Mass Spectrometry-based Metabolomics For The Investigation Of Climbazole-induced Reproductive Toxicity On Adult Zebrafish

As the main active ingredient for anti-dandruff,climbazole is commonly found in personal care products such as shampoos.After use,climbazole will directly or indirectly enter the receiving environment via domestic sewage.Climbazole was found at concentrations ranging from tens of ng/L to hundreds of ng/L levels in the aquatic environment.It has been reported that the residual azole fungicides may disrupt the fish endocrine system and cause potential reproductive toxicity.Climbazole is a typical azole fungicide;however,there is a lack of information on climbazole-induced reproductive toxicity on aquatic organisms.This study aims to select zebrafish(Danio rerio)as the target organism,to investigate the effects of climbazole(0.1,10,and 1000?g/L)on the reproductive system of zebrafish after 28 days'exposure by behavioral and morphological approaches,to screen the significantly changed endogenous metabolites and signaling pathways by mass spectrometry-based non-targeted metabolomics method,and to deeply explore the mechanism of climbazole-induced reproductive abnormality on adult zebrafish by combined biochemical methods.It should be noted that the low exposure concentration of 0.1?g/L is basically consistent with the real environmental level of climbazole.The main results of this study are shown as follows:(1)Based on solid-phase extraction combined with gas chromatography-mass spectrometry,the average concentrations of climbazole were 0.10,9.33,and 917?g/L in the exposure solution,suggesting the stable climbazole levels during the exposure period.(2)Based on the behavioral approach,chronic exposure to climbazole will inhibit adult zebrafish's egg production.Increasing the exposure concentrations of climbazole will lower the egg production of zebrafish and shorten the time that occurred significant difference in cumulative mean eggs between the exposure and control groups.Accordingly,climbazole showed a concentration-dependent effect on zebrafish fecundity impairment.Based on the morphological approach,climbazole was found to inhibit oocyte maturation and reduce the spermatogenic ability in the zebrafish ovary and testis by hematoxylin-eosin staining.(3)Based on the mass spectrometry-based non-targeted metabolomics method,the metabolome change was investigated in zebrafish gonadal tissues,including ovary and testis.The results showed that 26 significantly changed metabolites between the exposure and control groups were statistically selected and structurally identified.The levels of retinoic acid(p<0.05;FC=0.29-0.68),eicosapentaenoic acid(p<0.05;FC=0.27-0.67),and lipoxin A₄(p<0.05;FC=0.21-0.55)were downregulated,and the flavin adenine dinucleotide(p<0.05;FC=1.17-1.92)and the ratio of oxidized glutathione to reduced glutathione were upregulated in the zebrafish ovary.In addition,the metabonomics results were verified by matrix-assisted laser desorption/ionization mass spectrometry imaging,which was used to identify and visualize the significantly changed endogenous metabolites in the sagittal tissue section of zebrafish ovary.These findings suggested that exposure to climbazole caused significant disturbance in signaling pathways,including retinoic acid metabolism,riboflavin metabolism,inflammatory response,and glutathione metabolism.For the zebrafish testis,18 significantly changed metabolites between the exposure and control groups were statistically selected and structurally identified.The level of hypoxanthine was upregulated(FC=1.10-1.29),and that of reduced glutathione was downregulated(p<0.05;FC=0.69-0.85),indicating that climbazole could increase endogenous reactive oxygen species by upregulating purine metabolism in testes,thereby destroying glutathione homeostasis.(4)Based on the biochemical approach,including enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase d UTP nick end labeling,the concentrations of testosterone,11-ketotestosterone,and estradiol were significantly reduced(p<0.05)in both zebrafish ovarian and testicular tissues in the exposure group,indicating that climbazole could affect the steroidogenesis.In addition,various degrees of cell apoptosis was found in the ovary and testis of zebrafish exposed to climbazole.Accordingly,climbazole could inhibit the development of mature germ cells in adult zebrafish,reduce egg production,and finally cause reproductive dysfunction.According to the obtained results,two possible mechanisms of climbazole-induced reproductive toxicity are proposed as follows.On the one hand,climbazole disrupts sex hormone production,leading to endocrine dysfunction in zebrafish.On the other hand,climbazole triggers oxidative stress and inflammatory response,resulting in cell apoptosis in gonadal tissues.In this study,the potential toxicological signaling pathways are identified according to the change in metabolite profiling.Combined with the biochemical method for verification,the mechanism of climbazole-induced reproductive toxicity on zebrafish is systematically clarified.The obtained results have significant implications for assessing the ecological risk posed by climbazole in the aquatic environment.