Molecular Mechanism Of AFB₁-induced Nephrotoxicity And Neurotoxicity

Aflatoxin B₁,a well-known mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus,is a member of the aflatoxin family and a common contaminant in grain-based foods worldwide.Studies have shown that AFB₁has a strong genotoxicity,hepatotoxicity and immunotoxicity associated with various clinical symptoms in vivo,which have raised great concerns of its threatens on human and animal health.Long-term intake of food contaminated with AFB₁can induce carcinogenic,teratogenic and mutagenic effects.At present,studies on the toxicity of AFB₁mainly focus on the mechanism of DNA damage mutation and the induction of liver cancer.Although it has been reported that AFB₁can induce renal toxicity and neurotoxicity,but reports on the molecular mechanism of AFB₁-induced nephrotoxicity and neurotoxicity are still lacking.In order to investigate the mechanism of AFB₁-induced renal injury in vivo,the KM mice were intragastrically administered with 300μg/kg/day of AFB₁for 30 days.Notably,the kidney weight and body weight of KM mice were significantly reduced,as well as the body to brain weight ratio and the kidney weight to brain weight ratio,compared with the control group.To explore the mechanism of AFB₁-induced renal retardation,the residual amount of AFB₁and its metabolites in the kidney tissue of mice were detected using a high performance liquid chromatography.The results demonstrated that the AFBO peak in the kidney was significantly higher than AFB₁in the treatment group.Futhermore,the pathological tissue sections showed that the integrity of the kidney collecting duct of the mice was destroyed upon intragastric administration.The infiltration of red blood cells and inflammatory cells in the interstitial space indicated that AFB₁and its metabolite AFBO remained and induced pathological damage in the kidney of mice.To further investigate the pathological mechanism of AFB₁-induced renal injury,the human embryonic kidney cell line HEK293T was treated with different concentrations of AFB₁.The results of MTT assay showed that the IC₅₀of AFB₁treated HEK293T cells were 7.14μg/m L and 6.83μg/m L within 24 h and 48 h,respectively.Meanwhile,with the increase of AFB₁concentration and time treatment,remarkable decreased cell membrane integrity were observed by LDH release experiment,indicating that AFB₁causes severe renal cytotoxicity.Subsequencetly,we observed that the size of the nucleus were increased significantly after AFB₁treatment.Flow cytometry showed that AFB₁induced S-phase arrest in a dose-dependent manner.We futher investigated the uncovered mechanism of S-phase arrest induced by AFB₁in vitro and vivo.The result showed that AFB₁up-regulates p21 m RNA levels and protein expression,besides,the negative regulatory factors of p21（PLK1,MYC and PLD1）were significantly down-regulated within the indicated times upon AFB₁treatment.Of note,we identified that the interaction between PLK1 and MYC was decreased after AFB₁treatment which further enhances the stability of MYC protein.However,overexpression of PLK1,MYC and PLD1 significantly inhibited the induction of p21 by AFB₁and partially alleviated the S phase arrest caused by AFB₁.The results of this study indicated that AFB₁induces the up-regulation of p21 by inhibiting the expression of PLK1,PLD1 and MYC,and inhibiting the interaction of PLK1 and MYC,thereby significantly inducing renal damage and leading to S phase arrest.Using the established AFB₁-mediated cytotoxic model in vivo,we furtherinvestigated the nerve injury induced by AFB₁and its toxicology mechanism.We detected that AFB₁induced acute brain damage in mice,while AFB₁residues were observed in mice brain tissue.In order to analyze the mechanism of AFB₁-induced brain injury,cell membrane integrity,intracellular ROS levels,DNA damage,cell cycle arrest and apoptosis were detected using neuroblastoma cell line IMR-32 upon treatment with different concentrations of AFB₁.The results showed that the proliferation rate of cells decreased significantly with the increase of AFB₁concentration and time treatment.The IC₅₀values??of IMR-32 cells treated with AFB₁were 6.18μg/m L and 5.87μg/m L within 24 h and48 h,respectively.The damage of AFB₁to cell membrane integrity was also positively correlated with the time and concentration of AFB₁ treatment.Fluorescence microscopy and flow cytometry showed that ROS levels were significantly up-regulated in IMR-32cells after AFB₁treatment,and the m RNA levels of three antioxidant stress-related genes OXR1,SOD1 and SOD2 were notably down-regulated.Comet electrophoresis assay andγH2A staining showed that AFB₁induces significantly DNA damage in IMR-32,and the results of RT-PCR showed that the three DNA damage response genes PARP1,BRCA2 and RAD51 were significantly down-regulated after AFB₁treatment.Flow cytometry and RT-PCR showed that AFB₁induced S phase arrest of IMR-32 cells by promoting transcription of CDKN1A,CDKN1C and CDKN1D.Meanwhile,cell apoptosis is significantly enhanced in IMR-32 by promoting Caspase 3 transcription and activation.In this study,the damage effects of AFB₁and its metabolites in kidney and brain tissues were detected in vivo.The molecular mechanism of AFB₁inhibiting renal cell proliferation,inducing kidney damage and affecting kidney weight were further explored at the cellular level.The regulation pattern of AFB₁-induced S phase arrest by up-regulation of p21 in HEK293T cells was analyzed.On the other hand,this study examined AFB₁induces cellular damage effects such as ROS accumulation,DNA damage,S phase arrest and apoptosis in neuronal cells at the cellular level.The mechanism of AFB₁induces nerve cell damage was investigated at the transcriptional level.This study provides useful theoretical references for further understanding of nephrotoxicity and neurotoxicology of AFB₁,and prevention of renal and neurological damage of AFB₁.