please i am ganna post the article here and if you have note please let me know becuase this is big assighnment. check the citation and the reference. please do what ever u can to make this assighnment good. Thank you.<br />DDT Exposure in Utero and Breast Cancer<br />Barbara A. Cohn, Michele La Merrill, Nickilou Y. Krigbaum, Gregory Yeh, June-Soo Park, Lauren Zimmermann, and Piera M. Cirillo<br />Child Health and Development Studies (B.A.C., N.Y.K., L.Z., P.M.C.), Public Health Institute, Berkeley, California 94709; Department of Environmental Toxicology (M.L.M.), University of California, Davis, California 95616; Environmental Chemistry Laboratory (G.Y., J.-S.P.), California Department of Toxic Substances Control, Berkeley, California 94710; and Public Health Institute (G.Y.), Oakland, California 94607<br />Context:Currentlynodirectevidencelinksinuterodichlorodiphenyltrichloroethane(DDT)exposure to human breast cancer. However, in utero exposure to another xenoestrogen, diethylstilbestrol, predicts an increased breast cancer risk. If this finding extends to DDT, it could have far-reaching consequences. Many women were heavily exposed in utero during widespread DDT use in the 1960s. They are now reaching the age of heightened breast cancer risk. DDT exposure persistsandusecontinuesinAfricaandAsiawithoutclearknowledgeoftheconsequencesforthe next generation.<br />Hypothesis: In utero exposure to DDT is associated with an increased risk of breast cancer.<br />Design:Thiswasacase-controlstudynestedinaprospective54-yearfollow-upof9300daughters in the Child Health and Development Studies pregnancy cohort (n  118 breast cancer cases, diagnosed by age 52 y and 354 controls matched on birth year).<br />Setting and Participants: Kaiser Foundation Health Plan members who received obstetric care in AlamedaCounty,California,from1959to1967,andtheiradultdaughtersparticipatedinthestudy.<br />MainOutcomeMeasure:Daughters’breastcancerdiagnosedbyage52yearsasof2012wasmeasured.<br />Results:Maternalo,p-DDTpredicteddaughters’breastcancer(oddsratiofourthquartilevsfirst 3.7,95%confidenceinterval1.5–9.0).Mothers’lipids,weight,race,age,andbreastcancerhistory did not explain the findings.<br />Conclusions:ThisprospectivehumanstudylinksmeasuredDDTexposureinuterotoriskofbreast cancer.Experimentalstudiesareessentialtoconfirmresultsanddiscovercausalmechanisms.Findings support classification of DDT as an endocrine disruptor, a predictor of breast cancer, and a marker of high risk. (J Clin Endocrinol Metab 100: 2865–2872, 2015) Diethylstilbestrol (DES) is a synthetic estrogen, which wasprescribedtopregnantwomenuntilitwasbanned in the United States in 1971 and is a seminal example of a transplacentalcarcinogen(1).ThediscoverythatDESexposure in utero causes clear-cell carcinoma of the vagina and cervix(2)andalsopredictshigherriskforbreastcancer(3) raisesthepossibilitythatotherman-madechemicals,particularlythosethatdisruptnormalestrogen-relatedfunctions, couldcausebreastcancerinlaterlife.AlthoughDES(4)and otherexogenousestrogenicchemicals(5,6)havebeenshown tocausemammarycancerexperimentally,nootherinutero chemical exposures have been quantified and related prospectivelytoriskofbreastcancerinahumanpopulation.The present study addresses this gap. Theinfluenceoftheseinuteroexposuresincancerrisk informed an Endocrine Society scientific statement em<br />ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2015 by the Endocrine Society Received April 2, 2015. Accepted June 1, 2015. First Published Online June 16, 2015<br />Abbreviations: CCR, California Cancer Registry; CERLab, Clinical and Epidemiologic ResearchLaboratory;CHDS,ChildHealthandDevelopmentStudies;CI,confidenceinterval; DDE, dichlorodiphenyldichloroethylene; DDT, dichlorodiphenyltrichloroethane; DDTs, DDTcompoundssuchastheisomersp,p-DDTando,p-DDT;DES,diethylstilbestrol;DMV, Department of Motor Vehicles; OR, odds ratio.<br />O R I G I N A L A R T I C L E<br />doi: 10.1210/jc.2015-1841 J Clin Endocrinol Metab, August 2015, 100(8):2865–2872 press.endocrine.org/journal/jcem 2865<br />The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 September 2015. at 09:39 For personal use only. No other uses without permission. . All rights reserved.<br />phasizing that the timing of an exposure either to a hormoneoranendocrinedisruptordeterminesitseffects(7). For this reason, human studies of adult exposure to estrogenicchemicalsinrelationtobreastcancerriskarenot sufficient(8,9).Indeed,priorstudiesthatinvestigatedthe association between midlife exposure to the pesticide dichlorodiphenyltrichloroethane (DDT) and breast cancer were largely negative (10). Wereportedtheonepriorprospectiveandquantitative study to consider the implications of young age at DDT exposure on breast cancer risk. This study was based on incidentbreastcancerdiagnosedbeforeage50yearsinthe mothers’generationoftheChildHealthandDevelopment Studies (CHDS) pregnancy cohort over a 17-year follow-up(11).Weusedagein1945,whenDDTwaswidely introducedintotheUnitedStatesasaproxyforearliestage at DDT exposure to evaluate whether there was an increasedsusceptibilitytobreastcanceramongwomenwith early life exposure to DDT. We found that mothers who were exposed to DDT prior to age 14 years showed a 5-fold increase in risk of breast cancer (66th percentile of serump,p-DDTcomparedwiththe33rdpercentile);risk wasgreatestforwomenwhowereexposedevenearlier,by age 4 years, and the DDT association with breast cancer wasobservedonlyforwomenwhowereexposedpriorto age 14 years (P  .02 for interaction between p,p-DDT and age in 1945). These findings clearly support the need to further investigate the role of early-life DDT exposure in later breast cancer risk. Other than the present study, there is an absence of human breast cancer studies of in uteroexposuretotheDDTs(DDTcompoundssuchasthe isomers p,p-DDT and o,p-DDT that are found in technicalDDTandalsocompoundsthatarebreakdownproducts of DDT such as dichlorodiphenyldichloroethylene (DDE), the most prevalent and persistent metabolite of p,p-DDT)(12)dueinparttothelogisticbarriersofquantitative assessment of gestational exposure coupled with follow-up of at least 50 years needed to identify breast cancercases(13),giventhatthemedianageatdiagnosisis 61 years (14). DDTremainsrelevanttolivingpopulationsfornumerous reasons. First, most women born while DDT was extensivelyusedworldwidearestillalivetodayandarehence at risk for breast cancer. Second, DDT remains in active useforcontrolofmalariainAfricaandAsiainaccordance with World Health Organization recommendations despite intense debate (15, 16). Third, because of its persistent presence in the environment, people worldwide continuetobeexposedtoDDTsthatarealreadypresent(17). Environmental contamination and human exposure are greaterwhereusewasrecentandwheresaferuse,storage, and disposal are challenging (18), such as in China (19)<br />andareasinMexico(20,21)andAfrica(22,23).Indeed, DDThealtheffectswillremainrelevantfortheforeseeable future, given that the distribution of malaria vectors is predicted to expand with climate change (24). Melting glaciers release DDTs into arctic waters in which it is knowntobioaccumulateto1million-foldhigherconcentrations in people, reaching levels found among populations with endemic malaria in tropical regions in which DDT use remains in effect (25, 26). Hereweconductedthefirstprospectivestudytorelate quantitativemeasuresofinuteroDDTexposuretoriskof breast cancer in daughters.<br />Materials and Methods<br />Subjects Thisuniquestudyismadepossiblebya54-yearfollow-upof 20 754pregnancies,resultingin9300live-bornfemaleoffspring in the CHDS pregnancy cohort. TheCHDSwasdesignedtoexaminetheassociationbetween prenatal exposures and health and development over the life course for parents and children. The CHDS recruited women residing in the area of Oakland, California, who were members oftheKaiserFoundationHealthPlanandreceivedobstetriccare forpregnanciesbetween1959and1967(27).Morethan98%of alleligiblewomenenrolled.CHDSfoundingmothersvoluntarily participated in an in-person interview and gave permission to researchers for medical record access for themselves and their children. Their blood specimens were collected at several times through pregnancy and 1–3 days after delivery. The present study was reviewed and approved by the Institutional Review Board of the Public Health Institute (Oakland, California), and we have complied with all federal guidelines governing use of human subjects.<br />Breast cancer cases AllmembersoftheCHDScohortarelinkedtotheCalifornia DepartmentofMotorVehicles(DMV)filesonaregularbasisto determine California residence history, allowing us to update anynamechanges.AllnamesregisteredwiththeDMVareused inestablishingamatch.Simultaneouslinkageofmultiplefamily members enhances matching. The regular DMV matching provides a history of location for each subject, which is used to determinethepopulationatriskforcancer,correspondingwith geographic surveillance by California’s cancer registries. Breast cancer cases were identified by linkage to the California Cancer Registry and the California Vital Status Records as previouslydescribed(11,28)andbyself-reportduringasurvey ofCHDSdaughtersconductedfrom2010to2013.Allnamesfor each CHDS subject were submitted for cancer linkages using fixed (ie, birth date, sex, race, and name) and changeable (ie, address and patient record number) identifiers. A rigorous protocol was used to verify cases, comparing fixed vs changeable identifiers by manual review. The California Cancer Registry (CCR) is reported to be greater than 99% complete after a lag time of about 2 years (29). We ascertained 80% (n  94) of the cases via CCR linkage as of 2012 and 20% (n  24) via selfreport as of 2013. Due to the CCR lag time, the self-reported<br />2866 Cohn et al In Utero DDT and Breast Cancer J Clin Endocrinol Metab, August 2015, 100(8):2865–2872<br />The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 September 2015. at 09:39 For personal use only. No other uses without permission. . All rights reserved.<br />casesaremorerecentlydiagnosedthanthosefromCCR.Tumor characteristicswereavailablefor87%ofCCRcasesandfor50% of self-reported cases. Thus, we expect to have more complete informationontumorpathologywithcontinuingCCRlinkage. CasesweredefinedasCHDSdaughterswithincidentinvasive or noninvasive breast cancer diagnosed by age 52 years, identifiedthroughsurveillanceandthroughself-reportthroughMarch of 2013. There were 137 cases who met this case definition, diagnosedasof2012.Tobeincludedinthepresentstudy,cases were required to have a maternal perinatal blood sample for measurement of DDT exposure, resulting in inclusion of 118 cases(86%).Threecontrols,matchedonbirthyearandtrimester of maternal blood draw, were selected at random for each case from among those who were under cancer surveillance and known to be free of breast cancer at the age of diagnosis for the matchingcase.Inclusioninthepresentstudyalsorequiredavailabledataforthefollowingvariablesknowntobecorrelatedwith DDT exposure and potentially daughters’ breast cancer: maternallipids,age,race,earlypregnancyweight,height,andhistory of breast cancer and whether the daughter was breast-fed, resulting in a final sample size of 103 cases (87% of those with serum samples) and 315 controls (83% of those with serum samples).<br />Serum assays In 2014, we measured DDTs and serum lipids in nonfasting maternal perinatal serum samples that had been collected from 1959 through 1967. The mean age of subjects when blood was drawnwas26.9years.Wepreferredtousetheearlypostpartum samples(collectedwithin1–3dafterdelivery)whenavailableto conserveserumforfuturestudieswhentimingwithinpregnancy is more critical. Early postpartum samples were available and used for most case-control strata (77.8%); third-trimester samples were used for 17.7%; second-trimester samples were used for3.8%;andfirst-trimestersampleswereusedfor0.7%.Prior work has established that organochlorine levels are consistent acrossalltrimestersofpregnancyandsoonafterdeliverywithin women(30).Serumsampleshadbeenstoredat20ºCandwere firstthawedtoprepareanaliquotof1.5mLfororganochlorine assays.Aliquotswerethenshippedfrozentothelaboratoryofthe CaliforniaDepartmentofToxicSubstancesControlwherethey wereassayedforDDTs,includingp,p-DDTando,p-DDT,the primaryconstituentsoftechnicalDDT,andtheprimarymetaboliteofp,p-DDT,DDEusingmethodsdevelopedpreviously(31). Briefly, human serum samples (1 mL) spiked with surrogate standards(tetrachloro-m-xylene,polychlorinatedbiphenyls-14, -65,and-166)weredenaturedwithformicacid,extractedusing Oasis HLB SPE cartridges (Waters Corp) and subsequently cleaned up with 33% sulfuric acid silica using an automated sample extraction system (RapidTrace; Biotage). DDT compounds(o,p-DDT,p,p-DDT,andp,p-DDE)wereanalyzedon a DB-5ms column (30 m  0.25 mm inner diameter, 0.25  m film thickness; Agilent Technologies) installed in an Agilent gas chromatograph-tandem mass spectrometer (7890/7000B series). Chromatographic conditions included pulsed splitless injection at 250°C and helium carrier gas at 1 mL/min. The gas chromatographtemperatureprogramstartedwithaninitialtemperature of 90°C, a hold for 1 minute, a ramp of 50°C/min to 150°C,aholdfor1minute,arampof8°C/minto225°C,ahold for 6.5 minutes, and a final ramp of 14°C/min to 310°C, a hold for 6 minutes. The mass spectrometer was operated in electron<br />impact ionization mode using multiple reaction monitoring, source temperature of 275°C, ionization energy of 70 eV, and massresolutionof1.2amu.Acalibrationcurve,consistingoffive to eight standards with concentrations ranging from 0.1 to 30 pg/  L(DDTs)orfrom0.1to800pg/  L(DDE)andanR2 value of 0.990 or greater, was used for quantitation. Eachbatchof10sampleswasanalyzedusingastandardqualityassessmentandcontrolprotocol:alaboratorymethodblank (HyClonebovineserum;FisherScientific),amatrixspikeinbovine serum, and a standard reference material (1958; National InstituteofStandardsandTechnology).Matrixspikerecoveries frombovineserumforp,p-DDE,p,p-DDT,ando,p-DDTcongeners ranged from 93%  14%, 102%  19%, and 101%  12%, respectively. Precisions from 51 standard reference material samples were reasonable, eg, coefficients of variation were 12%, 19%, and 14% for p,p-DDE, p,p-DDT, and o,p-DDT, respectively. The method detection limits, calculated as 3 times the SD of the concentrations in method blanks (n  51), were 0.013 ng/mL o,p-DDT, 0.054 ng/mL p,p-DDT and 0.158 ng/mL p,p-DDE. Sample order was randomly assigned within and across batches. Case-control strata were analyzed in the samebatchestominimizedifferencesduetolaboratorydrift.The laboratorywasblindastocaseorcontrolstatusofthesamples. Interbatchandintrabatchcoefficientsofvariationwere15%and 5%forp,p-DDT,6%and5%forp,p-DDE,and11%and7% for o,p-DDT, respectively. The proficiency of the laboratory is demonstrated through regular successful participation in the ArcticMonitoringandAssessmentProgram(32)testingrounds (z-score  1). Using 150  L undiluted serum, total cholesterol and triglyceridesweremeasuredenzymaticallyonaRochePModularsystemusingreagentsandcalibratorsfromRocheDiagnosticsatthe Clinical and Epidemiologic Research Laboratory (CERLab) at BostonChildren’sHospital,whichiscertifiedbytheCentersfor Disease Control and Prevention/National Heart, Lung, and Blood Institute Lipid Standardization Program. For cholesterol the method combines the specificity of the enzymatic reaction with peroxidase/phenol-4-aminophenazone indicator reaction (33).Cholesterolestersarehydrolyzedbycholesterolesteraseto produce free cholesterol. In the presence of oxygen and cholesterol oxidase, cholesterol is oxidized to cholest-4-en-3-one and H2O2. The latter product then reacts with a dye to generate a quinoneimine dye. The intensity of the generated color is measuredat505nmandisdirectlyproportionaltotheconcentration ofcholesterolinthemeasuredsample.Atcholesterolconcentrations of 132.8 and 280.4 mg/dL, the day-to-day reproducibility in the CERlab, reflected by coefficient of variation, is 1.7% (SD  2.4 mg/dL) and 1.6%, respectively (n  693). Triglycerides were measured enzymatically with correction for endogenous glycerol (34). In a preliminary reaction, the endogenous glycerol is phosphorylated in the presence of glycerol kinaseandATP.Theformedglycerol-3-phosphateisoxidizedto generateH2O2,whichreactswith4-chlorophenoltoproducean oxidative product. Then in the actual assay reaction, triglycerides are hydrolyzed by lipase mixture to generate glycerol and fattyacids.Similarlytothepreliminaryreaction,glycerolisphosphorylated by the action of glycerol kinase and the generated glycerol-3-phosphate is oxidized to produce H2O2. The latter product reacts with a dye to generate a colored product. The intensity of the generated color is measured at 505 nm and is directlyproportionaltotheconcentrationoftriglyceridesinthe measured sample. Triglycerides at concentrations of 84.0 and<br />doi: 10.1210/jc.2015-1841 press.endocrine.org/journal/jcem 2867<br />The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 September 2015. at 09:39 For personal use only. No other uses without permission. . All rights reserved.<br />201.8mg/dLaredeterminedinintheCERLabwithaday-to-day reproducibility of 1.8% (SD 1.6 mg/dL) and 1.7% (SD 3.5 mg/ dL), respectively (n  675).<br />Statistical analysis Data analyses were performed using age-matched, conditional logistic regression. Each DDT variable (o,p-DDT, p,pDDT, and p,p-DDE) was categorized in quartiles of the logged distributionbasedonthecontrolpopulationandrepresentedas three nominal variables in models: quartile 2, quartile 3, and quartile 4 in which quartile 1 was the reference category. We used a likelihood ratio criterion (P  .05) to choose the bestmodelfromamongthefollowingnestedmodels:1)allthree DDT compounds were entered into the model, 2) terms for one of the three compounds was deleted beginning with the compoundshowingthehighestPvaluesandsmallesteffectsizes,and 3) models with only one DDT compound included. Trends acrossquartilesofDDTcompoundsinthebestmodelweretested using natural log-transformed continuous variables. All models were adjusted for serum total cholesterol and total triglycerides enteredasnaturallogs,maternalage(continuous),race(African American vs non-African American), overweight in early pregnancy(codedasoverweightvsnot,basedonabodymassindex 25 kg/m2), parity (primiparous vs multiparous), maternal history of breast cancer (yes vs no), and whether the daughter was breast-fed.Thefinalmodeldeletedadjustmentvariablesthathad littleornoinfluenceontheDDTpredictorsasevidencedbylessthan 10% change in the DDT coefficient(s) when removed from the model.Saturatedinteractionmodelswerealsotestedforthethree DDTvariables(codedascontinuouslogtransformedvariables)and for the DDT variables in the final model with each potential confounder. These were not significant and are not reported.<br />Results<br />Distributions of study variables are shown in Table 1. There were significant correlations among the DDT vari<br />ables. However, the correlation of o,p-DDT with p,pDDT was higher (Spearman rank correlation coefficient 0.78) than the correlation of either of these compounds withp,p-DDE(0.60and0.66,respectively).Comparison ofDDTassociationsinnestedmodelsareshowninTable 2, in which the best model included o,p-DDT and p,pDDE (model 4, Table 2). Independent of a maternal history of breast cancer, elevated maternal serum o,p-DDT significantly predicted a nearly 4-fold increase in the daughter’sriskofbreastcancer(Table3).Maternaloverweightinearlypregnancywasassociatedwithalowerrisk ofbreastcancerindaughtersbutwithmarginalstatistical significance. Maternal lipids, age, race, and parity and whether the daughter was breast-fed did not confound these findings and were not significant predictors of daughters’ breast cancer. No interactions were statistically significant. Most cases in this cohort were estrogen receptor positive (83%), progesterone receptor positive (76%), and HER2-negative (74%). In human breast cell lines, DDT activates HER2, a clinically relevant protein expressed in some breast cancers (35, 36). Therefore, we evaluated whether in utero DDT exposure was associated with HER2-positive breast cancers in this cohort of women with available stage at diagnosis (73%) and available HER2 status (59%). We found that o,p-DDT was significantly, positively associated with advanced stage at diagnosis (regional or distantdiseasevslocalorinsitu)andwiththeoccurrence of HER2-positive tumors, independent of maternal overweight, history of breast cancer, and p,p-DDE. Despite small sample sizes for these analyses, 22 advanced-stage tumors and 16 HER2-positive tumors, respectively, re<br />Table 1. Maternal Variables by Daughters’ Breast Cancer Status<br />Controls (n  315) Cases (n  103)<br />Percentile Percentile Maternal Variables 25th 50th 75th 25th 50th 75th Age, y 22 26 30 23 27 31 o,p-DDT, ng/mL 0.27 0.46 0.78 0.32 0.52 1.06 p,p-DDT, ng/mL 8.38 12.98 18.57 9.47 13.18 20.11 p,p-DDE, ng/mL 29.29 42.81 57.56 30.91 43.13 58.08 Cholesterol, mg/dL 182 222 278 188 227 283 Triglycerides, mg/dL 139 183 227 136 182 227 Percent n Percent n History of breast cancera 4.44 14 20.39 21 Primipara 29.84 94 26.21 27 African-American 25.08 79 23.30 24 Overweight (BMI  25 kg/m2) 25.08 79 17.48 18 Breast-fed her daughter 26.03 82 31.07 32 Abbreviation: BMI, body mass index. a P  .0001 for difference between controls and cases.<br />2868 Cohn et al In Utero DDT and Breast Cancer J Clin Endocrinol Metab, August 2015, 100(8):2865–2872<br />The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 September 2015. at 09:39 For personal use only. No other uses without permission. . All rights reserved.<br />sults were statistically significant. The estimated odds ratio(OR)fordiagnosisatanadvancedstagewas2.2[95% confidence interval (CI) 1.1–4.2, P  .02] for a doubling of o,p-DDT. The estimated OR for a HER2-positive tu<br />morwas2.2(95%CI1.0–4.6,P.04)foradoublingof o,p-DDT. Levels of o,p-DDT not only doubled but also tripledforwomeninthefourthquartileofthestudycompared with those in the first quartile (as seen in Table 1). Thecorrespondingriskofadvanced-stageandHER2-positivebreastcancerforthesewomenismorethan4-fold(for latestage,OR4.6;95%CI1.3–16.6forthefourthquartile of o,p-DDT vs the first quartile; for HER2 positive, OR 4.6, 95% CI 1.1–19.7). These results suggest a strong effect of in utero o,p-DDT on breast cancer stage, and HER2statusinthispopulation,andtherelevanceofthese findings is discussed below.<br />Discussion<br />Strengths Exposure timing Human and animal evidence establish the existence of developmental windows when the breast is more vulnerabletoxenoestrogens,suchasDDT(9).Oneofthesewindows is in utero. The present study is the first to quantify exposuretoDDTinuteroandlinkittosubsequentbreast cancer risk.<br />Quantifying exposure DDT was introduced in the general population in the United States in 1945 and was most heavily used worldwide in the late 1950’s and 1960’s (37). The CHDS enrolled pregnancies during 1959–1967, which, by coincidence, covered the years of highest DDT exposure (26, 38).Consistentwiththesedata,theCHDSmaternalserum samples, which were collected during the peak years of DDT use in the United States, show the highest levels of DDT compared with other studies of breast cancer in<br />Table 2. DDT Results for Nested Models<br />o,p-DDT p,p-DDT p,p-DDE<br />OR OR OR<br />Univariate models Model 1, o,p-DDT Quartile 2 1.8 Quartile 3 1.6 Quartile 4 2.8a P trend 0.054 Model 2, p,p-DDT Quartile 2 1.9 Quartile 3 1.5 Quartile 4 2.2b P trend 0.074 Model 3, p,p-DDE Quartile 2 1.3 Quartile 3 1.1 Quartile 4 1.3 P trend NS Co-adjusted models Model 4, o,p-DDT plus p,p-DDE Quartile 2 2.0 1.0 Quartile 3 1.8 0.7 Quartile 4 3.7c 0.7 P trend 0.048 NS Model 5, p,p-DDT plus p,p-DDE Quartile 2 2.0 1.1 Quartile 3 1.7 0.8 Quartile 4 2.9d 0.8 P trend 0.037 NS Model 6, o,p-DDT plus p,p-DDT plus p,p-DDE Quartile 2 1.9 1.7 1.0 Quartile 3 1.7 1.2 0.7 Quartile 4 3.5e 1.4 0.6 P trend NS NS NS Abbreviation: NS, not significant p  .15. All models are adjusted for maternal cholesterol and triglycerides, maternal overweight in early pregnancy, and maternal history of breast cancer. P trend is based on continuous log-transformed DDTs. Model 4, containing o,p-DDT and p,p-DDE, is chosen as the best model. The addition of p,p-DDT does not make a significant, independent contribution to risk beyond that shown for o,p-DDT (compare Model 4 with Model 6). This is likely due to the high correlation between o,p-DDT and p,p-DDT (see text). The association for o,p-DDT is partly masked by p,p-DDE (compare Model 1 with Model 4), which is why both terms are retained in the best model. Although p,p-DDE is not itself significant, it is inversely associated with daughters’ breast cancer while being positively correlated with o,p-DDT. a P  .007. b P  .058. c P  .004. d P  .054. e P  .019.<br />Table 3. Maternal Predictors of Daughters’ Breast Cancer<br />OR 95% CI<br />P Value History of breast cancera 6.4 2.8–14.3 .0001 Overweight in early pregnancya 0.6 0.3–1.1 .077 o,p-DDTb Quartile 2 2.0 0.9–4.3 .083 Quartile 3 1.8 0.8–4.0 .160 Quartile 4 3.7 1.5–9.0 .004 P trend .048<br />Quartile cut points are given in Table 1. This table is based on a single model adjusted for variables shown and also adjusted for the maternal serum p,p-DDE, total cholesterol, and total triglycerides, which were not significant predictors. Test for trend was based on log-transformed o,p-DDT entered as a continuous variable instead of quartile variables. a Yes versus No. b Quartile 1 is reference category.<br />doi: 10.1210/jc.2015-1841 press.endocrine.org/journal/jcem 2869<br />The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 September 2015. at 09:39 For personal use only. No other uses without permission. . All rights reserved.<br />which samples were collected in later decades (11, 13). Thus, the present study uses unique historic samples to quantify typical in utero exposure when DDT use was at its peak. Our laboratory methods were able to quantify threeDDTtargetcompoundsinallsamples,includingthe lower concentration o,p-DDT, a 15%–20% contaminant of technical DDT. This strength is due to state-ofthe-art laboratory methods, which optimized the detection of all compounds, but is also due to collection of serum samples in the 1960’s during a time period when there was high use of technical DDT.<br />Relevance to countries in which DDT is now banned DDTwasbannedinthe1970’sintheUnitedStatesand westernEurope(39).However,thewomenexposedmost heavily while in utero during the 1960’s are currently reaching the age of heightened breast cancer risk. Recent cases of breast cancer in daughters (F1) in the CHDS cohort represent the leading edge of birth cohorts heavily exposedtoDDTwheninutero.Thus,thefindingsofthis present study are relevant to breast cancer, even in countriesinwhichDDTisnotcurrentlyused.Inaddition,DDT remains a global environmental contaminant due to its environmental persistence and semivolatility (25).<br />Relevance to countries in which DDT is still used In those countries that continue to use DDT to control malaria,humanexposureremainshigh(26).Thus,thefindingsofthisstudyarerelevanttocurrentpopulationsinwhich highinuteroexposureiscertainlyoccurring,suchasinSouth Africa(23).TheimpactofDDTuseonunborngenerations hasbeenrecentlyraisedasanethicalconsideration(40).Our findings are relevant to this debate.<br />Plausibility DDTs have been studied as possible endocrine disruptors on the basis of observed deleterious reproductive effects in wildlife (39, 41) and based on demonstrated endocrine-active effects including estrogen activity, particularlyforo,p-DDT(42,43).Remarkably,independent of estrogen, there is likely a biological basis for the putative association of in utero DDT and HER2-positive cancers(36,44,45).Forexample,low-doseo,p-DDT(1 nM) enhanced the tyrosine kinase activity of HER2 in human MCF-7 breast cancer cells irrespective of tamoxifen exposure or estrogen depletion of culture media (36, 45).o,p-DDTalsogaverisetoincreasedMCF-7foci(abnormal concentric piling up of cells in postconfluent cultures) (45). The effects of o,p-DDT on tyrosine kinase activityofHER2andMCF7fociformationwereblocked by a mononuclear antibody specific to HER2. These experimental results are consistent with the hypothesis this<br />paper supports that o,p-DDT can cause human mammary tumor formation that depends on the activation of HER2 (36, 45). Although this hypothesized effect of in uteroDDTexposureonmammarycancerhasnotyetbeen tested experimentally in vivo, when exposure to its metabolitep,p-DDEwasinitiatedatweaning,thelatencyof HER2-positive mouse mammary tumors was shortened (46). Our nested analysis also suggested that high exposurewassignificantlyassociatedwithadvancedstage.Although no in vivo studies have evaluated this, a recent study of human breast cancer cell lines suggested o,pDDT caused estrogen-dependent invasion (47). PrioritizingtheacquisitionoftumorblocksandtheirimmunohistochemicalanalysisofestrogenreceptorandHER2inthe ongoing CHDS and in parallel experimental models will likely aid in resolving which receptor is the mechanistic target of in utero DDT on mammary carcinogenesis. DES is the most well-studied perinatal xenoestrogen exposure in humans and is known to be associated with increasedriskofbreastcancerinbothF0(exposedinpregnancy)(48)andF1(exposedinutero)(3).SimilartoDES, we found that maternal serum p,p-DDT was associated with an increased risk of breast cancer in CHDS mothers (F0)(11).Weusedbirthyearasaproxyforearliestageat DDT exposure and found the strongest association was observed in mothers who were initially exposed to DDT beforetheageof4years(11).Thepresentstudyprovides a direct, quantitative measure of exposure in utero for daughters(F1)intheCHDSandalsofindsanassociation between in utero DDT exposure and breast cancer.<br />Limitations Outcome window Thepresentstudyinvestigatesbreastcancerdiagnosed before age 52 years. Thus, these results do not address DDT associations with breast cancer diagnosed at a later age. One prior study of dioxin exposure to girls and women after a chemical explosion in Seveso, Italy, reported a significant association for breast cancers diagnosed within the first 20 years, which then declined over the subsequent 10 years of follow-up (49). Continuing follow-up in our cohort will be required to determine whetherinuteroDDTassociationswithbreastcancerare observed for cases diagnosed in the future.<br />Exposure window Whereas we have measured exposure directly relevant tofetallife,wecannotruleoutacontributionofpostnatal exposure. We did not observe a contribution or synergy with breast-feeding, even though this is a major maternal route of DDT exposure to offspring. However, this negative association may be partly explained by the low fre<br />2870 Cohn et al In Utero DDT and Breast Cancer J Clin Endocrinol Metab, August 2015, 100(8):2865–2872<br />The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 September 2015. at 09:39 For personal use only. No other uses without permission. . All rights reserved.<br />quency of breast-feeding in the CHDS and the relatively small sample size.<br />Unmeasured confounders We cannot rule out a contribution of other exposures that are correlated to DDT, including other unmeasured DDT metabolites. However, we were able to account for the DDTs that are present in the highest concentration in women.<br />Small sample This study investigates the first cases observed in CHDS daughters.Althoughthesamplesizewasadequatetoobserve a sizable association with in utero DDT, we are unlikely to have had the power to observe smaller associations with other risk factors or synergy among variables. To date, incompletedataontumorpathologylimitstheinterpretation ofthecorrelationbetweenDDTandtumorswithmoreaggressive features.<br />Summary Weobservedasizable,statisticallysignificantassociation betweeninuteroDDTexposureandriskofbreastcancerin youngwomenandapossibleassociationwithmoreaggressive tumors. These findings are the first ever reported for a prospectiveobservationofalargepregnancycohort.Experimentalstudiesareessentialtoconfirmfindingsanddiscover causalmechanisms.Ifconfirmed,thesefindingscouldleadto discoveryofbiomarkersandinterventionsforDDT-associatedbreastcancer.Ourfindingsarerelevanttotheinternational debate on the costs and benefits of DDT use for malaria control.<br />Acknowledgments<br />We acknowledge the late Jacob Yerushalmy, who founded the ChildHealthandDevelopmentStudiescohort;thelateBarbara van den Berg, the second Director of the Child Health and Development Studies; and her colleague, Roberta Christianson, who ensured its quality and longevity.<br />Addressallcorrespondenceandrequestsforreprintsto:Barbara A. Cohn, PhD, Child Health and Development Studies, 1683 Shattuck Avenue, Suite B, Berkeley, CA 94709. E-mail: bcohn@chdstudies.org. Thepointofviewandconclusionsexpressedinthispaperare thoseoftheauthorsanddonotnecessarilyrepresenttheofficial position or policies of the funding agencies. The ideas and opinions expressed herein are those of the author(s)andendorsementbytheStateofCalifornia,Department of Public Health the National Cancer Institute, and the Centers forDiseaseControlandPreventionortheircontractorsandsubcontractorsoranyofthefundersofthisresearchisnotintended nor should be inferred.<br /> 