Reductions in Blood Lead Overestimate Reductions in Brain Lead After Repeated Succimer Regimens in a Rodent Model of Childhood Lead Exposure

Diane E. Stangle; Myla S. Strawderman; Donald Smith; Mareike Kuypers; Barbara J. Strupp

Disclosures

Environ Health Perspect. 2004;112(3) 

In This Article

Results

Blood and brain levels were collected from 140 animals, from 25 litters. However, only data from a slightly smaller number of samples (136 blood samples and 131 brain samples) were available for statistical analyses because of collection and/or analytic problems. In addition, in the course of statistical analysis, it was discovered that five animals had unusually high lead levels in either blood (n = 1) or brain (n = 4), without correspondingly high values in the other sample from the animal. These values were orders of magnitude different from other animals receiving the same initial exposure, treatment, and follow-up time, suggesting an analytic problem. One possible explanation for the four high brain measurements is that the samples were contaminated during collection and/or processing for analysis, possibly by the inclusion of a small skull fragment. These five observations were excluded from the analyses presented below.

As noted above, three lead groups were designated for analysis based on the blood lead level of the littermate sacrificed on PND41. The resulting low, medium, and high lead exposure groups had average (± SD) blood lead levels of 24.4 (± 3.2), 49.5 (± 10.8), and 131.3 (± 26.0) µg/dL, respectively. Average (± SD) brain lead levels for the three groups were 1,825 (± 374), 2,867 (± 247), and 6,781 (± 1,863) ng/g dry weight, respectively.

For each of the three time points examined (immediately after the first chelation regimen at PND62, as well as PND90 and PND118), the animals treated with succimer had significantly lower lead levels in both blood and brain than did their vehicle-treated counterparts (all p < 0.0001). However, as indicated by a significant interaction among treatment, tissue, and time [F(2,156) = 6.68, p = 0.002], treatment efficacy varied as a function of both tissue type and time after chelation (Figure 2). At the end of the first chelation (PND62), the reduction in brain lead produced by succimer treatment (compared with the vehicle-treated animals, i.e., succimer/vehicle) was significantly smaller than that seen in blood (p = 0.0006). Across the following 8 weeks of the study, brain lead continued to drop in both the succimer- and vehicle-treated animals, with the relative difference between the two groups remaining relatively constant. In contrast, blood lead levels in the succimer-treated group increased significantly (i.e., rebounded) over the 4 weeks after chelation, whereas blood lead levels in the vehicle group continued to decrease. Between PND90 and PND118 (4 and 8 weeks after chelation), blood lead remained relatively constant in the succimer-treated animals (p = 0.9), at a level that was significantly lower than their vehicle-treated counterparts. These findings demonstrate that although succimer treatment was highly effective in removing lead from both tissues, the reduction in brain lead levels lagged significantly behind the reduction in blood lead levels.

The efficacy of one regimen of succimer (relative to vehicle treatment) in lowering blood and brain lead levels, assessed at various times after chelation ended on PND61. Tissue lead concentrations are expressed as parts per billion (ppb; for blood and brain this is equivalent to nanograms of lead per milliliter and nanograms of lead per gram dry weight, respectively). For comparison, 10 ppb blood lead equals 1 µg/dL.

A significant interaction was found between treatment and lead exposure group [F(2, 159) = 5.65, p = 0.004]. Although succimer treatment was significantly more effective than vehicle in removing lead from all three lead exposure groups (p < 0.0001), the magnitude of the succimer effect relative to vehicle varied by lead exposure group (Figure 3). The succimer effect relative to vehicle was largest in the low lead group and decreased with increasing lead exposure. However, as shown in Figure 3, the absolute amount of lead removed by succimer was larger in the high-lead group than in the two lower groups.

The efficacy of one regimen of succimer (vs. vehicle treatment) in lowering tissue lead levels (blood and brain levels combined), as a function of lead exposure, when assessed immediately after the first succimer regimen (PND62; see text for details).

The "rebound" analysis included data from the day immediately after chelation (PND62), as well as 1 and 4 weeks later (PND69 and PND90). There was a significant tissue type by time interaction (p < 0.0001). Averaged over lead exposure groups, blood lead levels exhibited a rebound 1 week after the cessation of treatment (p < 0.0001), but brain lead levels did not (p = 0.80; Figure 4). Further, whereas both blood and brain showed a decline in average lead levels from PND69 to PND90 (1-4 weeks after chelation), blood lead values at 4 weeks after chelation were still significantly higher than those seen immediately after chelation ended, as presented above. Thus, a rebound in lead levels was detected in blood but not in brain.

Levels of (A) brain and (B) blood lead across the 4 weeks after completion of the first succimer regimen.

This analysis included data from PND90 and PND118, corresponding to the day immediately after the second chelation regimen ended and 4 weeks later. The analysis was performed on 88 brain specimens and 97 blood specimens, contributed by 101 animals. As depicted in Figure 5, the animals that received two cycles of succimer had significantly less lead in both brain and blood than did animals that received either one succimer regimen or vehicle treatment (all p < 0.008). Therefore, two cycles of succimer offered a significant added benefit relative to one cycle throughout the time period examined.

Additional benefit of the second regimen of succimer treatment (vs. one regimen and vehicle treatments) in reducing blood and brain lead levels, as a function of time since the second regimen ended (see text for details).

However, treatment efficacy varied as a function of tissue type, time, and lead exposure, as indicated by two borderline three-way interactions involving treatment, as well as several significant underlying two-way interactions. A borderline interaction among treatment, tissue type, and time [F(2, 137) = 2.93, p = 0.06] reflected the fact that the magnitude of the added benefit of the second succimer regimen varied as a function of both tissue type and time since the second regimen ended. Immediately after the second chelation, the benefit of one cycle of succimer (relative to vehicle) and the added benefit of the second cycle (relative to only one cycle) were of similar magnitude in blood and brain tissue (Figure 5). However, 4 weeks later (PND118), the benefit of the second regimen (relative to vehicle) was significantly greater for brain than for blood (p = 0.002). This pattern of findings reflects the fact that during the final 4 weeks of the study, brain lead continued to decline in all treatment groups, whereas blood lead levels of the one-cycle succimer group remained relatively constant, and that of the two-cycle succimer group significantly increased (i.e., rebounded; p = 0.05).

A separate analysis was conducted on the blood samples from the animals receiving two cycles of succimer, based on the evidence (above) that the rebound occurred only in the blood, and because this approach decreased the residual error (the brain samples are more variable than the blood samples). This analysis showed that the average lead level in the blood was higher 4 weeks after the second chelation than immediately after cessation of the second chelation for the medium (p = 0.05) and high (p = 0.0003) exposure groups. For the low lead exposure group, the lead levels were very low at both time points and there was no difference in the average lead levels at the two time points. Thus, a significant rebound in blood lead levels, but not brain lead, occurred after the second chelation, similar to the pattern after the first succimer regimen.

A significant two-way interaction between treatment and lead exposure group was found [F(4, 144) = 5.77, p = 0.0002], as well as a borderline three-way interaction among treatment, lead exposure, and time [F(4, 137) = 2.93, p = 0.056]. When assessed immediately after the second regimen (PND90), all three exposure groups derived a significant benefit of the second regimen (vs. one regimen; all p < 0.007), although there were subtle differences in the reduction in tissue lead relative to vehicle as a function of lead exposure. When assessed 4 weeks later (PND118), the benefit of a second chelation regimen (vs. one regimen) was still apparent in the low and medium groups but not the high exposure group (Figure 6). This finding appears to reflect primarily the rebounding of blood lead over the 4 weeks after the second chelation, which was greatest for the high-lead group.

Additional benefit of the second regimen of succimer treatment (vs. one regimen and vehicle treatments) in reducing tissue lead (blood and brain combined), as a function of both the intensity of prior lead exposure and time since the second regimen ended (see text for details).

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