Studies On The Antioxidative Effect Of Alpha-Lipoic Acidon The Oxidative Damage Of L02Cell Induced By Cadmium Chloride

Cadmium is a kind of heavy metal that exisits extensively, it is easy to becomeenvironmental pollutants during the modern mining, smelting and other industrialactivities. Cadmium has been identified as ⅠA-level carcinogen-Namely humancarcinogens-by international cancer research center （IARC）in1994,and is listed as thesixth poisonous substance which endangers human health by ATSDR.。The acute toxic effect of cadmium is the oxidative damage to theorganisms.Cadmium is known as a toxin that is most easily accumulated within thebody and it often leads to oxidative stress, tumor, aging, and the reproductive systemdamage because its biological half-life period reaches10-30years, which is attributedto its strong cumulative effect and slow discharge rate. The DNA damage of cadmiumis related to the direct damage of DNA and the inhibition of DNA. The toxic effectsof cadmium chloride, the common inorganic compounds of cadmium, are to inducethe generation of ROS and to cause the process of lipid peroxidation. The latestresearch suggests that cadmium can induce various cells apoptosis, and there areobvious dose-effect relationship in specific cell lines. The chemical name of α-LA is1，2-dithiolane-3-pentanoic acid, the chemicalformula and molecular weight are C₈H₁₄O₂S₂and206.33, respectively. LA was firstlyextracted from the pig`s liver by REED in1951, since then researchers began to alarge study of it and gradually revealed the various antioxidant effect of LA on theremoval of ROS, chelating metal ions, regeneration of antioxidants, repairation ofoxidative damage and so on. The new theory ‘Antioxidant network’ reported byProfessor Packer suggests: the antioxidant effect of the body is mainly accompletedby five antioxidants Vitamin C, Vitamin E, CoQ<sub>10</sub>, GSH and LA. The fiveantioxidants are not separately play the role of protecting the body from oxidativedamage, but organically constitute into a complete and collaborative system ofantioxidant defense. LA becomes the core of ‘Antioxidant network’ because of itsfunction of regenerating other antioxidants.We take cadmium chloride as oxidative damage toxic, L02cells with relativelycomplete metabolic enzymes as target cells in this study. We begin with theinfluences of LA to L02cells`survival rate caused by cadmium chloride, set aboutfurther research from the influence to the levels of ROS, MDA of L02cells and theDNA damage repair, in order to preliminarily expounds the antioxidant effect of LA.PartⅠInfluences of LA to the survival rate of L02cells caused by cadmiumchlorideObjective: To investigate the antagonism effect of LA to the death of L02cellscaused by cadmium chloride.Methods: We use MTT experiment （colorimetric test） to determine the survivalrate of L02cells. We add6hours`pretreatment of LA in the process of L02cellscultivation. We also conduct real-time protection of LA during the exposure process of cadmium chloride, and observe the survival rate of L02cellsin each group after24hours.Results: Compared with the control group, cadmium chloride of25μmol/Lcould obviously lead to the deaths of L02cells（the survival rate of L02cells is55.81%, P＜0.05）; LA of10mg/ml could not lead to the deaths of L02cells（thesurvival rate of L02cells is103.61%, P＞0.05）; compared with separate functiongroup of cadmium chloride, LA of low concentration could not antagonize the deathof L02cells caused by cadmium chloride（the survival rate of L02cells is56.31%, P＞0.05）, LA of mediumand high concentrations could antagonize the death of L02cells caused by cadmium chloride（the survival rate of L02cells is73.82%,109.25%,P＜0.05）; compared with the control group, LA of high concentration couldcompletely antagonize the death of L02cells caused by cadmium chloride（thesurvival rate of L02cells is109.25%, P＞0.05）。Conclusions: LA could antagonize the death of L02cells caused by cadmiumchloride and rise the survival rates.PartⅡInfluences of LA to the ROS level of L02cells induced by cadmiumchlorideObjective: To investigate the scavenging effect of LA to the ROS level of L02cells induced by cadmium chloride.Methods: We take DCFH-DA as fluorescence probe, LA as a protective agent,L02cells as target cells to determine the ROS level in each group by DCFfluorescence intensity value. We add6hours`pretreatment of LA in the process ofL02cells cultivation. We also conduct real-time protection of LA during the exposureprocess of cadmium chloride, and observe the DCF fluorescence intensity value（ROSlevel）of L02cells in each group within2hours. Results: Compared with the control group, cadmium chloride of25μmol/Lcould obviously induce the generation of ROS of L02cells（the DCF fluorescenceintensity value is1652, P＜0.05）, LA of10mg/ml could not induce the generation ofROS of L02cells（the DCF fluorescence intensity value is1149, P＞0.05）; comparedwith separate function group of cadmium chloride, LA of low to high concentrationscould all reduce the ROS level induced by cadmium chloride（the DCF fluorescenceintensity value is1442,1162,822,P＜0.05）; Compared with the control group, LA ofmedium concentration could reduce the ROS level induced by cadmium chloride tothe level of normal cells（P＞0.05）, LA of high concentration could reduce the ROSlevel induced by cadmium chloride to a lower level than normal cells（P＜0.05）.Conclusions: LA could reduce the ROS level induced by cadmium chloride.PartⅢInfluences of LA to the oxidative damage product MDA level of L02cellsinduced by cadmium chlorideObjective: To investigate theantagonistic effect of LA to the oxidative damageof L02cells caused by cadmium chloride.Methods: We take cadmium chloride as oxidative damage toxic, LA as aprotective agent, L02cells as target cells. We add6hours`pretreatment of LA in theprocess of L02cells cultivation. We also conduct real-time protection of LA duringthe exposure process of cadmium chloride and continue to cultivate L02cells till24hours. Firstly we determine the total protein content in each group, and thendetermine the MDA content standardized respectively by the total protein content ineach group using TBA method.Results: Compared with the control group, cadmium chloride of25μmol/Lcould obviously cause oxidative damage of L02cells and rise the content of MDA （the content of MDA is14.05nmol/mgprot,P＜0.05）, LA of10mg/ml could not risethe content of MDA（the content of MDA is5.63nmol/mgprot,P＞0.05）; comparedwith separate function group of cadmium chloride, LA of low to high concentrationscould all reduce the content of MDA（the content of MDA is11.11,8.29,5.92nmol/mgprot, P＜0.05）; Compared with the control group, LA of high concentrationcould reduce the content of MDA to normal cells（P＞0.05）.Conclusions: LA could antagonize the oxidative damage of L02cells caused bycadmium chloride and reduce the content of MDA in cells.PartⅣInfluences of LA to the oxidative damage of DNA in L02cells induced bycadmium chlorideObjective: To investigate the antagonistic effect of LA to the oxidative damageof DNA in L02cells caused by cadmium chloride.Methods: We take cadmium chloride as oxidative damage toxic, LA as aprotective agent, L02cells as target cells. We add6hours`pretreatment of LA in theprocess of L02cells cultivation. We also conduct real-time protection of LA duringthe exposure process of cadmium chloride and continue to cultivate L02cells2hours.We use single cell gel electrophoresis （comet assay） to determine the Olive TailMoment Value of the nucleus under the fluorescence and then analyzed the degrees ofoxidative damage of DNA in each group.Results: Compared with the control group, cadmium chloride of25μmol/Lcould obviously cause oxidative damage of DNA in L02cells（the Olive Tail MomentValue is24.19,P＜0.05）, LA of10mg/ml could not cause oxidative damage of DNAon L02cells （the Olive Tail Moment Value is1.27, P＞0.05）;LA of low to highconcentrations could allprotect L02cells from oxidative damage of DNA caused bycadmium chloride （the Olive Tail Moment Value is18.09,10.62,5.17, P＜0.05）. Conclusions: LA could protect L02cells from oxidative damage of DNA causedby cadmium chloride.