Developmental Toxicology Of Isothiazolinone Derivant To Embryonic Zebrafish

Isothiazolinone and its'derivatives are artificially synthesized antiseptic with biological activity.Depending on the conditions used and the objects protected,it has a great variation in the range of structure and properties.Compared to other preservatives,isothiazolinone have the advantages in well water solubility,broad antibacterial spectrum,efficient,and especially the low toxicity.Based on this,the usage of those derivatives is facing a rapidly growing,by the way they are using,threaten to human and other biology is noticed,especially the aquatic organisms.In this study,Methylisothiazolinone?MI?,Methylchloroisothiazolinone?CMI?,Kathon compound with the mix rate of CMI/MI=3/1,which mainly used in personal care products such as cosmetics.Similarly,the Benzisothiazolinone?BIT?is famous in latex paint,paint and others chemical products for algal and mold inhibition.Zebrafish?Danio rerio?embryo as a model in toxicology,it is widely used in the toxicity test and evaluation of various water pollutants.In this study,zebrafish embryos at 3 hours post-fertilization?3hpf?was employed in acute toxicity test and other experiments.Healthy embryos were selected then exposed in different concentrations of isothiazolinone under static condition with tempture of 28?up to 96 h,subsequently,the hatchability,mortality and malformation rate of embryos was recorded and analysed.Results showed that the 96 h medial lethal concentration(96 h-LC₅₀)of MI,CMI,kathon and BIT were 6.09,4.79,5.55 and 3.34 mg/L,respectively.Moreover,the 96 h teratogenic concentration(96h-TC₅₀)of MI,CMI,kathon and BIT were 3.87,2.89,3.27 and 1.38 mg/L,respectively.Meanwhile,the embryos showed severely deformity in all exposure groups,including body axis bending,pericardial cyst,yolk cyst,tail dysplasia,developmental retardation and weaken in mobility.The incubation rate of embryos at 48 h inhibited significantly in a dose-dependent manner,especially at MI groups.According to the manual on global chemical classification and labelling coordination?GHS?,it concluded that MI,CMI and Kathon have high toxicity to the zebrafish embryos,while the BIT is very high.For further mechanism study,the expression of several related genes were detected.Up to 96 h,it showed that activity of GST and content of MDA and ROS increased significantly in a dose-dependent manner at all exposure groups.On the contrary,the activity of SOD and CAT decreased.The mRNA levels of cat,gstp-2,cox-1 and ucp-2 presented to increasing in a different degree.However,in the BIT exposure groups,those genes expression showed a trend of increasing in low concentration,while significant decreasing in high concentrations.The expression levels of anti-apoptosis gene bcl-2 and pro-apoptosis gene bax showed a trend of promoting apoptosis.The expression of caspase-3 and caspase-9 increased significantly,indicating that endogenous apoptosis program was been activated.Apoptotic cells were mainly found in the heart and tail areas after the acridine orange?AO?stain analysis for 48 h and 96 h exposure embryos.Metabolomics analysis showed that isothiazolinone have affect in the metabolism of amino acids,which related to energy transmission.Significantly change of metabolites were found in MI,CMI and BIT exposure groups,as 49 up-regulated,and 49 down-regulated in MI groups,45 up-regulated and 62down-regulated in CMI groups,45 up-regulated and 73 down-regulated in BIT groups,respectively.Results showing that isothiazolinone unable to be metabolized in zebrafish embryos,and has severely impact on amino acid metabolism and energy metabolism,altered cell signal,cellular transport processes in zebrafish embryos,which may lead to malformation,and growth retardation,enventually individual death.We also found that isothiazolinone have effect on cell proliferation,and have liver toxicity to zebrafish.HEC-1B cell exposed to isothiazolinone for 72 h,cell cycle was altered,cell proliferation index?PI?was declined significantly compared to control.Same with positive control groups,paclitaxel inhibited cell proliferation significantly,the PI index only at a rate of 7.68,cells gathered in G₀/G₁ phase.According to PI,the cell proliferation inhibition effects:MI>CMI>CMI>BIT.Additionally,HE stain showed that isothiazolinone caused liver toxicity to zebrafish larvae,resulted in loose structure of liver tissue,cavitation and swelling of liver cells.