| Objective:1-bromopropane (1-BP), a kind of highly volatile organic compound, has a relatively short half-life of about 15 days in the atmosphere. In middle latitude area,1-BP has a ozone depleting potential (ODP) of 0.013-0.018, lower than that of ozone-depleting solvents (ODS) such as chlorofluorocarbons (CFCs) and hydrochlorofluorocarbons (HCFCs). Therefore,1-BP could be used as an alternative of them in industry.1-BP can be used for cleaning metal, plastics and optical element as well as solvent for fat, wax and resin.1-BP is also intermediate product in manufacture of pesticides, Pharmaceuticals and flavors. In human patients and intoxicated animals,1-BP could induce deficits in peripheral nervous system (PNS) including sensory and motor dysfunction, and impairment in central nervous system (CNS) including memory loss, fatigue and depression. Studies showed that CNS symptoms occurred before PNS abnormality and that memory deficits could be accurately quantified by Morris Water Maze (MWM) in animal experiments. Thereby, study of dose-response relationship of 1-BP-induced CNS neurotoxicity and its possible mechanisms, through investigation of memory deficits, might be beneficial to early prevention, identification and treatment of 1-BP intoxication.From evidences in neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), it is possible that oxidative stress and memory deficits are causally related. In CNS, glutathione (GSH) is the major antioxidant, playing roles in elimination of reactive oxygen species (ROS) and detoxication of xenobiotics. So stable content of GSH is important in CNS function and GSH depletion could induce deficits. In addition, neuroglobin (Ngb), mainly distributed in brain, is endogenous antioxidant and neuroprotectant. Ngb is closely related to cognitive function. The amount of it and neuroprotective effects are dependent on intracellular redox state. Memory depends on the integration of neuron and memory dysfunctions are often accompanied by neuron injury or loss. The function of neurons is associated with neuron-astrocyte connection. Oxidative injury could activate astrocytes which would then release inflammatory mediators and ROS that could lead to neuronal injury and death. ROS and tumor necrosis factor-a (TNF-a) can be acted as inducers of astrocyte activation. In the current study, rats were randomly separated into 5 groups (n=17) and administered with 0,100,200,400 and 800 mg/kg.bw 1-BP orally for consecutive 12 days. Pathological changes in medial prefrontal cortex (mPFC), Ngb level, GSH metabolism and astrocyte activation were measured to investigate the involvement of oxidative stress and neuroinflammation in 1-BP-induced CNS neurotoxicity.Methods:1. Wistar rats (180-220 g) were randomly separated into 5 groups (n=17) and exposed to 0,100,200,400 and 800 mg/kg bw 1-BP orally everyday for consecutive 12 days. On the 13th experimental day,10 rats in each group were sacrificed and cerebral cortexes were dissected rapidly, frozen in liquid nitrogen and stored in-80℃.7 rats in each group were perfused intracardially with saline at first and then a solution of 4% paraformaldehyde for pathological detection.2. From 8th to 12th experimental day, rats were tested by MWM. In spatial navigation test, escape latency and distance travelled can reflect rat learning ability; in spatial probe trial, number of platform crossing and percentage of time spent in target quadrant are used for evaluation of rat memory.3. Neurons are labeled by anti-NeuN antibody through immunohistochemistry method. Prelimbic area (PL) is chosen for neuron counting.4. GSH content, oxidized glutathione (GSSG) content and glutamate cysteine ligase (GCL) activity are determined by kits. Lipid peroxidation indicators including 4-hydroxy-2-nonenal (4-HNE)-and n-epsilon-hexanoyl-lysine (HEL)-modified proteins, and Ngb are detected by western blot.5. Glial fibrillary acidic protein (GFAP)-positive astrocytes were identified by immunofluorescence. GFAP level is measured by western blot.6. TNF-α mRNA was quantified by qRT-PCR.7. Benchmark dose (BMD) method was applied to calculate BMD and BMD lower confidence limit (BMDL) of endpoints.Results1. 1-BP induced cognitive dysfunction in rats. Compared to control group,200,400 and 800 mg/kg bw 1-BP led to significant increase in escape latency and decrease in percentage of time spent in target quadrant (p<0.05, p<0.01); 400 and 800 mg/kg bw 1-BP resulted in significant increase in distance travelled and decrease in number of platform crossing (p<0.05, p<0.01).2. 1-BP caused neuron loss in layer 5 of PL in mPFC. Compared to control group,100, 200,400 and 800 mg/kg bw 1-BP decreased neuron population in layer 5 of PL in mPFC dose-dependently (p<0.01). There is no significant change in layer2/3 and layer 6.3. 1-BP resulted in oxidative stress in cerebral cortex. Compared to control group,100, 200,400 and 800 mg/kg bw 1-BP led to decrease in GSH content, GSH/GSSG ratio and GCL activity (p<0.01) with increase in GSSG content (p<0.01) significantly. 1-BP induced elevated levels of 4-HNE- and HEL-modified proteins (p<0.01). Ngb level decreased dramatically in rats of 200、400' 800 mg/kg bwl-BP group (p< 0.01).4. 1-BP led to astrocyte activation in cerebral cortex. GFAP-positive cells and GFAP level in cerebral cortex increased dramatically in 1-BP-treated group (p<0.01).5. 1-BP induced elevated level of TNF-α mRNA (p<0.05).6. The BMDL calculated from escape latency, distance travelled, percentage of time spent in target quadrant and number of platform crossing were 49.39、75.90、73.11 and 97.67 mg/kg.bw/day, respectively. The BMDL calculated from GSH content, GSH/GSSG ratio and GCL activity were 59.61、23.40 and 64.85 mg/kg.bw/day, respectively. Among them, GSH/GSSG ratio was the most sensitive endpoint.Conclusion1. 1-BP could induce learning and memory deficits in rats, which might be associated with neuron loss in layer 5 of PL in mPFC.2.1-BP could result in GSH depletion, lipid peroxidation and Ngb decrease, which might have important roles in neuron loss.3.1-BP could lead to astrocyte activation and elevation of mRNA expression of TNF-a, which might be involved in neuron loss as reactive astrocytes would release inflammatory mediators and ROS that could cause neuronal death.4. GSH/GSSG ratio in cerebral cortex is sensitive to 1-BP CNS neurotoxicity... |
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