Peripheral Neurotoxicity Induced By 1-bromopropane And Its Mechanism

1-Bromopropane(1-BP),an alternative to ozone depleting solvent(ODS),has been widely used as industrial organic solvent and dry cleaning agent.In recent years,the production and usage of 1-BP has kept rising in China,accordingly,the occupational populations exposed to 1-BP is also gradually increased.In addition to cases of poisoning from the United States and Japan,the 1-BP poisoning patients in China have gradually been reported.In 2013,1-BP poisoning was included in the new edition of the Classification and Catalogue of Occupational Diseases in China.Generally,most of the patients are hospitalized due to severe peripheral nerve injury,characterized by irreversible loss of vibration sense,anesthesia or dysesthesia in low extremities,ataxia,gait abnormality and even paralysis.However,the molecular mechanisms of 1-BP peripheral neurotoxicity are not clear,and no effective treatment is available at present.It is generally accepted that the axons are the primary targets for neurotoxic organic solvents.While based on the symptoms,signs,poor prognosis and sural nerve biopsy of 1-BP poisoning patients,it is speculated that the 1-BP may resulted in the neuronal damage,other than just axonal toxicity,which differ from other neurotoxic organic solvents induced peripheral neurotoxicity.Previous studies observed that the 1-BP could result in the granule cells and Purkinje cell degeneration in cerebellum,astroglia and microglia activation,as well as oxidative stress.We also fund that 1-BP exposure could induce neuroinflammation,accompanied with the neuron loss in prefrontal cortex and hippocampus,and cognitive dysfunction in expremental animals.Whether the 1-BP result in the neurons degeneration in motor cortex and the corresponding mechanisms have not been reported so far.Studies have shown that the metabolism of 1-BP is mainly catalyced by the cytochrome P4502E1(CYP2E1)and produced a variety of oxidative active metabolites.This study aims to explore the role of oxidative mechanisms in 1-BP neurotoxicity by using edaravone(EDV,free radical scavenger)and allyl sulfide(CYP2E1 specific inhibitor).It would be beneficial to intervention and treatment of 1-BP poisoning,to provid the reliable data for development and improvement of the protection measures for occupational population,as well as provide the ideas and clues for other organic solvents induced neuropathy.Part I The effects of free-radical scavenger edaravone on the peripheral neurotoxicity induced by 1-bromopropane in ratsObjectiveEDV,a free-radical scavenger,has been widely used in patients with Cerebral Ischemic Stroke(CIS)in clinic.EDV has also shown neuroprotective effects in animal models of neurodegenerative diseases such as Amyotrophic lateral sclerosis(ALS),Alzheimer's disease(AD)and Parkinson's disease(PD).To data,EDV exerts antioxidant effects by inhibiting hydroxyl radical-dependent and independent lipid peroxidation,reduces downstream neuroinflammation and neuronal loss in neurodegenerative injuries.Therefore,the present study aims to explore whether the removal of oxidative free radicals produced by 1-BP metabolism can improve the neurotoxicity of 1-BP by means of EDV.Methods1.1 Animal grouping and treatment:After acclimation for 7 days,a total of 72 SPF male Wistar rats(200±20 g)were randomly divided into six groups(n=12):200 mg/kg.bw 1-BP group,400 mg/kg.bw 1-BP group,800 mg/kg.bw 1-BP group,800 mg/kg.bw 1-BP+ 6 mg/kg.bw EDV group,6 mg/kg.bw EDV group and control group.EDV administration was 4-hour in advance of 1-BP.1-BP(orally)and EDV(intraperitoneally)were administrated once daily for 6 weeks.The hind limb grip strength of rats in each group were measured once a week.1.2 Morphologic observation:At the end of the experiment,the brain of rats was harvested and were sliced continuous coronal sections of 40 ?m thickness with freezing microtome.Nissl staining were used to observe the density of nissl body and the neuron injury.Immunohistochemistry staining was used to detect the neuron loss and microglia and astrocytes activation.Double-immunofluorescence staining was used to detect neuron apoptosis in motor cortex of rat brain.1.3 Biochemical index detection:The levels of nitric oxide(NO)and nitric oxide synthase(NOS)activity were determined respectively with commercial kits according to the manufacturer's instruction.1.4 Western blot analysis:The motor area of brain sample preparation and western blot were performed to detect the protein expression of NeuN,ionized calcium binding adapter molecule 1(Iba-1),glial fibrillary acidic protein(GFAP),cysteinyl aspartate specific proteinase-3(Caspase-3),thioredoxin 1(Trx 1),apoptosis signal-regulating kinase 1(ASK1),p-ASK1(Thr 845),p38,p-p38,3-nitrotyrosine(3-NT).Results1.1 Six weeks 1-BP exposure and EDV treatment showed no effect on the body weight of all rats.Compared with the control group,no significant changes in body weight were found among all groups.Rats intoxicated with 1-BP displayed progressive loss of hind-limb grip strength dose-dependently.Administration of EDV improved the behavioral performance of 1-BP-exposed rats by showing recovered hind-limb grip strength compared with 1-BP alone group.1.2 As shown in the nissl staining,the staining of nissl bodies of motor neurons in 400 and 800 mg/kg.bw 1-BP-treated rats was light,indicating damage of neurons.Administration of EDV significantly reduced neuronal damage induced by 1-BP as shown by recovered thionin staining in motor cortex.1.3 Neurons,microglial and astroglias in motor cortex among groups were further immunostained with antibody against NeuN,Iba-1 and GFAP.As the results shown,compared with vehicle controls,1-BP intoxication decreased the number of NeuN+cells dose-dependently,which was significantly attenuated by EDV.In 1-BP-treated rats,dose-dependently activated microglia and astrocytes were observed in motor cortex.Analysis of Iba-1+ and GFAP+ cell number supported these morphological observations.Compared with 1-BP group,EDV treatment markedly attenuated microglial and astrocytes activation,as shown by a ramified morphology and a reduced numbers of Iba-1+ and GFAP+ cells in motor cortex.Western blot analysis supported the immunofluorescence observations.1.4 Co-staining with NeuN antibody revealed a high expression of Cleaved Caspase-3 in NeuN+ cells in motor cortex of 1-BP-treated rats in the double-immunofluorescence staining.Treatment with EDV markedly reduced 1-BP-induced expression of Cleaved Caspase-3 and number of Cleaved Caspase-3+/NeuN+ cells compared with 1-BP alone group.Western blot analysis further confirmed the inhibitory effects of EDV on 1-BP-induced expression of Cleaved Caspase-3 in motor cortex of rats.1.5 As shown in the double-immunofluorescence staining,1-BP decreased the number of NeuN+ cells but increased the GFAP+ cells(characterized by larger cell body,more branches and glial scar formed)in the same arear of motor cortex.EDV intervention decreased 1-BP-induced neuron loss and astrocyte activation.1.6 1-BP intoxication dose-dependently reduced the expressions of Trx 1,and increased the expression of p-ASK1(Thr845),p-p38 and 3-NT,which were significantly mitigated by EDV in motor cortex of rats.In agreement with elevated Trx 1 expression,EDV administration abrogated 1-BP-induced activation of ASK1 and p38 in motor cortex by showing reduced phosphorylation of ASK1 and p38,decreased the expression of 3-NT in combined EDV and 1-BP rats compared with 1-BP alone group.1.7 Compared with vehicle controls,1-BP intoxication significantly increased the levels of NO in motor cortex of rats.Consistently,the contents of NOS in 1-BP-treated rats were increased dose-dependently.Administration of EDV significantly mitigated the alterations of NO and NOS in 1-BP-treated rats,indicating EDV attenuates 1-BP-induced oxidative stress.Conclusion1.1 Six weeks 1-BP exposure dose-dependently decreased the hindlimb grip strength coupled with dose-dependently neurons loss in motor cortex.EDV improves the neurobehavioral performances and protects against 1-BP-induced neurons loss.1.2 1-BP activated microglia and astrocytes dose-dependently in motor cortex.EDV suppresses 1-BP-induced glial activation in motor cortex.1.3 1-BP elevated NO levels,increased NOS activity and 3-NT expression,which evoked motor cortex oxidative/nitriding stress,activated Trxl-ASKl-p38 MAPK apoptotic signaling pathway and resulted in motor neuron apoptosis.EDV intervention can improve 1-BP-induced oxidation/nitriding stress,Trxl-ASK1-p38 MAPK apoptotic signaling pathway activation and motor neuron apoptosis.Part ? The effects of CYP2E1 inhibitor allyl sulfide on the peripheral neurotoxicity induced by 1-bromopropane in ratsObjectiveAccumulating evidence suggests that cytochrome P4502E1(CYP2E1)is critical for the metabolism of 1-BP.The C2 oxidation of 1-BP catalyzed by CYP2E1 yields the toxic metabolites in liver,1-bromo-2-propanol and 3-bromoacetone,which has been shown to be involved in 1-BP's toxicity.Active products of 1-BP liver metabolism are difficult to withstand long-distance transport and across the blood-brain barrier.The brain in physiological state expresses lower CYP2E1,but both mitochondria and microsomal CYP2E1 in brain can be induced by exogenous chemicals(eg,nicotine and ethanol),which are associated with it's neurotoxicity.As an organic solvent,it is very easy for 1-BP to enter the brain,however,whether the 1-BP entered into the brain could induce brain CYP2E1 expression?Is the inducible expression of CYP2E1 in brain and the brain in situ metabolism of 1-BP a "fuse" that initiates its neurotoxicity?No clear reports have yet been seen.In this experiment,we use allyl sulfide,a specific inhibitor of CYP2E1,to inhibit the activity of CYP2E1 and observe the effects in 1-BP-induced peripheral nerve injury,as well as changes in the expression level of brain CYP2E1.The changes of active metabolites in blood and brain were also analyzed to investigate the role of 1-BP in situ oxidative metabolism in brain in peripheral neurotoxicity.Methods2.1 Animal grouping and treatment:After acclimation for 7 days,a total of 48 SPF male Wistar rats(200±20 g)were randomly divided into four groups(n=12):1-BP group,1-BP+Allyl Sulfide group,Allyl Sulfide group and Control group.1-BP(800 mg/kg.bw)and allyl sulfide(100 mg/kg.bw)were orally treated to the rats in corresponding groups once daily for 9 continuous weeks,control rats were received equivalent volume of corn oil which used as dosing vehicle.Allyl sulfide administration was 4-hour in advance of 1-BP.The neurobehavioral performances,including hind limb grip strength and evaluation of gait score of all rats were measured once a week.At the end of the experiment,observe the paralysis of rats in each group.2.2 Morphologic observation:At the end of experiment,the brains of rat were harvested and were sliced continuous coronal sections of 40 ?m thickness with freezing microtome.Nissl staining was used to observe the density of nissl body and the neuron injury.Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)staining was used to detect neuron apoptosis in motor cortex of rat brain.Immunohistochemistry staining was used to detect the neuron loss and microglia and astrocytes activation.Double-immunofluorescence was used to detect neuron apoptosis,CYP2E1 expression in microglia,microglia M1 and M2 polarization and neuronal oxidative modification damage in motor cortex.2.3 Biochemical index detection:The Malondialdehyde(MDA)and Glutathione(GSH)levels were determined spectrophotometrically respectively with commercial kits according to the manufacturer's instruction.2.4 Western blot analysis:The motor area of brain sample preparation and western blot were performed to detect the protein expression of NeuN,Iba-1,GFAP,Cleaved Caspase-3,CYP2E1,iNOS,Arginase-1(Arg-1),Nuclear factor-KB(NF-?B),p-NF-?B,Trx1,ASK1,p-ASK1(Thr 845),p38,p-p38.2.5 qRT-PCR analysis:qRT-PCR analysis was used to determine the relative mRNA expression of M1(iNOS,Tumor necrosis factor-a(TNF-a),Interleukin-1?(IL-1?),IL-6)and M2(Arg-1,IL-10,IL-4,Transforming growth factor ?1(Tgfbl))marker.2.6 The metabolite of 1-bromopropane analysis by GC-MS:After acclimation for 7 days,a total of 24 SPF male Wistar rats(200± 20 g)were randomly divided into four groups(n=6):1-BP group,1-BP+Allyl Sulfide group,Allyl Sulfide group and Control group.1-BP(800 mg/kg.bw)and allyl sulfide(100 mg/kg.bw)were orally treated to the rats in corresponding groups once daily for 5 continuous days,control rats were received equivalent volume of corn oil which used as dosing vehicle.Allyl sulfide administration was 4-hour in advance of 1-BP.At the end of the experiment,analyze the active metabolites of C2 oxidative metabolism that is mainly catalyzed by CYP2E1,in the blood and brain samples by GC-MS.Results2.1 1-BP exposure slowed the increase of rats weight gain as compared to control rats since the 5th week significantly(p<0.01),which was significantly attenuated by allyl sulfide.2.2 As seen in nissl staining,nissl bodies of motor neurons in 1-BP-treated rats was very light,indicating damage of neurons,administration of allyl sulfide significantly reduced neuronal damage induced by 1-BP as shown by recovered thionin staining in motor cortex.Immunohistochemistry staining indicated that 1-BP intoxication decreased the number of NeuN+ cells,which was also significantly attenuated by allyl sulfide.TUNEL staining was employed to further confirm the motor neuronal apoptosis.1-BP-treated rats displayed an increased number of TUNEL-positive cells in motor cortex compared with vehicle controls.Treatment with allyl sulfide markedly reduced 1-BP-induced apoptosis of neurons as shown by decreased number of TUNEL-positive cells compared with 1-BP alone group.In agreement with morphological observation,western blot revealed that the reduced expression of NeuN in 1-BP-treated rats was recovered by allyl sulfide.2.3 Rats intoxicated with 1-BP displayed significant behavioral deficits including progressive loss of hind-limb grip strength,gait abnormality,and high paralysis rates compared with vehicle controls.Administration of allyl sulfide significantly improved the behavioral performance of 1-BP-exposed rats by showing recovered hind-limb grip strength and gait scores compared with 1-BP alone group.Consistently,1-BP intoxication resulted in 30%paralysis of rats,which was reduced to 10%when allyl sulfide was co-treated with 1-BP.2.4 Activation of microglia in motor cortex was morphologically observed by immunostaining with Iba-1.In 1-BP-treated rats,activated microglia,characterized by a hypertrophied morphology and intensified Iba-1,were observed in motor cortex.Analysis of Iba-1+ cell number supported these morphological observations.Compared with 1-BP group,allyl sulfide treatment markedly attenuated microglial activation,as shown by a ramified morphology and a reduced numbers of Iba-1+ cells in motor cortex.Western blot analysis also showed a reduced expression of Iba-1 in motor cortex in combined allyl sulfide and 1-BP-treated rats compared with 1-BP alone group.Since activated microglia are capable of inducing neurotoxic reactive astrocyte,astroglial activation was further determined.Consistently,astroglial activation was also mitigated by allyl sulfide.2.5 Double immunofluorescence staining by using antibodies against CYP2E1 and Iba-1 or GFAP,revealed a co-localization of CYP2E1 and Iba-1 but not GFAP,suggesting that CYP2E1 expressed in microglia but not astroglia.Compared with vehicle controls,1-BP intoxication elevated the expressions of CYP2E1 in microglia.However,the elevated expressions of CYP2E1 in microglia induced by 1-BP were not observed in combined allyl sulfide and 1-BP-treated rats.Western blot analysis supported the immunofluorescence observations.2.6 As shown by double immunofluorescence staining,compared with vehicle control,a high expression of iNOS was manifested in motor cortex of 1-BP-treated rats.In contrast to iNOS,the expression of Arg-1 was reduced by 1-BP,indicating an imbalanced ratio of M1/M2 microglia in 1-BP-treated rats.Interestingly,allyl sulfide treatment reversed the alterations of both iNOS and Arg-1 induced by 1-BP as shown by decreased iNOS and increased Arg-1 expressions in motor cortex of combined allyl sulfide and 1-BP-treated rats compared with 1-BP alone group.Western bolt and qRT-PCR analysis further supported these finds by showing reduced levels of iNOS and mRNA transcripts of iNOS,TNF-?,IL-1?,while elevated protein and mRNA levels of Arg-1 in motor cortex of allyl sulfide and 1-BP-treated rats compared with 1-BP alone group.2.7 1-BP significantly increased the levels of phosphorylated NF-?B.Whereas,rats co-treated with 1-BP and allyl sulfide displayed reduced phosphorylation of NF-?B,suggesting that allyl sulfide inhibits 1-BP-induced NF-?B activation.1-BP intoxication reduced the expressions of Trx1,which was significantly mitigated by allyl sulfide in motor cortex of rats.In agreement with elevated Trxl expression,allyl sulfide administration abrogated 1-BP-induced activation of ASK1 and p38 in motor cortex by showing reduced phosphorylation of ASK1 and p38 in combined allyl sulfide and 1-BP rats compared with 1-BP alone group.2.8 Compared with vehicle controls,1-BP intoxication significantly decreased the levels of GSH in motor cortex of rats.Consistently,the contents of MDA in 1-BP-treated rats were increased.Administration of allyl sulfide significantly mitigated the alterations of GSH and MDA in 1-BP-treated rats,indicating allyl sulfide attenuates 1-BP-induced oxidative stress.The double-immunofluorescence staining by using antibodies against NeuN and 4-HNE was initially performed.1-BP-treated rats displayed an elevated expression of 4-HNE in motor cortex of rats compared with vehicle controls.Co-staining with NeuN antibody revealed a high expression of 4-HNE in NeuN+ cells in motor cortex of 1-BP-treated rats.Treatment with allyl sulfide markedly reduced number of 4-HNE+/NeuN+ cells compared with 1-BP alone group.2.9 The results of GC-MS indicated that allyl sulfide decreased levels of 1-bromo-2-propanol in 1-BP exposure group.Conclusion2.1 Rats intoxicated with 1-BP for 9 weeks displayed significant behavioral deficits including progressive loss of hind-limb grip strength,gait abnormality,and high paralysis rates,coupled with neuronal apoptosis in motor cortex.It indicated that CNS motor cortex apoptosis is one of the mechanisms of 1-BP peripheral neuropathy.2.2.1-BP intoxication elevated the expressions of CYP2E1 in microglia,resulted in an imbalanced ratio of M1/M2 microglia.Activated microglia inducing neurotoxic reactive astrocyte and oxidative/nitrosative stress,which activated NF-?B and Trx-ASK1-p38 MAPK pathway,resulted in motor neuron apoptosis.2.3 1-BP exposure increased the levels of 1-bromo-2-propanol in the brain and blood of rats.2.4 Allyl sulfide suppresses the expressions of CYP2E1 in microglia,attenuates 1-BP-induced glial activation and M1/M2 imbalance in motor cortex,blocks 1-BP-induced oxidative/nitrosative stress and motor neuron apoptosis.Part ? The effects of exosomes released from microglia on the 1-bromopropane induced neuronal damagesObjectiveIn recent years,exosome-mediated microglia-neuron communication has been thought to be closely related to the progressive development of neurotoxicity in the brain.Studies have shown that alpha-synuclein oligomers associated with PD pathogenesis can be secreted extracellularly by exosomes or directly in free form,and that alpha-synuclein in exosomes is more likely to be affected somatic cell uptake leads to the development of PD.This study was to investigate whether exosomes are involved in neuronal damage caused by Ml polarization in microglia.Based on the above studies,this study treated 1-BP and allyl sulfide to the in vitro cultured BV-2 microglia,observed the M1/M2 polarization of BV-2 cells,and CYP2E1 expression in BV-2 cells.Thereafter,1-bromo-2-propanol,the 1-BP active metabolite,was used to treat BV-2 microglia in vitro.The secreted inflammatory exosomes from 1-bromo-2-propanol activated BV-2 microglia were collected and labeled and added to the primary cerebral cortex neuronal culture system of newborn rat,in order to observe the uptake of labeled exosomes by neurons and explore the mechanism of 1-BP neurotoxicity.Methods3.1 The M1/M2 polarization and CYP2E1 expression of BV-2 cells:Control,1-BP,1-BP+Allyl Sulfide,Allyl Sulfide,and LPS five administration groups were set.1-BP,allyl sulfide and LPS were added into the culture,and cell slides were prepared.After 24 hours of administration,double immunofluorescence was used to detect the M1/M2 polarization and the expression of CYP2E1 of BV-2 cells.3.2 Exosomes collection:A control group and a 1-bromo-2-propanol treatment group were set.24 hours after administration,serum-free medium containing 100 ?M ATP was exchanged and incubated at 37? for 30 minutes.The cells were washed twice with PBS and added to serum-free medium for further 48 hours.The culture solution was collected and transferred to a centrifuge tube.Exosomes were collected by gradient centrifugation.3.3 Exosomes identification:Western blot was used to detect the expression of positive marker proteins ALIX and Flotillin-2,and the expression of inflammatory factor IL-1? in exosomes.Exosome morphology was measured using a Hitachi HT-7700 transmission electron microscope.3.4 Exosomes labeled with fluorescent probe Dil:The isolated exosomes were labeled with a cell membrane red lipophilic fluorescent probe Dil.The above collected exosomes were suspended in sterile PBS and incubated with appropriate amount of Dil for 10 minutes.The exosomes were washed three times with sterile PBS to remove free DiI dyes and impurities.3.5 Exosomes uptake by neuron:The Dil-labeled exosomes(2 ?g/mL)extracted from the control group and the treatment group were added to the primary neuron culture medium,incubated with serum-free medium for 24 hours,washed three times with PBS to make cell slides.Then the neurons was labeled by MAP-2 and observed its morphologic changes.Results3.1 Immunofluorescence staining showed that 1-BP exposure resulted in M1 polarization of BV-2 microglia,and an increased expression of CYP2E1 was observed in M1 microglia.Allyl sulfide intervention reversed the polarization of M1/M2 in BV-2 microglia induced by 1-BP and reduced 1-BP-induced induction of CYP2E1 in Ml polarizated BV-2 microglia.3.2 1-Bromo-2-propanol exposure resulted in enhanced polarization of Ml but reduced polarization of M2 in BV-2 microglia.3.3 Western blot indicated that,compared with the control group,the expression of positive marker proteins ALIX,Flotillin-2 and the inflammatory factor IL-1? was higher in exosomes from 1-bromo-2-propanol group.3.4 Immunofluorescence staining showed that DiI-labeled exosomes were observed in primary cultured neurons.Compared with the control group,more exosomes from 1-bromo-2-propanol are observed in neurons,and showed damage to the cell body and neuronal axis.Conclusion3.1 Allyl sulfide inhibited the induction of CYP2E1 in BV-2 cells induced by 1-BP,and inhibited 1-BP induced BV-2 cells M1 polarization.3.2 1-Bromo-2-propanol,an active metabolite of 1-BP metabolized by CYP2E1,causes BV-2 microglia activation and M1 polarization.Ml-polarized BV-2 cells release a large amount of exosomes containing inflammatory mediators and harmful components,and exosomes in motion are taken up by neurons,resulting in neuronal damage.