| IntroductionPerfluorooctanesulfonate[PFOS;CF3(CF2)7SO3-],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 Methods1.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 PFOSForty-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 PFOSSixty 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 vitroSpleens 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 1×107 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% CO2-air mixture.(5).Spleen or thymus cells collectionMice 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 1×107 nucleated cells/ml.2.Experimental Methods(1).Serum concentration of PFOS0.5 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 SubpopulationsSpleen 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 ActivityThe splenocytes were washed and suspended in complete RPMI-1640 medium, then were counted and diluted to 1.0×106 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 assayThe concentration of splenocytes was adjusted to 5×106 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(5×105 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 NOThe 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 AssayThe 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;1×106 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 106 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 ELISpotFill 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(1×105/ml for IL-2 and 1×106/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-γAdd 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,IgMDispense 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).StatisticsData 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.Results1.Animal Body Weight,Food intake and Organ MassIn 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 ImmunophenotypesTreatment 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 ProliferationIn 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 NOFollowing 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 AssessmentsTreatment 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-γWith 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 IgMIn 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.Conclusion1.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.
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