Whitfield et al. erythrocytes from the blood samples. We estimated heritability of erythrocyte concen- tration for each element from the MZ and DZ twin-pair data after testing for effects of covariates [listed in Supplemental Material, Table 2 (doi:10.1289/ehp.0901541)], adjust- ing for covariate effects. Chromosomal locations of gene variants affecting element concentrations were evaluated using genome- wide linkage analysis. Results Covariate effects. We observed significant asso- ciations of sex with Cd, Cu, and Se (all with higher concentrations in women) and on Pb (with higher concentrations in men). Pb and Se increased significantly with age. Reported alcohol consumption was significantly associ- ated with increases in As, Hg, Pb, and Se [see Supplemental Material, Figure 1 (doi:10.1289/ ehp.0901541)]. Increasing self-reported alco- hol intake was also associated with increasing Cd, but this was due to the strong association between smoking and higher alcohol intake. Smoking had very substantial positive associa- tions with Cd, as expected, and with Hg, Pb, and Se. Of the demographic factors, body mass index and social class had no significant asso- ciations, whereas educational level was inversely associated with As, Cd, and Pb. Place of resi- dence was associated with Hg and Zn; Hg was higher in urban residents and Zn higher in rural residents. See Supplemental Material, Table 2 for a summary of significant effects of the cova- riates on element concentrations. Element correlations. We observed sig- nificant correlations between concentrations of several of the elements in both men and Sex Male Female Male Female Male Female Male Female Male Female Male Female Male Female Cu — 0.60 0.53 0.43 0.42 0.15 0.15 0.23 0.18 0.08 0.04 0.08 0.02 Zn — 0.49 0.47 0.15 0.17 0.21 0.22 0.05 0.07 0.06 0.03 women, after adjusting for day-of-analysis effects, quality control data, and sample hemoglobin concentration (Table 1). There were substantial associations (r ≈ 0.4–0.5) between Cu, Zn, and Se and between As and Hg. Correlations between Pb and Cu, Zn, and Cd and for Se with As and Hg were more moderate, but still significant (r ≈ 0.2). Results for men and women were similar. Sources of variation and covariation. The pairwise correlations for the measured ele- ments for MZ and DZ twin pairs are shown in Table 2. In each case the MZ correlation was greater than the DZ correlation, and for all except Cd the 95% confidence intervals (CIs) did not overlap. The proportion of variation in each element accounted for by additive genetic factors (A), environments shared by co-twins regardless of zygosity (C), and nonshared environments including measurement error and within-person biological variation (E), are shown in Table 2. These estimates of A, C, and E were derived under the ACE model allowing for all three sources of variation, but models containing A and E only did not show a significantly worse fit (all p > 0.05). Because of our interest in genetic or environ- mental sources of variation affecting more than one element, we re-estimated the genetic and environmental sources of variation in a multi- variate ACE model [see Supplemental Material, Table 3 (doi:10.1289/ehp.0901541)]. Minor differences from the univariate estimates of A, C, and E for each element arise from the mul- tivariate optimization procedure. Supplemental Material, Table 3, shows that 20% of the varia- tion in Zn concentration is estimated to be due to genetic factors (A) unique to Zn and another Se As Pb Hg Cd — 0.28 0.25 0.11 0.13 0.25 0.27 –0.03 –0.05 — 0.09 0.09 0.48 0.46 0.04 0.00 — 0.07 0.06 0.20 0.21 — –0.02 –0.02 — Table 1. Correlations between element concentrations after adjustment for analytical covariates. Element Cu Zn Se As Pb Hg Cd MZ pair DZ pair A C E 778 With ~ 2,900 subjects, any r value consistently > 0.07 for both men and women is highly significant (p < 0.001). Table 2. Twin pair correlations by zygosity [correlation (95% CI)] and estimated proportions of variance [percent (95% CI)] due to additive genetic (A), shared environmental (C), and nonshared environmental (E) factors. Cu 0.29 (0.22 to 0.36) 0.10 (0.02 to 0.18) 28 (14 to 34) 0 (0 to 11) 72 (66 to 79) Zn 0.41 (0.34 to 0.47) 0.25 (0.17 to 0.32) 30 (11 to 46) 10 (0 to 26) 60 (54 to 67) Se 0.36 (0.29 to 0.43) 0.20 (0.12 to 0.27) 31 (10 to 40) 2 (0 to 18) 67 (60 to 74) As 0.22 (0.14 to 0.29) 0.04 (–0.04 to 0.13) 19 (5 to 26) 0 (0 to 11) 81 (74 to 88) Pb 0.41 (0.34 to 0.47) 0.17 (0.09 to 0.25) 40 (26 to 45) 0 (0 to 11) 61 (55 to 67) Hg 0.32 (0.25 to 0.38) 0.13 (0.04 to 0.21) 30 (15 to 36) 0 (0 to 12) 70 (64 to 77) Cd 0.35 (0.28 to 0.41) 0.25 (0.17 to 0.32) 19 (0 to 31) 5 (0 to 22) 76 (69 to 84) ovlume 118 | number 6 | June 2010 • Environmental Health Perspectives 6% to genetic factors shared with Cu; 14% is due to shared environment (C), of which 11% is ascribed to unique shared environment and 3% common to both Zn and Cu. For unshared environment effects (E), about half of the varia- tion is unique and half shared with Cu. Overall, the unshared environmental variation is mainly unique for As, Pb, Hg, and Cd but shared by Cu, Zn, and Se. Shared (familial) environmen- tal effects are negligible, and additive genetic effects are partly shared between As and Hg. Estimation of the genetic correlations between pairs of elements (the correlation due to gene effects common to both) showed results similar to the phenotypic correlations in Table 1, except for the As/Hg pair, where the genetic correlation (rG) was 0.83 and the unique environmental correlation (rE) was 0.34. This shows that variation in some genes affects concentrations of both of these ele- ments and to a greater extent than environ- mental variation affects both. Linkage analysis. Results for linkage analy- sis on 501 DZ twin pairs for three chromo- somes are shown in Figure 1 (chromosome 3) and Figure 2 (chromosomes 2 and 4). Results for all chromosomes evaluated are sum- marized in Supplemental Material, Table 4 (doi:10.1289/ehp.0901541), and overall results are illustrated in Supplemental Material, Figure 2. As previously reported for 414 twin pairs (Whitfield et al. 2007), Pb gave the stron- gest linkage signal. The inclusion of extra twin pairs (for a total of 501 twin pairs) led to a revised LOD score of 4.21 on chromosome 3 (Figure 1), well within the significant range. The LOD score exceeded 4.21 in only 3 of 1,000 simulations, giving an empirical genome- wide p-value of 0.003. Suggestive linkage results (LOD > 1.6) were found for Zn on chromo- some 2, Se on chromosomes 4 and 8, Hg on chromosome 5, and Cd on chromosomes 2, 18, 20, and X. There was some overlap of link- age signals for different elements (Figure 2; see also Supplemental Material, Table 4 and Figure 2), notably Cd and Zn around 120 cM and Zn, Cd, and Se at around 220 cM on chro- mosome 2 (Figure 2A); and Se and Cu around 130 cM on chromosome 4 (Figure 2B). Discussion The main focus of this study was to assess whether, and to what extent, genetic variation between people affects individual differences in concentrations of toxic or trace elements.