NeuroToxicology 32 (2011) Contents lists available at ScienceDirect. NeuroToxicology

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1 NeuroToxicology 32 (2011) Contents lists available at ScienceDirect NeuroToxicology Maternal prenatal and child organophosphate pesticide exposures and children s autonomic function Lesliam Quirós-Alcalá a,b, Abbey D. Alkon c, W. Thomas Boyce d,1, Suzanne Lippert e, Nicole V. Davis c, Asa Bradman a, Dana Boyd Barr f, Brenda Eskenazi a, * a Center for Environmental Research and Children s Health (CERCH), School of Public Health, University of California, 1995 University Avenue, Suite 265, Berkeley, CA 94704, USA b United States Environmental Protection Agency STAR Fellow, USA c University of California, School of Nursing, 2 Koret Way, Box 0606, San Francisco, CA 94143, USA d School of Public Health and the Institute of Human Development, University of California, 1121 Tolman Hall #1690, Berkeley, CA , USA e Division of Emergency Medicine, Stanford University Hospitals and Clinics, 300 Pasteur Drive Alway Bldg., Room M121, Stanford, CA 94305, USA f Emory University, Rollins School of Public Health, 1518 Clifton Road, Atlanta, GA 30333, USA A R T I C L E I N F O Article history: Received 2 March 2011 Received in revised form 9 May 2011 Accepted 23 May 2011 Available online 29 June 2011 Keywords: Autonomic nervous system (ANS) Respiratory sinus arrhythmia (RSA) Preejection period (PEP) Heart rate Children Organophosphates Pesticides In utero Neurodevelopment A B S T R A C T Background: Organophosphate pesticides (OP), because of their effects on cholinergic fibers, may interfere with the functions of the autonomic nervous system (ANS). We conducted a study to assess the relation of in utero and child OP pesticide exposures and children s autonomic nervous system (ANS) dysregulation under resting and challenge conditions. We hypothesized that children with high OP levels would show parasympathetic activation and no sympathetic activation during rest and concomitant parasympathetic and sympathetic activation during challenging conditions. Methods: OP exposures were assessed by measuring urinary dialkylphosphate metabolites (DAPs, total diethyls-des, and total dimethyls-dms) in maternal and children s spot urine samples. ANS regulation was examined in relation to maternal and child DAPs in 149 children at 6 months and 1 year, 97 at 3 1/2 years and 274 at 5 years. We assessed resting and reactivity (i.e., challenge minus rest) measures using heart rate (HR), respiratory sinus arrhythmia (RSA), and preejection period (PEP) during the administration of a standardized protocol. Cross-sectional (at each age) and longitudinal regression models were conducted to assess OP and ANS associations. To estimate cumulative exposure at 5 years, we used an area-under-the-concentration time-curve (AUC) methodology. We also evaluated whether children with consistently high versus low DAP concentrations had significantly different mean ANS scores at 5 years. Results: Child DMs and DAPs were significantly negatively associated with resting RSA at 6 months and maternal DMs and child DEs were significantly positively associated with resting PEP at 1 year. No associations with resting were observed in 3 1/2- or 5-year-old children nor with reactivity at any age. There was no significant relationship between the reactivity profiles and maternal or child DAPs. Cumulative maternal total DEs were associated with low HR ( 3.19 bpm decrease; 95% CI: 6.29 to 0.09, p = 0.04) only at 5 years. In addition, there were no significant differences in ANS measures for 5- year-olds with consistently high versus low DAPs. Conclusion: Although we observe some evidence of ANS dysregulation in infancy, we report no consistent associations of maternal and child OP pesticide exposure, as measured by urinary DAPs, on children s ANS (HR, RSA, and PEP) regulation during resting and challenging conditions up to age 5 years. ß 2011 Elsevier Inc. All rights reserved. 1. Introduction * Corresponding author. Tel.: ; fax: addresses: lquiros@berkeley.edu (L. Quirós-Alcalá), abbey.alkon@nursing.ucsf.edu (A.D. Alkon), tom.boyce@ubc.ca (W.T. Boyce), slippert@stanford.edu (S. Lippert), nicolevdavis@gmail.com (N.V. Davis), abradman@berkeley.edu (A. Bradman), dbbarr@emory.edu (D.B. Barr), eskenazi@berkeley.edu, lesliamquiros@hotmail.com (B. Eskenazi). 1 Present address: College for Interdisciplinary Studies and Faculty of Medicine, East Mall University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Organophosphate (OP) pesticides are widely used in agriculture in the United States with more than 1.6 million lbs applied in California in 2008 (California Department of Pesticide Regulation, 2008). They are well-known acute neurotoxicants, which inhibit acetylcholinesterase, resulting in the buildup of acetylcholine in neuronal junctions (Eskenazi et al., 1999). OP pesticides may cause synaptic dysregulation and disrupt the establishment of neuronal architecture (Bigbee et al., 1999; Chanda and Pope, 1996; Dam X/$ see front matter ß 2011 Elsevier Inc. All rights reserved. doi: /j.neuro

2 L. Quirós-Alcalá et al. / NeuroToxicology 32 (2011) et al., 1999a,b; Dellinger and Mostrom, 1988; Slotkin et al., 2001). Recent studies have reported that low-level chronic in utero and child OP pesticide exposures are associated with poorer cognition and behavioral problems (e.g., attention problems) in children (Bouchard et al., 2010, 2011; Eskenazi et al., 2007; Grandjean et al., 2006; Marks et al., 2010; Rauh et al., 2006). No studies have assessed the effects of OP exposure on children s autonomic nervous system (ANS), although it might be potentially most susceptible to the toxic effects of organophosphates as acetylcholine is the key neurotransmitter at the pre- and post-ganglionic synapses in the parasympathetic nervous system (PNS) and the pre-ganglionic synapses in the sympathetic nervous system (SNS) (Fig. 1). OP pesticide acute toxicity can interfere with ANS functions, such as smooth muscle contraction, regulation of cardiac muscle and/or stimulation or inhibition of glandular secretions, and lead to autonomic dysregulation (Appendzeller and Oribe, 1997; Eskenazi et al., 1999; Rosas and Eskenazi, 2008). ANS dysregulation during childhood is related to physical and mental health problems. Studies have found that children with high resting parasympathetic measures, associated with activation, tend to show vagal withdrawal in response to stressors (Calkins et al., 2008; El-Sheikh, 2005). Children with high sympathetic reactivity display more externalizing behavior problems compared to children with lower sympathetic reactivity (Boyce et al., 2001a; Pearson et al., 2005). Furthermore, coactivation of both the parasympathetic and sympathetic nervous system during challenging conditions compared to resting states is associated with externalizing behavior problems for school-age children who live in families with high marital conflict (El-Sheikh et al., 2009). Respiratory sinus arrhythmia (RSA) and preejection period (PEP) are valid measures of the parasympathetic and sympathetic branches of the ANS respectively and, as peripheral, noninvasive measures, they offer a physiologic summary of the immensely complex processes that underlie autonomic responsiveness to a changing environment (Alkon et al., 2003). RSA measures the periodic oscillation in sinus rhythm occurring in synchrony with respiration and accounts for the decrease in heart rate during expiration and an increase during inspiration. PEP measures the duration of isovolumetric ventricular contraction and is a noninvasive and indirect measure of the SNS s influence on the cardiac cycle. Heart rate (HR) is also a valid measure of ANS regulation; it is an integrated measure of the PNS and SNS (Beauchaine, 2001; Berntson et al., 1993; Sherwood, 1993). ANS resting measures indicate a child s physiology during a calm state; challenging measures indicate a child s physiologic response to stressors; and reactivity measures indicate the physiologic response to a discrete environmental stimulus or challenge compared to resting state (Alkon et al., 2006). Thus, ANS function, as characterized by RSA, PEP, and HR in a developing young child, provides a potential index of the health of the ANS and the associated adaptive capabilities of individual infants to environmental stress (Boyce et al., 1998; Treadwell et al., 2010). Several hypotheses on the potential effects of OPs on children s ANS can be formulated. Inhibition of acetylcholinesterase by OP exposure would result in an acetylcholine excess and tonic Fig. 1. The autonomic nervous system.

3 648 L. Quirós-Alcalá et al. / NeuroToxicology 32 (2011) downstream stimulation within cholinergic neural pathways. OP exposure might induce excess baseline PNS stimulation, since acetylcholine remaining in the pre- and post-ganglionic synapses may prevent the withdrawal of PNS activation even in response to challenge. Thus, the PNS, which provides the predominant stimulation to the atrioventricular node of the heart, when excessively activated could result in a high resting RSA, but when challenged could result in a reactivity measure of small magnitude, because of an inability to de-activate or withdraw the PNS. In contrast, the SNS, which is largely inactive in the resting state, becomes active in response to a stressful challenge by releasing acetylcholine into the pre-ganglionic synapse. Thus in a resting state, high PEP values, corresponding to a longer interval of isovolumetric ventricular contraction, would reflect less SNS activity, but with OP exposure and the resulting accumulation of acetylcholine in the preganglionic synapses, excessive SNS activation may ensue, with shorter PEP times (i.e., negative PEP reactivity) and an inability to return to a resting state. Thus, we hypothesize that children with OP exposure would have PNS activation and minimal SNS activation under resting conditions and high sympathetic but minimal parasympathetic reactivity (i.e., negative SNS and minimally positive PNS reactivity) under challenging conditions. We conducted a study to assess the association of OP pesticide exposure and ANS regulation in children living in an agricultural region. We collected RSA, PEP, and HR measures under resting and challenging conditions by having children listen to relaxing stories or lullabies and complete a series of standardized tasks designed to elicit ANS responses. The main objectives were to: (1) assess the cross-sectional relationship of prenatal maternal and child OP pesticide exposure as measured by urinary dialkylphosphate (DAP) metabolites on children s ANS at 6 months, 1 year, 3 1/2 and 5 years of age; (2) determine whether cumulative measures of maternal and child DAPs were associated with ANS function at 5 years; and (3) determine whether children with consistently high versus consistently low OP pesticide exposures had significantly different mean ANS responses at 5 years. 2. Methods 2.1. Study population The children were participants in the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) longitudinal birth cohort study. The primary aim of this study was to evaluate the potential health effects of pesticides and other chemicals on maternal and child health in a population living in the agricultural community of Salinas Valley, California. Population recruitment details were described previously (Eskenazi et al., 2004). Briefly, we recruited expectant mothers between October 1999 and October 2000 who were: 18 years old, <20 weeks gestation, fluent in Spanish or English, qualified to receive government health insurance, receiving prenatal care at specific community clinics, and planning to deliver at the county hospital. The University of California Center for Protection of Human Subjects approved all study procedures and written informed consent was obtained from mothers for themselves and for their child. A total of 601 women were enrolled and 531 were followed to delivery of a liveborn neonate. We interviewed mothers during pregnancy (14 and 26 weeks), at delivery, and at the time of each child assessment to obtain demographic information on the families and participating children. Birth and pediatric medical records were abstracted by a registered nurse. Infants with an even identification number enrolled between April and October 2000 were eligible to participate in the ANS reactivity protocols during infancy (at 6 months and 1 year of age) and at 3 1/2 years of age. When children were five years of age, we offered the ANS reactivity protocol to all children enrolled. The ANS protocol was completed on 194 at 6 months, 181 at 1 year, 149 at 3 1/2 years, and 312 at 5 years. We excluded children who did not have scoreable and/or sufficient data on ANS measures (n = 33 at 6 months, n = 23 at 1 year, n = 10 at 3 1/2 years, and n = 11 at 5 years); were twins (n = 8 at 6 months, n = 2 at 1 year, n = 6 at 3 1/2 years, and n = 8 at 5 years); taking medications that could affect ANS responses e.g., seizure medications (n = 2 at 6 months, 1 and 3 1/2 years, and n = 5 at 5 years); missing complete maternal urinary pesticide metabolite data (n = 1 at 5 years); or missing child urinary pesticide metabolite data (n = 2 at 6 months, n = 5 at 1 year, n = 34 at 3 1/2 years, and n = 13 at 5 years). These exclusions resulted in a total of 149 children who provided data at 6 months, 149 at 1 year, 97 at 3 1/2 years and 274 at 5 years. Only 27 children had ANS and pesticide measurements at all four age time points. Overall, there were no significant differences in demographic characteristics or maternal prenatal DAP concentrations between the subsample of children who completed the ANS protocols at 6 months, 1 year and 3 1/2 years compared to the larger sample of children who completed the ANS protocol at 5 years ANS reactivity protocols and ANS data acquisition Protocols were administered to the child in his or her native language (English or Spanish) by trained bilingual staff at the research field office, in a recreational vehicle (RV), and/or at the participant s home. We administered a previously validated ANS reactivity protocol used on children age 3 1/2- and 5-years (Alkon et al., 2003, 2006; Boyce et al., 2001b); we modified this protocol for the 6-month and 1-year-olds (Alkon et al., 2003, 2006; Boyce et al., 2001b). We used age-appropriate social, physical, and emotional challenges and, in addition, for the 3 1/2- and 5-year-olds, we included a cognitive challenge task. These challenges were designed to represent common stressors for eliciting sympathetic and parasympathetic autonomic responses in children. Resting conditions at 6 months and 1 year were listening to digitally recorded lullabies and at 3 1/2 and 5 years, listening to a story read aloud. Challenging conditions at 6 months and 1 year were watching a jackin-the-box wound up and jump out of the box (social/startle), listening to a digitally recorded sick baby crying (emotion), and feeling a vibrator on the leg (physical). At 3 1/2 and 5 years, the challenging conditions were asking them to answer questions (social), watching a scary videoclip (emotion), tasting concentrated lemon juice on the tongue (physical), and repeating a series of numbers (cognitive). Further details on the ANS protocols are available elsewhere (Alkon et al., 2003, 2006; Boyce et al., 2001b). We obtained continuous measures of children s sympathetic and parasympathetic autonomic responses (RSA, HR, and PEP) during the resting conditions and challenge tasks by placing electrodes on the child s neck and thoracic region and then attaching the electrodes to an electrocardiograph (ECG), as detailed previously (Alkon et al., 2003, 2006; Boyce et al., 2001b). RSA scores were computed using: interbeat intervals on the ECG readings, respiratory rates derived from the impedance signal (dz/ dt), and a bandwidth range of Hz for the 6-month- and 1-year-olds and Hz for the 3 1/2- and 5-year-olds (Alkon et al., 2006; Bar-Haim et al., 2000). Heart rate was ascertained from the number of R-waves on the ECG and measured in beats per minute (bpm). PEP was measured in milliseconds as the interval between the ECG s Q-wave and the B notch on the dz/dt signal. After acquiring RSA, HR, and PEP measurements, data were then filtered, extracted, and scored using the ANS Suite software (National Instruments, Austin, TX) or Mindware ( (Alkon et al., 2003, 2006).

4 L. Quirós-Alcalá et al. / NeuroToxicology 32 (2011) ANS response measures and data reduction For the present analyses, we calculated six summary ANS measures: mean resting and reactivity scores for RSA, HR, and PEP (Alkon et al., 2003). Reactivity scores were calculated for each child as the difference in the mean score during the challenge tasks and the mean score of the first resting condition. Reactivity scores allowed us to evaluate the child s individual response to challenge tasks compared to their individual resting state. A negative RSA reactivity score indicates more parasympathetic withdrawal (inhibition) during the challenge tasks compared to resting, while a negative PEP reactivity score indicates more sympathetic activation during the challenge tasks compared to resting (Pearson et al., 2005). A negative RSA or PEP reactivity score also corresponds to an increase in HR; and a positive mean HR reactivity score indicates a higher HR during the challenge task compared to resting. We also generated ANS profiles to combine the parasympathetic (i.e., RSA) and sympathetic (i.e., PEP) reactivity scores into categories that reflect each child s ability to activate or inhibit their ANS responses under challenging conditions. RSA and PEP reactivity scores were dichotomized as positive or negative and then combined into four profiles: (1) coactivation (activation of both SNS and PNS during the challenge tasks compared to rest); (2) coinhibition (inhibition of both SNS and PNS during challenge tasks compared to rest); (3) reciprocal PNS activation and SNS withdrawal; or (4) reciprocal SNS activation and PNS withdrawal (Berntson et al., 1991; Salomon et al., 2000). ANS dysregulation has been defined as coactivation or coinhibition, which have been associated with behavior problems and emotional regulation (Beauchaine et al., 2007; El-Sheikh et al., 2009) Pesticide exposure assessment To assess prenatal OP pesticide exposure, we measured the DAP metabolite concentrations in maternal spot urine samples collected at the time of the prenatal interviews (i.e., at mean SD SD of and weeks gestation, respectively). To assess children s OP exposure, we measured DAP concentrations in spot urine samples collected from the children when they were approximately 6 months, 1, 2, 3 1/2 and 5 years of age. Samples were generally collected on the same day of the ANS assessments. Although no ANS assessments were conducted when children were 2 years old, urine samples collected at this time were used to contribute to the estimation of cumulative exposure to OPs during childhood as detailed below. All urine specimens were aliquoted and stored at 80 8C until shipment for laboratory analysis at the Centers for Disease Control and Prevention (National Center for Environmental Health, Atlanta, Georgia). Further details on the specimen collection methods and quality control procedures have been described elsewhere (Bradman et al., 2007; Eskenazi et al., 2007). We measured concentrations of six DAP urinary metabolites including three diethylphosphates (DE): diethylphosphate (DEP), diethylthiophosphate (DETP), diethyldithiophosphate (DEDTP); and three dimethylphosphates (DM): dimethylphosphate (DMP), dimethylthiophosphate (DMTP), and dimethyldithiophosphate (DMDTP). These metabolites represent the by-products of approximately 80% of the OP pesticides registered for use in the Salinas Valley (Bradman et al., 2007). DAP metabolite concentrations were measured using isotope dilution gas chromatography tandem mass spectrometry (Bravo et al., 2004). Limits of detection (LOD) for each DAP metabolite was as follows: 0.2 mg/l, 0.1 mg/l, and 0.1 mg/l for DEP, DETP, and DEDTP, respectively; and 0.6 mg/l, 0.2 mg/l, and 0.1 mg/l for DMP, DMTP, and DMDTP, respectively. Creatinine concentrations (mg/dl) in maternal and child urine samples were also determined using a commercially available diagnostic enzyme method (Vitros CREA slides, Ortho Clinical Diagnostics, Raritan, NJ, USA). Individual DAP metabolite concentrations below the LOD were imputed to LOD/H2. We summed the metabolite levels to create a measure of total DE (molar sum of DEP + DETP + DEDTP), total DM (molar sum of DMP + DMTP + DMDTP) and total DAPs (molar sum of total DEs + total DMs). We averaged the two pregnancy DAP measurements; the two prenatal measurements of total DAPs did not significantly differ (paired t-test = 0.69, p = 0.50). In 22 cases, there was only one prenatal pesticide measurement Data analyses To investigate the cross-sectional association between each of the six ANS measures and maternal prenatal and child OP pesticide exposures, we constructed separate linear regression models for each ANS outcome at each time point (i.e., at 6 months, and 1, 3 1/2 and 5 years of age). Because the prenatal and child DAP metabolite concentrations were not correlated (r = 0.04 to 0.03, p > 0.60), we placed both of these measures into each model as independent variables and also examined their interaction. Coefficients from these models were similar to those obtained in models containing either prenatal or child measures alone. There was no statistical interaction between prenatal and child DAP concentrations. Covariates were selected if they were potentially related to the ANS measures [i.e., exact age (in months) of the child at the time of the ANS assessment; location of the assessment (RV or field office or participant s home), and psychometrician]; or related (p < 0.10) to exposure and outcome (i.e., breastfeeding duration in months at the time of the ANS assessment). We adjusted for sex of the child and examined interaction by sex but found none. We included the same covariates in all regression models. In addition to the covariates listed, we also considered other variables (prenatal smoking or drinking, income, maternal education, birth weight and gestational age); however, these were not significantly associated with outcomes and/or exposure at any age. Additionally, we conducted a one-way analysis of variance (ANOVA) to determine if the mean prenatal or child total DAP concentrations significantly differed among the four ANS profiles at each time point. DAP metabolite concentrations were log10-transformed for statistical models. We also assessed whether cumulative pesticide exposures were associated with each of the six ANS outcomes in 223 (of 274) children assessed at 5 years who had: (1) at least one ANS measure at 5 years; (2) both prenatal DAP measurements (i.e., at 14 and 26 weeks gestation); (3) the six month and five year child DAP measurements; and (4) no more than one child DAP measurement missing between 12 months and 3 1/2 years. For those children with four child DAP measurements (i.e., missing at 12 months, or at 2 or 3 1/2 years), we imputed their missing value by taking the average of the preceding and subsequent untransformed DAP concentrations. To estimate cumulative exposure during the prenatal and early childhood periods, we used an area-underthe-concentration time-curve (AUC) methodology (Kaplan et al., 1997). Thus, our prenatal exposure variable consisted of the AUC for the untransformed DAP concentrations observed at approximately 14 and 26 weeks gestation; the child exposure variable consisted of the AUC for the untransformed DAP concentrations observed between 6 months and 5 years. Prenatal and child AUC values were log 10 transformed prior to inclusion in regression models. Covariates in these models included sex, location, psychometrician, and exact age at the 5-year assessment. In addition, we evaluated whether children with consistently high versus low DAP concentrations had significantly different mean ANS scores at 5 years of age. These t-tests were performed using maternal prenatal DAP concentrations only, child DAP

5 650 L. Quirós-Alcalá et al. / NeuroToxicology 32 (2011) concentrations only, and all DAP concentrations (prenatal and child). For each of these analyses, we first created a variable that categorized exposure at each time point by quartiles. We then created scores for each child by summing the quartile categories over the time points. There were few children who were in the lowest or highest quartiles jointly at all time points. Thus, we compared ANS scores for children whose scores fell in the highest 10% of DAP scores (representing children with consistently high DAP levels ) with those children with the lowest 10% of DAP scores (representing children with consistently low DAP levels ). Lastly, we also ran longitudinal mixed models to assess the relationship between prenatal and child DAPs on children s ANS measures; however, similar results were observed in these models as in the cross-sectional models and are thus not presented for ease of interpretation. All regression models were conducted using unadjusted urinary metabolite concentrations (nmol/l) and repeated using creatinineadjusted urinary metabolite concentrations (nmol/gcre) and with creatinine concentrations as covariates. Using creatinine as a covariate and creatinine-adjusted concentrations in the models revealed similar results. All statistical analyses were performed using STATA 10 for Windows (StataCorp LLC, College Station, TX) and statistical significance was set at p < 0.05 for main effects and p < 0.20 for interactions. 3. Results Less than 5% of the children weighed <2500 g at birth and 7% were born at <37 weeks gestation. Between 15% and 20% of children who participated in ANS protocols had mothers working in agriculture (n = mothers depending on the time of the ANS assessment). Two-thirds of children lived with at least one farmworker in the home and almost all mothers breastfed with the median total breastfeeding duration of six months (not shown). Mothers were primarily young (mean age 26 years), and relatively few had completed high school. Most mothers were born in Mexico (>86%) and had lived in the U.S. an average of approximately 8 years at the time of pregnancy. Additionally, over 60% of the participating families lived below the federal poverty threshold. Few women smoked during pregnancy (<5%) or drank alcohol (<26%). Details on the demographic characteristics of the mothers and children at each collection are available in Supplemental Table S1. Mean ANS resting measures and reactivity scores are presented in Table 1. Overall, the expected developmental changes in ANS were shown: mean HR resting measures decrease with age and RSA and PEP resting measures increased with age. The trends with age in reactivity scores were not as consistent but, in general, they showed an increase in reactivity with age: HR reactivity scores changed from negative to positive scores, indicating as children grew older, there was an increase in HR response to the challenging compared to resting conditions and RSA and PEP reactivity scores increased in negative reactivity scores, indicating greater PNS withdrawal and SNS activation during the challenging versus resting conditions. Maternal DE, DM and DAP geometric mean (confidence intervals, CI) concentrations for children who participated in the ANS protocols in at least one of the age time points were 22.8 (CI: ), (CI: ), and (CI: ) nmol/l, respectively (maternal GM concentrations for those children who participated at each age time point are available in Supplemental Table S2). Table 2 presents the geometric means (GM) and (CIs) for child DAP concentrations (unadjusted and creatinine-adjusted) at each time point. Child DM metabolite concentrations were higher than total DE concentrations. Additionally, maternal DAP concentrations were uncorrelated with any Table 1 Summary statistics for children s ANS resting and reactivity measures at each time point. a Collection time point Resting measures Reactivity measures n Mean (SD) n Mean (SD) 6 months RSA (0.8) (0.5) HR (10.8) (5.4) PEP (9.0) (3.6) 1 year RSA (0.9) (0.6) HR (12.3) (6.0) PEP (10.7) (4.1) 3 1/2 year RSA (1.1) (0.6) HR (9.8) (3.5) PEP (6.7) (1.8) 5 year RSA (1.2) (0.5) HR (10.8) (4.4) PEP (7.4) (1.7) a Values may vary slightly depending on the number of children with available information at the collection time point. RSA is an index, HR is measured in beats per minute and PEP is measured in milliseconds. Abbreviations: SD = standard deviation; RSA = resting respiratory sinus arrhythmia (index); HR = heart rate (beats per minute; bpm); PEP = preejection period (ms). of the child DAP concentrations, whether or not they were adjusted for creatinine (r = 0.08 to 0.05, p > 0.05, not shown) Resting measures When we examined the relation of child DAPs and ANS measurements at rest, we found a significant negative association between resting RSA and each ten-fold increase of child DM concentrations (b = 0.24, 95% CI: 0.42 to 0.05; p = 0.02; Table 3a) and total DAP concentrations (b = 0.27, 95% CI: 0.48 to 0.06; p = 0.01; Table 3a) at 6 months, which is suggestive of PNS withdrawal (corresponds with an increase in HR). However, this Table 2 Geometric Mean (GM) for child DAP concentrations and respective confidence intervals (CI) for those children with ANS and DAP measurements at each time point. a,. b Unadjusted concentration (nmol/l) Creatinine-adjusted concentration (nmol/gcre) GM (95% CI) GM (95% CI) 6 months (n = 149) Total DEs 10.4 (8.2, 13.1) 53.9 (42.4, 68.6) Total DMs 26.2 (20.1, 34.2) 136 (103.1, 179.2) Total DAPs 48.6 (38.5, 61.3) 252 (199.0, 319.0) 1 year (n = 149) Total DEs 10.7 (8.5, 13.3) 46.7 (37.6, 58.1) Total DMs 30.2 (22.5, 40.4) 132 (98.2, 178.6) Total DAPs 50.4 (38.9, 65.2) 221 (170.8, 285.9) 3 1/2 years (n = 97) Total DEs 6.6 (4.8, 9.1) 12.9 (9.3, 17.8) Total DMs 79.9 (57.8, 110.4) 155 (113.0, 211.2) Total DAPs 96.5 (70.9, 131.5) 187 (138.7, 251.4) 5 years (n = 274) Total DEs 7.6 (6.3, 9.3) 11.1 (9.1, 13.5) Total DMs 78.5 (65.6, 93.9) 114 (95.8, 136.0) Total DAPs (85.0, 119.6) 147 (124.2, 173.0) a Individual metabolites were converted to molar concentrations and then summed to produce total DEs, DMs, and DAPs (limits of detection for individual metabolites were as follows: DEP = 0.2 mg/l, DETP = 0.1 mg/l, DEDTP = 0.1 mg/l, DMP = 0.6 mg/l, DMTP = 0.2 mg/l, and DMDTP = 0.1 mg/l). b Sample size reflects subset of children at each time point with available ANS and DAP data. Abbreviations: DEs = diethyls; DM = dimethyls; DAPs = dialkylphosphates.

6 L. Quirós-Alcalá et al. / NeuroToxicology 32 (2011) Table 3a Relationship of maternal prenatal and child DAP levels and resting ANS measures in children at ages 6 months, and 1, 3 1/2 and 5 years (unadjusted for creatinine). a,b,. c Resting measures 6 months 1 year 3 1/2 years 5 years n b (95% CI) p n b (95% CI) p n b (95% CI) p n b (95% CI) p RSA (index) DEs (child) ( 0.34, 0.09) ( 0.29, 0.24) ( 0.37, 0.27) ( 0.17, 0.23) 0.77 DEs (PN) 0.06 ( 0.37, 0.25) ( 0.47, 0.21) ( 0.30, 0.74) ( 0.17, 0.51) 0.34 DMs (child) ( 0.42, 0.05) 0.02 * ( 0.26, 0.13) ( 0.46, 0.17) ( 0.34, 0.09) 0.26 DMs (PN) 0.07 ( 0.34, 0.19) ( 0.39, 0.20) ( 0.39, 0.64) ( 0.30, 0.34) 0.91 DAPs (child) ( 0.48, 0.06) 0.01 * ( 0.28, 0.16) ( 0.46, 0.20) ( 0.34, 0.12) 0.33 DAPS (PN) 0.05 ( 0.33, 0.24) ( 0.43, 0.21) ( 0.35, 0.73) ( 0.22, 0.49) 0.46 HR (bpm) DEs (child) ( 2.23, 3.47) ( 3.57, 3.53) ( 1.38, 4.53) ( 1.92, 1.65) 0.88 DEs (PN) 0.38 ( 4.51, 3.75) ( 6.12, 2.86) ( 8.51, 0.95) ( 3.87, 2.33) 0.63 DMs (child) ( 1.11, 3.94) ( 2.14, 3.02) ( 0.58, 5.14) ( 3.09, 0.84) 0.26 DMs (PN) 1.19 ( 4.71, 2.33) ( 6.39, 1.43) ( 7.81, 1.60) ( 0.93, 4.85) 0.18 DAPs (child) ( 1.43, 4.21) ( 2.58, 3.34) ( 0.83, 5.16) ( 3.21, 0.93) 0.28 DAPS (PN) 1.09 ( 4.96, 2.78) ( 6.72, 1.73) ( 8.74, 1.10) ( 1.89, 4.60) 0.41 PEP (ms) DEs (child) ( 3.15, 1.98) (1.24, 7.42) 0.01 * ( 2.87, 0.94) ( 0.53, 1.93) 0.26 DEs (PN) 1.25 ( 2.46, 4.96) ( 1.20, 6.68) ( 2.01, 4.07) ( 1.75, 2.53) 0.72 DMs (child) ( 2.27, 2.16) ( 1.99, 2.68) ( 1.11, 2.59) ( 1.10, 1.63) 0.70 DMs (PN) 0.77 ( 3.90, 2.35) (0.21, 7.33) 0.04 * 1.04 ( 2.01, 4.09) ( 3.18, 0.83) 0.25 DAPs (child) ( 2.83, 2.22) ( 1.39, 3.92) ( 1.38, 2.51) ( 1.09, 1.79) 0.63 DAPS (PN) 0.67 ( 4.11, 2.76) ( 0.09, 7.53) ( 1.92, 4.47) ( 3.11, 1.39) 0.45 a Cross sectional models adjusted for sex, exact age at time of ANS assessment (years), breastfeeding duration at the time of ANS assessment (months), location of ANS assessment, and psychometrician conducting ANS assessment. Prenatal and child DAPs are both included in the models. b To obtain a measure of prenatal DAPs exposure we calculated an average of DAPs collected at approximately 14 and 26 weeks of gestation and then log 10 transformed the average to generate prenatal averages. c Child DAP concentrations were log 10 transformed in models. Beta coefficients are provided for child and prenatal (PN) DAPs. * p < association was not observed at other ages nor was it observed when we used creatinine-adjusted concentrations. We also observed an increase in resting PEP score (less SNS activation or a decrease in HR or low activation at rest) in the 1-year-olds associated with prenatal DM concentrations (b = 3.77 ms per 10- fold increase, 95% CI: ; p = 0.04; Table 3a) and with child DE concentrations (b = 4.33 ms per 10-fold increase, 95% CI: ; p = 0.01; Table 3a). When the models included creatinineadjusted urinary metabolite concentrations, we found similar results although some associations were attenuated (results not shown). The only significant association in both the unadjusted and creatinine-adjusted models was for child DE concentrations Table 3b Relationship of maternal prenatal and child DAP levels and reactivity ANS measures in children at ages 6 months, and 1, 3 1/2 and 5 years (unadjusted for creatinine). a,b,. c Reactivity measures 6 months 1 year 3 1/2 years 5 years n b (95% CI) p n b (95% CI) p n b (95% CI) p n b (95% CI) p RSA (index) DEs (child) ( 0.14, 0.15) ( 0.09, 0.27) ( 0.16, 0.18) ( 0.11, 0.07) 0.67 DEs (PN) 0.09 ( 0.31, 0.12) ( 0.22, 0.24) ( 0.25, 0.29) ( 0.17, 0.14) 0.86 DMs (child) ( 0.21, 0.05) ( 0.14, 0.12) ( 0.19, 0.14) ( 0.04, 0.16) 0.21 DMs (PN) 0.15 ( 0.33, 0.03) (0.05, 0.45) 0.01 * 0.07 ( 0.21, 0.34) ( 0.18, 0.11) 0.63 DAPs (child) ( 0.24, 0.05) ( 0.14, 0.16) ( 0.20, 0.14) ( 0.04, 0.16) 0.26 DAPS (PN) 0.17 ( 0.36, 0.03) (0.03, 0.46) 0.03 * 0.06 ( 0.23, 0.34) ( 0.25, 0.08) 0.33 HR (bpm) DEs (child) ( 0.24, 2.71) ( 2.89, 0.71) ( 1.07, 1.08) ( 1.00, 0.41) 0.41 DEs (PN) 0.44 ( 1.69, 2.57) ( 2.16, 2.42) ( 2.41, 1.02) ( 1.01, 1.45) 0.73 DMs (child) ( 0.54, 2.09) ( 1.46, 1.19) ( 1.07, 1.02) ( 0.71, 0.86) 0.85 DMs (PN) 0.52 ( 1.31, 2.36) ( 2.34, 1.70) ( 2.16, 1.27) ( 0.96, 1.35) 0.74 DAPs (child) ( 0.26, 2.67) ( 1.88, 1.15) ( 1.01, 1.17) ( 0.91, 0.73) 0.83 DAPS (PN) 0.62 ( 1.37, 2.62) ( 2.38, 1.98) ( 2.31, 1.28) ( 0.87, 1.72) 0.52 PEP (ms) DEs (child) ( 1.10, 0.88) ( 0.50, 2.00) ( 0.77, 0.36) ( 0.05, 0.50) 0.11 DEs (PN) 0.07 ( 1.37, 1.51) ( 1.66, 1.49) ( 0.72, 1.07) ( 0.74, 0.22) 0.28 DMs (child) ( 0.80, 0.89) ( 0.53, 1.28) ( 0.74, 0.35) ( 0.16, 0.46) 0.35 DMs (PN) 1.21 (0.03, 2.40) ( 2.39, 0.38) ( 0.67, 1.13) ( 0.77, 0.14) 0.17 DAPs (child) ( 1.00, 0.93) ( 0.40, 1.67) ( 0.80, 0.34) ( 0.14, 0.50) 0.28 DAPS (PN) 1.23 ( 0.07, 2.54) ( 2.56, 0.41) ( 0.67, 1.21) ( 0.85, 0.16) 0.18 a Cross sectional models adjusted for sex, exact age at time of ANS assessment (years), breastfeeding duration at the time of ANS assessment (months), location of ANS assessment, and psychometrician conducting ANS assessment. Prenatal and child DAPs are both included in the models. b To obtain a measure of prenatal DAPs exposure we calculated an average of DAPs collected at approximately 14 and 26 weeks of gestation and then log 10 transformed the average to generate prenatal averages. c Child DAP concentrations were log 10 transformed in models. Beta coefficients are provided for child and prenatal (PN) DAPs. * p < 0.05.

7 652 L. Quirós-Alcalá et al. / NeuroToxicology 32 (2011) Table 4a Geometric mean OP urinary metabolite levels (total prenatal DAPs) by ANS profiles at each assessment (unadjusted for creatinine). a Profile SNS b PNS b n GM 95% CI F statistic, p value c 6 months Coactivation (143.6, 273.8) F = 1.53, p = 0.21 Coinhibition (69.9, 173.1) Reciprocal PNS activation and SNS withdrawal (113.8, 227.3) Reciprocal SNS activation and PNS withdrawal (69.9, 173.1) 12 months Coactivation (157.0, 298.4) F = 1.12, p = 0.34 Coinhibition (100.8, 199.5) Reciprocal PNS activation and SNS withdrawal (125.2, 240.3) Reciprocal SNS activation and PNS withdrawal (77.4, 264.6) 3 1/2 years Coactivation (101.9, 384.7) F = 1.58, p = 0.20 Coinhibition (117.0, 295.4) Reciprocal PNS activation and SNS withdrawal (72.4, 227.5) Reciprocal SNS activation and PNS withdrawal (96.1, 152.9) 5 years Coactivation (102.0, 186.6) F = 0.83, p = 0.48 Coinhibition (106.3, 164.3) Reciprocal PNS activation and SNS withdrawal (85.8, 154.1) Reciprocal SNS activation and PNS withdrawal (126.4, 177.3) a Total n at each time point may not add to the total number of children sampled at each designated time point as age categories at each time point were truncated as follows for the statistics reported in this table: 6 month time point (included children 5 8 months of age), 1 year time point (included children between 9 and 15 months of age), 3 1/2 year time point (included children 2 1/2 to 4 years of age), and 5 year time point ( years of age) and depends on the number of children with PN DAPs. For cross sectional, age at the time of assessment was included as a covariate so more children were included at each time point. b + indicates activation and indicates withdrawal or inhibition. c Results from ANOVA. and high PEP resting measures (less sympathetic activation) in 1- year-olds. No other associations were observed between DAP concentrations and mean PEP at other ages Reactivity measures When we examined the relation of child DAP concentrations and reactivity measures, we found a significant positive association between RSA reactivity scores in 1 year-olds with prenatal DM levels (b = 0.25 per 10-fold increase, 95% CI: ; p = 0.01; Table 3b) and with total DAP concentrations (b = 0.24 per 10-fold increase, 95% CI: ; p = 0.03; Table 3b), indicating higher RSA scores during challenge compared to rest (i.e., corresponding to lower HRs). Associations between reactivity scores and creatinine-adjusted prenatal DM and DAP levels were similar (not shown). No associations were observed with the other measures or at other ages ANS profiles We found that unadjusted maternal prenatal DAP metabolite concentrations did not significantly differ among ANS profiles at any age (p = 0.20 to p = 0.48; ANOVA; Table 4a); results were similar when using creatinine-adjusted prenatal concentrations Table 4b Geometric mean OP urinary metabolite levels (total child DAPs) by ANS profiles at each assessment (unadjusted for creatinine). a Profile SNS b PNS b n GM 95% CI F statistic, p value c 6 months Coactivation (28.2, 76.2) F = 1.79, p = 0.15 Coinhibition (39.5, 87.3) Reciprocal PNS activation and SNS withdrawal (24.9, 60.5) Reciprocal SNS activation and PNS withdrawal (40.3, 193.9) 12 months Coactivation (20.4, 58.2) F = 1.25, p = 0.29 Coinhibition (32.2, 104.5) Reciprocal PNS activation and SNS withdrawal (42.2, 111.8) Reciprocal SNS activation and PNS withdrawal (21.0, 105.0) 3 1/2 years Coactivation (23.2, 376.0) F = 0.87, p = 0.46 Coinhibition (45.2, 131.8) Reciprocal PNS activation and SNS withdrawal (37.1, 146.3) Reciprocal SNS activation and PNS withdrawal (78.9, 221.3) 5 years Coactivation (66.2, 149.4) F = 0.57, p = 0.63 Coinhibition (79.2, 153.0) Reciprocal PNS activation and SNS withdrawal (73.7, 211.4) Reciprocal SNS activation and PNS withdrawal (68.7, 119.0) a Total n at each time point may not add to the total number of children sampled at each designated time point as age categories at each time point were truncated as follows for the statistics reported in this table: 6 month time point (included children 5 8 months of age), 1 year time point (included children between 9 and 15 months of age), 3 1/2 year time point (included children 2 1/2 to 4 years of age), and 5 year time point ( years of age). For cross sectional, age at the time of assessment was included as a covariate so more children were included at each time point. b + indicates activation and indicates withdrawal or inhibition. c Results from ANOVA tests.

8 L. Quirós-Alcalá et al. / NeuroToxicology 32 (2011) (not shown). We also observed no significant differences in child DAP concentrations among profiles at any age using unadjusted child concentrations (p = ; ANOVA; Table 4b). However, we did find a significant difference in ANS profiles in 6-month-olds using the creatinine-adjusted child DAP concentrations (F = 3.18, p = 0.03; ANOVA; not shown) in that child DAP concentrations were highest among those with reciprocal sympathetic activation and parasympathetic withdrawal. There were no differences in maternal prenatal or child DAPs by ANS profiles for any other age group Analyses at 5 years using cumulative OP pesticide exposure measures When examining a cumulative measure of DAP levels during childhood, we found a negative association between prenatal DE levels and resting HR (b = 3.19 bpm per 10-fold increase in exposure; 95% CI: 6.29 to 0.09, p = 0.04; Supplemental Table S3); however, this association was no longer present when we used creatinine-adjusted concentrations and no other associations were significant. Lastly, we did not observe any significant differences for each of the six ANS response measures (p > 0.13, not shown) between children with consistently high versus consistently low DAP metabolite concentrations regardless of whether we considered prenatal and child measurements separately or jointly. 4. Discussion This is the first study to evaluate the association between prenatal and child OP pesticide exposures and ANS regulation in young children. Exposure to OP pesticides has been linked to neurodevelopmental and behavioral problems in children (Bouchard et al., 2010, 2011; Eskenazi et al., 2007; Grandjean et al., 2006; Marks et al., 2010; Rauh et al., 2006). ANS reactivity has been associated with behavioral and psychosocial problems in children (Boyce et al., 2001a; Treadwell et al., 2010), and we previously reported a significant association between prenatal DAP levels and attention problems and attention-deficit hyperactivity disorder in CHAMACOS children at 5 years of age (Marks et al., 2010). In addition, we expected that as acetylcholinesterase was inhibited, under resting conditions, the SNS would not be able to activate and the PNS would activate vagal tone; but during challenging conditions, the SNS would be excessively activated and PNS would not be able to withdraw vagal tone (Berntson and Quigley, 2007). Thus, we hypothesized that as OP pesticide exposures increased, we would observe more PNS activation and no SNS activation during rest, but more PNS and SNS activation during challenge. However, in this study, neither maternal prenatal DAP nor child DAP levels were consistently associated with ANS resting or reactivity measures in the children. Instead, we found that only maternal urinary DAP concentrations were associated with less SNS activation during rest (DM levels) and with less PNS withdrawal during challenge (DM and DAP levels) in one year olds and negatively associated with resting HR (DEs) at 5 years. No other measurements or time points were significantly related to prenatal maternal exposure. Similarly, child DAP concentrations (DMs and DAPs) were associated with more parasympathetic withdrawal during resting conditions and with reciprocal sympathetic activation and parasympathetic withdrawal at 6 months (total DAP levels), and with less sympathetic activation during resting conditions (DE levels) at 1 year. Again, no other time points or measures were associated with child exposure. Thus, although we hypothesized that OP pesticide exposure would be related to parasympathetic dominance at rest and ANS dysregulation, our hypotheses were only supported with an association between child exposure (i.e., in utero exposure to DM pesticides and child exposure to DE pesticides as measured by urinary DAP concentrations) and ANS functioning in infants at one year under resting conditions and did not persist at the older ages. Given that numerous comparisons were made, the few associations found are likely due to chance. Prior research on the effects of OP pesticide exposure on ANS regulation is limited but show a relationship between acute OP exposure and ANS. Two studies in dogs found no overall change in resting RSA (parasympathetic withdrawal) in OP-exposed dogs (Dellinger and Mostrom, 1988; Dellinger et al., 1987), although a single human study of 4 adults (Dellinger, 1985) reported an increase in resting RSA (less PNS withdrawal) as a result of postulated OP pesticide exposure in one subject but not in another. Studies in young animals show they exhibit greater susceptibility to OPs than adult animals (Padilla et al., 2000; Sheets, 2000). Our study is the first to examine this issue in a large sample size of young children, with an ANS protocol assessing both resting and challenging responses, and with an OP biomarker of exposure. This study had a number of limitations. One limitation is the use of DAP concentrations to estimate exposure to OP pesticides. Although DAP metabolites have been extensively used in epidemiologic and exposure studies as biomarkers of OP pesticide exposure (Duggan et al., 2003; Engel et al., 2007; Eskenazi et al., 2007), there are limitations to this biomarker. OP pesticide exposure likely varies from day to day and OP pesticides have short biological half-lives. Thus, DAP metabolites levels in spot urine samples may not reflect long-term exposure throughout periods of critical neurologic development (Eskenazi et al., 2007). We attempted to estimate exposure over critical prenatal and postnatal periods by using multiple metabolite measures. However, when we attempted to categorize children by having consistently low or high DAP concentrations, we found that prenatal and child DAP metabolite concentrations did not consistently fall into the same exposure quartiles over the various ages measured, pointing to the variability of exposure. Moreover, while adjustment for creatinine was conducted to account for kidney function and filtration of metabolites, this may not be appropriate for populations undergoing rapid physiologic changes, such as pregnant women and young children, due to the high intraindividual variability in creatinine concentrations (Ye et al., 2008). Lastly, recent studies suggest that DAP metabolite levels in urine may also represent exposure to preformed DAPs present in a person s environment (Lu et al., 2005; Weerasekera et al., 2009). There are also limitations to the ANS data collection protocol. The standardized protocol changed from infancy to preschool and thus may not have been equally challenging at each age; however, this should not affect the cross-sectional examination with DAPs. Although these measures represent the state-of-the-art for assessment of ANS functioning in children, it is possible (as mentioned above) that the measures or the challenges we selected were not sensitive to the potential effects of OP exposure. This study also has a number of strengths. Because we focused on children primarily living with farmworkers and in an agricultural region with intense OP pesticide use, we were able to focus on a population with potentially higher exposure to OP pesticides (Bradman et al., 2005). Additionally, we had relatively large sample sizes at each time point and were able to assess exposure and ANS regulation at multiple ages. Although having a demographically and ethnically homogenous population may limit generalizability of our findings, it may have reduced unmeasured confounders in our population. We also used a previously validated ANS protocol with reliable ANS response measures (Alkon et al., 2003, 2006; Boyce et al., 2001a). Our hypothesis that OP exposure would be associated with ANS dysregulation was based on biologic plausibility and on the animal literature describing the relationship between acute OP exposure