【作者】 李元锋；

【导师】 石年； 李涛；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 邻苯二甲酸二丁酯（Dibutyl phthalate，DBP）是目前用量较大的邻苯二甲酸酯类（Phthalateesters，PAEs）化合物的重要成员，是一种重要的内分泌干扰物。DBP主要用作纤维素树脂、聚氯乙烯、以及橡胶制品的增塑剂、软化剂，同时也是生产油漆、粘接剂、药物制剂等多种工业品的重要原料。在生产过程中，由于DBP并未与这些产品牢固结合，就会从中不断释放进入环境，对人群健康产生危害。据美国NTP估计，一般人群通过各种途径接受DBP的暴露水平为2-10μg／kg体重／天。当DBP进入机体后，可迅速代谢为邻苯二甲酸单丁酯（Mono-butyl phthalate，MBP），后者是DBP引起发育毒性效应的罪魁祸首。目前很多研究表明DBP的危害主要是其生殖和发育毒性。DBP的毒性机制主要源于其对内分泌的干扰作用，体内、体外实验表明DBP具有雌激素和／或抗雄激素的活性，并能影响体内甾体合成的多种酶的活性。然而DBP对神经行为发育的影响目前尚不清楚。中枢神经系统在发育时期对体内多种激素水平的变化非常敏感，若此期间暴露于内分泌干扰物DBP，其发育和功能很可能受到影响。流行病学资料提示DBP具有神经毒性，但由于是多种PAEs的混合暴露，其结果尚具有不确定性。神经行为指标不仅是反映内分泌干扰物对机体作用的敏感指标，也是化合物对多器官系统作用的综合反映。本研究主要从神经行为发育的角度，对DBP的毒性作用及其机制进行初步探讨，以进一步认识DBP的危害。研究以大鼠为对象，通过在妊娠期和哺乳期染毒，采用一系列神经行为测试组合，在多个时间点测试DBP对F₁大鼠神经行为发育的影响。多剂量组、大样本、跨代长期染毒是本研究的特点。由于发现DBP可影响子代雄性大鼠的空间学习记忆，机制研究中以其为代表，采用分子生物学方法检测在发育期间不同时间点仔鼠海马中与学习记忆相关的基因和蛋白质的表达，采用电生理的万法检测大鼠海马齿状回的长时程增强（long-term potentiation，LTP）能力的改变，初步探讨DBP对大鼠海马神经细胞的发育和功能的影响，进一步了解DBP的神经毒性作用机制，为DBP污染的防治工作提供重要的实验和理论依据。第一部分DBP对F₀雌性大鼠生殖和F₁仔鼠生长发育的影响目的：研究DBP对F₀雌性大鼠生殖和F₁仔鼠生长发育的影响。方法：Wistar妊娠大鼠60只，随机分为对照、低、次低、次高、高5个剂量组，每组11～13只。从GD6起，四个实验组分别喂饲给予质量百分比含量为0.037％、0.111％、0.333％和1％的DBP染毒孕、母鼠及F₁仔鼠至PND28，对照组饲料不含DBP。观察孕鼠的体重、进食量、染毒后的一般情况以及生殖情况；观察测量仔鼠的体重变化、早期生理发育指标（体长、尾长、肛殖距、张耳、出牙、开眼）；染毒结束时解剖检查各主要脏器情况并称量计算脏器系数。结果：（1）各实验组孕鼠的进食量和体重随妊娠天数逐渐增加，但与对照组比较，其增量未见明显改变（p＞0.05）。（2）各实验组孕鼠未见明显中毒症状，妊娠失败率、每窝活仔数、窝仔性别比与对照相比无明显差异（p＞0.05），但高剂量组的妊娠期较对照组稍延长（p＜0.05）。（3）高剂量组雌性和雄性仔鼠在PND0～28期间体重明显低于对照组的同性别仔鼠，差异有显著性（p＜0.01）。（4）高剂量且雄性仔鼠的肛殖距（体重立方根标准化）显著降低（p＜0.01），而各剂量组雌性仔鼠肛殖距未见明显改变（p＞0.05）。（5）各实验组仔鼠的张耳、出牙和开眼的天龄与对照组比较无显著差异（p＞0.05）。（6） PND28龄高剂量组仔鼠（雌和雄）肝脏系数显著增加（p＜0.01），高、次高剂量组雌性仔鼠脾脏系数和高剂量组雄性仔鼠睾丸系数显著降低（p＜0.01），各实验组脑、心、肺、肾和子宫脏器系数未见明显改变（p＞0.05）。结论：在本实验的染毒方式、时间和剂量下，未见DBP对F₀雌鼠的生殖能力产生明显的毒性作用，但高剂量DBP显著影响F₁仔鼠的生长发育并具有明显的脏器毒性，主要表现仔鼠体重减轻、肛殖距（雄）缩短、肝脏脾脏和睾丸的脏器系数的改变。第二部分DBP对F₁大鼠神经行为发育的影响目的：研究DBP暴露对F₁大鼠神经行为发育的影响。方法：孕鼠、仔鼠的分组染毒同第一部分。在出生后特定的天龄，每窝选择仔鼠进行平面翻正（PND4，7）、负趋地性（PND4，7）、悬崖回避（PND7）、前肢悬挂（PND10）、空中翻正（PND16）等行为功能测试，检测DBP对早期行为发育的影响；选择4周龄仔鼠进行开阔场实验，检测DBP对仔鼠活动度的影响；选择5周龄仔鼠进行Morris水迷宫实验，检测DBP对F₁仔鼠学习记忆能力的影响。结果：（1）早期行为发育指标：PND7高剂量组雄性仔鼠平面翻正的评分显著低于对照组，差异有显著性（p＜0.05），高剂量和低剂量组雄性仔鼠PND10前肢悬挂时间较对照组显著降低（p＜0.05），但在PND4的平面翻正以及各时间点的悬崖回避、负趋地性和空中翻正试验中不论是雄性还是雌性仔鼠都未见有显著的影响。（2）开阔场实验：各实验组仔鼠的跨格子数、跨格的分布、后腿站立次数以及停留超过0.5s的次数比较，既无处理效应的差异也无性别效应的差异（p＞0.05），合并性别比较也未见统计学显著的差异（p＞0.05）。（3）Morris水迷宫实验：①在隐蔽平台试验中，性别和训练天数有明显的交互作用（p＜0.01），高剂量组的雄性仔鼠在训练的第3-5天显示比对照组较好的空间学习能力（p＜0.05），但低剂量组雄性仔鼠在训练的第4-5天显示比对照组较差的空间学习能力（p＜0.05）；各实验组雌性仔鼠的学习能力与对照组比较无显著改变。②在空间探索试验中，低剂量组和次低剂量组雄性仔鼠的空间偏好（反映空间记忆）显著低于对照组（p＜0.05）；各实验组雌性仔鼠的空间偏好与对照组比较无显著改变。③可视平台试验：各实验组仔鼠（雄性和雌性）的逃避潜伏期和游泳路程与对照比较差异都无显著性（p＞0.05）。结论：在本实验条件下，DBP显露对F₁雄性大鼠的早期神经行为发育以及空间学习记忆有一定的影响，但对F₁雌性大鼠的影响却很少，其作用表现出明显的性别著异和非单调的剂量-反应关系。第三部分DBP对雄性仔鼠海马神经细胞发育有关的基因和蛋白质表达的影响目的：研究DBP对雄性大鼠海马神经细胞发育相关的基因和蛋白质表达的影响，探讨DBP的神经毒性作用机制。方法：姬娠Wistar大鼠，随机分为对照、低、次低、次高、高5个剂量组，每组8～10只。自GD6起，每日灌胃给予玉米油（溶剂）、25mg／kg，75 mg／kg、225 mg／kg、675mg／kg的DBP染毒孕、母鼠至PND21；取PND1，7，21雄性仔鼠的海马，用荧光定量RT-PCR检测c-fos、BDNF（all variants）、Spinophilin、PSD95、Synaptophysin、CREST、BDNF variantⅢ等基因的表达；用Western blot检测PND21雄性仔鼠海马BDNF、p-CREB、Spinophilin和芳香化酶蛋白质的表达。结果：（1）对海马基因表达的影响：①与神经细胞生长和分化以及学习记忆密切相关的即早基因：PND21高剂量组BDNF（all variants）基因的相对内参基因GAPDH的表达量较对照增加约30％，差异有显著性（p＜0.01），但各实验组c-fos基因的表达与对照比较无显著变化。②与树突和突触结构和功能相关的基因：PND21低剂量组Spinophilin基因相对表达量较对照组增加35.7％，差异有显著性（p＜0.05），但各实验组PSD95、Synaptophysin表达与对照比较无显著变化。③与CREB信号调控通路相关的基因：各实验组BDNF variantⅢ（尽管高剂量组占总BDNF mRNAs的比例降低）和CREST基因的相对表达量与对照相比无明显变化。（2）对海马蛋白质表达的影响：PND21高剂量组BDNF（成熟蛋白质分子）、Spinophilin和芳香化酶相对内参蛋白GAPDH的表达较对照组均显著增加（分别p＜0.01，p＜0.05和p＜0.01），但各实验组p-CREB蛋白的相对表达量与对照组相比未见明显改变。结论：妊娠期和哺乳期高剂量的DBP暴露可促进雄性仔鼠海马BDNF、Spinophilin和芳香化酶的表达，提示其可能促进海马神经细胞的生长与分化、树突棘增多；高剂量DBP暴露促进BDNF表达的作用与上游CREB的激活调控作用无关；DBP暴露可能不会影响雄性大鼠海马的突触结构和功能。第四部分DBP对雄性大鼠成年后学习记忆以及血清生化指标和海马LTP的影响目的：研究发育期间DBP暴露对雄性大鼠成年后学习记忆的影响，研究DBP长期暴露对雄性大鼠血清生化指标和海马齿状回LTP的影响。方法：第3部分中的剩余雄性大鼠，1只／窝／组，继续以同样的方式和剂量染毒至PND28，在1月龄和2月龄时进行Morris水迷宫实验；保留调剂量组、低剂量组和对照组继续染毒至3月龄，尾尖采血检测生化指标，在体测定海马齿状回LTP。结果：（1）水迷宫实验：与对照组相比，高剂量组雄性仔鼠1月龄时表现出较好的空间学习能力（p＜0.05）；染毒间断至仔鼠2月龄时仍表现出较好的空间学习记忆能力（p＜0.05）。（2）血清生化指标：与对照组相比，高剂量组雄性仔鼠血清白蛋白、总蛋白显著降低，而血清谷草转氨酶（AST）和血清胆碱酯酶（CHE）活性却显著增加（所有p＜0.05）。（3）海马齿状回LTP：各组大鼠海马齿状回LTP诱发的成功率均大于70％、PS增幅和EPSP斜率的增加都在100％左右，但与对照组比较，两个实验组的这3个指标均未见显著性差异（均p＞0.05）。结论：高剂量DBP染毒的雄性大鼠在近成年时仍然表现出较好的学习记忆能力，可能是源于早期DBP对神经系统发育的影响；高剂量DBP长期暴露对成年雄性大鼠有明显的肝脏毒性；DBP长期暴露对雄性大鼠海马齿状回颗粒细胞的突触传递功能影响不明显。综上4个部分研究所述，在本实验的研究条件下，发现高剂量的DBP具有明显的生殖毒性、发育毒性和肝脏毒性。对早期神经行为而言，DBP对雌性仔鼠的影响不明显，但对雄性仔鼠有一定的影响，表现为平面翻正、前肢悬挂等行为功能的异常。然而在高剂量时却促进了雄性仔鼠的空间学习记忆，海马基因和蛋白表达的检测提示可能与促进了神经系统的发育有关。尽管未见低剂量的DBP对生殖和发育产生明显的毒性效应，却发现其减少雄性仔鼠的前肢悬挂时间、降低空间学习记忆能力。虽然这种低剂量引起的效应在机制探讨中未得到恰当的解释，但由于这个剂量低于目前报道的基于形态学的NOAEL（50mg／kg／day），提示神经行为尤其是学习记忆更容易受到DBP暴露的影响，在今后的危险评价工作中应引起充分重视。更多还原

【Abstract】 Dibutyl phthalate （DBP） is an important representative phthalate esters （PAEs） usedubiquitously nowadays, and is also an important endocine disruptor. DBP is widely used as plasticizerand flexibilizer in production of cellulose resin, polyvinyl chloride and rubbers, and is also onekind of raw material for many industrial products, such as lacquers, agglomerant, pharmaceuticals.Due to no tight binding to the matrix of these products, DBP can migrate from them into theenvironment, and may impair human health.The National Toxicology Program （NTP） estimated exposure level of DBP was 2-10μg/kgBW/day for general population from all sotirces. Once into the body, DBP is quickly metabolizedto monobutyl phthalate （MBP）, which is responsible for the induction of developmental toxiceffects of DBP. The main impairments of DBP are reproductive and developmental toxicity,mechanism under which is the disruption of endocrine function. DBP has shown estrogenicactivtity in vitro and anti-androgenic activity in vivo, and can affect the activity of enzymecatalyzing sterides synthesis. However, the neurobehavioral effects of DBP still remain unclear.The central nevious system （CNS） is very sensitive to many hormones during development,and is susceptive to endocrine disruptors such as DBP. Epidemiological data suspected that DBPwas a neurotoxin; however, due to the combined exposure with other PAEs, the neuro effects ofDBP were not very certain. Neurobehaviors are sensitive indicators reflecting the effects of endocrinedisruptors on the nevious system and/or other systems. This study was design to investigate thetoxic effects on the development of neurobehaviors in rats, and to explore the undermining mechanismfor a better understanting of DBP’s health effects. Wistar rats were treated with DBP in gestational and lactational periods, and their neurobehaviorswere evaluated by a battery of tests on different postnatal days. Multi-levels of dose, large sample sizeand cross-generation exposure are features of the present study. Due to the effects of DBP on spatiallearning and memory in the male offspring rats, the mechanism focused on hippocampal region of thebrain. The effects of DBP on the developent and function of hipocampal neurons were evaluated bymeans of detecting gene and protein expression using real time RT-PCR and/or Western Blot, anddetecting in vivo long-term potentiation, with an intent to explore the mechanism of neurotoxicityand provide experimental and theoretical data for DBP comtamination prevention and regulation.PartⅠEffects of DBP on Reproduction of F₀ Femal Rats and on Growth and Development of F₁Generation RatsObjective: To explore the effects of DBP on reproduction of F₀ female rats and on growth anddevelopment of F₁ generation rats.Methods: Sixty pregnant wistar rats were randomly assigned to control, low, sub-low,sub-high and high dose groups, each were treated with DBP at doses of 0%, 0.037%, 0.111%,0.333% and 1% in a powdered diet from GD 6 to PND 28. Body weight, food intake, symptoms ofintoxicication and reproductive effects were observed; Indexs of growth and development of F₁generation, including body length, tail length, anogenital distance （AGD）, and ages at firstapperance of pinna detachement, incisor eruption and eye opening, were also measured or observed;Main organs of the F₁ generation were checked and weighted when the exposure expired onPND28 and organ coefficients were calculated.Results: （1） Food intake and body weight of the pregnant rats increased with the gestationaldays, but no alteration was observed as compared to control （p＞0.05）. （2） No obvious toxicsymptoms were observed in the treatment group pregnant rats, and no significant difference wasfound in pregnancy failure rate, live pups per litter or sex ratio of the exposure g as compared tocontrol （p＞0.05）, but the gestational period length of the high treatment group was prolongedslightly but significantly （p＜0.05）. （3） Body weight of the pups （male and female respectively）exposed to high dose DBP was significantly less than that of control from PND0 to PND 28（p＜0.01）. （4） AGD （adjusted by the cube root of body weight） of the male pups in the high dose group was significantly decreased, but that of the females was not changed （p＞0.05）. （5） The age atfirst appearance of pinna detachement, incisor eruption or eye opening of neither male of femalepups was affected by DBP exposure （p＞0.05）. （6） On PND 28, liver-to-body weight of both maleand female pups exposed to high dose DBP increased significantly （p＜0.01）; spleen-to-body weightof female pups in high and sub-high dose groups decreased significantly （p＜0.01）; testes-to-bodyweight of male pups in high dose group also decreased significantly （p＜0.01）; however, none ofbrain-, heart-, lung-, kidneys- or womb-to-body weight was altered obviously （p＞0.05） in anytreatment group （p＞0.05）.Conclusions: DBP treatments in the present study exhibited no significant toxic effect on thereproductive ability of the F₀ male rats, but the high treatment affected the growth and developmentof the F₁ generation rats and showed overt organ toxicity, including decreased body weight,shortened AGD and altered liver- spleen- and testes-to-body weight.PartⅡEffects of DBP on Neurobehaioral Development of F₁ Generation RatsObjective: To evaluate the effects of DBP on neurobehaioral development in the F₁ generationrats.Methods: The treatment was the same as in PartⅠ. Pups were selected to perform air rightingon PNDs 4 and 7, negative geotaxis on PNDs 4 and 7, cliff avoidance on PND 7, forepaw grip timeon PND 10 and air righting on PND 16 for evaluation of the Pups at the age of 4 weeks were aslosubmitted to a open field tests to measure the motional activity; Learning and memory capacity wasmeasureed by Morris water maze tests in the F₁ generation pups at the age of 5 weeks.Results: （1） Early neurobehaioral development: Male pups in the high-dose group had lowerscores in the surface righting reflex on PND 7 than control with significant difference （p＜0.05）, andmale pups in both the low and the high-dose groups showed shortened forepaw grip time on PND 10（p＜0.05）, but for the surface righting on PND 7, negative geotaxis on PNDs 4 and 7, cliff avoidanceon PND 7 and air righting on PND 16, no significant treatment effects were found in either themale or the female pups. （2） Open field test: No significant treatment effect or sex effect was foundin the numbers of crossings, rearrings or retentions for more than 5 seconds （p＞0.05）, and still notreatment effect was found when the upper data were collaped across sex （p＞0.05）. （3） Morris water maze:①5-day hidden platform trial: Sex X Day interaction were found significant （p＜0.01）,male pups in the high-dose group exhibited better performances in spatial acquisition in the last 3days （p＜0.05）, but male pups in the low-dose group showed depressed spatial acquisition learningin the last 2 days （p＜0.05）. However, no treatment effect was found in the female pups.②One-dayprobe trial: Male pups in the low- and sub-low-dose groups exhibited less preference for the targetzone, indicating worse spatial memory （p＜0.05）, however, there still no treatment effect on thepreference was found in the female pups.③Visible platform trial: No significant treatment effectson either the escape latency or swimming path length was witnessed in either the male or femalepups （p＞0.05）.Conclusions: Under the experiment conditions as ours, DBP exposure altered a fewneurobehavioral parameters and spatial learning and memory capacity in the F₁ generation malepups, but scarcely affected that of the females, indicating significant sex effect and non-monotonicdose-related responses.PartⅢEffects of DBP on the Expression of Development-related Genes and Proteins in theHippocampus of the Male PupsObjective: To investigate the effects of DBP on the expression of develpment-related genesand proteins for exploration into the neurotoxic mechanism.Methods: Pregant Wistar rats were randomly derided into five groups, each containing 8～10rats. They were treated daily by lavage with corn oil, 25, 75,225 or 675 mg/kg BW DBP from GD6 to PND 21; Male offspring rats aged 1, 7 and 21 days were sacrificed for determinating theexpression of hippicampal genes, including c-fos、BDNF（all variants）、Spinophilin、PSD95、Synaptophysin、CREST、BDNF variantⅢ, and of hippicampal proteins （PND 21 only）, such asBDNF, p-CREB, spinophilin and aromatase.Results: （1） Effects on hippocampal gene expression:①Immediate early genes related withcell growth and differentiation and learing and memory: BDNF expression in the high-dose group（PND21） increased by about 30%, and the differences were significant as compared to control（p＜0.01）, but the expression of c-fos gene was not significantly altered by the treatment,②Genesrelated with the structure and function of dendrite and synapse: spinophilin expression in the low-dose group （PND21） increased by 35.7% with significant differences as compaired to control（p＜0.05）. However, the expression of PSD95 and synaptophysin genes was scarcely affected by thetreatment.③Genes related with CREB singal pathway: the expression of BDNF variantⅢ（percaentage among all BDNF variants decreased in the high-dose group） and CREST was notaffected by DBP treatment. （2） Effects on hippocampal protein expression: the content of BDNFmature molecule, spinophilin and aromatase in the high-dose group （PND21） increasedsignificantly （p＜0.01, p＜0.05 and p＜0.01）, however, the relative level of p-CREB in thehippocampus was not altered by the treatment.Conclusions: In utero and lactational exposure to high-dose DBP increased the expression ofBDNF, spinophilin and aromatase, indicating that DBP may promote the growth and differentiationof hippocampal nerve cells and increase dendrite spine abundance. The increase in BDNFexpression may be unrelated with the upper stream regulation of p-CREB. DBP treatment may notaffect the synaptic structure and function in the hippocampus of the male rats.PartⅣEffects of DBP on learning and memory, serum biochemical indicators andhippocampal LTP in the adult male ratsObjective: To investigate the effects of DBP on learning and memory in adult male rats, onserum biochemical indicators and on hippocampal LTP.Methods: Male offsping rats, one/litter/group, were those remained in stuty PartⅢ, whichcontinuted to receive the same DBP treatment from PND 21 （wearing） to PND 28. Spatial learningand memory capacity was determined twice by water maze at the age of one and two monthsrespectively. The animals in the high- and low-dose groups then continued to receive DBPtreatment till 3 months old, which were then detrained for serum biochemical indicators and forhippocampal LTP.Results: （1） Water maze tests: male rats exposed to high-dose DBP demonstrated betteracquisition of spatial information （p＜0.05） at the age of one month, and still exhibited improvedmemory （p＜0.05） after a one-month exposure intermission. （2） Serum biochemical indicators: ALBand TP were decreased by high-dose DBP treatment in the male offspring rats, but the activities ofAST and CHE were significantly increased by the same treatment （both p＜0.05）. （3） LTP in hippocampal dentate gyrus: achievement ratio of LTP induction was more than 70%, andamplification of both PS wave and EPSP slope rate was about 100%. However, these indicatorswere not significantly affected by the high- or low-dose DBP exposure as compared to control （allp＞0.05）Conclusions: The male rats exposed to high-dose DBP still exhibited better learning andmemory capacity when grown tip, which may resulted from the alterations in the nervous systemdevelopment. Tiffs high-dose DBP also showed obvious hepatic toxicity to the adult F₁ generationmale rats. Exposure to this high-dose DBP scarcely affected the function of synaptic transmissionin the hippocampus dentate gyrus of the male rats.Collectively, under the present experimental conditions, the high-dose DBP exposure showedsignificant reproductive, developmental and hepatic toxicities. As for the early neurobehavioralreflexes, DBP treatment exhibited few influences on these indicators of the female pups, butaffected some reflexes of the males, such as surface righting and forepaw grip time. The toxichigh-dose DBP, however, promoted spatial learning and memory of the nlale pups, which may berelated with the improved expression of genes and protein accounting for neuro-development.While the low-dose DBP exhibited no overt toxicity of reproduction and development, it decreasedthe forepaw grip time and spatial learning and memory capacity of the male pups. In theexploration for mechanism, no appropriate explanation was derived for the effects caused by thelow-dose exposure; however, this level was lower than the reported NOAEL （50mg/kg/day） basedon morphological lesions, indicating neuro-behaviors, especially learning and memory are moresusceptive to DBP exposure and should be paid special consideration when been evaluated.更多还原