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Fetal Exposure to GlyphosatePaul Winchester1, Shahid Parvez2, Cathy Proctor1,Jun Ying3, Roy Gerona41Neonatology,Indiana University School of Medicine, Riley Hospital for Children, FranciscanHealth, Indianapolis, Indiana, US; 2Fairbanks School of Public Health, Indiana UniversityPurdue University, Indianapolis, Indiana, US; 3Department of Public Health Sciences,University of Cincinnati College of Medicine, Cincinnati, Ohio, US; 4Department of LaboratoryMedicine, University of California School of Medicine, San Francisco, California, US3/25/20171

DisclosureThe Investigators of this study have no financial relationshipsto disclose or Conflicts of Interest (COIs) to resolve.3/25/20172

Background 300 million pounds of glyphosate (GLY) (Roundup ) areapplied each year in the US. Though glyphosate has been in use since 1974 no previousmeasures of GLY exposure in US pregnancies have beenpublished. Rodent models of pesticide exposure in pregnancycorrelate with adult and transgenerational disease throughepigenetic mechanisms. We measured GLY in pregnant women to estimate fetalexposure and potential adverse effects on pregnancyoutcomes.3/25/20173

Environmentally Induced EpigeneticTransgenerational Disease Chemicals(or stressors) in Environment Induce DNA methylation of Genome(in child,adolescent or fetus) Which alters adult disease risk(gene expressionshifts) And is transmitted to future generations3/25/20174

Is Vinclozolin Safe?High doses don’t kill.High doses for 1 week don’t causeMichael Skinnerfetal loss or fetal anomalies, or lowbirth weight or preterm birth.Endocrine disruption does occur but male ratsaren’t deformed at birth.90% of V-exposed male rats had adult diseases:(immune, kidney, prostate,cancer,cholesterol,low sperm counts, accelerated aging, sexuallyundesirable)Diseases were heritable! DNA methylationpatterns changed gene expression acrossgenerations!U.Washington, Pullman

nExposure(PC days 8-14)F2F3Offspring

Failed Experiment !The Baby Rats Were Normal6 mosAdult Rats had Diseases

Fetal Exposure: Adult diseaseLow sperm countInfertilityCancerKidneyProstatePregnancy abnormalitiesImmune dysfunctionHigh cholesterolAccelerated agingNon “Sexy” ScentAnxiety Prone

Transgenerational Effects of FetalPesticide Exposure

The chromosome location and physical mappingof each candidate (numbers) are indicated for eachchromosome (Chr), with those unknown (UN) sequencesnot mapped indicated.

Vinclozolin ;Heritable AnxietyF3 generation males had a decrease inanxiety-like behavior,92 genes in the hippocampus and 276 genes in theamygdalaFemales had an increase in anxiety-likebehavior,1,301 genes in the hippocampus and 172 genes in the amygdala.Brain transcriptomemicroarray analysis from F3generation control (con) andvinclozolin (vin) animals.Skinner MK, Anway MD, Savenkova MI,Gore AC, Crews D (2008) TransgenerationalEpigenetic Programming of the BrainTranscriptome and AnxietyBehavior. PLoS ONE 3(11): e3745.doi:10.1371/journal.pone.0003745

Younger Age at Menarche

Declining Age of Menarche(Japan)BMC Women’s Health2012 Jul i

Males Developing 2 years Earlier Too!

Polycystic Ovarian DiseaseNilsson E, Larsen G, Manikkam M,Guerrero-Bosagna C, SavenkovaMI, Skinner M,et al. PLoS (2012)

Premature Ovarian Failure POFMenopause

DDT; Transgenerational ObesityDDT induced transgenerational obesity and associated disease through thefemale (egg) germline, Thus,female germline can also transmit transgenerationaldisease.1.Michael K Skinner, Mohan Manikkam, Rebecca Tracey, Carlos Guerrero-Bosagna, MuksitulHaque, Eric E Nilsson. Ancestral dichlorodiphenyltrichloroethane (DDT) exposure promotesepigenetic transgenerational inheritance of obesity. BMC Medicine, 2013; 11 (1): 228 DOI:10.1186/1741-7015-11-228

DNA Methylation

DNA Methylation at CpG Site

Contaminants Produced New DNAMethylation Sites

Prenatal Atrazine Causes Transgenerational Disease

ObjectivesThis is a prospective cross-sectional birth cohort study withthe following aims: How many Midwest pregnant women are exposed toglyphosate (GLY) ? Is drinking water an important exposure source? What risk factors and adverse pregnancy outcomescorrelate with exposure?3/25/201723

Design/Methods Pregnant women between ages 18-40 years and theirnewborn infants resulting from this pregnancy wereenrolled prospectively at a private obstetrical practice. Same day urine and household water samples werecollected during a subsequent clinical visit. Specific gravity in urine samples was measured by certifiedhospital laboratory technicians. Urine and water samples were frozen and stored at -800C . Urine and water samples were measured for GLY in ng/mLwith liquid chromatography-tandem mass spectrometry(LLOQ 0.5 ng/mL).*3/25/201724

Design/Methods Electronic medical records were reviewed and clinical andpregnancy outcome data were collected. Questionnaires relating to food and water consumptionduring pregnancy were administered electronically or paperform. Subjects received a 20 gift card compensation for theirtime and effort. Statistical linear models were used to assess relationshipsbetween GLY level and clinical outcomes of gestation ageand adjusted birth weight as well as pregnancy related riskfactors.3/25/201725

Study Population A total of 69 pregnant women with live-born infants were studied. 69 drinking water samples were tested. Mean maternal age was 29 years (range 18-39 years) Maternal race was 94.2% Caucasian, 7.8% Asian Maternal education, Household Income, Mean Pregnancy Length, Mean Birth Weight %ile, % NICU admissions are shown in Table 1.3/25/201726

Demographic Information of CasesVariableRes idential AreaMaternal RaceMaternal AgeMarital StatusPrim ipHypertens ion(CHTN & PIH)Diabetes(DM & GDM)Maternal Drug Us eMaternal Tobacco Us eMaternal Alcohol Us eRoute of DeliveryInfant SexNICU careHous ehold Incom eCounty of Res idenceMaternal Education3/25/2017ValueRuralSuburbanUrbanWhiteAs ianLes s than 20 years20-35 yearsGreater than 35 s ectionVaginalMaleFem aleNoYesLes s than 25,000 25,000 to 34,999 35,000 to 49,999 50,000 to 74,999 75,000 to 99,999 100,000 to 200,000 200,000 or m oreI don't knowHam iltonHendricksJohns onMarionMorgan High School Diplom a or GEDSom e college or As s ociate DegreeBachelors degreeN (%)14 (20)49 (71)6 (09)65 (94)4 (06)2 (03)63 (91)4 (06)19 (28)50 (72)49 (71)20 (29)51 (74)18 (26)64 (93)5 (07)68 (99)1 (01)57 (83)12 (17)66 (96)3 (04)20 (29)49 (71)36 (52)33 (48)66 (96)3 (04)6 (09)10 (14)8 (12)11 (16)14 (20)14 (20)1 (01)5 (07)1 (01)1 (01)23 (33)42 (61)2 (03)14 (20)23 (33)32 (46)27

Results 65 of 69 pregnant women (91%) tested for GLY ( LLOD) Mean GLY concentration was 3.6 0.12 ng/mL When GLY was compared with risk factors, it was foundnegatively correlated to women’s BMI both @ beginning andend of pregnancy (r’s -0.31, p’s 0.01) GLY levels were found higher in women who consumedmore than 24 ounces of caffeine per day (means of GLY4.61 vs. 3.33, p 0.001) GLY levels were found higher in women who lived in ruralareas (means of GLY 4.21 vs. 3.30, p 0.004) None of the drinking water samples had detectable GLY.3/25/201728

Maternal Weight vs. Glyphosate LevelsGLYPHOSATE VS. MATERNAL BMI(PREPREGNANCY)R .286p .0173.90p .069GLYPHOSATE (ng/mL)3.703.503.303.102.902.702.50healthy weightoverweightobeseMATERNAL BMI3/25/201729

Pre-pregnancy Obesity( 30BMI)vs.GLYGlyphosate vs Prepregnancy Obesity43.5p .02683GLY ng/mL2.521.510.5018.9-29.330-39.5Pre-pregnancy BMIObese mothers had lower Glyphosate levels.3/25/201730

Caffeine vs. GlyphosateGLYPHOSATE VS. DAILY CAFFIENE5.004.50GLYPHOSATE (ng/mL)p .021p .0044.003.503.002.50 8 oz16 oz 16 ozDAILY CAFFIENEDo you currently consume beverages containing caffeine (coffee, caffeinated soda, tea, energy drinks)?3/25/201731

Results 91% prenatal urines tested positive for glyphosate(LOD 0.1ng/mL) GLY levels were negatively correlated to gestation age(r -0.25, p 0.0352) GLY levels were negatively correlated to gestation adjustedbirth weight (r -0.24, p 0.045)3/25/201732

Pregnancy Length vs. GlyphosatePREGNANCY LENGTH VS. GLYPHOSATE278PREGNANCY LENGTH (DAYS)277276275274273272271lowmodhighGLYPHOSATE TERCILES3/25/2017* Only includes singleton 37week gestation infants.33

Gestation-Corrected Birth Weight(Bwt %Ile) vs. GlyphosateR .226p .062BIRTH WEIGHT PERCENTILEvs. GLYPHOSATEBIRTH WEIGHT TE TERCILES* Only includes singleton 37 weeks gestation infants3/25/201734

Summary The most heavily used pesticide in the US is found atquantifiable levels in over 90% of pregnancies in theMidwest. Higher GLY levels were associated with shorter gestationsand with lower gestation-adjusted birth weights. Since water samples were largely negative the source ofexposure is probably food. Maternal pre-pregnancy weight and Caffeine intake wereassociated with higher GLY but Organic food intake was NS.3/25/201735

Conclusion The majority of fetuses must be assumed to have exposureto glyphosate during critical periods of fetal development. It is incumbent upon policy makers to ensure that such alarge scale fetal exposure does not result in altered DNAmethylation and potential multigenerational disease.3/25/201736

Limitations Funding prohibited examination of GLY variability bytrimester and comparisons between blood, urine,concentrations. Small sample size and regional anddemographic differences are also not addressed. Fetalepigenetic changes related to maternal levels likewise werenot addressed. Food GLY residues and other exposuresources were not addressed. Specimen processing, transport etc. are also likely to affectmeasured levels. Laboratory validation of assays wasrigorously completed prior to this study.(RG)3/25/201737

AcknowledgementsFranciscan St. Francis Health––––Laboratory TechniciansOB/GYN PhysiciansLabor & Delivery NursesPost Partum Nurses– Neonatal Intensive Care NursesMedicineOB GYN of Indiana SouthOffice Manager– Medical Assistants– Physicians– Study Population3/25/2017Fairbanks Public Health- Grant Support- Study Design Collaboration- Co-PIUCSF Department of Laboratory- MedicineIndiana UniversityNeonatology Department- Clinical Research Coordinator38

OB GYN of Indiana,CollaboratorsScott Bowers, MDStephanie R. Young, MSNJamie Boyce, DOThomas Wisler Jr., MDTara Debikey, MDVicky Sherman,MDAGPCNP-C3/25/201739

*Glyphosate Assay Glyphosate (N-(phosphomethyl)glycine ) is directly analyzed using liquidchromatography- tandem mass spectrometry (LC-MS/MS). Water andurine samples are prepared for analysis by solid phase extraction usingan ion exchange column. Extracted samples are injected to the LCMS/MS and the analyte is separated using an Obelisc N column (SIELCTechnologies, Prospect Heights, IL) through isocratic elution. Ionizationof glyphosate is achieved using an electrospray ionization sourceoperated in negative polarity. The analyte is detected by multiplereaction monitoring using a 13C-labelled glyphosate as internalstandard. Quantification of the analyte is done by isotope dilutionmethod using an eight-point calibration curve. The assay has a limit of quantification of 0.1 ng/mL. The intra- andinter-day precision observed are 6-15% in concentrations that range0.1-80 ng/mL. Recoveries for glyphosate range 70-80% atconcentrations within the assay’s linear dynamic range.(RG, UCSF)3/25/201740

Drinking Water SourcesAll water samples LODSources of Drinking Water(N 69)1%39%Bottled waterPrivate wellPublic supply56%Don't know4%3/25/201741

Organic Food ConsumptionGLYPHOSATE VS. ORGANIC FOODOrganic Food ys56%Not significant3.70GLYPHOSATE ntlyORGANIC FOOD3/25/201742

Education of GLY Study ParticipantsMaternal EducationGlyphosate vs. Maternal Education3.99%20% High SchoolDiploma College DegreeMasters,PhD,Professional71%Mean Glyphoste (ng/mL)3.83.73.63.53.43.33.23.13.02.9 High School Diploma,GED, or equivalentSome College orAssociate DegreeBachelors or AdvancedDegreeMaximum Educational Attainment3/25/201743