Mechanism Of Immunotoxity And Regulation Of Immune Signaling Pathways On Oryzias Melastigma Under Benzo[a]pyrene Exposure

As a PAH-representative pollutant,Benzo[a]pyrene(BaP)is immunotoxic,carcinogenic and widely distributed in the ocean.In our previous study,BaP exposure,as well as lipopolysaccharide(LPS)challenge,could significantly modulate the expression of hepcidin-an antimicrobial peptide specific transcript in the red sea bream liver.Previous studies showed that the regulation of hepcidin by LPS challenge is associated with immune response,while the hepcidin regulated upon BaP exposure is lacking study.Thus,we chose a marine model fish Oryzias Tmelastigma to compare the effect of LPS challenge and environmental concentration BaP exposure by transcriptome.Whether the same signaling pathway participated in the modulation of hepcidin expression,the same immune associated genes modulating,and the same mechanism of immunoregulation by LPS challenge and upon BaP exposure were discussed.Similarity,based on the transcriptomic results that ROS associated genes were regulated upon BaP exposure for 3 days,we explored the relationship between the ROS generated by BaP exposure and its immunological effect.This study contributes to intensively understanding of the immune regulatory mechanism,the living conditions in complex environment of fish and the toxicological effects of pollutants.Moreover,it provides important theoretical value and guiding signifieance.The main studies are as follows:1.Referring to the regulation of antimicrobial peptide-hepcidin,transcriptomes of medaka by LPS challenge or upon BaP exposure were established.The transcriptome analysis showed that,456 unigenes were significantly modulated by LPS challenge,with 276 unigenes significantly up-regulated and 180 unigenes significantly down-regulated.In comparation,816 unigenes were significantly modulated upon BaP exposure for 2 days,with 228 unigenes significantly up-regulated and 588 unigenes significantly down,regulated.Besides,347 significantly modulated unigenes upon BaP exposure for 3 days,with 175 unigenes significantly up-regulated and 172 significantly down-regulated.2.The regulation of immune system was further analyzed based on the transcriptome by LPS challenge or upon BaP exposure.32,29,and 35 immune associated genes were significantly regulated by LPS challenge,BaP exposure for 2 days or 3 days respectively.Additional analysis revealed that hepcidin and SELE were the only two immune associated genes significantly regulated in three transcriptome groups,which indicated the signaling pathways participated in the immune associated genes regulation by LPS or upon BaP exposure may be difference.Interestingly,further transcriptomic analysis revealed that 11 immune associated pathways could be enriched by LPS challenge,while only one immune associated signaling pathway-the JAK/STAT pathway were significant enriched upon BaP exposure for 2 days.3.To further confirm this possible conclusion,the association between hepcidinl expression and the potentially involved JAK/STAT signaling pathway was evaluated on rmedaka using an inhibitor of JAK/STAT(NSC 74859)and on the cell line with a promoter plasmid of hepcidinl.As expected,the transcriptional expression of hepcidin1 was significantly down-regulated,according with the inhibition of JAK/STAT.In addition,the deletion of the STAT binding site in the hepcidinl promoter resulted in an inactivation of hepcidinl transcription.Additional experiments further supported this fact,as we observed that deletion of STAT-RE in the hepcidinl promoter diminished expression upon exposure to BaP in comparison with the control,suggesting that the transcriptional expression of hepcidinl was regulated or at least partially regulated by the JAK/STAT signaling pathway upon exposure to BaP.4.Interactive effects were found by LPS&BaP co-exposure.Based on that,RNA-seq was used to illustrate the gene expression pattern.Numerous immune pathways were significantly enriched according to four immune genes,including Gzmb,MhcI,C2 and C5.With the prolongation of treatment time,more metabolism pathways were significantly enriched.Ribosome and metabolism were significantly inhibited after co-exposure for 2 days which was different with single substance treatment or co-exposure for 12 h.5.A luminol-NaOCl system was built to detect H2O2 concentration in DIT-29 cell line medium which needs 20%FBS.Meanwhile,a H2O2 steady state generating system(H2O2ss)which could generate physiological concentration H2O2 for at least 24 hours was built by GOX and CAT in DIT-29 cell line medium.6.The interaction between BaP and NF-?B signaling pathway was revealed in vivo and in vitro.In this study,an in vivo examination of Oryzias melastigma demonstrated that BaP exposure led to a down-regulation of the NF-?B pathway and increased levels of ROS.Conversely,in vitro results using the medaka liver cell line DIT-29 and a widely applied H2O2 method showed the opposite:up-regulation of the NF-?B pathway.However,the down-regulation of NF-?B upon BaP exposure in vitro was inhibited by the addition of a ROS inhibitor,indicating ROS are involved in the modulation of NF-rcB.The discrepancy between in vivo and in vitro results of ROS impacts on NF-?B activation might be related to the concentration and persistence of ROS.Using the Iuminol-NaOC1 system,BaP was found to generate sustained physiological concentrations of ROS for 24 hours,while an H2O2 bolus generated ROS for less than 30 minutes.Furthermore,H2O2ss was used as a positive control of ROS.Comparative evaluation using H2O2,H2O2ss and BaP exposures on the medaka cell line with pGL4.32 demonstrated that the persistent physiological concentrations of ROS generated upon BaP exposure or treatnent with H2O2ss inhibited the NF-?B pathway,but direct H2O2 exposure had the opposite effect.Moreover,a western-blot assay and EMSA detection further confinned the modulation of the NF-?B pathway in DIT-29.