The Mechanism Of Neuronal Apoptosis In Brain Of Rats Induced By 1-Bromopropane

Objective1-Bromopropane (1-BP) has been widely used in the workplace as an alternative agent to ozone-depleting solvents such as chlorofluorocarbons (CFCs) and suspect carcinogens such as trichloroethylene, methylene chloride and tetrachloroethylene. It is mainly used as cleaning agent for metal, electronics, and optical instruments. Human and laboratory animals exposed to 1-BP display neurological disorders including peripheral nervous system (PNS) such as sensory and motor deficits and central nervous system (CNS) toxicity such as depression, anxiety as well as cognitive defects. Human cases of 1-BP poisoning have being continuously reported in the United States and Japan since 1999. Compared with other neurotoxic organic solvent such as hexane intoxicated cases, human with 1-BP intoxication are more likely to exhibit clinical symptoms of central nervous system (CNS). Until now, the initial target and early key event of CNS damage trigged by 1-BP was not clear yet. Evidences demonstrated that chronic inflammation always exists in brains during the onset and progression of human neurodegenerative diseases. The chronic neuroinflammation has been increasingly considered to be involved in the pathogenesis of many neurodegenerative diseases. Inflammatory response was also observed in brains of animals exposed to environmental nerve poison. As the main responder to CNS insults, the pivotal roles of astrocyte in inflammatory process have attracted more attention recently. But whether inflammatory response is the main mechanism of neurotoxicity of 1-BP has not been reported yet. Recently, reports suggested that docosahexaenoic acid (DHA) has a strong anti-inflammatory activity, and can cross the blood brain barrier. Dietary supplementation of DHA can significantly improve the level of DHA in the brain. DHA can alleviate 1-BP-induced neurotoxicity indicate that inflammation in brain may involve in the 1-BP-induced CNS toxicity. Meanwhile, ammonium pyrrolidinedithiocarbamate (PDTC) is a specific inhibitor of nuclear transcription factor κB (NF-κB) in inflammatory pathway. Inhibition of NF-Kb and blocking the inflammation can reverse 1-BP-induced neurotoxicity indicate that the role of inflammation in neurotoxicity mechanism of 1-BP. Therefore, first we observed the the dose response relationship of neuron injury and inflammatory changes in brain of 1-BP exposed rats, further we adopted DHA, an anti-inflammatory agent and PDTC, a NF-κB inhibitor to observe the reverse effects of 1-BP-induced neuron injury and evaluate the 1-BP-induced CNS toxicity and its mechanism. Thus we may provide a theoretical reference for effective treatment to 1-BP and other organic solvent neurotoxicity.MethodsThe studies were carried our in three batches of male Wistar rats, and the acclimation period of animals was 5 days prior to experiment. The first batch of 60 rats were randomly divided into 5 groups of 12 each, i.e. control,100,200,400 and 800mg/kg bw 1-BP (diluted in corn oil) respectively.1-BP was gavaged for consecutive 13 days. Animals in control group were treated equal volume solvent per day. At the following morning of termination of experiment, the tissues of prefrontal cortex (PFC) was quickly dissected after sacrificed and homogenized in ice-cold homogenizing buffer and then centrifuged at 15,000 xg for 30 min at 4℃. The caspase-3, glial fibrillary acidic protein (GFAP) and neuron nuclear protein (NeuN), GSK-3β and AKT in PFC of rat brains were investigated by western blot.4 rats in each group were perfused transcardially for preparing frozen tissue sections. The neurons and AS were observed with immunofluorescence double staining and cells apoptosis were dectected with TUNEL staining in PFC of rat brains. PFC of rat brains were homogenated frozen and the level of reactive oxygen species (ROS) in PFC were detected with 2’,7’-Dichlorofluorescein diacetate. The level of cyclooxygenase-2 (COX-2) mRNA in PFC of rat brains was detected with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR).The second batch of 60 male Wistar rats were randomly divided into four groups with 15 in each, i.e. control group,1-BP low-dose group, 1-BP+250mg/kg bw DHA group and 1-BP+500mg/kg bw DHA. After acclimation to the new environment for one week,1-BP dissolved in the corn oil was orally administered to rats in 1-BP group, 1-BP+low dose DHA group and 1-BP+high dose DHA group once a day for consecutive 13 days, at a dose of 800 mg/kg bw. DHA diluted in corn oil was given to rats by gavage once daily for a period of 19 days, beginning 7 days prior to 1-BP exposure, at the dosage of 250 mg/kg bw and 500 mg/kg bw for low dose and high dose intervention groups respectively. DHA was treated to rats 4h prior to 1-BP treatment. Rats in control group and 1-BP group were received accordingly equal volume of corn oil. After 7 days of 1-BP exposure, all rats were subjected to Morris Water Maze (MWM). After the MWM, the tissues of PFC and hippocampus (HPC) were quickly dissected after sacrificed and homogenized in ice-cold homogenizing buffer and then centrifuged at 15,000×g for 30 min at 4℃. The apoptosis related proteins including cytochrome c (cyt c), caspase-3, Bcl-2 and Bax, the redox related proteins including 4-HNE and arcolein adducts and the inactive glycogen synthase kinase 3β (GSK-3β) and protein kinase B (AKT) in PFC and HPC in rats were investigated by western blot.4 rats in each group were perfused transcardially for preparing paraffin tissue sections. The neurons injury was observed with Nissl staining and neuron apoptosis was dectected with TUNEL staining in the PFC and HPC of rat brains.The third batch of 48 rats was randomly divided into 4 groups of 12 each, i.e. control,800 mg/kg bw 1-BP (diluted in corn oil),100 mg/kg bw PDTC (dissolved in saline) and 100mg/kg bw PDTC+800 mg/kg bw 1-BP.1-BP was gavaged and PDTC was injected intraperitoneally 1 hour prior to 1-BP for consecutive 13 days. Animals in control group were treated equal volume solvent per day. After 7 days of 1-BP exposure, all rats were subjected to MWM. The following after the MWM was same as the second batch.ResultsIn experiment of first batch, compared with control group, the level of ROS was dose-dependently increased in PFC of 1-BP treated rats (p<0.05), and the level of COX-2 mRNA was dose-dependently increased in PFC of 1-BP treated rats (p<0.05). Compared with control group, the expression of caspase-3 and GFAP were dose-dependently up-regulated (p<0.05); the expression of NeuN was dose-dependently down-regulated (p<0.05) and the expression of p-GSK-3β and p-Akt were dose-dependently up-regulated (p<0.05) in PFC of rat brains after 1-BP exposure. Compared with control group, the activation of AS and the neuronal loss were observed with the immunofluroscence double staining and the neurons apoptosis was observed with TUNEL staining in PFC of 1-BP treated group rats.In experiment of second batch, the rats in all groups had tendency of reduced latency and distance to find the hidden platform as training progressed and no significant difference in the decreasing trends among the four groups was observed (p>0.05). The 1-BP-treated rats spent longer period of time and swimming distance in finding the platform than the control rats (p<0.05), and 1-BP-treated rats crossed though the original platform region fewer times in given time (p<0.01). However, compared with 1-BP group, rats pre-treatment with DHA decreased the latency and distance to find the submerged platform (p<0.05) and increased counts in crossing target region (p<0.05). In the PFC and HPC, compared with control group, the expression of Bcl-2 was significantly down-regulated (p<0.05), while cyt c, Bax, cleaved caspase-3 expressions and Bax/Bcl-2 ratio were up-regulated after 1-BP treatment (p<0.05); the levels of p-Akt and p-GSK-3β were significantly elevated (p<0.05); the level of 4-HNE and acrolein modified proteins were significantly increased (p<0.05). While compared with 1-BP group, DHA intervention groups dose-dependently reversed the changes of expressions of apoptosis-related proteins (p<0.05); inhibited the elevation of p-GSK-3β and p-Akt (p<0.05); decreased the 4-HNE and acrolein adducts (p<0.05) in 1-BP-intoxicated rats.1-BP intoxication resulted in decrease in density of Nissl bodies and cytoplasmic vacuolation of pyramidal cells in the CA3 region of HPC and PFC, which was attenuated by DHA pre-treatment. Almost no TUNEL-positive cell appeared in the brain of control rats, after exposed to 1-BP, TUNEL-positive cells were observed in the CA3 region of HPC and PFC, while DHA pre-treatment attenuated 1-BP-induced neuronal apoptosis.In experiment of third batch, the level of ROS and COX-2 mRNA was significantly increased in PFC of 1-BP treated rats (p<0.05). While PDTC intervention decreased the level of ROS and COX-2 mRNA.1-BP also induced the activation of AS accompanied with neuronal loss, and the increase of apoptosis positive cells. While PDTC intervention could alleviate the activation of AS, neuronal loss and neurons apoptosis. Compared with control group, the expression of cleaved caspase-3 and GFAP were significantly elevated (p<0.05); the expression of NeuN was significantly decreased (p<0.05) and the expression of p-GSK-3β and p-Akt were up-regμlated (p<0.05) in brains of 1-BP treated rats. While compared with 1-BP group, PDTC significantly decreased the expression of cleaved caspase-3 and GFAP (p<0.05), increased the expression of NeuN (p<0.05) and decreased the expression of p-GSK-3β and p-Akt (p<0.05).In MWM experiment of third batch, the 1-BP-treated rats spent longer period of time and swimming distance in finding the platform than the control rats (p<0.05), and 1-BP-treated rats crossed though the original platform region fewer times in given time (p<0.01). However, compared with 1-BP group, rats pre-treatment with PDTC decreased the latency and distance to find the submerged platform (p<0.05) and increased counts in crossing target region (p<0.05).Conclusions1. CNS is a sensitive target organ of 1-BP neurotoxicity effect,1-BP exposure could damage the cognitive function of rats.2. The activation of AS mediated neuroinflammation and the inactivation of GSK-3β may be involved in the neurotoxicity of 1-BP.3. Inhibiton of inflammation by the DHA and PDTC treatment could effectively alleviate the 1-BP-induced CNS injury.