全氟辛烷磺酸（PFOS）对小鼠免疫毒性效应研究
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摘要
前言
全氟辛烷磺酸(Perfluorooctane sulfonate,PFOS)是近年来引起科学家们关注的一种新型持久性有机化合物,该有机物被广泛用于机械润滑剂、涂料、农药和医药品、表面活性剂、纺织品和皮革制品表面防污剂、泡沫灭火剂等数百种工业和日用品生产领域。由于PFOS在自然界中的难降解性和生物体内的高蓄积性,其已经在生态系统中得到广泛的传播,并且正在通过食物链途径的生物浓缩和生物放大进行生物种群间的转移,最终高浓度地蓄积在食物链高位生物包括食肉动物和人体内。2004年杜邦公司“不粘锅事件”进一步引起了国内专家对PFOS的关注。
PFOS分子是由17个氟原子和8个碳原子组成的烃链,烃链末端碳原子上连接一个磺酰基。这种化学结构特点使分子中烃链碳原子间的共价键不容易受到外界作用而产生断裂。研究资料表明,PFOS即使在浓硫酸或浓硝酸溶液中煮沸1小时也不分解,或在98%硫酸中经过28天PFOS浓度也未见任何降低。与以往持久性有机污染物不同,PFOS具有疏水、疏油脂特性,虽然同属于持久性有机污染物,但是不蓄积在脂肪组织中,大部分与血浆蛋白结合存在于血液中,其余分布在动物肝脏、少量分布于肌肉和脑等其它组织中。研究表明,生物体内的PFOS主要来自食物、饮水、含PFOS泡沫灭火剂气溶胶吸入或体内含PFOS系列产品的降解。有研究表明,一个70kg体重的成年人通过饮食每天摄入PFOS的量约为62.5～74.2 ng。人群血清流行病学调查显示,各国各族人群血清中均检测出不同浓度的PFOS污染,其中职业暴露人群血清中PFOS浓度高达12.83mg/L;甚至新生儿血清中也存在PFOS污染。最近研究结果显示,人类血清中PFOS生物半衰期平均为8.67年(范围为2.29～21.3年,SD=6.12),最高达21.3年。而更值得关注的是,人体内PFOS负荷呈逐年增高趋势。日本学者的调查结果显示,在1983～1999年期间,人群血清中PFOS浓度以每年0.49 ug/L速度递增。我国沈阳地区人群血清PFOS浓度从1987年的0.03ug/L增加到2002年的22.4ug/L,15年间人群血清中PFOS浓度增加了747倍。
PFOS的大鼠一次经口半数致死剂量(LD_(50))为251mg/kg,按照WHO定义标准,PFOS应属于中等毒性物质。人群流行病学调查显示,PFOS可降低新生儿体重,并使职业人群患膀胱癌和前列腺癌的危险性增高。毒理学研究表明,PFOS可对机体的神经系统、生殖系统和内分泌系统产生损伤,并具有胚胎发育毒性和遗传毒性,表明PFOS可能为一种全身毒性物质。免疫系统是对外源性化合物最为敏感的系统之一,但是,目前对于PFOS材料引起的免疫毒性研究刚刚起步,资料甚少,现有的为数不多的研究主要集中在PFOS对某些有限的免疫指标的毒性效应上,而关于PFOS免疫毒性的系统研究未见报道。所以,全面研究PFOS的免疫毒性,了解PFOS对免疫系统的危害,阐明PFOS免疫毒性剂量—效应关系和毒作用机制,可使人们深入了解PFOS危害性,在PFOS环境安全性评价中提供科学依据。因此,本研究在观察PFOS短期染毒、亚慢性染毒和体外染毒对小鼠免疫系统毒性的基础上,应用ELISA、流式细胞分析等方法进一步探讨PFOS对小鼠细胞免疫、体液免疫、非特异性免疫等的影响,从分子水平上评价PFOS对小鼠免疫系统毒性效应。
材料与方法
一、试验动物染毒方式和样品制备
1、PFOS配制方法:
试验组配制:配制2%Tween 80溶液,按照设计剂量加入全氟辛烷磺酸钾盐(PFOS-K)配制PFOS溶液。对照组配制:配制2%Tween 80溶液。
2、短期染毒
8-10周的雄性C57BL/6小鼠48只,实验室内适应性喂养7天后按体重随机分为4组,每天一次经口灌胃染毒,试验组每天PFOS染毒剂量分别为5、20、40mg/kg(bw),对照组给予2%Tween-80。每天一次经口灌胃染毒7天。
3、亚慢性染毒
将8-10周的雄性C57BL/6小鼠60只,实验室内适应性喂养10天后按体重随机分为6组,每天一次经口灌胃染毒60天,试验组60天PFOS累计染毒剂量[TotalAdministered Dose(TAD)]分别为0.5、5、25、50、125mg/kg(bw),即每天染毒剂量分别为8.33、83.33、416.67、833.33、2083.33μg PFOS/kg(bw),对照组给予2%Tween-80。
4、体外染毒
10周雄性C57BL/6小鼠6只,无菌取脾制备脾细胞悬液,调整脾细胞终浓度至1×10~7/mL,取200μl/孔加入24孔培养板中。将PFOS(以DMSO为溶剂)按0、1、5、10、50、200、300μM的浓度加入24孔培养板中,37℃、5%CO_2的细胞培养箱内培养48小时,提取培养上清,检测细胞因子水平。
5、脾细胞采集
短期染毒后第8天或亚慢性染毒第61天,摘除眼球采血并收集血清,颈椎脱位处死小鼠,75%酒精浸泡,取出各组小鼠的胸腺、肾脏、肝脏、脾脏,称量质量,并计算器官指数。常规制备脾细胞悬液,用含2%热灭活FCS的PBS将脾细胞终浓度调至1×10~7/mL。
二、免疫指标测定方法
1、血清中PFOS浓度测定
0.5mL血清样品加入1.0mL 0.5mol/LTBAHS和2.5mL 0.25mol/L碳酸氢钠缓冲液,振荡混合20min后加入5.0mL MTBE进行萃取,离心后,收集上层MTBE相。沉淀物部分再次加入5.0mL MTBE,进行2次萃取。两次萃取合并液用高纯氮吹干后,准确加入1.0mL甲醇进行定容,进行HPLC/MS定量分析。
2、CD4~+、CD8~+细胞亚群检测
取0.1 mL脾细胞悬液,加入anti-CD3-FITC(553061)、anti-CD4-PE(557308)、anti-CD8-PERCP(553036)0.2μg,4℃下避光孵浴30min。加2mL PBS、混匀,离心5min(2000r/min),弃上清。将剩余液体转入流式管。流式细胞仪的激发波长为488nm,利用FACS CELLQUEST软件获取细胞,每个样品分析10,000个细胞;记录CD4~+T细胞、CD8~+T细胞的个数百分比。
3、NK细胞活性检测
以YAC-1细胞作为靶细胞,调整靶细胞浓度至1×10~5/ml备用。取脾细胞(1×10~6/ml)和靶细胞各0.1ml加入96孔细胞培养板中,每份标本设三个复孔,同时设靶细胞自然释放对照组和最大释放对照组。用酶联检测仪在490nm波长下读各孔OD值,计算NK细胞活性。
4、MTT法检测淋巴细胞增殖
用含10%小牛血清的RPMI-1640培养液调整脾细胞浓度为5×10~6/mL,加入96孔培养板,每孔100μl脾细胞悬液。每孔加入10μg/ml的ConA或LPS100μl,同时设只加培养液的阴性对照,每组设3复孔。在酶标仪上于570nm处测光吸收值(A),以下列公式计算淋巴细胞增殖指数(SI):SI=刺激孔A值/对照孔A值。
5、NO测定
取脾细胞培养上清接入96孔培养板中,100μl/孔,将100μmol/L NaNO_2在96孔培养板中进行倍比稀释。各孔中再加入0.1ml Griess试剂,室温静置10min,在550nm波长处测OD值。
6、B细胞溶血空斑试验测定
将绵羊红细胞(SRBC)免疫过的小鼠脾细胞制成2×10~6/ml悬液,与0.1ml SRBC和0.05ml的DEAE右旋糖酐补体在琼脂凝胶内混合,冷却凝固后放入37℃温箱1小时,加入豚鼠血清1.5ml。记录整个平皿中溶血空斑数。
7、ELISpot法检测IL-2和IL-10细胞数量
采用ELISpot法检测脾脏IL-2和IL-10的细胞数量,调整脾细胞浓度分别为1×10~5/ml(IL-2)和1×10~6/ml(IL-10)。每孔加入100μl脾细胞悬液和对照品,37℃CO_2温箱孵育1h。每孔加100μl生物素标记的IL-2或IL-10检测抗体,4℃过夜孵育。加入碱性磷酸酯酶标记链霉亲和素100μl/孔,室温孵育2h。加入BCIP/NBT显色液100μl/孔,避光孵育1h。ELISPOT自动分析仪读板计数IL-2或IL-10细胞数量。
8、ELIsA法细胞因子IL-2、IL-4、IL-10、IFN-γ测定
采用双抗体夹心ELISA法:每孔加入50μl倍比稀释的标准品,对照品或培养上清,混匀1min。室温孵育2h。每孔加入100μl各细胞因子多克隆抗体,室温孵育2h。100μl/孔加入酶标记溶液,室温避光孵育30min。加入100μl/孔终止液。用酶标仪在450nm波长读记吸光度OD值。
9、血清抗体IgG、IgM测定
采用ELISA检测血清抗体水平:100μl/孔标准品加入到96孔酶标板中,每孔加入100μl血清,加入50μl/孔酶标记溶液,36℃孵育1h,每孔加入底物呈色剂A、B液各50μl,36℃孵育15min,加入50μl/孔终止液。于450nm酶标仪读取OD值。
10、统计学分析
采用SAS8.2统计软件进行数据的分析。对数据进行正态性检验后,用均数(Mean)和标准误(SE)描述正态分布数据的描述指标。各组数据之间比较采用单因素方差分析,采用Dunnett(?)t检验进行各个试验组与对照组的多重比较。
结果
1、PFOS染毒对小鼠体重、饲料消耗量和器官指数的影响
短期染毒试验:对照组小鼠血清中PFOS浓度低于检测限(0.05mg/L),PFOS染毒5、20、40mg/kg剂量组小鼠血清中PFOS浓度分别为110.46±6.18mg/L、280.65±16.33mg/L、338.01±23.92mg/L。20、40mg PFOS/kg剂量组小鼠的体重和饲料消耗量呈显著的下降趋势(p<0.05),且脾脏指数和胸腺指数显著的低于对照组。各试验组小鼠肝脏指数均显著的高于对照组,肾脏指数变化并不显著。
亚慢性染毒试验:PFOS染毒0.5、5、25、50、125mg/kg(bw)TAD剂量组小鼠血清中PFOS浓度分别为0.67±0.17mg/L、7.13±1.04mg/L、21.64±4.41mg/L、65.43±11.73mg/L、120.67±21.76mg/L,均显著的高于对照组血清PFOS水平。25、50、125mg PFOS/kg TAD剂量组小鼠的体重和饲料消耗量呈显著的下降趋势,且脾脏指数和胸腺指数显著的低于对照组。≥5mg PFOS/kg TAD剂量组小鼠肝脏指数显著的高于对照组,并随着染毒剂量的增高,肝脏指数呈增高趋势。
2、PFOS对小鼠脾细胞和胸腺细胞总数及淋巴细胞亚群的影响
短期染毒试验:20、40mg PFOS/kg剂量组小鼠脾细胞和胸腺细胞总数显著的降低;40mg PFOS/kg剂量组小鼠CD4~+ CD8~-和CD4~-CD8~+细胞亚群的数量分别减少28%和21%。亚慢性染毒试验:25、50、125mg PFOS/kg TAD剂量组小鼠脾细胞和胸腺细胞总数显著的低于对照组,其中125mg PFOS/kg TAD剂量组小鼠脾细胞和胸腺细胞总数分别减少55%和70%。CD4~+CD8~-和CD4~-CD8~+细胞亚群的数量显著降低。
3、PFOS对淋巴细胞增殖功能的影响
短期试验中,当PFOS染毒剂量≥5mg/kg/day时,可显著的抑制T淋巴细胞增殖功能,而B淋巴细胞增殖功能的抑制开始于20mg/kg/day剂量组。亚慢性毒性试验中,随着PFOS染毒剂量的增高,T、B淋巴细胞增殖功能呈先增高后降低趋势。体外试验结果显示,当PFOS暴露剂量为≥200μM时,可显著抑制B细胞增殖功能。
4、PFOS对NK细胞活性的影响效应
短期试验中,PFOS染毒剂量20mg和40mg/kg/day组小鼠NK细胞的活性分别为18.04±1.42和13.08±1.11,显著的低于对照组水平(50.33±4.08)。亚慢性毒性试验中,5mg PFOS/kg TAD可显著的上调NK细胞的活性,当PFOS染毒剂量≥50mg/kg TAD时,NK细胞的活性显著降低。
5、PFOS对脾细胞培养上清NO产生水平影响
短期试验中,仅40mg/kg/day剂量组NO水平显著的低于对照组。亚慢性试验中,NO水平随着PFOS染毒剂量的增高呈先增高后降低趋势。体外实验显示,当PFOS暴露剂量为300μM时,可显著抑制NO水平。
6、B细胞溶血空斑试验测定
短期试验中,5mg、20mg、40mg PFOS/kg剂量组空斑形成细胞数分别减少63%、77%和86%。亚慢性毒性试验中,当PFOS暴露剂量为5mg/kg TAD时,空斑形成细胞数可显著的低于对照组,并随着染毒剂量的增高,空斑形成细胞数呈降低趋势。
7、细胞因子IL-2、IL-4、IL-10、IFN-γ测定
短期试验和亚慢性试验中,随着染毒剂量的增高,IL-2和IFN-γ的水平均呈降低趋势。短期试验中IL-4呈先增高后降低趋势;而亚慢性试验中PFOS可显著促进IL-4的水平。IL-10在各剂量组间的差异并没有达到统计学意义。体外实验显示,PFOS并没有显著的抑制细胞因子的表达。
8、血清抗体IgG、IgM测定
短期试验中,PFOS各暴露剂量组IgM水平均显著的低于对照组,而IgG水平在5mg/kg/day剂量组显著的增高,40mg/kg/day组显著的降低。亚慢性毒性试验中,当PFOS累计染毒剂量≥5mg/kg TAD时,随着染毒剂量的增高,IgM水平呈显著下降趋势。IgG水平呈先增高后降低趋势。
结论
1、PFOS是与机体免疫反应相关的外源性化学物质,以5mg PFOS/kg剂量连续7天染毒小鼠时即可抑制小鼠免疫功能。
2、PFOS对C57BL/6小鼠经口染毒60天可导致机体免疫系统的抑制,根据免疫指标的改变,其未观察到损害所用的剂量(NOAEL)和观察到损害作用的最低剂量(LOAEL)分别为0.5mg/kg TAD和5mg/kg TAD,对应血清中PFOS浓度分别为0.67±0.17mg/L和7.13±1.04mg/L。
3、体外试验结果比表明200μM PFOS可显著的抑制脾淋巴细胞的增殖功能,表明在较高剂量水平下PFOS可对淋巴细胞产生直接毒性效应。
4、根据自然人群血清中PFOS浓度不高于150μg/L计算,小鼠血清中PFOS浓度约为自然人群血清最高浓度的50倍时,可抑制机体免疫功能。
Introduction
Perfluorooctanesulfonate[PFOS;CF_3(CF_2)_7SO_3~-],which is produced synthetically or from the metabolism of other perfluorinated chemicals(PFCs),has extreme thermal, biological,and chemical stability as well as hydrophobic and lipophobic characteristics. These properties make it widely to be used as industrial surfactants and emulsifiers and in numerous consumer products.Nonstick pans,carpets,furniture,household cleaners, shampoos,shoes,clothing,and convenience food packaging are some of the products that can contain PFCs.PFOS has aroused scientists'great concern in recent years due to its widespread occurrence in the environment,in the wildlife and in humans. Furthermore,it has shifted among biological populations via biological concentration and magnification,leading to excessive accumulation among the higher trophic level of food chain(such as predator and human beings).
PFOS,a hydrocarbon chain composited with 17 F-atom and 8 carbon-atom and linked one sulphur acyl,has high surface tension-reducing properties,lower water and oil solubility,and is a relatively strong organic acid.The strength of carbon-fluorine bonds and the high electronegativity of perfluorinated alkyl acids contribute to the extreme stability and unique properties of PFOS which made it not expected to be metabolized when be seethed in oil of vitriol or nitric acid for one hour.Different from the other persistent organic pollutions,PFOS is the predominately PFCs found in both human and wildlife blood samples and accumulates primarily in the blood and liver for its lower water and oil solubility.Previously several exposure routes have been suggested to play an important role in the exposure to PFOS:food,drinking water,air borne dust,house dust or other PFCs which can be rapidly metabolized into PFOS via a perfluorooctane sulfonamide.One study showed that,on average,for a standard adult man(70 kg of body weight),the dietary intake of PFOS was estimated to be 62.5 or 74.2 ng/day.Several blood epidemiology investigaions reported regarding PFOS concentration in human serum,and human serum concentrations vary depending on the population evaluated.For occupational exposure,the serum value was 12.83mg/L. Even in infant,there was also PFOS contamination.One recent study showed that the arithmetic mean half-lives of serum elimination was 8.67 years(range:2.29～21.3 year, standard error=6.12 year),and the maximum biological half-life reaches up to 21.3 years.The historical samples collected from 1983 to 1999 in Japan demonstrated that the PFOS concentrations in Japanese have increased 4.4-fold at a rate of increase of 0.49ng/ml/year.One of our recent studies indicate that the serum PFOS level in un-occupationally exposed individuals from Shenyang of China increased significantly from 0.03μg/l in 1987 to 22.4μg/l in 2002,a 747-fold increase,and from 1999 to 2002, serum PFOS concentration also increased 13 fold.
For rat,the LD50 based on once oral exposure was 251mg PFOS/kg body weight. A hospital-based cross-sectional epidemiologic study observed small negative associations between both PFOS concentrations and birth weight and size.Another study conducted by 3M revealed an increase in bladder cancer and prostate cancer mortality among workers exposed to PFOS.A number of mammalian toxicology studies were conducted to assess the health effects of exposure to PFOS,recent studies indicated that PFOS can cause hepatotoxicity,tumors of the liver,and of the thyroid and mammary glands,developmental toxicity,reproductive toxicity,neurotoxicity and endocrine-disrupting.However,to our knowledge,few studies have assessed the effects of PFOS exposure on immune system.The purpose of our research is to study the immune toxicity induces by PFOS exposure in mice by flow cytometry assay,ELISA, et al.We therefore evaluated both humoral and cell-mediated immune function in experiments designed to provide scientific evidence in environmental safty evaluation and establish no observed adverse effect level(NOAEL) and lowest observed adverse effect level(LOAEL) values from dose-response studies of immune function.
Material and Methods
1.Animal samples
(1).PFOS solution:
Potassium PFOS suspensions were prepared in de-ionized water with 2%Tween(?) 80 at concentrations.Exposures consisted of oral administration of PFOS delivered in de-ionized water with 2%Tween(?) 80.Control mice received de-ionized water with 2%Tween(?) 80 only.
(2).7-day oral exposure to PFOS
Forty-eight adult C57BL/6 male mice were randomly divided by weight into four groups of 12/group.Once distributed into groups the mice acclimated to the new cage conditions and the new treatment room(12-h light/dark cycle,22±2℃,60-65% relative humidity) for 1 week before dosing was initiated.C57BL/6 mice were dosed once daily via oral gavage for 7 days(0,5,20,or 40 mg/kg body weight day).Food intake and body weight of all animals were measured daily for 7 days.
(3).60-day oral exposure to PFOS
Sixty adult C57BL/6 male mice were randomly divided by weight into six groups of 10/group.Once distributed into groups the mice acclimated to the new cage conditions and the new treatment room(12-hr light/dark cycle(light,0600-1800 hours; dark,1800-0600 hours),22.4±1.3℃,60-65%relative humidity) for 10 days before dosing was initiated.Exposures consisted of oral administration of PFOS delivered in de-ionized water with 2%Tween(?) 80.Control mice received de-ionized water with 2%Tween(?) 80 only.C57BL/6 mice were dosed once daily via oral gavage for 60 days (0,8.33,83.33,416.67,833.33 or 2083.33μg PFOS/kg body weight/day) to yield a targeted Total Administered Dose(TAD) over the 60 days of 0,0.5,5,25,50,or 125 mg PFOS/kg body weight.Food intake and body weight of all animals was measured daily for 60 days.
(4).PFOS exposure in vitro
Spleens from six adult C57BL/6 male mice were aseptically processed into single-cell suspensions by gentle grinding with the use of sterile,frosted microscope slides for functional immune endpoints.The splenocytes were washed three times in RPMI-1640 supplemented with 10%FBS and then resuspended in RPMI 1640 medium. The concentration of splenocytes was adjusted to 1×10~7 nucleated cells/ml.200μl aliquots of the resulting cell suspensions were dispensed into 24-well plates.The PFOS (dissolved in dimethylsulfoxide(DMSO)) was added to the medium at the indicated concentrations(0,1,5,10,50,200,300μM) at the beginning of the experiment.In this case,the same amount of DMSO alone(0.05%final concentration) was added to the control medium.The plates were incubated for 48 h at 37℃in a humidified 5% CO_2-air mixture.
(5).Spleen or thymus cells collection
Mice were bled by retro-orbital puncture under light diethyl ether anesthesia and subsequently sacrificedSpleen,thymus,liver,and kidneys were collected and weighed. All balances were calibrated,using standard weights,prior to use.Spleen and thymus were aseptically processed into single-cell suspensions by gentle grinding with the use of sterile,frosted microscope slides for functional immune endpoints and T-cell immunophenotype determinations.A Coulter Counter(model ZF;Hialeah,FL) was used to obtain cell counts from theses single-cell suspensions.Alterations in cell viability following treatment were assessed after red blood cell lysis.The concentration of splenocytes was adjusted to 1×10~7 nucleated cells/ml.
2.Experimental Methods
(1).Serum concentration of PFOS
0.5 ml of serum,1 ml of 0.5 M tetrabutylammonium hydrogen sulfate solution and 2 ml of sodium carbonate buffer(0.25 M,pH 10) were added to 15 ml polypropylene tube and thoroughly mixed.Following addition of 5 ml of MTBE to the solution,the organic and aqueous layers were separated by centrifugation,and the organic layer was removed.The aqueous mixture was rinsed with MTBE and separated twice.The solvent was evaporated at room temperature under a nitrogen gas flow,and the sample was then reconstituted in 0.5 mL of methanol.The sample was then passed through a nylon filter(Autovial R5 PUNYL;0.45-μm pore size;Whatman Japan,Tokyo) to remove any suspended materials and insoluble particles.
(2).Splenic and Thymic CD4/CD8 Subpopulations
Spleen or thymus cells were labeled with fluorescent(phycoerythrin or peridinin chlorophyl protein) rat IgG2 monoclonal antibodies specific for mouse CD4 or CD8 (rat anti-mouse).The antibody dilution used for FACS analysis was 1:5(v/v) for FITC conjugated rat-anti-mouse CD3,1:2(v/v) for PE conjugated rat anti-mouse CD4 and 1:2(v/v) for Percp conjugated rat anti-mouse CD8,respectively,following the manufacturer's instructions.Lastly,the cells were fixed with 1%paraformaldehyde and stored at 6℃in the dark.Flow cytometric analysis was performed using a Becton Dickinson flow cytometer(FACSCalibur;San Jose,CA,USA).Nonstained cells and isotypic antibody controls were used to establish gates for the CD4/CD8 subpopulations in splenic cells.Data are represented as absolute number of cells, determined by multiplying the percent gated cells by the total number of nucleated cells obtained by the Coulter Counter.
(3).Natural Killer(NK) Cell Activity
The splenocytes were washed and suspended in complete RPMI-1640 medium, then were counted and diluted to 1.0×10~6 nucleated cells/ml.The amount of the LDH released from the lysed target cells was determined to measure NK activity.The cell line Yac-1 was used as the target cell.The same volume of Yac-1 cells and splenocytes were added to the wells of 96 round-bottom microwell plates(the cell ratio of effector-to-target is 10:1).Three wells were used for every mouse.Finally,a microtiter plate reader(Bio-Rad,Modal 550) was used for evaluation of changes in the absorbance at a wavelength of 490 nm.The release of LDH from Yac-1 cells was expressed as absorbance.The percentage of NK cell activity was calculated by the formula:NK cell activity=[(E-S)/(M-S)]×100%.Where E represents the experimental release of LDH activity from target cells incubated in the presence of lymphocytes,M represents the maximum release of the LDH activity determined by lysing the target cells with 1%of NP-40,and S is the spontaneous release of the LDH activity from target cells incubated in the absence of lymphocytes.
(4).Lymphocyte proliferation assay
The concentration of splenocytes was adjusted to 5×10~6 nucleated cells/ml.The proliferation ability of the lymphocytes was determined by MTT stain assay.100μl aliquots of the resulting cell suspensions were dispensed into 96-well plates(5×10~5 cells/well) containing triplicate wells of either 10μg/ml mitogen Con A,10μg/ml LPS or supplemented RPMI-1640(unstimulated wells),the final culture volume was 200μl in each well.After incubation at 37℃in 5%CO2 atmosphere overnight,the microplate was read on a Bio-Rad microplate reader(Model 550) using test wavelength of 570 nm.The ratio of the optical density(OD) of stimulated to the OD of unstimulated cultures was used as the stimulation index.The proliferation index was calculated by the equation:proliferation index=A value of Con A or LPS-stimulated cells/A value of nonstimulated cells.
(5).Measurement of NO
The levels of NO metabolites(nitrite plus nitrate) were determined by enzymatically reducing the nitrate present with nitrate reductase.A standard nitrate curve was obtained by incubating sodium nitrate with reductase buffer.The total amount of nitrite was determined by the Griess method.Briefly,the samples were incubated with an equal volume of freshly prepared Griess reagent(1%sulfanilamide, 0.1%naphthylenediamine dihydrochloride in 5%phosphoric acid).Absorbance at 550nm was determined using a multi-well plate reader.
(6).Antibody Plaque-Forming Cell Assay
The number of plaque forming cells(PFCs) was determined using the Jerne plaque assay(Jerne and Nordin,1963).Briefly,Four days prior to euthanasia,mice were administered 0.1 mL of a 25%SRBC suspension in PBS via intraperitoneal injection.All SRBCs for the experiments were drawn from a single donor animal. Spleen cells collected from individual animals(0.1 mL;1×10~6 cells/0.1 mL),0.4 mL of 0.5%"low melting point" agarose(GIBCO,Grand Island,NY,USA) in RPMI-1640 medium,and 50μL of a suspension of 5%SRBC were added to test tubes at 37℃and poured onto microscope slides containing a bottom layer of 0.5%agarose in water.The slides were then incubated for 2 hr at 37℃and 5%CO2.Guinea pig serum diluted 1:4 in RPMI-1640 was added to the slides and after another 40 min(37℃and 5%CO2) incubation the number of plaques was counted and values were expressed as PFC per 10~6 cells.For the preparation of guinea pig serum as the complement source,animals were anesthetized before cardiac puncture and blood samples were collected.
(7).Measurement of IL-2,IL-10 by ELISpot
Fill all wells in the microplate with 200 L of sterile culture media and incubate for approximately 20 minutes at room temperature.When cells are ready to be plated, aspirate the culture media from the wells.Immediately add 100 L of the appropriate cells(1×10~5/ml for IL-2 and 1×10~6/ml for IL-10) and controls to each well.Incubate cells in a humidified 37°C CO2 incubator.Optimal incubation time for each stimuli should be determined by each investigator.Add 100 L of diluted Detection Antibody into each well and incubate at 2-8°C overnight.Add 100 L of diluted Streptavidin-AP into each well and incubate for 2 hours at room temperature.Add 100 L of BCIP/NBT Chromogen into each well and incubate for 1 hour at room temperature.The developed microplate can be analyzed by counting spots either manually using a dissection microscope or by using a specialized automated ELISpot reader.
(8).Measurement of IL-2,IL-4,IL-10,and IFN-γ
Add 50μL of Standard,Control,or sample per well.Mix by gently tapping the plate frame for 1 minute.Cover with the adhesive strip provided.Incubate for 2 hours at room temperature.Plate layouts are provided to record standards and samples assayed.Aspirate each well and wash,repeating the process four times for a total of five washes.Add 100μL of mouse IL-2,IL-4,IL-10 or IFN-γConjugate to each well. Cover with a new adhesive strip.Incubate for 2 hours at room temperature.Add 100μL of Substrate Solution to each well.Incubate for 30 minutes at room temperature. Protect from light.Add 100μL of Stop Solution to each well.Gently tap the plate to ensure thorough mixing.Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
(9).Measurement of IgG,IgM
Dispense 100μl of standards and specimens into appropriate wells,and then 50μl of Enzyme Conjugate Reagent into each well.Incubate at 36℃for 1 hour.Rinse and empty the microtiter wells 5 times,then dispense 50μl of color A and color B Reagent into each well.Incubate at 36℃for 15 minutes.Stop the reaction by adding 50μl of Stop Solution.Read the optical density at 450 nm within 30 minute.
(10).Statistics
Data were tested for normality(Shapiro-Wilks W-test) and homogeneity (Bartlett's test for unequal variances) and,if needed,appropriate transformations were made.Transformations when required are outlined in the figure legends.A one-way analysis of variance(ANOVA) was used to determine differences among doses for each endpoint using SAS software(Version 8.2;SAS Institute Inc.,Cary,NC) in which the standard error used a pooled estimate of error variance.When significant differences were detected by the F-test(p＜0.05),Dunnett's t-test was used to compare treatment groups to the control group.
Results
1.Animal Body Weight,Food intake and Organ Mass
In the 7-day study,body weight of mice exposure to 20 mg/kg and 40 mg/kg of PFOS showed significant deterioration from their own pre-exposed baseline.On the last day of the treatment,body,spleen,and thymus mass were signiWcantly decreased compared to the control following exposure to 20 mg/kg and 40 mg/kg of PFOS. Furthermore,liver mass was increased by 34,79,and 117%over control following treatment with 5,20,or 40 mg/kg,respectively.
In the 60-day study,there was a dose-dependent increase in the concentrations of PFOS in serum from exposed mice.At the last day of the treatment,body,spleen, thymus and kidney mass were significantly decreased compared to the control following exposure to 25 mg/kg TAD,50 mg/kg TAD and 125 mg/kg TAD of PFOS. Furthermore,liver mass was significantly increased at dose as low as 5 mg PFOS/kg TAD.
2.Splenic and Thymic Cellularity,Lymphocyte Immunophenotypes
Treatment with 5,20,or 40 mg PFOS/kg resulted in downtrend of splenic and thymic cellularity following 7 days of treatment.FACS analysis of spleen T lymphocytes demonstrated that the relative CD4+CD8- population of total splenocytes had decreased by 28%in the 40 mg/kg PFOS treatment and the relative change in the CD4-CD8+ population was decreased by 21%.Treatment with 25,50,or 125 mg PFOS/kg TAD resulted in downtrend of splenic and thymic cellularity following 60 d of treatment.Especially for the group of mice exposed to 125 mg PFOS/kg TAD,the splenic and thymic cellularity was found to be significantly decreased by 55%and 70% following the 60-d exposure compared with control mice.Splenic and thymic T lymphocytes demonstrated that the numbers of all T-cell CD4/CD8 subpopulations were significantly decreased beginning at 25 mg PFOS/kg TAD.
3.Effect of PFOS on Lymphocyte Proliferation
In the 7-day study,the average absorbances of T lymphocytes from≥5 mg/kg PFOS groups were lower than the control,and the B lymphocyte proliferation decreased beginning at≥20 mg/kg PFOS.In the 60-day study,with the level of PFOS exposure increasing,the average absorbances of T and B lymphocytes were first increasing and then decrasing.The effect of PFOS in vitro treatment on lymphocyte proliferation showed that the PFOS did not alter lymphocyte proliferation except for the condition of≥200μM PFOS.
4.Effect of PFOS on NK-cell Function
In the 7-day study,treatment with the dose of 20 mg/kg(18.04±1.42) and 40 mg/kg(13.08±1.11) PFOS resulted in a marked decrease in the levels of NK-cell activity compared with control(50.33±4.08).Treatment with the dose of 5 mg PFOS/kg TAD significantly increased the NK cell activity by 38%(45.43±4.74).In contrast,treatment with the dose of 50 mg/kg TAD(20.28±2.51) and 125 mg/kg TAD (15.67±1.52) PFOS resulted in a marked decrease in the levels of NK cell activity.
5.Effect of PFOS on NO
Following 7 days of treatment,NO level was significantly lower in group of 40 mg PFOS/ke/day compare control group.In the 60-day study,NO level was elevated at 5 mg PFOS/kg TAD and then decrased at 50 mg PFOS/kg TAD.The effect of PFOS in vitro treatment on NO levels showed that the PFOS did not alter NO level except for the condition of 300μM PFOS.
6.Plaque-Forming Cell Assessments
Treatment with 5,20,or 40 mg PFOS/kg resulted in significant suppression of the plaque-forming cell response following 7 days of treatment(63%,77%,and 86% respectively).There was a significant trend toward decreased PFC production with increasing PFOS exposure.The suppression of this response was dose-responsive beginning at exposures of 5 mg PFOS/kg TAD.
7.Effect of PFOS on IL-2,IL-4,IL-10 and IFN-γ
With the PFOS exposure increasing,there was a significant trend toward decreased IL-2 and INF-γproduction,and PFOS increased IL-4 synthesis.IL-10 was not statistically altered by exposure to the tested doses of PFOS.In vitro treatment,no other statistical changes in cytokine were observed exposed to PFOS.
8.Effect of PFOS on IgG and IgM
In the 7-day study,exposure to PFOS reduced IgM synthesis.IgG titers were elevated at 5 mg PFOS/kg/day and then decrased at 40 mg PFOS/kg/day.In the 60-day study,IgM synthesis was suppressed at exposures≥5 mg PFOA/kg TAD in a dose-dependent manner.
Conclusion
1.PFOS,as an exogenous chemical material,is related to immune reaction.PFOS exposure can suppress the immunity function in mice in a dose of 5 mg/kg bw for 7 d.
2.Based on the liver mass and PFC response,the no observed adverse effect level (NOAEL) and low observed effect level(LOAEL) for male mice exposed PFOS for 60 days was 0.5 mg/kg TAD and 5 mg/kg TAD,respectively.Measured PFOS serum concentrations at these dose levels were 0.67±0.17mg/L and 7.13±1.04mg/L, respectively.
3.In vitro treatment,PFOS exposure can suppress the lymphocyte proliferation which indicate that PFOS may have a direct toxicity to lymphocyte.
4.Human biomonitoring studies indicates that PFOS residues in adult blood or serum are generally present at levels lower than 150μg/L,that PFOS exposure can affect the immunity function in mice at levels approximately for 50-fold for highly exposed human populations.
全氟辛烷磺酸(Perfluorooctane sulfonate,PFOS)是近年来引起科学家们关注的一种新型持久性有机化合物,该有机物被广泛用于机械润滑剂、涂料、农药和医药品、表面活性剂、纺织品和皮革制品表面防污剂、泡沫灭火剂等数百种工业和日用品生产领域。由于PFOS在自然界中的难降解性和生物体内的高蓄积性,其已经在生态系统中得到广泛的传播,并且正在通过食物链途径的生物浓缩和生物放大进行生物种群间的转移,最终高浓度地蓄积在食物链高位生物包括食肉动物和人体内。2004年杜邦公司“不粘锅事件”进一步引起了国内专家对PFOS的关注。
PFOS分子是由17个氟原子和8个碳原子组成的烃链,烃链末端碳原子上连接一个磺酰基。这种化学结构特点使分子中烃链碳原子间的共价键不容易受到外界作用而产生断裂。研究资料表明,PFOS即使在浓硫酸或浓硝酸溶液中煮沸1小时也不分解,或在98%硫酸中经过28天PFOS浓度也未见任何降低。与以往持久性有机污染物不同,PFOS具有疏水、疏油脂特性,虽然同属于持久性有机污染物,但是不蓄积在脂肪组织中,大部分与血浆蛋白结合存在于血液中,其余分布在动物肝脏、少量分布于肌肉和脑等其它组织中。研究表明,生物体内的PFOS主要来自食物、饮水、含PFOS泡沫灭火剂气溶胶吸入或体内含PFOS系列产品的降解。有研究表明,一个70kg体重的成年人通过饮食每天摄入PFOS的量约为62.5～74.2 ng。人群血清流行病学调查显示,各国各族人群血清中均检测出不同浓度的PFOS污染,其中职业暴露人群血清中PFOS浓度高达12.83mg/L;甚至新生儿血清中也存在PFOS污染。最近研究结果显示,人类血清中PFOS生物半衰期平均为8.67年(范围为2.29～21.3年,SD=6.12),最高达21.3年。而更值得关注的是,人体内PFOS负荷呈逐年增高趋势。日本学者的调查结果显示,在1983～1999年期间,人群血清中PFOS浓度以每年0.49 ug/L速度递增。我国沈阳地区人群血清PFOS浓度从1987年的0.03ug/L增加到2002年的22.4ug/L,15年间人群血清中PFOS浓度增加了747倍。
PFOS的大鼠一次经口半数致死剂量(LD_(50))为251mg/kg,按照WHO定义标准,PFOS应属于中等毒性物质。人群流行病学调查显示,PFOS可降低新生儿体重,并使职业人群患膀胱癌和前列腺癌的危险性增高。毒理学研究表明,PFOS可对机体的神经系统、生殖系统和内分泌系统产生损伤,并具有胚胎发育毒性和遗传毒性,表明PFOS可能为一种全身毒性物质。免疫系统是对外源性化合物最为敏感的系统之一,但是,目前对于PFOS材料引起的免疫毒性研究刚刚起步,资料甚少,现有的为数不多的研究主要集中在PFOS对某些有限的免疫指标的毒性效应上,而关于PFOS免疫毒性的系统研究未见报道。所以,全面研究PFOS的免疫毒性,了解PFOS对免疫系统的危害,阐明PFOS免疫毒性剂量—效应关系和毒作用机制,可使人们深入了解PFOS危害性,在PFOS环境安全性评价中提供科学依据。因此,本研究在观察PFOS短期染毒、亚慢性染毒和体外染毒对小鼠免疫系统毒性的基础上,应用ELISA、流式细胞分析等方法进一步探讨PFOS对小鼠细胞免疫、体液免疫、非特异性免疫等的影响,从分子水平上评价PFOS对小鼠免疫系统毒性效应。
材料与方法
一、试验动物染毒方式和样品制备
1、PFOS配制方法:
试验组配制:配制2%Tween 80溶液,按照设计剂量加入全氟辛烷磺酸钾盐(PFOS-K)配制PFOS溶液。对照组配制:配制2%Tween 80溶液。
2、短期染毒
8-10周的雄性C57BL/6小鼠48只,实验室内适应性喂养7天后按体重随机分为4组,每天一次经口灌胃染毒,试验组每天PFOS染毒剂量分别为5、20、40mg/kg(bw),对照组给予2%Tween-80。每天一次经口灌胃染毒7天。
3、亚慢性染毒
将8-10周的雄性C57BL/6小鼠60只,实验室内适应性喂养10天后按体重随机分为6组,每天一次经口灌胃染毒60天,试验组60天PFOS累计染毒剂量[TotalAdministered Dose(TAD)]分别为0.5、5、25、50、125mg/kg(bw),即每天染毒剂量分别为8.33、83.33、416.67、833.33、2083.33μg PFOS/kg(bw),对照组给予2%Tween-80。
4、体外染毒
10周雄性C57BL/6小鼠6只,无菌取脾制备脾细胞悬液,调整脾细胞终浓度至1×10~7/mL,取200μl/孔加入24孔培养板中。将PFOS(以DMSO为溶剂)按0、1、5、10、50、200、300μM的浓度加入24孔培养板中,37℃、5%CO_2的细胞培养箱内培养48小时,提取培养上清,检测细胞因子水平。
5、脾细胞采集
短期染毒后第8天或亚慢性染毒第61天,摘除眼球采血并收集血清,颈椎脱位处死小鼠,75%酒精浸泡,取出各组小鼠的胸腺、肾脏、肝脏、脾脏,称量质量,并计算器官指数。常规制备脾细胞悬液,用含2%热灭活FCS的PBS将脾细胞终浓度调至1×10~7/mL。
二、免疫指标测定方法
1、血清中PFOS浓度测定
0.5mL血清样品加入1.0mL 0.5mol/LTBAHS和2.5mL 0.25mol/L碳酸氢钠缓冲液,振荡混合20min后加入5.0mL MTBE进行萃取,离心后,收集上层MTBE相。沉淀物部分再次加入5.0mL MTBE,进行2次萃取。两次萃取合并液用高纯氮吹干后,准确加入1.0mL甲醇进行定容,进行HPLC/MS定量分析。
2、CD4~+、CD8~+细胞亚群检测
取0.1 mL脾细胞悬液,加入anti-CD3-FITC(553061)、anti-CD4-PE(557308)、anti-CD8-PERCP(553036)0.2μg,4℃下避光孵浴30min。加2mL PBS、混匀,离心5min(2000r/min),弃上清。将剩余液体转入流式管。流式细胞仪的激发波长为488nm,利用FACS CELLQUEST软件获取细胞,每个样品分析10,000个细胞;记录CD4~+T细胞、CD8~+T细胞的个数百分比。
3、NK细胞活性检测
以YAC-1细胞作为靶细胞,调整靶细胞浓度至1×10~5/ml备用。取脾细胞(1×10~6/ml)和靶细胞各0.1ml加入96孔细胞培养板中,每份标本设三个复孔,同时设靶细胞自然释放对照组和最大释放对照组。用酶联检测仪在490nm波长下读各孔OD值,计算NK细胞活性。
4、MTT法检测淋巴细胞增殖
用含10%小牛血清的RPMI-1640培养液调整脾细胞浓度为5×10~6/mL,加入96孔培养板,每孔100μl脾细胞悬液。每孔加入10μg/ml的ConA或LPS100μl,同时设只加培养液的阴性对照,每组设3复孔。在酶标仪上于570nm处测光吸收值(A),以下列公式计算淋巴细胞增殖指数(SI):SI=刺激孔A值/对照孔A值。
5、NO测定
取脾细胞培养上清接入96孔培养板中,100μl/孔,将100μmol/L NaNO_2在96孔培养板中进行倍比稀释。各孔中再加入0.1ml Griess试剂,室温静置10min,在550nm波长处测OD值。
6、B细胞溶血空斑试验测定
将绵羊红细胞(SRBC)免疫过的小鼠脾细胞制成2×10~6/ml悬液,与0.1ml SRBC和0.05ml的DEAE右旋糖酐补体在琼脂凝胶内混合,冷却凝固后放入37℃温箱1小时,加入豚鼠血清1.5ml。记录整个平皿中溶血空斑数。
7、ELISpot法检测IL-2和IL-10细胞数量
采用ELISpot法检测脾脏IL-2和IL-10的细胞数量,调整脾细胞浓度分别为1×10~5/ml(IL-2)和1×10~6/ml(IL-10)。每孔加入100μl脾细胞悬液和对照品,37℃CO_2温箱孵育1h。每孔加100μl生物素标记的IL-2或IL-10检测抗体,4℃过夜孵育。加入碱性磷酸酯酶标记链霉亲和素100μl/孔,室温孵育2h。加入BCIP/NBT显色液100μl/孔,避光孵育1h。ELISPOT自动分析仪读板计数IL-2或IL-10细胞数量。
8、ELIsA法细胞因子IL-2、IL-4、IL-10、IFN-γ测定
采用双抗体夹心ELISA法:每孔加入50μl倍比稀释的标准品,对照品或培养上清,混匀1min。室温孵育2h。每孔加入100μl各细胞因子多克隆抗体,室温孵育2h。100μl/孔加入酶标记溶液,室温避光孵育30min。加入100μl/孔终止液。用酶标仪在450nm波长读记吸光度OD值。
9、血清抗体IgG、IgM测定
采用ELISA检测血清抗体水平:100μl/孔标准品加入到96孔酶标板中,每孔加入100μl血清,加入50μl/孔酶标记溶液,36℃孵育1h,每孔加入底物呈色剂A、B液各50μl,36℃孵育15min,加入50μl/孔终止液。于450nm酶标仪读取OD值。
10、统计学分析
采用SAS8.2统计软件进行数据的分析。对数据进行正态性检验后,用均数(Mean)和标准误(SE)描述正态分布数据的描述指标。各组数据之间比较采用单因素方差分析,采用Dunnett(?)t检验进行各个试验组与对照组的多重比较。
结果
1、PFOS染毒对小鼠体重、饲料消耗量和器官指数的影响
短期染毒试验:对照组小鼠血清中PFOS浓度低于检测限(0.05mg/L),PFOS染毒5、20、40mg/kg剂量组小鼠血清中PFOS浓度分别为110.46±6.18mg/L、280.65±16.33mg/L、338.01±23.92mg/L。20、40mg PFOS/kg剂量组小鼠的体重和饲料消耗量呈显著的下降趋势(p<0.05),且脾脏指数和胸腺指数显著的低于对照组。各试验组小鼠肝脏指数均显著的高于对照组,肾脏指数变化并不显著。
亚慢性染毒试验:PFOS染毒0.5、5、25、50、125mg/kg(bw)TAD剂量组小鼠血清中PFOS浓度分别为0.67±0.17mg/L、7.13±1.04mg/L、21.64±4.41mg/L、65.43±11.73mg/L、120.67±21.76mg/L,均显著的高于对照组血清PFOS水平。25、50、125mg PFOS/kg TAD剂量组小鼠的体重和饲料消耗量呈显著的下降趋势,且脾脏指数和胸腺指数显著的低于对照组。≥5mg PFOS/kg TAD剂量组小鼠肝脏指数显著的高于对照组,并随着染毒剂量的增高,肝脏指数呈增高趋势。
2、PFOS对小鼠脾细胞和胸腺细胞总数及淋巴细胞亚群的影响
短期染毒试验:20、40mg PFOS/kg剂量组小鼠脾细胞和胸腺细胞总数显著的降低;40mg PFOS/kg剂量组小鼠CD4~+ CD8~-和CD4~-CD8~+细胞亚群的数量分别减少28%和21%。亚慢性染毒试验:25、50、125mg PFOS/kg TAD剂量组小鼠脾细胞和胸腺细胞总数显著的低于对照组,其中125mg PFOS/kg TAD剂量组小鼠脾细胞和胸腺细胞总数分别减少55%和70%。CD4~+CD8~-和CD4~-CD8~+细胞亚群的数量显著降低。
3、PFOS对淋巴细胞增殖功能的影响
短期试验中,当PFOS染毒剂量≥5mg/kg/day时,可显著的抑制T淋巴细胞增殖功能,而B淋巴细胞增殖功能的抑制开始于20mg/kg/day剂量组。亚慢性毒性试验中,随着PFOS染毒剂量的增高,T、B淋巴细胞增殖功能呈先增高后降低趋势。体外试验结果显示,当PFOS暴露剂量为≥200μM时,可显著抑制B细胞增殖功能。
4、PFOS对NK细胞活性的影响效应
短期试验中,PFOS染毒剂量20mg和40mg/kg/day组小鼠NK细胞的活性分别为18.04±1.42和13.08±1.11,显著的低于对照组水平(50.33±4.08)。亚慢性毒性试验中,5mg PFOS/kg TAD可显著的上调NK细胞的活性,当PFOS染毒剂量≥50mg/kg TAD时,NK细胞的活性显著降低。
5、PFOS对脾细胞培养上清NO产生水平影响
短期试验中,仅40mg/kg/day剂量组NO水平显著的低于对照组。亚慢性试验中,NO水平随着PFOS染毒剂量的增高呈先增高后降低趋势。体外实验显示,当PFOS暴露剂量为300μM时,可显著抑制NO水平。
6、B细胞溶血空斑试验测定
短期试验中,5mg、20mg、40mg PFOS/kg剂量组空斑形成细胞数分别减少63%、77%和86%。亚慢性毒性试验中,当PFOS暴露剂量为5mg/kg TAD时,空斑形成细胞数可显著的低于对照组,并随着染毒剂量的增高,空斑形成细胞数呈降低趋势。
7、细胞因子IL-2、IL-4、IL-10、IFN-γ测定
短期试验和亚慢性试验中,随着染毒剂量的增高,IL-2和IFN-γ的水平均呈降低趋势。短期试验中IL-4呈先增高后降低趋势;而亚慢性试验中PFOS可显著促进IL-4的水平。IL-10在各剂量组间的差异并没有达到统计学意义。体外实验显示,PFOS并没有显著的抑制细胞因子的表达。
8、血清抗体IgG、IgM测定
短期试验中,PFOS各暴露剂量组IgM水平均显著的低于对照组,而IgG水平在5mg/kg/day剂量组显著的增高,40mg/kg/day组显著的降低。亚慢性毒性试验中,当PFOS累计染毒剂量≥5mg/kg TAD时,随着染毒剂量的增高,IgM水平呈显著下降趋势。IgG水平呈先增高后降低趋势。
结论
1、PFOS是与机体免疫反应相关的外源性化学物质,以5mg PFOS/kg剂量连续7天染毒小鼠时即可抑制小鼠免疫功能。
2、PFOS对C57BL/6小鼠经口染毒60天可导致机体免疫系统的抑制,根据免疫指标的改变,其未观察到损害所用的剂量(NOAEL)和观察到损害作用的最低剂量(LOAEL)分别为0.5mg/kg TAD和5mg/kg TAD,对应血清中PFOS浓度分别为0.67±0.17mg/L和7.13±1.04mg/L。
3、体外试验结果比表明200μM PFOS可显著的抑制脾淋巴细胞的增殖功能,表明在较高剂量水平下PFOS可对淋巴细胞产生直接毒性效应。
4、根据自然人群血清中PFOS浓度不高于150μg/L计算,小鼠血清中PFOS浓度约为自然人群血清最高浓度的50倍时,可抑制机体免疫功能。
Introduction
Perfluorooctanesulfonate[PFOS;CF_3(CF_2)_7SO_3~-],which is produced synthetically or from the metabolism of other perfluorinated chemicals(PFCs),has extreme thermal, biological,and chemical stability as well as hydrophobic and lipophobic characteristics. These properties make it widely to be used as industrial surfactants and emulsifiers and in numerous consumer products.Nonstick pans,carpets,furniture,household cleaners, shampoos,shoes,clothing,and convenience food packaging are some of the products that can contain PFCs.PFOS has aroused scientists'great concern in recent years due to its widespread occurrence in the environment,in the wildlife and in humans. Furthermore,it has shifted among biological populations via biological concentration and magnification,leading to excessive accumulation among the higher trophic level of food chain(such as predator and human beings).
PFOS,a hydrocarbon chain composited with 17 F-atom and 8 carbon-atom and linked one sulphur acyl,has high surface tension-reducing properties,lower water and oil solubility,and is a relatively strong organic acid.The strength of carbon-fluorine bonds and the high electronegativity of perfluorinated alkyl acids contribute to the extreme stability and unique properties of PFOS which made it not expected to be metabolized when be seethed in oil of vitriol or nitric acid for one hour.Different from the other persistent organic pollutions,PFOS is the predominately PFCs found in both human and wildlife blood samples and accumulates primarily in the blood and liver for its lower water and oil solubility.Previously several exposure routes have been suggested to play an important role in the exposure to PFOS:food,drinking water,air borne dust,house dust or other PFCs which can be rapidly metabolized into PFOS via a perfluorooctane sulfonamide.One study showed that,on average,for a standard adult man(70 kg of body weight),the dietary intake of PFOS was estimated to be 62.5 or 74.2 ng/day.Several blood epidemiology investigaions reported regarding PFOS concentration in human serum,and human serum concentrations vary depending on the population evaluated.For occupational exposure,the serum value was 12.83mg/L. Even in infant,there was also PFOS contamination.One recent study showed that the arithmetic mean half-lives of serum elimination was 8.67 years(range:2.29～21.3 year, standard error=6.12 year),and the maximum biological half-life reaches up to 21.3 years.The historical samples collected from 1983 to 1999 in Japan demonstrated that the PFOS concentrations in Japanese have increased 4.4-fold at a rate of increase of 0.49ng/ml/year.One of our recent studies indicate that the serum PFOS level in un-occupationally exposed individuals from Shenyang of China increased significantly from 0.03μg/l in 1987 to 22.4μg/l in 2002,a 747-fold increase,and from 1999 to 2002, serum PFOS concentration also increased 13 fold.
For rat,the LD50 based on once oral exposure was 251mg PFOS/kg body weight. A hospital-based cross-sectional epidemiologic study observed small negative associations between both PFOS concentrations and birth weight and size.Another study conducted by 3M revealed an increase in bladder cancer and prostate cancer mortality among workers exposed to PFOS.A number of mammalian toxicology studies were conducted to assess the health effects of exposure to PFOS,recent studies indicated that PFOS can cause hepatotoxicity,tumors of the liver,and of the thyroid and mammary glands,developmental toxicity,reproductive toxicity,neurotoxicity and endocrine-disrupting.However,to our knowledge,few studies have assessed the effects of PFOS exposure on immune system.The purpose of our research is to study the immune toxicity induces by PFOS exposure in mice by flow cytometry assay,ELISA, et al.We therefore evaluated both humoral and cell-mediated immune function in experiments designed to provide scientific evidence in environmental safty evaluation and establish no observed adverse effect level(NOAEL) and lowest observed adverse effect level(LOAEL) values from dose-response studies of immune function.
Material and Methods
1.Animal samples
(1).PFOS solution:
Potassium PFOS suspensions were prepared in de-ionized water with 2%Tween(?) 80 at concentrations.Exposures consisted of oral administration of PFOS delivered in de-ionized water with 2%Tween(?) 80.Control mice received de-ionized water with 2%Tween(?) 80 only.
(2).7-day oral exposure to PFOS
Forty-eight adult C57BL/6 male mice were randomly divided by weight into four groups of 12/group.Once distributed into groups the mice acclimated to the new cage conditions and the new treatment room(12-h light/dark cycle,22±2℃,60-65% relative humidity) for 1 week before dosing was initiated.C57BL/6 mice were dosed once daily via oral gavage for 7 days(0,5,20,or 40 mg/kg body weight day).Food intake and body weight of all animals were measured daily for 7 days.
(3).60-day oral exposure to PFOS
Sixty adult C57BL/6 male mice were randomly divided by weight into six groups of 10/group.Once distributed into groups the mice acclimated to the new cage conditions and the new treatment room(12-hr light/dark cycle(light,0600-1800 hours; dark,1800-0600 hours),22.4±1.3℃,60-65%relative humidity) for 10 days before dosing was initiated.Exposures consisted of oral administration of PFOS delivered in de-ionized water with 2%Tween(?) 80.Control mice received de-ionized water with 2%Tween(?) 80 only.C57BL/6 mice were dosed once daily via oral gavage for 60 days (0,8.33,83.33,416.67,833.33 or 2083.33μg PFOS/kg body weight/day) to yield a targeted Total Administered Dose(TAD) over the 60 days of 0,0.5,5,25,50,or 125 mg PFOS/kg body weight.Food intake and body weight of all animals was measured daily for 60 days.
(4).PFOS exposure in vitro
Spleens from six adult C57BL/6 male mice were aseptically processed into single-cell suspensions by gentle grinding with the use of sterile,frosted microscope slides for functional immune endpoints.The splenocytes were washed three times in RPMI-1640 supplemented with 10%FBS and then resuspended in RPMI 1640 medium. The concentration of splenocytes was adjusted to 1×10~7 nucleated cells/ml.200μl aliquots of the resulting cell suspensions were dispensed into 24-well plates.The PFOS (dissolved in dimethylsulfoxide(DMSO)) was added to the medium at the indicated concentrations(0,1,5,10,50,200,300μM) at the beginning of the experiment.In this case,the same amount of DMSO alone(0.05%final concentration) was added to the control medium.The plates were incubated for 48 h at 37℃in a humidified 5% CO_2-air mixture.
(5).Spleen or thymus cells collection
Mice were bled by retro-orbital puncture under light diethyl ether anesthesia and subsequently sacrificedSpleen,thymus,liver,and kidneys were collected and weighed. All balances were calibrated,using standard weights,prior to use.Spleen and thymus were aseptically processed into single-cell suspensions by gentle grinding with the use of sterile,frosted microscope slides for functional immune endpoints and T-cell immunophenotype determinations.A Coulter Counter(model ZF;Hialeah,FL) was used to obtain cell counts from theses single-cell suspensions.Alterations in cell viability following treatment were assessed after red blood cell lysis.The concentration of splenocytes was adjusted to 1×10~7 nucleated cells/ml.
2.Experimental Methods
(1).Serum concentration of PFOS
0.5 ml of serum,1 ml of 0.5 M tetrabutylammonium hydrogen sulfate solution and 2 ml of sodium carbonate buffer(0.25 M,pH 10) were added to 15 ml polypropylene tube and thoroughly mixed.Following addition of 5 ml of MTBE to the solution,the organic and aqueous layers were separated by centrifugation,and the organic layer was removed.The aqueous mixture was rinsed with MTBE and separated twice.The solvent was evaporated at room temperature under a nitrogen gas flow,and the sample was then reconstituted in 0.5 mL of methanol.The sample was then passed through a nylon filter(Autovial R5 PUNYL;0.45-μm pore size;Whatman Japan,Tokyo) to remove any suspended materials and insoluble particles.
(2).Splenic and Thymic CD4/CD8 Subpopulations
Spleen or thymus cells were labeled with fluorescent(phycoerythrin or peridinin chlorophyl protein) rat IgG2 monoclonal antibodies specific for mouse CD4 or CD8 (rat anti-mouse).The antibody dilution used for FACS analysis was 1:5(v/v) for FITC conjugated rat-anti-mouse CD3,1:2(v/v) for PE conjugated rat anti-mouse CD4 and 1:2(v/v) for Percp conjugated rat anti-mouse CD8,respectively,following the manufacturer's instructions.Lastly,the cells were fixed with 1%paraformaldehyde and stored at 6℃in the dark.Flow cytometric analysis was performed using a Becton Dickinson flow cytometer(FACSCalibur;San Jose,CA,USA).Nonstained cells and isotypic antibody controls were used to establish gates for the CD4/CD8 subpopulations in splenic cells.Data are represented as absolute number of cells, determined by multiplying the percent gated cells by the total number of nucleated cells obtained by the Coulter Counter.
(3).Natural Killer(NK) Cell Activity
The splenocytes were washed and suspended in complete RPMI-1640 medium, then were counted and diluted to 1.0×10~6 nucleated cells/ml.The amount of the LDH released from the lysed target cells was determined to measure NK activity.The cell line Yac-1 was used as the target cell.The same volume of Yac-1 cells and splenocytes were added to the wells of 96 round-bottom microwell plates(the cell ratio of effector-to-target is 10:1).Three wells were used for every mouse.Finally,a microtiter plate reader(Bio-Rad,Modal 550) was used for evaluation of changes in the absorbance at a wavelength of 490 nm.The release of LDH from Yac-1 cells was expressed as absorbance.The percentage of NK cell activity was calculated by the formula:NK cell activity=[(E-S)/(M-S)]×100%.Where E represents the experimental release of LDH activity from target cells incubated in the presence of lymphocytes,M represents the maximum release of the LDH activity determined by lysing the target cells with 1%of NP-40,and S is the spontaneous release of the LDH activity from target cells incubated in the absence of lymphocytes.
(4).Lymphocyte proliferation assay
The concentration of splenocytes was adjusted to 5×10~6 nucleated cells/ml.The proliferation ability of the lymphocytes was determined by MTT stain assay.100μl aliquots of the resulting cell suspensions were dispensed into 96-well plates(5×10~5 cells/well) containing triplicate wells of either 10μg/ml mitogen Con A,10μg/ml LPS or supplemented RPMI-1640(unstimulated wells),the final culture volume was 200μl in each well.After incubation at 37℃in 5%CO2 atmosphere overnight,the microplate was read on a Bio-Rad microplate reader(Model 550) using test wavelength of 570 nm.The ratio of the optical density(OD) of stimulated to the OD of unstimulated cultures was used as the stimulation index.The proliferation index was calculated by the equation:proliferation index=A value of Con A or LPS-stimulated cells/A value of nonstimulated cells.
(5).Measurement of NO
The levels of NO metabolites(nitrite plus nitrate) were determined by enzymatically reducing the nitrate present with nitrate reductase.A standard nitrate curve was obtained by incubating sodium nitrate with reductase buffer.The total amount of nitrite was determined by the Griess method.Briefly,the samples were incubated with an equal volume of freshly prepared Griess reagent(1%sulfanilamide, 0.1%naphthylenediamine dihydrochloride in 5%phosphoric acid).Absorbance at 550nm was determined using a multi-well plate reader.
(6).Antibody Plaque-Forming Cell Assay
The number of plaque forming cells(PFCs) was determined using the Jerne plaque assay(Jerne and Nordin,1963).Briefly,Four days prior to euthanasia,mice were administered 0.1 mL of a 25%SRBC suspension in PBS via intraperitoneal injection.All SRBCs for the experiments were drawn from a single donor animal. Spleen cells collected from individual animals(0.1 mL;1×10~6 cells/0.1 mL),0.4 mL of 0.5%"low melting point" agarose(GIBCO,Grand Island,NY,USA) in RPMI-1640 medium,and 50μL of a suspension of 5%SRBC were added to test tubes at 37℃and poured onto microscope slides containing a bottom layer of 0.5%agarose in water.The slides were then incubated for 2 hr at 37℃and 5%CO2.Guinea pig serum diluted 1:4 in RPMI-1640 was added to the slides and after another 40 min(37℃and 5%CO2) incubation the number of plaques was counted and values were expressed as PFC per 10~6 cells.For the preparation of guinea pig serum as the complement source,animals were anesthetized before cardiac puncture and blood samples were collected.
(7).Measurement of IL-2,IL-10 by ELISpot
Fill all wells in the microplate with 200 L of sterile culture media and incubate for approximately 20 minutes at room temperature.When cells are ready to be plated, aspirate the culture media from the wells.Immediately add 100 L of the appropriate cells(1×10~5/ml for IL-2 and 1×10~6/ml for IL-10) and controls to each well.Incubate cells in a humidified 37°C CO2 incubator.Optimal incubation time for each stimuli should be determined by each investigator.Add 100 L of diluted Detection Antibody into each well and incubate at 2-8°C overnight.Add 100 L of diluted Streptavidin-AP into each well and incubate for 2 hours at room temperature.Add 100 L of BCIP/NBT Chromogen into each well and incubate for 1 hour at room temperature.The developed microplate can be analyzed by counting spots either manually using a dissection microscope or by using a specialized automated ELISpot reader.
(8).Measurement of IL-2,IL-4,IL-10,and IFN-γ
Add 50μL of Standard,Control,or sample per well.Mix by gently tapping the plate frame for 1 minute.Cover with the adhesive strip provided.Incubate for 2 hours at room temperature.Plate layouts are provided to record standards and samples assayed.Aspirate each well and wash,repeating the process four times for a total of five washes.Add 100μL of mouse IL-2,IL-4,IL-10 or IFN-γConjugate to each well. Cover with a new adhesive strip.Incubate for 2 hours at room temperature.Add 100μL of Substrate Solution to each well.Incubate for 30 minutes at room temperature. Protect from light.Add 100μL of Stop Solution to each well.Gently tap the plate to ensure thorough mixing.Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
(9).Measurement of IgG,IgM
Dispense 100μl of standards and specimens into appropriate wells,and then 50μl of Enzyme Conjugate Reagent into each well.Incubate at 36℃for 1 hour.Rinse and empty the microtiter wells 5 times,then dispense 50μl of color A and color B Reagent into each well.Incubate at 36℃for 15 minutes.Stop the reaction by adding 50μl of Stop Solution.Read the optical density at 450 nm within 30 minute.
(10).Statistics
Data were tested for normality(Shapiro-Wilks W-test) and homogeneity (Bartlett's test for unequal variances) and,if needed,appropriate transformations were made.Transformations when required are outlined in the figure legends.A one-way analysis of variance(ANOVA) was used to determine differences among doses for each endpoint using SAS software(Version 8.2;SAS Institute Inc.,Cary,NC) in which the standard error used a pooled estimate of error variance.When significant differences were detected by the F-test(p＜0.05),Dunnett's t-test was used to compare treatment groups to the control group.
Results
1.Animal Body Weight,Food intake and Organ Mass
In the 7-day study,body weight of mice exposure to 20 mg/kg and 40 mg/kg of PFOS showed significant deterioration from their own pre-exposed baseline.On the last day of the treatment,body,spleen,and thymus mass were signiWcantly decreased compared to the control following exposure to 20 mg/kg and 40 mg/kg of PFOS. Furthermore,liver mass was increased by 34,79,and 117%over control following treatment with 5,20,or 40 mg/kg,respectively.
In the 60-day study,there was a dose-dependent increase in the concentrations of PFOS in serum from exposed mice.At the last day of the treatment,body,spleen, thymus and kidney mass were significantly decreased compared to the control following exposure to 25 mg/kg TAD,50 mg/kg TAD and 125 mg/kg TAD of PFOS. Furthermore,liver mass was significantly increased at dose as low as 5 mg PFOS/kg TAD.
2.Splenic and Thymic Cellularity,Lymphocyte Immunophenotypes
Treatment with 5,20,or 40 mg PFOS/kg resulted in downtrend of splenic and thymic cellularity following 7 days of treatment.FACS analysis of spleen T lymphocytes demonstrated that the relative CD4+CD8- population of total splenocytes had decreased by 28%in the 40 mg/kg PFOS treatment and the relative change in the CD4-CD8+ population was decreased by 21%.Treatment with 25,50,or 125 mg PFOS/kg TAD resulted in downtrend of splenic and thymic cellularity following 60 d of treatment.Especially for the group of mice exposed to 125 mg PFOS/kg TAD,the splenic and thymic cellularity was found to be significantly decreased by 55%and 70% following the 60-d exposure compared with control mice.Splenic and thymic T lymphocytes demonstrated that the numbers of all T-cell CD4/CD8 subpopulations were significantly decreased beginning at 25 mg PFOS/kg TAD.
3.Effect of PFOS on Lymphocyte Proliferation
In the 7-day study,the average absorbances of T lymphocytes from≥5 mg/kg PFOS groups were lower than the control,and the B lymphocyte proliferation decreased beginning at≥20 mg/kg PFOS.In the 60-day study,with the level of PFOS exposure increasing,the average absorbances of T and B lymphocytes were first increasing and then decrasing.The effect of PFOS in vitro treatment on lymphocyte proliferation showed that the PFOS did not alter lymphocyte proliferation except for the condition of≥200μM PFOS.
4.Effect of PFOS on NK-cell Function
In the 7-day study,treatment with the dose of 20 mg/kg(18.04±1.42) and 40 mg/kg(13.08±1.11) PFOS resulted in a marked decrease in the levels of NK-cell activity compared with control(50.33±4.08).Treatment with the dose of 5 mg PFOS/kg TAD significantly increased the NK cell activity by 38%(45.43±4.74).In contrast,treatment with the dose of 50 mg/kg TAD(20.28±2.51) and 125 mg/kg TAD (15.67±1.52) PFOS resulted in a marked decrease in the levels of NK cell activity.
5.Effect of PFOS on NO
Following 7 days of treatment,NO level was significantly lower in group of 40 mg PFOS/ke/day compare control group.In the 60-day study,NO level was elevated at 5 mg PFOS/kg TAD and then decrased at 50 mg PFOS/kg TAD.The effect of PFOS in vitro treatment on NO levels showed that the PFOS did not alter NO level except for the condition of 300μM PFOS.
6.Plaque-Forming Cell Assessments
Treatment with 5,20,or 40 mg PFOS/kg resulted in significant suppression of the plaque-forming cell response following 7 days of treatment(63%,77%,and 86% respectively).There was a significant trend toward decreased PFC production with increasing PFOS exposure.The suppression of this response was dose-responsive beginning at exposures of 5 mg PFOS/kg TAD.
7.Effect of PFOS on IL-2,IL-4,IL-10 and IFN-γ
With the PFOS exposure increasing,there was a significant trend toward decreased IL-2 and INF-γproduction,and PFOS increased IL-4 synthesis.IL-10 was not statistically altered by exposure to the tested doses of PFOS.In vitro treatment,no other statistical changes in cytokine were observed exposed to PFOS.
8.Effect of PFOS on IgG and IgM
In the 7-day study,exposure to PFOS reduced IgM synthesis.IgG titers were elevated at 5 mg PFOS/kg/day and then decrased at 40 mg PFOS/kg/day.In the 60-day study,IgM synthesis was suppressed at exposures≥5 mg PFOA/kg TAD in a dose-dependent manner.
Conclusion
1.PFOS,as an exogenous chemical material,is related to immune reaction.PFOS exposure can suppress the immunity function in mice in a dose of 5 mg/kg bw for 7 d.
2.Based on the liver mass and PFC response,the no observed adverse effect level (NOAEL) and low observed effect level(LOAEL) for male mice exposed PFOS for 60 days was 0.5 mg/kg TAD and 5 mg/kg TAD,respectively.Measured PFOS serum concentrations at these dose levels were 0.67±0.17mg/L and 7.13±1.04mg/L, respectively.
3.In vitro treatment,PFOS exposure can suppress the lymphocyte proliferation which indicate that PFOS may have a direct toxicity to lymphocyte.
4.Human biomonitoring studies indicates that PFOS residues in adult blood or serum are generally present at levels lower than 150μg/L,that PFOS exposure can affect the immunity function in mice at levels approximately for 50-fold for highly exposed human populations.
引文
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