Residential proximity to agricultural fumigant use and respiratory health in 7-year old children.

OBJECTIVES: To examine the relationship between residential proximity to agricultural fumigant use and respiratory symptoms and lung function in 7-year old children. METHODS: Participants were 294 children living in the agricultural Salinas Valley, California and enrolled in the Center for the Health Assessment of Mothers and Children Of Salinas (CHAMACOS) study. We obtained information on respiratory symptoms and asthma medication use from maternal questionnaires and children performed spirometry to determine the forced expiratory volume in one second (FEV1), forced vital capacity (FVC), and forced expiratory flow 25-75% (FEF25-75) at 7-years of age. We estimated agricultural fumigant use within 3, 5 and 8 km of residences during pregnancy and from birth to age 7 using California's Pesticide Use Report data. We evaluated the association between prenatal and postnatal residential proximity to agricultural use of methyl bromide, chloropicrin, metam sodium and 1,3-dichloropropene with respiratory symptoms and use of asthma medication with logistic regression models and continuous lung function measurements with linear regression models adjusted for confounders. RESULTS: There were no significant associations between residential proximity to use of fumigants and respiratory symptoms or use of asthma medication. We did not observe any adverse relationships between residential proximity to fumigant use and lung function measurements. Unexpectedly, we observed suggestive evidence of improved FEV1 and FEF25-75 with higher use of methyl bromide and chloropicrin during the prenatal period. For example, for each 10-fold increase in methyl bromide use during the prenatal development period we observed higher FEV1 (ß = 0.06 L/s; 95% CI: 0.00, 0.12) and higher FEF25-75 (ß = 0.15 L/s; 95% CI: 0.03, 0.27). Maternal report of child allergies (runny nose without a cold during the previous year) modified the relationship between FEV1 and prenatal proximity to methyl bromide use (p = .07) and we only observed higher FEV1 among children without allergies (ß = 0.08 L/s; 95% CI: 0.02, 0.14 for a 10-fold increase in methyl bromide use during the prenatal period). CONCLUSIONS: Residential proximity to agricultural fumigant use during pregnancy and childhood did not adversely affect respiratory health in the children through 7 years of age. These findings should be explored in larger studies.

Residential proximity to agricultural fumigant use and IQ, attention and hyperactivity in 7-year old children.

OBJECTIVES: Our objective was to examine the relationship between residential proximity to agricultural fumigant use and neurodevelopment in 7-year old children. METHODS: Participants were living in the agricultural Salinas Valley, California and enrolled in the Center for the Health Assessment of Mothers and Children Of Salinas (CHAMACOS) study. We administered the Wechsler Intelligence Scale for Children (4th Edition) to assess cognition and the Behavioral Assessment System for Children (2nd Edition) to assess behavior. We estimated agricultural fumigant use within 3, 5 and 8km of residences during pregnancy and from birth to age 7 using California's Pesticide Use Report data. We evaluated the association between prenatal (n = 285) and postnatal (n = 255) residential proximity to agricultural use of methyl bromide, chloropicrin, metam sodium and 1,3-dichloropropene with neurodevelopment. RESULTS: We observed decreases of 2.6 points (95% Confidence Interval (CI): -5.2, 0.0) and 2.4 points (95% CI: -4.7, -0.2) in Full-Scale intelligence quotient for each ten-fold increase in methyl bromide and chloropicrin use within 8km of the child's residences from birth to 7-years of age, respectively. There were no associations between residential proximity to use of other fumigants and cognition or proximity to use of any fumigant and hyperactivity or attention problems. These findings should be explored in larger studies.

Factors associated with serum polybrominated diphenyl ether (PBDE) levels among school-age children in the CHAMACOS cohort.

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants historically used in textiles, furniture, and electronic products. Recent studies have documented widespread PBDE exposure to humans, with higher levels measured in children than adults. We analyzed 10 tri- to hepta-BDE congener levels in blood collected from 7-year old Mexican-American children living in an agriculture community in California (n = 272). The most frequently detected PBDE congeners in child serum were BDE-47, -99, -100, and -153, all of which were measured in >99% of the children. We used multiple linear regression models to examine associations between child total PBDE levels (ng/g lipid) and determinants of exposure. Factors positively associated with higher PBDE levels in the children were total PBDE levels in maternal serum during pregnancy, duration of exclusive breastfeeding, and having no safe places to play in their neighborhood. Child BMI was inversely associated with serum PBDE levels (regression p-values <0.05). Our findings confirm that exposure to the penta-BDE mixture is ongoing, and that Mexican-American children living in California may be experiencing higher PBDE exposure from their environment compared to children sampled from the general U.S. population. Additional research is needed to assess the health impacts of these exposures.

Cellular function of neuropathy target esterase in lysophosphatidylcholine action.

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.

Cytokines and other immunological biomarkers in children's environmental health studies.

Environmental exposures (e.g. pesticides, air pollution, and environmental tobacco smoke) during prenatal and early postnatal development have been linked to a growing number of childhood diseases including allergic disorders and leukemia. Because the immune response plays a critical role in each of these diseases, it is important to study the effects of toxicants on the developing immune system. Children's unique susceptibility to environmental toxicants has become an important focus of the field of immunotoxicology and the use of immune biomarkers in molecular epidemiology of children's environmental health is a rapidly expanding field of research. In this review, we discuss how markers of immune status and immunotoxicity are being applied to pediatric studies, with a specific focus on the various methods used to analyze T-helper-1/2 (Th1/Th2) cytokine profiles. Furthermore, we review recent data on the effects of children's environmental exposures to volatile organic compounds, metals, and pesticides on Th1/Th2 cytokine profiles and the associations of Th1/Th2 profiles with adverse health outcomes such as pediatric respiratory diseases, allergies, cancer and diabetes. Although cytokine profiles are increasingly used in children's studies, there is still a need to acquire distribution data for different ages and ethnic groups of healthy children. These data will contribute to the validation and standardization of cytokine biomarkers for future studies. Application of immunological markers in epidemiological studies will improve the understanding of mechanisms that underlie associations between environmental exposures and immune-mediated disorders.

In utero and childhood DDT, DDE, PBDE and PCBs exposure and sex hormones in adolescent boys: The CHAMACOS study.

Dichlorodiphenyltrichloroethane (DDT), polybrominated diphenyl ether (PBDE) flame retardants, and polychlorinated biphenyls (PCBs) are believed to be endocrine-disrupting chemicals (EDCs) in humans and animals. The purpose of this study is to examine the relationship of in utero and childhood exposure to these purported EDCs and reproductive hormones in adolescent boys who participated in CHAMACOS, an ongoing birth cohort in California's Salinas Valley. We measured o,p'- and p,p'-DDT, p,p'-DDE, PBDEs and PCBs in serum collected from mothers during pregnancy or at delivery and from their sons at 9 years. We measured concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and total testosterone (T) from 234 of their sons at 12 years. In adjusted models, we found that a 10-fold increase in maternal prenatal serum concentrations of BDE-153 was associated with a 22.2% increase (95% CI: 1.0, 47.9) in FSH, a 96.6% increase (95% CI: 35.7, 184.7) in LH, and a 92.4% increase (95% CI: 20.9, 206.2) increase in T. Similarly, BDE-100 concentrations were associated with increases in boys' LH levels. A 10-fold increase in total prenatal ΣPCBs was associated with a 64.5% increase (95% CI: 8.6, 149.0) in FSH, primarily driven by non- dioxin-like congeners. Boys' hormone levels were only marginally associated with prenatal DDT or DDE in primary models, but when boys' Tanner stage at age 12 was added to models, prenatal maternal DDT levels were associated with decreases in LH (adjusted percent change per 10-fold increase=-18.5%, 95% CI: -29.8, -5.4) and T (percent change=-18.2%, 95% CI: -30.2, -4.2) and DDE with LH (percent change=-18.3%, 95% CI: -32.9, -0.6). Exposures measured in the children's serum at 9 years also showed associations between BDE-153 and ΣPCBs. However, there is evidence that these associations appear to be mediated by child BMI. This study suggests associations on male hormones of 12year old boys related to exposure to certain EDC exposure prenatally. The implications on future reproductive function in puberty and adulthood should be determined.

Early environmental exposures and intracellular Th1/Th2 cytokine profiles in 24-month-old children living in an agricultural area.

BACKGROUND: Children who reside in agricultural settings are potentially exposed to higher levels of organophosphate (OP) pesticides, endotoxin, and allergens than their urban counterparts. Endotoxin and allergens stimulate maturation of the immune response in early childhood, but little is known about the effect of exposures to OPs or to the three combined. OBJECTIVES: In this study, we investigated the relationships between these exposures and T-helper 1 (Th1) and T-helper 2 (Th2) cytokines, biomarkers of allergic asthma, in the subjects of CHAMACOS (Center for the Health Assessment of Mothers and Children of Salinas), a longitudinal birth cohort in Salinas Valley, California. Exposures were ascertained by interviewer-administered questionnaires and by home visits, and clinical diagnoses were abstracted from medical records. Blood samples were collected at 12 and 24 months of age and analyzed for Th1/Th2 status by flow cytometric detection of intracellular interferon-gamma/interleukin-4 cytokine expression. FINDINGS: Mean Th2 levels were significantly higher in children with doctor-diagnosed asthma and children with wheezing at 2 years of age. In a multiple linear regression model, exclusive breast-feeding at 1 month and pet ownership were associated with 35.3% (p < 0.01) and 34.5% (p = 0.01) increases in Th1, respectively. Maternal agricultural work and presence of gas stove in the home were associated with a 25.9% increase (p = 0.04) and 46.5% increase (p < 0.01) in Th2, respectively. CONCLUSIONS: Asthma and wheeze outcomes in children at 24 months of age are associated with elevated Th2 status in children at an early age. Our data further suggest that early exposures to an agricultural environment, breast-feeding, pets, and gas stoves affect the development of children's Th1/Th2 immune response.

Prenatal Exposure to Organophosphorous Pesticides and Fetal Growth: Pooled Results from Four Longitudinal Birth Cohort Studies.

BACKGROUND: Organophosphorous (OP) pesticides are associated with reduced fetal growth in animals, but human studies are inconsistent. OBJECTIVES: We pooled data from four cohorts to examine associations of prenatal OP exposure with birth weight (n = 1,169), length (n = 1,152), and head circumference (n = 1,143). METHODS: Data were from the CHAMACOS, HOME, Columbia, and Mount Sinai birth cohorts. Concentrations of three diethyl phosphate (ΣDEP) and three dimethyl phosphate (ΣDMP) metabolites of OP pesticides [summed to six dialkyl phosphates (ΣDAPs)] were measured in maternal urine. Linear regression and mixed-effects models were used to examine associations with birth outcomes. RESULTS: We found no significant associations of ΣDEP, ΣDMP, or ΣDAPs with birth weight, length, or head circumference overall. However, among non-Hispanic black women, increasing urinary ΣDAP and ΣDMP concentrations were associated with decreased birth length (ß = -0.4 cm; 95% CI: -0.9, 0.0 and ß = -0.4 cm; 95% CI: -0.8, 0.0, respectively, for each 10-fold increase in metabolite concentration). Among infants with the PON1192RR genotype, ΣDAP and ΣDMP were negatively associated with length (ß = -0.4 cm; 95% CI: -0.9, 0.0 and ß = -0.5 cm; 95% CI: -0.9, -0.1). CONCLUSIONS: This study confirms previously reported associations of prenatal OP exposure among black women with decreased infant size at birth, but finds no evidence of smaller birth weight, length, or head circumference among whites or Hispanics. Contrary to our hypothesis, we found stronger inverse associations of DAPs and birth outcome in infants with the less susceptible PON1192RR genotype. The large pooled data set facilitated exploration of interactions by race/ethnicity and PON1 genotype, but was limited by differences in study populations. CITATION: Harley KG, Engel SM, Vedar MG, Eskenazi B, Whyatt RM, Lanphear BP, Bradman A, Rauh VA, Yolton K, Hornung RW, Wetmur JG, Chen J, Holland NT, Barr DB, Perera FP, Wolff MS. 2016. Prenatal exposure to organophosphorous pesticides and fetal growth: pooled results from four longitudinal birth cohort studies. Environ Health Perspect 124:1084-1092; http://dx.doi.org/10.1289/ehp.1409362.

Prenatal Organophosphorus Pesticide Exposure and Child Neurodevelopment at 24 Months: An Analysis of Four Birth Cohorts.

BACKGROUND: Organophosphorus pesticides (OPs) are used in agriculture worldwide. Residential use was common in the United States before 2001. OBJECTIVES: We conducted a pooled analysis of four birth cohorts (children's centers; n = 936) to evaluate associations of prenatal exposure to OPs with child development at 24 months. METHODS: Using general linear models, we computed site-specific and pooled estimates of the association of total dialkyl (ΣDAP), diethyl (ΣDEP), and dimethylphosphate (ΣDMP) metabolite concentrations in maternal prenatal urine with mental and psychomotor development indices (MDI/PDI) and evaluated heterogeneity by children's center, race/ethnicity, and PON1 genotype. RESULTS: There was significant heterogeneity in the center-specific estimates of association for ΣDAP and ΣDMP and the MDI (p = 0.09, and p = 0.05, respectively), as well as heterogeneity in the race/ethnicity-specific estimates for ΣDAP (p = 0.06) and ΣDMP (p = 0.02) and the MDI. Strong MDI associations in the CHAMACOS population per 10-fold increase in ΣDAP (ß = -4.17; 95% CI: -7.00, -1.33) and ΣDMP (ß = -3.64; 95% CI: -5.97, -1.32) were influential, as were associations among Hispanics (ß per 10-fold increase in ΣDAP = -2.91; 95% CI: -4.71, -1.12). We generally found stronger negative associations of ΣDAP and ΣDEP with the 24-month MDI for carriers of the 192Q PON1 allele, particularly among blacks and Hispanics. CONCLUSIONS: Data pooling was complicated by center-related differences in subject characteristics, eligibility, and changes in regulations governing residential use of OPs during the study periods. Pooled summary estimates of prenatal exposure to OPs and neurodevelopment should be interpreted with caution because of significant heterogeneity in associations by center, race/ethnicity, and PON1 genotype. Subgroups with unique exposure profiles or susceptibilities may be at higher risk for adverse neurodevelopment following prenatal exposure. CITATION: Engel SM, Bradman A, Wolff MS, Rauh VA, Harley KG, Yang JH, Hoepner LA, Barr DB, Yolton K, Vedar MG, Xu Y, Hornung RW, Wetmur JG, Chen J, Holland NT, Perera FP, Whyatt RM, Lanphear BP, Eskenazi B. 2016. Prenatal organophosphorus pesticide exposure and child neurodevelopment at 24 months: an analysis of four birth cohorts. Environ Health Perspect 124:822-830; http://dx.doi.org/10.1289/ehp.1409474.

Methodologic and logistic issues in conducting longitudinal birth cohort studies: lessons learned from the Centers for Children's Environmental Health and Disease Prevention Research.

In anticipation of the National Children's Study, lessons can be learned from the smaller birth cohort studies conducted by five Centers for Children's Environmental Health and Disease Prevention Research funded by the National Institute of Environmental Health Sciences and the U.S. Environmental Protection Agency. The populations studied are diverse in ethnicity and social class and reside in urban and rural environments. Although almost all of the centers chose to enroll participants through medical care facilities, they had to develop independent staffs and structures because of the overburdened medical care system. Some of the lessons learned by the centers include the importance of continuous funding, building community partnerships to conduct culturally appropriate research, hiring bilingual and bicultural staff from the community, prioritizing research goals, developing biorepositories to ensure future utility of samples, instituting quality control procedures for all aspects of specimen and data collection, maintaining frequent contact with study participants, ensuring ethical conduct of the research in a changing medical-legal climate, and communicating results in a timely and appropriate manner to participants and the wider community. All centers underestimated the necessary start-up time, staff, and costs in conducting these birth cohort studies. Despite the logistical complexity and added expenses, all centers emphasize the importance of studying the impact of environmental exposures on those children most at risk, those living in minority and low-income communities. These centers present barriers encountered, solutions found, and considerations for future research, with the hope that the lessons learned can help inform the planning and conduct of the National Children's Study.

Association between in utero organophosphate pesticide exposure and abnormal reflexes in neonates.

The detrimental effects of organophosphate pesticide (OP) exposure on neurodevelopment have been shown in animals. The present study aimed to assess the relationship between in utero and early postnatal OP exposure and neonatal neurobehavior in humans, as measured by seven clusters (habituation, orientation, motor performance, range of state, regulation of state, autonomic stability, and reflex) on the Brazelton Neonatal Behavioral Assessment Scale (BNBAS). We assessed 381 infants < or = 2 months old and born to women participating in the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) study, a longitudinal, birth cohort study of low-income, Latina women living in the agricultural community of the Salinas Valley, California. Exposure to OP pesticides was determined by urinary levels of dialkylphosphate (DAP) metabolites, including dimethyl and diethylphosphate metabolites, measured twice during pregnancy (M = 14 and 26 weeks gestation) and once post-delivery (M = 7 days postpartum). The relationship between exposure and BNBAS performance was examined for the entire sample and stratified by the median age at assessment, 3 days. We observed a significant association between exposure and the reflex cluster for the entire sample and for infants >3 days old (n = 184). Among the >3 day old infants, increasing average prenatal urinary metabolite levels were associated with both an increase in number of abnormal reflexes (total DAP: adjusted beta = 0.53, 95% CI = 0.23, 0.82; dimethyls: adjusted beta = 0.41, 95% CI = 0.12, 0.69; diethyls: adjusted beta = 0.37, 95% CI = 0.09, 0.64), and the proportion of infants with more than three abnormal reflexes (total DAP: adjusted OR = 4.9, 95% CI = 1.5, 16.1; dimethyls: adjusted OR = 3.2, 95% CI = 1.1, 9.8; diethyls: adjusted OR = 3.4, 95% CI = 1.2, 9.9). No detrimental associations were found between postnatal urinary metabolite levels and any of the BNBAS clusters for infants < or = 3 or >3 days old at assessment. Whether neonatal reflex functioning is predictive of neuropsychological functioning as the child matures will continue to be evaluated in this birth cohort.

Effect of Organic Diet Intervention on Pesticide Exposures in Young Children Living in Low-Income Urban and Agricultural Communities.

BACKGROUND: Recent organic diet intervention studies suggest that diet is a significant source of pesticide exposure in young children. These studies have focused on children living in suburban communities. OBJECTIVES: We aimed to determine whether consuming an organic diet reduced urinary pesticide metabolite concentrations in 40 Mexican-American children, 3-6 years of age, living in California urban and agricultural communities. METHODS: In 2006, we collected urine samples over 16 consecutive days from children who consumed conventionally grown food for 4 days, organic food for 7 days, and then conventionally grown food for 5 days. We measured 23 metabolites, reflecting potential exposure to organophosphorous (OP), pyrethroid, and other pesticides used in homes and agriculture. We used linear mixed-effects models to evaluate the effects of diet on urinary metabolite concentrations. RESULTS: For six metabolites with detection frequencies > 50%, adjusted geometric mean concentrations during the organic phase were generally lower for all children, and were significant for total dialkylphosphates (DAPs) and dimethyl DAPs (DMs; metabolites of OP insecticides) and 2,4-D (2,4-dichlorophenoxyacetic acid, a herbicide), with reductions of 40%, 49%, and 25%, respectively (p < 0.01). Chemical-specific metabolite concentrations for several OP pesticides, pyrethroids, and herbicides were either infrequently detected and/or not significantly affected by diet. Concentrations for most of the frequently detected metabolites were generally higher in Salinas compared with Oakland children, with DMs and metolachlor at or near significance (p = 0.06 and 0.03, respectively). CONCLUSION: An organic diet was significantly associated with reduced urinary concentrations of nonspecific dimethyl OP insecticide metabolites and the herbicide 2,4-D in children. Additional research is needed to clarify the relative importance of dietary and non-dietary sources of pesticide exposures to young children.

Flow cytometric detection of intracellular TH1/TH2 cytokines using whole blood: validation of immunologic biomarker for use in epidemiologic studies.

Few biological markers of immune function have been thoroughly validated for use in epidemiologic studies that involve delayed sample processing and analysis. Here, we report our validation results for flow cytometric detection of intracellular T-helper 1/T-helper 2 (Th1/Th2) cytokines using 500 microL of whole blood obtained from children and adults. The detection of Th1/Th2 cytokine profiles by flow cytometry is a practical and mechanistically relevant assay because dysregulated cytokine production has been observed in many immune-mediated disorders, including cancer. We evaluated the intraassay and intraindividual and interindividual variability and the effects of a 24- to 72-hour delayed analysis on Th1 and Th2 end points. We compared the distributions of %CD4 lymphocytes, %Th1, and %Th2 in young children (age 1 year, n = 50) and adults (age 25-52 years, n = 16). Subjects sampled monthly for up to 1 year showed minimal variation in CD4, Th1, and Th2 end points. Delayed analysis of samples (up to 24 hours) resulted in no significant differences in the expression of CD4, Th1, and Th2; however, at 48 and 72 hours, all end points differed significantly from baseline (P < 0.01). A random effects model confirmed that interindividual variability was much greater than intraindividual variability for CD4 and Th1. Compared with adults, children had marginally higher %CD4, similar %Th2, but significantly lower %Th1 (P < 0.01). These results show that flow cytometric detection of CD4, Th1, and Th2 markers using whole blood is reproducible and that these biomarkers can be effectively used in human population studies that involve transported samples, delayed processing and analysis, and limited blood volumes.

Association of in utero organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population.

Although pesticide use is widespread, little is known about potential adverse health effects of in utero exposure. We investigated the effects of organophosphate pesticide exposure during pregnancy on fetal growth and gestational duration in a cohort of low-income, Latina women living in an agricultural community in the Salinas Valley, California. We measured nonspecific metabolites of organophosphate pesticides (dimethyl and diethyl phosphates) and metabolites specific to malathion (malathion dicarboxylic acid), chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphoro-thioate], and parathion (4-nitrophenol) in maternal urine collected twice during pregnancy. We also measured levels of cholinesterase in whole blood and butyryl cholinesterase in plasma in maternal and umbilical cord blood. We failed to demonstrate an adverse relationship between fetal growth and any measure of in utero organophosphate pesticide exposure. In fact, we found increases in body length and head circumference associated with some exposure measures. However, we did find decreases in gestational duration associated with two measures of in utero pesticide exposure: urinary dimethyl phosphate metabolites [beta(adjusted) = -0.41 weeks per log10 unit increase; 95% confidence interval (CI), -0.75 -- -0.02; p = 0.02], which reflect exposure to dimethyl organophosphate compounds such as malathion, and umbilical cord cholinesterase (beta(adjusted) = 0.34 weeks per unit increase; 95% CI, 0.13-0.55; p = 0.001). Shortened gestational duration was most clearly related to increasing exposure levels in the latter part of pregnancy. These associations with gestational age may be biologically plausible given that organophosphate pesticides depress cholinesterase and acetylcholine stimulates contraction of the uterus. However, despite these observed associations, the rate of preterm delivery in this population (6.4%) was lower than in a U.S. reference population.

Biological sample collection and processing for molecular epidemiological studies.

Molecular epidemiology uses biomarkers and advanced technology to refine the investigation of the relationship between environmental exposures and diseases in humans. It requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples, and minimizing future research costs by use of banked samples. Many factors, such as tissue type, time of collection, containers used, preservatives and other additives, transport means and length of transit time, affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for cytogenetic, immunological and biochemical analyses. Given the multiple uses of the samples in molecular epidemiology studies, appropriate informed consent must be obtained from the study subjects prior to sample collection. Use of barcoding and electronic databases allow more efficient management of large sample banks. Development of standard operating procedures and quality control plans is a safeguard of the samples' quality and of the validity of the analyses results. Finally, specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples, as well as communication of study results.Here, we focus on the factors affecting the quality and the potential future use of biological samples and some of the provisions that must be made during collection, processing, and storage of samples, based on our experience in the Superfund Basic Research Program and Children's Environmental Health Center, at the University of California, Berkeley.

Micronucleus frequency and proliferation in human lymphocytes after exposure to herbicide 2,4-dichlorophenoxyacetic acid in vitro and in vivo.

Widespread use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its association with non-Hodgkin's lymphoma (NHL) and other cancers has raised public concern. Here, micronucleus (MN) formation has been used as a biomarker of genotoxicity, and replicative and mitotic indices (MIs) as biomarkers of cell cycle kinetics in human lymphocytes. Cells were cultured either as whole blood or isolated lymphocytes and treated with pure or commercial forms of 2,4-D at doses between 0.001 and 1 mM for 48 h. Exposure to 2,4-D produced a minimal increase in MN in whole blood and even smaller one in isolated lymphocyte cultures. This induction took place only at levels approaching cytotoxicity and was accompanied by a significant inhibition of replicative index (RI). At a low (0.005 mM) dose of commercial 2,4-D, a small, marginally significant increase in RI (12-15%) was found in two independent sets of experiments (P=0.052). Additionally, we found that lymphocyte RI was more affected by commercial 2,4-D containing 9.4% of the chemically pure 2,4-D, than with an equal concentration of the latter suggesting that other ingredients present in the commercial pesticide may be responsible or may enhance the effect of 2,4-D. Mitotic index, however, did not show any significant change with either commercial or pure 2,4-D. The lymphocytes of 12 male applicators exposed solely to 2,4-D during a 3-month period had a significantly higher RI than the same group prior to exposure and than a control group (P<0.01), in accordance with the in vitro finding of increased RI at low doses.

Maternal urinary bisphenol a during pregnancy and maternal and neonatal thyroid function in the CHAMACOS study.

BACKGROUND: Bisphenol A (BPA) is widely used in the manufacture of polycarbonate plastic bottles, food and beverage can linings, thermal receipts, and dental sealants. Animal and human studies suggest that BPA may disrupt thyroid function. Although thyroid hormones play a determinant role in human growth and brain development, no studies have investigated relations between BPA exposure and thyroid function in pregnant women or neonates. OBJECTIVE: Our goal was to evaluate whether exposure to BPA during pregnancy is related to thyroid hormone levels in pregnant women and neonates. METHODS: We measured BPA concentration in urine samples collected during the first and second half of pregnancy in 476 women participating in the CHAMACOS (Center for the Health Assessment of Mothers and Children of Salinas) study. We also measured free thyroxine (T4), total T4, and thyroid-stimulating hormone (TSH) in women during pregnancy, and TSH in neonates. RESULTS: Associations between the average of the two BPA measurements and maternal thyroid hormone levels were not statistically significant. Of the two BPA measurements, only the one taken closest in time to the TH measurement was significantly associated with a reduction in total T4 (ß = -0.13 µg/dL per log2 unit; 95% CI: -0.25, 0.00). The average of the maternal BPA concentrations was associated with reduced TSH in boys (-9.9% per log2 unit; 95% CI: -15.9%, -3.5%) but not in girls. Among boys, the relation was stronger when BPA was measured in the third trimester of pregnancy and decreased with time between BPA and TH measurements. CONCLUSION: Results suggest that exposure to BPA during pregnancy is related to reduced total T4 in pregnant women and decreased TSH in male neonates. Findings may have implications for fetal and neonatal development.

Prenatal and postnatal bisphenol A exposure and body mass index in childhood in the CHAMACOS cohort.

BACKGROUND: Bisphenol A (BPA), a widely used endocrine-disrupting chemical, has been associated with increased body weight and fat deposition in rodents. OBJECTIVES: We examined whether prenatal and postnatal urinary BPA concentrations were associated with body mass index (BMI), waist circumference, percent body fat, and obesity in 9-year-old children (n = 311) in the CHAMACOS longitudinal cohort study. METHODS: BPA was measured in spot urine samples collected from mothers twice during pregnancy and from children at 5 and 9 years of age. RESULTS: Prenatal urinary BPA concentrations were associated with decreased BMI at 9 years of age in girls but not boys. Among girls, being in the highest tertile of prenatal BPA concentrations was associated with decreased BMI z-score (ß = -0.47, 95% CI: -0.87, -0.07) and percent body fat (ß = -4.36, 95% CI: -8.37, -0.34) and decreased odds of overweight/obesity [odds ratio (OR) = 0.37, 95% CI: 0.16, 0.91] compared with girls in the lowest tertile. These findings were strongest in prepubertal girls. Urinary BPA concentrations at 5 years of age were not associated with any anthropometric parameters at 5 or 9 years, but BPA concentrations at 9 years were positively associated with BMI, waist circumference, fat mass, and overweight/obesity at 9 years in boys and girls. CONCLUSIONS: Consistent with other cross-sectional studies, higher urinary BPA concentrations at 9 years of age were associated with increased adiposity at 9 years. However, increasing BPA concentrations in mothers during pregnancy were associated with decreased BMI, body fat, and overweight/obesity among their daughters at 9 years of age.

Utility of urinary Clara cell protein (CC16) to demonstrate increased lung epithelial permeability in non-smokers exposed to outdoor secondhand smoke.

The objective of this study was to assess the utility of urinary Clara cell protein (CC16) as a biomarker of increased lung epithelial permeability in non-smokers exposed to outdoor secondhand smoke. Twenty-eight healthy non-smoking adults visited outdoor patios of a restaurant and a bar where non-participants smoked and an open-air control with no smokers on three weekend days in a crossover study; subjects visited each site once for 3 h. Urine samples were collected at baseline, immediately post exposure and next morning, and analyzed for CC16. Changes in CC16 across location types or with cigarette count were analyzed using mixed-effect models, which included all subjects and stratified by gender. Urinary CC16 was higher in males (n=9) compared with females (n=18) at all measurement occasions (P<0.002), possibly reflecting prostatic contamination. Urinary CC16 from pre-exposure to post-exposure was higher following visits to restaurant and bar sites compared with the control among females but this increase did not reach statistical significance. Post-exposure to pre-exposure urinary CC16 ratios among females increased with cigarette count (P=0.048). Exposure-related increases in urinary CC16 were not seen among males. In conclusion, urinary CC16 may be a useful biomarker of increased lung epithelial permeability among female non-smokers; further work will be required to evaluate its applicability to males.

Variability of organophosphorous pesticide metabolite levels in spot and 24-hr urine samples collected from young children during 1 week.

BACKGROUND: Dialkyl phosphate (DAP) metabolites in spot urine samples are frequently used to characterize children's exposures to organophosphorous (OP) pesticides. However, variable exposure and short biological half-lives of OP pesticides could result in highly variable measurements, leading to exposure misclassification. OBJECTIVE: We examined within- and between-child variability in DAP metabolites in urine samples collected during 1 week. METHODS: We collected spot urine samples over 7 consecutive days from 25 children (3-6 years of age). On two of the days, we collected 24-hr voids. We assessed the reproducibility of urinary DAP metabolite concentrations and evaluated the sensitivity and specificity of spot urine samples as predictors of high (top 20%) or elevated (top 40%) weekly average DAP metabolite concentrations. RESULTS: Within-child variance exceeded between-child variance by a factor of two to eight, depending on metabolite grouping. Although total DAP concentrations in single spot urine samples were moderately to strongly associated with concentrations in same-day 24-hr samples (r ≈ 0.6-0.8, p < 0.01), concentrations in spot samples collected > 1 day apart and in 24-hr samples collected 3 days apart were weakly correlated (r ≈ -0.21 to 0.38). Single spot samples predicted high (top 20%) and elevated (top 40%) full-week average total DAP excretion with only moderate sensitivity (≈ 0.52 and ≈ 0.67, respectively) but relatively high specificity (≈ 0.88 and ≈ 0.78, respectively). CONCLUSIONS: The high variability we observed in children's DAP metabolite concentrations suggests that single-day urine samples provide only a brief snapshot of exposure. Sensitivity analyses suggest that classification of cumulative OP exposure based on spot samples is prone to type 2 classification errors.