ENVIRONMENTAL RESEARCH, SECTION A 78, 168—176 (1998)ARTICLE NO. ER983854

Efficacy of Succimer Chelation for Reducing Brain Pb Levelsin a Rodent Model

Donald Smith,* Lorna Bayer,- and Barbara J. Strupp-,‡

*Department of Environmental Toxicology, University of California, Santa Cruz, California 95064; -Department of Psychology,Cornell University, Ithaca, New York 14853; and ‡Department of Nutritional Sciences, Cornell University, Ithaca, New York 14853

Received February 6, 1998

Increasing evidence indicates that early low-levellead (Pb) exposure produces enduring cognitive im-pairment in children, underscoring the need to de-velop improved therapeutic intervention. Althoughchelating agents have been shown to effectively re-duce body Pb levels, it is not yet known whether thistreatment ameliorates Pb-induced cognitive dys-function. Clinical research in this area is hamperedby the need to rely on reductions in blood Pb levelsas the index of treatment efficacy, despite the factthat brain Pb level is the exposure parameter ofgreatest relevance to neurocognitive outcomes. Thepresent studies were designed to provide informa-tion that will aid future research in this area in bothhuman and animal models. The objectives of thesestudies were (1) to evaluate the efficacy of differentdoses and durations of succimer (meso-2,3-dimer-captosuccinic acid; DMSA) chealtion for reducingbrain and blood Pb levels and (2) to determine theextent to which blood Pb can serve as a surrogate ofbrain Pb following chelation. Long–Evans hoodedrats were exposed to Pb from birth until day 31(Study 1) or day 40 (Study 2) of life, followed by oraltreatment with a vehicle or one of two succimerregimens for a duration of either 7 or 21 days. Re-sults indicated that 7 days of succimer treatmentproduced a 1.5- to 2.5-fold greater reduction of Pb inblood than in brain, relative to time-matched ve-hicle groups. Prolonged treatment (21) days did notfurther reduce blood Pb levels (relative to 7-daysuccimer treatment), but did produce further re-ductions in brain Pb level compared to time-matched vehicle groups. Thus, chelation-mediatedreductions in brain Pb did not parallel reductionsin blood Pb over the course of treatment. While therelevance of these data to humans may be con-founded by anatomical and physiological differ-ences between rodents and primates, as well asdifferences in the metabolism of succimer (DMSA),they suggest that clinical studies should exercisecaution when using blood Pb as an index of the

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0013-9351/98 $25.00Copyright ( 1998 by Academic PressAll rights of reproduction in any form reserved.

efficacy of chelation tratment for reducing brain Pblevels. ( 1998 Academic Press

Key Words: lead; chelation; succimer; brain;neurotoxicity.

INTRODUCTION

There is increasing evidence that even moderateelevations of lead (Pb) during early developmentproduce enduring cognitive impairment (e.g., seeRice, 1996, for a review). As research has identifiedadverse effects at progressively lower levels of Pbexposure, the number of children known to be at riskfor cognitive impairment has grown. This has under-scored the need to develop improved interventionstrategies. Chelation treatment using succimer(meso-2,3-dimercaptosuccinic acid; DMSA) has beenshown to reduce Pb levels in blood, brain, and othersoft tissues (Pappas et al., 1995; Jones et al., 1994;Smith and Flegal, 1992; Fournier et al., 1988; Cory-Slechta, 1988; Graziano, 1986), as well as somesymptoms of Pb intoxication (Glotzer, 1993; Frum-kin and Gerr, 1993). However, very few studies haveevaluated the efficacy of chelation treatment for re-ducing Pb-induced neurocognitive deficits, althoughongoing studies of succimer treatment are now ad-dressing this issue in humans, nonhuman primates,and rodents.

Succimer is considered to be a safe and effectivechelating agent for the treatment of Pb poisoning inhumans (Goyer et al., 1995; Smith et al., 1994;Chisolm, 1992; Aposhian and Aposhian, 1990;Fourier et al., 1988; Xu and Jones, 1988; Graziano,1986; Graziano et al., 1978). Moreover, it offersmany advantages over CaNa2EDTA chelation treat-ment, since it is administered orally and it does notappear to cause a significant diuresis of other essen-tial metals (e.g., iron and zinc). However, a review of

EFFICACY OF SUCCIMER CHELATION FOR REDUCING BRAIN Pb 169

the relevant studies suggests that information onthe efficacy of succimer for reducing Pb from brain,bone, blood, and other soft tissues is incomplete andin some cases conflicting (Pappas et al., 1995; Joneset al., 1994; Smith and Flegal, 1992; Fournier et al.,1988; Cory-Slechta, 1988; Graziano, 1986; Cantilenaand Klaassen, 1982; Friedheim et al., 1978). Forinstance, Pappas et al. (1995) observed no clear ad-ded benefit of an elevated succimer dose (32 mg/kg/day versus 16 mg/kg/day]21 days) in reducingeither blood or brain Pb levels. In contrast, Cory-Slechta (1988) did observe a significant dose effect ofsuccimer (DMSA at 50 mg/kg/day versus 25 mg/kg/day]5 days) in reducing blood Pb levels, but notbrain Pb levels. These and other studies suggest thatthe magnitude of tissue Pb reduction with chelationmay be dependent on both the dose and the durationof chelation and possibly also on the body Pb burden(Goyer et al., 1995). However, this latter suggestionis not substantiated by a recent clinical study(Liebelt et al., 1994), which reported no difference inthe relative efficacy of succimer treatment for reduc-ing blood Pb levels in children with moderately(group mean Pb"31lg/dL) versus more elevated(group mean Pb"51 lg/dL) blood Pb levels.

It is also apparent that succimer treatment mayremove Pb from various target organs (e.g., brain,blood, bone) with differing success. For example,several studies have suggested that succimer treat-ment is less effective in removing Pb from brain thanfrom blood (Pappas et al., 1995; Smith and Flegal,1992; Xu and Jones, 1988; Cory-Slechta, 1988). Inaddition to experimental differences, these observa-tions may reflect important differences in the factorscontrolling the uptake and release of Pb from thesetwo body compartments. Consideration of these tis-sue differences in the reduction of Pb is important inevaluating the efficacy of treatment, particularlysince clinical studies must rely on measured bloodPb levels to estimate reductions in brain Pb, theorgan of greatest relevance to neurocognitive out-comes. The present studies utilized a rodent model ofchildhood Pb exposure in order to: (1) assess theextent to which blood Pb can serve as a surrogate ofbrain Pb levels following succimer treatment, usingtreatment regimens similar to those used clinically;and (2) examine the efficacy of different succimerregimens (i.e., different doses and durations of treat-ment). Previous studies in this laboratory (B.J.S.)have demonstrated cognitive impairment in rats ex-posed to Pb early in life, using Pb treatment regi-mens comparable to those described here (Hilsonand Strupp, 1997; Alber and Strupp, 1996). An addi-tional goal of the present study was to serve as the

first of a series of ongoing studies to determine thesuccimer treatment regimen most efficacious for re-ducing brain Pb levels. This information will in turnbe used in subsequent studies to specifically evalu-ate the efficacy of succimer treatment for reducingneuroognitive impairment in this rodent model.

METHODS

Study Design

The studies described here were performed withinthe context of a larger ongoing study that is evaluat-ing the efficacy of succimer treatment for reducingneurocognitive toxicity in a rodent model of child-hood Pb toxicity. Two studies (Studies 1 and 2) wereconducted to evaluate: (i) the utility of whole bloodPb level as a surrogate of brain Pb level followingsuccimer treatment and (ii) the efficacy of differentsuccimer treatment regimens for reducing brain andblood Pb levels. Study 1 evaluated two doses of suc-cimer, each for a single 7-day treatment duration,whereas Study 2 evaluated a single dose regimen ofsuccimer for two treatment durations, 7 and 21 days.The following description of methodology pertains toboth studies unless otherwise indicated.

Animals

Adult male and nulliparous female Long—Evansrats were obtained from Harlan Sprague—Dawley(Indianapolis, IN). Adult breeding animals werehoused in stainless-steel hanging cages and main-tained on a semipurified rodent diet (PROLAB RMH1000, PMI Feeds, Inc.) and Milli-Q (MQ) water adlibitum. Following breeding and conception, damswere housed individually. At parturition, dams wereswitched to a pelleted semipurified rodent diet (AIN-93G, Dyets, Inc.). On postnatal day 1 (P1), each litterwas sexed, weighed, and culled to 10 pups, preserv-ing a constant male : female ratio within all litters(only female offspring were utilized here). At wean-ing (P21), one female offspring from each litter wasrandomly assigned to a treatment group. Weanlingswere given food and MQ water ad libitum. All ani-mals were maintained on a regular light—dark cyclethroughout the experiment. The use of only one ani-mal per litter for each treatment condition wasnecesary to maintain statistical independence of theobservations in the ongoing parent study.

Pb and Succimer Treatments

Pb in drinking water was made fresh weekly overthe course of the Pb exposure duration by dissolving

170 SMITH, BAYER, AND STRUPP

2.4 g Pb-acetate (Pb(Ac), Sigma) in 4 L of MQ waterto a desired Pb concentration of 325 lg Pb/mL (w/v)(primary Pb stock). Water bottles were filled withfresh Pb-drinking water daily in order to minimizethe loss of Pb in the water bottles due to adsorptiononto bottle surfaces, etc.

Succimer, as Chemet (DMSA) was obtained fromBock Pharmacal Co. (St. Louis, MO). The daily suc-cimer dose was administered in two equally divideddoses given 12 h apart. Immediately prior to eachuse, succimer was dissolved in apple juice (vehicle)to the appropriate dose level and administered bygavage at 5:00 AM and 5:00 PM with a tuberculin syr-inge fitted with a 20-gauge stainless steel feedingneedle (Popper and Sons, Inc.).

Study I. Thirty neonatal Long—Evans rats wereexposed to Pb via nursing dams receiving 325lgPb/mL in drinking water from P1 to P21, then dir-ectly via drinking water containing 30lg Pb/mL(prepared via diluting the primary Pb stock with MQwater) from P21 to P30. Pups (aged P31) weredivided into three treatment groups (n"10/group)and treated for 7 days with (i) vehicle, (ii) 30 mgsuccimer/kg/day, or (iii) 60 mg succimer/kg/day. Allanimals were sacrificed following the 7-day treat-ment, and whole blood and brain samples were col-lected, as described below.

Study II. Forty neonatal rats were exposed to Pbas described above for Study I, with the exceptionthat the Pb exposure duration continued until P40,and the concentration of Pb in the drinking waterover the duration P21—P40 was maintained at325lg/mL. On P41, pups were randomly assignedto one of the following five treatment groups(n"8/group): (i) time 0 control (represents body Pblevels prior to the start of chelation or vehicle treat-ment), (ii) vehicle for 7 days, (iii) 50 mg succimer/kg/day for 7 days, (iv) vehicle for 21 days, and(v) 50 mg succimer/kg/day for 7 days followed by25 mg succimer/kg/day for 14 days. Animals weresacrificed at the end of their respective treatments,and whole blood and brain samples were collected,as described below.

Sample Collection and Analyses

Sample collections. With the exception of theblood collection vacutainers, all sample collection,storage, and laboratory-ware (i.e., Teflon, polyethyl-ene, polypropylene, and stainless steel) were acid-cleaned using established procedures (Flegal andSmith, 1992; Smith et al., 1992). Reagents used insample processing and Pb analyses were double sub-

boiling quartz distilled high-purity grade, and thewater (MQ) was ultrapure grade ([18 Mohm-cm2).

Animals were euthanized via sodium-pentobar-bital overdose (50 mg/kg) and exsanguination. A 0.8-ml sample of whole blood was collected into a poly-propylene syringe via cardiac puncture from surgi-cally exposed hearts of anesthetized animals anddispensed into a Microtainer vacutainer (EDTA anti-coagulent). Whole brain was excised using acid-washed stainless-steel dissecting tools, rinsed withMQ water, and deposited into polypropylene storagecontainers. All tissue sampling was conducted usingtrace metal-clean procedures. Dissecting instru-ments (stainless steel) were cleaned prior to eachdissection and rinsed frequently within a dissectionprocedure to avoid contamination from nonbrain tis-sues. All samples were stored frozen.

Analyses. Lead concentrations were measured inwhole blood and brain tissue. Blood Pb analyseswere conducted using graphite furnace atomic ab-sorption spectrometry by the Wisconsin State Lab-oratory of Hygiene (WSHL, Madison, WI). TheWSHL administers the Nationwide Blood Lead Pro-ficiency Testing Program as a cooperative programwith the Centers for Disease Control and the Bureauof Maternal and Child Health, thus ensuring re-liable and accurate analyses. For whole brain Pbdeterminations, samples were thawed, transferredto Teflon vials, dried at 65°C to a constant weight,digested for 8 h in 3 ml hot 16 N HNO3, evaporated todryness, and redissolved in 1 N HNO3 for analyses(Smith et al., 1992). Sample Pb was measured usinga Finnigan Element inductively coupled plasma(ICP) high-resolution mass spectrometer in multi-isotope counting mode, measuring masses 208Pb and209Bi, with 209Bi used as an internal standard(Woolard et al., 1998). External standardization wasvia a certified Pb standard that had been indepen-dently isotopically characterized via thermal ioniz-ation mass spectrometry. National Institute ofStandards and Technology (NIST) Standard Refer-ence Materials (SRM) 955A (Blood) and 1577 (Bo-vine Liver) were used to evaluate proceduralaccuracy. This methodology has been demonstratedto yield a measurement precision of \$0.5% forsample Pb concentrations [0.05ppb (Woolard et al.,1998). The analytical detection limit was 0.005 ppb.

Statistical analyses of the data were performedusing pairwise comparisons of treatment groups (ttest, one-tailed, unless otherwise stated) following,F tests for variance quality. Calculated t statisticswith probability P\0.05 were considered signifi-cant.

FIG. 1. Study 1 blood Pb (solid bars) and brain Pb (hatchedbars) concentrations in vehicle and succimer-treated juvenile rats(treatment duration, 7 days). Values are averages$SE (n"10per group). * indicates significantly different from vehicle group(P40.05, t test). Note: Average brain Pb in the 30 and 60 mg/kg/day succimer groups tended to differ from the vehicle group, butthese differences (P"0.051 and P"0.077) did not achieve con-ventional levels of statistical significance.

EFFICACY OF SUCCIMER CHELATION FOR REDUCING BRAIN Pb 171

RESULTS

Study 1—Evaluation of Two Succimer DoseRegimens

Blood Pb. Succimer treatment at a dose of either30 or 60 mg/kg/day for 7 days significantly (P\0.001, t test) reduced blood Pb levels, producing anB50% reduction compared to the vehicle-treatedgroups (Table 1, Fig. 1). There was no apparentbenefit (P[0.19, t test) of the increased succimerdose (60 mg/kg/day versus 30 mg/kg/day) for reduc-ing blood Pb levels.

Brain Pb. Succimer treatment was less effectivein reducing brain Pb levels over this 7-day treatmentperiod, producing only a 20—25% reduction relativeto the vehicle groups (P"0.051, 30 mg/kg/day treat-ment; P"0.077, 60 mg/kg/day treatment). As withblood Pb levels, there was no apparent benefit of theincreased succimer dose (60 mg/kg/day versus30 mg/kg/day) for reducing brain Pb levels (P[0.37)(Table 1, Fig. 1).

Study 2—Evaluation of Two Succimer TreatmentDuration Regimens

Blood Pb. Relative to the group sacrificed priorto chelation (T"0 control group), blood Pb levelsdecreased significantly (P\0.001) in both the ve-hicle and the succimer-chelated groups after both7 and 21 days of treatment (Fig. 2). Also, succimersignificantly reduced blood Pb levels relative to the

TABLE 1Average (6SD) Pb Concentrations in Whole Blood and

Brain of Pb-Treated Juvenile Rodents from Study 1(n 5 10/Treatment) and Study 2 (n 5 8/Treatment)

Treatment Duration Blood Pba Brain Pba

Study 1 Vehicle 7 days 18.8$5.0 1.07$0.30Succimer 7 days at

30 mg/kg/day8.89$3.69 0.85$0.25

7 days at60 mg/kg/day

7.33$3.67 0.79$0.50

Study 2 T0

control 48.5$5.4 2.35$0.34Vehicle 7 days 24.3$3.65 1.81$0.21

21 days 21.0$2.75 1.78$0.30Succimer 7 days at

50 mg/kg/day9.38$2.16 1.11$0.18

21 days at50 mg/kg/day

9.88$2.36 0.57$0.16

Note. Study 1 and Study 2 utilized different Pb-exposure regi-mens (see Methods for details).

a Blood Pb units, lg/dL; brain Pb units, lg/g dry wt.

vehicle-treated groups after both 7 days (P\0.005)and 21 days (P\0.005) of treatment. However, 21days of succimer treatment provided no additionalbenefit (P[0.05) in reducing blood Pb levels relativeto 7 days of treatment (Table 1, Fig. 2).

Brain Pb. Brain Pb levels were reduced signifi-cantly after both 7 days (P\0.02) and 21 days(P\0.002) of treatment relative to the time-matchedvehicle-treated groups (Fig. 2). Moreover, 21 days ofsuccimer treatment provided significant (P\0.05)additional benefit in reducing brain Pb levels com-pared to 7 days of succimer treatment, while brainPb in the vehicle group did not decrease significantlybeyond 7 days (P[0.05). Relative to the T"0 con-trol (prechelation), brain Pb levels decreased signifi-cantly in the succimer groups after 7 days (P\0.002) and 21 days (P\0.001) of treatment. How-ever, vehicle group brain Pb levels did not decreasesignificantly after either 7 or 21 days, relative to thegroup sacrificed prior to treatent (T"0 control).

Relationship between Blood and Brain Pb. A sig-nificant linear relationship existed between blood Pband brain Pb levels in the combined 7- and 21-dayvehicle-treated groups (r"0.574, P\0.05, n"15),while this relationship did not exist in the corres-ponding succimer-treated groups (r"0.224, P[0.2,n"16) (Fig. 3). This suggests that, while blood Pblevels may be somewhat useful in predicting brain

FIG. 2. (a) Study 2 blood Pb and (b) brain Pb concentrationsin Day 0 control (open bars), vehicle (hatched bars), and succimer(filled bars) treated juvenile rats following 7 or 21 days of treat-ment. Values are averages$SE (n"8 per group). * indicatessignificantly (P\0.05, t test) and ** indicates highly significantly(P40.005, t test) different from paired group. NS, not significant(P[0.05).

172 SMITH, BAYER, AND STRUPP

Pb levels in the absence of chelation, this is not truefollowing succimer treatment.

Collectively, these data indicate that succimerchelation was significantly more effective in remov-ing Pb from both blood and brain than was thevehicle treatment (i.e., simple cessation of Pb expo-sure). Moreover, 7 days of succimer treatment wasB1.5- to 2.5-fold more effective in removing Pb fromblood than from brain, whereas after 21 days oftreatment, succimer chelation appeared equally effi-cacious in the two tissues (Table 1). This was due tothe fact that 21 days of succimer chelation did notfurther reduce blood Pb levels beyond those mea-sured after 7 days of treatment, while brain Pb levelscontinued to decline with the extended 21 days oftreatment (Table 1, Fig. 2).

Because the Pb exposure and resultant body Pbburden of the animals in Study 2 was greater than inStudy 1 (see Table 1), these studies combined mayshed light on the relationship between body Pb bur-den and succimer efficacy. An examination of the7-day treated groups in Study 1 versus those inStudy 2 indicates that there were no significant dif-

ferences in blood Pb levels in either the succimer(P[0.5, two-tailed t test) or vehicle (P[0.1) groupsor in brain Pb levels (P[0.1) in the succimer-treatedgroups (Table 1). In contrast, there was a significantdifference (P\0.005) in brain Pb levels of the 7-dayvehicle-treated groups in Study 1 versus those inStudy 2 (Table 1). These data suggest that succimerwas relatively more efficacious at reducing bothblood and brain Pb levels in animals with higherbody Pb burdens (i.e., Study 2).

An assessment of the benefit of succimer treat-ment compared to the cessation of Pb exposure (i.e.,vehicle group) can be made in Study 2 by comparingblood and brain Pb levels between the 21-day treatedgroups (vehicle and succimer) with the T"0 controlgroup (Fig. 2). These comparisons indicate that bloodPb levels were reduced by 80 and 57% in the suc-cimer and vehicle groups, respectively, relative tothe T"0 control group, while brain Pb levels werereduced by 76 and 24% in the succimer and vehiclegroups. Thus, the benefit of succimer treatment (21days) relative to the cessation of Pb exposure (i.e., Pbabatement) appears greater when one considers re-ductions in brain Pb levels rather than blood Pblevels.

DISCUSSION

Pb neurotoxicity in children is considered to berelated to past or current Pb levels in the CNS.Consequently, there is substantial interest in evalu-ating the efficacy of therapeutic treatment regimenssuch as succimer chelation for reducing brain Pblevels and measurable Pb toxocity of the CNS. Sinceit is not possible to measure brain Pb levels in hu-mans exposed to Pb, an alternate biological markerof exposure and treatment efficacy must be evalu-ated, and most commonly that marker is blood Pblevel. However, there is a paucity of informationconcerning the degree to which Pb levels in bloodreflect levels in the brain, particularly followingchelation treatment. Similarly, the benefit of chela-tion treatment for specifically reducing brain Pblevels is poorly understood.

The present studies explored the efficacy of suc-cimer treatment for reducing brain and blood Pblevels in a rodent model of childhood Pb poisoning.Overall, succimer was highly effective in removingPb from both blood and brain tissue. Blood Pb levelsin particular were readily reduced by 53—61% afteronly 7 days of succimer treatment, although con-tinued treatment for 21 days provided no additionalbenefit relative to time-matched vehicle-treatedgroups. A comparable relative reduction (68%) in

FIG. 3. Blood Pb versus brain Pb concentrations in vehicle(d, n"15) and succimer (K, n"16) treated juvenile rats fromStudy 2. Data are from 7- and 21-day treatment durations. Linearregression statistics: Vehicle (——), y-int"0.745, slope"0.046,r"0.574 (P\0.05); Succimer (- - - - - -), y-int"0.647, slope"0.020, r"0.224 (P[0.2).

EFFICACY OF SUCCIMER CHELATION FOR REDUCING BRAIN Pb 173

brain Pb levels was also observed, but only aftera full 21 days of treatment. There was no addedbenefit of the increased succimer dose (60 mg/kg/dayversus 30 mg/kg/day) for reducing either blood orbrain Pb levels. This latter observation is consistentwith Pappas et al. (1995), who also noted no appar-ent benefit of increasing the succimer dose from 16 to32 mg/kg/day in reducing either blood or brain Pblevels.

The present study also examined the efficacy ofprolonged chelation treatment duration for reducingblood and brain Pb levels. These data indicate thatextending the treatment period from 7 to 21 dayssignificantly increased the benefit derived from suc-cimer treatment with respect to brain Pb levels.However, this benefit of prolonged treatment wasnot seen with respect to blood Pb (Table 1, Fig. 2).Jones et al. (1994) also observed an added benefit ofprolonged succimer treatment (8 days versus 4 days)in reducing brain Pb levels in mice using a dose of\180mg/kg/day (blood Pb levels were not reported).These observations may collectively reflect impor-tant differences in the therapeutic efficacy of suc-cimer in the circulation versus the CNS, as well asdifferences in the transfer rates of Pb between thesetwo compartments. This latter suggestion is consis-tent with stable Pb isotope tracer studies in rodents,which have indicated that the rates of brain Pbuptake and loss are much slower relative to thecirculatory and other soft tissue compartments (Sea-ton et al., 1998; Lasman et al., 1997; Smith andFlegal, 1992).

Several aspects of these data suggest that meas-ured blood Pb levels may be a relatively poor indi-cator of the efficacy of chelation treatment forreducing brain Pb levels. After 7 days of succimertreatment, blood Pb levels were substantially re-duced (53—61%), while there was a smaller reduction(20—39%) in brain Pb levels, relative to the time-matched vehicle groups. Also, prolonged treatmet(21 days versus 7 days) was effective in furtherreducing levels of Pb in brain but not in blood, asnoted above. This disparity between reductions inbrain and blood Pb is further substantiated by thepresence of a significant correlation between bloodPb and brain Pb levels in the nonchelated groups(T"0 control and vehicles), versus the lack of a cor-relation in the succimer-treated groups (combined 7-and 21-day treated groups) (Fig. 3). Collectivelythese results indicate that chelation-mediated re-ductions in blood Pb do not accurately reflect cha-nges in brain Pb, particularly after intermediate(e.g., 7 days) treatment durations. This raises con-cerns, since blood Pb levels are the most commonly

used index of treatment efficacy in clinical studies.This concern is further substantiated by laboratorystudies suggesting that blood Pb level is a relativelypoor marker of neurotoxicity (Goldstein et al., 1974)and by a recent clinical study reporting delays inimprovements of cognitive scores in children follow-ing chelation-mediated reductions in blood Pb levels(Ruff et al., 1993). Consequently, the data presentedhere on the relationship between succimer-mediatedchanges in blood and brain Pb provide an importantadjunct to the nonhuman primate and human clini-cal studies of succimer now underway.

Because the Pb exposure and resultant body Pbburdens of the animals in Study 2 were significantlygreater than those in Study 1, these data may pro-vide insight into the relationship between body Pbburden and succimer efficacy. After 7 days of suc-cimer treatment, blood and brain Pb of the animalsin the two studies were reduced to comparablelevels, despite the higher prechelation Pb burdens ofthe animals in Study 1 (as verified by the 7-dayvehicle-treated animals in both studies) (Table 1).This suggests that the benefit derived by succimertreatment relative to vehicle, in terms of reductionin brain Pb, was greater in animals with higherbody Pb burdens (i.e., Study 2). This suggestion is

174 SMITH, BAYER, AND STRUPP

consistent with a recent clinical study employingCaNa2EDTA chelation, in which the apparent bene-fit of treatment for reducing body Pb levels wasrelated to the initial body Pb burden, with the lowerPb group showing less benefit than the higher Pbgroup (Markowitz et al., 1993). However, it is incon-sistent with another recent clinical study with suc-cimer (Liebelt et al., 1994), which reported nodifference in the relative efficacy of treatment forreducing blood Pb levels in children with moderately(group mean Pb"31lg/dL) versus more elevated(group mean Pb"51 lg/dL) blood Pb levels. Reasonsfor this discrepancy are not apparent, although itmay be related to the fact that these studies useddifferent therapeutic chelating agents and theirstudy cohorts possessed somewhat different blood Pblevels.

The relative benefit of chelation treatment com-pared to cessation of Pb exposure (i.e., Pb abate-ment) is of considerable interest in the clinicalmanagement of Pb poisoning. One of the few clinicalstudies that has addressed this issue found thatchelation treatment using CaNa2EDTA was no moreeffective than Pb abatement in reducing body Pbburdens and toxicity, when prechelation Pb levelswere considered (Markowitz et al., 1993). Resultsfrom Study 2 in the present investigation show thatblood Pb levels declined significantly even withoutsuccimer treatment, while brain Pb levels did not(comparison of the T"0 control group versus the 7-and 21-day vehicle groups, Fig. 2). These data pro-vide additional support for the important differencesin Pb transfer rates (i.e., rate of loss) between thecirculation versus the CNS. They also indicate thatthe benefit of succimer treatment compared tosimple Pb abatement (i.e., vehicle group) wasgreater when reductions in brain Pb levels wereconsidered, rather than reductions in blood Pb. Inother words, if only blood Pb levels were considered,one could erroneously conclude that succimer treat-ment did not offer substantial benefit over simple Pbabatement. However, when considering reductionsin brain Pb levels, the benefit of succimer treatment(versus vehicle) was readily apparent.

The studies reported here utilized doses and dura-tions (7 and 21 days) of succimer treatment compa-rable to those used clinically. However, therelevance of these data to humans may be con-founded by anatomical and physiologic differencesbetween rodents and primates, including differencesin the general metabolic rates and the kinetics of Pbmetabolism (Smith et al., 1992; O’Flaherty, 1991,1993, 1995). It is possible that the treatment dura-tions used here would extrapolate to longer dura-

tions in humans. Thus the observations here on theefficacy of succimer for reducing brain Pb levels mayoverestimate the efficacy of the comparable treat-ment duration in humans. Further, differences havebeen reported in the metabolism of succimer(DMSA) in rodents compared to primates (Aposhianand Aposhian, 1990; Maiorino et al., 1990; McGownet al., 1984), although how or if these differencesaffect the efficacy of succimer for reducing body Pblevels in rodent models versus humans is not known.

SUMMARY

Although substantial progress has been made inthe U.S. to reduce both Pb emissions and humanexposures, the Pb discharged to the environmentover past decades continues to pose a source of hu-man exposure and toxicological impact (Patterson,1965, 1994; Flegal and Smith, 1995; Smith andFlegal, 1995). In particular, the effects of Pb onneurocognitive impairment in children continue torepresent a significant health threat (NRC, 1980,1993). This, coupled with the fact that there may beno lower threshold for the neurotoxic effects of Pb incontemporary populations, strongly substantiatescontinued efforts for reducing human exposures andresultant Pb levels in sensitive target organs (NRC,1993).

The present results provide additional evidence ofthe benefit of succimer treatment in reducing tissuePb levels. Succimer treatment was highly effectivein removing Pb from both blood and brain, althoughthe benefits of prolonging the treatment period from7 and 21 days were evident only in terms of a furtherreduction in brain Pb and not blood Pb levels. Fur-ther, succimer-mediated reductions in brain Pblevels were larger in Study 2 than in Study 1, whichmay suggest a greater relative efficacy of chelationat the higher Pb exposures used in Study 2. Thepresent findings also indicate that succimer-me-diated reductions in blood Pb provide a poor proxyfor treatment efficacy on brain Pb, the target organof greatest interest with respect to Pb neurotoxicity.While the relevance of these data to humans may beconfounded by anatomical and physiologic differ-ences between rodents and primates, as well as dif-ferences in the metabolism of succimer (DMSA),they suggest that clinical studies should exercisecaution when using blood Pb as an index of theefficacy of chelation treatment for reducing brain Pblevels. Additional studies are in progress to furtherclarify the effects of Pb exposure parameters (expo-sure duration and magnitude) on the efficacy of suc-cimer for reducing brain and blood Pb levels.

EFFICACY OF SUCCIMER CHELATION FOR REDUCING BRAIN Pb 175

ACKNOWLEDGMENTS

We are indebted to Dr. Clair Patterson for his tireless effortsand monumental contributions in establishing the magnitude andimpact of the anthropogenic contamination of the biosphere withPb. We also thank Mareike Kuypers and Doug Woolard for theirimportant technical and analytical contributions to this study.This study was supported by NIEHS Grants ES07457, ES06918,and ES07535 and EPA U914718.

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