The Effect Of Maternal BDE-209 Exposure On Acetylcholine Neurotransmitter And Nitrogen Monoxidum Neurotransmitter In The Hippocampus Of Offsping Rats

1.About Polybrominated diphenly ethers(PBDEs) and the current studyPolybromine diphenyl ethers(PBDEs) is a series of aromatic compounds including bromine.At first extensively used as fire retardant chemicals,they have been considered as the the global environmental pollutant and danger to health recently.For the stable structure and the character of high biological activity, liposolubility,they can ingress in the atmosphere,water resource,soil and the food chain of human with the dumped electricity,so people call them as Persistent Organic Pollutants(POPs) and some experts claimed it as the "chemical time bomb". The can do potentially harm to the environment and human being enomously.As PBDEs exist in the air,soil and chain food,they can ingress into organism by respiration,skin and digestive tract,even into the fetals and newborns by the placenta and maternal milk.They accumulate in the fat tissue,and part of PBDEs gather in the target organs,such as liver,the adrenal cortex,ovary,lungs and the brain(initial),which the concentration increase gradually by the biology accumulation.At present animal experiments have demonstrated that PBDEs have harmful effects to the organic health.For example,they can disrupt the hormones in thyroid,decrease the ability of learning and study,alter the behaviour,destroy the sence of heating,postpone the adolescence,reduce the sperma amount,result in the fetus anomalies and cancer,influence the function of target organs,including testicle,ovary, and so on.Recent years it is reported that the concentration of PBDEs has been increased largely in the environment and organic body with the years,and double every two to five years,meanwhile they double in the ocean mammals such as seals every one point eight years.It is claimed that the concentration of PBDEs in the meternal milk had have five times between the year of 1992 and 2002.In the year of 2008,the scholars in Belgium indicated that the density of PBDEs in the human livers and fat tissues have arrived to 5.3ng/g/lw and 3.6ng/g/lw,which has been five to ten times over past.The study of public health center in Texas US,have manifestated that the concentration of PBDEs in the meternal milk of North America has reached to6—419 ng/g,which has the three to ten times in the order of magnitude and dominate the first in the wideworld.2.About BDE-209 and the current studyBDE-209 belongs to ten bromines associations phenylate(deca-BDE),includes ten bromines atoms.As one congener of the deca-bromine biphenyl ethers,BDE-209 is the most widely used,in commercial field are five bromines associations phenylate,eight bromines associations phenylate and ten bromines associations phenylate.BDE-209 has the photochemical degradation the characteristic,after can pass through the ultraviolet ray illumination to decompose the debrominate but finally to decompose into low bromine PBDBs(penta-BDE,octa-BDE).For ten bromines associations phenylate(deca-BDE is BDE-209) potential toxic damage of the study are unclear and limited information.The overseas scholar discovered ten bromines PBDEs(deca-BDE is BDE-209) has the carcinogenicity,and presents the correlation with the deca-BDE dosage.Thought BDE-209 mainly causes the liver malignant tumor,the non- white courage and uprightness leukemia,breast cancer and the thyroid cancer and so on.At present some country environmental protection organizations thought deca-BDE has the characteristic of the potential hazard to the human health and the environment and it is chemical substance which one kind receives pays attention highly.The health and the environment risk scientific committee(SCHER) proposed ten bromines diphenyl oxide(deca-BDE) to environment discharging,will be possible in the future to constitute serious issue. European Union later has come to reach the agreement about the comprehensive prohibition of using PBDE and gathering the bromine biphenyl(PBB) last year on July 1,2006.The PBDE-209 commercialization produced and applies still has the dispute until now.3.OBJECTIVE:BDE-209 is possibly one kind of POPs matter(durable,biological accumulation, Toxic),this research information hints of its potential hazards(including the ecological environment and human health).People from all over the world began to pay close attention to PBDEs,which is called the persistent organic pollutant substance(POPs).In recent studies we have shown that PBDE congeners BDE47,99,and 153can induce persistent neurotoxic effects in adult mice.In addition, neonatal PBDE 99 exposure can affect adult spontaneous behaviour and the cholinergic system in rats.H.Viberg et al have shown that neonatal exposure to BDE-209 can induce persistent behavioural neurotoxic effects in adult mice and that there effects get worse woth increasing age.Congener specific knowledge about the toxicity of PBDEs is limited.No complete toxicological evaluation is currently available on any PBDE individual congeners or any of the commercial mixtures.The effects still have been being discussed on the acute/chronic toxicity,irritation, sensitization,genotoxicity,carcinogenicity and teratogenicity.Some observations do indicate that PBDEs might act as potential endocrine disrupters and interfere with brain development.The people mainly through the edible meats,the domesticated fowl,the fish and the dairy products expose in this kind of chemical material.And PCBs were found in organisms exist as a result of greater toxicity,Serious health hazards to the next generation.Internationally banned.At present correlation harm correlation research day by day increases regarding the structure with PCBs the similar PBDEs,But high bromine biphenyl ethers(especially ten Bromine biphenyl ethers or PBDEs-209) toxicity studies are relatively limited,And therefore its potential for toxicity is not yet clear role.Thus,the study on the rodentia animals from the scholar in Canada,has indicated that PBDEs can impact on the cholinergic nerve system to be developmental neurotoxicity.Viberg et al find the changes in spontaneous behaviour and altered response to nicotine in the adult rat,after neonatal exposure to the brominated flame retardant,decabrominated diphenyl ether(BDE 209),but they never study the effect of BDE-209 exposure to the rats prior to births from the meternal point,and did not verify the further mechanism.Based on the protophase experiment of our topic group,we evaluate the effect of maternal BDE-209 in different dosage on the offsprings' learning and memory ability and its relashionship with the dosage;To discover the mechanisms of the developmental neurotoxicity of BDE-209 from diacetyl-cholinergic system and nitric oxide neurotransmitter conduction and to establish the foundation of corresponding intervention,prevention and therapeutic treasure.This study has been dividied into 3 parts as following.PartⅠEFFECT Of mAternal BDE-209 EXPOSURE ON LEARNING AND MEMORY ABILITY and the microstructure in hippocampus of offspring rats.[Objective]To evaluate the effect of prenatal and lactational BDE-209(brominated Diphenyl Ethers-209) in different dosage on the offspring's learning and memory ability through animal experiments,there is no correlated report so far.The study is to compare the learning and memory ability in each group after make the model of deca-BDE exposure in different dosage by oral gavage,and to observe the changes of the microstructure in hippocampus of offspring rats accounting for the development neurotoxicity induced by BDE-209 and the effect-dosage relationship.[Material and Methods]1.Establish the model of maternal BDE-209 exposure:Three-month-old rats 75 totally were obtained from the southern medical university(the body weight vary from 250 to 320g,including female 50 and male 25), according to two and one,female and male rats were housed individually in plastic cages in a room.The success of mate was determined by the slough plug from the vagina in the female rats.The study time is from the success of mate of the maternal rats to lactation ends of the offspring ratsGroup:Peanut oil suspensions of commercial BDE-209 was given in doses of 100(A),300(B),600(C),1200(D)mg/kg·d by oral gavage for the experimental groups.The control group was administered only with the same capacity of Peanut oil at the same time.The animals were supplied with standardized pellet food with an ambient temperature of 22℃,and a 12/12h cycle of light and dark.The time of oral gavage is at dust.The offspring rat were grouped A,B,C,D,E correspongding.Group A(the group exposed to BDE-209 100mg/ kg·d in gestation and lactation);Group B(the group exposed to BDE-209 300mg/ kg·d in gestation and lactation);Group C(the group exposed to BDE-209 600mg/ kg·d in gestation and lactation);Group D(the group exposed to BDE-209 1200mg/ kg·d in gestation and lactation)Group E(The control group)2.100 of the offspring rats will be examined of their learning and memory ability after lactation by the method of Morris water maze:Numbers the animals,picked up randomly the first generation offspring male rats,which contained twenty rats after the period of delactation in each group.Train the rats four times at the fixed time everyday for five days,and put the rats into the water from four different entrance and make the rats face the lateral wall.Let the rats stay on the platforms for thirty seconds after they found the platforms,record the time to search the stage,which is called the escaped latency.If the rats cannot find the stage,then we took them to the stage with hands,and the time is recorded 120 seconds.The interval between the two train is 60 seconds at least.To evaluate the learning and memory ability of the adult rats,by comparing the escaped latency of each group.3.Make the pathological section of Hippocampus:After the Morris water maze, picked 5 of the first offspring rats,which had participated the behavior test.Throuth the process of drawing material,fixation,washings,dehydration,transparence, immersing in the wax,embedment,slicing and sticking,sheding the wax,dyeing and so on,to observe the microstructure by HE staining through light microscope in the CA1 zone of the Hippocampus.[Result]1.The effect of BDE-209 on the learning and memory ability of the offspring rats:1.1 The main effect:there was significant difference in time factor,which indicated that the latency was time-dependent(F=37.351,P=0.000);the recipro-effect between time and group had no difference,which mean the time don not work as the group factor(F=0.163,P=1.000).1.2 there was significant difference in group factor,which indicated that the group works(F=10.971,P=0.000).1.3 Contrast of groups at every time:There was no significant difference among five groups in the first one-two days(P＞0.05),which indicated that the rats each group have the similar learning and memory ability.In the next three to five days, there was no significant difference between group A and the control group(group E) (P＞0.05),which indicated low dosage of BDE-209 have not influenced the learning and memory ability of the offspring rats compared with control group;The latency of group B,C,D have prolonged compared with control group(P＜0.05),which indicated that BDE-209 in high dosage have diminish the learning and memory ability of the offspring rats.2.The comparison of pathology in the CA1 zone of hippocampus each group: There are obvious histomorphology changs between the C,D groups and control group E by HE staining using light microscope.[Conclusions]1.maternal BDE-209 in low dosage did not affect the special learning and memory ability of the offspring rats,when it got to a certain concentration,maternal BDE-209 exposure diminished the offspring rats' learning and memory ability.It is shown that the effect of maternal BDE-209 on the learning and memory ability of the offspring rats has the close relation with the exposure dosage.2.A certain dose of maternal BDE-209 exposure can affect the the microstructure of nerve cell(number and structure) in hippocampus,which is possible material ground of the impair to the nervous system.PartⅡEFFECT OF MATERNAL BDE-209 EXPOSURE ON THE CHOLINERGIC SYSTEM IN HIPPOCAMPUS OF OFFSPING RATS[Objective]Previous work has shown that the maternal BDE-209 had affect the learning and memory ability in the offspring rats.In the present study we contrasted the levels of cholinergic system parameter involved in of hippocampus tissue,such as acetylcholine amount,acetylcholinesterase activity,the expression of M-receptor and N-receptor,and approached the potential mechanism of the impair to the offspring rats′learning and memory ability.So as to settle the base for the next intervention and treatment.[Material and Methods]1.Handling the animals and collecting the specimens:The animal model came from part one.After the Morris water maze test,picked up randomly ten offspring rats each group for the study objection(All of the specimens come from the experimental animals based on the groupA;B;C;D;E of the part 1).Fix the animals,anesthetize them through intraperitoneal injection with chloral hydrate at the 4ml/kg dosage,then open the chest to reveal the heart,intubate through cor sinistrum,cut auricle of right atrium and pour quickly into normal saline with 4% Polyoxymethylene(PFA)stationary liquid about 200ml,get the brain on the ice cap after the decapitation,then dissociate the bilateral hippocampus.Wash the blood in the precooling 4℃normal saline,add nine times as much as the volume,then make the 10%tissue homogenate of brain tissue with the glass machine of homogenate at high speed.Take the homogenate to measure,according to the requirement and step of the correlated kit strictly.2.The levels of cholinergic system parameter in hippocampus tissue of offspring rats were detected using sandwich ELISA procedure,such as acetylcholine amount,acetylcholinesterase activity,the expression of M-receptor and N-receptor.[Result]1.The ACH quantity in hippocampus was decreased with increased dosage of the exposure to BDE-209.The B,C,D groups had significant difference from control group(P＜0.05);There was no significant difference between group A and the control group E(P=0.637);Among the experiment group,the groups A,B,C had significant difference from group D(P＜0.05).2.There were obviously significant decrease of acetylcholinesterase activity in hippocampus of the offspring rats in the group B,C,D,comparing with the control group E(P＜0.05).but there was no significant difference between the group A and the control group E(P=0.173).Among the experiment group,the groups A,B,C had significant difference from group D(P＜0.05).3.The expression content of M-Receptor in hippocampus were decreased in the groups of C,D,comparing with the control group E(P＜0.05),but there was no significant difference between the group A,B and the control group(P=0.161, P=0.116);Among the groups,there were significant difference of expression of M-Receptor in hippocampus between the group D and the other experimental groups (P＜0.05)except group C.4.The expression of content N-Receptor in hippocampus were decreased in the groups of D,comparing with the control group(P=0.001),but there was no significant difference between other experimental groups and the control group (P＞0.05);Among the groups,there were significant difference of expression of M-Receptor in hippocampus between the group D and the other experimental groups (P＜0.05).[Conclusions]The present results showed that the exposure of BDE-209 may destroy cholinergic system index involved in the CA1 zone of hippocampus tissue,such as acetylcholine amount,acetylcholinesterase activity,the expression of M-receptor and N-receptor,and the cholinergic neurotransmitter was the possible mechanism of the impair to the offspring rats′learning and memory ability.PartⅢEEFFECT OF MATERNAL BDE-209 EXPOSURE ON THE NITRIC OXIDE NEUROTRANSMITTER IN HIPPOCAMPUS OF OFFSPRING RATS[Objective]Our initial studies have proved that the maternal BDE-209 exposure can damage the learning and memory of the offspring rats.The mechanism by which BDE-209 induce these alterations remain unclear.In This study,by contrasting the levels of Nitric Oxide(NO),neuro-nitricoxide synthase(nNOS) and the cyclic guanylic acid (cGMP)in the hippocampus of the first offspring rats in each group,we approached the possible mechanism of the impair to the offspring rats′learning and memory ability.So as to settle the base for the next intervention and treatment.[Material and Methods]1.Handling the animals and collecting the specimens:The animal model came from part one.After the Morris water maze test,picked up randomly ten offspring rats each group for the study objection(All of the specimens come from the experimental animals based on the groupA;B;C;D;E of the part 1).Fix the animals,anesthetize them through intraperitoneal injection with chloral hydrate at the 4ml/kg dosage,then open the chest to reveal the heart,intubate through cor sinistrum,cut auricle of right atrium and pour quickly into normal saline with 4%Polyoxymethylene(PFA)stationary liquid about 200ml,get the brain on the ice cap after the decapitation,then dissociate the bilateral hippocampus.Wash the blood in the precooling 4℃normal saline,add nine times as much as the volume, then make the 10%tissue homogenate of brain tissue with the glass machine of homogenate at high speed.Take the homogenate to measure,according to the requirement and step of the correlated kit strictly.2.Accoriding to the request and procedure of the respective assay knit,we measure the levels of Nitric Oxide(NO),neuro-nitricoxide synthase(nNOS) and the cyclic guanylic acid(cGMP) in hippocampus tissue of offspring rats[Result]1.The NO content in the BDE-209 treated groups increased by the dosage of BDE-209 exposure concentrations(F=0.6323,P=0.000);the content of C,D groups were higher than the control group(P＜0.05),while there were no significant difference in the group A and group B,respectively comparing with the control group E (P=0.515,P=0.135).Among the experimental groups,there was the highest content of NO in group D,though there was no significant difference between group A and group D.2.The nNOS activity in the BDE-209 treated groups also was increased by the dosage of exposure to BDE-209(F=11.928,P=0.000);the activity of nNOS in the group C and group D were higher than the control group(P＜0.05),while there were no significant difference in the group A and group B,respectively comparing with the control group E(P=0.965,P=0.112).Among the experimental groups,there was the highest activity of nNOS in group D,though there was no significant difference in group A and group B,respectively comparing with the data of the group D.3.The cGMP content in the BDE-209 treated groups increased by the dosage of BDE-209 exposure concentrations(F=4.119,P=0.00);the content in group B,group C and group E were significantly higher than the control group E(P＜0.05),while there were no significant difference between group A and the control group E (P=0.133).Among the experimental groups,there was significant content between group D and group A inclusively.[Conclusions]The study suggested that BDE-209 can influence the levels of nitric Oxide(NO),the activity of neuro-nitric oxide synthase(nNOS) and the levels of cyclic guanosine monophosphate(cGMP) in hippocampus of offspring rats.It indicated that the Nitric Oxide as a neurotransmitter,may be the possible mechanism of the impair of BDE-209 exposure to the offspring rats′learning and memory ability,especially the NMDAR-NO-CGMP pathway in the nerve system.SUMMARY1.We successfully established the model of BDE-209 exposure in different dosage by oral gavage in the spur period of the brain development of rats,settle the base for the study on the special behavior test of the offspring rats and on the possible mechanism of the impair to the learning and memory ability in offspring rats.The model can be manipulated easily and emulated successfully..2.It is reasonable to conclude that maternal BDE-209 in certain dosage cant affect the spatial learning and memory ability of the offspring rats,and change the microstructure of the hippocampus cells,meanwhile the effect of maternal BDE-209 on the learning and memory ability has close relation with the exposure dosage.3.A certain dose maternal BDE-209 exposure can affect the the microstructure of nerve cell(number and structure),which is possible material ground of the impair to the nervous system.4.The exposure of BDE-209 may effect acetylcholine content,acetylcholinesterase activity,the expression content of M-receptor and N-receptor involved in the hippocampus tissue of offspring rats,and the cholinergic neurotransmitter was the possible mechanism of the impair of BDE-209 exposure to the offspring rats′learning and memory ability.5,The exposure of BDE-209 may influence the levels of nitric oxide(NO),the activity of neuro-nitric oxide synthase(nNOS) and the levels of cyclic guanosine monophosphate(cGMP) in hippocampus of offspring rats.It indicated that the Nitric Oxide as a neurotransmitter,may be the possible mechanism of the impair of BDE-209 exposure to the offspring rats′learning and memory ability,especially the NMDAR-NO-CGMP pathway in the nerve system.In summary,we elucidate that the exposure of BDE-209 may effect the learning and memory ability of offspring rats by the possible mechanism cholinergic neurotransmitter and nitric oxide neurotransmitter pathway.But so far the threshold dose of the BDE-209 to induce the biotoxicity remain unclear.It is still need to be study whether there is other mechanism and the association of all the mechanisms. Because the biological accumulation of BDE-209,with the further study of the toxicity and danger,people will pay more attention to the use of BDE-209,including the developed countries.