Research Article Differential Immunotoxicity Induced by Two …downloads.hindawi.com/journals/ad/2014/982073.pdf · 2019-07-31 · list of hazardous chemicals. One of the predominant

Research ArticleDifferential Immunotoxicity Induced by Two Different Windowsof Developmental Trichloroethylene Exposure

Kathleen M Gilbert William Woodruff and Sarah J Blossom

University of Arkansas for Medical Sciences Arkansas Childrenrsquos Hospital Research Institute 13 Childrenrsquos WayLittle Rock AR 72202 USA

Correspondence should be addressed to Kathleen M Gilbert gilbertkathleenmuamsedu

Received 1 October 2013 Revised 19 November 2013 Accepted 20 November 2013 Published 20 February 2014

Academic Editor Aristo Vojdani

Copyright copy 2014 Kathleen M Gilbert et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Developmental exposure to environmental toxicants may induce immune system alterations that contribute to adult stageautoimmune disease We have shown that continuous exposure of MRL++ mice to trichloroethylene (TCE) from gestationalday (GD) 0 to postnatal day (PND) 49 alters several aspects of CD4+ T cell function This window of exposure correspondsto conception-adolescenceyoung adulthood in humans More narrowly defining the window of TCE developmental exposurecauses immunotoxicity that would establish the stage at which avoidance andor intervention would be most effective The currentstudy divided continuous TCE exposure into two separate windows namely gestation only (GD0 to birth (PND0)) and early-lifeonly (PND0-PND49) The mice were examined for specific alterations in CD4+ T cell function at PND49 One potentially long-lasting effect of developmental exposure alterations in retrotransposon expression indicative of epigenetic alterations was foundin peripheral CD4+ T cells from both sets of developmentally exposed mice Interestingly certain other effects such as alterationsin thymus cellularity were only found in mice exposed to TCE during gestation In contrast expansion of memoryactivation cellsubset of peripheral CD4+ T cells were only found in mice exposed to TCE during early life Different windows of developmentalTCE exposure can have different functional consequences

1 Introduction

The chlorinated hydrocarbon and industrial solvent trich-loroethylene (TCE) is a widespread environmental contam-inant As noted in a 2011 IRIS report the EPA has con-cluded that ldquothere is substantial potential for environmentalexposure to TCE as its improper disposal has resulted inthe widespread contamination of groundwater and soilrdquo [1]Contact with TCE may be elevated for people living nearwaste facilities where TCE is released residents of someurban or industrialized areas or individuals using TCE-containing products Regardless of whether TCE exposureis oral dermal or inhalation-based the chemical is rabidlyabsorbed and distributed Based on the likelihood of expo-sure together with likely negative health impact TCE isconsistently ranked 16th out of 275 chemicals on theCERCLAlist of hazardous chemicals

One of the predominant human health effects associatedwith TCE exposure is immunotoxicity most notably thedevelopment of autoimmunity and other types of hyper-sensitivity diseases Chronic TCE exposure in adults (bothoccupational and environmental) has been linked to avariety of autoimmune diseases including systemic lupuserythematosus scleroderma hepatitis and diabetes [2ndash10]In addition there are many cases in recent years of a TCE-induced hypersensitivity disorder that targets the skin andliver [11 12] Alterations in CD4+ T cells are often found to bean effect biomarker in patients suffering from TCE-inducedimmunotoxicity [2 13ndash15]

Most studies of TCE-induced autoimmunity or hyper-sensitivity in humans have focused on adult exposure tothe higher concentrations of TCE that are most commonlyfound in the workplace The type of low-level TCE exposure

Hindawi Publishing CorporationAutoimmune DiseasesVolume 2014 Article ID 982073 12 pageshttpdxdoiorg1011552014982073

2 Autoimmune Diseases

people may experience through drinking water contamina-tion is generally thought to be risk-free However there isevidence that the developing immune system is especiallysensitive to even low-level immunotoxicants A recent reviewcompared early versus adult exposure to several immuno-suppressive toxins including lead and tributyltin in animalmodels [16] In all cases sensitivity was greater if exposureoccurred during development In fact immune suppressionin developmentally-exposed offspring oftenoccurred at dosesthat were ineffective in adults Developmental sensitivityto toxicants has also been found in humans [17 18] Thisincludes evidence that adult onset autoimmune disease canbe triggered by pre- and early postnatal toxicant exposure[19 20]

Developmental exposure to TCE in humans is notuncommon one study showed that 100 of breast milksamples from 4 US urban areas had detectable levels of TCE[21] Gestational and early life TCE exposure has primarilybeen examined for its neurotoxicity [22] However childrencontinuously exposed for 3ndash19 years beginning in utero to awater supply contaminatedwith solvents [with TCE being thepredominant toxicant (267 ppb)] had altered ratios of T cellsubsets and increased levels of autoantibodies [2]

We have studied a direct cause and effect relationshipbetween TCE exposure and immunotoxicity in a mousemodel Adult exposure to TCE primarily altered effectorCD4+ T cells with little effect on CD8+ T cells B cells or 119879regcellsThe effects of TCEonCD4+ T cells were seen after only 4weeks and included expansion of activatedmemory (CD44hi

CD62Llo) CD4+ T cells and altered cytokine production [23ndash25] Adult TCE exposure seemed to have minimal effecton thymus cellularity After chronic adult exposure (26ndash32weeks) the TCE-induced alterations in CD4+ T cells led toT cell-mediated liver inflammation identical to that seen inidiopathic autoimmune hepatitis (AIH) in humans [24 26]

We have also examined mice following continuous TCEexposure beginning in utero and then encompassing lactationas well as an additional 4 weeks of direct exposure Thiscontinuous exposure to TCE at concentrations lower thanthat used for adult exposure altered thymocyte cellularity andmodified the phenotype and function of peripheral CD4+ Tcells [27]

Although informative studies of continuous toxicantexposure do not reveal which windows of exposure havethe most impact on the developing immune system Forexample it has been shown that early gestational exposureto certain toxicants such as lead are not as likely to sup-press Th1 function as late gestational exposure [28] Thissuggests that within the developmental period there existsparticular windows of relative susceptibility and resistance[29] Thus knowing more about the functional outcomesof particular developmental windows of TCE exposure isneeded to accurately estimate risk and to plan interventionsThis study was designed to compare central and peripheralimmune system alterations in mice exposed to TCE duringtwo crucial windows of immune development and toxicantsensitivity namely gestation [GD0 to birth (PND0)] andearly life (PND0 to PND49)

2 Materials and Methods

21 Mice and TCE Exposure Developmental exposure toTCE has been described [30] Basically breeding pairs of 8-week-old MRL++ mice (Jackson Laboratories Bar HarborME)were established In one experiment immediately follow-ing detection of pregnancy GD0 the females were divided(following stratified randomization) into 3 groups and givenwater with 0 001 or 01mgmL TCE Controls were givenwater containing only 1 Alkamuls EL-620 the reagent usedto solubilize the TCE All the drinking water was Ultrapureunchlorinated to assure that chlorination by-products do notconfound the results The TCE-containing drinking waterwas changed 3 timesweek to offset degradation of TCEMaternal exposure to TCE-containing drinking water endedat birth in this experiment The female pups that underwentthe gestation only exposure to TCEwere examined at PND49As observed previouslymaternal toxicity was not noted at thelevel of body weight mating index fertility index sex ratio oflitters gestational length and food and water consumption

In the second experiment the female breeders were notexposed to TCE during pregnancy but were given TCE-containing drinking water immediately after giving birthThus the pups were exposed to TCE from PND0 to PND21via lactation Once the female pups were weaned at PND21they were exposed to TCE directly in their drinking water forthe duration of the experiment The female pups from thisearly life exposure were sacrificed at PND49

Both female breeders and resulting pups in both exper-iments were weighed weekly and water consumption wasmonitored All studies were approved by the Animal CareandUse Committee at the University of Arkansas forMedicalSciences

When the female pups were sacrificed at PND49 pooledspleen cell suspensions and thymus cell suspensions frompaired mice from each litter (119899 = 5ndash7 litterstreatmentgroup) were examined by flow cytometry In addition CD4+T cells were isolated from the pooled spleen cell suspensionsand stimulated with immobilized anti-CD3 antibody andanti-CD28 antibody for 24 hours as described [25] Culturesupernatants were then collected for cytokine evaluation andthe activated CD4+ T cells were frozen for subsequent qRT-PCR analysis In addition adherent macrophages isolatedfrom pooled peritoneal exudates from 2-3 micelitter wereincubated for 20 hours in medium alone or in the presenceof LPS (1 120583gmL) and IFN-120574 (100 unitsmL) Approximately80 of adherent peritoneal exudate cells (PEC) regardlessof treatment group expressed the transmembrane proteinF480 a marker of mature macrophages (data not shown)Culture supernatants from the peritoneal macrophages werethen collected for cytokine evaluation RLT Lysis Buffer (Qia-gen Sciences Germantown MD) was then added directly tothe remaining adherent cells before freezing for subsequentqRT-PCR analysis

22 qRT-PCR Fluorescence-based quantitative reverse tran-scriptase polymerase chain reaction (qRT-PCR) was con-ducted using RNA isolated from activated CD4+ T cells

Autoimmune Diseases 3

Table 1 Primer sequences used for qRT-PCR

Gene Primer sequences 51015840 to 31015840 Tm (∘C)

Aire Sense AGATCGCGGTGGCCATAG 574Antisense TCGTGGTCGGCTAGAGCAT 584

Bax Sense TTGCTGATGGCAACTTCAACTGGG 602Antisense TGTCCAGCCCATGATGGTTCTGAT 604

Bim Sense CTGTGTAATGTGCCCTACTGTTTC 558Antisense GGAAGAGAACCAGCCACTACC 573

CD70 Sense TGCTGGTGGTGTTTATTACTGTG 554Antisense CTCTGGTCCGTGTGTGAAGG 577

Cdkn1a Sense AATCCTGGTGATGTCCGACCTGTT 602Antisense GTGACGAAGTCAAAGTTCCACCGT 594

Dnmt1 Sense TGATAAGGAGGACAAGGAGAATGC 564Antisense CACCGCCAAGTTAGGACACC 583

Dnmt3a Sense CAGCACCATTCCTGGTCATGCAAA 602Antisense TCCTGTGTGGTAGGCACCTGAAAT 602

Fas Sense CGCCCGCTGTTTTCCC 576Antisense GCAAGCACCAGAGGCAGG 594

FasL Sense GGCTGGGTGCCATGCA 594Antisense GGCACTGCTGTCTACCCAGAA 592

IAP Sense GCACCCTCAAAGCCTATCTTAT 546Antisense TCCCTTGGTCAGTCTGGATTT 558

Ifng Sense AGCTCATCCGAGTGGTCCAC 591Antisense AGCAGCGACTCCTTTTCCG 578

Il2 Sense CCCAAGCAGGCCACAGAATTGAAA 602Antisense AGTCAAATCCAGAACATGCCGCAG 599

Il4 Sense AGCCATATCCACGGATGCGACAAA 608Antisense AATATGCGAAGCACCTTGGAAGCC 600

Il6 Sense AGAGGAGACTTCACAGAGGATACC 571Antisense CATTTCCACGATTTCCCAGAGAAC 561

Muerv Sense TGGTGGTCGAGATGGAGGTTA 575Antisense CCGTGAATGGTGGTTTTAGCA 558

peritoneal macrophages or thymocytes The details for con-ducting qRT-PCR including quality control and referencegene selection have been described previously [31] Theprimers used are described in Table 1

23 Phenotypic Analysis of Spleen and Thymus Cells Thephenotypic analysis of 30000 events per sample was carriedout using a CyFlow ML (Partec GmbH Munster Germany)as described previously using monoclonal antibodies fromBD Biosciences or eBioscience [32] and the data werepresented as mean percentage plusmn standard error FluorescenceMinus One controls and isotype Ig controls were included

24 Cytokine Analysis The culture supernatants from theactivated CD4+ T cells or peritoneal macrophages wereexamined using READY-SET-GO ELISA kits for mouse IL-2 IL-6 or TNF-120572 from eBioscience San Diego CA

25 Statistics The data are presented as means and standarddeviations Assays were conducted using samples from 5ndash7individual litters per treatment group Comparisons betweenvalues obtained from controls and different treatment weremade using a Studentrsquos t-test The threshold for statisticalsignificance was set at 120572 = 005

3 Results

31 Gross Changes Induced by TCE Exposure Two windowsof TCE developmental exposure were examined in femaleMRL++ mice The mice were either exposed to TCE duringgestation (GD0-PND0) or during early life (PND0-PND49)All of the mice were assessed at 7 weeks of age (PND49) fora variety of immune parameters TCE was added at 001 or01mgmL to the drinking water of the dams andor the pupsBased on water consumption the resulting TCE exposureswere described in Figure 1 Many of the alterations in CD4+ T


Cel

l num

ber (times108)

Cel

l num

ber (times108)

100

075

050

025

000

120

080

040

000

10

20

30

40

35

30

25

20

0

10

20

30

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0

Control

(mg

kgd

ay)

(mg

kgd

ay)

(g)

(g) lowast

lowast

Control

Control Control

Control

Control

45

40

35

30

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001 TCE 01TCE 001

GestationEarly life TCE exposure

Direct Maternal

Body weight

Spleen size

Figure 1 Characteristics of two windows of developmental TCE exposure Female mice were exposed to TCE (001 or 01mgmL) duringgestation only or during early life only (lactation and 4 weeks of direct exposure) All of the developmentally-exposed mice were examinedat PND49 For the gestation only exposure maternal TCE ingestion based on water consumption was presented For the early life exposuredirect TCE ingestion of the pups postweaning based on water consumption was presented Body weight and spleen cell numbers (mean plusmnSD) were determined at PND49 lowastSignificantly different (120572 lt 005) compared to control values

cells associated with adult-only direct exposure to TCE werefound after 4 weeks [23 25 33] Thus the PND49 end pointcan be used to test how the effects of gestational or early lifeexposure compared to the effects of a direct 4-week adult-only exposure

Similar to adult exposure to TCE the early life exposurein the current study did not alter the weight of the femalepups at PND49 (Figure 1) In contrast female pups exposedto the highest concentration of TCE during gestation weresignificantly smaller than controls Spleen cell numbers in thesame mice were also lower than those of controls Spleen cellnumbers were not altered in mice exposed to TCE duringearly life only Thus TCE exposure during gestation ratherthan early life did induce some gross changes in body weightand spleen size

32 Developmental TCEExposure andChanges inMacrophageFunction Potential TCE-induced alterations in peripheralimmune function were examined These included spleencellularity and functional activity of key cellular componentsof chronic inflammation namely macrophages and CD4+ Tcells IL-6 is a pleiotropic cytokine that has proinflammatoryanti-inflammatory and growth factor properties Adult expo-sure to TCE has been shown to suppressmacrophage produc-tion of IL-6 [34] In the current study early life exposure toTCE at 01mgmL similarly inhibited IL-6 production at boththe protein and gene expression level (Figure 2) In contrastgestational exposure toTCEdid not suppress IL-6 productionby peritoneal macrophages

33 Developmental TCE Exposure and Changes in CD4+ TCell Gene Expression Early-occurring changes in peripheral


1200

1000

800

600

400

200

0Control Control

Control

20

15

10

5

0

Fold

chan

ge

Fold

chan

ge

NoneLPS

NoneLPS

NoneLPS

NoneLPS

lowast

lowast

6000

5000

4000

3000

2000

1000

0

60

40

20

0

(pg

mL)

(pg

mL)

lowast

Control


TCE 01TCE 001TCE 01TCE 001

Early life Gestation

IL-6 protein

Il6 expression

Figure 2 TCE inhibited IL-6 production and Il6 expression in macrophages Peritoneal macrophages were incubated with LPS and IFN-120574following isolation from untreated control mice or from mice exposed to TCE (001 or 01mgmL) during gestation only or during early lifeonly Culture supernatants were examined for IL-6 (mean plusmn SD) Il6 gene expression was examined in the same peritoneal macrophages Thedata represents the mean plusmn SD lowastSignificantly different (120572 lt 005) compared to control values

CD4+ T cells have been observed following adult and con-tinuous exposure to TCE These include expansion of theactivationmemory subset of peripheral CD4+ T cells andincreases in the production of T cell-derived cytokines [24]These potential alterations were assessed in the current studyTCE did not alter the percentages of total splenic CD4+ Tcells CD8+ T cells or B cells regardless of concentration orwindow of exposure (data not shown) However similar toboth adult and continuous TCE exposure early life exposureto TCE at 001 or 01mgmL did increase the percentage ofCD44hi CD62Llo CD4+ T cells the population of CD4+ Tcells considered to represent amemoryactivated CD4+ T cell

phenotype (Figure 3) Gestational exposure did not increasethe percentage of activatedmemory CD4+ T cells in theperiphery at PND49

Splenic CD4+ T cells isolated from control and TCE-treated mice were activated in vitro prior to an examinationof gene expressionCompared to baseline gene expressionin unstimulated CD4+ T cells activation in vitro increasedexpression of almost all genes in all CD4+ T cells (datanot shown) To simplify comparison the gene expressionof in vitro stimulated CD4+ T cells from TCE-treated micewas compared to that of similarly activated CD4+ T cellsfrom control mice This comparison yielded some subtle but


30

20

10

0

CD4

()

30

20

10

0

CD4

()

lowast

lowast

Control TCE 01Control TCE 01 TCE 001TCE 001


6244hi CD T cells in spleenCD4

+ CD Llo

Figure 3 TCE enhanced the percentage ofmemoryactivated CD4+ T cells following early life exposure Spleen cell suspensions fromPND49mice that had been exposed to TCE during gestation only or during early life only were examined by flow cytometry to determine thepercentage of CD4+ T cells that expressed high levels of CD44 and low levels of CD62L lowastSignificantly different (120572 lt 005) compared tocontrol values

significant differences As observed previously followingadult exposure early life exposure to TCE increased expres-sion of Ifng and Il2 but had little effect on Il4 (Table 2) Ges-tational exposure unlike early life exposure did not increasethe expression of these cytokine genes in activated peripheralCD4+ T cells Thus once again early life exposure unlikegestational exposure mirrored what has been observed inadult and continuous TCE exposure

Although CD4+ T cells from the two windows ofdevelopmental TCE exposure differed in their expressionof cytokine genes they demonstrated remarkably similarexpression profiles for Iap and Muerv These two genesencode for retrotransposons Intracisternal A particle andmurine endogenous retrovirus The expression of these tworetrotransposons is kept in check by epigenetic processespredominantly DNA methylation [35] Events that causeglobal DNA hypomethylation tend to increase expressionof Iap and Muerv while DNA hypermethylation tends tosuppress baseline expression of the retrotransposons In thisstudy both gestational and early life exposure to the lowerconcentration of TCE inhibited expression of Iap andMuERVin peripheral CD4+ T cells

DNA methylation is regulated by DNA methyltrans-ferases a family of enzymes that catalyze the transfer of amethyl group toDNAThis family encompassesDnmt1 that isthought to participate inmaintenanceDNAmethylation andDnmt3a involved in de novo DNA methylation in responseto external stimuli [36] At PND49 neither TCE exposureappreciably altered expression of Dnmt1 or Dnmt3a in CD4+T cellsThus the downregulation of Iap andMuerv in CD4+ Tcells from mice developmentally exposed to TCE regardlessof gestation or early life could not be explained by an increasein the expression of Dnmt1 or Dnmt3a

34 Developmental TCE Exposure and Changes in CD4+T Cell Function In addition to gene expression cytokine

production was examined as a potential marker of TCE-induced alterations in CD4+ T cell function Early lifeexposure to TCE similar to adult exposure increased theproduction of certain cytokines including IL-2 by CD4+ Tcells (Figure 4) Gestational exposure alone was not sufficientto increase cytokine production in the peripheral CD4+ Tcells

35 Developmental TCE Exposure and Thymus CellularityThe effects of developmental TCE exposure on centralimmune function were also examined The number of totalthymocyteswas not altered byTCE exposure during gestationor early life (data not shown) However the composition ofthymic subsets was altered in one set of the TCE-exposedmice Exposure to the lower concentration of TCE duringgestation increased the percentage of thymic single positiveCD4+ T cells (Figure 5) The same mice also demonstratedan increase in the percentage of double negative population 1(DN1) thymocytes as well as a decrease in percentage of DN4thymocytes Unlike gestational exposure early life exposureto TCE did not change the percentages of the differentthymocyte subsets measured Thus in terms of thymocytecellularity early life exposure generated results that resembledthat of adult exposure while gestational exposure generatedresults that most resembled that of previously documentedcontinuous exposure [27] The thymocytes were then incu-bated for 18 hours to promote gene expression Interestinglyneither gestational nor early life exposure to TCE alteredIap expression in the thymus unlike peripheral CD4+ Tcells (Figure 6) This decreases the likelihood that the alteredthymus cellularity of the gestationally exposed mice could beattributed to global changes in DNA methylation

36 Developmental TCE Exposure and Thymus Gene Expres-sion The expression of genes that regulate thymic apoptosisand development were examined in an attempt to explain


Table 2 Gene expression in CD4+ T cells from mice exposed to TCE during gestation or early life

Early life GestationControl TCE 001 TCE01 Control TCE 001 TCE 01

Iap 10 plusmn 017 012 plusmn 002 042 plusmn 036 100 plusmn 01 043 plusmn 005 072 plusmn 019Muerv 095 plusmn 026 06 plusmn 011 076 plusmn 023 10 plusmn 015 044 plusmn 004 095 plusmn 026

Il2 10 plusmn 01 149 plusmn 02 131 plusmn 01 091 plusmn 01 097 plusmn 029 066 plusmn 01Il4 10 plusmn 017 11 plusmn 035 14 plusmn 034 11 plusmn 043 147 plusmn 075 144 plusmn 093

Cdkn1a 10 plusmn 003 09 plusmn 007 092 plusmn 007 105 plusmn 038 162 plusmn 064 141 plusmn 11

Ifng 091 plusmn 016 095 plusmn 011 131 + 022 132 plusmn 10 10 plusmn 081 142 plusmn 111

Dnmt1 10 plusmn 024 105 plusmn 012 089 plusmn 022 111 plusmn 051 158 plusmn 036 173 plusmn 071

Dnmt3a 098 plusmn 002 108 plusmn 007 109 plusmn 007 098 plusmn 051 112 plusmn 035 12 plusmn 087

Gene expression was examined in splenic CD4+ T cells incubated with anti-CD3 and anti-CD28 Abs for 24 hours following isolation from untreated controlmice or from mice exposed to TCE during gestation only or during early life only The data represents the mean plusmn SD lowastSignificantly different (120572 lt 005)compared to control valuesThe bolded text represents values that are significantly different from control values (120572 lt 005)

400

80

320

240

160

0

lowast

lowast

Control TCE 01TCE 001


100

80

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40

20

0

lowast


140

112

84

56

28

0

(pg

mL)

(pg

mL)


1500

1200

900

600

300

0

TNF-120572

IL-2Early life Gestation

(pg

mL)

(pg

mL)

Figure 4 TCE altered cytokines in CD4+ T cells from mice exposed to TCE during early life Cytokine levels in culture supernatants fromCD4+ T cells prepared as described in Figure 4 were examined lowastSignificantly different (120572 lt 005) compared to control values

the altered cellularity in the thymi of mice gestationallyexposed to the lower concentration of TCE In line with theunaltered Iap expression in the thymi of mice gestationallyexposed to TCE the expression of enzymes that regulateDNA methylation namely Dnmt1 and Dnmt3a were alsounaltered by TCE in the thymus (Table 3) Deficiencies inthymic expression of the gene for the proapoptotic protein

Bim or its downstream protein Bax can cause a defect inthe negative selection process [37] Neither of these geneswas altered by TCE exposure in the thymus Survivin aprotein originally described as an inhibitor of apoptosis hasmore recently been shown to be required for maturationof DN thymocytes to DP thymocytes [38] Even thoughgestational TCE exposure did seem to block an early stage


100

10

1

01

DN

thym

ocyt

es (

)

DN1 DN2 DN3 DN4

100

10

1

01

lowast

lowast

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (

)

80

70

60

50

40

30

20

10

0

CD4+ CD4

minusCD8minusCD8

+ CD4+CD8

+

lowast Thym

ocyt

es (

)

70

60

50

40

30

20

10

0CD4

+ CD4minusCD8

minusCD8+ CD4

+CD8+

Thym

ocyt

es (

)

No TCETCE (001mgmL)TCE (01mgmL)





Figure 5 TCE altered thymus cellularity in mice exposed to TCE during gestationThymocyte suspensions from PND49mice that had beenexposed to TCE during gestation only or during early life only were incubated for 18 hours and then examined by flow cytometry to determinethe percentage of single positive double negative and double positive cells In addition after gating on the double negative cell populationthe cells were stained with anti-CD44 and anti-CD25 Abs and labeled as DN1 (CD44+CD25minus) DN2 (CD44+CD25+) DN3 (CD44minusCD25+)and DN4 (CD44minusCD25minus) lowastSignificantly different (120572 lt 005) compared to control values

of DN thymocyte differentiation it did not alter expressionof Survivin Gestational exposure to TCE did not alterexpression of Fas However both concentrations of TCE didsuppress expression of Fasl the gene that encodes for theligand for Fas and an important mediator of activation-induced apoptosisThus the increase in SPCD4+ thymocytesand DN1 thymocytes may be attributable to a decreasein apoptosis mediated by a TCE-induced decrease in theexpression of Fasl However since the altered cellularitywas only found in thymi of mice gestationally exposed to001mgmL TCE while the decrease in Fasl was found inmice exposed to either concentration of TCE the role of Faslin the altered cellularity needs to be clarified

4 Discussion

The concordance rate for developing an autoimmune diseasein identical twins demonstrates the involvement of an ill-defined genetic susceptibility [39] Tomimic this requirementldquoautoimmune-pronerdquoMRL++micewere used in our studiesLate in life MRL++ mice can spontaneously develop a rela-tively mild lupus-like disease as well as other autoimmunedisorders such as Sjogrenrsquos syndrome and T cell-infiltratingpancreatitis [40 41] Young female MRL++ mice with theirpropensity for autoimmunity but absence of overt diseasemake a good model to test the immunostimulatory capacityof developmental TCE exposure Others have similarly used


Fold

chan

ge

Fold

chan

ge

Control TCE 001 TCE 01

10

1

01

10

1

01


Early life GestationIap Iap

Figure 6 TCE did not inhibit Iap in thymocytes from either window of developmental TCE exposure Gene expression was examined inthymocytes incubated for 18 hours following isolation from untreated control mice or from mice exposed to TCE during gestation only orduring early life only The data represents the mean plusmn SD lowastSignificantly different (120572 lt 005) compared to control values

Table 3 Gene expression in thymi of mice exposed to TCE duringgestation

GestationControl TCE 001 TCE 01

Bax 104 plusmn 014 078 plusmn 024 106 plusmn 075

Bim 102 plusmn 015 082 plusmn 03 087 plusmn 021

Dnmt1 10 plusmn 03 116 plusmn 05 103 plusmn 025

Dnmt3a 114 plusmn 056 117 plusmn 046 093 plusmn 026

Survivin 13 plusmn 097 053 plusmn 047 068 plusmn 042

Aire 11 plusmn 058 071 plusmn 009 076 plusmn 051

Fas 096 plusmn 049 083 plusmn 046 078 plusmn 028

Fasl 106 plusmn 042 04 plusmn 025 045 plusmn 028Gene expression was examined in thymocytes incubated for 18 hoursfollowing isolation from untreated control mice or from mice exposed toTCE during gestation onlyThe data represents the mean plusmn SD lowastSignificantly different (120572 lt 005)compared to control valuesThe bolded text represents values that are significantly different from controlvalues (120572 lt 005)

ldquoautoimmune-pronerdquomouse strains to test the developmentalimmunotoxicity of chemicals such as dioxin bisphenol Aand mercury [42ndash44]

The exposure associated with the highest TCE con-centration of 001mgmL (10 ppm) is considerably lowerthan acceptable human occupational exposure The EPArsquosMaximum Contaminant Level for public drinking water is5 ppb but TCE has been found at levels up to 14 ppm [45]In humans exposed to TCE-contaminated water the amountingested is only a fraction of the TCE absorbed via inhalationand dermal contact [46] Since TCE exposure in the micewill be limited to ingestion it could be argued that mouseexposure to 10 ppm is within the range of possible humanenvironmental exposure

For essentially all of the parameters tested the early lifeexposure to TCE induced immune system changes similarto adult exposure albeit at much lower concentrationsOn the other hand the results obtained from gestationalonly exposure to TCE were similar to those documented

after continuous exposure to TCE Gestational exposure toTCE caused alterations in thymic cellularity and effects onbody weight and spleen size In contrast gestational TCEexposure had little effect on peripheral CD4+ T cells atleast the parameters measured at PND 49 Unlike gestationalexposure early life exposure to TCE had no effect onthymic cellularity but was able to increase the percentage ofactivatedmemory CD4+ T cells in association with increasedproduction of cytokines This implies that exposure to TCEeven as early as PND0 had more of an impact on deployedperipheral CD4+ T cells than on the thymus It is possiblethat lactational exposure to TCE at the concentrations usedwas not sufficient to impact the still-developing thymus andthat the effects observed following early life exposure wereprimarily attributable to the direct TCE exposure begun atPND21 An additional study of lactational only exposure toTCE is needed to resolve this question Immune developmentinmice correlates well with immune development in humansalbeit with different kinetics [47] One difference is that theperinatal immune system in mice is even more immaturethan that of humansThismeans that the immunematurationthat occurs during human gestation is commensurate withthat found in mice at weaning at PND21Thus lactation onlyexposure in mice would correspond with late gestation inhumans

Taken together it would seem that the individuals mostsusceptible to the most wide-ranging effects of TCE on thecentral and peripheral immune system are those exposedto the chemical during gestation as well as early life Sucha scenario in humans could occur when contaminateddrinking water for both mother and child results in indirectTCE exposure during gestation and lactation and then directexposure after weaning However it should be noted thatalthough the two different windows of TCE developmentalexposure had distinct effects on peripheral and central Tcells both windows of exposure inhibited expression ofretrotransposons in peripheral CD4+ T cells This impliesthat both gestation and early life are times during whichepigenetic changes can be induced Whether these changesmanifest themselves in functional alterations that promote


TCE-induced hypersensitivity later in life needs to be deter-mined Epigenetic processes can regulate several aspects ofnormal CD4+ T cell function including Th1Th2 differen-tiation [48] cytokine production [49] and maintenance ofTreg cells [50] There are also several pieces of evidencethat epigenetic processes regulate the activity of self-reactiveCD4+ T cells that mediate autoimmune disease [51ndash53] Inview of the connection between epigenetic alterations andCD4+ T cell autoreactivity the need to further investigatethe capacity of TCE to induce these alterations becomes evenmore important

How TCE impacts CD4+ T cells is still being definedEarlier studies showed that the CD4+ T cell-altering effectsof TCE were most likely mediated by its primary oxida-tive metabolite trichloroacetaldehyde hydrate (TCAH) [3233] As an aldehyde TCAH can form a functionally activechemical interaction known as a Schiff base with amineson the surface of the CD4+ T cells [54] The possibilitythat TCAH forms a Schiff base with a costimulator receptorsuch as CD28 and thus provides bystander costimulation forantigen-activatedCD4+ T cells is being investigatedHow thismechanism would impact thymocytes during developmentis unclear In adults TCE metabolism to TCAH is primarilymediated by cytochrome P450 2E1 (CYP2E1) and to a lesserextent by CYP1A1 and occurs primarily in the liver Howeversince CYP1A1 unlike CYP2E1 can be detected in GD7 in thethymus [55] it is possible that CYP1A1 generates TCAH inthe thymus of mice developmentally exposed to TCE

Characterizing TCE-induced developmental immuno-toxicitymay be part of an important big picture Immune dys-function in the form of hypersensitivity disorders are amongthe most common medical conditions affecting children inthe USAMany of these appear to be increasing For examplemore children are being diagnosed with type I diabetes[56] childhood Gravesrsquo disease [57] and lupus nephritis[58] than ever before Similarly the CDC reports that theprevalence of food allergies increased from 34 in 1997ndash1999to 51 in 2009ndash2011 [59] The prevalence of skin allergiesalso increased from 74 in 1997ndash1999 to 125 2009ndash2011It is possible that the increase in childhood hypersensitivityis linked to increased exposure to environmental chemicalswith potential developmental toxicity More than 85000synthetic chemicals have been developed in the past 75 yearsand only about 20 of the 3000 most widely used chemicalshave ever been tested for general developmental toxicitylet alone developmental immunotoxicity [60] As part ofNHANES 2003-2004 it was found that after adjusting forcovariates levels of many chemicals in pregnant women wereincreased compared to nonpregnant women [61] Certainpolychlorinated biphenyls organochlorine pesticides poly-cyclic aromatic hydrocarbons andmany other chemicals weredetected in 99-100 of pregnant women Some of thesechemicals cross the placenta and mediate developmentalimmunotoxicity Developmental immunotoxicity is based onthe premise that during thematuration of the immune systemtoxicant exposure results in a qualitative or quantitativedifference in the effect or a greater persistence of effectThis increased sensitivity may be attributed to (i) a greaterchemical exposure per pound of body weight (ii) immature

metabolic systems unable efficiently clear toxicants (iii)developing immune system that is easily disrupted and (iv)more time to develop chemical-induced immune-mediateddiseases Studying developmental immunotoxicity of chemi-cals such as TCE with proinflammatory effects may provideimportant clues to the etiology of idiopathic autoimmunedisease

5 Conclusion

Early life exposure to TCE induced changes in peripheralCD4+ T cell commensurate with those found inmice exposedto TCE as an adult Gestational TCE exposure inducedchanges in central immune function similar to that observedfollowing continuous exposureHowever the twowindows ofexposure did have one effect in common namely the down-regulation of retrotransposon expression in CD4+ T cellsSince the effects induced by the two developmental windowsof TCE exposure were associated with TCE concentrationslower than those effective in adult exposure the differentialresponses are functionally important

Conflict of Interests

There is no conflict of interests regarding the publication ofthis paper

Acknowledgments

The authors would like to gratefully acknowledge the excel-lent technical assistance of Brannon Broadfoot Kirk Westand Rachel Lee and the support of the UAMS Trans-lational Research Institute (National Institutes of HealthUL1RR029884) This work was supported by Grants fromthe Arkansas Biosciences Institute the National Institutes ofHealth (1R01ES017286 1R01ES021484 R21ES017311) and theOrganic Compounds Property Contamination class actionsettlement (CV 1992-002603)

References

[1] US EPA Toxicological Review of Trichloroethylene In Suppot ofSummay Information on the integrated Risk Information System(IRIS) US Environmental Protection AgencyWashington DCUSA 2011

[2] V S Byers A S Levin D M Ozonoff and R W Bald-win ldquoAssociation between clinical symptoms and lymphocyteabnormalities in a population with chronic domestic exposureto industrial solvent-contaminated domestic water supply and ahigh incidence of leukaemiardquoCancer Immunology Immunother-apy vol 27 no 1 pp 77ndash81 1988

[3] S Yanez Diaz M Moran P Unamuno and M Armijo ldquoSilicaand trichloroethylene-induced progressive systemic sclerosisrdquoDermatology vol 184 no 2 pp 98ndash102 1992

[4] B L Hansen andH Isager ldquoA scleroderma-resembling disease-exposure to trichloroethylene and trichloroethane is there acausal connectionrdquo Ugeskrift for Laeger vol 150 no 13 pp805ndash808 1988


[5] E M Saihan J L Burton and K W Heaton ldquoA new syndromewith pigmentation scleroderma gynaecomastia Raynaudrsquosphenomenon and peripheral neuropathyrdquo British Journal ofDermatology vol 99 no 4 pp 437ndash440 1978

[6] H Flindt-Hansen and H Isager ldquoScleroderma after occu-pational exposure to trichloroethylene and trichlorethanerdquoToxicology Letters vol 95 pp 173ndash181 1987

[7] L Czirjak E Pocs andG Szegedi ldquoLocalized scleroderma afterexposure to organic solventsrdquo Dermatology vol 189 no 4 pp399ndash401 1994

[8] J E Lockey C R Kelly and G W Cannon ldquoProgressive sys-temic sclerosis associated with exposure to trichloroethylenerdquoJournal of Occupational Medicine vol 29 no 6 pp 493ndash4961987

[9] R Dubrow and D M Gute ldquoCause-specific mortality amongRhode Island jewelry workersrdquo American Journal of IndustrialMedicine vol 12 no 5 pp 579ndash593 1987

[10] G L Gist and J R Burg ldquoTrichloroethylene-a review ofthe literature in view of the results of the trichloroethylenesubregistryrdquo Toxicology and Industrial Health vol 11 no 3 pp253ndash307 1995

[11] G R Bond ldquoHepatitis rash and eosinophilia followingtrichloroethylene exposure a case report and speculation onmechanistic similarity to halothane induced hepatitisrdquo Journalof Toxicology vol 34 no 4 pp 461ndash466 1996

[12] X Xu R Yang N Wu et al ldquoSevere hypersensitivity dermatitisand liver dysfunction induced by occupational exposure totrichloroethylenerdquo Industrial Health vol 47 no 2 pp 107ndash1122009

[13] J Yi Y X Teng D Zang et al ldquoAnalysis of subgroups of lym-phocyte in peripheral blood among dermatitis medicamentosa-like of trichloroethylene patients and healthy exposed workersrdquoZhonghua Yu Fang Yi Xue Za Zhi vol 45 pp 1017ndash1021 2011

[14] K H Kilburn and R H Warshaw ldquoPrevalence of symptomsof systemic lupus erythematosus (SLE) and of fluorescentantinuclear antibodies associated with chronic exposure totrichloroethylene and other chemicals in well waterrdquo Environ-mental Research vol 57 no 1 pp 1ndash9 1992

[15] I Iavicoli A Marinaccio and G Carelli ldquoEffects of occupa-tional trichloroethylene exposure on cytokine levels in work-ersrdquo Journal of Occupational and Environmental Medicine vol47 no 5 pp 453ndash457 2005

[16] RW LuebkeDH Chen RDietert Y YangMKing andM ILuster ldquoThe comparative immunotoxicity of five selected com-pounds following developmental or adult exposurerdquo Journal ofToxicology and Environmental Health B vol 9 no 1 pp 1ndash262006

[17] C Heilmann E Budtz-Joslashrgensen F Nielsen B Heinzow PWeihe and P Grandjean ldquoSerum concentrations of antibodiesagainst vaccine toxoids in children exposed perinatally toimmunotoxicantsrdquo Environmental Health Perspectives vol 118no 10 pp 1434ndash1438 2010

[18] M M Leijs J G Koppe K Olie W M C Van Aalderen PDe Voogt and G W Ten Tusscher ldquoEffects of dioxins PCBsand PBDEs on immunology and hematology in adolescentsrdquoEnvironmental Science and Technology vol 43 no 20 pp 7946ndash7951 2009

[19] A N Colebatch and C J Edwards ldquoThe influence of early lifefactors on the risk of developing rheumatoid arthritisrdquo Clinicaland Experimental Immunology vol 163 no 1 pp 11ndash16 2011

[20] P Langer ldquoThe impacts of organochlorines and other persistentpollutants on thyroid and metabolic healthrdquo Frontiers in Neu-roendocrinology vol 31 no 4 pp 497ndash518 2010

[21] US Environmental Protection Agency Third National Healthand Nutrition Examination Survey U S Environmental Protec-tion Agency Washington DC USA 1994

[22] G L Gist and J A R Burg ldquoTrichloroethylenemdasha review ofthe literature from a health effects perspectiverdquo Toxicology andIndustrial Health vol 11 no 3 pp 253ndash307 1995

[23] J M Griffin S J Blossom S K Jackson K M Gilbertand N R Pumford ldquoTrichloroethylene accelerates an autoim-mune response by Th1 T cell activation in MRL++ micerdquoImmunopharmacology vol 46 no 2 pp 123ndash137 2000

[24] J M Griffin K M Gilbert L W Lamps and N R PumfordldquoCD4+ T-cell activation and induction of autoimmune hepatitisfollowing trichloroethylene treatment in MRL++ micerdquo Toxi-cological Sciences vol 57 no 2 pp 345ndash352 2000

[25] KMGilbertN R Pumford and S J Blossom ldquoEnvironmentalcontaminant trichloroethylene promotes autoimmune diseaseand inhibits T-cell apoptosis in MRL++ micerdquo Journal ofImmunotoxicology vol 3 no 4 pp 263ndash267 2006

[26] K M Gilbert B Przybyla N R Pumford et al ldquoDelineatingliver events in trichloroethylene-induced autoimmune hepati-tisrdquo Chemical Research in Toxicology vol 22 no 4 pp 626ndash6322009

[27] S J Blossom and J C Doss ldquoTrichloroethylene alters cen-tral and peripheral immune function in autoimmune-proneMRL++ mice following continuous developmental and earlylife exposurerdquo Journal of Immunotoxicology vol 4 no 2 pp129ndash141 2007

[28] T L Bunn P J Parsons E Kao and R R Dietert ldquoExposureto lead during critical windows of embryonic developmentdifferential immunotoxic outcome based on stage of exposureand genderrdquoToxicological Sciences vol 64 no 1 pp 57ndash66 2001

[29] R R Dietert J-E Lee and T L Bunn ldquoDevelopmental imm-unotoxicology emerging issuesrdquo Human and ExperimentalToxicology vol 21 no 9-10 pp 479ndash485 2002


[31] K M Gilbert B Przybyla N R Pumford et al ldquoUse of tra-nscriptomics and metabolomics to delineate liver events asso-ciated with trichloroethylene-induced autoimmune hepatitisrdquoSubmitted 2008

[32] S J Blossom J CDoss andKMGilbert ldquoChronic exposure toa trichloroethylene metabolite in autoimmune-prone MRL++mice promotes immune modulation and alopeciardquo Toxicologi-cal Sciences vol 95 no 2 pp 401ndash411 2007

[33] J M Griffin K M Gilbert and N R Pumford ldquoInhibition ofCYP2E1 reverses CD4+ T-cell alterations in trichloroethylene-treated MRL++ micerdquo Toxicological Sciences vol 54 no 2 pp384ndash389 2000

[34] K M Gilbert B Reisfeld and T Zurlinden ldquoModeling mecha-nism by which trichloroethylene inhibits liver repair in mousemodel of autoimmunityrdquo 2013

[35] T Takabatake H Ishihara Y Ohmachi et al ldquoMicroarray-based global mapping of integration sites for the retrotranspo-son intracisternal A-particle in the mouse genomerdquo NucleicAcids Research vol 36 no 10 p e59 2008


[36] X Zhang R Fu J Yu and X Wu ldquoDNA demethylation wheregenetics meets epigeneticsrdquo Current Pharmaceutical Design2013

[37] C A Chougnet P Tripathi C S Lages et al ldquoA major role forBim in regulatory T cell homeostasisrdquo Journal of Immunologyvol 186 no 1 pp 156ndash163 2011

[38] Z Xing E M Conway C Kang and AWinoto ldquoEssential roleof survivin an inhibitor of apoptosis protein in T cell devel-opment maturation and homeostasisrdquo Journal of ExperimentalMedicine vol 199 no 1 pp 69ndash80 2004

[39] X S He A A Ansari and M E Gershwin ldquoXenobiotic con-siderations for the development of autoimmune liver diseasesbad genes and bad luckrdquo Reviews on Environmental Health vol16 no 3 pp 191ndash202 2001

[40] K Skarstein A C Johannessen R Holmdahl and R JonssonldquoEffects on sialadenitis after cellular transfer in autoimmuneMRLlpr micerdquo Clinical Immunology and Immunopathologyvol 84 no 2 pp 177ndash184 1997

[41] W M Qu T Miyazaki M Terada et al ldquoA novel autoim-mune pancreatitis model in MRL mice treated with polyi-nosinicpolycytidylic acidrdquoClinical and Experimental Immunol-ogy vol 129 no 1 pp 27ndash34 2002

[42] A Mustafa S D Holladay M Goff et al ldquoDevelopmentalexposure to 2378-tetrachlorodibenzo-p-dioxin alters postna-tal T cell phenotypes and T cell function and exacerbatesautoimmune lupus in 24-week-old SNF1 micerdquo Birth DefectsResearch A vol 85 no 10 pp 828ndash836 2009

[43] J Bodin B A Kocbach R Becher F Kuper M Lovik and UC Nygaard ldquoTransmaternal bisphenol A exposure acceleratesdiabetes type 1 development in NOD micerdquo ToxicologicalSciences 2013

[44] Y Zhang V J Bolivar and D A Lawrence ldquoMaternal exposureto mercury chloride during pregnancy and lactation affectsthe immunity and social behavior of offspringrdquo ToxicologicalSciences vol 133 no 1 pp 101ndash111 2013

[45] ATSDR Progress Report Survey of Specific Childhood Cancersand Birth Defects Amoung Children Whose Mothers WerePregnantWhile Living at US Marine Corps Base Camp LejeuneNorth Carolina 1968ndash1985 Agency for Toxic Substances andDisease Registry Atlanta Ga USA 2003

[46] L J Lee C Chan C Chung Y Yee-Chung G Wang andJ Wang ldquoHealth risk assessment on residents exposed tochlorinated hydrocarbons contaminated in groundwater of ahazardous waste siterdquo Journal of Toxicology and EnvironmentalHealth A vol 65 no 3-4 pp 219ndash235 2002

[47] S D Holladay and R J Smialowicz ldquoDevelopment of themurine and human immune system differential effects ofimmunotoxicants depend on time of exposurerdquo EnvironmentalHealth Perspectives vol 108 supplement 3 pp 463ndash473 2000

[48] B R Winders R H Schwartz and D Bruniquel ldquoA distinctregion of the murine IFN-120574 promoter is hypomethylated fromearly T cell development through mature naive and Th1 celldifferentiation but is hypermethylated in Th2 cellsrdquo Journal ofImmunology vol 173 no 12 pp 7377ndash7384 2004

[49] D Bruniquel and R H Schwartz ldquoSelective stable demethy-lation of the interleukin-2 gene enhances transcription by anactive processrdquo Nature Immunology vol 4 no 3 pp 235ndash2402003

[50] S Floess J Freyer C Siewert et al ldquoEpigenetic control of thefoxp3 locus in regulatory T cellsrdquo PLoS Biology vol 5 no 2 pe38 2007

[51] R Yung D Powers K Johnson et al ldquoMechanisms of drug-induced lupus II T cells overexpressing lymphocyte function-associated antigen 1 become autoreactive and cause a lupuslikedisease in syngeneic micerdquo Journal of Clinical Investigation vol97 no 12 pp 2866ndash2871 1996

[52] M A Jeffries M Dozmorov Y Tang J T Merrill J DWren and A H Sawalha ldquoGenome-wide DNA methylationpatterns in CD4+ T cells from patients with systemic lupuserythematosusrdquo Epigenetics vol 6 no 5 pp 593ndash601 2011

[53] B M Javierre A F Fernandez J Richter et al ldquoChanges in thepattern of DNAmethylation associate with twin discordance insystemic lupus erythematosusrdquoGenome Research vol 20 no 2pp 170ndash179 2010

[54] KMGilbert A BWhitlow andN R Pumford ldquoEnvironmen-tal contaminant and disinfection by-product trichloroacetalde-hyde stimulates T cells in vitrordquo International Immunopharma-cology vol 4 no 1 pp 25ndash36 2004

[55] D Choudhary I Jansson I Stoilov M Sarfarazi and J BSchenkman ldquoExpression patterns of mouse and human CYPorthologs (families 1ndash4) during development and in differentadult tissuesrdquo Archives of Biochemistry and Biophysics vol 436no 1 pp 50ndash61 2005

[56] T H Lipman L E Levitt Katz S J Ratcliffe et al ldquoIncreasingincidence of type 1 diabetes in youth twenty years of thePhiladelphia pediatric diabetes registryrdquo Diabetes Care vol 36no 6 pp 1597ndash1603 2013

[57] GWWong andP S Cheng ldquoIncreasing incidence of childhoodGravesrsquo disease in Hong Kong a follow-up studyrdquo ClinicalEndocrinology vol 54 no 4 pp 547ndash550 2001

[58] K H Costenbader A Desai G S Alarcon et al ldquoTrends inthe incidence demographics and outcomes of end-stage renaldisease due to lupus nephritis in the US from 1995 to 2006rdquoArthritis and Rheumatism vol 63 no 6 pp 1681ndash1688 2011

[59] A M Branum and S L Lukacs ldquoFood allergy among USchildren trends in prevalence and hospitalizationsrdquo NCHSData Brief no 10 pp 1ndash8 2008

[60] P J Landrigan and L R Goldman ldquoChemical safety health carecosts and the affordable care actrdquoAmerican Journal of IndustrialMedicine vol 57 no 1 pp 1ndash3 2013

[61] T J Woodruff A R Zota and J M Schwartz ldquoEnvironmentalchemicals in pregnant women in the united states NHANES2003-2004rdquo Environmental Health Perspectives vol 119 no 6pp 878ndash885 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


people may experience through drinking water contamina-tion is generally thought to be risk-free However there isevidence that the developing immune system is especiallysensitive to even low-level immunotoxicants A recent reviewcompared early versus adult exposure to several immuno-suppressive toxins including lead and tributyltin in animalmodels [16] In all cases sensitivity was greater if exposureoccurred during development In fact immune suppressionin developmentally-exposed offspring oftenoccurred at dosesthat were ineffective in adults Developmental sensitivityto toxicants has also been found in humans [17 18] Thisincludes evidence that adult onset autoimmune disease canbe triggered by pre- and early postnatal toxicant exposure[19 20]

Developmental exposure to TCE in humans is notuncommon one study showed that 100 of breast milksamples from 4 US urban areas had detectable levels of TCE[21] Gestational and early life TCE exposure has primarilybeen examined for its neurotoxicity [22] However childrencontinuously exposed for 3ndash19 years beginning in utero to awater supply contaminatedwith solvents [with TCE being thepredominant toxicant (267 ppb)] had altered ratios of T cellsubsets and increased levels of autoantibodies [2]

We have studied a direct cause and effect relationshipbetween TCE exposure and immunotoxicity in a mousemodel Adult exposure to TCE primarily altered effectorCD4+ T cells with little effect on CD8+ T cells B cells or 119879regcellsThe effects of TCEonCD4+ T cells were seen after only 4weeks and included expansion of activatedmemory (CD44hi

CD62Llo) CD4+ T cells and altered cytokine production [23ndash25] Adult TCE exposure seemed to have minimal effecton thymus cellularity After chronic adult exposure (26ndash32weeks) the TCE-induced alterations in CD4+ T cells led toT cell-mediated liver inflammation identical to that seen inidiopathic autoimmune hepatitis (AIH) in humans [24 26]

We have also examined mice following continuous TCEexposure beginning in utero and then encompassing lactationas well as an additional 4 weeks of direct exposure Thiscontinuous exposure to TCE at concentrations lower thanthat used for adult exposure altered thymocyte cellularity andmodified the phenotype and function of peripheral CD4+ Tcells [27]

Although informative studies of continuous toxicantexposure do not reveal which windows of exposure havethe most impact on the developing immune system Forexample it has been shown that early gestational exposureto certain toxicants such as lead are not as likely to sup-press Th1 function as late gestational exposure [28] Thissuggests that within the developmental period there existsparticular windows of relative susceptibility and resistance[29] Thus knowing more about the functional outcomesof particular developmental windows of TCE exposure isneeded to accurately estimate risk and to plan interventionsThis study was designed to compare central and peripheralimmune system alterations in mice exposed to TCE duringtwo crucial windows of immune development and toxicantsensitivity namely gestation [GD0 to birth (PND0)] andearly life (PND0 to PND49)

2 Materials and Methods

21 Mice and TCE Exposure Developmental exposure toTCE has been described [30] Basically breeding pairs of 8-week-old MRL++ mice (Jackson Laboratories Bar HarborME)were established In one experiment immediately follow-ing detection of pregnancy GD0 the females were divided(following stratified randomization) into 3 groups and givenwater with 0 001 or 01mgmL TCE Controls were givenwater containing only 1 Alkamuls EL-620 the reagent usedto solubilize the TCE All the drinking water was Ultrapureunchlorinated to assure that chlorination by-products do notconfound the results The TCE-containing drinking waterwas changed 3 timesweek to offset degradation of TCEMaternal exposure to TCE-containing drinking water endedat birth in this experiment The female pups that underwentthe gestation only exposure to TCEwere examined at PND49As observed previouslymaternal toxicity was not noted at thelevel of body weight mating index fertility index sex ratio oflitters gestational length and food and water consumption

In the second experiment the female breeders were notexposed to TCE during pregnancy but were given TCE-containing drinking water immediately after giving birthThus the pups were exposed to TCE from PND0 to PND21via lactation Once the female pups were weaned at PND21they were exposed to TCE directly in their drinking water forthe duration of the experiment The female pups from thisearly life exposure were sacrificed at PND49

Both female breeders and resulting pups in both exper-iments were weighed weekly and water consumption wasmonitored All studies were approved by the Animal CareandUse Committee at the University of Arkansas forMedicalSciences

When the female pups were sacrificed at PND49 pooledspleen cell suspensions and thymus cell suspensions frompaired mice from each litter (119899 = 5ndash7 litterstreatmentgroup) were examined by flow cytometry In addition CD4+T cells were isolated from the pooled spleen cell suspensionsand stimulated with immobilized anti-CD3 antibody andanti-CD28 antibody for 24 hours as described [25] Culturesupernatants were then collected for cytokine evaluation andthe activated CD4+ T cells were frozen for subsequent qRT-PCR analysis In addition adherent macrophages isolatedfrom pooled peritoneal exudates from 2-3 micelitter wereincubated for 20 hours in medium alone or in the presenceof LPS (1 120583gmL) and IFN-120574 (100 unitsmL) Approximately80 of adherent peritoneal exudate cells (PEC) regardlessof treatment group expressed the transmembrane proteinF480 a marker of mature macrophages (data not shown)Culture supernatants from the peritoneal macrophages werethen collected for cytokine evaluation RLT Lysis Buffer (Qia-gen Sciences Germantown MD) was then added directly tothe remaining adherent cells before freezing for subsequentqRT-PCR analysis

22 qRT-PCR Fluorescence-based quantitative reverse tran-scriptase polymerase chain reaction (qRT-PCR) was con-ducted using RNA isolated from activated CD4+ T cells























3 Results



Cel

l num

ber (times108)

Cel

l num

ber (times108)

100

075

050

025

000

120

080

040

000

10

20

30

40

35

30

25

20

0

10

20

30

40

0

Control

(mg

kgd

ay)

(mg

kgd

ay)

(g)

(g) lowast

lowast

Control

Control Control

Control

Control

45

40

35

30

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001



Direct Maternal

Body weight

Spleen size







1200

1000

800

600

400

200

0Control Control

Control

20

15

10

5

0

Fold

chan

ge

Fold

chan

ge

NoneLPS

NoneLPS

NoneLPS

NoneLPS

lowast

lowast

6000

5000

4000

3000

2000

1000

0

60

40

20

0

(pg

mL)

(pg

mL)

lowast

Control




IL-6 protein

Il6 expression






30

20

10

0

CD4

()

30

20

10

0

CD4

()

lowast

lowast




+ CD Llo



















400

80

320

240

160

0

lowast

lowast



100

80

60

40

20

0

lowast


140

112

84

56

28

0

(pg

mL)

(pg

mL)


1500

1200

900

600

300

0

TNF-120572


(pg

mL)

(pg

mL)





100

10

1

01

DN

thym

ocyt

es (

)

DN1 DN2 DN3 DN4

100

10

1

01

lowast

lowast

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (

)

80

70

60

50

40

30

20

10

0

CD4+ CD4

minusCD8minusCD8

+ CD4+CD8

+

lowast Thym

ocyt

es (

)

70

60

50

40

30

20

10

0CD4

+ CD4minusCD8

minusCD8+ CD4

+CD8+

Thym

ocyt

es (

)








4 Discussion



Fold

chan

ge

Fold

chan

ge


10

1

01

10

1

01
























5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































3 Results



Cel

l num

ber (times108)

Cel

l num

ber (times108)

100

075

050

025

000

120

080

040

000

10

20

30

40

35

30

25

20

0

10

20

30

40

0

Control

(mg

kgd

ay)

(mg

kgd

ay)

(g)

(g) lowast

lowast

Control

Control Control

Control

Control

45

40

35

30

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001



Direct Maternal

Body weight

Spleen size







1200

1000

800

600

400

200

0Control Control

Control

20

15

10

5

0

Fold

chan

ge

Fold

chan

ge

NoneLPS

NoneLPS

NoneLPS

NoneLPS

lowast

lowast

6000

5000

4000

3000

2000

1000

0

60

40

20

0

(pg

mL)

(pg

mL)

lowast

Control




IL-6 protein

Il6 expression






30

20

10

0

CD4

()

30

20

10

0

CD4

()

lowast

lowast




+ CD Llo



















400

80

320

240

160

0

lowast

lowast



100

80

60

40

20

0

lowast


140

112

84

56

28

0

(pg

mL)

(pg

mL)


1500

1200

900

600

300

0

TNF-120572


(pg

mL)

(pg

mL)





100

10

1

01

DN

thym

ocyt

es (

)

DN1 DN2 DN3 DN4

100

10

1

01

lowast

lowast

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (

)

80

70

60

50

40

30

20

10

0

CD4+ CD4

minusCD8minusCD8

+ CD4+CD8

+

lowast Thym

ocyt

es (

)

70

60

50

40

30

20

10

0CD4

+ CD4minusCD8

minusCD8+ CD4

+CD8+

Thym

ocyt

es (

)








4 Discussion



Fold

chan

ge

Fold

chan

ge


10

1

01

10

1

01
























5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cel

l num

ber (times108)

Cel

l num

ber (times108)

100

075

050

025

000

120

080

040

000

10

20

30

40

35

30

25

20

0

10

20

30

40

0

Control

(mg

kgd

ay)

(mg

kgd

ay)

(g)

(g) lowast

lowast

Control

Control Control

Control

Control

45

40

35

30

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001

TCE 01TCE 001



Direct Maternal

Body weight

Spleen size







1200

1000

800

600

400

200

0Control Control

Control

20

15

10

5

0

Fold

chan

ge

Fold

chan

ge

NoneLPS

NoneLPS

NoneLPS

NoneLPS

lowast

lowast

6000

5000

4000

3000

2000

1000

0

60

40

20

0

(pg

mL)

(pg

mL)

lowast

Control




IL-6 protein

Il6 expression






30

20

10

0

CD4

()

30

20

10

0

CD4

()

lowast

lowast




+ CD Llo



















400

80

320

240

160

0

lowast

lowast



100

80

60

40

20

0

lowast


140

112

84

56

28

0

(pg

mL)

(pg

mL)


1500

1200

900

600

300

0

TNF-120572


(pg

mL)

(pg

mL)





100

10

1

01

DN

thym

ocyt

es (

)

DN1 DN2 DN3 DN4

100

10

1

01

lowast

lowast

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (

)

80

70

60

50

40

30

20

10

0

CD4+ CD4

minusCD8minusCD8

+ CD4+CD8

+

lowast Thym

ocyt

es (

)

70

60

50

40

30

20

10

0CD4

+ CD4minusCD8

minusCD8+ CD4

+CD8+

Thym

ocyt

es (

)








4 Discussion



Fold

chan

ge

Fold

chan

ge


10

1

01

10

1

01
























5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1200

1000

800

600

400

200

0Control Control

Control

20

15

10

5

0

Fold

chan

ge

Fold

chan

ge

NoneLPS

NoneLPS

NoneLPS

NoneLPS

lowast

lowast

6000

5000

4000

3000

2000

1000

0

60

40

20

0

(pg

mL)

(pg

mL)

lowast

Control




IL-6 protein

Il6 expression






30

20

10

0

CD4

()

30

20

10

0

CD4

()

lowast

lowast




+ CD Llo



















400

80

320

240

160

0

lowast

lowast



100

80

60

40

20

0

lowast


140

112

84

56

28

0

(pg

mL)

(pg

mL)


1500

1200

900

600

300

0

TNF-120572


(pg

mL)

(pg

mL)





100

10

1

01

DN

thym

ocyt

es (

)

DN1 DN2 DN3 DN4

100

10

1

01

lowast

lowast

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (

)

80

70

60

50

40

30

20

10

0

CD4+ CD4

minusCD8minusCD8

+ CD4+CD8

+

lowast Thym

ocyt

es (

)

70

60

50

40

30

20

10

0CD4

+ CD4minusCD8

minusCD8+ CD4

+CD8+

Thym

ocyt

es (

)








4 Discussion



Fold

chan

ge

Fold

chan

ge


10

1

01

10

1

01
























5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















30

20

10

0

CD4

()

30

20

10

0

CD4

()

lowast

lowast




+ CD Llo



















400

80

320

240

160

0

lowast

lowast



100

80

60

40

20

0

lowast


140

112

84

56

28

0

(pg

mL)

(pg

mL)


1500

1200

900

600

300

0

TNF-120572


(pg

mL)

(pg

mL)





100

10

1

01

DN

thym

ocyt

es (

)

DN1 DN2 DN3 DN4

100

10

1

01

lowast

lowast

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (

)

80

70

60

50

40

30

20

10

0

CD4+ CD4

minusCD8minusCD8

+ CD4+CD8

+

lowast Thym

ocyt

es (

)

70

60

50

40

30

20

10

0CD4

+ CD4minusCD8

minusCD8+ CD4

+CD8+

Thym

ocyt

es (

)








4 Discussion



Fold

chan

ge

Fold

chan

ge


10

1

01

10

1

01
























5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























400

80

320

240

160

0

lowast

lowast



100

80

60

40

20

0

lowast


140

112

84

56

28

0

(pg

mL)

(pg

mL)


1500

1200

900

600

300

0

TNF-120572


(pg

mL)

(pg

mL)





100

10

1

01

DN

thym

ocyt

es (

)

DN1 DN2 DN3 DN4

100

10

1

01

lowast

lowast

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (

)

80

70

60

50

40

30

20

10

0

CD4+ CD4

minusCD8minusCD8

+ CD4+CD8

+

lowast Thym

ocyt

es (

)

70

60

50

40

30

20

10

0CD4

+ CD4minusCD8

minusCD8+ CD4

+CD8+

Thym

ocyt

es (

)








4 Discussion



Fold

chan

ge

Fold

chan

ge


10

1

01

10

1

01
























5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

10

1

01

DN

thym

ocyt

es (

)

DN1 DN2 DN3 DN4

100

10

1

01

lowast

lowast

DN1 DN2 DN3 DN4

DN

thym

ocyt

es (

)

80

70

60

50

40

30

20

10

0

CD4+ CD4

minusCD8minusCD8

+ CD4+CD8

+

lowast Thym

ocyt

es (

)

70

60

50

40

30

20

10

0CD4

+ CD4minusCD8

minusCD8+ CD4

+CD8+

Thym

ocyt

es (

)








4 Discussion



Fold

chan

ge

Fold

chan

ge


10

1

01

10

1

01
























5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Fold

chan

ge

Fold

chan

ge


10

1

01

10

1

01
























5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion




Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article Differential Immunotoxicity Induced by Two …downloads.hindawi.com/journals/ad/2014/982073.pdf · 2019-07-31 · list of hazardous chemicals. One of the predominant