Effect Of PCBs Extracted From Sediments In Zhanjiang Offshore On Histological Structure,Microflora And Cytokines Of Zebrafish Intestine

Polychlorinated biphenyls(PCBs)are released into the environment from a wide range of sources.They do not degrade easily and therefor 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.Thus PCBs accumulate in organisms including marine species and this can lead to contamination of seafood and fishmeal and thus cause harm to human and animal health.There are no studies on the use of histology,microbiology and immunology of zebrafish(Danio rerio)gut uesd as a biomarker to indirectly detect PCBs in the marine sediment.In this study,the Zhanjiang offshore sediments were collected,PCBs were extracted and purified and the following tests were performed:(1)Gas chromatograph-mass spectrometry(GC-MS)analysis of Zhanjiang seashore sediments to detect PCB contamination in the area.(2)Histopathology of gastrointestinal tract of zebrafish.This was uesd as a biomarker of exposure to PCBs.Zebrafish were exposed to 4 different doses(0,5,10,15?g/L)of PCB extracts and 1 dose(10 ?g/L)of PCB 81 and sacrificed at different time periods(1,3,5,7 d).(3)High-throughput sequencing of the V4 region of the 16 s rDNA of intestinal microorganisms in zebrafish exposed to the four PCB concentrations(0,5,10,15 ?g/L of PCB extracts)and sacrificed at different time periods(1,3,5,7 d).This was to explore the effects of PCBs in marine sediments on zebrafish intestinal microflora.(4)Expressionof zebrafish intestinal mucosal immune cytokines-TNF-?(tumor necrosis factor ?),IL-1?(interleukin-1?)and chemokine IL-8(interleukin-8)mRNA following exposure of zebrafish to 4 different doses(0,5,10,15 ?g/L)of PCB extracts and 1 dose(10 ?g/L)of PCB 81 and sacrificed at different time periods(1,3,5,7 d).This was to explore the effects of PCBs on intestinal mucosal immunity.The results were as follows:(1)The GC-MS analysis of PCBs extracted from Zhanjiang seashore sediments following ultrasonic extraction and silica gel column purification showed that PCBs had contaminated(27~35 ?g/kg)the sediments in the offshore waters of Zhanjiang.The PCBs concentration in the sediments near Binhu Park wasthe highest at 35 ?g/kg.(2)The intestinal epithelial cells of the zebrafish exposed to PCBs extracts of the sediment showed evidence of dose responsive degeneration,necrosis and cellular detachment.(3)High-throughput sequencing analysis intestinal microflora of zebrafish revealed that the dominant phyla of zebrafish were Proteobacteria,Fusobacteria,Firmicutes,and Bacteroidetes.On exposure of zebrafish to PCB extracts,the average abundance of Proteobacteria declinesd and Fusobacteria increased significantly(P<0.05)on day 5>7>3>1.Thus it appears that Fusobacteria intially incerase upto 5 d but with the prolonged exposure,it declines.No significant changes were evident in the abundance of Firmicutes(P>0.05)but the abundance of Bacteroidetes decreased at 3 d and then return to nomal.However,as the exposure concentration increased,Bacteroidesin the treatment groups was significantly lower(P<0.05)than in the control group.There was no significant change in allthe other bacteria tested.The predominant bacteria genus in the zebrafish gut are Cetobacterium.spp.,Aeromonas sp.,Vibrio spp.,and Gemmobacter spp..On exposure of zebrafish to PCB extracts,the abundance of Cetobacterium were increased on days 3 and 5 but declined on day 7.However,the abundance of Cetobacterium was still significantly(P<0.05)higher than day 1.The abundance of Aeromonas and Gemmobacter showed fluctuating changes.There was no significant changed in the abundance of Vibrio spp.at each sampling period(P>0.05).Thus,at the genus level,the exposure dose had no significant effect on the abundance of each genus type.This indicates that at the level of the phylum or genus,the abundance of dominant microflora are affected by the length of exposure rather than the dose exposed to.(4)The zebrafish cytokines(IL-1?,IL-8 and TNF-?)showed a significant(P<0.05)'dose-and time-response' relationship.The relative expression levels of IL-1?,IL-8 and TNF-? increased significantly with increasing concentrations and prolonged time.In summary,Zhanjiang coastal area sediments were contaminated by PCBs and could possibly be calssed as moderatly polluted.PCB extracts of sediments induced zebrafish intestinal lesions such as necrosis and loss of epithelial cells and such changes will affect nutrient digestion and absorption by the intestine.Zebrafish exposed to PCBs extracts from the sediments caused changes in the composition and abundance of the intestinal microflora.This study showed that changes in the dominant of the phyla and the genus of gastrointestinal microbes change significantly especially in zebrafish exposed to PCBs for a prolonged period of time,independent of the exposure concentration.PCBs in sediments significantly increased the relative expression levels of IL-1?,IL-8 and TNF-? mRNA in zebrafish,in a dose responsive manner,an indicative of induction of an intestinal inflammatory response.