Effect And Mechanism Of Lycopene On Liver AFB₁ Metabolism And Mitochondrial Damage In AFB₁-exposed Mice

Aflatoxin B1(AFB1)is one of the most toxic and carcinogenic mycotoxins with the most widespread contamination,which has great harm to human health,animal and plant food safety as well as economic and trade development.AFB1 could metabolize in the liver and produce toxic products,causing liver injury,even cancer and death.However,the specific mechanism of AFB1-induced liver injury is still inconclusive,and there is no systematic study.Furthermore,there is no effective treatment for AFB1-induced liver injury.Lycopene(LYC)is an internationally recognized food-derived nutrient,which has the functions of anti-hepatocarcinoma,regulating detoxifcation enzymes,anti-oxidation and protecting mitochondria,and can promote the metabolism of AFB1 in the liver and make it become a non-toxic product.Therefore,it is feasible that LYC can be used to protect the liver from AFB1 toxicity.To investigate the toxic mechanism of AFB1-induced liver injury,80 male Kunming mice were randomly divided into four groups,which were given 0(control group,CG),0.375(low dose group,LG),0.75(medium dose group,MG)and 1.5(high dose group,HG)mg/kg AFB1 by intragastric administration,respectively.The model of subchronic AFB1 poisoning in mice was established for 30 days.Liver structure and function,AFB1 metabolism,mitochondrial structure,energy metabolism,mitochondrial biogenesis,as well as mitochondrial fusion and division were detected for verifying the effects of subchronic AFB1 exposure on AFB1 metabolism and mitochondrial damage of liver in mice,and screen the optimal toxic dose of AFB1 used in LYC intervention experiment.After that,another 80 male Kunming mice were randomly divided into four groups: control group(CG,0.2 m L olive oil),AFB1 treated group(AG,0.75 mg/kg AFB1),LYC intervention group(ALG,5 mg/kg LYC + 0.75 mg/kg AFB1),and LYC control group(LYC,5 mg/kg LYC).The model that LYC intervention of AFB1-induced mice liver injury was established by having LYC and/or AFB1 with intragastric administration for 30 d.Liver structure and function,AFB1 metabolism,mitochondrial structure,energy metabolism,mitochondrial biogenesis,as well as expressions of key factors and target genes in Nrf2 pathway we re detected for evaluating the effect and mechanism of LYC on AFB1 metabolism and mitochondrial damage in mice liver exposed to AFB1.(1)The results of AFB1 poisoning were as follows:1)The body weight,liver weight and liver coefficient in the MG and HG were significantly decreased compared with the CG(P<0.05,P<0.01),indicating subchronic AFB1 exposure inhibits the growth of mice.2)Under optical microscope,intact lobuli hepatis and neatly arranged hepatocytes were observed in the CG;relatively neatly arranged hepatocytes and a small amount of scattered red blood cells were observed in the LG;hepatocytes disarrangement,steatosis,sinus hepaticus congestion and bile duct hyperplasia were observed in the MG and HG.Under electron microscope,normal subcellular structure was observed in the CG;nuclear membrane invagination,nucleoplasmolysis and increased nuclear pores were observed in the LG;nuclear shrinkage,nucleoplasmolysis,mitochondrial swelling,decreased rough endoplasmic reticulum,increas ed lysosomes and lipid droplets were observed in the MG and HG,indicating subchronic AFB1 exposure damages the liver structure of mice.3)In the AFB1-treated group,AST,ALT,ALP and LDH activities were significantly increased as compared to the CG(P<0.05;P<0.01),indicating subchronic AFB1 exposure damages the liver function of mice.4)In the AFB1-treated group,the content of AFB1-DNA adduct was significantly increased compared with the CG(P<0.01).Furthermore,total CYP450 content and CYP3A11 m RNA expression in the LG were significantly raised,whereas total CYP450 content and the m RNA expressions of CYP1A2 and CYP3A11 in the MG and HG were significantly reduced as compared to the CG(P<0.01).Besides,in the AFB1-treated group,GST activity,GSH content and GSH/GSSG were significantly inhibited(P<0.01),whereas GSSG content was significantly augmented compared with the CG(P<0.05;P<0.01).These results suggest that subchronic AFB1 exposure disrupts I phase and II phase metabolism,resulting in the accumulation of AFB1 toxic product in the liver of mice.In the AFB1-treated group,ROS,H2O2 and MDA content were significantly raised,whereas T-AOC,SOD and CAT activities were significantly inhibited as compared to the CG(P<0.05;P<0.01),indicating subchronic AFB1 exposure induces oxidative stress in the liver of mice.5)In the AFB1-treated group,hepatocytes mitochondria swelling,membrane and cristae structure blurred were observed,and MMP was significantly decreased(P<0.05;P<0.01),cytoplasmic Cyt-c,Bax and p53 protein expression,Bax,p53 and Caspase-3/9 m RNAexpression,as well as hepatocytes apoptosis were significantly elevated(P<0.05;P<0.01),whereas Bcl-2 protein and m RNA expression was significantly reduced(P<0.01)as compared to the CG,indicating subchronic AFB1 exposure results in mitochondrial structural damage and activation of mitochondria-mediated apoptosis in the liver of mice.6)In the AFB1-treated group,PA content was significantly increased(P<0.01),whereas ICDHm,respiratory chain complexes I,II and IV activities were significantly inhibited(P<0.05;P<0.01)compared with the CG.Besides,in MG and HG,PDH,α-KGDH,SDH,respiratory chain complexes III and V activities,as well as ATP content were significantly restrained(P<0.05;P<0.01)as compared to the CG,indicating subchronic AFB1 exposure inhibits mitochondrial energy metabolism in the liver of mice.7)In the AFB1-treated group,PGC-1α protein expression,Nrf1 and Tfam protein and m RNA expressions were significantly decreased(P<0.05;P<0.01),whereas PGC-1α m RNA expression were significantly increased(P<0.05;P<0.01)compared with the CG,indicating subchronic AFB1 exposure inhibits mitochondrial biogenesis in the liver of mice.8)In the AFB1-treated group,Mfn1 and Fis1 protein expression were significantly reduced(P<0.05;P<0.01),whereas the protein and m RNA expressions of Opa1 and Drp1,as well the m RNA expression of Fis1 were significantly raised(P<0.05;P<0.01)as compared to the CG.In addition,Mfn1 m RNA expression in LG was significantly raised(P<0.05),whereas it was significantly reduced in MG and HG(P<0.01)compared with the CG,indicating subchronic AFB1 exposure disturbs the mitochondrial fusion and fission balance in the liver of mice.(2)The results of LYC intervention in AFB1 hepatotoxicity were as follows:1)LYC significantly relieved the decrease in body weight,liver weig ht and liver coefficient induced by AFB1(P<0.05;P<0.01),suggesting LYC alleviates AFB1-induced growth disorder in mice.2)LYC relieved the histopathological and ultrastructural changes of hepatocytes,and significantly mitigated the increase of liver function enzymes activities(P<0.01)induced by AFB1,suggesting LYC alleviates AFB1-induced structure and function injury of liver in mice.3)LYC significantly reduced the content of AFB1-DNA adduct(P<0.01),and significantly relieved the decrease in GST activity,GSH content and GSH/GSSG,as well as the increase in GSSG content induced by AFB1(P<0.01).However,LYC had no significant effect on the decrease of total CYP450 content and m RNA expression of CYP1A2 and CYP3A11 induced by AFB1(P>0.05),suggesting LYC reduces the accumulation of AFB1 toxic products in the liver by enhancing the II phase metabolism mediated by GST.LYC significantly relieved the increase of ROS,H2O2 and MDA content(P<0.01),and the decrease of T-AOC,SOD and CAT activities(P<0.05;P<0.01)induced by AFB1,suggesting LYC inhibits AFB1-induce doxidative stress in the liver of mice.LYC significantly relieved the decrease of Keap1 and Nrf2 expression(P<0.05;P<0.01),as well the decrease of target gene GSTA3,GSTM1,GSTP1,GSS,GCLC,GCLM,NQO1,SOD1,CAT m RNA expression(P<0.05;P<0.01)induced by AFB1,suggesting LYC promotes GST-mediated II phase metabolism and enhances anti-oxidation potential of liver by activating Nrf2 pathway.4)LYC relieved the ultrastructural changes of mitochondria in hepatocytes,and significantly mitigated the decrease of MMP and anti-apoptotic factors expressions(P<0.01),the increase of pro-apoptotic factors expressions(P<0.01),and hepatocytes apoptosis induced by AFB1,suggesting LYC alleviates AFB1-induced mitochondrial structure damage and apoptosis in liver of mice.5)LYC significantly relieved the increase of PA content(P<0.01),the inhibition of PDH,ICDHm,α-KGDH,SDH and respiratory chain complexes I-V activities(P<0.05;P<0.01),as well the reduction of ATP content(P<0.01)induced by AFB1,suggesting LYC alleviates AFB1-induced mitochondrial energy metabolism disorder in liver of mice.6)LYC significantly relieved the reduction of PGC-1α protein expression,Nrf1 and Tfam protein and m RNA expression(P<0.05;P<0.01)induced by AFB1,and significantly increased the m RNA expression of PGC-1α(P<0.01),suggesting LYC alleviates AFB1-induced mitochondrial biogenesis inhibition in liver of mice.In conclusion,subchronic AFB1 exposure could inhibit I phase and II phase metabolism,destroy mitochondrial structure,energy metabolism and biogenesis,and disrupt mitochondrial fusion and fission,finally causing liver injury in mice.Besides,LYC could alleviate AFB1-induced II phase metabolism inhibition and oxidative stress by activating Nrf2 pathway,and could relieve AFB1-induced mitochondria structural and functional disorder through promoting PGC-1α-regulated mitochondrial biogenesis.