Disturbing Effects Of Veterinary Antibiotic Florfenicol On Lipid Metabolism In Zebrafish (Danio Rerio) And Its Underlying Mechanisms

Florfenicol(FF),as a substitute for chloramphenicol,is widely used in the prevention and treatment of bacterial disease of farmed animals.FF can be released into recipient environments via urine and feces of farmed animals,and persist in the environment for a long period.FF was predicted as high as 10.76 μg/L in surface water,and was measured up to67.71 μg/L in urines of children.Thus,it poses a potential threat to human health.A positive correlation between FF exposure and the incidence of obesity has been revealed by epidemiological investigation.However,specific influences of FF on animal’s lipid metabolism and its underlying mechanisms still remain largely unknown.Hence,in this study,zebrafish(Danio rerio)were exposed to 1,10,or 100 μg/L of FF from 2 hours past fertilization(hpf)to 15 days past fertilization(dpf)(early-life exposure)or from 2 hpf to 120dpf(whole-life exposure).Then,body mass index(BMI),visceral fat accumulation levels,levels of important lipid macromolecules(including triacylglycerol(TAG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),and fatty acids(FFA))were measured to reveal the obesogenic effects of FF.Further,pathways through which FF disturbed lipid homeostasis were further explored by analyzing expression patterns of genes encoding key proteins regulating lipid metabolism.Finally,the underlying mechanism through which FF interfered with lipid metabolism was investigated,by analyzing influences of FF exposure on the structure and function of gut microbiome,and on the contents of intestinal short-chain fatty acids(SCFA)and its downstream signaling pathways.The results indicated that:(1)1,10,and 100 μg/L FF exposure significantly increased TAG contents and induced lipid accumulation in zebrafish.After early-life exposure to FF,larvae exhibited significant obesogenic effects at the individual,tissular,and molecular levels: BMI values of larvae were significantly increased;areas of abdominal fat stained by Oil red O and Nile red were significantly increased;TAG,TC,HDL-C,and LDL-C levels in the whole-body homogenate were significantly elevated and FFA levels were significantly decreased.After whole-life exposure to FF,for males,BMI values,TAG levels in liver,and TAG,TC levels in plasma were significantly increased,while HDL-C,LDL-C,and FFA levels in plasma were significantly decreased;for females,TAG levels in liver were significantly increased,while TAG、TC、HDL-C、LDL-C and FFA levels were significantly decreased.Early-life exposure to FF induced a more pronounced obesogenic effect in zebrafish larvae than adults exposed for whole-life.Following whole-life exposure,TAG and TC levels were also significantly changed in males and females in a gender-specific manner: for males,plasma and hepatic TAG contents and plasma TC contents were all significantly increased;for female fish,hepatic TAG contents were significantly elevated.(2)1,10,and 100 μg/L FF exposure significantly altered expression levels of genes encoding key proteins related to lipid synthesis,transport,storage,and decomposition in zebrafish.After early-life exposure to FF,real-time PCR(RT-PCR)results showed that FF significantly increased the m RNA levels of genes(dgat2)related to lipid synthesis,and significantly decreased the expression of genes(hsl,mgll,and acox1)related to lipid decomposition in larvae.After whole-life exposure to FF,in male,FF significantly increased the m RNA levels of genes(fitm2)related to lipid storage and significantly decreased the m RNA levels of genes(hsl,mgll,and acox1)related to lipid decomposition;in female,FF significantly up-regulated the expression of genes related to lipid synthesis(dgat2 and agpat4),storage(fitm2)and decomposition(hsl,mgll and acox1).In larvae,FF induced obesogenic effects mainly by enhancing TAG de novo synthesis in liver,inhibiting FFA oxidation,and promoting fat storage.In adults under a whole-life exposure,FF disturbed TAG metabolism in a gender-specific pattern: FF induced TAG accumulation mainly by promoting fat storage and inhibiting lipid decomposition in males,and by by increasing hepatic TAG de novo synthesis in females.(3)1 and 100 μg/L FF exposure altered the composition and function of gut microbiota and reduced the contents of intestinal SCFA in zebrafish.In order to obtain enough samples to further explore the acting mechanism underlying FFtoxicity,adult zebrafish were exposed to 1 and 100 μg/L FF for 28 d,and 16 s r RNA gene sequencing and targeted metabolomics were used to analyze the effects of FF exposure on the composition of the gut microbiota and intestinal SCFA contents in male and female fish.The results of 16 s r RNA gene sequencing showed that the diversity of gut microbiota was significantly reduced in the treatment groups,and the composition of microbiota in the gut changed significantly at both phylum and genus levels: the ratio of Bacteroidetes/Firmicutes related to obesity was significantly decreased,the abundance of Proteobacteria and Actinobacteria related to obesity was significantly increased,and the abundance of Bacteroidetes and Fusobacterium related to SCFA production was significantly decreased.The results of targeted metabolomics showed that levels of most SCFA(including acetic acid,propionic acid,butyric acid,isovaleric acid,valeric acid,and caproic acid)in the gut of male and female fish were significantly reduced.Since SCFA was reported to regulate lipid homeostasis by activating the AMPK signaling pathway,we speculated that the bactericidal substance FF may reduce the contents of SCFA by changing the structure and function of the gut microbiota,and then disrupt its downstream AMPK signaling pathway to interfere with lipid synthesis,decomposition and storage,leading to occurance of obesity.In conclusion,our data provided experimental evidences,at individual,tissular,and molecular levels,on the obesogenic effects of FF exposure on zebrafish larvae and adults.FF induced TAG accumulation by interfering with the lipid metabolism pathway,and disturbed the metabolism of other lipid macromolecules such as TC.The disturbing effects of FF on lipid metabolism may be related to the disruption of the structure and function of gut microbiota,which then resulting in changes of the contents of SCFA,an upstream regulator of lipid homeostasis.The results of this study may inform the evaluation of potential health risks of FF.