NP at the high level of 10^-3mol/L compared with the control group. The result from the cell morphological observation by microscope showed that NP at the high level of 10^-3mol/L had a significant cytotoxicity on the hepatocytes, which caused a obvious morphological alteration. The trend of MT content were like a"U",MT expression level significantly increased in treatment groups of 10^-7,10^-5mol/L (P<0.05,P <0.01), but MT content of 10^-6mol/L group significantly decreased (P<0.01).MT level in other groups had no different with control group. Based on cell viability and MT level, we chosed 10^-6,10^-4μmol/L NP for further tests. Fluorescence intensity of Ca²⁺ was the higher and the activity of Ca²⁺-ATPase significantly increased(P<0.01)in the treatment groups than in control group. Ca²⁺-ATPase activity in treatment group were 6.2, 4.6 times higher than that in the control group respectively. The results indicated that cadmium increased levels of Ca²⁺ in hepatocytes of Carassius auratus, which may be one of the mechanisms that cause cellular injury of NP.The fourth part: The combination toxicity of cadmium and nonylphenol. Three methods were used to assess combined toxicity of Cd and NP, which were Toxic Unit, Additional Index, Mixture Toxic Index. The result showed that Cd and NP have synergistic effect, the order of toxicity was 20μmol/L Cd + 10^-4mol/L NP group>15μmol/L Cd +10^-4mol/L NP group>20μmol/L Cd +10^-6mol/L NP group>10μmol/L Cd +10^-4mol/L NP group > 15μmol/L Cd +10^-6mol/L NP group > 5μmol/L Cd +10^-4mol/L NP group > 10μmol/L Cd +10^-6mol/L NP group > 5μmol/L Cd +10^-6mol/LNP group,The synergistic effect is strongest in 5μmol/L Cd +10^-6mol/L NP group and was lowest in 20μmol/L Cd+10^-4 mol/L NP group. The level of MT expression din not decrease when treated with CdCl₂ or 10^-4mol/L NP alone, but decreased when treated together. Treated with CdCl₂ and NP,we found that the activity of Ca²⁺-ATPase decreased than only CdCl₂。Based on our experiment,the content of MT and Ca²⁺-ATPase could use as biomarkers for CdCl₂ and NP.