* Department of Environmental Toxicology, University of California, Santa Cruz, California 95064; and
Harlow Center for Biological Psychology, University of Wisconsin, Madison, Wisconsin 53175
Received October 7, 1999; accepted November 15, 1999
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ABSTRACT |
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Key Words: essential elements excretion; rhesus monkeys; lead (Pb); succimer; blood Pb levels; chelating agents.
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INTRODUCTION |
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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, 1988; Cremin et al., 1999
; Pappas et al., 1995
; Smith et al., 1998
; Smith and Flegal, 1992
; Xu and Jones, 1988
). 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, 1995
; TLC, 1998
). 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.
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MATERIALS AND METHODS |
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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, 1996).
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 3545 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 3545 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 (2045 µg/dL) participating in the succimer clinical trials (TLC, 1998). 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 3545 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., 1992). 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., 1992
).
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 (12 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:
In all cases, approximately 130190 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 distilledOptima 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 48 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., 1992).
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., 1998). 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., 1998
).
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.
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RESULTS |
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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, 1982; Friedheim et al., 1978
; Graziano et al., 1988
, 1985
). 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 2
). 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 2
, Fig. 5
).
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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.
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DISCUSSION |
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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 2). 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., 1988
, 1992
, 1985
). 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. 1
). 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 15 for all elements were considered (Table 1). 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. 4
), although none of these relative increases reached statistical significance for any particular element (Table 1
). 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 1
). 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 1
).
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., 1989; Friedheim et al., 197; Graziano et al., 1988
, 1985
). 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 2
, Fig. 5
). 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 (3049 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, 1993; O'Flaherty, 1995
, 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., 1988).
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ACKNOWLEDGMENTS |
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NOTES |
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