Interaction Between CYP450 Of Fish And Organophosphorus Insecticides

Cytochrome P450s are a diverse array of multifunctional heme-thiolate protein which have absorb peak at 450nm when binding with CO. Cytochrome P450 enzyme can not only catalyze some important physiological function of endogenous substances such as hormones, fatty acids, terpenoids and others, but also participate in many exogenetic material biological oxidation including herbicides, insecticides, fungicides, drug, environmental pollutants and other toxic substances which distributes widely in animal, plant and microorganism. Drug biotransformation catalyzed by P450 is an important step in drug metabolism, meanwhile P450 enzyme activity can be stimulated or inhibited by drug. At this study, using three insecticides, i.e. parathion, chlorpyrifos and malathion as test drug, four species of fish, i.e. Danio rerio, Xiphophorus helleri, Pseudorasbora parva, and Lepomis gibbosus as test animal, microsomal as carrier, acute toxicity of the three insecticides to the four fish, oxidative activation of the three insecticides catalyzed by cytochrome P450 system, and the influence of insecticide to the P450 system were investigated. This experiment aimed to provide a theoretical basis for ecological safety assessment of insecticide to environment. The details was list below:Danio rerio, Xiphophorus helleri, Pseudorasbora parva, and Lepomis gibbosus were used as the test organism to determine the acute toxicity of parathion, chlorpyrifos and malathion. The results showed that the 96h LC50 of parathion to each fish was 2.60 mg/L,0.0559 mg/L,1.78 mg/L and 1.02mg/L, chlorpyrifos to each fish was 1.94 mg/L, 0.171 mg/L,0.0273 mg/L and 0.0681mg/L, and malathion to each fish was 7.07 mg/L, 0.907 mg/L,6.03 mg/L,0.172mg/L.The concentration of paraoxon, chlorpyrifos-oxon, malaoxon were determined by HPLC or GC. The concentration was compared with sensitivity of corresponding fish towards the insecticides. Result of the study indicated that desulfuration activity of hepatic cytochrome P450, as scaled with Vmax/Km, was estimated to be 1.67×104,2×104, 5×104, and 2×104L·min-1·mg-1, respectively, for chlorpyrifos, and the activity was estimated to be 2×104,5×105, 1.11×105, and 1.43×105L·min-1·mg-1, respectively, for parathion. For malathion, the activity was estimated to be 5×103,5×104,2.5×103, and 1.67×104L·min-1·mg-1, respectively. A negative correlation was found between LC50 and Vmax/Km. The equation of linear regression was Y=-196710X+561808, Y=-35406X+ 99130, Y=-7241.3X+66969, respectively for parathion, chlorpyrifos, and malathion. The value of R2 was estimated to be 0,8635,0.3854, and 0.9845, respectively for parathion, chlorpyrifos, and malathion. Result of the study suggested that cytochrome P450 played a negative role in creating tolerance of fish to organothiophosphorus insecticides.The content or activity of cytochrome P450 enzyme system was determined by biochemical methods or UV spectrophotometry. Results showed that total content of cytochrome P450 is 0.309±0.037nmol/mg,0.415±0.032 nmol/mg,0.316±0.042nmol/mg, 0.378±0.030 nmol/mg, respectively for Danio rerio, Xiphophorus helleri, Pseudorasbora parva, and Lepomis gibbosus. The activity of NCCR was 2.214±0.165nmol/min/mg, 10.217±1.772 nmol/min/mg,1.921±0.179 nmol/min/mg,5.147±0.767 nmol/min/mg, respectively for each fish. The activity of APND was 1.082±0.284 nmol/min/mg, 2.461±0.094 nmol/min/mg,1.820±0.237 nmol/min/mg,1.468±0.408 nmol/min/mg, respectively for each fish. The activity of ERND was 2.226±0.140nmol/min/mg, 3.972±0.394 nmol/min/mg,1.725±0.214 nmol/min/mg,2.114±0.025 nmol/min/mg, respectively for each fish. The activity of AH was 0.075±0.007 nmol/min/mg, 0.114±0.010 nmol/min/mg,0.041±0.013 nmol/min/mg,0.086±0.013 nmol/min/mg, respectively for each fish. The results suggested that the established method for determining the contents and activities of cytochrome P450 enzyme system in four fish was sensitive, reliable with good reproducibility.Using three insecticides, i.e. parathion, chlorpyrifos and malathion as test drug, the influence of insecticides to the activities of cytochrome P450 enzyme system in four fish was researched. The results suggested that total content of cytochrome P450, the activities of NCCR, APND and ERND in fish treated with the three insecticides had no statistically significant difference in parameters compared with the solvent control group (P>0.05). However, a significant difference in the activity of AH in fish treated with three insecticides was observed(P<0.05).