| Objectives: Acrylonitrile(AN) is one of the important products and raw materials in organic synthesis industry. It is widely used in acrylic, nitrile rubber, synthetic fiber, resin, etc. With the rapid development of relative industries in recent years, the exposure opportunities to AN in workplace and daily have been increasing. To protect exposed people’s health, domestic and foreign scholars conduct many studies on toxicity, especially acute toxicity, while the toxic mechanism of AN remains unclear. According to two metabolic pathways of AN in vivo, AN can bined with sulfydryl-containing proteins or compounds and consume more sulfydryl, which is necessary for the synthesis of hydrogen sulfide(H2S). H2 S, a newly-found endogenous gasotransmitter, has important biological activities and is widely involved in a variety of physiological and pathological process. Studies found that H2 S is also involved in the toxicity of several environmental, occupational chemicals and drugs. It has been suggested that exogenous H2 S has antagonistic effect against chemical toxicity. The preasent study aims to build AN poisoning animal models and mainly explore: 1. the time and dose-effect relationship between AN and endogenous H2 S metabolism in rat; 2. the potential mechanism of H2 S metabolic changes by regulating CYP2E1, which is the key enzyme of AN metabolism; the present study aims to explain AN toxic mechanism, providing theoretical basis for the prevention and treatment of AN poisoning.Methods:Study 1: 60 male SD rats were randomly divided into the control group(injected with normal saline), 25 mg/kg AN group, 50 mg/kg AN group and 75 mg/kg AN group. Each group was further divided into 3 subgroups depending on the time of treatment.After intraperitoneal injection for 1 h, 6 h and 24 h, 5 rats from each group were executed with anesthesia. Liver and brain cortex was isolated. Study 2: a totle of 48 male SD rats were divided into 4 groups: control group(injected with normal saline), AN alone group, DCE alone group and AN-treated with DCE pretreatment group(DCE+AN). AN alone group was treated by intraperitoneal injection of the dosage of 50 mg/kg AN. DCE alone group was injected with 100 mg/kg DCE. AN-treated with DCE pretreatment group was injected 50 mg/kg AN after pretreatment with 100 mg/kg DCE for 2.5h. After infection with AN for 1 h, 6 h and 24 h in all AN-treated groups, 6 rats from each group were executed after anesthesia, the liver and the brain cortex was isolated. Study 3: a totle of 48 male SD rats were divided into 4 groups: control group(injected with normal saline), AN alone group, acetone alone group and AN-treated with acetone pretreatment group(acetone + AN).AN was infected by intraperitoneal injection of 50 mg/kg. acetone + AN group was treated by drinking water involving 1% acetone for a week, then injected with 50 mg/kg AN. After infection with AN for 1 h, 6 h and 24 h in all AN-infected groups, 6 rats from each group were executed after anesthesia, the liver and the brain cortex was isolated. The rats were observed for cholinomimetic signs; detecting endogenous H2 S content and the activity of H2 S synthase both in the liver and the brain cortex by methylene blue spectrophotometer method; detecting the protein expression of CBS、CSE、3-MPST in the liver and brain cortex by western blot. The first set of experiments determined the oxidative stress markers: reduced glutathione(GSH), malondialdehyde(MDA) and reactive oxygen species(ROS) content; The second and the third set examined CYP2E1 enzyme activity and CYP2E1 protein expression in the liver microsoms.Results: Study 1:Behavioral observations show all AN treated rats exhibited simiar cholinomimetic signs;Compared with control group, both the endogenous H2 S content and enzyme activity in the rat liver and brain cortex of the AN-treated groups decreased, and in time and dose-dependent manner;The results of western blot showed that CBS, CSE and 3-MPST protein expression in liver of various doses of AN-treated groups was significantly decreased(p<0.05). CBS, 3-MPST protein content in the cerebral cortex also declined significantly, and CSE is not defected in cerebral cortex; Results of oxidative stress markers showed that GSH concentration in AN-infected group was significantly lower than the control group(p<0.05) and the concentration of MDA and ROS significantly increased,three of which all showed the time and dose-response relationship. Study 2: DCE significantly inhibited CYP2E1 protein expression and enzyme activity(p<0.05);Compared with AN alone group, endogenous H2 S content in rat liver and brain cortex of DCE pretreatment group was significantly higher in the point of 24h(p< 0.05); H2 S synthase activity in liver of DCE pretreatment group significantly increased compared with AN alone group in the point of 24h(p<0.05);H2S synthase activity in cerebral cortex in 6 h and 24 h significantly increased. Study 3: Acetone can significantly induce CYP2E1 protein expression and enzyme activity(p<0.05);Compared with control group, the endogenous H2 S content and the activity of hydrogen sulfide synthase in the liver and brain cortex of the acetone pretreatment group showed no significant difference; CBS and 3-MPST protein expression in the liver and brain cortex of acetone pretreatment group declined, which was statistically significant in the point of 6h(p<0.05). CSE protein expression in the liver also fell, but showed no significant difference.Conclusion: AN significantly inhibited endogenous H2 S metabolism; Inhibiting or inducing CYP2E1 activity significantly altered endogenous H2 S metabolism induced by AN, which was related to the roles of AN metabolites.It implicated that inhibition of H2 S metabolism may affribute to the toxicity of AN and H2 S may be a new target for treatment of AN poisoning. |
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