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

Materials and Methods

The present study was a 3 × 3 factorial design, involving three levels of lead exposure and three levels of chelation therapy (vehicle, one 3-week succimer regimen, two 3-week succimer regimens). Animals were followed for a total of 11 weeks after cessation of lead exposure, corresponding to 8 weeks after the first chelation regimen ended and 4 weeks after the second regimen ended. As depicted in Figure 1, animals were sacrificed at various time points throughout the study to assess the efficacy of succimer in reducing blood and brain lead levels. This lengthy follow-up period allowed assessment of succimer efficacy in both blood and brain across various postchelation time periods, as well as an assessment of the potential "rebounding" of blood and brain lead levels after cessation of chelation.

Study design. Lead exposure occurred from PND0 through PND40, followed by either one 3-week regimen of succimer, two 3-week regimens of succimer, or vehicle treatment. Arrows represent days at which rats from each lead exposure group were sacrificed and blood and brain lead levels analyzed.

Nulliparous Long-Evans rats (Harlan Sprague Dawley, Indianapolis, IN) were housed in animal facilities accredited by the Association for the Assessment and Accreditation for Laboratory Animal Care, with a 12:12-hr light:dark cycle. Food (Pro-Lab RMH 1000; Nutrition International LLC, Brentwood, MO) and water were available ad libitum. Rats were mated at approximately 10 weeks of age. Pregnancy was determined by the presence of a sperm plug and subsequent weighing of the females at approximately 5-day intervals. Dams were maintained on tap water and laboratory chow during gestation.

Lead was administered from birth until postnatal day (PND) 40. Until weaning on PND21, the pups received lead via nursing from lead-exposed dams, whereas from PND21 through PND40, the pups directly consumed lead from their water supply. At birth, each dam was randomly assigned to a lead exposure condition. All dams received lead via drinking water (600 µg/mL lead as lead acetate) during PND1-18, followed by daily oral administration of an equivalent lead dose to the dams via eye dropper until weaning on PND21. After weaning, the lead exposure diverged into two groups as follows: a) 20 µg/mL lead in the drinking water during PND21-28, followed by 30 µg/mL during PND29-40, or b) 40 µg/mL lead in the drinking water during PND21-28, followed by 60 µg/mL during PND29-40. Lead exposure in all animals was terminated at PND40.

The decision to give all dams the same lead dose during lactation (PND1-21) was based on prior studies in our laboratory showing that pup blood lead levels remain relatively low and constant across a wide range of maternal intakes (15-25 µg/dL) (Garavan et al. 2000; Morgan et al. 2001) and do not increase until very large doses are used that produce maternal toxicity (unpublished data). The selection of lead doses after weaning was also based on prior studies in our lab (unpublished data). The lower-dose regimen produces relatively consistent, low blood lead levels, whereas the higher-dose regimen produces a wider range of blood lead levels. These two doses together therefore allowed us to examine low, moderate, and high exposure in the pups.

Twenty-four hours after birth, the litters were culled to 10 pups. Within each lead treatment condition, 10 subgroups were defined by the five sacrifice dates and three succimer treatment conditions (vehicle, one succimer regimen, two succimer regimens; see Figure 1). When possible, one pup per litter was assigned to each of these 10 subgroups, with the goal of providing littermate comparisons for these 10 conditions, and to avoid having more than one animal per litter in a given subgroup.

Succimer (Chemet; Sanofi-Synthelabo Inc., New York, NY) was administered via oral gavage at a dose of 50 mg/kg/day for 1 week followed by 25 mg/kg/day for an additional 2 weeks, for a total treatment duration of 3 weeks per regimen. Succimer was dissolved in apple juice and administered within 15 min of mixing. The vehicle groups received equivolume apple juice carrier, also by gavage. The daily dose of succimer or vehicle was divided and administered as two equal doses given 10-12 hr apart.

Animals were sacrificed at five time points throughout the study (Figure 1) to assess the efficacy of the various treatments in lowering blood and brain lead levels, as follows: a) One animal per litter was sacrificed at PND41, immediately after cessation of lead exposure, and immediately before the start of chelation. Sacrifice at this time point provided a representative measure of the tissue lead levels for each litter. b) On PND62, immediately after the first round of chelation treatment, both succimer- and vehicle-treated animals from each lead exposure condition were sacrificed to assess the immediate efficacy of one chelation regimen. c) An additional group of rats that received one round of succimer treatment were sacrificed at PND69, 1 week after treatment ended and immediately before the second round of chelation treatment began, to examine whether a rebound in blood and/or brain lead levels occurred after cessation of succimer treatment. d) Rats from each chelation treatment group (vehicle, one succimer regimen, two succimer regimens) were sacrificed at PND90, immediately after the second round of chelation (and 4 weeks after the first round of chelation ended). Sacrifice at this time served a dual purpose to examine the long-term effects of one round of succimer treatment and the added benefit of a second round of succimer treatment compared with one round of treatment. e) Finally, animals from each of the three chelation conditions were sacrificed at PND118, 8 weeks after the first chelation regimen and 4 weeks after the second regimen. Sacrifice at this time point also served a dual function: to assess the long-term efficacy of one and two succimer regimes relative to vehicle treatment and relative to each other, and whether a rebounding occurred in blood or brain levels after the second chelation regimen.

At the time of sacrifice, a 2- to 3-mL sample of whole blood was collected into a polypropylene syringe via cardiac puncture from surgically exposed hearts of anesthetized animals and deposited into Vacutainers specified for trace metal (no. 367734, Becton Dickson, Research Triangle Park, NC). Animals were given a sodium pentobarbital overdose (50 mg/kg) and exsanguinated. Whole brain was removed using acid-washed stainless-steel dissecting tools, rinsed with Milli-Q water, and deposited into polypropylene storage containers. All tissue sampling was conducted using trace-metal-clean procedures. Dissecting instruments (stainless steel) were cleaned before each dissection and rinsed frequently within a dissection procedure to avoid contamination from nonbrain tissues. All samples were stored frozen.

Lead concentrations were measured in whole blood and brain tissue. Blood lead levels were determined using graphite furnace atomic absorption spectroscopy at the Wisconsin State Laboratory of Hygiene (WSLH; Madison, WI). The WSLH administers the Nationwide Blood Lead Proficiency Testing Program in cooperation with the Centers for Disease Control and Prevention and the Maternal and Child Health Bureau. Brain lead levels were measured at the University of California, Santa Cruz, using a Finnegan-element inductively coupled plasma (ICP)/high-resolution mass spectrometer (MS) in multi-isotope counting mode, measuring masses 208Pb and 209Bi, with 209Bi used as an internal standard (Smith et al. 2000a, 2000b; Woolard et al. 1998). External standardization was via a certified lead standard that had been isotopically characterized independently via thermal ionization MS. National Institute of Standards and Technology (Gaithersburg, MD) Standard Reference Materials 955A (blood) and 1577 (bovine liver) were used to evaluate procedural accuracy. This ICP/MS methodology has been demonstrated to yield a measurement precision of < ± 0.5% for sample lead concentrations of ≥ 0.05 ppb (Woolard et al. 1998). The analytic detection limit was 0.01 ppb.

All statistical analyses were conducted using SAS 8.2 (SAS Institute, Inc., Cary, NC). The present data contained correlated observations due to the use of two observations from each animal (blood and brain) and due to the use of multiple animals per litter. Therefore, a repeated-measures analysis of variance model (PROC MIXED; Littell et al. 1996) was used. To analyze the data for both tissues in a single analysis, blood and brain lead concentration data were first converted to comparable units of parts per billion (i.e., nanograms per milliliter and nanograms per gram, respectively). However, box plots indicated that the scale and skewness were larger in the brain samples than in the blood samples. A natural log transformation was applied to normalize the residuals and reduce the scale of the random effects. Taking a log transformation is often successful when the effects in the model are proportional rather than additive.

The estimated least-squares means produced in the analysis of log-transformed data are expressed as geometric means after back-transforming to the original units. Exponentiation of the difference in least-squares means on the analysis scale is equivalent to the ratio of geometric means on the original scale. Therefore, the usual tests of effects involving differences in means on the analysis scale (i.e., succimer minus vehicle) are actually assessing proportional, or relative, changes in the original units (i.e., succimer/vehicle). Figures are presented in the original scale for ease of interpretation. Standard errors for means are not included on the figures because the appropriate standard error of a difference in means cannot be derived directly from the individual mean standard errors because of the covariance terms, which vary by the particular groups being compared.

The "one succimer regimen" analysis investigated vehicle versus one cycle of succimer treatment at three time points: PNDs 62, 90, and 118. The "two succimer regimens" analysis investigated the efficacy of vehicle, one cycle of succimer, and two cycles of succimer at the two time points after the second chelation regimen: PNDs 90 and 118. The initial models were constructed containing the main effects of tissue type (blood or brain), lead exposure group, succimer treatment (vehicle, one cycle, or two cycles), and time (age at sacrifice). Preliminary analysis of the animals sacrificed immediately after cessation of lead exposure (PND41), before chelation, confirmed that the higher exposure regimen produced a much wider range of blood lead levels (range, 25-160 µg/dL) than did the lower exposure regimen (range, 20-30 µg/dL). Therefore, for the two analyses of the chelation treatment effects, a three-level lead exposure variable was created, in which each animal's lead exposure designation was based on the blood lead level of the littermate that was sacrificed at PND41. This three-level designation explained much more of the variation in the data than did a two-level designation, based on the Akaike Information Criterion (Akaike 1974; Bozdogan 1987; Wolfinger 1993). The initial models also included all relevant two-, three-, and four-way interactions involving the main effects. Each model was reduced by removing the least significant higher-order term first, reevaluating the model, and repeating the process until all effects were either significant at approximately the 5% level or included in a higher-order term that was significant.

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