Effects Of Cadmium Stress On Oxidative Stress And Apoptosis In Drosophila Melanogaster Larvae

With the increase of human activities,cadmium(Cd)pollution has become a global environmental problem affecting biological metabolism in ecosystems.As a result of,it has an extremely long half-life(approximately 20 to 30 years in humans and excreted slowly in organs.Thus,it poses a serious threat to human health.Studies have shown that Cd stress induces high reactive oxygen species of cells and then the increased reactive oxygen species interact with the DNA damage response system,which affects the stability of the organism genome.At the same time,it impairs the inherent anti-oxidant defense ability and induces oxidative stress of cells.When the damage induced by Cd exceeds the cell's ability to repair itself,it activates signaling pathways to initiate apoptosis.In addition,organisms may regulate gene expression through epigenetic pathways to combat toxic stress from Cd stress.In this study,the third instar larvae of Drosophila melanogaster Canton-S strain were used as experimental materials.Based on the determination of the LC50 of Drosophila larvae,the nitrogen blue tetrazolium reduction experiment was used to determine the active oxygen content;Trypan blue staining test and larva crawling test to determine the damage of mid-gut cells and mechanical motor neurons and analyze the oxidative damage of cells by Cd stress;comet assay to detect DNA damage of mid-gut cells;Real-time quantitative PCR to detect the relative expression levels,including apoptosis-related genes Debcl,hid,rpr,Diap1,p53,Sce,mitochondrial adenine nucleotide transposase(ANT)encoding genes Ant2,sesB,and Cd stress resistance genes Cdc42 and Mekk1,BSP detected the methylation site of Mekk1 gene,to explored the effects of Cd stress on oxidative damage and apoptosis of Drosophila third instar larvae.The findings are as follows:(1)The LC50 of Cd on Drosophila larvae was 4.449 mg/kg,and the Cd concentrations were 1.125 mg/kg,2.25 mg/kg,4.5 mg/kg,and 9 mg/kg for subsequent research.(2)The results of reduction experiments of nitroblue tetrazolium and trypan blue staining showed that the active oxygen content of Drosophila larvae increased and the mid-gut cells were damaged.At the same time,the larvae exhibited certain behavioral abnormalities,such as increased number of turns and twists,and abnormal body wall contraction.(3)The results of comet experiments showed that compared with the control group,the percentage of tail DNA(Tail DNA%)increased in the DNA of the mid-gut cells of Drosophila larvae in the Cd-treated group.That is to say,DNA damage occurred in the mid-gut cells.It is speculated that the increase in ROS content may induce cell oxidation caused by stress.(4)The expression of ANT protein-encoding genes Ant2 and sesB showed an overall downward trend,indicating that Cd stress may impair mitochondrial function of Drosophila,thereby inducing oxidative stress and oxidative damage.The expression of the pro-apoptotic genes Debcl,hid,rpr and p53 increased with the increase of Cd concentration,and the expression of the suppressive apoptotic gene Sce showed a trend of firstly increase and then decrease.The expression of Diap1 gene down-regulated,indicating that Cd stress promoted pro-apoptosis gene expression which inhibits the expression of anti-apoptotic genes.Therefore,the concentration increases,at the same time,the mid-gut cells and DNA damage increases.In addition,this study found that Cd stress induced changes in the morphology and size of Drosophila wings,especially in the high-concentration treatment group(9 mg/kg)"residual wing" Drosophila was found,and Sce as an apoptosis inhibitor was mainly used in fruit.The expression in fly wing discs indicates that the change in Sce expression induced by Cd is involved in the regulation of wing morphological development,resulting in a change in wing phenotype of Drosophila.(5)The expression of stress resistance gene Mekk1 was up-regulated,and BSP sequencing results showed that its DNA methylation level was reduced.This result is consistent with the results of whole genome methylation sequencing and digital gene expression profiling of Drosophila.At the same time,there was a significan't negative correlation between gene expression levels and the methylation rate of the gene(r=-1.000).The results showed that Cd stress induced changes in the DNA methylation level of the Drosophila Mekk1,thereby regulating the expression of the gene,and further showed that the toxic effect of Cd on Drosophila was related to the epigenetic pathway.In summary,the high concentration of Cd increased reactive oxygen content of the third instar larvae and induced oxidative stress;changes in the expression of apoptosis-related genes eventually led to apoptosis.In addition,the methylation rate of stress-resistance genes in Drosophila decreased,and gene expression was up-regulated,indicating that Cd induced changes in Drosophila DNA methylation modification to regulate the expression of related genes.In this study,we studied the effects of Cd on the wing phenotype,mid-gut cell activity,larval motility,cellular DNA integrity,expression of apoptosis-related genes,and methylation levels of stress-resistant genes in Drosophila.The aim of toxic effects on early exposure to was to explore the related mechanisms of Cd-induced apoptosis,to clarify the role of epigenetics in Cd-induced Drosophila toxic stress,and to provide theoretical and experimental basis for Cd pollution control.