C Pharmacology & Toxicology 2003, 93, 180–185. Copyright CPrinted in Denmark . All rights reserved

ISSN 0901-9928

Ascorbic Acid Supplementation Does Not ImproveEfficacy of meso-Dimercaptosuccinic Acid Treatment in

Lead-Exposed Suckling RatsVeda Marija Varnai, Martina Piasek, Maja Blanusa, Dijana Juresa, Marija Saric and Krista Kostial

Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia

(Received March 17, 2003; Accepted August 12, 2003)

Abstract: It was suggested that ascorbic acid as a natural chelating agent can influence lead toxicokinetics and improvechelating properties of dimercaptosuccinic acid (DMSA) in adult rats. In this paper potential benefits of ascorbic acidsupplementation, alone or combined with DMSA, in decreasing lead retention in suckling rats were evaluated. Such datain young mammals are not available. L-Ascorbic acid (daily dose 650 mg/kg b.wt.) and/or DMSA (daily dose 91 mg/kgb.wt.) were administered orally to suckling Wistar rats either during ongoing 8-day oral lead exposure (as acetate; dailydose 2 mg lead/kg b.wt.) or after 3-day lead exposure (total dose 12 mg lead/kg b.wt.). Lead concentrations were analysedin the carcass (skeleton), liver, kidneys and brain by atomic absorption spectrometry. By ascorbic acid supplementationlead retention was not reduced under either lead exposure condition. Lead concentration was even increased in the carcass.Treatment with DMSA under both exposure conditions significantly reduced lead in all analysed tissues. Combinedtreatment with ascorbic acid and DMSA during ongoing lead exposure was substantially less effective than DMSAtreatment alone, and did not affect DMSA efficacy when administered after lead exposure. It was concluded that ascorbicacid administered either during or after lead exposure in suckling rats has no beneficial effect on either lead retention orDMSA chelation effectiveness.

In many parts of the world, in both developed and undevel-oped countries, lead exposure during childhood is still animportant public health problem (Bogden et al. 1997; Hiltset al. 1998; Matte & Jacobs 2000; Lanphear et al. 2002).Even at a very low level, lead exposure in young childrencan induce subtle but irreversible impairments, especiallyon neurodevelopment (Schwartz 1994). In order to preventlead absorption, enhance its elimination and mitigate leadtoxicity, nutritional measures are still actual research topics(Hu et al. 1995; Mahaffey 1995; Matte 1999; Houston &Johnson 2000; Tandon et al. 2001; Hsu & Guo 2002; Srokaet al. 2002).

The role of ascorbic acid under conditions of lead ex-posure has been investigated in both man and animals forseveral decades with contradictory results, especially con-cerning lead toxicokinetics. It has been suggested that as-corbic acid is a natural chelating agent capable of com-plexing lead by forming a poorly ionized but soluble leadcompound (Flora & Tandon 1986; Tandon & Singh 2000).Back in 1940 Dannenberg et al. described an unsuccessfulattempt to treat with ascorbic acid a 27-month-old childsuffering from lead encephalopathy. Studies in lead-exposedworkers did not show beneficial effect of ascorbic acid onblood lead concentration, lead elimination and/or biochem-ical markers of lead toxicity (Evans et al. 1943; Lauweryset al. 1983). Dietary ascorbic acid supplementation also did

Author for correspondence: Veda M. Varnai, Institute for MedicalResearch and Occupational Health, P.O. Box 291, HR-10001 Zag-reb, Croatia (fax π385 1 4673 303, e-mail vvarnai/imi.hr).

not affect lead, cadmium, and mercury concentrations inblood and hair in a non-occupationally exposed population(Calabrese et al. 1987). On the other hand, some recent hu-man data have shown inverse relationship between ascorbicacid intake and blood lead concentration, both in childrenand adults (Cheng et al. 1998; Dawson et al. 1999; Tandonet al. 2001). Analysing data from the Third National Healthand Nutrition Examination Survey (NHANES), Simon &Hudes (1999) found an inverse association of serum ascor-bic acid concentrations and the prevalence of elevated bloodlead concentrations, in both children and adults. It must betaken into account that substantial number of confoundingfactors, such as socio-economic status and lifestyle, can in-fluence the results and their interpretation in humanstudies. As authors themselves suggested, ascorbic acid in-take, especially through supplements, is often positively cor-related with healthier lifestyle, including better nutrition,safer life environment and higher socio-economic status.There is also a possibility that lead in the blood may lowerserum ascorbic acid concentration (Houston & Johnson2000).

Certain animal experiments, however, also showed bene-ficial effects of ascorbic acid on lead metabolism and toxic-ity. Studies on rats have shown that ascorbic acid can de-crease lead absorption (Flora & Tandon 1986), promotelead elimination (Goyer & Cherian 1979; Flora & Tandon1986; Dhawan et al. 1988; Tandon & Flora 1989; Vij et al.1998; Tandon & Singh 2000), and alleviate toxic effects oflead (Flora & Tandon 1986; Fisher et al. 1998; Hsu et al.1998; Vij et al. 1998; Patra et al. 2001). Lower lead incor-

181ASCORBIC ACID AND MESO-DMSA IN LEAD-EXPOSED SUCKLING RATS

poration and reduced lead toxicity has been reported byFisher et al. (1998) in Chinese hamster peritoneal cell cul-tures concomitantly exposed to lead and ascorbic acid.However, some other experimental studies showed oppositeresults (Suzuki & Yoshida 1979; McNiff et al.1978; Patra etal. 2001).

The effectiveness of ascorbic acid to enhance the anti-dotal potency of chelating agents has also been tested. Theexperiments in animals showed beneficial effect of ascorbicacid combined with calcium disodium ethylendiaminetetra-acetate (EDTA), a-mercapto-b-(2-furyl) acrylic acid(MFA), and 2,3-dimercaptosuccinic acid (DMSA) (Goyer &Cherian 1979; Dhawan et al. 1988; Tandon & Flora 1989).In the experiment of Goyer & Cherian (1979) one-month-old Sprague-Dawley rats that received the combination ofascorbic acid and EDTA excreted more than twice theamount of lead than those treated with either EDTA orascorbic acid alone. Dhawan et al. (1988) reported that as-corbic acid enhanced MFA efficacy by increasing urinarylead elimination.

In all experiments mentioned above, ascorbic acid sup-plementation and chelating agents were administered toeither post-weaning or adult rats. However, little is knownabout the effects of ascorbic acid on lead absorption andretention, as well as about its influence on the efficacy ofDMSA chelating properties during the suckling period,when lead exposure is much more harmful than in adult-hood due to age-related differences in lead toxicokineticsand health effects, especially regarding neurodevelopment(Ziegler et al. 1978; Mykkanen et al. 1979; Aungst & Fung1981; Klein & Koch 1981; Kostial et al. 1991a; Revich 1994;Schwartz 1994; Wasserman et al. 1997; Oskarsson et al.1998). In this paper we evaluated potential benefits of ascor-bic acid supplementation, alone or combined with DMSA,on lead retention in suckling rats under two experimentalconditions, either during ongoing oral lead exposure orafter lead exposure. Similar data in literature are not avail-able.

Materials and Methods

Animals. Experiments were performed on suckling rats of both gen-ders, Wistar strain (bred in Laboratory Animals Unit of the Insti-tute for Medical Research and Occupational Health, Zagreb). Atthe beginning of the experiments the pups were six days old withinitial average body weight 15.8 g (12.6–18.4 g). The animals werekept with their mothers, eight per litter (number of pups reducedon second day of life) in individual polycarbonate cages (26¿20¿14cm) with stainless steel lids and bottoms (Ehret, Austria). Cageswere cleaned and pine-shaving bedding was changed daily. Bodyweights of pups were recorded every morning. In each experiment40 pups were used: five litters of eight pups. Pups in each litter wererandomly assigned into four experimental groups; two pups perlitter for each group.

All research procedures were performed according to the nationallaw on the care and use of laboratory animals and were officiallyapproved by the Croatian Ministry of Agriculture and Forestry.

Experimental design. The effects of ascorbic acid supplementation,alone or combined with DMSA, on concentration of lead in se-

lected rat tissues were evaluated in two separate experiments. Inthe first experiment ascorbic acid and/or DMSA treatment wereadministered during ongoing lead exposure, and in the second ex-periment ascorbic acid and/or DMSA treatment were applied afterlead exposure. Pups were designated to one of the four experimentalgroups with 10 pups in each: untreated (control), treated with ascor-bic acid, treated with DMSA, or treated with DMSA and ascorbicacid.

Lead, ascorbic acid and DMSA were administered orally usingthe method of artificial feeding that closely resembles bottle-feedingof infants, introduced by Kostial et al. (1967). Each morning pupswere separated from mothers, placed in labelled boxes, and keptwarmed during the treatments that begun one hour later. Solutionswere given drop by drop by an automatic pipettor allowing pups toswallow the drops. After treatments pups were returned to mothers.

Ongoing lead exposure (first experiment). Pups were separatedfrom their mothers twice a day, that is, in the morning (at 8:00 a.m.)and in the afternoon (at 1:00 p.m.). All animals were orally exposedto lead (as lead acetate, p.a. grade, ‘‘Kemika’’ Co., Zagreb, Croatia,dissolved in distilled water) during 8 consecutive days (day 6through 13 after birth) in a total dose of 16 mg lead/kg body weight.Daily dose (2 mg lead/kg b.wt.) was administered in two drops perday (1 mg lead/kg b.wt. in each), first in the morning (at 9:00 a.m.)and second in the afternoon (at 2:00 p.m.), immediately after ascor-bic acid administration. L-(π)-Ascorbic acid (p.a. grade, ‘‘Kemika’’Co., Zagreb, Croatia, dissolved in distilled water) was administeredin four drops a day during 8 consecutive days at a daily dose of 650mg/kg b.wt. Two drops were given in the morning and two in theafternoon, instantly before lead administration. Meso-DMSA (Ald-rich Chemical Co., Milwaukee, WI, USA, dissolved in 0.05 ml of5% NaHCO3) was given orally at a dose of 45.5 mg/kg b.wt. in twodrops twice a day (91 mg/kg b.wt./day, i.e. 0.5 mmol/kg b.wt./day),immediately after lead administration, for six days; day 6 through8 and 11 through 13 after birth (total dose 3 mmol DMSA/kg, i.e.547 mg/kg).

Previous lead exposure (second experiment). Pups were separatedfrom their mothers only once a day, at 8:00 a.m. All animals wereorally exposed to lead at the beginning of the experiment, once aday during three consecutive days (day 6 through 8 after birth) ata daily dose of 4 mg lead/kg b.wt. (total dose 12 mg lead/kg) in fourdrops (at 9:00 am). Seventy-two hr after the last lead administrationascorbic acid and/or DMSA treatment begun. Pups were treatedonce a day (at 9:00 am) during 6 consecutive days (day 11 through16 after birth) at the same doses as in the first experiment (ascorbicacid at a daily dose of 650 mg/kg b.wt. and DMSA at a daily doseof 91 mg/kg b.wt.), in four drops per day each. Ascorbic acid wasadministered first, and immediately followed by DMSA treatment.

In our experiments lead exposure was at doses previously shownnot to affect pups’ growth and development (Varnai et al. 2001a),and at sufficient concentration for valid lead tissue analysis. Groupsthat did not receive ascorbic acid and/or DMSA were administeredthe vehicle, i.e. distilled water and/or 5% NaHCO3.

Suckling rats in our experiments were orally supplemented withascorbic acid at a dose (650 mg/kg/day) approximate to those thatwere reported by other authors to enhance DMSA effectiveness inadult rats (Dhawan et al. 1988; Tandon & Flora 1989).

Since for technical reasons faecal and urinary samples could notbe collected in sucklings, the values of tissue lead concentrationswere taken as indicators of the efficacy of treatments under particu-lar exposure condition.

Analysis of tissue lead. Twenty-four hr after the last treatment dayin the first experiment or 48 hr after in the second experiment, pupswere killed by exsanguination from the abdominal aorta in etheranaesthesia. Liver, brain, both kidneys and carcass (whole bodyafter removal of the organs, total gastrointestinal tract and skin)were dissected and used for further analyses. Fresh organ weightswere recorded. Carcass and organ samples were dry ashed at 450 æin a muffle furnace and dissolved in 10% nitric acid (Blanusa &

VEDA MARIJA VARNAI ET AL.182

Breski 1981). Concentrations of lead in the carcass, liver and kid-neys were analysed by flame atomic absorption spectrometry(Varian AA-375, Australia) and in the brain by electrothermalatomic absorption spectrometry (Varian SpectrAA 300, Australia).To verify methods applied certified standard reference material(SRM) was measured: Bovine Liver SRM 1577b from NIST (USA).Preparation of reference material for AAS measurement was identi-cal as for experimental samples (Blanusa et al. 1999). The followingvalue of lead concentration (arithmetic mean∫S.D. of three repli-cates) was obtained (certified reference value in brackets):0.132∫0.003 mg/g dry weight (0.129∫0.004).

Hypotheses and statistical analysis. The study was designed to evalu-ate the effect of ascorbic acid supplementation, DMSA treatment,and ascorbic acid supplementation combined with DMSA on tissuelead concentrations in pups exposed to lead during or prior to treat-ments. Planned comparisons were conducted to examine each hy-pothesis. F tests were used to evaluate each of three planned com-parisons (at level of significance of P,0.05): Ascorbic acid versusUntreated; DMSA versus Untreated; and DMSA versus DMSAπAscorbic acid. To eliminate heterogeneity of variances, data werelogarithmically transformed before the analyses. Since two-wayANOVA indicated no gender effect in any of the examined variablesin each experiment, gender was excluded from further analysis.Statistica for Windows program (StatSoft 1995 package, release 5.0)was used for all statistical analyses.

Results presented in tables are given as absolute values of arith-metic means and standard errors of the mean (S.E.M.), expressedin micrograms per gram wet tissue weight.

Results

Effects of ascorbic acid supplementation and/or DMSA treat-ment during ongoing lead exposure (first experiment). By

Table 1.

Effect of ascorbic acid supplementation and/or DMSA treatment on tissue lead concentrations in suckling rats during ongoing oral leadexposure.

Tissue lead (mg/g wet wt.)

Group Carcass Liver Kidneys Brain

Untreated 6.34∫0.295 2.65∫0.099 2.62∫0.094 0.215∫0.038Ascorbic acid 7.85∫0.297* 2.54∫0.118 2.63∫0.125 0.236∫0.013DMSA 4.28∫0.130* 0.439∫0.023* 0.815∫0.065* 0.046∫0.006*DMSAπAscorbic acid 6.45∫0.384** 0.713∫0.035** 2.60∫0.172** 0.069∫0.012**

ANOVA resultsAll groups

F (dfΩ3; 36) 34.0 377.8 105.7 48.0P ,0.001 ,0.001 ,0.001 ,0.001

Planned comparisonsAscorbic acid versus Untreated

F (dfΩ1;36) 12.6 0.452 0.001 1.04P 0.001 0.506 0.979 0.314

DMSA versus UntreatedF (dfΩ1;36) 40.0 741.8 213.6 78.4P ,0.001 ,0.001 ,0.001 ,0.001

DMSA versus DMSAπAscorbic acidF (dfΩ1;36) 42.5 53.9 206.3 4.38P ,0.001 ,0.001 ,0.001 0.044

All suckling rats received daily orally 2 mg/kg lead (as acetate), through 8 consecutive days (day 6 through 13 after birth) in total dose of16 mg lead/kg. During this period ascorbic acid was given orally at a daily dose of 650 mg/kg. DMSA was given orally at a daily dose of91 mg/kg for six days (day 6–8 and 11–13 after birth) in total dose of 547 mg/kg.Results are presented as arithmetic means∫S.E.M. and F test. There were 10 animals in each group.*Statistically significant differences (P,0.05) for comparisons: Ascorbic acid versus Untreated, and DMSA versus Untreated.

**Statistically significant difference (P,0.05) for comparison: DMSA versus DMSAπAscorbic acid.

ascorbic acid supplementation, tissue lead concentrations insuckling rats concomitantly exposed to lead were not re-duced (table 1). On the contrary, an increase of carcass leadconcentration was observed. Treatment with DMSA aloneefficiently lowered lead in all analysed tissues when com-pared to untreated control (table 1). However, in combi-nation with ascorbic acid supplementation DMSA was sig-nificantly less effective. In the carcass and kidneys, ascorbicacid even completely inhibited the effect of DMSA treat-ment.

Effects of ascorbic acid supplementation and/or DMSA treat-ment following previous lead exposure (second experiment).Ascorbic acid supplementation did not enhance lead decor-poration (table 2). By DMSA treatment, lead concentrationin all analysed tissues was reduced as expected. Combinedtreatment with ascorbic acid and DMSA was equally effec-tive as the treatment with DMSA alone.

Supplementation with ascorbic acid, as well as DMSAtreatment, had no effect on pups’ body weight gain andorgan wet weights. The average daily body weight gain wasabout 2 g in both experiments.

Discussion

Beneficial effects of ascorbic acid supplementation on leadabsorption and elimination have been repeatedly observedin animal experiments (Goyer & Cherian 1979; Flora &Tandon 1986; Dhawan et al. 1988; Dalley et al. 1989; Tan-

183ASCORBIC ACID AND MESO-DMSA IN LEAD-EXPOSED SUCKLING RATS

don & Flora 1989; Vij et al. 1998; Tandon & Singh 2000).Flora & Tandon (1986) reported that ascorbic acid supple-mentation at a daily dose of 25 mg/kg b.wt. led to signifi-cantly decreased lead uptake by blood, liver and kidneys inrats concomitantly orally exposed to lead (10 mg/kg/day),in comparison to the non-supplemented control. In our ex-periments, however, supplementation with ascorbic acid ata daily dose of 650 mg/kg had no beneficial effect on eitherlead retention or lead elimination in suckling rats. More-over, the elevation of lead concentration in the carcass wasobserved in the pups supplemented with ascorbic acid dur-ing ongoing lead exposure, indicating that ascorbic acid canaffect lead toxicokinetics in sucklings in a non-desirableway.

An increase in lead absorption caused by ascorbic acidadministration was reported previously by Conrad & Bar-ton (1978). Ascorbic acid added to a test dose of lead radio-isotope increased lead absorption from an isolated rat’sduodenal loop. In vitro tests have shown that ascorbic acidincreases the solubility of lead at neutral and alkaline pH.Authors postulated that ascorbic acid binds lead in vitroand thus maintains lead in a physical state that makes itmore available for absorption in the duodenum. Otherauthors reported that ascorbic acid supplementation didnot enhance lead elimination as well (McNiff et al. 1978;Suzuki & Yoshida 1979; Patra et al. 2001). McNiff et al.(1978) found no notable increase of lead elimination in ratsreceiving dietary supplement containing 1% of ascorbic acid

Table 2.

Effect of ascorbic acid supplementation and/or DMSA treatment on tissue lead concentrations in suckling rats previously exposed to lead.

Tissue lead (mg/g wet wt.)

Group Carcass Liver Kidneys aBrain

Untreated 1.98∫0.102 0.371∫0.021 0.799∫0.048 0.073∫0.004Ascorbic acid 1.98∫0.114 0.378∫0.025 0.767∫0.051 –DMSA 1.21∫0.062* 0.160∫0.015* 0.292∫0.031* 0.037∫0.002*DMSAπAscorbic acid 1.30∫0.077 0.189∫0.011 0.306∫0.013 0.041∫0.004

ANOVA resultsAll groups

F (df) 23.1 (3;36) 46.2 (3;36) 73.1 (3;36) 29.0 (2;22)P ,0.001 ,0.001 ,0.001 ,0.001

Planned comparisonsAscorbic acid versus Control

F (df) 0.001 (1;36) 0.010 (1;36) 0.249 (1;36) –P 0.982 0.920 0.621 –

DMSA versus ControlF (df) 39.6 (1;36) 83.5 (1;36) 123.2 (1;36) 48.0 (1;22)P ,0.001 ,0.001 ,0.001 ,0.001

DMSA versus DMSAπAscorbic acidF (df) 0.726 (1;36) 3.87 (1;36) 0.643 (1;36) 0.658 (1;22)P 0.400 0.057 0.428 0.426

All suckling rats received for 3 days (6–8 after birth) lead (as acetate) orally at a daily dose of 4 mg/kg (total dose 12 mg lead/kg). After thecompletion of lead exposure, ascorbic acid was given at a daily dose of 650 mg/kg orally and DMSA at a daily dose of 91 mg/kg orallyduring 6 consecutive days (day 11–16 after birth).Results are presented as arithmetic means∫S.E.M. and F test. There were 10 animals in each group.aAll brain samples in the Ascorbic acid group, two brain samples in the DMSA group and three brain samples in the DMSAπAscorbicacid group were lost during preparation.

*Statistically significant differences (P,0.05) for comparisons: Ascorbic acid versus Untreated and DMSA versus Untreated.There was no statistically significant difference (P,0.05) for comparison DMSA versus DMSAπAscorbic acid.

after a single intravenous lead administration (1 mg lead/kgb.wt., as acetate). Suzuki & Yoshida (1979) have reportedin male growing Wistar rats that supplementation with 1%ascorbic acid in the feed with concomitant oral lead ex-posure (500 ppm in diet, as acetate) did not prevent lead-induced growth retardation and decrease in haemoglobinvalues, and had no effect on lead concentrations in the liverand kidney. Similarly, Patra et al. (2001) observed that sup-plementation with 100 mg/kg b.wt. of ascorbic acid did notreduce lead concentration in the liver, kidney and brain,although it lowered the lipid peroxidation levels in rats pre-viously parenterally exposed to lead for 4 weeks (1 mg lead/kg b.wt./day, as acetate), confirming its beneficial antioxi-dant potential. Authors even observed an increase in kidneylead concentration by approximately 50% when comparedto lead-exposed non-supplemented controls.

In our experiments meso-DMSA was effective in decreas-ing tissue lead when applied either during oral lead ex-posure, or after discontinuation of lead exposure. These re-sults confirm our previous findings on effectiveness ofDMSA treatment during ongoing oral lead exposure insuckling rats (Varnai et al. 2001b). Combination of ascorbicacid with DMSA treatment, however, did not improveDMSA efficacy. Quite opposite, ascorbic acid supplementa-tion reduced or completely abolished DMSA efficacy in re-ducing tissue lead in suckling rats during ongoing lead ex-posure. The effect of DMSA on previously incorporatedlead was not affected by ascorbic acid. These results differ

VEDA MARIJA VARNAI ET AL.184

from the results of other authors which reported enhancedDMSA efficacy when this chelator was combined with as-corbic acid supplementation in adult rats previously ex-posed to lead (Dhawan et al. 1988; Tandon & Flora 1989).Dhawan et al. (1988) have found that ascorbic acid en-hanced efficacy of DMSA (given at a daily dose of 0.5mmol/kg b.wt.) in increasing lead elimination by urine andin reducing lead concentrations in the liver and kidney inrats supplemented with 1% ascorbic acid in drinking water(approximate daily intake of 650 mg/kg b.wt.). Tandon &Flora (1989) also have found that combined treatment withDMSA (at a dose of 0.1 mmol/kg b.wt./day) and ascorbicacid (1% in drinking water; approximate daily intake of 650mg/kg b.wt.) caused higher urinary lead elimination as wellas greater decrease of lead concentration in the blood, kid-ney, and brain than DMSA treatment alone.

Among the variables that can account for contradictoryresults on efficacy of ascorbic acid supplementation in thestudies described above including ours, are age and differ-ences in experimental design. Gastrointestinal absorptionof lead, as well as lead retention in tissues, are significantlyhigher in sucklings compared to adults (Kostial et al.1991a), and pharmacokinetics and chelator efficacy couldalso differ from those in adult, mature organisms (Kostialet al. 1991b). Differences in experimental design, such asascorbic acid dosage may also play a role. Daily doses ofascorbic acid shown to have beneficial effect on lead toxic-okinetics and DMSA chelating properties varied in a widerange, from 25 mg/kg b.wt. (Flora & Tandon 1986) to 2000mg/kg b.wt. (Goyer & Cherian 1979). On the other hand,ascorbic acid supplementation at a daily dose of 100 mg/kgb.wt. had no desirable effect on lead toxicokinetics (Patraet al. 2001), although duration of supplementation (4 to 7days) as well as tissue lead concentrations in lead-exposedcontrol groups did not differ much from previously men-tioned studies. To clarify the influence of dose of ascorbicacid on observed parameters it would be necessary to per-form experiments with various dosages under uniform ex-perimental conditions and on the same strain of experimen-tal animals.

Since the relationship between ascorbic acid and lead ex-posure seems to be an interesting topic – not yet clarified –further investigations in this field are justified. Our resultsin sucklings indicate that age is an important variable thatis not investigated in previous work of other authors. Wewill therefore continue with our investigations in sucklingrats by using different doses of ascorbic acid. Experimentsalong these lines are already in progress.

In conclusion, ascorbic acid supplementation in sucklingrats had no beneficial effect on either lead retention or leaddecorporation under described experimental conditions. Onthe contrary, it even increased concentration of lead in thecarcass. Ascorbic acid was also ineffective in enhancing che-lation properties of DMSA. When administered during on-going lead exposure it seriously impaired DMSA efficacy.Similar results were not previously reported. Our resultssuggest that special concern is necessary if ascorbic acid

supplementation is considered in case of lead exposure dur-ing the suckling period, especially if DMSA therapy is incourse.

AcknowledgementsThe authors gratefully acknowledge the excellent techni-

cal assistance of Mrs. Marija Ciganovic, Durd–a Breski andJasna Milekovic.

This study was financially supported by the CroatianMinistry of Science and Technology (grant No. 220102).

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