Toxicological Effects Of Dydrogesterone In Zebrafish Based On Metabolomics

Progestins with a higher consumption than synthetic estrogens and androgens have attracted increasing attention in environmental research over the past two decades.Many progestins have shown adverse effects on sex differentiation and reproduction in fish,including oogenesis,spermatogenesis,egg production,and hormone levels.Dydrogesterone(DDG)is a synthetic progestin used in a wide range of gynecological conditions,including hormone replacement therapy and recurrent miscarriage.DDG is among the most consumed synthetic progestins in many countries,and it has been frequently detected in various aquatic environments.Recent studies have showed that environmental DDG could be an endocrine disruptor to fish,which can increase percentages of postovulatory and atretic follicles,impair egg production,promote spermatogenesis,cause a male-biased sex ratio and perturb circadian rhythm in zebrafish.Therefore,DDG might have a potential ecological risk to fish populations.Several studies have proven that DDG exhibits adverse effects in zebrafish,but most of them were based on histological and transcriptomic approaches.However,the data on the effects of DDG generated using some post-transcriptional approaches are very scarce.Metabolites are the end products of cellular physiological processes,and their changes can be regarded as the ultimate response of an organism to either genetic or environmental changes.Metabolomics,which can provide a comprehensive profile of the metabolites in a biological sample,has become a useful approach to pinpoint toxicological mechanisms.Thus it makes sense to study the metabolic changes and explore the pathways induced by DDG through metabolomics approach.The objective of this study is to understand how DDG affects zebrafish using metabolomic,histological and Fourier transform infrared spectroscopy(FTIR)analyses.We exposed zebrafish embryos to 0(C),2.8(L),27.6(M),and 289.8(H)ng/L of DDG.Part of the juvenile zebrafish were collected for metabolomics and the FTIR analysis at 35 days post fertilization(dpf).The rest of the fishes were exposed to DDG until sexual maturity(totally 140 days)for the evaluation of the metabolomic,histological,and FTIR alterations in fish gonads,brains and livers to explore the mechanisms of the effects of DDG on the interference of various physiological functions in zebrafish.The main findings are as follows:(1)DDG affected the physiological processes of zebrafish fry and increased the percentage of male zebrafish in a dose-dependent manner.FTIR results indicated that DDG resulted in lipid and protein accumulation in juvenile zebrafish.DDG increased certain free fatty acids(especially n-3 polyunsaturated fatty acids(PUFAs)),monoglycerides,acylcarnitines and organic acids,while decreased lysophospholipids,uric acid and bile acids in zebrafish fry during sex differentiation.DDG exposure also reduced nucleoside monophosphate and UDP-sugars,but increased nucleoside and its bases.These metabolites suggested inhibition of NF-?B/COX-2 and Wnt/?-catenin pathways,or activation of p53 pathway,which might induce oocytes apoptosis,trigger the female-to-male sex reversal and result in male-biased fish population.(2)DDG affected the physiological processes and reproductive function of zebrafish gonad.After 140 days of exposure,DDG promoted zebrafish oogenesis and spermatogenesis.DDG induced more postovulatory follicles and atretic follicles in the ovary of the female fish.The results of FTIR showed that DDG led to a decrease of lipid and an increase of DNA level in testis and protein accumulation in ovary.Ovarian metabolome showed that DDG increased the concentrations of free amino acids,urea,putrescine,free fatty acids,acylcarnitines,lysophospholipids,and other metabolites catabolized from proteins and lipids.DDG induced oocyte maturation,ovulation and oocytes over-ripening due to these metabolites.Arachidonic acid(AA)induced oocyte maturation,while its 5-LOX metabolite 5-oxo-6,8,11,-eicosatetraenoic acid(5-oxo-ETE)and purinergic signaling supported ovulation.However,DDG inhibited PGF2? production,which resulted in blockage of spawning,oocytes over-ripening,and post-ovulatory follicles and atretic follicles digestion failure.(3)DDG affected the physiological state and circadian function of zebrafish brain.The results of FTIR showed a decrease of nucleic acid and carbohydrate level and changes of the secondary structure of proteins in zebrafish brain from the exposure group.The exposed females also showed lipid accumulation.In both female and male zebrafish brain,the contents of cholesterol,saturated fatty acids,and nucleoside monophosphates were increased,while the levels of polyunsaturated fatty acids,lysophosphatides,and nucleosides were decreased by DDG.The changes in the levels of metabolites induced by DDG showed close correlations with circadian clock genes alterations induced by DDG in our previous study.Thus it was probably that the metabolite profile of DDG-treated zebrafish brain might be a reflection of circadian clock disturbances,and changes of some metabolites,such as saturated fatty acids,cholesterol,and n-3 PUFAs,might be a way for DDG to disrupt the normal circadian clock.(4)DDG affected the physiology of zebrafish liver.Histological examination showed that DDG induced morphological changes in the liver of female zebrafish but had no effect on the liver of male zebrafish.FTIR results showed that DDG exposure resulted in decreased lipid-and protein-related absorption and increased carbohydrate absorption in the liver of male fish,but the effect on the liver of female fish was different.Metabolomic results showed that the constitutive concentration of metabolites in zebrafish liver was different between sexes.Moreover,many metabolites in the liver of female fish were elevated by DDG,while most metabolites in the liver of male fish were decreased by DDG.These results indicated that the effect of DDG on zebrafish liver was significantly different between sexes.Genes related to metabolism in zebrafish liver inherently have sex differences,which may lead to differences in constitutive metabolites between males and females.The gender difference in metabolic response to DDG may be related to CYP enzymes,because CYP3A65 and other enzymes can metabolize both DDG and endogenous substances such as fatty acids.In conclusion,DDG exposure led to changes in zebrafish tissue histology,biological macromolecules and small molecular metabolites.DDG interfered with zebrafish physiological functions such as sex differentiation,reproduction and circadian rhythm mainly through fatty acids(including saturated fatty acids,n-6 PUFAs and n-3 PUFAs)and metabolites of purinergic pathway.This study helps to understand and deal with the hazards of progesterone substances to aquatic organisms.