| n-Hexane is an organic solvent commonly used in industry.Long-term exposure can cause occupational poisoning of n-hexane in workers.Patients show paresthesia,numbness,and even paralysis of the lower extremities.Since the 1990s,the amount of n-hexane produced and used in China has increased year by year,and n-hexane intoxicaiton once became one of the occupational diseases with the highest incidence in China.In recent years,although new solvents have been used instead of n-hexane,there have been still reported cases of n-hexane intoxicaiton.At present,there is no specific therapy for patients with n-hexane intoxicaiton,and nutritional support therapy is the main treatment.It takes a long time for patients to recover.Therefore,the establishment of biological exposure limit and exploring the mechanism of n-hexane-induced peripheral neuropathy are of great significance for the prevention and clinical treatment of occupational n-hexane intoxication.n-Hexane enters the liver through blood circulation,and is eventually metabolized to 2,5-hexanedione(2,5-HD)under the catalysis of the Cytochrome oxidase CYP2E1 in the liver.Subsequently,2,5-HD can react with amino acid groups in vivo to form 2,5-dimethylpyrrole adducts,which are accumulated in various organs,among which the concentration of pyrrole adducts in the sciatic nerve is the highest,as one of the main target organs.In order to prevent n-hexane-induced peripheral neuropathy,occupational health has established the maximum allowable concentration of n-hexane in the air and 2,5-HD biological exposure thresholds in urine,but none of these indicators can reflect the nerve damage.Non-specific indicators such as nerve injury indicators in serum and electrophysiological examinations used in clinical diagnosis have no significant changes in the early stages of pathogenesis.Therefore,it is necessary to find specific biomarkers that can reflect the nerve injury and can be monitored for a long time.In our previous research,we found that the pyrrole adducts in serum and urine of n-hexane-exposed rats had a good correlation with nerve damage.This study used rats as experimental model to establish a method for measuring hair pyrrole adducts,to monitor the relationship between pyrrole adducts and nerve damage in rats with long-term n-hexane exposure,and to compare the correlation coefficients of serum,urine,and hair pyrrole adducts to nerve damage,to calculate the NOAEL of hair pyrrole adducts and explore its feasibility to be biological exposure limits for prevention of n-hexane intoxicaiton.In order to determine the relationship between pyrrole adducts and n-hexane intoxicaiton,we used CYP2E1 specific inhibitors Diallyl Sulfide to reduce the metabolic activation of n-hexane in rats,and to explore the causal relationship between the accumulations of pyrrole adducts in nerve tissues and neurotoxicity.Meanwhile,early events,key regulatory molecules and injury mechanisms of peripheral neurotoxicity induced by n-hexane were explored,and effective intervention and treatment targets were found,which provided experimental basis for occupational prevention and clinical treatment of people exposed to n-hexane.Meanwhile,this study may also provides ideas and new sight for other organic solvents induced peripheral neuropathyChapter 1 Hair pyrrole adducts serve as a biomarker of peripheral neuropathy induced by n-hexaneObjectiveThe pathogenesis of peripheral nerve injury induced by n-hexane is a chronic process.In previous studies,the concentration of pyrrole adducts in serum and urine samples showed a good correlation with nerve damage in exposed animals.Although the half-life of pyrrole adducts in serum and urine was longer than 2,5-HD,they were not suitable for the prevention and clinical diagnosis of poisoning.Hair samples have good stability and can reflect the long-term accumulation of pyrrole adducts in the body,and may become better biomarkers than blood and urine.Therefore,this study aimed to establish a method for the determination of pyrrole adducts in hair,to monitor changes in rat hair pyrrole adducts and behavioral changes in rats chronically exposed to n-hexane,and to explore hair pyrrole adducts as biomarkers for the prevention of n-hexane-induced peripheral neuropathy.MethodSection 1 Determination of hair pyrrole adducts concentrationUse 2,5-HD to soak rat hair and human hair to establish hair models containing pyrrole adducts;use different methods to digest hair and determine the concentration of pyrrole adducts,select the most suitable and stable method,and calculate the precision and recovery rate;after verifying the method was feasible,the pyrrole adducts in 2,5-HD soaked hair model was measured.Besides,the rat hair were collected and hair pyrrole adducts concentration was measured in the rats exposed to n-hexane chronically.Besides,Spearman’s correlation analysis was performed to calculate the correlation coefficient between gait score and hair pyrrole adducts.Section 2 Determination of no-observe-adverse-effect-level in hair pyrrole adductsThe rats were exposed to different dosages of n-hexane chronically to establish n-hexane-induced peripheral neuropathy models;the changes in gait scores,rota-rod latency,and nerve conduction velocity were monitored during the treatment period and the recovery period;changes in the concentrations of pyrrole adducts in serum,urine and hair were observed;satellite groups for toxicokinetics studies were established to calculate the half-life of pyrrole adducts in bio-samples;multiple regression analysis were performed to select the most appropriate behavioral indicators for correlation analysis,then the partial correlation coefficients of pyrrole adducts in serum,urine,and hair to nerve damage indices were calculated,and the biological samples with the highest partial correlation coefficient were screened to calculate the NOAEL value.ResultSection 1 Determination of hair pyrrole adducts concentrationMeasurement of hair pyrrole adducts was feasible,with high precision and recovery rate;in 2,5-HD soaked hair model,the concentration of pyrrole adducts in hair was in a dose-dependent manner with the 2,5-HD concentration used(P<0.05);there was no difference in the concentrations of pyrrole adducts between human hair and rat hair treated with the same concentration of 2,5-HD;there was no difference between n-hexane soaked hair and the normal control group;there was an accumulation of pyrrole adducts in hair of rats exposed to n-hexane,which was dose-dependent and positively correlated with nerve damage,r=0.8507(P<0.05).Section 2 Determination of NOAEL in hair pyrrole adductsThe concentration of pyrrole adducts in the hair of rats exposed to long-term n-hexane changed slowly,and the half-life(24.9 ± 7.5 week)was longer than that of serum and urine;the nerve injury was positively correlated with the pyrrole adducts concentration(rT=0.683,rR=0.702)(P<0.05),and the partial correlation coefficient of hair pyrrole adducts(treatment phase 0.324,recovery phase 0.322)was higher than that of serum(treatment phase 0.193,recovery phase 0.050)and urine pyrrole adducts(treatment phase 0.120,recovery phase 0.115);based on the absence of any nerve injury in rats treated at dosages of 0.5 g/kg,the NOAEL value of hair pyrrole adducts were calculated as 275.2±61.5 nmol/g.proteinConclusion1.The determination method of hair pyrrole adducts is stable and feasible,and suitable for the determination of n-hexane intoxication;2.The concentration of pyrrole adducts in the hair of the rats changes slowly,with the longest half-life and the strongest correlation with nerve damage,suggesting that it can be used as a specific,sensitive and stable bio-marker;3.The NOAEL value of hair pyrrole adducts is set as 275.2 ± 61.5 nmol/g.protein,providing a reference value for the biological exposure threshold limit in human.Chapter 2 Study on the effect of pyrrole adducts accumulation in n-hexane-induced peripheral neuropathyObjective1.To verify the causal correlation between pyrrole adducts and n-hexane induced neuropathy;2.To verify the feasibility of NOAEL of hair pyrrole adducts.3.To study the effect of calcium related signal pathway in n-hexane-induced peripheral neuropathy.MethodSection 1 Study on mechanism of diallyl sulfide attenuating n-hexane induced neuropathyAn animal model was established by using n-hexane to treat rats,and DAS was administered at a dose of 50 or 100 mg/kg 2 hours in advance.The behavioral changes of rats in each group were monitored to evaluate the effect of DAS.The concentrations of pyrrole adducts in rat serum,urine,hair,and sciatic nerve were detected to verify the feasibility of hair pyrrole adducts NOAEL;a toxicokinetics study was conducted in the medium of the treatment to investigate effect of DAS on metabolism of n-hexane in vivo.After the treatment,the sciatic nerves of rats were collected to make pathological sections to observe the sciatic nerve injury,and the livers of rats were collected to detect the expression and activity of related metabolic enzymes.Section 2 Study on mechanism of diallyl sulfide enhancing 2,5-HD neurotoxicityModels were established by exposing rats to n-hexane or 2,5-HD,and pre-treatment of DAS at a dosage at 100 mg/kg was administrated in advance;the changes in gait score and rota-rod latency of rats were monitored;a satellite group was set for toxicokinetics studies to investigate the effect of DAS on the metabolism of n-hexane and 2,5-HD;at the end point of the treatment,rat liver,kidney,spinal cord,and sciatic nerve measurements were collected and the concentrations of pyrrole adducts were measured;sciatic nerve pathological sections were made to check nerve damage and the expression of myelin-related proteins were detected;the expression of calpain and calcium-dependent autophagy related proteins in spinal cord and sciatic nerve were detected;2,5-HD treated N2a cell models were established,DAS and chloroquine were pre-treated for intervention treatment.Cell damage was observed,and cell survival rate,axon length,and calcium ion concentration were measured.ResultSection 1 Study on mechanism of diallyl sulfide attenuating n-hexane induced neuropathyBehavioral changes:compared with the model group,the behavioral performances of rats in DAS intervention group were significantly improved,the gait score was decreased,the latency was increased,and the nerve conduction velocity was increased(P<0.05);Pathological examination:DAS intervention reduced the sciatic nerve injury in rats,which was manifested as the vacuole-like lesions were significantly reduced in sciatic nerves,and the tissue structure was more dense and complete;Pyrrole adducts concentration:compared with the n-hexane model group,the concentrations of serum,urine,hair,and sciatic nerve pyrrole adducts in the DAS intervention group were decreased in a dose-dependent manner.(P<0.05);Metabolic changes:DAS intervention reduced the metabolic activation of n-hexane in rats,by which the levels of 2,5-HD and pyrrole adducts in serum decreased,besides,2,5-HD and pyrrole adducts in the serum of DAS intervention group showed a shorter half-life and are dose-dependent(P<0.05);Metabolic enzyme expression and activity:compared with the n-hexane model group,DAS intervention reduced the expression and activity of CYP2E1 in the liver,showing a dose-dependent manner(P<0.05);compared with the n-hexane model group,DAS intervention increased the expression and activity of NQO1 in the liver in a dose-dependent manner(P<0.05);compared with the n-hexane model group,DAS intervention also increased the expression and activity of GSTT1 in the liver in a dose-dependent manner(P<0.05).Section 2 Study on mechanism of diallyl sulfide enhancing 2,5-HD neurotoxicityBehavioral changes:compared with the 2,5-HD model group,the nerve damage in the DAS plus 2,5-HD group developed faster in the DAS intervention group,and the animals were completely paralyzed in the third week,while the behavioral performance of the DAS plus n-hexane group did not appear abnormal changes;Pyrrole adducts concentration:the concentration of pyrrole adducts in the sciatic nerve of rats in the DAS plus 2,5-HD group was twice that of the 2,5-HD model group,while the concentration of pyrrole adducts in the sciatic nerve of the rats form DAS plus n-hexane group decreased;Metabolic changes:DAS reduced the metabolic activation of n-hexane and reduced the formation of 2,5-HD and pyrrole adducts,but showed no direct effect on 2,5-HD;Pathological examination:the sciatic nerve of 2,5-HD and n-hexane model group showed structural disturbances and vacuole-like changes,the structure of rats form DAS plus 2,5-HD group was more disordered,while the DAS plus n-hexane group showed no significant damage;compared with that of 2,5-HD group,DAS intervention increased the myelin-related protein MBP in the DAS plus 2,5-HD group by 95.8%;Calcium related pathway protein expression:in 2,5-HD and n-hexane model group,calpain was activated by the system,and DAS intervention further increased m-calpain,μ-calpain protein expression;calcium-dependent autophagy in the 2,5,HD model group and n-hexane model group sciatic nerve was activated,and the intervention of DAS further increased the expression of autophagy-related proteins;the results of cell experiments showed that pre-treatment with DAS caused a shorten axon in N2a cells and a elevated calcium concentrations in DAS plus 2,5-HD groups in a dose-dependent manner(P<0.05),while axon length was increased in the chloroquine plus 2,5-HD group(P<0.05).Conclusion1.The accumulation of pyrrole adducts in the sciatic nerve leads to nerve damage in a dose-dependent manner;2.DAS protects the sciatic nerve by inhibiting the expression and activity of CYP2E1 in the liver of the rat,reducing the formation of 2,5-HD in the body,thereby reducing the accumulation of pyrrole adducts,and show a potential to prevent n-hexane-induced neuropathy,but the usage of DAS must be ahead of n-hexane exposure.3.Pyrrole adducts accumulation may increases the intracellular calcium and activate of calpain system in sciatic nerves to cause impairments,over-activation of calcium dependent autophagy may aggravate the impairments.Chapter 3 The role of phosphorylated TDP43 accumulation in peripheral nerve injury induced by n-hexaneObjectiveThis study aimed to detect the accumulation of pyrrole adducts and expression of TDP43 and other related proteins in the sciatic nerve of rats,and to explore the signal pathway and pathogenic mechanism of pyrrole adducts in n-hexane-induced peripheral neuropathy,supporting occupational prevention and clinical treatment.MethodA model of rats was built by treating different dosages of 2,5-HD;changes in gait scores and rota-rod latency of rats in each group were monitored during the treatment;after the treatment,pyrrole adducts in serum,hair,and sciatic nerve were determined;pathological sections of rat sciatic nerve were made to detect nerve damage,and immunohistochemical staining was used to detect p-TDP43 accumulation;western blots were used to detect the expression of TDP43,p-TDP43,endoplasmic reticulum stress related proteins,calpains and axon damage proteins;correlation analysis of rat gait scores,sciatic nerve pyrrole adducts,and p-TDP43 protein expression,were performed.ResultBehavioral changes:compared with the rats of control group,the 2,5-HD treated rats showed an increase in gait score and a decrease in the latency time,which were dose-dependent(P<0.05);Pyrrole adducts changes:compared with the rats of control group,the pyrrole adducts concentration in serum,hair and sciatic nerve of 2,5-HD treated rats increased in a dose-dependent manner(P<0.05);Sicatic nerve injury:the sciatic nerve sections of the experimental group showed structural disorders and p-TDP43 accumulation in a dose-dependent manner(P<0.05);TDP43 expression:compared with the control group,the expression of TDP43,p-TDP43 in the sciatic nerve in the 2,5-HD exposure group increased,besides,the proportion of poly p-TDP43/mono TDP43 also increased,in a dose-dependent manner(P<0.05);ER stress related protein:compared with the control group,the expression of PERK in the medium-and high-dose 2,5-HD treated groups increased,the expression of ATF4 and CHOP also increased(P<0.05);Calpain expression:compared with the control,the p,-calpain in the high-dosage group increased by 23.1%,the m-calpain in the 2,5-HD groups increased,and the calpastain in the 2,5-HD groups decreased(P<0.05);Axon damage protein:compared with the control:The expression of axon damage protein SARM1 in the medium and high dosage group increased,and the expression of axon protection protein STMN2 and NMNAT2 decreased in all 2,5-HD groups(P<0.05);Correlation analysis:p-TDP43 expression in the sciatic nerve was positively correlated with sciatic nerve pyrrole adducts concentration and gait score(P<0.05)Conclusion1.An abnormal accumulation of p-TDP43 is observed in the sciatic nerve of 2,5-HD-expostreated rats and is positively correlated with nerve damage.2.The accumulation of p-TDP43 is dose-dependent and also positively correlated with pyrrole adducts in the sciatic nerve.There is an interaction between the abnormal accumulation of p-TDP43 and the pyrrole adducts.3.Pyrrole adduct may trigger the abnormal p-TDP43 aggregation in sciatic nerves,which induced endoplasmic reticulum stress and increased intracellular calcium concentration,further activates the calpain system to cause damage to nerve cells. |
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