Effects Of Subchronic Benzo(a)Pyrene Exposure On Rat Cholinergic System

Polycyclic Aromatic Hydrocarbon (PAH), a product of incomplete combustion of coal, petroleum,wood, tobacco and other organics at high temperature, widely existed in environment. Benzo [a] Pyrene (B[a]P), main component part of polycyclic aromatic hydrocarbons, was a confirmed carcinogen.Besides of its carcinogenicity,its neurotoxicity is being laid stress on by researchers. Some experiments have shown that B[a]P could damage animal’s nerve tissue and make their spatial memory significantly decline. The role of B[a]P and B [a] P metabolites in plasma and brain were significantly related. Cholinergic system played an important role in learning and memory course. Ach,as an important neurotransmitter, was related with learning and memory capacity. But now it is still unclear that whether neurotoxicity induced by B[a]P is related with cholinergic system. This stuy will investigate the neurotoxic mechanisms of benzo [a] pyrene through the detection of rat peripheral blood and hippocampal acetylcholine (ACh) level, cholinesterase (AChE) activity and expression of acetylcholine receptor a7subtype (nAChRa7)mRNA and protein after subchronic exposure to benzo [a] pyrene.Objective:To investigate the neurotoxic mechanisms of benzo [a] pyrene through the observation of rat neural behavior, detection of peripheral blood and hippocampal acetylcholine (ACh) level, cholinesterase (AChE) activity and expression of acetylcholine receptor α7subtype (nAChRa7)mRNA and protein after subchronic exposure to benzo [a] pyrene.Methods:50healthy male SD rats were randomly divided into blank control group, solvent control group and testing group by giving dose of1.0,2.5,6.25mg/kg KW B[a]P individually after exposure for90days by intraperitoneal injection every other day. The learning and memory ability of the rats were analyzed by Morris water maze and platform assay. The peripheral blood and hippocampal Ach level was detected by alkaline hydroxylamine method, and the AChE activity by DNTB method. The expression level of hippocampal nAChRa7mRNA was detected by Q-PCR and of protein by Western-blot.Results:50healthy male SD rats were randomly divided into blank control group, solvent control group and testing group by giving dose of1.0,2.5,6.25mg/kg KW B[a]P individually after exposure for90days by intraperitoneal injection every other day. The learning and memory ability of the rats were analyzed by Morris water maze and platform assay. The peripheral blood and hippocampal Ach level was detected by alkaline hydroxylamine method, and the AChE activity by DNTB method. The expression level of hippocampal nAChRa7mRNA was detected by Q-PCR and of protein by Western-blot. The Morris water maze results showed that the average escape latency and the total distance of the rats exposed to B[a]P increased with the increase of B[a]P exposure dose,when the platform quadrant retention time decreasd with the increase of B[a]P exposure dose.The average escape latency in6.25mg/kg B[a]P group were significantly higher than the blank control, solvent control and l.Omg/kg B [a]P group (17.38±1.82s,25.12±1.95s and22.91±1.86s, P<0.05).The platform quadrant retention time in6.25mg/kg B[a]P group were significantly lower than the blank control, solvent control, l.Omg/kg B [a]P and2.5mg/kg B[a]P group (48.37±10.6s,41.49±11.44s,43.15±10.24s and38.92±13.31s, P<0.05). There were no significant diferrence among other groups. The total distance in the2.5mg/kg、6.25mg/kg B[a]P group were significantly higher than the blank control, solvent control and1.0mg/kg B [a]P group (P<0.05).The platform assay showed that the first error numbers(EN1) of6.25mg/kg B[a]P group were significantly higher than the blank control, solvent control and1.0mg/kg B [a]P group (P<0.05).The second error numbers(EN2) of6.25mg/kg B[a]P group were significantly higher than the blank control, solvent control,1.0mg/kg B [a]P and2.5mg/kg B[a]P group (P<0.05); The diferrence was statistically significant.There were no statistically significance among every group in rat peripheral blood Ach content. The AChE activity in rat peripheral blood in2.5mg/kg and6.25mg/kg B[a]P group respectively were14.3±6.68�'�12.3±4.96U/ml, significantly lower than the blank control (17.9±5.92U/ml). solvent control (17.2±4.84). The diferrence was statistically significant (P<0.05). Tendency can be seen that The rat hippocampal Ach content decrease with the increase of B[a]P exposure dose. Among these, the2.5.6.25mg/kg B[a]P groups (43.4±26.8and34.9±12.3μg/ml) were significantly lower than the blank control, solvent control and1.0mg/kg B [a]P group(60.8±6.46、55.6±16.9�'�53.7±17.8μg/ml).The diferrence was statistically significant (P<0.05). The rat hippocampal AChE activity in6.25mg/kg B[a]P group was3.01±1.32U/ml,and significantly lower than the blank control, solvent control,1.0mg/kg and2.5mg/kg B [a]P group (5.32±1.66、5.12±1.62、5.37±1.68�'�5.23±1.02U/ml) There were statistically significance (P<0.05).Q-PCR and Western-blot results showed that there were no significant difference about nAChRa7mRNA and protein expression levels among all groups (P>0.05).Conclusion:1. The subchronic exposure to benzo[a] pyrene can damage the rat learning and memory ability;2. The subchronic exposure to benzo[a] pyrene can inhibit AchE activities in rat peripheral blood and hippocampus;3. The subchronic exposure to benzo[a] pyrene can result in the decrease of rat hippocampal Ach level, and they had dose relations.4. The subchronic exposure to benzo[a] pyrene does not affect hippocampal nAChRa7mRNA and protein expression level;5. The rat hippocampal Ach content decreasing might be the probable machanism that subchronic exposure to benzo[a] pyrene damage the rat learning and memory ability.