Succimer and the Urinary Excretion of Essential Elements in a Primate Model of Childhood Lead Exposure

D. R. Smith*,1, C. Calacsan*, D. Woolard*, M. Luck{dagger}, J. Cremin* and N. K. Laughlin{dagger}

* Department of Environmental Toxicology, University of California, Santa Cruz, California 95064; and {dagger} Harlow Center for Biological Psychology, University of Wisconsin, Madison, Wisconsin 53175

Received October 7, 1999; accepted November 15, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Succimer is considered to be a safe and effective treatment for lead (Pb) poisoning, since it reduces body Pb levels without an apparent diuresis of other essential elements. However, while existing clinical data indicate that succimer does not significantly increase the excretion of non-target elements, those studies have also reported a wide range of outcomes. Therefore, we investigated whether succimer treatment measurably increased the urinary excretion of essential elements in a primate model of childhood Pb exposure. Infant rhesus monkeys (Macaca mulatta) were exposed to Pb from birth through one year of age, and presented blood Pb levels of {approx}40–50 µg/dL at the start of treatment. Subsequently, they were treated with succimer (30 mg/kg/day x 5 days followed by 20 mg/kg/day x 14 days, n = 15) or vehicle (n = 14) for 19 days. Complete urine samples were collected over the first 5 days of treatment, and were analyzed for levels of calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), magnesium (Mg), manganese (Mn), nickel (Ni), and zinc (Zn), using trace metal-clean techniques and magnetic sector–ICP-MS. Succimer treatment significantly (p < 0.05) reduced blood Pb levels when compared to the vehicle group over the treatment period, and concomitantly produced a significant >4-fold increase in urinary Pb excretion. Succimer treatment also significantly (p < 0.05, multivariate ANOVA) increased the urinary excretion of essential elements, but only when the cumulative total excretion over treatment days 1–5 for all elements were considered. None of these relative increases reached statistical significance for any particular element x day, although increases in Zn (day 3) excretion were only marginally non-significant (0.1 > p > 0.05). Multivariate analyses of a subset of elements (Cu, Fe, Mn, Zn) similarly indicated no significant effect of succimer treatment overall, although the urinary excretion of Mn was significantly increased on day 3 of treatment. Collectively, these data indicate that succimer does contribute to an increase in the urinary excretion of essential elements, although not significantly for any single element considered here. This may be important in Pb-exposed children, who can possess reduced trace element reserves due to nutritional deficiencies.

Key Words: essential elements excretion; rhesus monkeys; lead (Pb); succimer; blood Pb levels; chelating agents.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The clinical management of lead (Pb) poisoning in children may utilize treatment with a number of possible therapeutic chelating agents such as succimer (meso-2,3-dimercaptosuccinic acid, DMSA, Chemet®) or CaNa2EDTA (CaNa2ethylenediaminetetraacetic acid, Versenate®). Succimer is considered to be a particularly safe and effective treatment for lowering blood Pb levels, as well as for some symptoms of Pb intoxication (Chisolm, 1992Go; Fournier et al., 1988Go; Glotzer, 1993Go; Goyer et al., 1995Go; Graziano, 1986Go; Liebelt et al., 1994Go; Smith et al., 1994Go; Xu and Jones, 1988Go). Moreover, it offers some advantages over CaNa2EDTA chelation, since it is administered orally and it does not appear to cause a significant diuresis of other essential elements.

The existing clinical studies indicating that succimer does not significantly affect non-target essential elements have reported a wide range of outcomes. For example, Fournier et al. (1988) evaluated the effect of DMSA treatment on plasma levels of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), and zinc (Zn) in heavy metal (Pb, arsenic, mercury)-poisoned subjects, and found that only plasma Zn decreased significantly. Consistent with this, Graziano et al. (1985) reported that succimer treatment of 6 Pb-poisoned adult males led to a statistically significant increase in urinary Zn excretion, and to trending but non-significant increases in Cu, but not Ca, Fe, or Mg. A lower succimer-dosing regimen in a different cohort of 6 subjects led to statistically significant increases in Ca, Cu, and Zn, but not in Fe or Mg. However, in a similar study in Pb-poisoned children, Graziano et al. (1988) reported that their highest succimer-dosing regimen (1050 mg/m2/day, equivalent to 30 mg/kg/day) led to trending but non-significant increases in urinary Ca, Cu, and Fe, but not in Zn or Mg.

Further, several laboratory studies, including a recent non-human primate study, have indicated that succimer is less effective in removing Pb from brain tissues than from blood (Cory-Slechta, 1988Go; Cremin et al., 1999Go; Pappas et al., 1995Go; Smith et al., 1998Go; Smith and Flegal, 1992Go; Xu and Jones, 1988Go). This suggests that more prolonged or repeated treatment regimens may be necessary to effectively reduce brain Pb levels than would otherwise be indicated by reductions in blood Pb levels. Moreover, if succimer treatment is proven to alleviate neurocognitive impairment from moderate Pb exposure, treatment may be pursued at lower blood Pb levels than currently considered (AAPCD, 1995Go; TLC, 1998Go). In light of these issues, there is additional need to thoroughly understand any non-target effects of succimer treatment, such as on physiologic levels and/or the diuresis of essential elements. These may be considered in evaluating succimer's overall utility in cases of prolonged treatment of moderately Pb-exposed subjects.

Therefore, we investigated the effect of succimer treatment on the urinary excretion of essential elements in a primate model of childhood Pb exposure. Infant rhesus monkeys were exposed to Pb from birth through one year-of-age, and treated with a succimer regimen comparable to that used clinically. Complete urine samples were collected over the first 5 days of treatment and analyzed for levels of Ca, cobalt (Co), Cu, Fe, Pb, Mg, manganese (Mn), nickel (Ni), and Zn, using trace metal-clean techniques and a sensitive ICP-MS methodology. These data will contribute to a more thorough understanding of the non-target effects of succimer treatment in moderately Pb-poisoned pediatric populations.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study design/treatments.
The present study was comprised of a standard 1 x 2 design (Pb exposure x succimer or placebo treatment). Animals were exposed to Pb orally for one year, then treated with succimer or vehicle for a period of 19 days, as described below. Complete 24-h urine samples were collected over 6 consecutive days, beginning 1 day prior to treatment (t-1) and concluding after 5 days of treatment (t5). This study was conducted as part of a larger ongoing study of the efficacy of succimer treatment for alleviating neurocognitive impairment and reducing tissue (blood, liver, bone) Pb levels. Those complete data will be reported elsewhere.

Animals.
Twenty-nine female infant rhesus monkeys (Macaca mulatta) born at the Harlow Center for Biological Psychology`s breeding colony were used in this study. These animals were recruited in 2 cohorts over 2 consecutive years. Treatment groups (succimer vs. vehicle) were balanced by cohort, with the vehicle group (n = 14) containing 7 animals from cohort I and 7 from cohort II, and the succimer group (n = 15) containing 8 animals from cohort I and 7 from cohort II. This animal recruitment strategy was necessitated by the large number of animals (n = 72) used in the parent study. Complete details of the animal recruitment and care procedures will be reported elsewhere. Briefly, the infants were housed in a single cage with their respective mothers until weaning at age 26 weeks, then subsequently housed in groups of 5 females and 1 non-study male in group cages. Animals were breast-fed until 26 weeks, then maintained on Purina Monkey Chow (#5037) ad libitum thereafter. While in the metabolic cages, animals were given free access to food and water. All procedures related to animal care conformed to the guidelines set forth in the Guide for the Care and Use of Laboratory Animals (NRC, 1996Go).

Lead exposure.
Animals were exposed to Pb through one year-of-age. Beginning on the eighth postpartum day and continuing through weaning at age 26-weeks, Pb was given directly to the infant orally, as Pb acetate (Sigma Chemical Co., St. Louis, MO), in a solution of Similac® milk formula. Post-weaning, the monkeys received their oral-Pb dose dissolved in apple juice. Lead was administered daily in amounts targeted to produce blood Pb levels of approximately 35–45 µg/dL. The Pb dose was adjusted biweekly as needed to maintain the target blood Pb level. Oral Pb exposure was discontinued immediately prior to succimer treatment. Whole blood Pb levels were measured biweekly throughout Pb exposure.

The target blood Pb level of 35–45 µg/dL was chosen because it is within the range of Pb levels in which succimer is being given to children, and it is also within the range of the blood Pb levels in children (20–45 µg/dL) participating in the succimer clinical trials (TLC, 1998Go). In previous studies, we (NKL) have observed no overt signs of Pb toxicity (e.g., decreased weight gain, reduced hematocrit) in infant rhesus monkeys at blood Pb levels of 35–45 µg/dL.

Succimer treatment.
Treatment with oral succimer or vehicle commenced at 1 year-of-age. Succimer as Chemet® (Bock Pharmcol, St. Louis, MO) was orally administered in apple juice vehicle via syringe within 15 min of dissolving the succimer, to ensure maximum activity. Succimer was administered at 30 mg/kg/day and divided into 3 doses/day for 5 days (administered at 9:00 A.M., 4:00 P.M., and 11:00 P.M.). It was then continued for an additional 14 days at 20 mg/kg/day, divided into 2 doses/day (administered at 9:00 A.M. and 11:00 P.M.), for a total 19-day treatment regimen. The vehicle group received a succimer placebo in the apple juice carrier at the same intervals. All laboratory staff who administered the treatments and collected samples were blinded to the treatments.

Sample collection.
All sample collection, storage, and laboratory-ware (i.e., Teflon®, polyethylene, polypropylene, and stainless steel) were acid-cleaned using established procedures (Smith et al., 1992Go). Reagents used in sample processing and analyses were double sub-boiling, quartz-distilled, high-purity grade, and the water was ultrapure grade (>18 Mohm-cm2).

Twenty-four h prior to the start of chelation (t-1d), individual animals were placed in stainless steel metabolic cages, where they remained throughout the first 5 days of treatment (t-1d -> t5) for a total of 6 consecutive days, in order to facilitate complete urine collections. Urine collection bottles were changed at 24-h intervals. The floor of each metabolic cage was modified for this study by fitting 2 stainless steel mesh screens (1 cm2 and 0.5 mm2 mesh, respectively) over a steep angle (45°) urine-collection pan with a 1-cm diameter outlet tube that drained directly into a 250-mL polyethylene sample-collection bottle. This configuration minimized the transfer of food and fecal material into the collected urine sample. Cages were cleaned daily via rinsing with ultrapure water. Cages were cleaned thoroughly weekly (i.e., between placing new animals in the cages) using soap and water, acid-washing with dilute HNO3, and rinsing with ultrapure water, in order to minimize sample contamination with Pb and other metals. All samples were collected using trace metal-clean techniques (Smith et al., 1992Go).

Immediately following collection of a 24-h urine sample, the total volume was recorded, and an 8-ml sub-sample of urine was removed and filtered through an acid-cleaned 0.45 µm Teflon® syringe filter to remove any suspended particles. Samples were filtered into a polyethylene bottle and stored frozen until analyses. Whole blood samples (1–2 mL) were collected by femoral venipuncture into evacuated collection tubes (Vacutainer® #367734, Becton-Dickinson, Franklin Lakes, NJ) containing heparin anticoagulant. Blood samples were collected biweekly over the Pb-exposure period, and approximately every other day over the chelation period. However, only the blood Pb data relevant to the present study are reported here (t0, t1, t3, t5, and t20 days, relative to the start of chelation).

Sample collection blanks.
Urine sample collection blanks were collected to evaluate the possibility of sample contamination with Pb and essential elements from food, feces, and the metabolic cages. Three types of sample collection blanks were collected in duplicate to measure various possible forms/stages of contamination:

  1. Ultrapure water rinse, unoccupied clean-cage blank. Following thorough weekly cleaning of the metabolic cage as described above (inter-animal), ultrapure water (pH 5.5) was rinsed around the bottom of the unoccupied cage and collected into a sample bottle. This blank represents the minimum contamination of urine due solely to the cage and sample processing (collection -> storage) at the beginning of sample collecting.
  2. Ultrapure water rinse, occupied clean-cage blank. Following the moderate daily cleaning of the metabolic cage as described above, ultrapure water (pH 5.5) was rinsed around the bottom of the occupied cage and collected into a sample bottle. This blank represents a likely level of contamination of urine throughout the duration of sample collection.
  3. Ultrapure water rinse, occupied, dirty-cage blank. Prior to the daily cleaning of the metabolic cage, ultrapure water (pH 5.5) was intentionally rinsed over feces and food remains around the floor of the occupied cage and collected into a sample bottle. This blank represents the unlikely maximum possible contamination of urine samples.

In all cases, approximately 130–190 mL of ultrapure water was rinsed around the cage and collected. Following collection, the blank samples were processed using procedures identical to those used for the urine samples.

Sample processing and analyses.
All sample processing and analyses was conducted using trace metal-clean techniques under HEPA-filtered air conditions to minimize contamination of the samples. Prior to analyses, urine samples were thawed and acidified (pH < 2) with 16 N Q-HNO3 (Quartz distilled–Optima grade, Fisher Scientific, U.S.) to prevent loss of trace elements to the walls of the polyethylene storage bottles. For blood Pb measurements, samples were thawed, transferred to Teflon® vials, dried, and digested for 4–8 h in 2 mL of hot 16 N Q-HNO3, evaporated to dryness, and redissolved in 1 N Q-HNO3, for analyses (Smith et al., 1992Go).

Urine Ca, Co, Cu, Fe, Mg, Mn, Ni, and Zn were measured using a Finnigan MAT Element magnetic sector-inductively coupled plasma (ICP) mass spectrometer in multi-isotope counting mode, measuring masses 44Ca, 59Co, 65Cu, 56Fe, 24Mg, 55Mn, 60Ni, 45Sc, and 64Zn with 45Sc (scandium-45) used as an internal standard for measurement of these 8 elements. Lead concentrations in urine and blood were determined independently, measuring masses 201Hg (used to correct for 204Hg interference of 204Pb), 204Pb, 206Pb, 207Pb, 208Pb, and 209Bi with 209Bi (bismuth-209) used as an internal standard (Woolard et al., 1998Go). External standardization for the 8 essential elements was via certified standards (Spex Industries Inc., Edison, NJ, U.S.). National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2670 (toxic metals in freeze-dried urine) was used to evaluate procedural accuracy. For sample Pb, external standardization was via an NIST SRM 981 isotopic standard, and a certified SPEX Pb standard that had been independently characterized, isotopically, via thermal ionization mass spectrometry. NIST SRM 955A (Pb in blood), 1577 (bovine liver), and 2670 were used to evaluate procedural accuracy of the Pb analyses. The measurement accuracy for the essential elements in the urine SRM 2670 averaged 94% ± 8% RSD, with analytical detection limits as follows: Ca 0.03 ppm, Co 0.004 ppb, Cu 0.37 ppb, Fe 0.16 ppb, Mg 0.02 ppm, Mn 0.16 ppb, Ni 0.11 ppb, and Zn 4.1 ppb. The measurement accuracy for Pb in urine (SRM 2670) and blood (SRM 955a) was 101% ± 5% RSD for analyzed Pb concentrations >0.05 ppb, with an analytical detection limit of 0.01 ppb. (Woolard et al., 1998Go).

Data analysis.
Measured urine element levels were calculated as element concentrations (e.g., µg element/mL urine), total element excreted per 24 h (e.g., µg element/day), and total element excreted over 5 days of treatment (e.g., µg element/5 days, t1 -> t5), using the measured urine excretion volumes.

Statistical differences in urine element levels (e.g., µg/mL) and total element levels (e.g., µg) between treatment groups (vehicle vs. succimer) were evaluated with multivariate ANOVA, using the General Linear Models procedure of SAS (1989). The variable "urine volume" was included as a covariate to remove this source of variation from the analyses. Subsequently, data for each element were analyzed separately using univariate ANOVA to assess which specific elements approached/reached significance between treatments. Elements identified by these analyses were then re-analyzed together, with multivariate ANOVA. In all cases, values of p <= 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blood and Urine Pb Levels
Blood Pb values declined dramatically with treatment in both the succimer- and vehicle-treated groups, although levels in the succimer-treated group declined significantly more (Fig. 1Go). Notably, blood Pb levels at the beginning of treatment were higher in the succimer group (49 ± 4 µg/dL SE) compared to the vehicle group (40 ± 2 µg/dL SE), although this difference was not significant (p > 0.05, t-test). By day-5 of treatment (the duration over which urine samples were collected), the succimer group blood Pb levels were significantly lower than those in the vehicle group (p < 0.01, t-test), and this significant difference between treatments persisted through the end of treatment (day 20) (p < 0.05, t-test). A complete evaluation of the efficacy of succimer for reducing blood- and tissue-Pb levels in all animals of the parent study will be presented elsewhere.



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FIG. 1. Blood Pb concentrations (µg/dL) in vehicle (triangle, n = 14 animals/group) and succimer (filled circle, n = 15 animals/group)-treated monkeys over the treatment period. All values are averages (± SE). Also indicated is the duration that animals were housed in metabolic cages for the collection of complete 24-h urine samples.

 
Succimer rapidly and significantly increased the urinary excretion of Pb by approximately 4–6-fold compared to the vehicle group, over the first 2 days of treatment, and levels of Pb excretion remained significantly increased by approximately 3-fold over the vehicle group over days 3–5 of treatment (Fig. 2aGo). Collectively, the total urinary diuresis of Pb in the succimer group was increased significantly by >4-fold (p < 0.001, t-test) above the vehicle group over the first 5 days of treatment (Fig. 2bGo).



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FIG. 2. (a) Complete daily (24-h) excretion of Pb (µg) in urine of vehicle (square, n = 14 animals/group) and succimer-treated monkeys (circle, n = 15 animals/group) prior to treatment (day 0), and over the first 5 days of treatment (days 1–5). (b) Total cumulative excretion of Pb (µg) in urine of vehicle (open bar) and succimer (filled bar)-treated groups over treatment days 1–5. All values are averages (± SE); *** indicates significant difference from vehicle group (p < 0.001, t-test).

 
Urine Volume
Since the urinary excretion of essential elements may be biased by the urine output, the volume of urine excreted by the vehicle vs. succimer-treated groups was statistically evaluated by univariate ANOVA. Those analyses indicated that the urinary volume output of the vehicle and succimer groups were not significantly different over the 24 h prior to the start of treatment (day 0), or over the first 5 days of treatment (days 1–5) (Tables 1 and 2GoGo). There was a trend for the succimer group to excrete more urine over the treatment period, although this trend was also evident starting on day 0 before treatment began. This substantiated the use of urine volume as a covariate in all statistical ANOVA analyses of the effects of succimer treatment on the urinary excretion of the essential elements.


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TABLE 1 P-value Results of Multivariate and Univariate ANOVA Analyses to Evaluate the Effect of Succimer vs. Vehicle Treatment on the Urinary Excretion of Essential Elements
 

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TABLE 2 P-value Results of Multivariate and Univariate ANOVA Analyses to Evaluate the Effect of Succimer vs. Vehicle Treatment on the Urinary Excretion of Cu, Fe, Mn, and Zn
 
Diuresis of Essential Elements
The succimer group trended towards the excretion of higher cumulative levels of Ca, Fe, Mn, Ni, and Zn, but not of Co, Cu, and Mg, over the first 5 days of treatment (days 1–5 total). However, none of the apparent increases in these individual elements reached statistical significance (Figs. 3 and 4GoGo; Table 1Go). Multivariate ANOVA analyses of the daily urinary excretion of all 8 essential elements collectively (i.e., all elements x treatment day) indicated marginally non-significant increases in the succimer group on treatment days 1, 2, and 3 (0.1 > p > 0.05, Table 1Go). However, when the cumulative total excretion over treatment days 1–5 were considered, succimer treatment did significantly (p < 0.05, multivariate ANOVA) increase the urinary excretion of these elements, compared to the vehicle (all elements, day 1 -> 5 total; Table 1Go). This latter outcome reflects the fact that the 5-day total amount of essential elements excreted in urine tended to be higher in the succimer-treated group for 7 of the 8 elements measured (excepting Co), indicating that succimer treatment had a small but measurable effect on increasing the diuresis of these elements.



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FIG. 3. Total daily (24-h) urinary excretion of Ca, Co, Cu, Fe, Mg, Mn, Ni, Zn (µg or mg per day) by vehicle (hatched bar, n = 14 animals/group) and succimer (solid bar, n = 15 animals/group)-treated monkeys over days relative to the start of treatment (day 0). All values are averages (± SE). See Table 1Go for results of statistical analyses.

 


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FIG. 4. Total 5-day cumulative urinary excretion of essential elements (µg or mg) by vehicle (solid bars, n = 14 animals/group) and succimer (hatched bars, n = 15 animals/group)-treated monkeys over treatment days 1–5. All values are averages (± SE). See Table 1Go for results of statistical analyses.

 
Subsequent univariate analyses of each element separately indicated no significant differences between treatments, although increases in Zn (day 3) excretion with succimer treatment were only marginally not significantly different (0.1 > p > 0.05) from the vehicle-treated group (Fig. 3Go, Table 1Go). There was an exception to this, in that the succimer group excreted significantly less Co than the vehicle group (days 1–5 total, Table 1Go, Fig. 4Go). However, this significant decrease is likely biased by a significant pre-existing difference between groups in Co excretion that was present prior to beginning treatment (day 0, p = 0.039, Table 1Go).

Additional ANOVA analyses were performed that considered only data for Cu, Fe, Mn and Zn, since (1) there were at times trending differences between treatments for these elements, and (2) previous studies have suggested that the diuresis of these elements may be increased with succimer treatment (Cantilena and Klaassen, 1982Go; Friedheim et al., 1978Go; Graziano et al., 1988Go, 1985Go). Multivariate ANOVA analyses of all 4 of these elements collectively indicated no significant effect of succimer treatment, although treatment differences on day 3 were only marginally not significant (p = 0.055, Table 2Go). Subsequent univariate analyses of each of these 4 elements indicated that succimer treatment significantly increased the urinary excretion of Mn on day 3 of treatment (Table 2Go, Fig. 5Go).



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FIG. 5. Daily urinary excretion of Mn (µg/day) by vehicle (square, n = 14 animals/group) and succimer (circle, n = 15 animals/group) treated monkeys over treatment days 0–5. All values are averages (± SE); * indicates significantly different from time-matched vehicle group (p < 0.05, ANOVA followed by post hoc comparisons).

 
Sample Collection Blanks
A complete range of possible urine-sample contamination was provided by the collected metabolic cage blanks. Cage blanks type (1) (see Materials and Methods, ultrapure water rinse, unoccupied clean cage) were typically <4% of the average daily excretion for all elements. Elemental levels in the cage blanks considered most representative of possible urine sample contamination [i.e., type (2), ultrapure water rinse, occupied clean cage] were also typically low (<15% of the average daily excretion) for all elements. In contrast, element levels in the cage blanks representing the unlikely maximum sample contamination [i.e., type (3), ultrapure water rinse, occupied dirty cage], where ultrapure water was intentionally rinsed directly over food and feces on the cage floor, yielded blank values that were highly variable across elements, from <5% for Ca and Mg, to >=100% for Co, Mn, and Zn. Due to the impossibility in this study for evaluating the true level of urine sample contamination for these elements, no blank correction of the samples was attempted. Instead, these blank values were used to indicate the likelihood of unacceptable sample contamination. In all cases, the likely level of contamination [blank type (2)] was <15%, which we considered acceptable.

It is noteworthy that, while contamination of urine samples may have occurred, this would have had the effect of increasing the levels of some elements in the samples of both the vehicle and succimer treatment groups. Moreover, since cages were cleaned daily, the magnitude of contamination would have varied daily. The possible presence of variable contamination between samples may have reduced our ability to detect measurable differences in essential element diuresis between the succimer and vehicle groups, due to an increase in within-group variability. Thus, observed differences in urine element levels between treatment groups, which reached or approached traditional levels of statistical significance (i.e., p < 0.05 or p < 0.10, respectively) likely reflect actual differences.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Succimer is considered to be a safe and effective treatment for Pb poisoning in humans (Chisolm, 1992Go; Fournier et al., 1988Go; Goyer et al., 1995Go; Graziano, 1986Go; Liebelt et al., 1994Go; Smith et al., 1994Go; Xu and Jones, 1988Go). Numerous laboratory studies have demonstrated its effectiveness in reducing Pb levels in blood and most other soft tissues (Cory-Slechta, 1988Go; Jones et al., 1994Go; Pappas et al., 1995Go; Smith et al., 1998Go; Smith and Flegal, 1992Go). However, some studies have suggested that more prolonged or repeated treatment regimens may be necessary to reduce Pb (and presumably toxicity) in target organs such as the CNS and the skeleton (Cory-Slechta, 1988Go; Cremin et al., 1999Go; Pappas et al., 1995Go; Smith et al., 1998Go; Smith and Flegal, 1992Go; Xu and Jones, 1988Go). In light of this, there is a need to thoroughly understand any non-target outcomes of succimer treatment, such as effects on the diuresis of essential elements.

The efficacy of succimer for reducing blood Pb levels and substantially increasing Pb diuresis is clearly demonstrated in this non-human primate model of childhood Pb exposure (Figs. 1 and 2GoGo). Both the reductions in blood Pb levels and the concomitant 4-fold increase in Pb diuresis with chelation are comparable to effects observed clinically (Graziano et al., 1988Go, 1992Go, 1985Go). Succimer treatment for 19 days increased both the rate and final magnitude of blood Pb decline compared to the vehicle group, although most of this benefit was realized within the first 5 days of treatment (Fig. 1Go). When adjusted to their starting (t0) values, blood Pb levels in the succimer-treated group were approximately 40% lower than the vehicle group after 5 days of treatment, and about 36% lower than the vehicle group at the end of treatment.

However, succimer treatment also significantly increased the urinary excretion of the essential elements considered here, when their cumulative total excretion over treatment days 1–5 for all elements were considered (Table 1Go). This outcome reflects the trend for the succimer group to excrete higher 5-day total amounts for 7 of the essential elements measured (Ca, Cu, Fe, Mg, Mn, Ni, Zn, excepting Co) (Fig. 4Go), although none of these relative increases reached statistical significance for any particular element (Table 1Go). The greatest effect of succimer treatment appeared to occur over the first 3 days of chelation, based on the multivariate ANOVA analyses x treatment day (Table 1Go). The subsequent univariate ANOVA analyses, separately for each element x treatment day, similarly indicated no significant differences between treatments, although an increase in Zn excretion (day 3) with succimer treatment was suggested by the analyses (0.1 > p > 0.05) (Table 1Go).

In light of the above outcomes, a multivariate ANOVA analyses of a reduced data set containing only Cu, Fe, Mn, and Zn was performed, in order to maximize the detection of a succimer effect on the diuresis of these elements. This was justified, based on several previous clinical studies that reported significant increases in Cu, Fe, and Zn excretion with succimer treatment (Aposhian et al., 1989Go; Friedheim et al., 197; Graziano et al., 1988Go, 1985Go). This analysis similarly indicated no significant effect of succimer treatment, although treatment differences on day 3 were only marginally not significant (p = 0.055). However, the univariate analyses of each of these 4 elements separately indicated that succimer treatment significantly increased the urinary excretion of Mn on day 3 of treatment (Table 2Go, Fig. 5Go). Collectively, these data indicate that succimer does in fact contribute to an increase in the overall urinary excretion of essential elements, although not significantly for any single element considered here. While the magnitude of this overall increase in these nutritionally replete monkeys does not appear to be physiologically important, it may be important in Pb-poisoned children, who can possess reduced trace element reserves due to nutritional deficiencies.

These results are somewhat consistent with previous clinical studies (e.g., type="bib">Aposhian et al., 1989; Friedheim et al., 1978; Graziano et al., 1988, 1985). Nevertheless, those studies that have specifically addressed element diuresis using complete urine collections over the first week or so of succimer treatment in Pb poisoned children and adults have reported mixed results. For example, Graziano et al. (1985) reported that treatment of 6 Pb-poisoned adult males with a 5-day succimer regimen of 30 mg/kg/day led to a statistically significant increase in urinary Zn (81%) excretion, and trending but non-significant increases in Cu (48%), but not in Ca, Fe, or Mg. However, a lower 20-mg/kg/day dosing regimen in a different cohort of 6 subjects led to statistically significant increases in Ca (75%), Cu (200%), and Zn (66%), but not in Fe and Mg, compared to pre-treatment baseline levels. In a subsequent study of Pb-poisoned children who were recruited based on presenting blood Pb levels (30–49 µg/dl) and a positive CaNa2EDTA Pb mobilization test, Graziano et al. (1988) reported that succimer treatment (1050 mg/m2/day, equivalent to 30 mg/kg/day) for 5 days led to trending but non-significant increases in urinary Ca (76%), Cu (36%), and Fe (18%), but not in Zn or Mg.

Others have also reported succimer-mediated increases in the urinary excretion of some essential elements. Aposhian et al. (1989) investigated the patterns of urinary elimination of DMSA and its metabolites, along with the urinary excretion of Pb, Cu, Zn, and more than 20 additional essential elements, in 6 normal healthy (i.e., non-Pb-poisoned) young adult males following treatment with a single 10 mg/kg dose of DMSA. Notably, the diuresis of Pb, Cu, and Zn were all increased following DMSA treatment, with the peak excretion coinciding with peak excretion of DMSA metabolites, although increases in Cu and Zn were not considered clinically significant. No measurable differences in excretion for the other elements measured (e.g., Ca, Co, Mg, Mn, Ni) were observed, although many of those measurements were limited by analytical detection limits. In an earlier study, Friedheim et al. (1978) treated 5 Pb-poisoned smelter workers with DMSA doses ranging from 8.4 mg/kg/day to 42 mg/kg/day over 6 days. This study resulted in statistically significant but clinically unremarkable increases in the urinary excretion of Cu, but no measurable increases in Ca, Fe, Mg, or Zn.

The present study possessed several notable benefits over previous studies investigating the effects of succimer on element diuresis, including (1) standardized rearing, housing, nutrition, and Pb-dosing regimens across treatments, and (2) the use of trace metal-clean techniques and a sensitive ICP-MS methodology in sample collection, processing, and analyses. Moreover, the primate animal model utilized here allows these results to be readily extrapolated to the treatment of Pb-poisoned children (Leggett, 1993Go; O'Flaherty, 1995Go, 1996, 1998). However, the outcomes of this study may also be qualified by several factors, including (1) the limited duration of urine collections (5 days) relative to the complete treatment regimen (19 days), and (2) the possible contamination of urine samples due to food and fecal debris, etc. Although sample contamination during collection would not have produced any treatment bias, it would have likely increased variability in element levels between samples (i.e., increased within-group variance), thereby reducing the power of detection for a treatment effect. Consequently, results from the present study may underestimate the true effects of succimer on the diuresis of essential elements.

In summary, this study indicates that succimer treatment contributes to an overall increase in the urinary excretion of essential elements, although these increases do not appear to be physiologically remarkable. However, this latter statement may be qualified in the extrapolation of these data to Pb-poisoned children, who can possess reduced trace element reserves due to nutritional deficiencies. Moreover, the extent to which these observations may be extrapolated to cases requiring repeated succimer treatment regimens, or treatment at lower blood Pb levels is not known. Finally, these data may not reflect the effects of succimer on the endogenous levels or distribution of essential elements, although those outcomes may be physiologically important (Fournier et al., 1988Go).


    ACKNOWLEDGMENTS
 
This study was supported by NIEHS grant #ES06918.


    NOTES
 
1 To whom correspondence should be addressed at Environmental Toxicology, Applied Sciences Building, University of California, 1156 High Street, Santa Cruz, CA 95064. Fax: (831) 459-4719. E-mail: dsmith{at}biology.ucsc.edu. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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