Study On Biochemical And Molecular Toxicology Mechanism Of Substituted Benzenes To Detoxifying Enzymes Of Chironomus Kiensis Tokunaga

In this study,we selected ten substituted benzenes according to Black List of China's Priority Pollutants in water and earlier researches by our group,and Chironomus kiiensis as our research targets.We determined the acute and joint toxicity of substituted benzenes to 4th-instar C.Kiiensis larvae,and evaluated joint toxicity effects of binary substituted benzenes with the four methods of AI,TU,MTI and X.These results showed that the substituted benzenes were low toxic to 4th-instar Chironomus kiiensis larvae and the LC50 values of 10 kinds of substituted benzenes ranged from 40 mg/L to 500 mg/L.The toxicity of substituted benzenes depended on its types and the position of substituted group in the benzene ring.The decreasing order of substituted group toxicity was-OH>-NO2>-CH3>-H.And the order of toxicity among isomers of dimethybenzene was 1,2-dimethylbenzene>1,3-dimethylbenzene>1,4-dimethyl benzene while the toxicity of p-phenylenediamine was higher to m-phenylenediamine.The results were not completely consistent when the joint toxities of eight the binary substituted benzenes mixed with the same toxic unit using four different evaluation methods.However,the results were completely consistent with the evaluation method of TU and MTI,and the results were completely consistent with the evaluation method of AI and ?.The results were consistent in the synergistic and simple additional toxic effect among the four evaluation methods,while the evaluation results were different in the partial addition and antagonism toxic effect.The results were inconsistent with each of the four evaluation methods when the joint toxic effect of p-chlorophenol and nitrobenzene in different proportion was assessed indicating that the joint toxic effect of substituted binary benzenes was related to the concentration of substituted benzenes.In order to have a better understanding of the toxicological mechanisms of substituted benzenes to 4th-instar C.kiiensis larvae,we selected the typical substituted benzenes based on the acute toxicity results to systemically study their in vivo and in vitro effects on the main detoxification enzymes including AChE,CarE and GSTs.When chironomids exposed in p-chlorophenol,phenol,p-phenylenediamine and m-phenylenediamine with the sublethal concentrations of 0.4,4 and 40 mg/L,and exposed in p-chlorophenol+phenol and p-phenylenediamine+ m-phenylenediamine with the concentrations of LC5,LC10 and LC20,both AChE and CarE in C.kiiensis larvae differently responded to these stresses.The in vivo GSTs activities in chironomid larvae were inhibited under the 1,10 and 100 mg/Lof phenol stresses.According to time-and dose-effects and response of enzymes activities,CarE was the most sensitive to the stresses of substituted benzenes.Among the three main detoxifying enzymes,the CarE and GSTs were sensitive to substituted benzenes and only sensitive to p-phenylenediamine,m-phenylenediamine and p-phenylenediamine+m-phenylenediamine.According to time-and dose-effects and responsive extent of enzyme activities,the sensibility of three kinds of in vitro detoxifying enzymes to substituted benzenes were CarE>GSTs>AChE.In order to further explore molecular toxicology mechanism of phenol,we analyzed characteristic of GSTs genes of chironomid,and studied the expressions of GSTs genes in 4th instar C.kiiensi larvae during different developmental stages and under phenol stresses by Real-time quantitative PCR.In present study,the 12 GSTs genes in chironomid were identified from transcriptome library.These GSTs genes belong to Theta,MAPEG,Delta and Sigma familys,respectively,and encoding 149?225 amino acid residues with the molecular mass of 17.31?26.34 kDa and theoretical pI of 4.94?7.78.The transcription profiling of 12GSTs genes during different development stages revealed significant variations.At the postembryonic stage,most of the genes that belong to Theta family,MAPEG family and Delta family were mainly downregulated while Sigma family genes were mainly upregulated compared to the egg stage.Most of GSTs genes in 4th-instar larval chironomid were downregulated by phenol treatment with the concentrations of 1,10 and 100 mg/L.The result was consistent with GSTs activities were decreased under phenol stresses.In this paper,we studied biochemical and molecular toxicology mechanisms of typical substituted benzenes to Chironomus kiiensis at macrocosm and microcosm levels.These results provided insight into eco-toxicity of substituted benzenes and theoretical basis for water quality standard determination and the ecological risk assessment.