Effect And Mechanisms Of Benz[a]Pyrene On Learning And Memory In Rats

Benz[a]pyrene(B[a]P),a product of incomplete combustion at temperatures between 300℃and 600℃,widely existed in ecosystem,is one of key hazardous chemicals that can harm human health on the earth.A vast number of studies over the previous three decades have documented links between B[a]P and cancers.According to the chemical structure and physicochemical properties of B[a]P,scientists infer that it might be a neurotoxin.Animal studies showed that B[a]P induce impairment of brain and alterations in behavior,and epidemiology researches also found that B[a]P is a neurotoxin certainly,especially impacting on learning and memory behavior.Evidences that B[a]P cause neurotoxicity,however,are still not complete.The need for more research on neurotoxicity of B[a]P becomes increasingly important as the combustion of fossil fuel increases to keep pace with the world's energy needs.Therefore the aim of this study is to acquire systemic knowledge about the effect of B[a]P on learning and memory behavior in rats and mechanisms involved in it.PARTⅠEFFECTS OF BENZ[A]PYRENE ON HIPPOCAMPUS MORPHOLOGY AND CAPABILITY OF LEARNING AND MEMEORY IN RATSAccording to the results of water maze test,one hundred twenty weaned male Wistar rats were divided into 5 groups,including a control group,a solvent control group and three B[a]P -treated groups,which were administered intra-peritoneally with three doses from 1.0,2.5 to 6.25 mg/kg body weight(BW) for 13 weeks(90 d).During 90 d,there is no change of rats' behavior and no animal died.Model of rat exposed to Benz[a]pyrene for 90 d is established successfully.After 90 d,Morris water maze test is applied to assess the effects of B[a]P on capability of the learning and memory in rats.The test results showed,with the increasing of the dose exposed to B[a]P,that the average escape latency of rats increases,the frequency of crossing platform decreases and the time of swimming in the target area decreases.There are significant differences between the treated groups and the control groups and among three treat groups(P＜0.05).Results of pathological sections stained with hematoxylin and eosin showed that hippocampal neurons from all groups exhibited a typical normal appearance.Results of pathological sections stained with thionine for Nissl bodies showed that cytoplasm of hippocampal neuron was blue and Nissl bodies were very numerous in all groups,except slight chromatolysis appeared in 2.5 and 6.25 mg/kg B[a]P treat group.Electron micrographs confirmed the normal appearance of hippocampal neurons, except a few swelled mitochondrias appearing in 6.25 mg/kg treat group.The study of this part suggested that low dose of B[a]P for 90 d toxicological test could impair the capability of learning and memory in rats,but did not impair the morphology and structure of hippocampal neuron.PARTⅡEFFECTS OF BENZ[A]PYRENE ON HIPPOCAMPUS NEUROBIOLOGYAfter the Morris water maze test,hippocampus tissues were obtained from decapitated rats.Nitric oxide(NO),nitric oxide synthase(NOs), superoxide dismutase(SOD),malondialdehyde(MDA),choline cetyltransferase(CHAT),acetylcholine esterase(ACHE) were detected by commercially available kits.Acetylcholine(Ach) was detected by alkaline hydroxylamine method.Norepinephrine(NE),adrenaline(A), 3,4-dihydroxyphenylacetic acid(DOPAC),dopamine(DA), 5-hydroxyindoleacetic acid(5-HIAA),5-hydroxytryptamine(5-HT) were detected by high-performance liquid chromatography and electrochemical detection.Gliutamic acid(Glu) andγ-aminobutyric acid(GABA) were detected by immunohistochemistry.CA1 hippocampal synapse morphology was observed by transmission electron microscopy.Results of transmission electron microscopy showed that length of postsynaptic dlensity(PSD) and length of active zone were significant shorter than those of control groups(P＜0.05),and radius of modeling circular of synaptic interface was significant larger than those of controls(P＜0.05). With the increasing of the dose exposed to B[a]P,activity of SOD decreasing,concentration of MDA increasing,there were significant differences between the treat groups and the control groups and among three treat groups(P＜0.05).Activity of NOs and concentration of NO in treat groups were lower than those of control groups(P＜0.05),and were a decreasing trend companied with the increasing of the dose.Compared with control groups,activity of ChAT decreased,activity of AChE increased,concentration of Ach decreased(P＜0.05).Concentration of NE, DA,DOPAC and 5-HT in treat groups were lower than those of control groups(P＜0.05),and there were an increasing trend companied with the increasing of the dose and significant differences among three treat groups (P＜0.05).The study of this part suggested that low dose of B[a]P for 90 d toxicological test could alter the concentration of neurotransmitter to inhibit the inducement and sustainment of long-term potentiation(LTP), further leading to impair the capability of learning and memory in rats. The decreasing concentration of NO/NOs and Ach,and impairment of lipid oxidation could be related to effect the capability of learning and memory.PARTⅢEFFECTS OF BENZ[A]PYRENE ON WHOLE HIPPOCAMPUS GENE EXPRESSIONOur study original applied microarray technique to analyze the whole hippocampus gene expression exposed to B[a]P,then confirmed a part of results of microarray by RT-qPCR.The results of microarray showed that 440 genes were up-regulated by≥2 folds and 582 genes were down-regulated by≥2 folds,when treat group compared to control. Function genes were involving in 14 types,including in cell cycle regulatory factors,stress response proteins,ion channel and transporters, intracellular signal transduction regulatory factors,apoptosis proteins, DNA synthesis protein,tumor suppressor gene,neurotransmitter receptors, cell surface antigens,intercellular communication protein,cytoskeleton and movement protein,transcription factor,DNA connexin and proto-oncogene.Among these,genes perhaps associated with mechanisms of learning and memory involved:ion channel gene and neurotransmitter gene related to transmission(Kcnh4,Kcnab3,Kcnn4,Syt2,Synpr down-regulated;Htr1a,Htr5b,Gabra5,Nrxn1,Calb3 up-regulated);genes related to signal transduction(Prkcd,Prkch,Snf1lk,Ppm1f,Rab27a, Htr1d,PSD,G naz down-regulated;Trpc4,Mapk2 up-regulated);genes related to transcription(c-Fos,Nr2f6,Egr2 down-regulated;Erg,BNDF up-regulated);genes related to cell structure/cytoskeleton(Myl1,Mybpc, Gcnt2,MyoⅠc down-regulated;Myh4 up-regulated). The study of this part suggested that decreasing capability of learning and memory in rats might be associated with the alteration of polygene, such as up/down-regulated genes related to ion channel and neurotransmitter,signal transduction,transcription and cell structure/cytoskeleton.PARTⅣEFFECTS OF BENZ[A]PYRENE ON WHOLE HIPPOCAMPUS PROTEIN EXPRESSIONRecent years,proteomics is widely applied to study mechanisms of learning and memory.Then our study focus on investigating any possible mechanisms of B[a]P impairing learning and memory by proteomics technique(Spring's Master Antibody Microarray).According to change of protein expression in antibody microarray,results showed that 26 proteins were up-regulated by≥1.5 folds and 23 protein were down-regulated by≥1.5 folds,when treat group compared to control.Among these,proteins perhaps associated with mechanisms of learning and memory involved: retinoic Acid Receptor b(RAR b) down-regulated≥2.6 folds,brain derived neurotrophic factor(BDNF) down-regulated≥2.1 folds, synaptotagmin iosfomrs 1(Sty1) down-regulated≥1.6 folds.The study of this part suggested that it is possible that B[a]P impair the capability of learning and memory in rats by down-regulated RAR b, BDNF and Sty1 expression.CONCLUSIONSThis study suggested that low dose of B[a]P exposure for 90 d toxicological test could impair the capability of learning and memory in rats.It is possible that impairment is related to decreasing of hippocampus amino acid neurotransmitters,NO/NOs and concentration of Ach, increasing of monoamine neurotransmitters and impairment of lipid oxidation.Decreasing capability of learning and memory in rats exposed to B[a]P might be associated with the regulation of polygene together and down-regulated RAR b,BDNF and Sty1 protein expression.It is worthy further research on concrete molecular mechanism of B[a]P impairing the capability of learning and memory in rats.