research article mercury, autoimmunity, and environmental...

Research ArticleMercury Autoimmunity and Environmental Factors onCheyenne River Sioux Tribal Lands

Jennifer Ong1 Esther Erdei1 Robert L Rubin2 Curtis Miller1 Carlyle Ducheneaux3

Marcia OrsquoLeary4 Bernadette Pacheco1 Michael Mahler5 Patricia Nez Henderson6

K Michael Pollard7 and Johnnye L Lewis1

1 College of Pharmacy Community Environmental Health Program University of New Mexico Health Sciences Center905 Vassar NE Albuquerque NM 87106 USA

2 School of Medicine University of New Mexico Health Sciences Center 1 University of New Mexico Albuquerque NM 87131 USA3Department of Natural Resources Cheyenne River Sioux Tribe PO Box 590 East Highway 212 Eagle Butte SD 57625 USA4Missouri Breaks Industries Research Inc Hc 64 Box 52 Timber Lake SD 57656 USA5 INOVA Diagnostics Inc 9900 Old Grove Road San Diego CA 92131 USA6Black Hills Center for American Indian Health 701 St Joseph Street Suite 204 Rapid City SD 57701 USA7 Scripps Research Institute 10550 North Torrey Pines Road La Jolla CA 92037 USA

Correspondence should be addressed to Johnnye L Lewis jlewiscybermesacom

Received 7 November 2013 Accepted 17 February 2014 Published 23 April 2014

Academic Editor Aristo Vojdani

Copyright copy 2014 Jennifer Ong et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Mercury (Hg) shown to induce autoimmune disease in rodents is a ubiquitous toxicant throughout Cheyenne River Sioux Tribe(CRST) lands CRST members may be exposed to Hg through fish consumption (FC) an important component of native culturethat may supplement household subsistence Our goals were to ascertain whether total blood Hg levels (THg) reflect Hg exposurethrough FC and smoking and determine whether THg is associated with the presence of anti-nuclear antibody (ANA) and specificautoantibodies (sAuAb)We recruited 75 participants who regularly consume fish fromCRSTwaters Hg exposure through FC andsmoking were assessed via questionnaires Whole blood samples were collected from participants and THg was measured usingICP-MS ANA and sAuAb in serumwere modeled using demographic and exposure information as predictors Female gender ageand FC were significant predictors of THg and sAuAb self-reported smoking was not 31 of participants tested positive for ANAge 2+ Although ANAwas not significantly associated with Hg the interactions of gender with Hg and proximity to arsenic depositswere statistically significant (119875 lt 005) FC resulted in a detectable body burden of Hg but THg alone did not correlate with thepresence of ANA or sAuAb in this population

1 Introduction

Formore than a century mining from greater than 900minesin the Black Hills including gold mines in which Hg wasused for amalgamation purposes has released contaminantsinto watersheds draining onto CRST lands [1] Addition-ally approximately one ton of airborne Hg is emitted peryear from coal power plants in Montana Wyoming NorthDakota and South Dakota [2] and carried downwind toCRST lands where precipitation and dust wash this mercuryout of the air into water and soilThus Hg is virtually ubiqui-tous throughout the CRST reservation Studies over the last

decade conducted by the tribe United States EnvironmentalProtection Agency (USEPA) and University of Colorado [3]have documented high mercury concentrations in mid-flowwater samples and sediment [4] invertebrates [5] and fish[5ndash7] As a result of the widespread presence of Hg in theenvironment fish consumption warnings have been postedalong the Cheyenne River since 1974 yet no comprehensivehealth studies have ever been conducted in the CRST pop-ulation to assess the health effects of consuming fish fromtribal waters In spite of postedwarnings CRSTmembers stillconsume locally caught fish for complex reasons Fishing andfish consumption are not only important in Lakota culture

Hindawi Publishing CorporationAutoimmune DiseasesVolume 2014 Article ID 325461 12 pageshttpdxdoiorg1011552014325461

2 Autoimmune Diseases

but high rates of poverty (sim50) [8 9] and unemployment(88) [10] on the CRST reservation increase the communityrsquoslikelihood of using fish to supplement household subsistenceTherefore the safety of eating mercury-contaminated fishcaught on tribal lands was a prime concern for CRSTmembers To address the CRSTrsquos environmental healthconcerns a research partnership Environmental Justice onCheyenne River was established in 2003 among the CRSTDepartment of Environment andNatural Resources (DENR)the Black Hills Center for American Indian Health andthe University of New Mexico Community EnvironmentalHealth Program (UNMCEHP)Through community forumsand discussions with tribal leaders the partnership identifieda major concern that a perceived increase in autoimmunedisease (AD) prevalence in the CRST population might berelated to Hg exposures through fish consumption as wellas a widespread frustration that actual health studies hadnot occurred in spite of Hg warnings posted for nearly 40years Although deidentified numbers of autoimmune caseswere obtained from IndianHealth Service (IHS) data sourcesinterpretation of the prevalence is difficult in identificationof an appropriate denominator and determination of anappropriate comparison figure for Native American popu-lations Data on antinuclear antibody (ANA) prevalence inNative populations has not been evaluated Prevalence ofANA in other US populations was recently derived fromNational Health and Nutrition Survey data (NHANES) [1112] but values for Native American populations could not beextracted due to no representation in that sample Referencevalues for specific AD in tribal populations relative to the UStotal population are also not readily accessible

Since tribal populations are comparativelymore homoge-nous than other studied US populations it may be tempt-ing to ascribe any elevations in AD in the CRST merelyto genetics However while genetic susceptibility has longbeen acknowledged as an important causative factor in thedevelopment of AD and evidence [13 14] exists that geneticcomposition may predispose CRST members to AD it isestimated that genetic factors only account for one-third ofdisease risk and that gene-environmental interactions playa vital role in the onset of autoimmunity [15] The growingrole of environmental factors including aluminum metalcompounds and thimerisol in vaccines as adjuvants to thepathogenesis of autoimmunity has been studied extensively[16] In addition studies [17 18] indicate that Hg toxicity andautoimmunity may be synergistically enhanced by variousinfectious and noninfectious triggers It is reasonable thatchronic stimulation of the immune system by environmentalHg may act through similar mechanisms To address thecommunityrsquos concerns and begin to address existing gapsin knowledge about the effects of chronic low-level envi-ronmental exposures to metals we sought to systematicallyexamine the relationships among fish consumption THgand basic immune system markers in the CRST populationin this study

Existing knowledge about the effects of metals on theimmune system comesmainly from the use of rodentmodelsIn these models relatively high doses of inorganic mercuryadministered to genetically susceptible mouse strains lead to

the development of lupus-like autoimmune syndrome whichincludes increased circulating antibodies to nuclear targets(antinuclear autoantibodies ANA) [16 17] Further exposureto inorganic or organic mercury exacerbates and acceler-ates the development of lupus-like disease in susceptiblemouse strains [18ndash21] Rodent models of mercury-inducedautoimmunity [22ndash24] as well as their consistency withsex differences in autoimmune disease incidence observedin humans suggest it is biologically plausible that Hg andother metals contribute to autoimmune pathogenesis inhumans Yet with the exception of a few epidemiologicstudies investigating the role of mercury amalgam fillings inmultiple sclerosis [25 26] and studies of ANA and cytokinesin mercury-exposed Amazonian Brazil populations [27ndash30]too few [31 32] have investigated the potential role of chronicenvironmental metal exposures as risk factors in the develop-ment of AD in humans While relationships between metalexposure and immune dysfunction have been demonstratedin animals limited data exist in humans Since Hg haslong been linked to development of AD-like symptoms inanimal models [17] we hypothesized that increased mercuryexposure primarily through fish consumption would beassociated with higher levels of circulating autoantibodiesin the CRST population In order to test this hypothesisand respond to community concerns we modeled ANA andspecific autoantibody concentrations in blood collected fromCRST community members using THg fish consumptionsmoking age gender and proximity to high-concentrationarsenic sediment deposits as predictors

2 Materials and Methods

21 Human Subjects The protocol and study design wereapproved by the Executive Committee of the CheyenneRiver Sioux Tribe Tribal Council (Tribal Resolution num-ber E-302-08-CR and extended under E-343-2009-CR) andby the University of New Mexico Health Sciences CenterHumanResearch ProtectionOffice (HRPOnumber 08-486)As deidentified serum samples were sent to the ScrippsResearch Institute Department ofMolecular and Experimen-tal Medicine the Scripps Research Institutersquos InstitutionalReview Board provided approval for an analysis of serumANA and specific autoantibodies

Participants were recruited by using community-basedcommunication tools and procedures previously developedby this team and applied in the Environmental Justice onCheyenne River study Outreach enrollment and samplingwere conducted in conjunction with local collaboratorsnotably Missouri Breaks Industries Research Inc (MBIRI)who were crucial contributors in several previous federallyfunded research projects among Cheyenne River Tribalcommunities and collaborating staff from the CRST DENRThe recruitment was targeted toward fishermen and theirfamily members who were known to local collaborators asregular consumers of fish caught from the Cheyenne Riverand its tributaries

Written informed consent was obtained from a total of75 adults living on the CRST Lands during the peak offishing seasonThe study population includes members from

Autoimmune Diseases 3

Bridger

Red Scaffold

TakiniCherry Creek

Dupree

Eagle Butte

Sampling community

Moreau River

Cheyenne River

Timber Lake

0

0

5

5 10

10

15

15

20

20

25 30

(miles)(km)

Sampling community in closeproximity to high-concentrationas sediment deposits

TN MN7∘

44∘3599840000998400998400N

44∘4199840000998400998400N

44∘4799840000998400998400N

44∘5399840000998400998400N

44∘5999840000998400998400N

45∘0599840000998400998400N

45∘1199840000998400998400N

45∘1799840000998400998400N

45∘2399840000998400998400N

44∘3599840000998400998400N

44∘4199840000998400998400N

44∘4799840000998400998400N

44∘5399840000998400998400N

44∘5999840000998400998400N

45∘0599840000998400998400N

45∘1199840000998400998400N

45∘1799840000998400998400N

45∘2399840000998400998400N

102∘ 05998400 00998400998400

W

101∘ 55998400 00998400998400

W

101∘ 45998400 00998400998400

W

101∘ 35998400 00998400998400

W

101∘ 25998400 00998400998400

W

101∘ 15998400 00998400998400

W

100∘ 55998400 00998400998400

W

100∘ 45998400 00998400998400

W

100∘ 35998400 00998400998400

W

100∘ 25998400 00998400998400

W

100∘ 07998400 00998400998400

W

WG

SB4

WG

SB4

102∘ 05998400 00998400998400

W

101∘ 55998400 00998400998400

W

101∘ 45998400 00998400998400

W

101∘ 35998400 00998400998400

W

101∘ 25998400 00998400998400

W

101∘ 15998400 00998400998400

W

101∘ 05998400 00998400998400

W101∘ 05998400 00998400998400

W

100∘ 55998400 00998400998400

W

100∘ 45998400 00998400998400

W

100∘ 35998400 00998400998400

W

100∘ 25998400 00998400998400

W

100∘ 07998400 00998400998400

W

091309

TOPO Map printed on 091309

Figure 1 Map of Cheyenne River Sioux Tribal lands and sampling communities

multiple communities including Eagle Butte Cherry CreekDupree Timber Lake Red Scaffold Bridger Takini andHowes (Figure 1) At each location enrollmentwas conductedand biological samples were collected in community centersThese communities some of which are in close proximity torivers lakes and ponds onCRST lands encompass both com-mercial centers and rural areas as well as members whoseprimary source of food is store-bought versus acquired fromthe local environment (subsistence lifestyle) and thereforereflect a wide range of potential exposures to Hg throughfish consumption Smoking status was a concern as analternate contributor to THg based on previosly reportedincreases in smoking on the CRST reservation [33] and thedemonstrated contributions to THg from cigarette smoking[34 35] MBIRI team interviewers collected demographic(eg age gender) health condition fishing and smokinghabit information through personal interviews conductedin English using a Centers for Disease Control- (CDC-)

developed fish consumption survey and our own shortsmoking exposure questionnaire When participants neededinformation or clarification spoken in their native languagethe community-certified nurse interviewers provided theanswers

22 Surveys Used in the Study

221 Fish Consumption A CDC questionnaire as well aslocal collaboratorsrsquo knowledge of CRST community mem-bersrsquo fishing habits was used to assign a categorical ratingof 1 2 or 3 to each participantrsquos fish consumption with 1designating minimal to no fish consumption and 3 corre-sponding to high fish consumption For reference to the localenvironment and consumption patterns the safe amount offish intake per month was previously recommended by ourEnvironmental Justice on Cheyenne River study using DENRHgmeasurements from local fish andUSEPA guidelines [37]


Cornfields picnic area Orchards

CeremonialGrounds

Roping Arena

SS-CR-BR-0811-01

SS-CR-BR-0811-04 SS-CR-BR-0811-02

SS-CR-BR-0811-03As = 121 (ppm)

As = 178 (ppm)SS-CR-DR-0811-02

SB-CR-BR-0811-02SB-CR-BR-0811-04

SS-CR-FR-0811-02SB-CR-FR-0811-01SS-CR-FR-0811-01

SS-CR-CCW-0811-01

SS-CR-CCR-0811-01

SS-CR-DR-0811-01

SB-CR-DR-0811-01SS-CR-CCR-0811-02

SS-CR-MN-0811-01

SB-CR-MN-0811-01

SS-CR-CRC-0811-04

SS-CR-CRC-0811-03

SS-CR-CRC-0811-02

SS-CR-CRC-0811-01

SS-CR-H63-0811-01

SS-CR-H63-0811-02SS-CR-MN-0811-02

As = 128 (ppm)

As = 135 (ppm)

As = 103 (ppm)

SB-CR-BR-0811-01As = 221 (ppm)

Figure 2 Map of arsenic sampling conducted by USEPA and CRST DENR Concentrations of arsenic in sediment exceeding 100 ppm aremarked with a burst pattern Exposure-relevant sites are labeled with activities frequently conducted in those areas

One monthly recommended serving was defined as onenorthern pike two bass or perch three walleye or fourcatfish A rating of (1) denotes consumption of lt1 servingof fish per month (2) denotes 1-2 servingsmonth and (3)denotes gt2 servingsmonth

222 Smoking To account for smoking as both a potentialsource of Hg and contributor to immune system effectsparticipant smoking data were collected via questionnaireThe questionnaire was based on coauthor PNHrsquos previouswork [33] on smoking among tribal members and wasgiven to all participants in order to obtain self-reportedinformation regarding smoking exposuresThequestionnaireencompassed both direct and second-hand exposure tocigarette smoke There were seven questions total smokingscore was coded as low (1) when fewer than two questionswere answered affirmatively medium (2) when 3-4 questionswere answered affirmatively and high (3) when greater thanfive questions were answered affirmatively A participant wasconsidered an ldquoactive smokerrdquo if heshe answered ldquoyesrdquo to theincluded question ldquoDo you smoke currentlyrdquo

223 Arsenic Proximity During the analytic phase of thisstudy elevated sedimentary arsenic deposits were discov-ered in land-use areas in close proximity to several of thesampling-site communities in this study (Figure 2) Ongoingcollaborations among DENR Dr Lewis and USEPA Region8 are surveying residents and characterizing exposure path-ways frequencies and duration However as these depositswere identified subsequent to consent for this study noarsenic biomonitoring data were obtained from the popu-lation in the original design nor were exposure activitiesinvolving these sedimentary deposits identified Due to stud-ies in humans and animals indicating that arsenic suppressesautoimmunity [38 39] while mercury may either suppress

or increase autoimmune response [28 32] a surrogate ofparticipant arsenic exposure was incorporated intomodels toaddress potentially competing exposures A binary surrogatefor arsenic exposure was derived the designations of ldquonearrdquoor ldquofarrdquo proximity to knownquantified environmental arsenicdeposits by USEPA were given according to self-reportedparticipant residence data The designation of ldquonearrdquo wasgiven to participants who live in the communities of CherryCreek Takini Bridger and Red Scaffold (Figure 1) Surveys ofresidents have identified potential exposure pathways whichinclude common land-use practices such as fishing herbfruit and firewood gathering inhalation ofwood combustionproducts during sweat lodge and ceremonial practices andropingother horseback riding activities along the CheyenneRiver near the identified alluvial arsenic deposits (Figure 2)(personal communication C Ducheneaux and J Lewis)Participants residing in the Eagle Butte Dupree and TimberLake (Figure 1) communities more distal to the arsenicdeposits were given a designation of ldquofarrdquo for arsenic prox-imity in this pilot assessmentThis binary variable was incor-porated to determine if further studies on the relationship ofthese exposures to AD were warranted

23 Biological Sample Collection

231 Blood and Serum Samples Venous blood samples werecollected by venipuncture at community centers or duringhome visits by a trained and certified phlebotomist orregistered nurse One red top (9mL) for serum collectionand one purple top (7mL) Vacutainer tube were collected forbiomonitoring from each participant After clotting serumsamples were spun at 2500 rpm for 10 minutes and separatedinto cryovials and placed into a minus80∘C freezer At a later timepoint sera were shipped to the UNM HSC laboratory andsubsequently to the Scripps Research Institute


(a) (b)

Figure 3 Examples of ANA determination by immunofluorescence Human sera were incubated with HEp-2 cells followed by fluorescentanti-human IgGThe sample on the left (a) is ANA negative while the sample on the right (b) was considered 2+ ANA positive showing finespeckled nuclear staining sparing the nucleolus

24 Experimental Use of Collected Biological Samples

241 Biomonitoring The EDTA-containing whole bloodsamples were transported to the CDC ONDIEHNCEHEnvironmental Health Laboratory where inductively coupledplasma mass spectrometry (ICP-MS) was used to determineTHg concentrations The limit of detection was 032 120583gL

242 Detection of Autoantibodies

(1) Antinuclear Antibodies (ANA) The presence of ANAwas determined by indirect immunofluorescence (IIF)microscopy using HEp-2 cells as substrate (MBL-BION DesPlaines IL) and Alexa Fluor 488 Goat Anti-Human IgG (H+ L) (Life Technologies NY USA) as detecting reagent Serawere diluted 1 100 in serum diluent and detecting reagent1 200 with anti-Ig diluent as previously described [40] Slideswere viewed by a single observer (KMP) blinded to par-ticipant identity on a BH2-RFCA fluorescence microscope(Olympus Lake Success NY) Intensity of fluorescence wasgraded on a scale of 0ndash4+ A reading of ge2+ was consideredsignificant and further used in our statistical modeling Thiscut-off value reflects a stricter value based on the literature[36 41] Example immunofluorescence images for ANAdetermination can be found in Figure 3 for negative (0) andANA ge 2+ readings

(2) Specific Autoantibodies (sAuAb) Commercially availablekits (INOVA Diagnostics San Diego CA) were used asdescribed by the manufacturer to detect and quantify serumautoantibodies to the following antigens chromatin SmRNP SSA SSA-52 SSB Scl-70 RNA Pol III CENP-ABRibo-P Jo-1 M2 EP (MIT3) and primary biliary cirrhosis(PBC) screen a panel of antigens (M2 EP gp210 and sp100IgGIgA) Assay-specific positive controls were used to con-vert optical density values to units in order to determinewhether the results of assays for Sm RNP SSA SSA-52 SSB

Scl-70 RNA Pol III Ribo-P and Jo-1 were negativeequivocal(lt20 units) weakly positive (20ndash39 units) moderately posi-tive (40ndash80 units) or strongly positive (gt80 units) The testsfor M2 EP and the PBC screen were interpreted as beingequivocal from 201 to 249 units and positive for gt25 unitsCentromere-AB (CENP-AB) has negativeequivocal resultsfor lt20 units weak positive for 20ndash30 units and strongpositive for gt30 units Chromatin has a negativeequivocalreading lt20 units moderate positive between 20 and 60units and a strong positive gt60 units

Additional assays to chromatin denatured DNA (single-stranded dDNA) native DNA (nDNA) and histones werequantified by enzyme-linked immunosorbent assays (ELISA)as previously described in [42 43] Briefly Immulon 2HBmicrotiter plates (Dynex Laboratories Inc Alexandria VA)were coated with antigen at 25 120583gmL concentrations Forthe antichromatin assays in-house-prepared H1-strippedchromatin was used as the solid-phase antigen S1-nuclease(Invitrogen-) treated DNA (Calbiochem) was used in theantinative DNA assay and DNA was heated for 10minand then quickly cooled for preparation of the dDNAantigen Prior to coating plates with nDNA or dDNAplates were pre-coated at 2 120583gmL with the synthetic 20ndash50kDA polypeptide poly(lys-phe) (Sigma-Aldrich) comprisinga co-polymer of lysine and phenylalanine at a 11 ratio aspreviously described [43] Total histone was from Wor-thington Serum samples were diluted 1 200 and incubatedon the plate for 2 hours at room temperature with gentleshaking Each sample was run in duplicate The boundantibodies were detected with peroxidase-conjugated anti-human IgG (Southern Biotech AL) and 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (MPBioproducts) as thesecondary substrate Optical densities (OD) beyond the rangeof direct measurement at 1 h in the ELISA were extrapolatedfrom OD at earlier time-points as described [44] Positiveand negative control sera were always included in each assayand values determined in different assays were normalized by


multiplying by the ratio of the reactivity of the positive controlsera tested in each assay

25 Statistical Analysis Modeling Total blood mercuryresults lower than the limit of detection (LOD = 032 120583gL)were analyzed with the value of LODradic2 because fewerthan 50 of participants had a biomonitoring value lt LODTotal blood mercury results are presented as median valueswith the interquartile range since the median is a betterindicator of the true population value for the distribution ofthe collected dataThemean and 95 confidence interval forTHg are also presented for ease of comparisonwith publishedNHANES population data When comparing groups (egmale versus female) in relation to THg and ANA statusFisherrsquos exact test was used

To characterize the complex exposures on CRST landsand their relationships to immune system responses andautoantibody production several statistical models incorpo-rating biomonitoring data fish consumption score smok-ing exposure score distance to arsenic contamination andimmune system markers were developed The approachesincluded multiple linear logistic and Poisson regressionmodels to evaluate relative contributions of environmentalexposures to circulating autoantibodies They also includedaccepted risk factors such as age and gender Multiple linearregression was used to model THg in relation to environ-mental exposure and risk factors while logistic regressionwas used to model ANA ge 2+ in relation to predictorsPoisson regression was used tomodel the numbers of specificautoantibodies (both determined via INOVA and additionalassays)with environmental exposure and risk factors Poissonmodels accommodate count information with nonnormaldistribution thereby enhancing the analytical capacity tounderstand the exposure factorsrsquo underlying contributionto risk Full models were fitted using all demographicbiomonitoring and exposure data as predictors Reducedmodels were selected using the Akaike information criterion(AIC) which is ameasure of the relative quality of a statisticalmodelThe openly available statistical software R [45] and thestepAIC function from the package MASS [46] were used tocomplete this AIC model selection where

AIC = 2119896 minus 2 ln (119871) (1)

And 119896 = 2 and 119871 is the likelihood of each model TheAIC selection criterion minimizes the distance between thepredicted values of the model and the true values while alsofavoring models with fewer parameters

251 Specific Approach to Analyze Specific AutoantibodyResults from INOVA Assays Since positivity for individual-specific autoantibodieswas expected to be lower in frequencywe pooled all participants who tested positive for any specificautoantibodies to examine an overall prevalence of specificautoantibodies and the contributing exposure factors bysumming the number of specific autoantibodies for whicheach participant tested positive and then conducting Poissonregression on the count variable generated Biomonitoringdata and exposure data were incorporated as well This count

variable makes biological sense because it follows establishedclinical ADdiagnosis criteria individuals diagnosedwithADpresent with varying combinations of specific autoantibodies

252 Specific Approach to Analyze Specific AutoantibodiesResults fromAdditional Autoantibody Assays Poisson regres-sion was used to model several combinations of autoantibod-ies that would otherwise be rarely detectedThe combinationsmodeled were selected in order to examine different possiblescenarios of positive autoantibody response Individuals withdetectable autoantibody response were classified into groupsaccording to the following scenarios based on the literature[42ndash44]

(a) presence of any autoantibody response (nDNAdDNA histone chromatin)

(b) detectable levels of potentially environmentallyrelated autoantibodies (dDNA and histone)

(c) disease-associated autoantibodies (nDNA and chro-matin)

Results of these models were summarized in several tablespresented in the next section

Because anti-chromatin autoantibodies were detectedusing both INOVA and in-house assays we evaluated thereproducibility of this antigen We applied a nonparametriccorrelation (Spearman 119903-value) and used a 119911-score

253 Reporting of Significant Results While our primaryresults will follow a more standard reporting cutoff of 119875 lt005 we will report those with probabilities up to 01 to guardagainst Type 2 error and to ensure comprehensive consid-eration of predictors in designing follow-up investigationsThis decision is warranted given (1) the importance of theresults to the communities (2) the lack of prior studies in thisarea and in this population and (3) the lack of biomonitoringdata at this time on other potential environmental exposuresincluding arsenic resulting in imprecise measures for thatvariable

3 Results

31 Mercury Exposure and Population Characteristics Popu-lation characteristics including gender smoking score fishconsumption score community size and proximity to iden-tified high-concentration sedimentary arsenic deposits aresummarized in Table 1 Total blood mercury concentrations(THg) ranged from below the limit of detection (LOD032 120583gHgL) to 414 120583gHgL with a median lower than theLOD (Figure 4) For most population characteristic cate-gories the median THg was below the LOD with the excep-tion of males (037 120583gHgL) and participants with ldquomediumrdquoor ldquohighrdquo fish consumption scores (035 and 054 120583gHgLresp)

Total blood mercury in the CRST depended on genderage and fish consumption but not smoking The reducedmultiple linear regression modeling results for THg as aresponse with demographic and exposure information as


Table 1 Biomonitoring and ANA ge2+ results linked with study participant characteristics

Population characteristic 119873Participants withTHg gtLODlowast

Hg biomarker median(interquartile range)

Hg biomarkermean (95 CI)

ANA readingge2+ (119899 )

All participants 75 36 (36) ltLOD (ltLODndash087) 075 (055ndash095) 23 (31)Gender

Male 38 23 (61) 037 (ltLODndash181) 101 (067ndash137) 2 (5)Female 37 13 (35) ltLOD (ltLODndash056) 048 (032ndash063) 9 (24)

Smoking score(1) Low 32 17 (53) 037 (ltLODndash074) 067 (041ndash092) 8 (25)(2) Medium 18 8 (44) ltLOD (ltLODndash124) 082 (036ndash128) 3 (17)(3) High 25 11 (44) ltLOD (ltLODndash121) 081 (039ndash123) 7 (28)

Active smokerYes 42 19 (45) ltLOD (ltLODndash096) 077 (049ndash105) 4 (10)No 31 16 (52) 037 (ltLODndash087) 076 (040ndash106) 7 (23)

Fish score(1) Low 41 17 (59) ltLOD (ltLODndash060) 059 (036ndash082) 7 (17)(2) Medium 18 10 (56) 035 (ltLODndash153) 090 (038ndash143) 2 (11)(3) High 16 9 (56) 054 (ltLODndash189) 099 (051ndash148) 2 (13)

Arsenic proximityYes 23 7 (30) ltLOD (ltLODndash052) 045 (025ndash064) 7 (30)No 52 29 (56) 037 (ltLODndash127) 089 (062ndash116) 4 (8)

lowastLOD = 032120583gL

4

3

2

1

0

1140956

Tota

l blo

od m

ercu

ry (T

Hg120583

gL)

Figure 4 Scatterplot of total blood mercury (THg) for samplepopulation with median denoted by an encircled ldquoXrdquo and meandenoted by an encircled cross The reference lines at 0956 120583gL and114120583gL indicate the 95 CI for THg in the US population fromNHANES [36]

predictors are summarized in Table 2 Male gender and olderage were significant predictors of THg (119875 = 00084 and0022 resp) fish consumption approached significance as apredictor for THg (119875 = 0053)

32 Prevalence of ANA in the CRST Population Anti-nuclearantibodies (ANA) were analyzed in serum samples from allparticipants Data are presented in Table 1 and representativeimages of negative and ANA ge 2+ readings are shown inFigure 3 Approximately thirty-one percent of participantshad an ANA reading of ge2+ For readings ge2+ ANAprevalence was significantly higher in women than in men

Table 2 Reduced model (multiple linear regression) for total bloodmercury

Estimate 119875 value Std errorIntercept 0412 046 056Genderlowast minus0545 00084 02Age 00181 0022 00077Smoking score minus00127 091 012Fish score 0246 0053 013Arsenic proximity minus0526 0020 022119875 values less than or equal to 005 are italicizedlowastMale gender was used as the reference so the estimate describes the effectof being a female

(24 versus 5 119875 = 0025) ANA prevalence was alsolarger in community members living in proximity to high-concentration sedimentary arsenic deposits (30 versus 8119875 = 0028)

Gender and fish consumption were significant predictorsof ANA ge2+ and gender modifies the effects of environ-mental exposures with respect to ANA The logistic regres-sion model information is shown in Table 3 Age and fishconsumption are borderline predictors (119875 lt 010) of ANAge 2+ (119875 = 0081 and 119875 = 0092 resp) with age andfish consumption positively associated with the probabilityof ANA ge 2+ level of circulating ANA Gender THg andproximity to arsenic by themselves do not strongly correlatewith the probability of ANA ge 2+ however the interactionsof gender with THg and arsenic proximity are significant and


160

140

120

100

80

60

40

20

0

Activ

ity u

nits

Autoantibodies to SSA and SSA52

SSA52SSA

80-strong

39-moderate20-weak

(a)

140

120

100

80

60

40

20

0

Activ

ity u

nits

Autoantibodies to CENP-AB

30-strong

20-weak

(b)

350

300

250

200

150

100

50

0

Activ

ity u

nits

25-strong

PBC ScreenM2 EP

Autoantibodies to M2 EP PBC screen antigens

(c)

Figure 5 Dot plots of detectable autoantibodies in participant serum measured in activity units with labels for clinical cutoffs Note Inclinical practice there are no ldquomoderaterdquo positive readings for CENP-AB there are no ldquoweakrdquo or ldquomoderaterdquo positive readings for M2 EPand the PBC Screen

Table 3 Point estimates and 95 confidence intervals (CI) forcoefficients and odds ratios (OR) for fitting logistic regressionmodels for ANA ge2+

OR 95 CI 119875 valueIntercept 0011 NA 093Age 11 096ndash117 0081Gender 24 0046ndash6455 037THG 04 0045ndash175 089Fish score 29 056ndash2070 0092Gender THG 138 097ndash4878 0026Gender fish score 01 00013ndash112 097Gender arsenic proximity 271 068ndash2101 0040Arsenic proximity 03 0015ndash426 082119875 values less than or equal to 005 are italicized

their odds ratios are greater than one (OR = 1383 119875 = 0026and OR = 2771 119875 = 004 resp)

33 Specific Autoantibodies in the CRST Population Of thespecific autoantibodies for which participant sera were testedSSA SSA52 CENP-AB M2 EP and the autoantibodiesdetected by the primary biliary cirrhosis (PBC) panel werenoteworthy These results are summarized in Figure 5 andTable 4 Fifteen percent of participants tested positive forautoantibodies toM2 EP while 24were positive for autoan-tibodies to the PBC panel

The number of specific autoantibodies detectable byINOVA kit increased with female gender and fish con-sumption score Information for the reduced Poisson modelfor the number of detectable specific autoantibodies usingINOVA assays can be found in Table 5(a) The number ofspecific autoantibodies detectable from INOVA assays wasassociated significantly with female gender (119875 = 00064)The model indicated that the mean number of specificautoantibodies detectable in serum is increased by a factorof 65 in female versus male community members Age andfish consumption had significant (119875 = 0012 and 119875 =00073 resp) but smaller effects on the number of specificautoantibodies in the collected serum samples In particular

the mean number of specific autoantibodies detectable byINOVA assay was 26 times greater in participants with ahigh (3) versus a low (1) fish consumption scoreThe numberof participants positive for autoantibodies to native DNAhistone and chromatin using in-house assays was small inour study No significant associations were found betweenany demographic or exposure predictors including smokingand various combinations of in-house autoantibodies exceptin the case of dDNA and histone The values for the reducedmodel can be seen in Table 5(b) Fish score was a significantpredictor (119875 = 0035) of the number of subjects with elevatedanti-dDNA and anti-histone autoantibodies detectable usingin-house assays An increase of one fish score categorypredicts a 25-factor increase in the number of dDNA andhistone autoantibodies Smoking was a borderline predictor(119875 = 0065) with a 04-factor decrease in the number ofdDNA and histone autoantibodies for an increase of onesmoking category

Antichromatin positivity and reproducibility betweenINOVA and additional assays were confirmed in all positiveserum samples (575) there was 100 agreement in detectionusing INOVA and in-house assays

4 Discussion

We assumed that due to consumption of locally caught fishcommunity members would have elevated levels of totalblood mercury (THg) We hypothesized that THg wouldcorrespond to an increased level of autoantibodies as hasbeen shown in animal models [17] Contrary to expectationsalthough Hg deposition in fish tissue had been documentedin CRST sources by DENR the detected THg levels inparticipants were low with a median THg lt LOD despitesampling during the middle of fishing season when fishconsumption was considered to be maximal The medianTHg for all participants (ltLOD)was lower than the publishedresults of the NHANES survey [47] NHANES reported amean THg of 0944 120583gL for those 12 years and older [47]Native American populations were not stratified in thatstudy the data were compiled under ldquootherrdquo ethnicity Thelow levels of blood mercury among the CRST members


Table 4 Results of selected specific autoantibody results from the CRST population sample

119899 = 75 Negative Moderate positive Strong positive Total positiveAutoantibodySSA 72 (96) 2 (3) 1 (13) 3 (4)SSA-52 70 (93) 2 (3) 3 (4) 5 (7)CENP-AB 72 (96) 0 (0) 3 (4) 3 (4)M2 EP 64 (85) 11 (15)PBC panel 57 (76) 18 (24)

Table 5 (a) Model (Poisson regression) for the number of detectedspecific autoantibodies using INOVA assays (b) Model (Poissonregression) for number of detected specific denatured DNA andhistone autoantibodies from in-house assays

(a)

Factor of change 95 CI 119875 valueIntercept 00 000ndash018 00012Genderlowast 65 169ndash2478 00064Age 10 101ndash104 0012Fish score 16 113ndash216 00073Smoking score 19 060ndash577 029Gender smoking score 05 025ndash101 0053119875 values less than or equal to 005 are italicized lowastMale gender was used asthe reference thus the factor of change reflects the effect of being female

(b)

Factor of change 95 CI 119875 valueIntercept 02 0004ndash9831 0406Age 1 0919ndash1055 0658Smoking 04 0127ndash1064 0065Fish score 25 1067ndash5980 0035Arsenic proximity 0 0000ndash3752 0117Age arsenic proximity 11 0984ndash1244 0092119875 values less than or equal to 005 are italicized

found in our study confirmed the THg levels reported in a2008 collaborative study between our team and CDC [4849] Potential reasons for the observed low THg includevariation due to raceethnicity and possible physiological andmetabolic changes among CRST community members orpossible alterations in deposition and clearance with repeatedexposure in this population

While gender age and fish consumption showed animpact on THg levels in the CRST population smokingdid not (Table 2) This finding is puzzling since 56 of theparticipants reported current smoking yet only 45 of thatgroup had THg above the detection level The trends in ourdata parallel those seen in the NHANES survey [50] Maleshave greater mean THg versus females and THg increaseswith age Males may consume larger quantities of locallycaught fish or engage in activities that increase dust andparticulate exposures to mercury (eg agricultural workhorse-tending)The age-dependent increase in THg found inthis study as well as in Wolkin et al [48] and the NHANES

survey [50] is likely due to the accumulation of metals in thebody over time

Thirty-one percent of participants had an ANA readingof ge2+ ANA production could be associated with chronictoxicant exposure which introduces self-antigens to antigenpresenting cells resulting in the breakdown of T-cell toler-ance While no single predictor was significantly associated(119875 lt 005) with ANA ge 2+ fish score was a borderlinepredictor (119875 = 0092) A larger proportion of ANA-positiveparticipants were female which concurs with the literatureand clinical findings about autoimmune diseases [24] andmay support a possible role for female hormones in ADand immune dysregulation Although THg and proximity tohigh-concentration arsenic deposits by themselves did notcorrelate with the probability of ANA ge 2+ the interactionsof female gender with THg and female gender with arsenicproximity are significant (119875 = 0026 and 119875 = 0040 resp)and the odds ratios were large (OR = 138 and 271 resp)Gender differences may reflect alterations in the molecularmechanisms by which gender-specific detoxification occurswithin the human body

Another interesting finding is that current smokers wereless likely to have ANA ge 2+ results (Table 1) Additionallythe specific autoantibody model estimates for smoking werenegative with ORs less than one (Tables 5(a) and 5(b))suggesting a protective effect of smoking The fact that fewerautoantibodies were detected in this subgroup of smokerssheds light on probable molecular mechanisms by whichsmoking induces immunosuppressive effects

There were significant associations between predictorexposure variables and the presence of autoantibodies todDNA and histone (Table 5(b)) This is potentially similarto previously observed instances of xenobiotic-induced anti-body responses such as drug-induced lupus [42] Autoanti-body production to dDNA and histone may also be linked toepigenetic changes triggered by environmental stimuli

The CRST population exhibited strong positivity for M2EP autoantibodies and autoantibodies detectable with thePBC screen both ofwhich are associatedwith liver diseases Itis possible that themedical problemof high rates of idiopathicliver cirrhosis in Sioux communities (personal communica-tion J Henderson) may have environmental etiology Similarfindings were reported among Alaskan Natives [51] As withANA ge 2+ models fish score was a significant predictor ofspecific autoantibodies using both detection methods (119875 lt001 for INOVA kit detection and 119875 lt 005 for in-houseELISA)


As to the specific mechanisms responsible for mercuryand metalmetalloid-induced autoimmune responses in theCRST population several mechanisms should be consid-ered In susceptible individuals environmental metals maybehave as adjuvants that prolong or enhance antigen-specificimmune response through various mechanisms such asmolecular mimicry [52] polyclonal activation of B cells [53]bystander activation [54] and epitope spreading [55] Addi-tionally chronic exposures to environmental metals includ-ing Hg and arsenic are well known to induce oxidative stressAs has been characterized with thimerisol [56] this oxidativestress could lead to sensitization of inositol 145-triphospate(IP3) receptors resulting in enhanced intracellular calciumrelease and subsequently the dysregulation of immune cellsand autoimmunity Another possibility includes the role ofchronic gut exposures to ingested dietary nanoparticles of soiland minerals which induce inflammasome production andthe breakdown of immune tolerance via enhanced gastroin-testinal antigen presentation Since fish consumption was animportant predictor of antibody production in this studydietary exposure may be one potential pathway throughwhich molecular markers of autoimmunity are generatedespecially among native community members who are morelikely to inhale and ingest large quantities of dust and metalsdue to their rural location cultural practices and subsistenceand agricultural activities

We hypothesize that fish consumption reflects multipleexposures including coexposures to mercury arsenic andother environmental toxicants such as pesticides pharma-ceuticals and infectious agents In animal and cell studiesHg toxicity and autoimmunity are synergistically enhancedby coexposure to additional xenobiotics These ideas willbe explored in future studies and additional activities thatincrease inadvertent exposure to toxicants will also beexamined Future studies will include a larger sample sizeparticipant AD medical record history and biomonitoringfor arsenic and cotinine (indicator of smoking exposures) inorder to address this studyrsquos limitations We also acknowl-edge that technical issues with indirect immunofluorescenceassays (IIFA) for the detection ofANA limit the comparabilityof these data to other population information and previouspublications However IIFA is the gold-standard techniquefor ANA detection [57] and we attempted to minimizevariability by using only one evaluator of staining (KMPcoauthor)

In this study compelling evidence that the CRST pop-ulation exhibited elevated levels of both ANA and specificautoantibodies was found The observed results highlightedenvironmental toxicants that may contribute to autoantibodyproduction in this population and also underscored the needto characterize the CRST communitiesrsquo lifestyles and behav-iors to better understand how complex exposures contributeto autoimmune health effects There is a large knowledge gapconcerning environmental influences on the developmentof AD and it is imperative that they be addressed withinthe context of environmental health disparities issues par-ticularly in tribal communities Information will empowerCRST community members and leaders by aiding them inmaking informed decisions about health health services the

environment and the preservation of their culture in whichfishing plays a vital role

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jennifer Ong and Esther Erdei contributed equally to thiswork

Acknowledgments

Colleen Martin and the Centers for Disease Control areacknowledged for their helpwith data collection andbiomon-itoring Dr Jeffrey A Henderson from the Black Hills Centerfor American Indian Health is acknowledged for his con-tinuing support close relationship with CRST communitymembers and clinical knowledge Esther Erdei receivedCommunity Engaged Research Pilot Grant from Universityof New Mexico (UNM) (2008) Center for ParticipatoryResearch under the Institute for Public Heath Departmentof Family and Community Medicine in partnership with theCommunity Engagement and Research Unit of the Clinicaland Translational Science Center (CTSC) Johnnye Lewisreceived theUniversity of New Mexico Clinical and Trans-lational Science Center Pilot Grant NIEHS R25 ES013292Environmental Justice on Cheyenne River (2010)

References

[1] H Kirkemo W L Newman and R P Ashley Gold USGeological Survey Denver Colo USA 2001

[2] S Vinyard and R Lauren ldquoDirty Energyrsquos Assault on OurHealth Mercuryrdquo 2011

[3] G C Bryner ldquoCoalbed methane development the costs andbenefits of an emerging energy resourcerdquo Natural ResourcesJournal vol 43 no 2 pp 519ndash560 2003

[4] TWMay R HWiedmeyer J Gober and S Larson ldquoInfluenceofmining-related activities on concentrations ofmetals inwaterand sediment from streams of the Black Hills South DakotardquoArchives of Environmental Contamination and Toxicology vol40 no 1 pp 1ndash9 2001

[5] J BigEagle ldquoDevelopment processes of consumption advisoriesfor the Cheyenne River Sioux Indian Reservationrdquo in Proceed-ings of theNational Forum onContaminants in Fish pp 142ndash144Baltimore Md USA 2005

[6] J M Johnston D Hoff R Hoogerheide R Edgar D Walland C Ducheneaux Mercury Risk Management in LivestockPonds on the Cheyenne River Sioux Reservation Science ForumWashington DC USA 2003

[7] A T Byrne ldquoFish Consumption Survey for the Cheyenne RiverBasin within the Cheyenne River Indian Reservation SouthDakotardquo Eagle Butte SD USA 2002

[8] US Census Bureau ldquoDistribution of Income by Family andHouseholdrdquo 2000

[9] US Department of Health ampHuman Services ldquoPoverty Guide-lines Research and Measurement ndrdquo


[10] United States Department of the Interior Bureau of IndianAffairs Office of Indian Services ldquo2005 American Indian Popu-lation and Labor Force Reportrdquo 2005

[11] M Satoh E K L Chan L A Ho et al ldquoPrevalence andsociodemographic correlates of antinuclear antibodies in theUnited Statesrdquo Arthritis amp Rheumatism vol 64 no 7 pp 2319ndash2327 2012

[12] C M Gallagher A E McElroy D M Smith M G Golightlyand J R Meliker ldquoPolychlorinated biphenyls mercury andantinuclear antibody positivity NHANES 2003-2004rdquo Interna-tional Journal of Hygiene and Environmental Health vol 216 no6 pp 721ndash727 2013

[13] M S Leffell M D Fallin W H Hildebrand J W Cavett B AIglehart andA A Zachary ldquoHLA alleles and haplotypes amongthe lakota sioux report of the ASHI minority workshops partIIIrdquo Human Immunology vol 65 no 1 pp 78ndash89 2004

[14] R C Williams L T H Jacobsson W C Knowler et al ldquoMeta-analysis reveals association between most common class IIhaplotype in full-heritage native Americans and rheumatoidarthritisrdquo Human Immunology vol 42 no 1 pp 90ndash94 1995

[15] J Heward and S C Gough ldquoGenetic susceptibility to thedevelopment of autoimmune diseaserdquo Clinical Science vol 93no 6 pp 479ndash491 1997

[16] K M Pollard P Hultman and D H Kono ldquoImmunologyand genetics of induced systemic autoimmunityrdquoAutoimmunityReviews vol 4 no 5 pp 282ndash288 2005

[17] D Germolec D H Kono J C Pfau and KM Pollard ldquoAnimalmodels used to examine the role of the environment in thedevelopment of autoimmune disease findings from an NIEHSExpert Panel Workshoprdquo Journal of Autoimmunity vol 39 no4 pp 285ndash293 2012

[18] S Havarinasab and P Hultman ldquoAlteration of the spontaneoussystemic autoimmune disease in (NZB times NZW)F1 mice bytreatment with thimerosal (ethyl mercury)rdquo Toxicology andApplied Pharmacology vol 214 no 1 pp 43ndash54 2006

[19] P Hultman A Taylor J M Yang and K M Pollard ldquoTheeffect of xenobiotic exposure on spontaneous autoimmunityin (SWR times SJL)F1 hybrid micerdquo Journal of Toxicology andEnvironmental Health A vol 69 no 6 pp 505ndash523 2006

[20] KM Pollard D L Pearson P Hultman T N Deane U Lindhand D H Kono ldquoXenobiotic acceleration of idiopathic system-atic autoimmunity in lupus-prone BXSB micerdquo EnvironmentalHealth Perspectives vol 109 no 1 pp 27ndash33 2001

[21] K M Pollard D L Pearson P Hultman B Hildebrandt andD H Kono ldquoLupus-prone mice as models to study xenobiotic-induced acceleration of systemic autoimmunityrdquoEnvironmentalHealth Perspectives vol 107 supplement 5 pp 729ndash735 1999

[22] D Fairweather S Frisancho-Kiss and N R Rose ldquoSex differ-ences in autoimmune disease from a pathological perspectiverdquoThe American Journal of Pathology vol 173 no 3 pp 600ndash6092008

[23] J B Nielsen and P Hultman ldquoMercury-induced autoimmunityin micerdquo Environmental Health Perspectives vol 110 no 5 pp877ndash881 2002

[24] K M Pollard ldquoGender differences in autoimmunity associatedwith exposure to environmental factorsrdquo Journal of Autoimmu-nity vol 38 no 2-3 pp J177ndashJ186 2012

[25] K K Aminzadeh and M Etminan ldquoDental amalgam and mul-tiple sclerosis a systematic review and meta-analysisrdquo Journalof Public Health Dentistry vol 67 no 1 pp 64ndash66 2007

[26] M N Bates J Fawcett N Garrett T Cutress and T KjellstromldquoHealth effects of dental amalgam exposure a retrospectivecohort studyrdquo International Journal of Epidemiology vol 33 no4 pp 894ndash902 2004

[27] J F Nyland M Fillion F Barbosa Jr et al ldquoBiomarkersof methylmercury exposure immunotoxicity among fish con-sumers in amazonian Brazilrdquo Environmental Health Perspec-tives vol 119 no 12 pp 1733ndash1738 2011

[28] R M Gardner J F Nyland I A Silva A M Ventura JM de Souza and E K Silbergeld ldquoMercury exposure serumantinuclearantinucleolar antibodies and serum cytokine levelsin mining populations in Amazonian Brazil a cross-sectionalstudyrdquo Environmental Research vol 110 no 4 pp 345ndash3542010

[29] E K Silbergeld I A Silva and J F Nyland ldquoMercury andautoimmunity implications for occupational and environmen-tal healthrdquo Toxicology and Applied Pharmacology vol 207 no2 supplement pp 282ndash292 2005

[30] I A Silva J F Nyland A Gorman et al ldquoMercury exposuremalaria and serum antinuclearantinucleolar antibodies inamazon populations in Brazil a cross-sectional studyrdquo Environ-mental Health A Global Access Science Source vol 3 article 112004

[31] R M Gardner J F Nyland and E K Silbergeld ldquoDifferentialimmunotoxic effects of inorganic and organic mercury speciesin vitrordquo Toxicology Letters vol 198 no 2 pp 182ndash190 2010

[32] R M Gardner J F Nyland S L Evans et al ldquoMercury inducesan unopposed inflammatory response in human peripheralblood mononuclear cells in vitrordquo Environmental Health Per-spectives vol 117 no 12 pp 1932ndash1938 2009

[33] P N Henderson S Kanekar Y Wen et al ldquoPatterns of cigarettesmoking initiation in two culturally distinct American Indiantribesrdquo American Journal of Public Health vol 99 no 11 pp2020ndash2025 2009

[34] F Pragst and S Leo ldquoSmoking trials with mercury contam-inated cigarettes studies of attempted poisoningrdquo Archiv furKriminologie vol 188 no 3-4 pp 77ndash86 1991

[35] M R Fresquez R S Pappas and C HWatson ldquoEstablishmentof toxic metal reference range in tobacco from US cigarettesrdquoJournal of Analytical Toxicology vol 37 no 5 pp 298ndash304 2013

[36] J S Smolen B Butcher M J Fritzler et al ldquoReference serafor antinuclear antibodies II Further definition of antibodyspecificities in international antinuclear antibody referencesera by immunofluorescence and western blottingrdquo Arthritis ampRheumatism vol 40 no 3 pp 413ndash418 1997

[37] United States Environmental Protection Agency ldquoConsump-tion Advice Joint Federal Advisory for Mercury in Fish ndrdquo

[38] A S Andrew D A Jewell R A Mason M L Whitfield J HMoore and M R Karagas ldquoDrinking-water arsenic exposuremodulates gene expression in human lymphocytes from a USpopulationrdquo Environmental Health Perspectives vol 116 no 4pp 524ndash531 2008

[39] R Biswas P Ghosh N Banerjee et al ldquoAnalysis of T-cellproliferation and cytokine secretion in the individuals exposedto arsenicrdquo Human and Experimental Toxicology vol 27 no 5pp 381ndash386 2008

[40] J-M Yang B Hildebrandt C Luderschmidt andKM PollardldquoHuman scleroderma sera contain autoantibodies to proteincomponents specific to the U3 small nucleolar RNP complexrdquoArthritis amp Rheumatism vol 48 no 1 pp 210ndash217 2003


[41] E M Tan T E W Feltkamp J S Smolen et al ldquoRange ofantinuclear antibodies in ldquohealthyrdquo individualsrdquo Arthritis ampRheumatism vol 40 no 9 pp 1601ndash1611 1997

[42] R L Rubin ldquoDrug-induced lupusrdquo Toxicology vol 209 no 2pp 135ndash147 2005

[43] R W Burlingame and R L Rubin ldquoSubnucleosome structuresas substrates in enzyme-linked immunosorbent assaysrdquo Journalof Immunological Methods vol 134 no 2 pp 187ndash199 1990

[44] G Schett J Smolen C Zimmermann et al ldquoThe autoimmuneresponse to chromatin antigens in systemic lupus erythemato-sus autoantibodies against histone H1 are a highly specificmarker for SLE associated with increased disease activityrdquoLupus vol 11 no 11 pp 704ndash715 2002

[45] R Core Team R A Language and Environment for StatisticalComputing R Foundation for Statistical Computing ViennaAustria 2013

[46] B D Ripley B Venables D M Bates K Hornik A Gebhardtand D Firth ldquoSupport Functions andDatasets for Venables andRipleyrsquos MASSrdquo 2013

[47] Centers for Disease Control ldquoFourth National Report onHuman Exposure to Environmental Chemicals UpdatedTables September 2013rdquo 2013

[48] A Wolkin D Hunt C Martin K L Caldwell and M AMcGeehin ldquoBloodmercury levels among fish consumers resid-ing in areas with high environmental burdenrdquo Chemospherevol 86 no 9 pp 967ndash971 2012

[49] C Martin A Wolkin C Ducheneaux J Lewis and J Hender-son Blood Mercury (Hg) Levels Among Consumers of LocallyCaught Fish Residing on the Cheyenne River Sioux Tribe (CRST)Reservation American Public Health Association 2009

[50] K L Caldwell M E Mortensen R L Jones S P Caudill and JD Osterloh ldquoTotal blood mercury concentrations in the USpopulation 1999ndash2006rdquo International Journal of Hygiene andEnvironmental Health vol 212 no 6 pp 588ndash598 2009

[51] K J Hurlburt B J McMahon H Deubner B Hsu-TrawinskiJ L Williams and K V Kowdley ldquoPrevalence of autoimmuneliver disease in Alaska nativesrdquo The American Journal of Gas-troenterology vol 97 no 9 pp 2402ndash2407 2002

[52] E Israeli N Agmon-Levin M Blank and Y ShoenfeldldquoAdjuvants and autoimmunityrdquo Lupus vol 18 no 13 pp 1217ndash1225 2009

[53] O Barzilai M Ram and Y Shoenfeld ldquoViral infection caninduce the production of autoantibodiesrdquo Current Opinion inRheumatology vol 19 no 6 pp 636ndash643 2007

[54] K Murali-Krishna J D Altman M Suresh et al ldquoCountingantigen-specific CD8 T cells a reevaluation of bystander activa-tion during viral infectionrdquo Immunity vol 8 no 2 pp 177ndash1871998

[55] P V Lehmanann T Forthuber A Miller and E E SercazldquoSpreading of T-cell autoimmunity to cryptic determinants ofan antigenrdquo Nature vol 358 no 6382 pp 155ndash157 1992

[56] D Yeter and R Deth ldquoITPKC susceptibility in Kawasakisyndrome as a sensitizing factor for autoimmunity and coro-nary arterial wall relaxation induced by thimerosalrsquos effects oncalcium signaling via IP3rdquo Autoimmunity Reviews vol 11 no12 pp 903ndash908 2012

[57] N Agmon-Levin J Damoiseaux C Kallenberg U Sack TWitte M Herold et al ldquoInternational recommendations for theassessment of autoantibodies to cellular antigens referred to asanti-nuclear antibodiesrdquo Annals of the Rheumatic Diseases vol73 no 1 pp 17ndash23 2014

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


but high rates of poverty (sim50) [8 9] and unemployment(88) [10] on the CRST reservation increase the communityrsquoslikelihood of using fish to supplement household subsistenceTherefore the safety of eating mercury-contaminated fishcaught on tribal lands was a prime concern for CRSTmembers To address the CRSTrsquos environmental healthconcerns a research partnership Environmental Justice onCheyenne River was established in 2003 among the CRSTDepartment of Environment andNatural Resources (DENR)the Black Hills Center for American Indian Health andthe University of New Mexico Community EnvironmentalHealth Program (UNMCEHP)Through community forumsand discussions with tribal leaders the partnership identifieda major concern that a perceived increase in autoimmunedisease (AD) prevalence in the CRST population might berelated to Hg exposures through fish consumption as wellas a widespread frustration that actual health studies hadnot occurred in spite of Hg warnings posted for nearly 40years Although deidentified numbers of autoimmune caseswere obtained from IndianHealth Service (IHS) data sourcesinterpretation of the prevalence is difficult in identificationof an appropriate denominator and determination of anappropriate comparison figure for Native American popu-lations Data on antinuclear antibody (ANA) prevalence inNative populations has not been evaluated Prevalence ofANA in other US populations was recently derived fromNational Health and Nutrition Survey data (NHANES) [1112] but values for Native American populations could not beextracted due to no representation in that sample Referencevalues for specific AD in tribal populations relative to the UStotal population are also not readily accessible

Since tribal populations are comparativelymore homoge-nous than other studied US populations it may be tempt-ing to ascribe any elevations in AD in the CRST merelyto genetics However while genetic susceptibility has longbeen acknowledged as an important causative factor in thedevelopment of AD and evidence [13 14] exists that geneticcomposition may predispose CRST members to AD it isestimated that genetic factors only account for one-third ofdisease risk and that gene-environmental interactions playa vital role in the onset of autoimmunity [15] The growingrole of environmental factors including aluminum metalcompounds and thimerisol in vaccines as adjuvants to thepathogenesis of autoimmunity has been studied extensively[16] In addition studies [17 18] indicate that Hg toxicity andautoimmunity may be synergistically enhanced by variousinfectious and noninfectious triggers It is reasonable thatchronic stimulation of the immune system by environmentalHg may act through similar mechanisms To address thecommunityrsquos concerns and begin to address existing gapsin knowledge about the effects of chronic low-level envi-ronmental exposures to metals we sought to systematicallyexamine the relationships among fish consumption THgand basic immune system markers in the CRST populationin this study

Existing knowledge about the effects of metals on theimmune system comesmainly from the use of rodentmodelsIn these models relatively high doses of inorganic mercuryadministered to genetically susceptible mouse strains lead to

the development of lupus-like autoimmune syndrome whichincludes increased circulating antibodies to nuclear targets(antinuclear autoantibodies ANA) [16 17] Further exposureto inorganic or organic mercury exacerbates and acceler-ates the development of lupus-like disease in susceptiblemouse strains [18ndash21] Rodent models of mercury-inducedautoimmunity [22ndash24] as well as their consistency withsex differences in autoimmune disease incidence observedin humans suggest it is biologically plausible that Hg andother metals contribute to autoimmune pathogenesis inhumans Yet with the exception of a few epidemiologicstudies investigating the role of mercury amalgam fillings inmultiple sclerosis [25 26] and studies of ANA and cytokinesin mercury-exposed Amazonian Brazil populations [27ndash30]too few [31 32] have investigated the potential role of chronicenvironmental metal exposures as risk factors in the develop-ment of AD in humans While relationships between metalexposure and immune dysfunction have been demonstratedin animals limited data exist in humans Since Hg haslong been linked to development of AD-like symptoms inanimal models [17] we hypothesized that increased mercuryexposure primarily through fish consumption would beassociated with higher levels of circulating autoantibodiesin the CRST population In order to test this hypothesisand respond to community concerns we modeled ANA andspecific autoantibody concentrations in blood collected fromCRST community members using THg fish consumptionsmoking age gender and proximity to high-concentrationarsenic sediment deposits as predictors

2 Materials and Methods

21 Human Subjects The protocol and study design wereapproved by the Executive Committee of the CheyenneRiver Sioux Tribe Tribal Council (Tribal Resolution num-ber E-302-08-CR and extended under E-343-2009-CR) andby the University of New Mexico Health Sciences CenterHumanResearch ProtectionOffice (HRPOnumber 08-486)As deidentified serum samples were sent to the ScrippsResearch Institute Department ofMolecular and Experimen-tal Medicine the Scripps Research Institutersquos InstitutionalReview Board provided approval for an analysis of serumANA and specific autoantibodies

Participants were recruited by using community-basedcommunication tools and procedures previously developedby this team and applied in the Environmental Justice onCheyenne River study Outreach enrollment and samplingwere conducted in conjunction with local collaboratorsnotably Missouri Breaks Industries Research Inc (MBIRI)who were crucial contributors in several previous federallyfunded research projects among Cheyenne River Tribalcommunities and collaborating staff from the CRST DENRThe recruitment was targeted toward fishermen and theirfamily members who were known to local collaborators asregular consumers of fish caught from the Cheyenne Riverand its tributaries

Written informed consent was obtained from a total of75 adults living on the CRST Lands during the peak offishing seasonThe study population includes members from


Bridger

Red Scaffold

TakiniCherry Creek

Dupree

Eagle Butte

Sampling community

Moreau River

Cheyenne River

Timber Lake

0

0

5

5 10

10

15

15

20

20

25 30

(miles)(km)


TN MN7∘

44∘3599840000998400998400N

44∘4199840000998400998400N

44∘4799840000998400998400N

44∘5399840000998400998400N

44∘5999840000998400998400N

45∘0599840000998400998400N

45∘1199840000998400998400N

45∘1799840000998400998400N

45∘2399840000998400998400N

44∘3599840000998400998400N

44∘4199840000998400998400N

44∘4799840000998400998400N

44∘5399840000998400998400N

44∘5999840000998400998400N

45∘0599840000998400998400N

45∘1199840000998400998400N

45∘1799840000998400998400N

45∘2399840000998400998400N

102∘ 05998400 00998400998400

W

101∘ 55998400 00998400998400

W

101∘ 45998400 00998400998400

W

101∘ 35998400 00998400998400

W

101∘ 25998400 00998400998400

W

101∘ 15998400 00998400998400

W

100∘ 55998400 00998400998400

W

100∘ 45998400 00998400998400

W

100∘ 35998400 00998400998400

W

100∘ 25998400 00998400998400

W

100∘ 07998400 00998400998400

W

WG

SB4

WG

SB4

102∘ 05998400 00998400998400

W

101∘ 55998400 00998400998400

W

101∘ 45998400 00998400998400

W

101∘ 35998400 00998400998400

W

101∘ 25998400 00998400998400

W

101∘ 15998400 00998400998400

W

101∘ 05998400 00998400998400

W101∘ 05998400 00998400998400

W

100∘ 55998400 00998400998400

W

100∘ 45998400 00998400998400

W

100∘ 35998400 00998400998400

W

100∘ 25998400 00998400998400

W

100∘ 07998400 00998400998400

W

091309









CeremonialGrounds

Roping Arena

SS-CR-BR-0811-01

SS-CR-BR-0811-04 SS-CR-BR-0811-02

SS-CR-BR-0811-03As = 121 (ppm)

As = 178 (ppm)SS-CR-DR-0811-02

SB-CR-BR-0811-02SB-CR-BR-0811-04


SS-CR-CCW-0811-01

SS-CR-CCR-0811-01

SS-CR-DR-0811-01

SB-CR-DR-0811-01SS-CR-CCR-0811-02

SS-CR-MN-0811-01

SB-CR-MN-0811-01

SS-CR-CRC-0811-04

SS-CR-CRC-0811-03

SS-CR-CRC-0811-02

SS-CR-CRC-0811-01

SS-CR-H63-0811-01

SS-CR-H63-0811-02SS-CR-MN-0811-02

As = 128 (ppm)

As = 135 (ppm)

As = 103 (ppm)

SB-CR-BR-0811-01As = 221 (ppm)









(a) (b)













AIC = 2119896 minus 2 ln (119871) (1)











3 Results
















4

3

2

1

0

1140956

Tota

l blo

od m

ercu

ry (T

Hg120583

gL)









160

140

120

100

80

60

40

20

0

Activ

ity u

nits


SSA52SSA

80-strong

39-moderate20-weak

(a)

140

120

100

80

60

40

20

0

Activ

ity u

nits


30-strong

20-weak

(b)

350

300

250

200

150

100

50

0

Activ

ity u

nits

25-strong

PBC ScreenM2 EP


(c)









4 Discussion






(a)


(b)


















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Bridger

Red Scaffold

TakiniCherry Creek

Dupree

Eagle Butte

Sampling community

Moreau River

Cheyenne River

Timber Lake

0

0

5

5 10

10

15

15

20

20

25 30

(miles)(km)


TN MN7∘

44∘3599840000998400998400N

44∘4199840000998400998400N

44∘4799840000998400998400N

44∘5399840000998400998400N

44∘5999840000998400998400N

45∘0599840000998400998400N

45∘1199840000998400998400N

45∘1799840000998400998400N

45∘2399840000998400998400N

44∘3599840000998400998400N

44∘4199840000998400998400N

44∘4799840000998400998400N

44∘5399840000998400998400N

44∘5999840000998400998400N

45∘0599840000998400998400N

45∘1199840000998400998400N

45∘1799840000998400998400N

45∘2399840000998400998400N

102∘ 05998400 00998400998400

W

101∘ 55998400 00998400998400

W

101∘ 45998400 00998400998400

W

101∘ 35998400 00998400998400

W

101∘ 25998400 00998400998400

W

101∘ 15998400 00998400998400

W

100∘ 55998400 00998400998400

W

100∘ 45998400 00998400998400

W

100∘ 35998400 00998400998400

W

100∘ 25998400 00998400998400

W

100∘ 07998400 00998400998400

W

WG

SB4

WG

SB4

102∘ 05998400 00998400998400

W

101∘ 55998400 00998400998400

W

101∘ 45998400 00998400998400

W

101∘ 35998400 00998400998400

W

101∘ 25998400 00998400998400

W

101∘ 15998400 00998400998400

W

101∘ 05998400 00998400998400

W101∘ 05998400 00998400998400

W

100∘ 55998400 00998400998400

W

100∘ 45998400 00998400998400

W

100∘ 35998400 00998400998400

W

100∘ 25998400 00998400998400

W

100∘ 07998400 00998400998400

W

091309









CeremonialGrounds

Roping Arena

SS-CR-BR-0811-01

SS-CR-BR-0811-04 SS-CR-BR-0811-02

SS-CR-BR-0811-03As = 121 (ppm)

As = 178 (ppm)SS-CR-DR-0811-02

SB-CR-BR-0811-02SB-CR-BR-0811-04


SS-CR-CCW-0811-01

SS-CR-CCR-0811-01

SS-CR-DR-0811-01

SB-CR-DR-0811-01SS-CR-CCR-0811-02

SS-CR-MN-0811-01

SB-CR-MN-0811-01

SS-CR-CRC-0811-04

SS-CR-CRC-0811-03

SS-CR-CRC-0811-02

SS-CR-CRC-0811-01

SS-CR-H63-0811-01

SS-CR-H63-0811-02SS-CR-MN-0811-02

As = 128 (ppm)

As = 135 (ppm)

As = 103 (ppm)

SB-CR-BR-0811-01As = 221 (ppm)









(a) (b)













AIC = 2119896 minus 2 ln (119871) (1)











3 Results
















4

3

2

1

0

1140956

Tota

l blo

od m

ercu

ry (T

Hg120583

gL)









160

140

120

100

80

60

40

20

0

Activ

ity u

nits


SSA52SSA

80-strong

39-moderate20-weak

(a)

140

120

100

80

60

40

20

0

Activ

ity u

nits


30-strong

20-weak

(b)

350

300

250

200

150

100

50

0

Activ

ity u

nits

25-strong

PBC ScreenM2 EP


(c)









4 Discussion






(a)


(b)


















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















CeremonialGrounds

Roping Arena

SS-CR-BR-0811-01

SS-CR-BR-0811-04 SS-CR-BR-0811-02

SS-CR-BR-0811-03As = 121 (ppm)

As = 178 (ppm)SS-CR-DR-0811-02

SB-CR-BR-0811-02SB-CR-BR-0811-04


SS-CR-CCW-0811-01

SS-CR-CCR-0811-01

SS-CR-DR-0811-01

SB-CR-DR-0811-01SS-CR-CCR-0811-02

SS-CR-MN-0811-01

SB-CR-MN-0811-01

SS-CR-CRC-0811-04

SS-CR-CRC-0811-03

SS-CR-CRC-0811-02

SS-CR-CRC-0811-01

SS-CR-H63-0811-01

SS-CR-H63-0811-02SS-CR-MN-0811-02

As = 128 (ppm)

As = 135 (ppm)

As = 103 (ppm)

SB-CR-BR-0811-01As = 221 (ppm)









(a) (b)













AIC = 2119896 minus 2 ln (119871) (1)











3 Results
















4

3

2

1

0

1140956

Tota

l blo

od m

ercu

ry (T

Hg120583

gL)









160

140

120

100

80

60

40

20

0

Activ

ity u

nits


SSA52SSA

80-strong

39-moderate20-weak

(a)

140

120

100

80

60

40

20

0

Activ

ity u

nits


30-strong

20-weak

(b)

350

300

250

200

150

100

50

0

Activ

ity u

nits

25-strong

PBC ScreenM2 EP


(c)









4 Discussion






(a)


(b)


















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)













AIC = 2119896 minus 2 ln (119871) (1)











3 Results
















4

3

2

1

0

1140956

Tota

l blo

od m

ercu

ry (T

Hg120583

gL)









160

140

120

100

80

60

40

20

0

Activ

ity u

nits


SSA52SSA

80-strong

39-moderate20-weak

(a)

140

120

100

80

60

40

20

0

Activ

ity u

nits


30-strong

20-weak

(b)

350

300

250

200

150

100

50

0

Activ

ity u

nits

25-strong

PBC ScreenM2 EP


(c)









4 Discussion






(a)


(b)


















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















AIC = 2119896 minus 2 ln (119871) (1)











3 Results
















4

3

2

1

0

1140956

Tota

l blo

od m

ercu

ry (T

Hg120583

gL)









160

140

120

100

80

60

40

20

0

Activ

ity u

nits


SSA52SSA

80-strong

39-moderate20-weak

(a)

140

120

100

80

60

40

20

0

Activ

ity u

nits


30-strong

20-weak

(b)

350

300

250

200

150

100

50

0

Activ

ity u

nits

25-strong

PBC ScreenM2 EP


(c)









4 Discussion






(a)


(b)


















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























4

3

2

1

0

1140956

Tota

l blo

od m

ercu

ry (T

Hg120583

gL)









160

140

120

100

80

60

40

20

0

Activ

ity u

nits


SSA52SSA

80-strong

39-moderate20-weak

(a)

140

120

100

80

60

40

20

0

Activ

ity u

nits


30-strong

20-weak

(b)

350

300

250

200

150

100

50

0

Activ

ity u

nits

25-strong

PBC ScreenM2 EP


(c)









4 Discussion






(a)


(b)


















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















160

140

120

100

80

60

40

20

0

Activ

ity u

nits


SSA52SSA

80-strong

39-moderate20-weak

(a)

140

120

100

80

60

40

20

0

Activ

ity u

nits


30-strong

20-weak

(b)

350

300

250

200

150

100

50

0

Activ

ity u

nits

25-strong

PBC ScreenM2 EP


(c)









4 Discussion






(a)


(b)


















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















(a)


(b)


















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article mercury, autoimmunity, and environmental...

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