Toxic Effects And Mechanism Of Perchlorate On Primary Cultured Hepatocytes Of Carassius Auratus

Due to the rapid mobility, stability, and nonreactivity in environment, perchlorate (ClO4- in aquatic systems can persist for years to decades. To determine the toxic effects of ClO4-, experiments in vivo and pathological studies were used frequently. Studies have reported that ClO4- in human body can result in improper regulation of metabolism for adults, and can cause developmental and behavioral problems for infants and children etc. In addition, treatment with high concentration of ClO4- significantly influenced the growth of preantral follicles and antral follicles of fish, as well as other organisms. However, based on the toxicity of ClO4- on living organism, there are relatively few studies on toxic effects of ClO4- in vitro, or has not been investigated in-depth. Moreover, little is known about the mechanism by which ClO4- act on cellular level or subcellular level. Due to the property of ClO4-, fish in contaminated water may be easily affected by ClO4-. The liver is known to be the major site of contaminants accumulation and metabolism in fish. Consequently, primary cultured hepatocytes of fish were used here to reveal the toxic effects of ClO4-. The main content and frame work is as follows:(1) Primary cultured cell lines, isolated from carassius auratus liver, were established, and which were manifested fit for toxicity study with ClO4-.(2) TAMⅢmulti-channel microcalorimeter was used to evaluated the short-term effects of ClO4- at different doses on mitochondria and primary cultured hepatocytes isolated from carassius auratus under the closed, static experimental environment. The results showed that the inhibition effect on hepatocytes associated with the increasing of ClO4-concentration. No significant toxic effect observed when mitochondria incubated with low concentration of ClO4-(0.001-0.01 mM). The metabolic activity of mitochondria was stimulated by 0.1-1 mM ClO4-, but inhibited by 10 mM ClO4-.(3) The morphology of hepatocytes after incubated with ClO4- were observed. It was found that in the presence of high concentration of ClO4-, cells started to shrink and became irregular in shape, displayed nuclear fragmentation, membrane blebbing, endoplasmicreticulum dilation, and mitochondrial swelling or vacuolization. In addition, flow of Ca2+ from mitochondria was also detected. When treated with ClO4-, the membrane permeabilization was slightly changed, as the release of LDH increased but no change about AST and ALT were observed. A better relationship of ClO4- and inhibition of Na+/K+-ATPase and Ca2+/Mg2+-ATPase were observed. The membrane fluidity was also decreased by 10 mM ClO4. In addition, the results possibly indicated that the oxidative damage on DNA is one high-dose intake groups, which was consistent with the damage of membrane.(4) The lipid peroxidation (MDA) content, ROS content, activities of CAT, POD, GSH-Px, SOD were determined after cells treated with various concentrations of C1O4- for 12h. The results showed that when exposure to ClO4-, the content of ROS and MDA increased, the activity of CAT and POD inhibited, and no changes observed with GSH-Px. Activity of SOD decreased at low concentration ClO4-, and increased at high.(5) Apoptosis was studied on hepatocytes when incubated with ClO4- for 12 h. It was found that in the presence of high concentration of ClO4, the percentage of apoptosis cell increased, and DNA fragmentation were also observed.(6) The inhibition of complexⅠandⅣactivity were also obseerved when motichondria incubated with ClO4-.(7) Analysis of RT-PCR and immuocytochemistry revealed that expression of Bax caspase-3、caspase-9、Cytc increased, and Bax/Bcl-2 also increased with the inceased of ClO4- concentration. No obvious change about the expression of AIF and Bcl-2. The results manifested that ClO4- induced apoptosis of primary cultured hepatocytes isolated from carassius auratus by path of mitochondrion.