Chronic Cr⁶⁺Exposure Induces Growth And Developmental Toxicity In Bufo Gargarizans Embryos And Tadpoles

Hexavalent chromium(Cr⁶⁺)is one of the major and most detrimental heavy metal pollutants.The Cr⁶⁺penetrates biological membranes easily and causes cellular damage by oxidative stress.Several anthropogenic activities such as electroplating,manufacturing industries and ferrochrome production are responsible for the pronounced increase in the concentration of Cr⁶⁺in aquatic environments,their accumulation and persistence constitutes a potential threat to aquatic organisms.B.gargarizans embryos and larvae have been proved to be ideal study organisms to assess negative consequences of aquatic contaminants,because their embryos and larvae show high-sensitivity to water pollution.In this study,the chronic toxic effects of Cr⁶⁺exposure on Bufo gargarizans embryos and larval were evaluated.The results of this study can provide valuable information about the toxicity and safe use of Cr⁶⁺that have the potential to enter aquatic systems.The main findings were as follows:1.To determine the potential developmental and metabolic abnormalities caused by Cr⁶⁺exposure on B.gargarizans embryos,embryos at Gosner stage 2?G2?were treated with different concentrations of Cr⁶⁺(0,13,52,104,208,and 416?g Cr⁶⁺L^-1)during embryogenesis.Morphological abnormalities,total length,weight and developmental stage were monitored.The transcript levels of several genes associated with lipid metabolism,oxidative stress,and thyroid hormones signaling pathways were also determined.Our results showed a time-dependent inhibitory effect of Cr⁶⁺on the growth and development of B.gargarizans embryos.On day 4,total length,weight,and developmental stage of embryos were significantly lower at416?g Cr⁶⁺L^-1 relative to control embryos.On day 6,significant reductions in total length,weight,and developmental stage were observed at 104,208,and 416?g Cr⁶⁺L^-1.Malformed embryos were found in all Cr⁶⁺treatments,which were characterized by axial flexures,yolk sac edema and rupture,surface tissue hyperplasia,stunted growth,wavy fin and fin flexure.RT-qPCR results showed that exposure to Cr⁶⁺down-regulated TR?and D2 mRNA expression and up-regulated D3 mRNA level at 416?g Cr⁶⁺L^-1.The transcript levels of SOD and GPx were upregulated at 52,208,and 416?g Cr⁶⁺L^-1,while the transcript level of HSP90 was downregulated at 52,208,and 416?g Cr⁶⁺L^-1.Also,mRNA expression of lipid synthesis-related genes?FAE and ACC?were significantly downregulated in embryos exposed to 208 and416?g Cr⁶⁺L^-1,but mRNA expression of fatty acid?-oxidation-related genes?ACOX,CPT,and SCP?was significantly upregulated at 416?g Cr⁶⁺L^-1.2.To examine the effects of chronic exposure to Cr⁶⁺on growth and development,thyroid and skeletal development in B.gargarizans tadpoles,B.gargarizans tadpoles were treated with different concentrations of Cr⁶⁺(0,13,52,104,208,and 416?g Cr⁶⁺L^-1)from G2 to completion of metamorphosis?G46?.Total length,snout-vent length,weight,developmental stage,hind limb length and fore limb length were monitored,and thyroid histological examinations were employed at G32,G38,G42.The skeletal systems of tadpoles at G42 and G46 were investigated by using double staining methodology.Results showed that significantly reductions in total length,weight and developmental stage were observed at 104,208 and 416?g Cr⁶⁺L^-1 on day30.The 104,208 and 416?g Cr⁶⁺L^-1 inhibited the body size of B.gargarizans larvae at G42and metamorphosis was inhibited by Cr⁶⁺exposure at all concentrations.In addition,104,208and 416?g Cr⁶⁺L^-1 inhibited the body size of B.gargarizans larvae at G46.Histological examinations showed 416?g Cr⁶⁺L^-1 caused follicular cell hyperplasia and depletion of partial colloid in thyroid gland.Moreover,104,208 and 416?g Cr⁶⁺L^-1 suppressed the bone mineralization of larvae at G42 and G46.3.To evaluate the effects of chronic exposure to Cr⁶⁺on histological changes,lipid metabolism and fatty acids composition in the liver of B.gargarizans tadpoles.B.gargarizans tadpoles were treated with different concentrations of Cr⁶⁺(0,13,52,104,208,and 416?g Cr⁶⁺L^-1)from G2 to G42.Histological alterations in liver were examined.The transcript levels of several genes associated with lipid metabolism signaling pathways as well as fatty acids composition and content were also determined.Results showed that histological changes characterized by an increase in the number of melanomacrophage,nucleus pyknosis,and deposition of lipid droplets were observed in liver at 416?g Cr⁶⁺L^-1.RT-qPCR results showed that mRNA expression of lipid synthesis-related genes?SCD,MECR,TECR and ELOVL1?were significantly downregulated in larvae exposed to 416?g Cr⁶⁺L^-1.Also,mRNA expression of fatty acid?-oxidation-related genes?ECHS1,HADHA and ACAA1?were significantly upregulated in larvae treated with 13,52,104,208 and 416?g Cr⁶⁺L^-1,but mRNA expression of fatty acid?-oxidation related genes?ACOT1,PPT1,HADH and ACAA2?were significantly downregulated at 416?g Cr⁶⁺L^-1.In situ hybridization revealed that BSEP was expressed in hepatocyte nucleus and plasma membrane,and HSD17B12 was abundantly expressed in the plasma membrane.The positive signals of HSD17B12 in plasma membrane of 416?g Cr⁶⁺L^-1treatment group were weaker than that of control group.An increase in polyunsaturated fatty acids?PUFAs?and a decrease monounsaturated fatty acids?MUFAs?were found in the 52,104and 416?g Cr⁶⁺L^-1 groups.