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Background: In the light of the ongoing debate about lowering the cut-off for acceptable blood lead level to <5 μg/dL from the currently recommended level of <10 μg/dL, we considered whether prenatal exposure to varying levels of lead is associated with similar or disparate effects on neonatal behavior. Methods: Using Brazelton's Neonatal Behavioral Assessment Scale (NBAS), an epidemiological approach and robust statistical techniques like multivariate linear regression, logistic regression, Poisson regression and structural equations modeling analyses we estimated the simultaneous indirect effects of umbilical cord blood lead (CBL) levels and other neonatal covariates on the NBAS clusters. Results: We observed that when analyzed in all study subjects, the CBL levels independently and strongly influenced autonomic stability and abnormal reflexes clusters. However, when the analysis was restricted to neonates with CBL <10 μg/dL, CBL levels strongly influenced the range of state, motor and autonomic stability clusters. Abnormal walking reflex was consistently associated with an increased CBL level irrespective of the cut-off for CBL, however, only at the lower cut-offs were the predominantly behavioral effects of CBL discernible. Conclusion: Our results further endorse the need to be cognizant of the detrimental effects of blood lead on neonates even at a low-dose prenatal exposure. at low doses of exposure, environmental lead continues to Background There is an ongoing debate over the appropriate cut-off of be a biological and social toxicant [4,5,7,8]. Recently, blood lead concentration to detect lead poisoning [1-6]. there is a burgeoning recognition that even at low doses Starting from 60 μg/dL the cut-off recommended by the exposure to lead has serious implications on a child's Centers for Disease Control (CDC) receded to 25 μg/dL behavior pattern. For example, lead exposure in low doses and then to the currently used value of 10 μg/dL. This has been convincingly implicated in juvenile delinquency was essentially due to a series of studies showing that even [9,10], intelligence quotient (IQ) patterns [4,11-18] and Page 1 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 crime rates [19,20]. In the light of these findings, Needle- scale consists of the 28 behavior-related items scored on a man and others recommend that the time has arrived to 9-point scale, 18 reflexes and 7 supplementary items. Two lower the CDC recommended cut-off for blood lead to 5 trained pediatricians administered the scale. Before the μg/dL . study began, these two investigators independently and together evaluated a separate set of 20 neonates to ensure Blood lead has also been considered for a long time to be concordance of observations. The NBAS was administered a behavioral teratogen. Interestingly, however, literature within three days of birth. Since the arousal state can on the putative association of the prenatal blood lead influence a newborn's performance on the individual exposure with the behavioral prototypes in the newborns items of the NBAS scale , we noted the initial state is scant and inconsistent . For example, Ernhart et al (the state of the newborn at the beginning of the NBAS , Rothenberg et al  and more recently Emory et al evaluation) and predominant state (the state which the  could not demonstrate any striking association newborn was most commonly in over the duration of between umbilical cord blood lead level and neonatal NBAS assessment and which was recorded at the end of behavior. In contrast, two recent prospective studies have the NBAS evaluation) of the newborn. We converted the – using the Mental Development Index (MDI) – shown raw scores on the NBAS items into the following seven association of low-exposure to lead with the neurobehav- clusters as recommended by Lester et al : habituation, ioral development in early life [24,25]. Additionally, since orientation, motor, range of state, regulation of state, neonatal behavior is a multi-dimensional construct with autonomic stability and abnormal reflexes. The associa- several hard-to-measure and correlated domains, the ana- tion of the predictor variables was then assessed with the lytical strategy to test the association between blood lead cluster scores. levels and behavioral indicators is not always straightfor- ward [2,26]. Blood lead measurement Cord blood samples (5 ml) were obtained for each We therefore undertook this study to address two research neonate in a metal-free K3 EDTA bulb and analyzed questions: a) Do umbilical cord blood lead (CBL) levels within 48 hours of sample collection for blood lead by independently correlate with the early neonatal neurobe- flameless atomic absorption spectrophotometry (Hitachi havioral pattern? b) Do these neurobehavioral associa- Z-8000) in parts per billion at a wavelength of 283.3 nm tions, if any, continue to be present in neonates with CBL with a slit width of 1.3 nm using the method described by levels below 10 μg/dL? We hypothesized that the behavio- Lagesson et al . The detection rate of lead for the ral archetypes of neonates are influenced by the level of instrument was 1 μg/l, with an average error rate of 5% for prenatal exposure to lead even at relatively low doses of reproducibility of results. The samples were analyzed for exposure. To test this hypothesis, we conducted a cross- estimation of the lead concentration within 48 hours of sectional study assessing the association between umbili- collection. cal cord blood lead levels and the neonatal neurobehavio- Covariates ral responses using appropriate measurement scales and statistical models. Table 1 describes the characteristics of the study subjects. In multiple linear regression analyses (described below), Methods we used the following covariates: maturity, hours of birth, Study subjects sex, birth weight, head circumference, fetal and maternal The present cross-sectional study was conducted at the obstetric problems, specific disorder in fetus/newborn, Government Medical College and Hospital, a tertiary care problem noted during labor, use of oxytocic agents, rup- hospital in Nagpur, India. The data were collected over a ture of membranes before onset of labor, tobacco intake four-month period starting from January 1998. All con- by the mother and alcohol intake by the mother. The secutively born neonates at the study center whose meaning and description of some of these covariates is mother gave an informed consent were included in the provided in details in Supplementary Table 1 (see addi- study. Overall, 230 children were included. However, tional file 1, supplementary table 1). The covariates were blood lead measurements were available on 176 (~77%) measured based on the antenatal medical records, labor of the neonates who comprised our study sample. The notes and by interviewing the mothers. study was approved by the Ethical Committee of the Gov- ernment Medical College, Nagpur, India. Statistical analysis Our general strategy for statistical analysis was to test the Study variables association between cord blood lead levels and each Outcomes NBAS cluster score in univariate and multivariate con- We measured the neonatal behavior using Brazelton's texts. Since, in theory, the NBAS clusters represent essen- Neonatal Behavioral Assessment Scale (NBAS) . The tially orthogonal i.e. uncorrelated factors, we used the Page 2 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 Table 1: Characteristics of the neonatal study subjects (n = 176) Maturity in weeks (mean ± S.D) 38.93 ± 3.45 Hours of birth (mean ± S.D) 45.78 ± 15.15 Sex Male (n, %) 99 (56.3) Female (n, %) 77 (43.7) Birth weight (g, mean ± S.D) 2644.35 ± 413.15 Head circumference (cm, mean ± S.D) 32.47 ± 2.13 Umbilical cord blood lead level (μg/dL, mean ± S.D) 5.15 ± 12.65 Fetal obstetrical problem (n, %) Yes 6 (3.4) No 157 (89.2) Unknown 13 (7.4) Specific disorder in fetus/neonate (n, %) Yes 17 (9.6) No 145 (82.4) Unknown 14 (8.0) Maternal obstetrical problem (n, %) Yes 44 (25.0) No 119 (67.6) Unknown 13 (7.6) Problem noted during labor (n, %) Yes 59 (33.5) No 109 (61.9) Unknown 8 (4.6) Use of oxytoxic agents during labor (n, %) Yes 44 (25.0) No 127 (72.2) Unknown 5 (2.8) Rupture of membranes before labor onset (n, %) No 135 (76.7) Less than 24 hours 29 (16.5) Page 3 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 Table 1: Characteristics of the neonatal study subjects (n = 176) (Continued) 24 to less than 72 hours 5 (2.8) 72 to less than 120 hours 1 (0.6) More than 120 hours 1 (0.6) Unknown 5 (2.8) Maternal medical problem during this pregnancy (n, %) Yes 30 (17.1) No 137 (77.8) Unknown 9 (5.1) Tobacco intake by mother (n, %) No 164 (93.2) Yes 8 (4.6) Unknown 4 (2.2) Alcohol intake by mother (n, %) Yes 3 (1.8) No 169 (96.0) Unknown 4 (2.2) House painted (n, %) No or white wash 98 (55.7) Yes, some 35 (19.9) Yes, complete 39 (22.2) Unknown 4 (2.2) Age of house paint (n, %) < 5 years 62 (83.8) 5 – 10 years 7 (9.5) Unknown 5 (6.7) NBAS cluster scores (mean ± S.D) Habituation 28.91 ± 3.29 Orientation 43.06 ± 8.19 Motor 26.60 ± 3.69 Range of state 16.05 ± 3.83 Regulation of state 18.69 ± 5.38 Autonomic stability 14.12 ± 3.29 Abnormal reflexes 2.37 ± 1.98 Page 4 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 score for each NBAS cluster as an outcome. For estimating alpha error rate of 0.05 was used to test statistical signifi- the unadjusted influence, we used only CBL level as the cance. predictor. Subsequently in a multiple linear regression model we estimated the adjusted influence of CBL for Results each NBAS cluster score by including the covariates men- The characteristics of the study subjects are described in tioned above, the initial and predominant states of Table 1 and Supplementary Table 1 (see additional file 1, arousal (Table 2). It was essential to include both initial supplementary table 1). Only two (1.1%) neonates were and predominant states in the multiple regression models premature (<32 weeks), 10 (11.3%) had head circumfer- because there two variables were not completely collinear ence less than 30 cm, eight (4.6%) were small (birth with each other indicating that in a given infant often the weight < 2 kg), three (1.7%) were very small (birth weight initial state was not the same as the predominant state < 1.5 kg) and 14 (8.0%) had cord blood lead exceeding 10 (Spearman's rho = 0.093, p = 0.1938). Lastly, only for the μg/dL. In general, therefore our study sample mostly "abnormal reflexes" cluster we used single and multiple included healthy neonates. This was also reflected by the Poisson regression analyses because the scores for this mean scores for each of the NBAS clusters as shown in cluster actually represent the count of the number of Table 1. During the NBAS evaluation, the most common abnormal reflexes. initial states were light sleep (65 neonates, 36.9%), deep sleep (43 neonates, 24.4%) and alertness (35 neonates, Our next step of analysis was to assess the association of 19.9%) while the most common predominant states were the CBL levels with the NBAS cluster scores in a multivar- alertness (70 neonates, 39.7%), open eyes (49 neonates, iate context. For this purpose, we first conducted analysis 27.8%) and crying (31 neonates, 17.6%). of covariance (ANCOVA) using each NBAS cluster as the outcome and CBL as the predictor – first alone (unad- CBL and NBAS cluster scores justed analysis) and then using initial and predominant The results shown in Table 2 indicated that when the anal- states as covariates (adjusted analysis). Using the results yses were conducted in all study subjects, the CBL levels from these analyses, we tested for the influence of blood significantly correlated with the autonomic stability and lead on multiple outcomes using the Multiple Indicator abnormal reflexes clusters even after adjustment for the Multiple Causes (MIMIC) model under the umbrella of aforementioned covariates. However, when the same Structural Equations Modeling (SEM). The details of the analyses were performed in neonates with CBL levels <10 MIMIC model that we employed in our analyses are μg/dL, the unadjusted analyses identified the association described below. of the CBL levels with the range of state and regulation of state clusters but the adjusted model identified the associ- Additionally, we used Poisson regression to test the asso- ation with orientation and regulation of state clusters. We ciation between blood lead and the number of abnormal also considered whether the association of CBL with each reflexes and multiple logistic regression analysis to test the NBAS cluster is specifically influenced by the potential association between various reflexes and dichotomized effect of the initial and predominant states of the newborn values of blood lead as described in the succeeding sec- on the NBAS cluster scores and found, using ANCOVA, tions. We used Stata 8.0 (Stata Corp, College Station, TX) that it was not (see additional file 1, supplementary table and Amos 5.0 (Amos Development Corp, Spring House, 2). This first pass analysis through the multiple regression PA) for statistical analyses. Unless specified otherwise, an models and ANCOVA thus indicated that i) The CBL lev- Table 2: Results of regression analyses for prediction of NBAS cluster scores based on CBL and other covariates in all neonates (left column) and neonates with CBL levels <10 μg/dL. NBAS cluster All Neonates Neonates with CBL < 10 μg/dL Unadjusted (coefficient, p) Adjusted (coefficient, p) Unadjusted (coefficient, p) Adjusted (coefficient, p) Habituation 0.0145, 0.468 0.0292, 0.213 -0.0432, 0.812 -0.0057, 0.988 Orientation 0.0092, 0.853 0.0176, 0.753 0.2823, 0.518 1.5972, 0.053 Motor 0.0188, 0.446 0.0108, 0.724 -0.2733, 0.136 0.4154, 0.282 Range of state -0.0304, 0.196 -0.0419, 0.085 -0.5135, 0.008 -0.1957, 0.548 Regulation of state 0.0030, 0.930 0.0458, 0.336 -0.7138, 0.010 -1.2912, 0.036 Autonomic stability -0.0567, 0.008 -0.0506, 0.077 -0.1219, 0.462 -0.3156, 0.507 -5 Abnormal reflexes* 0.0118, 6.8 × 10 0.0073, 0.084 -0.0487, 0.163 -0.1049, 0.168 List of the covariates is provided in the Methods section, Study Variables subsection * Estimated using Poisson regression analysis Page 5 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 els correlated with specific NBAS clusters; ii) The CBL lev- Simultaneous effects of CBL on neonatal behavior: els were differentially associated with NBAS clusters in all specification of the MIMIC model To be parsimonious, we wanted to select the most signifi- subjects versus subjects with CBL levels below 10 μg/dL and iii) The association of CBL levels with NBAS clusters cant NBAS clusters that were least likely to be correlated varied between the unadjusted and adjusted analyses in with each other. For this purpose, using a reverse neonates with low-dose prenatal lead exposure. approach, we first used CBL levels as the outcome and the NBAS cluster scores as the predictors. We conducted step- Correlation among NBAS cluster scores wise linear regression with a strict retention probability Even though the NBAS clusters are theoretically uncorre- criterion of 0.05. The clusters that were retained in the lated, we assessed if the correlations among these clusters final model (see additional file 1, supplementary table 4) were dataset-specific. To consider this possibility and the were motor, autonomic stability and abnormal reflexes in implications thereof, we first assessed the correlation all neonates and range of state in neonates with CBL levels structure of the seven NBAS clusters in all neonates as well below 10 μg/dL. Therefore, we chose these four clusters as as in neonates with CBL levels below 10 μg/dL (Figure 1 outcomes for modeling the simultaneous effects of CBL. and additional file 1, supplementary table 3). Not surpris- This choice of the NBAS clusters was also consistent with ingly, we observed that there were a number of statistically the observed correlation structure since habituation and significant correlations between pairs of NBAS clusters. orientation were strongly correlated with the motor clus- Specifically, the habituation, orientation and motor clus- ter while regulation of state was moderately correlated ters were strongly correlated with each other while the with the range of state. range of state and regulation of state clusters showed a trend towards a significant correlation with each other in We then chose four neonate-related predictors which we all neonates as well as in neonates with CBL levels below modeled as the covariates – CBL levels, head circumfer- 10 μg/dL. Arguably, this correlation structure can alter the ence, maturity and birth weight. There were three reasons interpretations regarding the simultaneous influence of for choosing this set of covariates. First, there exists litera- the predictors on the NBAS clusters. Therefore, we chose ture support for a putative association of these covariates to conduct further analyses in which we modeled the with NBAS cluster scores. Second, in a series of stepwise influence of CBL levels and other covariates simultane- regression models in our dataset, these variables were con- ously on the NBAS clusters. sistently associated with one or more of the NBAS clusters (see additional file 1, supplementary table 5). Finally, as these variables can be considered to be of a continuous disposition, the correlation matrices to be used in struc- tural equations modeling are more reliable and easier to construct and require no preprocessing of the data. The path diagram of our model (Figure 2A) thus con- tained four predictors and four outcomes. In SEM, a model of this nature is referred to as the Multiple Indica- tor Multiple Causes (MIMIC) model . In the proposed MIMIC model, none of the predictors directly influences any of the outcomes, that is, there exists no direct arrow in the path diagram (Figure 2A) from any predictor to any outcome – they all pass through a conceptual, latent and Correlation structure of the n dL (B) Figure 1 eonates (A) and neonates with NB the CBL levels below 10 AS cluster scores in all μg/ unmeasured variable. We argue that these four predictors Correlation structure of the NBAS cluster scores in influence a latent (unobserved) trait which we refer to as all neonates (A) and neonates with the CBL levels the "neonatal behavior". In our model, the NBAS clusters below 10 μg/dL (B). The color codes at the bottom pro- were thus considered as indicators of the neonatal behav- vide a reference for the magnitude and significance of the ior. Pearson correlation coefficients. Open boxes represent sta- tistically non-significant correlation coefficient. The actual We modeled the influence of the predictors on neonatal estimates of correlation coefficients and their significance val- behavior and on the four outcomes within the framework ues are shown in Supplementary Table 3 (see additional file 1, of structural equations modeling (SEM). The regression supplementary table 3). The NBAS clusters shown here are: weights (parameters labeled as r to r in Figure 2A) thus habituation (HAB), orientation (ORI), motor (MOT), range 1 7 measure the influence of the predictors on each outcome of state (RAN), regulation of state (REG) and autonomic sta- bility (AUT) and abnormal reflexes (REF). in a multivariate context. The random errors of measure- ment associated with all observed variables – four predic- Page 6 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 Figure 2 Structural equations modeling of the influence of neonatal predictors on the NBAS clusters Structural equations modeling of the influence of neonatal predictors on the NBAS clusters. (A) The MIMIC model. The details of this model are given in text. Rectangles represent observed variables, circles represent latent variables, one-headed arrows represent influence and double-headed arrows represent covariance. The numbers or identifiers along the arrows are the model parameters. For ease of identification, the one-headed arrows of interest are color coded. Parameters e –e represent the errors in measurement of observed variables. (B and C) Standardized regression coefficients for the color 1 8 coded influences shown in panel A. Numbers indicate the statistical significance. The analysis was first conducted in all neonates (B) and then in neonates with CBL levels below 10 μg/dL (C). (D and E) Standardized indirect effects of the neonatal predic- tors (x-axis) on the NBAS cluster scores (y-axis). The z-axis represents the magnitude of the effect. Red cylinders indicate a negative effect while green cylinders indicate a positive effect. The analysis was conducted in all neonates (D) and then in neonates with CBL levels below 10 μg/dL (E). Complete results of SEM are shown in Supplementary Table 6 (see additional file 1, supplementary table 6). Abbreviations for the NBAS clusters are: motor (MOT), range of state (RAN), autonomic stability (AUT) and abnormal reflexes (REF). Page 7 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 Table 3: Association of NBAS items with risk of possessing high comes were not influenced. This analysis thus recaptured CBL levels: results from final models using stepwise multiple the observations from the previous analysis that even in a logistic regression analyses. multivariate and multiple-outcome context the independ- ent influence of CBL on autonomic stability and abnor- NBAS Item Risk of CBL levels > the shown cut-off point mal reflexes was discernible. OR 95% CI P When the analysis was restricted to neonates with CBL lev- els below 10 μg/dL, we observed a notable shift in the pat- 5 μg/dL tern of association. The CBL levels were now the only statistically significant predictor and the influence on the Range of State cluster neonatal behavior was limited to the motor, range of state Peak of excitement 0.60 0.37 – 0.98 0.042 and autonomic stability clusters. Thus, concordant with Autonomic stability the earlier results, our results of MIMIC modeling reaf- cluster Tremors 0.77 0.63 – 0.94 0.012 firmed that the dominant effects of CBL were different in Abnormal reflexes all neonates compared to neonates with low-dose expo- Moro's reflex 3.37 1.13 – 10.04 0.029 sure to lead. Walking reflex 3.55 1.24 – 10.15 0.018 Our results of the SEM modeling indicated that the model 10 μg/dL fit was not adequate either for all neonates or for neonates with CBL <10 μg/dL. We further investigated the reason Autonomic stability for this apparent lack of fit for which purpose we assessed cluster Tremors 0.75 0.58 – 0.96 0.023 the predictive performance of 10 other models nested Abnormal reflexes within the model shown in Figure 2A. While constructing Babinski sign 4.26 1.01 – 17.8 0.047 the nested models, we considered all combinations of the Walking reflex 5.99 1.44 – 24.9 0.014 four predictors taken three at a time and then taken two at a time. These 10 models and their performance is shown 25 μg/dL (see additional file 1, supplementary table 7) in Supple- mentary Tables 7A (for all neonates) and 7B (for neonates Abnormal reflexes with CBL <10 μg/dL). A close look at the model fits for Babinski sign 11.3 1.89 – 68.1 0.008 these nested models revealed the following: i) Removal of Walking reflex 8.17 1.36 – 49.2 0.022 CBL as a predictor from the MIMIC model always wors- ened the model fit; ii) Inclusion of maturity and head cir- tors and four outcomes – were included as shown cumference was most of the times associated with a poor (parameters labeled as e to e in Figure 2A). Since the fitting model; iii) The best model for all neonates was 1 8 measurements of NBAS clusters are correlated, we assume with two predictors: CBL and maturity; and iv) The best that the measurement errors associated with these varia- model for neonates with CBL <10 μg/dL contained CBL bles will also be correlated (shown by the curved arrows and head circumference. Thus, the full model with all four in the model and the parameters labeled as c to c ). predictors was associated with a poor model fit but we 1 6 Finally, to make the model identifiable, we constrained have shown it here only because it permitted us to study the head circumference → neonatal behavior regression the effects of CBL adjusted for other potential confound- weight to unity. ers. Results from the MIMIC model Association of increased CBL levels on items within the Figure 2B–E and Supplementary Table 6 (see additional significantly associated NBAS clusters file 1, supplementary table 6) show the results of SEM Given the significant multivariate effects of CBL levels on analyses using the MIMIC model. As the predictor and the four NBAS clusters included in the MIMIC model outcome variables are measured on different metrics, we analyses in the previous step, we next considered whether present the data in the form of standardized estimates of there were any specific items within these clusters that the regression coefficients (Figure 2B and 2C). We were associated with the risk of increased CBL levels. For observed that when the analysis was conducted in all this purpose, we dichotomized the CBL levels into high neonates, CBL levels and maturity independently influ- and low using three different cut-off points: 5, 10 and 25 enced neonatal behavior – more mature neonates had a μg/dL. Using each of these binary outcomes we used back- better behavior score. Interestingly, this influence of CBL ward stepwise unconditional multiple logistic regression and maturity was detectable only with respect to auto- analyses with a probability criterion of 0.05 to identify the nomic stability and abnormal reflexes – the other two out- NBAS items most significantly associated with the likeli- Page 8 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 hood of possessing CBL levels exceeding these cut-offs. of locomotion control. Our findings that the motor, range The results of these analyses are shown in Table 3. of state, autonomic instability and the abnormal reflexes NBAS clusters were specifically associated with the CBL: i) We observed that not all items within each cluster were corroborate the conjecture that all domains of neonatal significantly associated with the risk of an increased CBL behavior will not be equally influenced by exposure to level. For example, if a high value (>25 μg/dL) for the CBL lead; and ii) are consistent with the known behavior- cut-off was used then only two abnormal reflexes – Babin- related functions of those areas in the human brain that ski's sign and walking reflex – were significantly associ- have been shown in animal studies to be the primary tar- ated. At the currently used cut-off of >10 μg/dL, tremors gets for the effects of exposure to lead. were additionally identified to be significantly associated, while at a lower cut-off of >5 μg/dL, the peak of excite- Second, and more interestingly, we found that the NBAS ment also was significantly associated. Moreover, Moro's clusters associated with CBL levels in all neonates were reflex rather than Babinski's sign was the significant not the same as the NBAS clusters identified by restricting abnormal reflex. the analyses to low levels of exposure. In neonates with CBL <10 μg/dL, we did not observe an association of the varying CBL levels with the abnormal reflexes cluster but Discussion In the process of human brain development the perinatal did uncover an association with the motor cluster. These period characterizes a critical interval during which there data indicate that relatively higher values of CBL will be is highest rate of brain development, rampant genesis of needed for lead to demonstrate its influence on the abnor- new synapses, widespread neuronal proliferation, and mal reflexes; however at a relatively milder dose it may maximum density of the N-methyl-D-aspartate (NMDA) continue to demonstrate an association with the motor, receptors [31-37]. The last of these facts bears a special rel- autonomic instability and range of state clusters. Evidence evance to lead neurotoxicity since it has been argued that to support the deleterious effects of low-dose lead expo- ++ the Ca permeable NMDA receptors also act as the neuro- sure on human neonatal behavior is continuously increas- ++ nal gateway for Pb . Therefore the newborn brain is ing [24,25,52,53] however a novel finding of the present especially prone to the toxic effects of environmental neu- study is that the patterns of behavior are different in rotoxicants  and can be expected to be sensitive to neonates with CBL <10 μg/dL as compared to those with even low doses of lead exposure. Based on this biological a higher dose of exposure. rationale, using the NBAS administered within three days of birth and employing multivariate statistical approaches Study limitations for analysis, we observed that umbilical cord blood lead Our study suffers from three limitations. First, for the rea- levels were significantly associated with different aspects sons explained earlier, the main focus of our study was the of the neonatal behavior even at relatively low doses of behavioral patterns in the newborn which we assessed exposure. using NBAS. However, this is a cross-sectional study design – a fact that does not permit inferences about the Study findings potential causal role of low-dose lead exposure We observed that the association of CBL levels with NBAS [25,54,55]. clusters was differential in two respects. First, not all NBAS clusters were equally associated with the CBL levels. Bio- Second, a single measurement of umbilical cord blood logically, since the development of the newborn brain is lead is unlikely to faithfully capture the overall cumulative neither simultaneous nor equivalent across all areas exposure to lead  thereby making our measurement of [26,39-41]; it can be expected that the influence of lead lead exposure questionable. We did not have data on may not be alike on all areas of the developing brain. serial measurements of the lead concentrations in Indeed, several experimental studies have demonstrated mother's blood over the entire duration of pregnancy. that in the rat models of lead toxicity, the predominantly Our rationale for using umbilical CBL was based on the affected brain areas include the hippocampus [42,43], the following observations: i) As reported by previous studies, hypothalamus , the prefrontal cortex , the tempo- the correlation coefficient between maternal and umbili- ral cortex  and the cerebellum . In humans, the cal cord blood lead levels ranges between 0.55 to 0.92 posterior hippocampus has been shown to be associated [56,57]; ii) All through gestation, lead is known to cross with behavior , the prefrontal cortex is known to con- the placenta and is considered to be the most important trol cognitive functions like language, abstract reasoning, source of umbilical cord blood lead ; and iii) inde- problem solving, social interactions, and planning pendent of the maternal bone lead – an index of the [49,50], the temporal lobe along with portions of hippoc- cumulative lead exposure – umbilical cord blood lead has ampus and prefrontal cortex has been implicated in object been shown to be a significant predictor of child develop- working memory  while cerebellum is the known seat ment . Considering these pieces of evidence from the Page 9 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 literature and the absence of serial measurements of Another area of interest in the field of lead poisoning maternal blood lead in our study, we used umbilical cord relates to the policies and practice of screening. Sargent blood lead as a surrogate for the cumulative lead exposure and others  argue that in order to reduce the false pos- of the newborn. itive error rate, it may be unwarranted to screen for chil- dren with blood lead levels between 10 and 15 μg/dL. As Third, we did not have data on co-exposure of the new- an alternative, Binns et al  suggested high-risk popula- born to other toxicants like cadmium and polychlorinated tion screening. In situations where blood lead tests may biphenyls which can also imitate some of the effects of not be easily or inexpensively available, it has also been lead [54,59,60]. In the absence of this data, our study will thought to consider the use of blood lead questionnaires not be able to definitively point towards a causal role of [68,69]. In that vein, we identified only a few NBAS items lead, however the compatibility of our findings with the to be specifically correlated with the risk of possessing existing literature and the robust analytical methods used high CBL levels. Our findings imply that peak of excite- in this study urge the consideration of a plausible role of ment, tremors and abnormal Babinski's sign and walking low-dose lead exposure in determining the patterns of reflexes may together serve as a potential initial screen to neonatal behavior. identify neonates possessing moderate to high CBL levels. While our study was not designed to address the issue of Study implications screening for lead toxicity, our results suggest that With the caveats mentioned in the preceding section, we neonates with the aforementioned characteristics may believe that our study has three important implications. need a further evaluation with a special emphasis on lead First, it is not currently known whether the neonates who poisoning. are affected by the low levels of lead exposure grow into children more likely to be affected with regards to their Conclusion overall mental health. However, it has been observed that Needleman  believes that we are now into a "fifth children exposed to low doses of lead show suboptimal cycle" of understanding the effects of this commonest cognitive functioning and reduced intelligent quotients environmental toxicant. Our findings concur with the [12-14]. Further, the following observations indirectly observation that the effects of reduced levels of blood lead suggest a strong link between the events in early neonatal only indicate a possible avoidance of the physical presen- life and childhood development: gestational low-dose tation of lead poisoning; they may not however preclude exposure to lead in rats can lead to a significant future risk the more subtle behavioral repercussions that can con- of alterations in monoaminergic metabolism during tinue to have a high impact on the social realm of the dis- adulthood ; neonatal infection can result in robust ease. Therefore efforts to reduce exposure to this hippocampal-dependent memory impairment in adult- physiologically redundant but environmentally toxic hood ; neonatal prefrontal cortex lesions can manifest metal need to continue. in adult animals as behavioral disturbances ; and early life does have an influence on the behavioral pat- Abbreviations terns in later life . Considering all these observations CBL Cord Blood Lead together, it is conceivable that neonates demonstrating behavioral disturbances secondary to lead exposure may NBAS Neonatal Behavioral Assessment Scale continue to manifest these disturbances in childhood. SEM Structural Equations Modeling We believe that, among others, a possible reason for the discordance in the results and interpretations of the effects MIMIC Multiple Indicators, Multiple Causes of low-dose lead exposure on neonatal behavior can be attributed to a lack of a standardized analytical protocol. ANCOVA Analysis of covariance Theoretically, lead can have multiple and simultaneous effects and we suggest that future studies need to incorpo- CDC Centers for Disease Control rate statistical techniques like SEM to handle the data more efficiently and accurately. The use of Generalized IQ Intelligence quotient Estimating Equations (GEE) for regressing the predictors  on multiple outcomes is another attractive alterna- Competing interests tive. In either case, the emphasis needs to be laid on the The author(s) declare that they have no competing inter- measurement and identification of a concomitant influ- ests. ence of blood lead – alone or with other predictors – on multiple outcomes related to behavior. Page 10 of 12 (page number not for citation purposes) Behavioral and Brain Functions 2006, 2:22 http://www.behavioralandbrainfunctions.com/content/2/1/22 8. Shen X, Wu S, Yan C: Impacts of low-level lead exposure on Authors' contributions development of children: recent studies in China. Clin Chim ABP conceptualized the study, participated in data collec- Acta 2001, 313:217-220. tion, data management and the review of the manuscript. 9. Dietrich KN, Ris MD, Succop PA, Berger OG, Bornschein RL: Early exposure to lead and juvenile delinquency. Neurotoxicol Teratol MRM contributed to the statistical analyses, created the 2001, 23:511-518. illustrations, and contributed to the manuscript writing 10. Pabello NG, Bolivar VJ: Young brains on lead: adult neurological consequences? Toxicol Sci 2005, 86:211-213. and review. TPT contributed to the review of the manu- 11. Bellinger DC, Hu H, Kalaniti K, Thomas N, Rajan P, Sambandam S, script. HRK conducted the statistical analyses and wrote Ramaswamy P, Balakrishnan K: A pilot study of blood lead levels the manuscript. All authors read and approved the final and neurobehavioral function in children living in Chennai, India. Int J Occup Environ Health 2005, 11:138-143. version of the manuscript. 12. Canfield RL, Gendle MH, Cory-Slechta DA: Impaired neuropsy- chological functioning in lead-exposed children. Dev Neuropsy- Additional material chol 2004, 26:513-540. 13. Canfield RL, Kreher DA, Cornwell C, Henderson CR Jr: Low-level lead exposure, executive functioning, and learning in early childhood. Neuropsychol Dev Cogn C Child Neuropsychol 2003, Additional file 1 9:35-53. This MS Word file contains the seven Supplementary Tables alluded to in 14. Chen A, Dietrich KN, Ware JH, Radcliffe J, Rogan WJ: IQ and blood this manuscript. Supplementary Table 1 describes some covariates listed in lead from 2 to 7 years of age: are the effects in older children the residual of high blood lead concentrations in 2-year-olds? Table 1 of the main text in more details. Supplementary Table 2 shows the Environ Health Perspect 2005, 113:597-601. results of ANCOVA analyses. Supplementary Table 3 shows the correla- 15. Chiodo LM, Jacobson SW, Jacobson JL: Neurodevelopmental tion coefficients between pairs of NBAS clusters and their statistical sig- effects of postnatal lead exposure at very low levels. Neuro- nificance. The analysis is first shown in all neonates and then in neonates toxicol Teratol 2004, 26:359-371. with CBL <10 mg/dL. Supplementary Table 4 shows the results of final 16. Ozmert EN, Yurdakok K, Soysal S, Kulak-Kayikci ME, Belgin E, Ozmert E, Laleli Y, Saracbasi O: Relationship between physical, models from stepwise multiple linear regression models predicting the CBL environmental and sociodemographic factors and school levels based on the NBAS cluster scores. Supplementary Table 5 shows the performance in primary schoolchildren. J Trop Pediatr 2005, results of the final models from stepwise multiple linear regression analyses 51:25-32. of the covariates on each of the four NBAS clusters chosen for SEM anal- 17. Pocock SJ, Smith M, Baghurst P: Environmental lead and chil- ysis. Supplementary Table 6 shows the results of the full SEM model. dren's intelligence: a systematic review of the epidemiologi- Lastly, Supplementary Table 7 shows the model fit indices for a total of 11 cal evidence. BMJ 1994, 309:1189-1197. 18. Schwartz J: Low-level lead exposure and children's IQ: a meta- nested models. In Supplementary Tables 3 and 5 – 7, the analyses are first analysis and search for a threshold. Environ Res 1994, 65:42-55. shown for all neonates and then for neonates with CBL <10 μg/dL. 19. Holmes SE, Slaughter JR, Kashani J: Risk factors in childhood that Click here for file lead to the development of conduct disorder and antisocial [http://www.biomedcentral.com/content/supplementary/1744- personality disorder. Child Psychiatry Hum Dev 2001, 31:183-193. 9081-2-22-S1.doc] 20. Stretesky PB, Lynch MJ: The relationship between lead expo- sure and homicide. Arch Pediatr Adolesc Med 2001, 155:579-582. 21. Ernhart CB, Wolf AW, Kennard MJ, Erhard P, Filipovich HF, Sokol RJ: Intrauterine exposure to low levels of lead: the status of the neonate. Arch Environ Health 1986, 41:287-291. 22. Rothenberg SJ, Schnaas L, Cansino-Ortiz S, Perroni-Hernandez E, de Acknowledgements la Torre P, Neri-Mendez C, Ortega P, Hidalgo-Loperena H, Svends- This study was supported by grant 1004-94-6305 from International Clini- gaard D: Neurobehavioral deficits after low level lead expo- cal Epidemiology Network (INCLEN), Philadelphia, USA. We thank the sure in neonates: the Mexico City pilot study. Neurotoxicol National Environmental Engineering Research Institute (NEERI) Nagpur, Teratol 1989, 11:85-93. 23. Emory E, Pattilo R, Archibold E, Bayorh M, Sung F: Neurobehavio- India, for their help in analyzing the blood samples. We also thank Dr. ral effects of low-level lead exposure in human neonates. Am Nandini Gokulchandran, Dr. Leena Dhande, the Department of Pediatrics J Obstet Gynelcol 1999, 181:S2-S11. and the Department of Gynecology and Obstetrics at Government Medical 24. Shen X-M, Yan C-H, Guo D, Wu S-M, Li R-Q, Huang H, Ao L-M, College, Nagpur, India for their assistance. Lastly, the authors gratefully Zhou J-D, Hong Z-Y, Xu J-D, Jin X-M, T J-M: Low level prenatal lead exposure and neurobehavioral development of children acknowledge the critical reviews by two reviewers that lead to a significant in the first year of life: A prospective study in Shanghai. Envi- improvement in the manuscript. ron Res 1998, 79:1-8. 25. Gomaa A, Hu H, Bellinger D, Schwartz J, Tsaih SW, Gonzalez-Cossio References T, Schnaas L, Peterson K, Aro A, Hernandez-Avila M: Maternal bone lead as an independent risk factor for fetal neurotoxic- 1. Ernhart CB: Lead levels and confounding variables. Am J Psychi- ity: a prospective study. Pediatrics 2002, 110:110-118. atry 1982, 139:1524. 26. 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