The Effect Of Arsenic Exposure During Pregnancy/lactation Period On Central Nervous System Development In The Offspring And Study On Its Mechanism

Background and ObjectiveChronic arsenic intoxication is a global health problem affecting many millions ofpeople. China is one of most seriously affected countries with the exposed populationabout3million. The detrimental impact of chronic exposure to arsenic on healthincludes pathological damages on skin, digestive system, and peripheral and centralnervous systems, as well as some kinds of cancers. Because the nerve cell has notregeneration capacity and is more sensitive to arsenic toxicity than any other kinds ofcells, increasing concerns have been focused on arsenic toxic effects on the nervoussystem. Developing organisms are undergoing rapid cell growth and extensivedifferentiation and have poor ability to clear toxicant, so are especially sensitive to thetoxic effects of arsenic. Up to now, only five (eg, lead, methylmercury, polychlorinatedbiphenyls, arsenic, and toluene) of the thousands known chemicals has been proven tocause developmental neurotoxicity in humans. Arsenic is one of them. Arsenic easilypasses the blood-brain and placenta barriers in mammal. Thus, the fetus is faced withthe risk of neurodevelopmental toxicity of arsenic from the intrauterine growth stage. Afew epidemiological and animal studies have indicated that exposure to arsenic at earlystage of development led to dysplasia of the central nervous system and abnormalappearance of advanced neural function, such as learning and memory. However, theinfluencing degree and exact mechanism of arsenic neurodevelopmental toxicity isunclear. It is well known that gestation and postnatal early stage are the critical periodsfor brain development. Exposure to harmful substances during the periods mentionedabove may affect the development of neurons and normal construction of neuralnetworks. The mechanism may lie in the interaction of toxicant and key proteins relatedto brain development. More and more evidence showed that Rho proteins played a key role in neuronal morphology and neural networks constitute. It has been documentedthat during rapid brain developing period exposure to alcohol could induce abnormalneural network construction and result in decline of learning and memory. Themechanism describing the above changes has been shown to be associated with alcoholdisturbing the normal regulation effects of Rho proteins. Therefore, whether Rhoproteins are involved in the neurodevelopmental toxicity mechanism of arsenictriggered our great interest. In this study, Kunming mice were chose as experimentalmodel and these experimental animals were exposed to arsenic through drinking waterduring whole pregnancy and lactation. The morphological changes, as well as theimpacts on learning and memory, were observed in offspring of arsenic exposedmothers. The potential target molecules of arsenic neurodevelopment toxicity, Rhoproteins, were explored at gene and protein levels in our study so as to better understandeffect of arsenic on developing nervous system.Methods(1) Animal model of arsenic exposure during pregnant/lactating period: Pregnantmice were randomly divided into four groups. Group1received distilled water(control)，the other three groups received1,4,16mg/L As2O3aqueous solution throughfree drinking water. Mother mice were exposed to arsenic from gestation day0untilweaning. The pops weaned at postnatal21day.(2) Pathological observation on brain tissue of offspring: The brain tissues ofneonatal and weaned pups were removed, paraffin-embedded, sliced, stained with HE,and observed under inverted fluorescence microscope. The developmental conditions ofneurite in arsenic exposed pups were observed by growth-associated protein (Gap-43)immunohistochemical method.(3) Neurobehavioral tests: The directional navigation experiment by Morris WaterMaze test was used to examine ability of learning and memory of exposed offspring.(4) Gene Chip was used to detect genes associated with brain development.(5) Determination of expression of Rho family in brain tissue of arsenic exposedoffspring: Real-time RT-PCR, and Western-blot detection were used to determineexpression of Rho family members at mRNA and protein levels.Results(1) Impact of arsenic exposure during pregnancy/lactation period on braindevelopment: Under microscope, exposed offspring showed thinner cerebral andcerebellar cortical. In hippocampus of exposed neonatal mice, pyramidal cells appeared edema, enrichment nuclear, and pyramidal cell layer became thinner compared withcontrol group.(2) Impact of arsenic exposure during pregnancy/lactation period on morphologyof neurite: Observing by growth-associated protein (Gap-43) immunohistochemicalmethod, there were some changes in pyramidal cells of cerebral cortex in exposedoffspring compared with the control group, such as decreased baby, reduction in thenumber of axon and dendritic, and uneven stained neuropil. These phenomenasuggested that arsenic exposure during pregnancy and lactation could causes retardedneural growth and poor-constructed neural network.(3) Impact of arsenic exposure during pregnancy/lactation period on ability oflearning and memory: During the observation period, the escape latency in exposedoffspring of4mg/L and16mg/L groups is significantly longer than the control group.After hiding platform, there is no significant difference in escape latency between allgroups. However, after the platform showing, the escape latency in offspring of16mg/L group is significantly longer than control group,1mg/L and4mg/L exposedgroup.(4) Impact of arsenic exposure during pregnancy/lactation period on genes andproteins related to brain development: In Gene Chip, expressions of Rho A、Rac1andCdc42gene were significantly increased in brain tissue in offspring of exposed groupcompared with those of the control group. The ratios of Rho A/Rac1and Rho A/Cdc42were both greater than1in exposed group. The genes of downstream effect factorsof Rho, Creb and Arp, were significantly decreased. Expressions of Cbln4, Chd andGjb1gene were significantly increased，and expressions of Ncam, Crim, Fgf14, Grik2,Rnf103, Rps6ka3and Sim1were significantly decreased in exposed group. Comparedwith the control group, expressions of Rho A and Rac1in mRNA and protein levelswere significantly decreased in neonatal offspring, while significantly increased inweaned offspring by Real-time RT-PCR and Western-blot detection. Particularly, Theratios of Rho A/Rac1and Rho A/Cdc42both increased with arsenic exposure doseeither in neonatal or in weaned offspring. Compared with the control group, expressionsof Cdc42in mRNA and protein levels had no significant changes in exposed offspring.Conclusions(1) Arsenic exposure during pregnancy/lactation period can cause abnormalmorphology of cerebral and cerebellar tissues in offspring;(2) Arsenic exposure during pregnancy/lactation period can cause decreased ability of learning and memory in offspring;(3) Morphological changes in neurons and processes in brain of arsenic exposedoffspring might be the anatomical basis of arsenic neurodevelopmental toxicity;(4) Arsenic exposure during pregnancy/lactation period can cause expressions ofRho A and Rac1in mRNA and protein levels down regulated in neonatal offspring;(5) Arsenic exposure during pregnancy/lactation period can cause expressions ofRho A and Rac1in mRNA and protein levels up regulated in weaned offspring;(6) Arsenic-induced expression imbalance of Rho A/Rac1may be an importantmechanism for slowed brain development and dysfunction of learning and memory.