A complete description of the CCCEH NYC cohort and study design appears elsewhere (Perera et al. 2003
). Briefly, African-American and Dominican women who resided in Washington Heights, Harlem, or the South Bronx in New York City, USA, were recruited between 1998 and 2003 into a prospective cohort study (Perera et al. 2003
). To reduce the potential for confounding, we limited enrollment to women who were in the age range of 18–35 years; non–cigarette smokers; nonusers of other tobacco products or illicit drugs; free of diabetes, hypertension, or known HIV; and who initiated prenatal care by the 20th week of pregnancy. We complied with all applicable requirements of the United States, and the institutional review board of the New York Presbyterian Medical Center approved the study. All women provided written informed consent prior to study initiation and at each visit; children provided assent starting at 7 years of age.
Personal interview and HOME inventory.
A trained bilingual interviewer administered a 45-min questionnaire during the last trimester of pregnancy to obtain demographic information, residential history, and health and environmental data such as active and passive smoking. ETS exposure was self-reported as having at least one smoker in the home, dichotomized as a yes/no variable (Perera et al. 2003
). The questionnaire also elicited information on dietary PAH (consumption of broiled, fried, grilled, or smoked meat), and socioeconomic information related to income and education (Perera et al. 2003
). Postnatal maternal interviews were administered in person when the child was 6 months and annually thereafter to determine any changes in residence, exposure to ETS, and other health or environmental conditions. The Home Observation for Measurement of the Environment (HOME) Inventory (Bradley 1994
) was used to assess the quality of the home caretaking environment. Self-reported maternal demoralization was measured by the Psychiatric Epidemiology Research Instrument Demoralization Scale (Dohrenwend et al. 1978
). We administered the Test of Nonverbal Intelligence-Second Edition (TONI-2), a language-free measure of intelligence (Brown et al. 1990
; Caldwell and Bradley 1979
) to the mothers at about child age 3 years.
Umbilical cord blood (30–60 mL) was collected at delivery (Perera et al. 2004
). The nuclease P1 digestion enhancement procedure of the 32
P-postlabeling assay was used to analyze bulky/hydrophobic DNA adducts in umbilical cord blood samples having a sufficient yield of DNA (≥ 12 µg). The 32
P-postlabeling assay is highly sensitive and can be used to detect various DNA adducts with multiple structures (Phillips and Arlt 2007
). It can detect adducts in the range of one per 108
nucleotides using a 4-µg DNA sample. The bulky/hydrophobic DNA adducts detected by the assay include those formed by PAHs and other genotoxic carcinogens, for example, nitro-PAH and aromatic amines.
In this assay, tissue DNA is degraded enzymatically to mononucleotides; these are then 32
P-labeled via T4 polynucleotide kinase-catalyzed [32
P]phosphate transfer from [γ-32
P]ATP to form 5´-32
P-labeled 3´,5´-bisphosphate derivatives. The labeled products are resolved by multidirectional thin-layer chromatography and detected and quantified as described (Phillips and Arlt 2007
; Randerath et al. 1989
). An aliquot (4 µg) of each DNA sample was analyzed on three separate occasions in batches of between 20 and 28 samples. For each assay, a positive control consisting of DNA modified with BaP diol-epoxide was also analyzed.
Lead was measured at the Centers for Disease Control and Prevention (CDC) in a subset of cord bloods (n
= 153) using inductively coupled plasma mass spectrometry (CDC/Division of Laboratory Science 2003). To adjust for postnatal exposure to PAH, urinary PAH metabolites were measured in a subset of children at 5 years of age (n
= 102). Urine was analyzed, as DNA from blood samples was not available for this assay and urinary PAH metabolites are a well-validated internal dosimeter for these compounds. The CDC measured a suite of 22 PAH metabolites, using enzymatic deconjugation, followed by automated liquid–liquid extraction and quantified by gas chromatography/isotope dilution high-resolution mass spectrometry (Li et al. 2008
Research workers trained in neurodevelopmental testing administered the CBCL to the mothers, using the 99-item CBCL for children < 6 years of age (Achenbach and Rescorla 2000
) and the 118-item CBCL for children ≥ 6 years (Achenbach and Rescorla 2001a
). The syndrome scores were computed for each domain of a priori
interest (Anxious/Depressed and Attention Problems) by summing the scores on the specific items, which yields a raw score. This raw score can be converted to a standardized T-score. We report results from two versions of the CBCL administered at two different ages; the two versions have different numbers of items and distributions of scores. T-scores were generated by the CBCL software according to the procedure of Abramowitz and Stegun (1968)
. A T-score of 50 is assigned to children with percentiles of raw scores ≤ 50 based on a reference population (Achenbach and Rescorla 2000
), whereas those with percentiles of raw scores > 50 are assigned the actual T-score.
The CBCL also yields scales derived from the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) (American Psychiatric Association 2000
), that are intended to approximate clinical diagnoses. The DSM-IV scores are dichotomized using a borderline or clinical cut point corresponding to the 93rd percentile for each domain (Achenbach and Rescorla 2000
). Based on our a priori
hypothesis, we focused on the DSM-oriented Anxiety Problems in our analysis.
Statistical methods. Cord DNA adducts (range 0.2–24.8 adducts/108 nucleotides) were dichotomized at the upper quartile (2.7 adducts/108 nucleotides) of the 461 children with data on DNA adducts and maternal prenatal questionnaire data. Among the 215 children in the present analysis, we identified 58 and 157 subjects as high exposed and low exposed, respectively. Although the dichotomized adduct variable is our independent variable of choice, we have also used logarithm-transformed adducts as a continuous variable. Covariates were selected based on whether they were significant contributors to the model (here at p ≤ 0.1) for at least one of the outcomes. Although ethnicity (African American or Dominican) was not a significant covariate in the model, it was included because it is highly correlated with the CBCL scores and there is a significant difference in ethnicity between the subjects included and those not included. Covariates in the models included ETS exposure during pregnancy, sex of child, gestational age of the child, intelligence of mother as measured by TONI-2, completed years of education of the mother before birth of the child, age of child at assessment in months, quality of the early home caretaking environment measured at 3 years of age, and season of the last trimester (either during the heating season from 1 October to 30 April, when ambient PAH levels are higher, or not). Gestational age was based on medical record data for almost all subjects. Covariates are defined in .
Characteristics of the sample (mean ± SD or %).
With respect to CBCL syndrome scores, both the continuous raw scores and the dichotomized T-scores were analyzed. Because the raw scores of each domain of interest are counts data that sum the scores on the specific items within each syndrome scale, we applied the Poisson model on the raw scores for the syndromes. We further analyzed the syndrome T-scores dichotomized at 65 (the cutoff for scores for the borderline and clinical range) (Achenbach and Rescorla 2000
) using a logistic model. We used logistic regressions for analysis of dichotomized DSM-oriented Anxiety Problems (cutoff at 93rd percentile for the borderline and clinical range).
To estimate associations between PAH and behavior outcomes at different ages, two separate analyses were done on CBCL measures at (mean) 4.8 years (n = 96) and (mean) 7 years (n = 205). Note that 86 children had measures at both time points.
To check for the possible confounding effect of postnatal PAH exposure, we fit a separate model with CBCL measurements of children at 7 years adjusting for PAH metabolites in child urine collected at 5 years of age. Because of incomplete data on postnatal PAH exposure, the model with postnatal PAHs had a smaller sample size (n = 100 at age 7 years). Moreover, to check the possible confounding effect of postnatal ETS exposure, we fit separate models adjusting for postnatal ETS exposure measured at 2 years of age. Similarly, because of incomplete data on postnatal ETS exposure, the separate models had smaller sample sizes (n = 85 at age 4.8 years and n = 182 at age 7 years). We also fit a model that included an interaction term between adducts and sex of the child to check for possible interactions.
All effect estimates, 95% confidence intervals (CIs), and p-values (α set at 0.05) were generated using SAS (version 126.96.36.199; SAS Institute Inc., Cary, NC, USA).