Biochemical And Molecular Toxicologic Mechanism Of Cytochrome P450 In Chironomus Kiiensis Responding To Substituted Benzenes Stress

Substituted benzenes of organic pollutants could make damage to the aquatic ecosystem with the agricultural and industrial wastewater draining, it will cause the irreversible hazard to the human health with long pollution. Chironomid can be used as a good indicator organism, and cytochrome P450 as biomarker in chironomid was applied to warning for environmental pollutant. In present study, we used 4th-instar Chironomus kiiensis larvae as target to study acute and joint toxicity of phenol, p-chlorophenol and p-phenylenediamine and their effects on cytochrome P450 activity and CYP6 family genes. The detailed results showed as follow.(1) The acute toxicity of 3 substituted benzenes to the chironomid larve is in decreasing order of p-chlorophenol>p-phenylenediamine>phenol. The median lethal concentration of 48 h are ranged from 10 mg/L to 130 mg/L showing low toxicity, which indicated that the substituted group toxicity is-C1>-NH2>-OH. The four evaluation methods were used to evaluate joint toxicity of nine binary substituted benzenes including the phenol+p-chlorophenol (1:1,2:1,1:2), phenol+p-phenylenediamine(1:1,2:1,1:2), p-chlorophenol+p-phenylenediamine (1:1,2:1,1:2). The results showed that the evaluation results of A were completely consistent with the X while the results of M were completely consistent with the MTI. The different substituted benzenes with toxic units (1:1,2:1,1:2) were mainly synergistic effect, which indicated the joint toxic effects of substituted binary benzenes were not related to the concentration changes of substituted benzenes.(2) Three single substituted benzenes (1～100μmol/L) mainly showed inhibitory effect to the P450 activity of the chironomid larvae. About phenol, the inhibition of in vivo P450 activity in 4th instar C. kiinensis larvae gradually increased with the increase of treatment time and inhibition concentration. In contrast, the inhibition of in vivo P450 activity decreased with the p-chlorophenol treatment time increased, while the induction of P450 activity increased with the increase of p-phenylenediamine treatment time and concentration. The three substituted benzenes, phenol, p-chlorophenol and p-phenylenediamine, were low sensitive to the in vitro P450 activity with the 576.85 mg/L,466.15 mg/L and 594.43 mg/L of the median value of inhibitory concentration IC50, respectively. The sensitivity in decreasing order is p-chlorophenol> phenol>p-phenylenediamine. The P450 activity in phenol+p-chlorophenol (1:1) and phenol+p-phenylenediamine (1:1) treatments was lower than the control, which mainly showed inhibitive effect. The in vivo P450 activity of the sublethal concentration LC20 treatment was higher than those of LC10 treament, which showed enzymatic activity increased in the high concentration.(3) The ten CYP6 genes could encode 421～528 amino acid residues with the molecular mass of 49.01～61.94 kDa and 6.01～8.50 of theoretical isoelectric point (PI). Under LC10 and LC20 of the sublethal concentration of phenol+p-chlorophenol, phenol+p-phenylenediamine stresses, the CYP6GB1 has been induced with 1.10～45387.27-fold of the control. However, the LC10 of sublethal concentration of phenol+p-chlorophenol showed inhibition effect on CYP6 genes with inhibition effect declined along with the increase of concentration and time.Under LC10 of phenol+-p-phenylenediamine stress,, the 10 CYP6 genes showed induction effects, but the induction effect gradually declined along with the increase of treatment time. The 10 CYP6 genes could be clustered into three groups. The CYP6EV10, CYP6EW3, CYP6EV9, CYP6FX1, CYP6FY1 clustered into one group while the CYP6FW1 and CYP6EY2 were in a group. The third group was CYP6FV1, CYP6FV2, CYP6GB1. The CYP6FV1, CYP6FV2 and CYP6GB1 genes showed induction and activation effect while CYP6EV10, CYP6EW3, CYP6EV9, CYP6FX1, CYP6FV1, CYP6FW1, CYP6EY2 showed inhibition effects.These results showed that the substituted benzenes could inhibit the P450 enzymes activity and the express of CYP6 genes in the chironomid larve indicating that the larval chironomid could respond to the stress of substituted benzenes in a concentration- and time-dependent way. Therefore, These results provide information for environmental quality monitoring.