Increased Oxidatively Modified Proteins In Nervous Tissue Of Rats Intoxicated With1-Bromopropane

Objective1-Bromopropane (1-BP) has been introduced as a substitute for ozone depleting substance (ODS) which could damage the ozone layer, because it can possesses favorable physical properties characterized by high volatility, low flammability, and photochemical breakdown that allows it to have a relatively short atmospheric half-life, thus possibly decrease its ozone-damaging capacity. Now1-BP widely applied in cleaning agent for metal, electronics and optical instruments. It is also used for intermediates in the synthesis of pharmaceuticals, insecticides, quaternary ammonium compounds, flavors, fragrances, and as a solvent for fats, waxes or resins. The neurotoxic effects of this agent have been described in humans and experimental animals. As a result, with its growing rapidly use, the number of workers exposed occupationally to1-BP expands quickly. However, there is no sufficient toxic data for1-BP risk characterization until now.The underlying mechanism of1-BP neurotoxicity in humans is not well understood. Growing animals experimental study indicates that oxidative stress and lipid peroxidation is possibly associated with the neurotoxicity induced by1-BP. The depletion of GSH in liver and brain of rats by1-BP had been reported. Recently protein genome analysis found that the protein expression profile in hippocampus of rats associated with oxidative stress changed due to1-BP exposure.But there was a few studies on the protein adduct levels of4-hydroxiynonenal (4-HNE), 4-hydroxy-2-hexenal (4-HHE),malondialdehyde (MDA) and arcolein of cerebral cord, spinal cord and sciatic nerve in rats in the neuropathy caused by1-BP.To observe the peripheral neurotoxicity of1-BP, we develop the animal model of peripheral neuropathy induced by1-BP, and neurobehavioral indexes were detected at regular intervals.The supernatant of their homogenate was collected to detect the changes of the lipid peroxidation adducts respectively. The aim of our study is to illustrate that the dysfunction of oxidative stress and lipid peroxidation are attributed to the damage of peripheral neuropathy.MethodK40Male Wistar rats were randomly divided into four groups with10in each, i.e.1-BP low-dose group (200mg/kg.bw), medium-dose group (400mg/kg.bw), high-dose group (800mg/kg.bw) and control group. Animals in each group were given1-BP by gavage in accordance with their own dosage in volume0.2mL/100g.bw for16weeks. Neurobehavioral indexes including gait score, hindlimb grip strength and hindlimb landing foot splay was recorded periodically. Hematological and biochemical parameters were also determined.2> The tissues of cerebral cord, spinal cord and sciatic nerve, were quickly dissected after sacrificed and homogenized in ice-cold homogenizing buffer and then centrifuged at150,000×g for30min at4℃. The protein adduct levels of4-HNE^4-HHE、MDA and arcolein of cerebral cord, spinal cord and sciatic nerve in rats were investigated by western blotting.3、The relative levels of4-HNE protein adduct of cerebral cord, spinal cord and sciatic nerve after adding in1-BP and4-HNE in vitro in rats were also determined by immunoblotting.Results1、The changes of neurobehavioral indexesAfter treatment with1-BP,gait scores of animals in1-BP treatment groups were higher than those of the control group, with the first appearance of significant differences in high-dose, medium-dose and low-dose groups at the end of8,14and15weeks respectively (P<0.05,P<0.01), and these differences continued to the end. Rats exposure to high-dose1-BP resulted in the significant decrease of hindlimb grip strength since the end of9week (P<0.05, P<0.01), and progressed into hindlimb paralysis at the end of16week. The hindlimb grip strength of rats in medium-dose and low-dose groups were decreased to72.6%and91.2%of the control value at the end of16week respectively (P<0.01, P<0.05). Hindlimb landing foot splay of rats in high-dose group and medium-dose group were significantly higher than those of control group since the end of12and14weeks respectively (P<0.05,P<0.01). Rats in high-dose and medium-dose group at the end of8week and rats in all three1-BP exposure groups at the end of16week showed decrease of ALT respectively, and there were significant differences compared to the control group (P<0.01).2、The relative levels of protein adductThe results of western blotting showed that compared with control group, the4-HNE protein adduct levels of the high-dose1-BP group was significantly increased by27.57%(P<0.01), while medium-dose and low-dose groups were only incresed by11.82%and20.94%which had no significant differences with the control group (P>0.05) in cerebral cord of rats. While the4-HNE protein adduct levels of low-dose, medium-dose and high-dose groups in spinal cord of rats respectively incresed by5.37%,16.82%,17.82%which had no significant differences with the control group. But in sciatic nerve, the4-HNE protein adduct levels of the low high-dose group was only increased by17.83%, while medium-dose and high-dose groups were significantly incresed by64.64%(P<0.01) and117.59%(P<0.01)Compared with control group, the MDA protein adduct levels of the high-dose1-BP group was significantly increased by179.38%(.P<0.01), while medium-dose and low-dose groups were only incresed by24.08%and30.14%which had no significant differences with the control group (P>0.05) in cerebral cord of rats. While the MDA protein adduct levels of low-dose, medium-dose and high-dose groups in spinal cord of rats respectively incresed by4.02%、33.48%、53.34%which had no significant differences with the control group. But in sciatic nerve, the MDA protein adduct levels of the low high-dose group has no difference with the control group, while medium-dose and high-dose groups were significantly incresed by46.03%(P<0.01) and70.13%(P<0.01)Compared with control group, the4-HHE protein adduct levels of the low-dose1-BP group was trimming slightly down which had no significant differences with the control group, the increase of4-HHE protein adduct levels in medium-dose group was not apparently, while the high-dose group were incresed by16.40%(P<0.01) in cerebral cord of rats. While the4-HHE protein adduct levels of low-dose, medium-dose and high-dose groups in spinal cord of rats respectively incresed by54.78%,44.21%,45.52%which had no significant differences with the control group. But in sciatic nerve, the4-HHE protein adduct levels of the low high-dose group was only increased by37.26%, while medium-dose and high-dose groups were significantly incresed by67.58%(P<0.01) and59.34%(P<0.01)Compared with control group, the arcolein protein adduct levels of low-dose, medium-dose and high-dose groups in cerebral cord of rats respectively incresed by7.97%(P<0.05),22.09%(P<0.01),29.99%(P<0.01). While in spinal cord, the arcolein protein adduct levels of low-dose, medium-dose and high-dose groups in cerebral cord of rats respectively incresed by54.78%、44.21%、45.52%which had no significant differences with the control group. But the arcolein protein adduct levels of low-dose, medium-dose and high-dose groups in sciatic nerve were reduced by38.65%(P<0.01)、35.07%(P<0.01)、49.05%(P<0.01)3. The effects of1-BP on the formation of4-HNE protein adduct of rat nervous tissue in vitroThe4-HNE protein adduct is nearly undetectable in experimental system in vitro with lmg/ml protein coming from cortex, spinal cord and sciatic nerve or rat. When1mg/ml cortex, spinal cord and sciatic nerve protein were cultured with50uM1-BP, there was a little of4-HNE protein adduct formation. Compared with4-HNE group, the4-HNE protein adduct levels in4-HNE+25uM1-BP、4-HNE+50uM1-BP and4-HNE+100uM1-BP groups respectively incresed by18.87%,51.01%(P<0.01) and58.08%(P<0.01)of cerebral cord. While in spinal cord, the4-HNE protein adduct levels in4-HNE+25uM1-BP、4-HNE+50uM1-BP and4-HNE+100uM1-BP groups respectively incresed by83.25%(P<0.01)、116.11%(P<0.01) and211.73%(P<0.01). And in sciatic nerve, the4-HNE protein adduct levels in4-HNE+25uM1-BP,4-HNE+50uM1-BP and4-HNE+100uM1-BP groups respectively incresed by35.32%、72.21%(P<0.01) and128.76%(P<0.01) Conclusion1、Nervous system might be the most sensitive target of the1-BP, and1-BP exposure could induce peripheral neuropathy of rats.2、1-BP could significantly improve the protein adduct levels of4-HNE4-HHH、MDA of cerebral cord, spinal cord and sciatic nerve in rats.3.1-BP could enhance the formation of4-HNE protein adducts in nervous protein of rats with dose dependency.