Study On Hepatotoxicity And Its Underlying Mechanisms Of Ethyl Carbamate

Ethyl carbamate(EC)is a toxin widely existing in fermented food and beverages.EC has been classified as Group 2A by the international agency for research on cancer(IARC),which means agents probably carcinogenic to human.Liver is regarded as one of the critical target organs of EC.Our lab previously found that EC could cause severe liver cell death.However,underlying mechanism of hepatotoxicity caused by EC is still obscure.Therefore,this thesis is aimed at investigating EC-induced hepatotoxicity and potential mechanisms by fluorescence labeling,gene transfection,western blotting,immunohistochemistry and other technologies.The main results were shown as follows:(1)EC caused oxidative damage in hepatocytes and autophagy process manipulated oxidative stress.60 mM and 80 mM EC decreased viability of hepatocytes to 76%and 57%respectively,accompanied by excessive production of ROS(118%and 170%)and GSH depletion(87%and 74%).The antioxidant NAC inhibited ECinduced oxidative damage,which indicated that oxidative damage was the main cause of EC-induced cell death.In addition,EC increased the level of autophagy marker LC3Ⅱ and the number of autolysosomes,the autophagy turnover assay using CQ further verified that EC promoted the process of autophagy.Furthermore,NAC pretreatment decreased the expression of LC3-Ⅱ protein and the number of autophagic lysosomes,indicating that EC promoted the process of autophagy by triggering oxidative stress.On the contrary,autophagy inhibitor CQ pretreatment exacerbated oxidative damage,indicating that autophagy alleviated the occurrence of oxidative stress under EC exposure.Neither EC exposure nor CQ intervention affected the number of mitochondria,indicating that EC failed to initiate mitophagy in order to recover redox homeostasis.Mechanistically,EC promoted translocation of transcription factor TFEB from cytoplasm to nucleus and increased activity of TFEB,indicating that TFEB plays a critical role in regulating autophagy and lysosome-related pathways under EC exposure.(2)Vinyl carbamate,a critical metabolite of ethyl carbamate,was synthesized by m-chloroperbenzoic acid(m-CPBA)oxidation.On this basis,the mechanism of VCinduced hepatotoxicity was investigated.2.5 mM VC decreased cell viability to 50%.However,VC did not affect ROS level,GSH content and autophagy process,which was completely different from those changes induced by EC.In addition,VC exposure induced lysosomal alkalization and lysosomal dysfunction.cAMP intervention promoted lysosomal re-acidification and increased cell viability under EC exposure.However,pretreatment of H-89 and CFTRinh172,inhibitors of cAMP downstream proteins PKA and CFTR,respectively blocked the protective effects of cAMP on lysosomal alkalization and cellular damage induced by ethyl carbamate,indicating that lysosomal alkalization contributed to VC-caused hepatotoxicity(3)Ferroptosis is a newly discovered ROS-dependent programmed cell death.Therefore,whether EC triggered ferroptosis in liver cells was detected.EC exposure increased intracellular iron content and levels of lipid peroxidation final product MDA.Besides,EC also decreased reduced GSH and expressions of GPx4 and Ferritin,which indicated that ferroptosis is a crucial form of EC-induced cell death.Ferrostain-1,a ferroptosis inhibitor,significantly reduced iron content and MDA level,as well as increased reduced GSH content and cell viability,compared with EC individually treated cells.Antioxidant NAC pretreatment also inhibited EC-induced ferroptosis,which was similar with ferrostain-1.EC effectively inhibited the expression of GCLC and SLC7A11,two key proteins involved in GSH biosynthesis.In addition,EC also reduced the activity of Nrf2,a critical negative regulator of ferroptosis,by inhibiting the phosphorylation modification of Nrf2 and the expression of Nrf2 in nucleus and cytoplasm,which further resulted in reduced levels of downstream targets HO-1 and NQO1.These results showed that the inhibition of Nrf2 activity and GSH synthesis was the main reason for EC-caused ferroptosis.(4)EC induced hepatic ferroptosis in BALB/c mice.According to the organization for economic cooperation and development(OECD)Test Guideline 407,a subacute(28 day)exposure model was established to analyze the in vivo toxicity caused by EC.Intragastric administration of 150 mg/kg and 450 mg/kg EC for 28 days resulted in decreases of body weight and food intake,accompanied by the reduction of liver index,abnormal liver structure and dysfunctions(the decrease of serum AST and ALT).EC also caused significant inflammation in liver by observing increased expressions of NLRP3 and IL-1β and TNFα.In addition,EC exposure caused liver oxidative stress by decreasing SOD enzyme activity and reduced GSH content,as well as increasing the contents of MDA and 4HNE,two final products of lipid peroxidation.EC also increased the level of autophagy marker protein LC3-Ⅱ and decreased the expression of p62 protein.Besides,EC enhanced number of autophagosomes.Meanwhile,EC upregulated the iron contents in serum and liver,and decreased the expression of GPx4 and Ferritin.Mitochondrial damage in liver was observed by detection of mitochondrial swelling,membrane damage and ridge disappearance.Furthermore,EC treatment decreased GSH synthesis-related proteins SLC7A11 and GCLC levels in liver and inhibited the expression of Nrf2 protein,suggesting that EC triggered ferroptosis by inhibiting GSH synthesis and Nrf2 protein in liver.In conclusion,this thesis elucidated that EC could cause oxidative damage and autophagy process manipulated oxidative stress in liver cells.Besides,VC,a critical metabolite of EC,induced hepatotoxicity by enhancing lysosomal alkalization,which was completely different from EC.Furthermore,EC inhibited glutathione synthesis pathway and inactivated ferroptosis negative regulator Nrf2,which ultimately induced ferroptosis in liver cells and mouse liver.These results revealed underlying mechanisms of EC induced liver toxicity,which provides a theoretical basis for possible food safety concerns caused by EC and new perspectives for future studies on regulation and prevention of EC-induced toxicity.