The Relationship Between Neurotoxicity Induced By Benzo[a]pyrene And CYP1A1 Gene Polymorphism

Benzo(a)pyrene(B[a]P),one of polycyclic aromatic hydrocarbon(PAH)compounds,is product of incomplete combustion of organic matters and is widespread in the environment.The carcinogenicity of B[a]P is well established in human and animal model.Due to the widespread distribution of B[a]P in the environment and the potential exposure for human,the adverse effects of these compounds deserved to be investigated.Although the neurotoxic effect of B[a]P has not drawn much attention,.there were some related data implying that B[a]P showed the neurotoxic effects.when exposed to B[a]P by oral gavage,rats were induced behavioral impairments,and there was a significant correlation between neurotoxic effects and plasma B[a]P concentration,and its metabolite concentration in brain tissue.Coke-production plant workers in Poland developed neurotic syndromes with vegetative disregulation,and a loss of short-term memory;their prevalence depended on the level of exposure to B[a]P.B[a]P is metabolised to active metabolites that are tumourigenic.The bioactivation of B[a]P is catalyzed by some cytochrome P450(CYP)enzymes,particularly CYP 1A1.The brain tissues of various species contain almost all CYP-enzymes found in liver,including CYP1A1.CYP-enzymes in brain were associated with neurotoxic effects of some chemicals,such as lindane and manganes.The correlation between the neurotoxicity of B[a]P and B[a]P metabolite concentrations in brain implied that CYP1A1 may mediate the neurotoxicity of B[a]P.CYP1A1 gene polymorphisms have been studied in relation to various kinds of cancers.Our hypothesis is that CYP1A1 gene polymorphisms wound modulates the neurotoxicity of B[a]P.The neurotoxicity of B[a]P may be a important occupational risk like its carcinogenicity for occupational exposed populations.In this research,occupational exposed population,experimental animals and primarily cultured neuron exposed to B[a]P were investigated to preliminarily elucidate the mechanism of neurotoxicity of B[a]P and its association with CYP1A1 gene polymorphisms.The research was divided into three parts.PartⅠNeurotoxicity study on occupational population exposed to B[a]P1.neurobehavioral function of coke oven wokers200 healthy adult male coke oven workers were selected from coke plant of a state-owned enterprise in Taiyuan City.88 controls unexposed to polycyclic aromatic hydrocarbons(PAHs) occupationally were selected from the same enterprise.All the subjects participated this investigation freely according to their consent.Concentration of B[a]P in the working environment was monitored by High Performance Liquid Chromatography(HPLC).Urine samples were collected immediately after working shift.The level of urinary 1-hydroxypyrene was determined by High Performance Liquid Chromatography(HPLC).General informations of workers correlated to the investigation were collected by query and filled in the questionnaire according to the same criteria by well-trained investigators.WHO recommended neurobehavioral core test battery(NCTB)was performed on coke oven workers and controls to test the neurobehavioral changes and the mood state.The concentration of B[a]P at oven bottom,oven side and oven top were 0.0195μg/m3,,0.186μg/m3 and 1.624μg/m3 respectively,and Concentration of B[a]P at oven side and oven top were higher than the occupational hygiene criterion.Urinary 1-hydroxypyrene were significant difference between exposure group(3.42±0.98μmol/mol creatinine)and control group(2.75±1.09μmol/mol creatinine).No significant differences were found between exposure group and control group on age,length of service,smoking,drinking and unhealthy food consumption;however,compared to the controls,the scores of the coke oven workers were lower in the total digital span,the forward digital span,right dotting and total dotting,and the differences showed statistical significance(P＜0.05).Significant difference of the total digital span,the forward digital span, backward digital span,digit symbol and Benton visual retentions existed in different urinary 1-hydroxypyrene concentration groups and showed a dose-response tendency.Results of multiple stepwise regression analysis and correlation analysis showed that the level of urinary 1-hydroxypyrene affect memory and perception of coke oven workers and there wre negative correlations between the levels of urinary 1-hydroxypyrene and changes in neurobehavioral function.It was suggested that B[a]P wound mainly causes decrease of memory and perception in coke oven workers..2.Function of autonomic nervous system(ANS)in coke oven wokersFunction of autonomic nervous system(ANS)of 288 subjiects were measured with the method recommended by Ewing DJ,which consists of 4 tests:Valaslva Manoeuvre heart rate variation(HR-V),variation of heart rate when deeply breathing(HR-DB),variation of heart rate when instantly standing up(HR-IS,including RR30:15 and RRmax:min)and variation of blood pressure when instantly standing up(BP-IS).Compared to controls,HR-V decreased significantly in coke oven workers(p＜0.01),which represents modulation of parasympathetic nervous function.But no statistical differences were found in HR-DB,RR30:15,RRmax:min and BP-IS between exposed groups and control group(p＞0.05).Significant difference of HR-V existed in different urinary 1-hydroxypyrene concentration groups.Results of multiple linear stepwise regression demonstrated that the level of urinary 1-hydroxypyrene and level of education affect HR-V.The results indicated that B[a]P affected autonomic nervous function of coke oven workers by mainly down- regulating parasympathetic nervous function.3.Changes of electroencephalogram and brain electrical activity mapping in coke oven wokers45 subjects,as a exposed group,were randomly selected from exposed groups according to their type of work in production and age,25 controls were chosen from unexposed group according to the same principal.All subjects were conducted electroencephalogram and brain electrical activity mapping.Compare with controls,the ratio of abnormal EEG increased,brain electrical activity mapping showed all frequency bands energy values were lowered,andαfrequency band distribution was diffuse,sometimes dissymmetrical.The results displayed B[a]P increased ratio of abnormal EEG.4.Changes of conduction velocity of peripheral nerve and evoked potential in coke oven wokersThe conduction velocity of peripheral nerve and evoked potential were monitored by neuropack M1,(Nihon Kohden coporation),compared with controls,there is no significant difference in sensory conduction velocity and motorial conduction velocity of ulnar nerve and median nerve.The sensory evoked potencial,auditory evoked potencial and pattern-visual evoked potencial in two groups also showed no significant diffrence.It was suggested that B[a]P shoud have no effect on conduction velocity of peripheral nerve and evoked potential.5.Analysis of CYP1A1 gene polymorphisms in coke oven wokersCYP1A1 gene polymorphisms including MspⅠsite and Ile-Val site were monitored by PCR-RFLP and ASA-PCR,respectively.The results revealed that there were no statistic difference of the genotype frequencies of m1/m1,m1/m2,m2/m2 of MspⅠsite and that of Iie/Iie,Ile/Val,Val/Val of Ile-Val site between exposed group and contol group,In exposed group the level of urinary 1-hydroxypyrene of Val/Val was significantly higher than Ile/Ile wild homozygote.Although there was a tendency of m1/m1＜m1/m2＜m2/m2 in urinary 1-hydroxypyrene,there was no statistically significant difference.The scores of Dignity Span Backward with Val/Val gene type were higher than those with Iie/Iie gene type.Other index of NTCB and ANS showed no difference among three gene types of Ile-Val site.All index of NTCB and ANS showed no difference among three gene type of MspⅠsite.The present data revealed CYP1A1 Ile-Val site gene polymorphism modulats the neurobehavioral toxicity of B[a]P.PARTⅡstudy ofneurotoxicity of B[a]P in vivo1.single intracerebroventricular injection of benzo[a]pyrene induces morphological changes in ratsForty male SD rats,adult and healthy,were divided into 5 groups randomly:normal control, DMSO control,B[a]P 0.5mmol/L,B[a]P 5mmol/L,B[a]P50mmol/L,all groups were administered by intracerebroventricular injection,and the injectant volume was 10μl.The morphology of neural cells in rats' hippocampus and cerebellum were observed by optical microscope.In cerebellum and hippocampus,arrangement and numbers of the neural cells were normal in control group,but with the increasing of the doses of B[a]P exposed,the arrangement of neural cells became disorderly,the quantity of cells became diminished,and the cells showed necrosis appearance.The results displayed that benzo[a]pyrene damaged brain tissues of rats,and the higher the dose of B[a]P is,the more serious the damage of brain is.2.Subchronical intracerebroventricular injection of benzo[a]pyrene decreased the capacity of learning and memory in ratsForty male SD rats,adult and healthy,were intubated,after one week,all incubated rats were divided into 4 groups randomly:normal control,olive oil control,B[a]P2.5mmol/L,B[a]P 5mmol/L,and B[a]P10mmol/L,all groups were administered by intracerebroventricular injection, and the injectant volume was 10μl.Step-down and Morris water maze tests were applied to assess the capacity of learning and memory in rats.With the dose of B[a]P increasing,the step down latency was shortern,and times of stepping down increased,there existed significant differences in three B[a]P -treated groups compared with controls.The results indicated that B[a]P can induce impairment of passive avoidance learning and short-term memory.In Morris water maze tests the mean escape latency was prolonged and the length of staying in the original quadrant was shorterned in treated groups while dose was increasing.There was an evident dose -response relationship between the dose of B[a]P and the data of Morris water maze tests,which suggested that B[a]P would affecte the spatial learning and memory functions.3.Subchronicai intracerebroventricular injection of benzo[a]pyrene induces hippocampus morphological changes and apoptosisThe observation under light microcope showed:arrangement of the neural cells in rats' hippocampus was orderly in control group,but with the increasing of the doses of B[a]P exposed,the cells displayed disorderly arranged,the connections among the neural cells were loosened,the karyon shrinked,some rings around the cells were observed.Picture under electron microscope displayed:in control group,the karyotin distributed symmetrically,and its form was regular,the form of mitochondria was regular,the arrangement of bars was orderly,and with the increasing of the doses of B[a]P exposed,the karyon shrinked,the karyotin assembled in the edge,karyotheca was broken,mitochondria swelled,broken,the bars were reduced and disappeared.With the increasing of the dose of B[a]P exposed,the apoptosis indices(AI)of neural cells in rats' hippocampus increased,and AI in 5mmol/L B[a]P group and 10mmol/L B[a]P group were significantly higher than that of controls(P＜0.05),and which in B[a]P 10mmol/L group were significantly higher than that of 2.5mmol/L B[a]P group.4.Subchronicai intracerebroventricular injection of benzo[a]pyrene induces neurochemical changes of hippocampus in ratsTo explore the mechanism of B[a]P induced neurotoxicity,amino acid transmitters, monoamine transmitters,SOD and MDA were determined.Compared with controls,GLU was decreased in three treated groups,and other amino acid transmitters showed no changes,while there was an increasing trend of 5-HT in treated groups,no differences of DA and 5-HIAA were found between treated groups and contol group.SOD activity decreased and content of MDA increased in three treated group compared with the conrols.All these data implied the potential mechanism of neurotoxicity of B[a]P.5.Subchronical intracerebroventricular injection of benzo[a]pyrene induces CYP1A1 expression changes of hippocampus in ratsTo explore the relationship between neurotoxicity of B[a]P and CYP1A1 expression, RT-PCR was used to determine CYP1A1 mPNA,and immunohistochemical staining and western bloting were performed to determine CYP1A1 protein.CYP1A1 mRNA and CYP1A1 protein increased significantly compared with the control group and in a dose dependent manner, a positive correlation between AI and the expression of CYP1A1mRNA,the expression of CYP1A1 protein were observed.Considering the data mentioned above and the capacity of learning and memory in rats and neurochemical changes prehensively,CYP1A1 expression may be thought to be the critical step in neurotoxicity of B[a]P.PartⅢstudy of neurotoxicity of B[a]P in vitro1.Changes of cell morphology and apoptosis of primary cultured neurons induced by B[a]Pprimary neurons were dissociated from cerebral cortex of 1-3 days old SD rat and cultured with DMEM incubator at 37℃,cell density was adjusted at 1×10~5cells/ml,after 5 days' cultivation,the astrocyte GFAP was identified by immunohistochemical staining.If the astrocyte was less than 10%,the cultured cells was thought to be pure neurons.Then,they were divided into two groups,+S9 and -S9,and all the two groups of neurons were cultivated with B[a]P at 0,10,20,40μmol/L,respectively,for 40 hours.The morphology was observed,and apoptosis was measured.Under light microscope,with the increment of B[a]P dose,the numbers of neurites and dendrites of neurons and the conjunctions among cells were reduced evidently.The changes were more serious at 40μmol/L in +S9 group.The neurites of some neurons became shorter,even disappeared,and the cell body became round.Stained with AO/EB,under fluorescence light,live neuron showed a bright green color,while the dead one was red,as apoptotic cell was orange.Based on the color of neuron,we can distinguish living conditions of the neurons.With B[a]P dose increasing,the ratios of orange colored cells increased,and the numbers of neurites and dendrites of neurons and the conjunctions among cells decreased,+S9 group showed more severity,at 40μmol/L,the cell body became round and anumber of neurons were stained orange even red,and the neurite of some neuron became shorter,even disappear,the karyon shrinked or aggregated in the edge or cleaved.With AnnexinⅤand PI staining,the apoptosis ratios of neurons were measured,early-onset apoptosis ratio,late -onset apoptosis ratio and total apoptosis ratio increased in a dose dependent manner,and +S9 group showed more serious situation.The data suggested that B[a]P could induce apoptosis of neurons,and bioactivation of B[a]P was important to exert its adverse effects.2.Changes of cell viability and the mitochondrial membrane potential(MMP)and lipid peroxidation induced by B[a]PNeuron vilability decreased in—S9 group at dose of 20μmol/L compared with controls,in +S9 group there existed significant differences in three treated grous.The determination of MMP in neuron showed that MMP decreased in—S9 group at dose of 20μmol/L compared with controls,while in+S9 group,the MMP decresed in a dose dependent manner.The SOD activity decreased and MDA increased in treated group compared with controls.These results suggested that liquid peroxidation induced by BaP could lead to MMP changes,which may initiate neuron apoptosis and cell viability decline.3.CYP1A1 expression induced by B[a]P in neuronWith the dose of B[a]P increasing,CYP1A1 mRNA and CYP1A1 protein increased,there existed significantly difference in treated groups compared with control group.A positive correlation between neuron apoptosis ratio and the expression of CYP1A1mRNA,as well as the expression of CYP1A1 protein were observed.The results indicated that CYP1A1 expression is related to cytotoxicity of B[a]P.summary1.B[a]P-related neurotoxicity in B[a]P exposed wokers are characterized by poor memory and down regulated parasympathetic nervous function.2.The neurobehavioral effect of B[a]P is related to CYP1A1 gene polymorphism.3.It had been proved that CYP1A1 expression induce by B[a]P may be the critical step in neurotoxicity of B[a]Rin vivo and in vitro.4.The impairment of learning and memory by B[a]P was also found in rats,which may be related to neural cell apoptosis,variations of GLUand 5-HT and statue of lipid peroxidation in hippocampus.5.B[a]P may induce neural cell apoptosis through mitochodria pathway.