| Objective Polychlorinated biphenyls(PCBs)are released into the environment from a wide range of sources.They do not degrade easily and therefore persist in the environment.The PCBs exhibit a variety of properties including biological toxicity,high fat solubility,accumulation in sediments of water bodies,and bioaccumulation in the food chain.In the ocean,it poses a threat to aquatic plants and organisms including fish,shrimp,shellfish,and other aquatic species.The food resources in the ocean are abundant,and aquatic species including fish,shellfish are important sources of raw material for fishmeal.If animals ingest feed containing PCBs,it may accumulate and cause harm to aquaculture species,farm animals,and humans.The immune system is the most important line of defense to prevent disease and thereby maintain health.Damage to the immune system can result in a variety of diseases and impair human and animal health.In this study,PCBs pollution of the Zhanjiang mangrove sediments was investigated,and zebrafish was used as a model organism to examine the effects of the PCBs extracted from the Zhanjiang mangrove sediments on the immune function of zebrafish.Materials and methods The sediments were collected from 3 mangrove forest points in Zhanjiang and subjected to ultrasonic-assisted extraction using sulfuric acid and potassium permanganate in combination with the Florisil column purification method,and the types and concentration of PCBs were measured using a gas chromatography electron capture detector(GC-ECD).Zebrafish were randomly assigned to respective treatment groups and were placed into the aerated,oxygenated water tank(10 L)containing 40 zebrafish/tank.The zebrafish were distributed into 5 groups with 4 replicates containing 40 zebrafish/replicate,as were control group(CG),positive control group(PC: Aroclor1254;10 μg/l),low dose group(LD;0.6 μg/l),medium-dose group(MD;3.0 μg/l)and high dose group(HD;15 μg/l).The formal experiment was started after 30 days of adaptive feeding.The PCBs exposure conducted for an experimental period of 14 days,and fish were sacrificed on the day 1,7,and 14.The immune organs(intestine,liver)were collected on day 1,7,and 14 of the experiment.The intestine and liver indices were calculated by measuring the weight of immune organs on weighing balance.The histopathological changes of adult zebrafish immune organs were investigated by HE staining.The effects of PCBs on adult zebrafish immune cells(red blood cell,white blood cells,and neutrophils count)was analyzed on day 1,7,and 14 of the experiment.The gene expression of zebrafish intestinal and liver cytokines,TNF-α(tumor necrosis factor α),IL-1β(interleukin-1β),and chemokine IL-8(interleukin-8)m RNA following exposure to different doses(0,0.6,3.0,15 μg/l)of PCBs extract was analyzed by q RT-PCR.Results After pre-treatment of 10 kinds of PCBs,the recovery rate was between 67.4%-90.6%,the relative standard deviation(RSD)was between 24.15%-78.72% and the detection limit of each monomer of PCBs was between 0.029-0.068 ng/g.In this experiment,the concentration of PCBs in the sediments at three points of Guangdong Zhanjiang Mangrove National Nature Reserve(MNNR),Gaoqiao Mangrove Reserve(GMR),and Leizhou Peninsula(LP)was investigated.The results of the present study showed that PCB153 was detected in the sediments of MNNR and GMR while PCB101,PCB112,PCB155,and PCB198 were detected in the sediments of LP.The total amounts of PCBs in MNNR,GMR,and LP sediments were 1.29 ng/g,1.19 ng/g,and 16.49 ng/g respectively.As compared to the fish in CG,on day 1 of PCBs exposure,the body weight decreased significantly(P<0.05)in the PC and HD groups,while the body weight of the fish in the MD group decreased significantly on day 7(P<0.05)as compared to the CG.In addition,the body weight of the LD group decreased significantly on day 14(P<0.05)than CG.The intestine index of the zebrafish in the PC and the HD groups was significantly lower(P<0.05)on day 1,and was highly significant on day 14(P<0.01)as compared to the CG.In the LD and MD groups,a significant decrease(P<0.05)was observed on day 14 than CG.The change in the liver index was reverse.After the PCBs exposure to day 1,the intestinal villi were slightly damaged,and the shedding of enterocytes(intestinal cells)was observed in the HD group.After PCBs exposure to day 7,the intestinal villi showed serious damage,cup-shaped cells,and columnar-absorbent epithelium necrosis and shedding of enterocytes in the HD and PC groups,while in the MD group the intestinal villi were slightly damage.On day 14 of PCBs exposure,the intestinal villi in the HD group were not obvious.The damage was serious,the epithelial lining disappeared with mucosa layer cells necrosis and shedding of enterocytes.The gap between the intestinal villi in the HD group was not obvious.The MD and PC groups had similar lesions,while in the LD group only slight damage to intestinal tissue was observed on day 14.In CG the liver tissue presented with a normal lobular structure with normal hepatocytes and hepatic cords.In the PCBs-treated groups,there was confirmation of hepatic cord disorganization,swollen hepatocytes with condensed levels of cytoplasm,moderate infiltration of chronic inflammatory cells mainly lymphocytes,dilatation of sinusoidal capillaries,dilatation of central vein,and necrosis of hepatic lobule.It was found that the effect of PCBs on immune cells and gene expression of inflammatory cytokines in zebrafish was as follow: As compared to the CG,the number of red blood cell in the PC and HD groups decreased significantly(P<0.05)on day 1 while in LD and MD groups the number of red blood cell significantly decreased(P<0.05)on day 7 when compared with CG.The white blood cell number significantly decreased(P<0.05)in the PC and HD groups on day 7,and highly decreased on day 14(P<0.01)as compared to the CG,while in LD and MD groups the number of white blood cell significantly decreased(P<0.05)on day 14 than CG.The neutrophils count in zebrafish increased significantly(P<0.05)on day 1 and was highly significant from day 7 onward in all treatment groups when compared with CG.The zebrafish intestinal cytokines(IL-1β,IL-8,and TNF-α)showed a significant(P<0.05)‘dose and time dependent’ relationship.On day 1 of PCBs exposure,there was no significant change in IL-1β m RNA expression except for a significant increase in the PC and HD groups(P<0.05)as compared to the CG,while in the LD and MD groups the significant change in IL-1β m RNA expression was observed on day 7(P<0.05)than CG.On day 1 of PCB exposure,in the HD and PC groups,the IL-8 m RNA expression increased significantly(P<0.05)than CG.On day 14,all the treatment groups have a significant difference as compared to the CG(P<0.01).On day 1,TNF-α m RNA expression significantly increased(P<0.05)in the PC and HD while in the LD and MD groups the same increase was observed on day 7 as compared to the CG.Similar results were obtained for relative expression of IL-1β,IL-8,and TNF-α m RNA in zebrafish liver tissue.Conclusion In conclusion,Zhanjiang mangrove forest sediments were contaminated by PCBs and could be classified as mild pollution.PCBs exposure can significantly reduce the body weight and intestinal index of zebrafish,while the results of the liver index were reverse.PCBs extracts of sediments induced zebrafish intestinal lesions such as necrosis and loss of epithelial cells.The liver tissue also showed histopathological changes like hepatic cord disorganization and swollen hepatocytes with condensed levels of cytoplasm.PCBs extract from sediments significantly decreased the red and white blood cells but neutrophils count increased significantly after exposure to PCBs.The relative expression levels of IL-1β,IL-8 and TNF-α m RNA in zebrafish intestinal and liver tissue increased significantly in a dose and time responsive manner which is revealing the induction of inflammatory response... |
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