The Relationship Between Rodent Offspring Blood Lead Levels and Maternal Diet

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1 The Relationship Between Rodent Offspring Blood Lead Levels and Maternal Diet Allison Crawford, Xiahong Li, Mira Shapiro 1, Ruitao Zhang Introduction A study was undertaken to understand the effect of maternal lead ingestion, from food, on fertility in mice offspring. Twenty-four mothers were given feed at eight lead levels ranging from 0.02 to 40 ppm. Four offspring were chosen, at random, from each of the mothers and their blood lead concentration was measured. The purpose of this report is to evaluate the blood lead concentration levels of the mouse offspring with respect to the maternal lead level ingestion. Material and Methods Data Twenty-four mothers were randomized to receive a rodent diet with a specified level of lead (0.02, 0.06, 0.11, 0.2, 2, 4, 20, or 40 ppm). These exposure levels were classified as follows: 20 ppm and 40 ppm are considered high or relatively high exposure, 2 ppm and 4 ppm are low exposure, 0.2 ppm is the control level, 0.06 ppm and 0.11 ppm are very low exposure, and.02 ppm is considered lead free. In the initial study, 0.2 ppm was specified as the control group given that this is the usual background level of lead in the rodent feed. Since in this analysis the goal is to assess offspring lead level with respect to the lead level in the maternal diet, we will use the lowest level 0.02 ppm as our referent group. Ninety-six offspring, four randomly selected from each mother, constituted the subjects for this study. Model In order to assess the relationship between the lead level of the feed given to the mothers and the blood lead concentration in the offspring, a mixed effects model of the following form was evaluated using SAS Proc Mixed was used to fit the model, exploiting this procedure s capability to include both fixed and random effects. 1 Primary author

2 Model: Yijk = µ.. + τi + Sj(i) + εijk where: i: indicates the treatment, lead level in the rodent feed j: indicates the mother k: indicates the individual offspring µ.. : is a constant representing the overall mean blood level in the offspring Yijk : Blood lead level of individual offspring τi : fixed treatment effect, where τi = 0 Sj(i) : Represents the random maternal effect for mother i, independent with N(0, σ 2 ). The error is nested within treatment since the ith treatment was given to more than one of the of j mothers. εijk : Represents the between subjects, is independent with N(0, σ 2 ) Sj(i) and εij are independent. Results The mean level of lead concentration measured in the offspring s blood, for each level of maternal exposure, along with the standard error for the estimate, is shown in Table 1. The mean level of lead concentration in the blood of the offspring increases along with maternal lead exposure at all levels. Table 1: Estimated Means for Each Level of Lead Exposure Standard Effect pblevel Estimate Error pblevel 1.high pblevel 2.low pblevel 3.control pblevel 4.very low pblevel 5.lead free Evaluating the differences in mean levels of offspring blood lead concentrations between each of the groups results in significant differences at the <.0001 level for High versus all of the other exposure levels. The difference in means

3 between low and very low, and low and lead free exposure levels resulted in p- values of and respectively. The mean differences were not statistically significant for any of the other group comparisons. These results are displayed in Appendix 2. The mixed model was designed with the lead free group as the referent group. Examining the fixed effects solution (Table 2) reveals that, with respect to a lead free diet, only the low and high lead rodent feed categories result in a statistically significant difference in offspring blood lead concentrations. Table 2: Solution for Main Effects Standard Effect pblevel Estimate Error DF t Value Pr > t Intercept pblevel 1.high <.0001 pblevel 2.low pblevel 3.control pblevel 4.very low pblevel 5.lead free 0 The results of the mixed model include the breakdown of the variance into the between-mother and within-mother components, and are shown in Table 3. In this model, since the mother is nested in lead exposure level, the betweenmother variance component includes the variance associated with the lead level exposure and that associated with the mother. It makes sense that the variation within lead exposure level is a smaller portion of the overall variance than the between component. Table 3: Covariance Parameter Estimates Estimate Mother(pblevel) between Residual within Conclusion It is clear from these results that maternal consumption of lead is associated with offspring blood lead concentration, and not surprisingly the highest concentration causes the most significant effects. An interesting follow-up study might include evaluating the effect of blood lead level concentration on offspring behavior and development.

4 Appendix 1: SAS Code ***************************************************************************; * Bioepi 743 Spring 2006; * Group Project: Allison Crawford, Xiaohong Li, Mira Shapiro, Ruitao Zhang; * Problem 2.7 * Date: 2/12/06; * Program Source: z:\bioepi74027.sas; * Data Source: ffindings4; ****************************************************************; libname hw27 "z:\"; data mice(keep=pblevel mother bpb pb); length pblevel $ 12; set hw27.ffindings4; * create categorical variable representing lead (Pb) levels; select(pb); when(40) pblevel='1.high'; when(20) pblevel='1.high'; when(2) pblevel='2.low'; when(4) pblevel='2.low'; when(0.06) pblevel='4.very low'; when(0.11) pblevel='4.very low'; when(0.2) pblevel='3.control'; when(0.02) pblevel='5.lead free'; otherwise; end; run; * use proc mixed to assess the effect of Pb ingestion by the mother on; * the offspring; * Nest Pblevel in Mother and make a random effect; * PBlevel is a surrogate for offspring in model; * fixed effect: Pb level; * LSmeans to obtain mean and variance estimates; proc mixed data=mice ; class pblevel Mother; model bpb=pblevel/s ; random Mother(pblevel); lsmeans pblevel/diff; run;

5 Appendix 2: Mean Differences

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