Toxicological Effects Of Single And Combined Exposure Of Cr And Pb On Rana Chensinensis Tadpoles

The gradually increased concentrations of Cr(VI)and Pb in the water environment had imposed widespread adverse effects on the health of aquatic organisms due to the extensive use of Cr(VI)and Pb in the production of industrial products.Rana chensinensis is a widely distributed amphibian group of northern China and has been proposed as a bioindicator of river pollution by heavy metals.The objective of this study was to determine the effects of 104 ?g/L Cr,50 ?g/L Pb,both single and mixture exposure,on R.chensinensis tadpoles.The main findings were as follows:1.The effects of single and combined chronic exposure of Cr and Pb on the growth and development indexes of Rana chensinensis tadpoles of Gs 26 stage were analyzed,and the effects of exposure on the thyroid tissue and bone ossification of tadpoles at Gs 42 and Gs 46 were also analyzed.The results showed that the total length,body length and body weight of tadpoles were significantly inhibited by combined exposure at 18 days.Cr and combined exposure inhibited the total length,body length,body weight and hindlimb length of tadpoles at Gs 42.Both Cr and combined exposure could cause the high reduction of epithelial cells and shrinkage of thyroid tissue in tadpoles.In addition,Cr and combined exposure can partially inhibit the bone ossification of tadpoles at Gs 42 and Gs 46,while Pb can promote the bone ossification to a certain extent.RT-q PCR analysis results showed that the expression levels of ossification related genes(FGF13,MMP13,TR?,SOX9,BMP3)were significantly upregulated in Pb-treated group compared with the control group.2.The effects of single and combined chronic exposure of Cr and Pb on the growth,intestinal histology,digestive enzymes activity and intestinal microbiota in R.chensinensis tadpoles at Gs 38 were investigated.Our results revealed that the obvious histopathologic alterations such as blurring of cell borders and irregular nucleus were observed in mixtureexposed group.Mixture exposure significantly induced growth retardation,intestinal histological lesions and digestive enzymes activities.Lipase,amylase in mixture-exposed group and pepsin in Cr-exposed group were significantly changed compared with the control.Moreover,Chao 1 and Shannon indexs were significantly increased in Cr-,Pb-and mixtureexposed groups compared to the control.Beta diversity showed three treatment groups separation with the control.At the phylum level of intestinal microbial community,the relative abundances of Proteobacteria in mixture-exposed group and the relative abundances of Verrucomicrobia and Actinobacteria in Cr-exposed group were significantly decreased.At the genera level,the relative abundances of Akkermansia and Alpinimonas were significantly decreased in Cr-exposed group,while the relative abundances of Anaerotruncus were significantly increased in mixture-exposed group.3.The effects of single and combined chronic exposure of Cr and Pb on transcriptomics and gene expressions in intestine of R.chensinensis tadpoles at Gs 38 were investigated.We depicted the characterization of gene expressions with R.chensinensis tadpoles using highthroughput profiling of gene expression.Moreover,6 genes related to fat digestion and absorption(CLPS,LIPASE,NPC1L1,PAP,SCARB1,L-FABP)were selected in the c DNA library of intestine by using RNA-Seq technology,and the expression profiles of these genes in intestine of tadpoles at Gs 38 between the control group and Cr,Pb,mixture exposure group were analyzed.Among these genes,three genes(LIPASE,NPC1L1,PAP)up-regulated and 2genes(CLPS,L-FABP)down-regulated in response to Cr,Pb and mixture exposure.In conclusion,single and combined chronic exposure of Cr and Pb at environmental related concentrations have effects on R.chensinensis tadpoles,but the combined effects of Cr-Pb are not simply enhanced or weakened.This work lays a foundation for further research on the mechanism of action.