Meso-2,3-dimercaptosuccinic acid (DMSA) affects maternal and fetal copper metabolism in Swiss mice

Toxwology, 72 (1992) 2 7 - 4 0 Elsewer Scientific Pubhshers Ireland Ltd

27

Meso-2,3-dimercaptosuccinic

acid (DMSA) affects maternal and fetal copper metabolism in Swiss mice Marie Weldon Taubeneck a, Jose L. Domlngo b, Juan M. Llobet b and Carl L. Keen a,c aDepartment of Nutrttton, Umverstty of Cahforma, Darts, CA 95616 ( U S .4 ), bLaboratory of Toxtcology and Btoehemlstry, School of Medteme, Umverstty of Barcelona, Reus ( Spam) and 'Department of Internal Medwme, Umverstty of Cahforma, Davts, CA 95616 (U S .4 ) (Received August 12th, 1991, accepted December 4th, 1991)

Summary Meso-2,3-dlmercaptosuccimc acid (DMSA) is a chelating agent used to treat heavy metal Intoxication. D M S A has been reported to be teratogenic in the mouse, and it has been suggested that th~s teratogeniclty may be secondary to DMSA-induced alterations in Zn metabolism. In the present study, 0, 400 or 800 m g DMSA/kg body weight were administered on gestation days 6-15 to pregnant Swiss mice by gavage (PO) or subcutaneous lnjecuon (SC) Mice were fed a diet containing 14 #g Zn, 10/~g Cu, 120 #g Fe, 1175 #g Mg and 6 8 mg Ca/g &et A sub-group of mice in the 800 mg DMSA/kg SC group was fed a diet containing 250 #g Zn/g D M S A administration &d not result in overt maternal toxicity There was no effect of the drug on fetal or placental weight, or on crown-rump length However, some fetuses from DMSA-treated dams were characterized by skeletal abnormalities including supernumerary ribs, unoss~fied anterior phalanges and malformed sternebrae Drug exposure was not assooated with consistent changes m tissue Zn, Fe, Ca or Mg levels Supplemental Zn had no marked effects on the fetus Fetal hver Cu concentrations exhibited dose-dependent decreases with increasing DMSA dose This finding suggests that the developmental tOXlOty of D M S A may be mediated through disturbed maternal/fetal copper m e t a b o h s m Key words Copper, Meso-2,3-dimercaptosuccmic acid (DMSA), Pregnant mice, Oral admlmstratlon, Subcutaneous administration, Zinc

Introduction The metal complexing agent meso-2,3-dimercaptosuccmlc acid (DMSA) has been known as an effective antidote for heavy metal poisoning since 1957 [1], with this drug bemg compared favorably with British Anti-Lewisite (BAL). DMSA has been reported to be of value m the treatment of lead [2-4], cadmium [5-7], mercury [8,9] and arsenic intoxication [10-12] in humans and animals. A potential advantage of DMSA over most other metal complexing agents is its Correspondence to Carl L Keen, Department of Nutrition, University of California, Davis, CA 95616, U.S A 0300-483X/92/$05 00 © 1992 Elsevier Scientific Pubhshers Ireland Ltd Printed and Published in Ireland

28 relatively high water solubility and low lipid solubility. For example, compared to BAL, the drug of choice in the United States for arsenic (As) poisoning since the 1940s, D M S A can be given orally and it is more hydrophlhc and less hpophihc [13]. In addition, signs of toxicity associated with BAL (nausea, vomiting, and pain) are minimal with D M S A [13,14]. While it is evident that D M S A can be of value in the treatment of metal intoxication, concern has been raised over the possibility that the drug may induce essential trace element deficiencies simultaneously with the removal of the target metal. For example, Cantilena and Klaassen [15] reported that in male mice endogenous copper (Cu) excretion was significantly increased after D M S A treatment. Frledhelm et al. [2] reported that 5 men treated with D M S A for lead intoxication had an approximately 2-fold increase in urinary Cu excretion compared to pretreatment values It has been suggested that DMSA-induced alterations m mineral metabolism may result in pregnancy complications. Domlngo et al. [16] evaluated the maternal and fetal toxicity of D M S A in pregnant Swiss mice injected subcutaneously (SC) with 410, 820 or 1640 mg DMSA/kg body weight on gestation days (GD) 6-15. Effects on fetal development were primarily seen in the 1640 mg DMSA/kg group, with the drug resulting in a significant increase in prenatal death and fetal growth retardation (lower body weight and shorter crown-rump length) Fetal body weight was also significantly lower in the 820 mg D M S A group compared with controls. Soft tissue abnormalities were observed in fetuses in both the 1640 and 820 mg DMSA/kg per day group. Skeletal anomalies were observed at a higher than normal frequency in the 820 and 1640 mg D M S A groups. In a subsequent study of the effects of oral D M S A exposure, Domlngo et al. [17] reported that 800 mg DMSA/kg per day resulted in postnatal growth retardation in mice. Consistent with the above, in rats oral D M S A exposure on G D 6-15 resulted in a lower than normal maternal weight gain and low fetal weights [17,18]. In these studies, it was reported that D M S A resulted in low maternal liver Cu and calcium (Ca) concentrations and high liver iron (Fe) concentrations. Fetal liver Ca, Cu and zinc (Zn) concentrations were reported to be lower In the D M S A exposed groups than in controls, while fetal liver Fe concentrations were higher in the drug exposed groups. Based on the report that maternal D M S A administration can result in low fetal Zn concentrations [18], and the observation that even transitory embryonic/fetal Zn deficiency can be teratogenic [19], in the current study we tested the hypothesis that the teratogenicity of D M S A is due in part to an induced Zn deficiency and that its teratogenicity could be ameliorated by dietary Zn supplementation. Materials and methods

Adult female Swiss mice (NIHS, 20-25 g body weight) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Animals were housed in plexlglass cages in a temperature controlled facility with a 12:12 h light cycle and acclimated to a semipurified casein-based diet [20]. The diet contained 14/~g Zn, 10 #g Cu, 120 #g Fe, 1175 t~g magnesium (Mg) and 6.8 mg Ca/g. Dams were mated overnight with males of the same strain. The presence of a vaginal plug the next morning was considered

29 evidence of mating The day on which the plug was found was designated GD 0. Mxce were ear-punched for identification and pregnant females were housed three per cage. All mice were fed the 14 ~g Zn/g casein diet (Zn-adequate) ad hbltum The mice were distributed among 6 treatment groups of 8-13 animals each Dams were dosed with 0, 400, or 800 mg DMSA/kg body weight per day, either by SC injection or by gavage (PO) A seventh group of 9 females was fed a 250 #g Zn/g casein-based diet (Zn-supplemented) and dosed on G D 6-15 SC with 800 mg DMSA/kg body weight Dams' body weights were recorded dally throughout pregnancy A plastic mesh platform was placed in each plexiglass cage to minimize coprophagy Restricted retake groups were not included in this study, as previous investigations have shown no effect of the drug on food intake. Meso-2,3-dlmercaptosuccimc acid obtained from Sigma Chemical Co. was dissolved in distilled delonazed water, and sodmm bicarbonate ( N a H C O 0 added until the solution turned clear and a pH of 7.0-7 2 was achieved A NaHCO3 solution devoid of DMSA, but containing approximately the same concentration of NaHCO3 and of the same pH as the DMSA solution, was used as the vehicle Ahquots of prepared solutions were frozen and then thawed as needed DMSA or the vehicle was given on G D 6-15 by either PO or by SC injection. Gavaged animals were fasted 2 - 4 h prior to dosing at 1200 h each day. InJected animals were dosed at 1200 h. On G D 18, dams were anesthetized with metaphane Blood was collected by cardiac puncture using non-heparlnized 1-ml syringes. Serum was obtained after centrifugation of the blood at 14 000 rev./mln for 5 mln Maternal liver, kidneys, heart, spleen, thymus and intestine were quickly excised and frozen The intestine was perfused prior to freezing with 10 ml of 0.9% saline, and all tissues frozen The gravld uterus was excised and weighed The number of implantation sites, resorption sites, live/dead fetuses, and fetus locations were noted. Fetuses were weighed, sexed, measured for crown-rump length and inspected for gross anomalies. Two fetuses and all placentae per litter were saved for assessment of mineral concentrations All other fetuses in the litter were eviscerated and stained with Alizarin red S and Alclan blue for skeletal examination [21]. Maternal tissues and products of conception were wet ashed with nitric acid and assayed for Zn, Cu, Fe, Ca, and Mg concentrations as described by Clegg et al. [22] Data are shown as mean 4- S.E M Data from the Zn-supplemented 800 mg SC DMSA group were contrasted with the control Zn 800 mg SC DMSA group using Student's t-test. All other data were analyzed by A N O V A and Fisher PLSD post hoc test when a significant F-value was observed The latter was considered the statistical umt of measure [23]. Data in Table III were compared using blnomml coefficient statistics [24]. Results

Treatment with DMSA had no consistent effect on maternal health, or on litter size or resorption frequency (Table I) Although comparison of the 800 mg SC DMSA Zn-supplemented group and the Zn-adequate 800 mg SC group showed an

REPRODUCTIVE

DATA

PO800 DMSA P values Dose Route

1 8 5 +- 1 6 0 6741

0 1087 31 1 ± 0 8 0 0468

0 0748

0 2532 0 2485

± 07

308 ± 06 3 0 2 +- 0 6 289 ± 07 31 1 ± 0 9 2 9 4 +- 0 7

0 8083 0 8001

l l 15 1 1 10 314

± ± -4±

Maternal body we,ght minus gravtd u t e r u s (g)

2 1 7 +- I 0

21 2 194 200 209

2 2 5 +- 0 8

GestaUon w e i g h t gain (g)

+± ± +-4-

83±10 0 5201

0 5723 0 5145 0 2027

106 92 92 101 101

06 10 06 05 08

107 + 03

No of implantation sites

++± ± +-

70+10 0 1453

0 7331 0 1341 0 2485

99 91 82 94 92

06 09 07 05 06

1 0 2 4- 0 4

No of fetuses

± +++ +

155+-62 0 0359

2 I a'b 11 b 3 Ia 25 ab 31 d

+ 2 2 ~b

0 5657 0 0269 0 3289

70 1 1 102 73 86

41

Resorption f r e q u e n c y (%)

++± +±

004 002 003 002 003

126+003 0 8137

0 1173 0 0629 0 3682

1 20 125 1 33 t29 126

126 ± 003

Fetus weight (g)

± +± ± +-

0004 0003 0003 0002 0002

0089+0005 0 2790

0 4214 0 0542 0 3154

0086 0083 0084 0078 0077

0 0 8 4 4- 0 0 0 2

Placenta weight (g)

± -4± -4±

004 004 003 002 002

256+002 0 0878

0 1386 0 0048 0 9401

249 247 262 256 256

254-4-003

a.b b b a ab a,b

Crown-rump length (cm)

D M S A , 0, 400 o r 800 m g d l m e r c a p t o s u c c m l c a c l d / k g b o d y weight, SC, s u b c u t a n e o u s l n j e c u o n , P O , g a v a g e by m o u t h

d'bValues with different s u p e r s c r i p t s w~thm a c o l u m n are significantly different at P < 0 05 C o m p a r e d w~th the 14 ~g Z n SC 800 D M S A g r o u p

9

9

SC400 DMSA SC800 DMSA PO0 DMSA PO400 DMSA

D x R 250 lzg Zn/g diet SC 8 0 0 D M S A P values c

9

10 9 10 8

14 ~,~ Zn/g diet SCODMSA

No of htters

Values are m e a n ± S E M A b b r e w a t l o n s

MATERNAL

TABLE I

31

association of supplementation with an increased termmal maternal body weight (corrected for the gravid uterine weight), Zn supplementation was also associated with an increased resorption frequency. There was no influence of D M S A on fetal or placental weight (Table I), or on the ratio of males to females (data not shown). There was no consistent effect of treatments on crown-rump length. However, route of drug admmistratlon influenced crown-rump lengths among the 14 #g Zn/g diet groups, with values being lower in the SC groups. Route of drug administration had no influence on fetal or placental wetght. In order to assess potential overt toxicity of D M S A to organ systems, changes in maternal organs as a percent of corrected terminal maternal body weight were exammed (Table II). Kidney, cardiac and spleen hypertrophy were associated with high D M S A dosages, with the effect most pronounced in the SC group. Zinc supplementation had no consistent effect on maternal organ tissue weights Fetal skeletons were evaluated for degree of ossification and presence of normal structures. Only the presence of left anterior phalanges seemed to be affected by the combination of route and dose of D M S A N o changes were observed between the Zn-adequate and Zn-supplemented groups in any parameter (Table III). Table III shows the incidence of skeletal vartatlons observed within treatment groups, both by percentage of litters in which more than one fetus was affected and by percentage of the total number of fetuses scored in each group which were affected. The incidence of both additional ossification sites in the vertebral column, and supernumerary ribs (SNR), were significantly increased in the SC 800 D M S A group when compared with all other 14/zg Zn diet groups. When the 800 SC Zn-supplemented

T A B L E I1 M A T E R N A L O R G A N S AS P E R C E N T A G E O F C O R R E C T E D BODY WEIGHT - GRAVID UTERUS WEIGHT)

BODY WEIGHT (MATERNAL

Values are mean 4- S E M A b b r e v i a t i o n s D M S A , 0, 400 or 800 mg dlmercaptosuccmlc acld/kg body weight, SC, subcutaneous injection, PO, gavage by mouth Liver (%)

Kidney (%)

Heart (%)

Spleen (%)

Thymus (%)

14 I~g Zn/g dtet SC 0 D M S A

6 98 -4- 0 24

24 + 0 0 2 a

0 4 3 ± 0 0 2 a'b

0 3 8 -4- 0 0 2 4

0 0 4 + 001

SC SC PO PO

6 6 6 6

24 31 27 35

043 047 041 042

033 050 031 039

012 005 004 004

400 D M S A 800 D M S A 0 DMSA 400 D M S A

PO 800 D M S A

72 43 39 87

44-4-4-

0 0 0 0

34 28 17 22

6 66 4- 0 21

444+

003 004 003 005

a a,b.~ a'c bc

36 -4- 0 0 2 b

4± ± -4-

0 0 2 ab 002 d 001 b

001 b

44-44-

003 004 001 004

a b a a

+ ± 4-4-

007 001 001 001

0 4 2 4- 001 b

0 3 6 4- 0 0 2 a

0 0 4 4- 001

0 1725 00311 0 4558

0 0074 00212 0 0036

0 4381 0 1875 0 3710

041 -4- 0 0 1 0 0162

045 ± 003 0 2676

0 0 7 -4- 0 0 3 0 6422

P values

Dose Route

0 6247 0 7267

D x R

0 2057

0 0358 0 0192 0 4229

6 16 + 0 13 0 4002

1 36 -4- 0 0 3 0 3828

250 tzg Zn/g dwt SC 800 D M S A

P values d

a b,CValues with &fferent superscripts within a column are significantly different at P < 0 05 dCompared with the 14 #g Zn SC 800 D M S A group

78 65 60 59 65

46

9

74

l0 9 10 8 9

9

No pups examined

± 4444-

0 10" 002 'Lb 0 03 "b 001 b 0 04" b

3 93 4- 0 04 0 3444

0 5109 0 9226 0 0322

384 397 3 94 399 3 86

399 + 001 b' 44444-

0 12 002 0 03 002 0 05

3 95 4- 0 03 0 7061

0 4297 0 9453 0 0810

382 396 3 94 396 3 85

398 ± 001 ~

3 (7) 0 0002

1 (l) 3 (5) 2 (3) 112) 1(2)

1110) 3 (33) 2 (20) 1113) 1111)

3 (33) 0 0042

4 (5) e

3 (33) d

Pups

Litter

Left

Right

Bipartite sternebrae

No of anterior phalanges

d bValues w~th &fferent superscripts within a column are slgmficantl) different at P < 0 05 bValues are mean 4- S E M dNo of litters w~th ~_ 1 affected fetuses (% of total litters ~ 1 affected fetuses) eNo of examined fetuses affected (% of examined fetuses affected) fCompared with the 14 #g Zn SC 800 DMSA group

SC 40 0 DMSA SC 800 DMSA PO 0 DMSA PO 400 DMSA PO 800 DMSA P valuer Dose Route D x R 250 ~g Zn/g diet SC 800 DMSA P values f

14 ~g Zn/g diet SC 0 DMSA

No litters examined

133) d (20) (44) (10) (25) (22)

0 (0) 0 0244

3 2 4 1 2 2

L~ttcr

0 (0) 0 0030

4 (5) "~ 3 (4) d 11 (17) b 1 (2) a 2 13) d 2 (3)"

Pups

Supernumerary rlbs

Abbreviations DMSA, 0, 400 or 800 mg dlmercapto,,ucclmC acld,kg body sselght. SC subcutaneous injection, PO, gavage by mouth

FETAL SKELETAL P A R A M E T E R S

TABLE 111

33

and Zn-adequate groups were compared, both litters and fetuses showed decreased incidences of extra ossification s~tes on the vertebral column and SNR in the Znsupplemented group No other abnormahtles were noted. Maternal serum and hver mineral concentrations were similar among the groups (Tables IV and V). Kidney mmeral concentrations were not affected by DMSA However, mice m the SC groups had consistently higher kidney Zn concentrations than mice in the PO groups (Table VI) Route of drug admmtstratton and diet had varied effects on maternal intestine mineral concentrations (Table VII) Iron and Ca concentrattons tended to be higher and lower, respectwely, m the 14 t~g Zn/g diet SC groups compared to the PO groups. When Zn-adequate and Zn-supplemented groups were compared, Zn concentrations were significantly higher, and Cu and Mg concentrations were sigmficantly lower, m the supplemented group than m controls. Placenta Zn, Cu and Fe concentrations were Influenced by DMSA and/or route of administration (Table VIII). Differences an Zn and Fe levels were observed according to dose, and Cu vaned with route of administration m the Zn-adequate groups Placenta Cu concentrations were significantly lower an the Zn-supplemented group than m controls. Fetal whole body (minus hver) mineral concentrattons were not consistently influenced by DMSA (data not shown). Fetal liver Cu concentrations (Table IX) were significantly lower in the high D M S A dosage groups, independent of route DMSA had no consistent effect on the concentrations of the other elements tn the fetal liver.

T A B L E IV MATERNAL SERUM MINERALS Values are s h o w n as t~moles m m e r a l / g wet weight t,ssue a n d as mean 4- S E M A b b r e v i a t i o n s D M S A , 0, 400 or 800 mg & m e r c a p t o s u c c m l c acld/kg b o d y weight, SC, s u b c u t a n e o u s rejection, PO, gavage b? mouth Zn

14 ~g Zrt/g dwt SC0 DMSA SC400 DMSA SC 800 D M S A POODMSA PO 400 D M S A P O 800 D M S A P values Dose Route D × R 250 ~g Zn/g dtet SC 800 D M S A P values a

0011 0011 0012 0010 0010 0009

Cu

± 4± ± ± ±

0001 0001 0002 0002 0001 0001

0019 0020 0024 0019 0019 0017

Fe

+ ± ± 444-

0001 0001 0003 0002 0002 0002

0071 0074 0060 0071 0059 0066

Ca

+ ± ± 444-

0010 0006 0004 0008 0007 0008

337 3 13 371 427 348 382

Mg

+ 4444±

040 047 056 013 036 032

1009 1001 1039 1026 1012 1013

± 444± 4-

022 027 019 028 017 011

0 9741 0 2128

0 7045 0 1246

0 5409 0 6313

0 3829 0 1767

0 6604 0 9701

0 8008

0 2441

0 3176

0 6017

0 5724

0015 ± 0002 0 2315

0 0 1 9 4- 0 0 0 2 0 1521

0057 ± 0002 0 5445

381 ± 0 1 4 0 8657

1041 ± 0 1 7 0 9261

a C o m p a r e d w~th the 14 #g Z n SC 800 D M S A g r o u p

34

TABLE V MATERNAL

LIVER MINERALS

Values are s h o w n as # m o l e s m m e r a l / g wet w e i g h t h s s u e a n d as m e a n s + S E M A b b r e v m h o n s

DMSA,

0, 400 o r 800 m g d l m e r c a p t o s u c c m l c a c l d / k g b o d y weight, SC, s u b c u t a n e o u s rejection, P O , g a v a g e by mouth Zn

Cu

Fe

Ca

Mg

14 ~g Zn/g dwt SCODMSA

046

± 002

0 0 5 7 + 0 0 0 2 ~'b~

158 ± 020

047

SC400 DMSA SC 800 D M S A POODMSA PO400 DMSA

045 048 046 047

± ± ± +

0054 0063 0061 0058

I 66 192 236 1 58

0 18 019 027 022

049 ± 002 0 5 0 4- 0 0 3 048 ± 003 041 ± 003

100 104 103 101

P O 800 D M S A

0 43 4- 0 01

0 053 4- 0 002 b

2 42 ± 0 40

0 33 + 0 02

101 ± 0 1

0 9510 0 5252 0 1660

0 5214 0 7045 0 0123

0 1110 0 0625 0 2385

0 4056 0 0596 0 2756

0 6604 0 9701 0 5724

046 + 004 0 6901

0057 + 0002 0 1620

233 ± 021 0 1677

055 ± 005 0 4336

104 ± 02 0 9261

002 002 002 002

± ± ± ±

0001 0003 0002 0004

c a a'c a'b'c

± ± 4±

± 003

101 + 0 2 ± ± ± ±

02 02 03 02

P values Dose Route D × R

250 ~g Zn/g dwt SC 800 D M S A

P values d

a'b'CValues w~th & f f e r e n t s u p e r s c n p t s w~thm a c o l u m n are s~gmficantly & f f e r e n t at P < 0 05 a C o m p a r e d w i t h the 14 ~ g Z n SC 800 D M S A g r o u p .

T A B L E VI MATERNAL

KIDNEY

MINERALS

Values are s h o w n a s / ~ m o l e s m m e r a l / g wet w e i g h t t~ssue a n d as m e a n s ± S E M A b b r e v i a t i o n s D M S A , 0, 400 o r 800 m g d l m e r c a p t o s u c o m c a c l d / k g b o d y weight, SC, s u b c u t a n e o u s mject~on, P O , g a v a g e b y mouth Zn

Cu

Fe

Ca

Mg

14 I~g Zn/g dwt SC 0 D M S A

0 3 3 ± 0 0 1 a'c

0072 ± 0003

33 ± 0 0 9

086 + 001

72 ± 02

SC 400 D M S A SC 800 D M S A POODMSA P O 400 D M S A

033 035 026 0 28

0069 0071 0067 0 067

P O 800 D M S A

0 26 ± 0 02 b

0 067 ± 0 002

34 46 52 25 22

089 087 087 100 086

72 ± 74 + 73 + 72 ± 69±01

0 7773 0 0001 0 5617

0 8676 0 0870 0 8507

0 5001 0 6449 0 1815

0 2806 0 6750 0 4546

0 8795 0 5096 0 4285

0 35 ± 0 01 0 7195

0 069 ± 0 003 0 5489

1 39 + 0 0 5 0 6188

085 + 002 0 5627

74 + 02 0 9404

± ± ± ±

0 0 1 a'c 001 a 002 b 0 02 b'c

+ ± ± ±

0003 0003 0003 0 003

+ ± ± ± +

015 013 0 II 010 008

± ± ± ± 4-

003 003 003 018 002

03 04 03 02

P values Dose Route D × R

250 t~g Zn/g dtet SC 800 D M S A

P values d

a'b'CValues with & f f e r e n t s u p e r s c r i p t s w i t h i n a c o l u m n a r e significantly & f f e r e n t at P < 0 05 d C o m p a r e d wLth the 14 /~g Z n S C 800 D M S A g r o u p

35

T A B L E VII MATERNAL

INTESTINE

MINERALS

Values are s h o w n as t~moles m l n e r a l / g w e t w e i g h t tissue a n d as m e a n s ± S E M A b b r e v i a t i o n s D M S A , 0, 400 o r 800 m g & m e r c a p t o s u c c m ~ c ac~d/kg b o d y wetght, SC, s u b c u t a n e o u s mject~on, P O , g a v a g e b y mouth Zn

Cu

Fe

Ca

Mg

14 p.g Zn/g dtet SC 0 D M S A SC 400 D M S A SC 800 D M S A PO0 DMSA P O 400 D M S A P O 800 D M S A

031 033 034 032 0 32 0 31

± ± ± ± ± 4.

001 002 001 001 0 01 0 02

0028 0033 0034 0028 0 032 0 025

± 4. 4. 44. ±

0002 0004 0003 0001 0 002 0 001

057 081 075 109 1 18 1 16

444. 4-44.

009 a 013 ab 0 1 2 a'b 017 b 0 24 b 0 15 b

46 40 45 22 2 7 2 4

4. ± 444. 4.

10 a 1 0 a'b 1 7 a'b 03 b 0 4 a'b 0 4 a'b

81±06 994.16 8 8 4. 0 6 78 + 03 7 8 4. 0 3 75 ± 02

P values Dose Route

0 6794 0 4210

0 2154 0 1465

0 5211 0 0011

0 9982 0 0150

0 5174 0 0564

D × R

0 3095

0 2608

0 8865

0 8420

0 4814

047 + 004 0 0043

0026 + 0002 0 0407

099 + 014 0 2026

1 8 + 03 0 1452

6 9 -4- 0 3 00115

250 l~g Zn/g dwt SC 800 D M S A

P values c

d,bValues w i t h & f f e r e n t s u p e r s c r i p t s w i t h i n a c o l u m n a r e s l g m f i c a n t l y different at P < 0 05 c C o m p a r e d with the 14/~g Z n SC 800 D M S A g r o u p

T A B L E VIII PLACENTA

MINERALS

Values a r e s h o w n as txmoles m m e r a l / g wet w e i g h t tissue a n d a r e m e a n s 4. S E M A b b r e v t a t ~ o n s D M S A , 0, 400 o r 800 m g d ~ m e r c a p t o s u c c l m c a c l d / k g b o d y weight, SC, s u b c u t a n e o u s rejection, P O , g a v a g e b y mouth Zn

Cu

Fe

Ca

1 59 4. 0 12 d'b 1 7 8 ± 0 12 b 1 4 2 4- 0 09 a 179-)-010 b 1 8 6 4- 0 17 b

4 5 4. 0 4 6 ± 0 4 9 4. 0 494-03 4 6 4. 0

Mg

14 ttg Zn/g dzet SC SC SC PO PO

0 DMSA 400 D M S A 800 D M S A 0 DMSA 400 D M S A

P O 800 D M S A

0 30 0 32 0 33 031 0 34

± 0 01 a ± 0 01 a'b'c ± 0 02 a'b'c ± 0 0 1 a'c ± 0 01 b

0 046 0 038 0 046 0033 0038

± 4. + + ±

0 004 a 0 0 0 3 a'b 0 005 a 0003 b 0 0 0 6 a'b

3 4 7 3

62 53 58 54 54

44444.

08 01 06 02 03

0 33 -4- 0 01 b,c

0 027 4. 0 004 b

I 61 4- 0 09 a'b

52 ± 0 6

5 4 4. 0 2

Dose Route

0 0165 0 3305

0 6999 0 0033

0 0408 0 0988

0 4859 0 5487

D × R

07634

00650

08279

09125

0 6137 0 3685 0 5713

0 3 0 4. 0 0 2 0 2545

0 0 2 4 4. 0 0 0 4 0 0030

1 56 4- 0 0 8 0 2481

5 8 4. 0 9 0 4313

56±01 0 7706

P values

250 i-tg Zn/g dtet SC 800 D M S A

P values d

a'b'CValues w~th d i f f e r e n t s u p e r s c r i p t s w i t h i n a c o l u m n a r e s l g m f i c a n t l y different at P < 0 05 d C o m p a r e d w i t h the 14 ~g Z n SC 800 D M S A g r o u p

36

T A B L E IX FETAL LIVER MINERALS

Values are shown as #moles mmeral/g wet weight t~ssue and are means + S E M A b b r e v i a t i o n s D M S A , 0, 400 o r 800 mg &mercaptosuccm~c dod/kg body weight, SC, subcutaneous injection, P O , gavage by mouth Zn

Cu

Fe

Ca

Mg

14 ~tg Zn/g dtet SCODMSA

029

0 1 6 6 + 0 0 1 2 ad

2 6 2 ~= 0 0 8

050 + 005

6 6 ± 0 3 db

SC400DMSA SC800DMSA POODMSA PO400DMSA

032 + 002 034 ~ 002 029 + 001 0 3 0 :t: 0 0 1

0151 + 0016 0126 + 0019 0188 + 0007 0136 + 0010

273 269 231 262

010 018 013 011

052 ± 004 053 + 007 044+003 056 + 006

67 68 57 63

PO800DMSA P values

029 + 001

0089 ± 0009 c

2 5 2 a= 0 1 8

046 + 003

6 2 + 0 2 db

Dose Route

0 1085 0 0504

0 0001 0 3610

0 3101 0 0834

0 3663 0 4315

0 5158 0 0169

D x R 250 #g Zn/g dwt SC800DMSA P value~ e

0 2454

0 0948

0 7433

0 4271

0 8449

036 + 002 0 4034

0Ill

2 8 0 =~ 0 1 1 0 6168

048 + 004 0 4963

61 +03 0 2625

+ 001

0 4938

+0012

b'd b'c 't bd

~: ~: ~= a:

=~ ± + ±

04 a 05 d 028 0 3 d,b

a,b,c'dValues with different superscripts within a column are significantly different at P < 0 05 eCompared w~th the 14/~g Z n SC 800 D M S A group

Discussion The results of the present investigation show that D M S A at relatively high levels is not a potent developmental toxicant to the pregnant mouse However, alterations in fetal Cu nutriture and skeletal features suggest that D M S A may exert a subtle influence on fetal development. Dietary Zn supplementation did not improve pregnancy outcome and may have contributed to altered Cu metabolism in DMSA-treated dams. Fetal development (Table I) was only slightly affected by maternal D M S A exposure Only crown-rump length was significantly different between the SC and PO dosed groups fed the 14 /~g Zn diet. Fetuses from SC dosed dams tended to be shorter than those from PO dosed dams. In addition, there was a tendency toward lower fetal weight an SC and lower placenta weight in PO dosed groups. The number of sternebrae, caudal vertebrae, left and right metacarpals and anterior phalanges in the forelimbs, were found to be unaffected by drug treatment (data not shown), although the number of left anterior phalanges was slightly lower in high D M S A groups. Sternal variations of several types were observed, but only the incidences of extra ossification sites in the vertebral column and S N R were slgmficantly increased by the treatments. It IS known that pregnant animals dosed by various routes, with very different toxicants, often produce litters which exhibit one developmental variation in common, an increased incidence of SNR. Chernoff et al. [25] lmmobihzed pregnant CD-1 mice and Sprague-Dawley rats for 12 h during the critical period of fetal rib development

37 (GD 9 and 10 in the mouse and rat, respectively [26]). They found an increase in SNR in the late fetal mouse, but not in the rat. The authors suggested that glucocortlcold hormones, which are known to be teratogenic at high levels [27,28] and are also known to be increased by stresses, like those enumerated above [29], were induced by the stress of lmmoblhzation. Ftlrthermore, experimentation has shown that briefly handling a pregnant rat once results in an Increase in glucocorticolds which does not occur with daily handling. However, dally handling combined with a SC Injection of saline produced a tripling of circulating glucocortlcoids even after 14 days of such treatment [30,31]. If increased incidences of fetal skeletal variations are indicators of generalized maternal stress, our results suggest that SC injection was a greater stress than PO gavage during G D 6-15. By whatever mechamsm SNR is produced, their incidence both within litters and among fetuses was slgmficantly lower in the Zn supplemented group. This finding suggests that the effect on the SC 800 mg DMSA/kg group is, In part, due to altered mineral metabolism, consistent with the hypothesis of Domlngo et al [17]. While there was no effect of DMSA or diet on maternal or fetal tissue Ca, Mg, or Fe concentrations, an effect of DMSA dose on Cu balance and of DMSA route of administration on Zn balance was observed Consistent with this observation, Cantilena and Klaassen reported increased excretion of endogenous Cu in the mouse subsequent to DMSA administration, whereas Fe, manganese (Mn) and Mg excretion was unaffected by the drug [15]. Adult human males treated with DMSA for lead (Pb) poisoning have also been reported to have increased urinary Cu excretion

[21 Maternal kidney Cu concentrations did not vary significantly with treatment However, Zn concentrations were affected by route of administration. Zinc was significantly higher in SC groups and lower in PO groups, independent of dose of DMSA (Table VI). Changes of the same magnitude in kidney Zn levels have been reported by others Amemlya et al found that kidney Zn of untreated pregnant mice fed ad libltum a semi-purified diet adequate in all nutrients was 21.0/~g/g wet weight, while it was 18.7/zg/g in untreated mice fed the same diet at a restricted level. When ad libitum fed mice were injected with a drug vehicle carboxymethylcelluiose (CMC), kidney Zn was 19 7/~g/g wet weight [32] The above illustrates that kidney Zn concentratIons can be markedly affected by a number of variables associated with drug treatment. Maternal intestine Fe and Ca concentrations (Table VII) were affected by the route of administration. While Zn and Cu concentrations were within expected ranges [32] and Fe values in the SC dosed treatment groups were comparable to previously reported values in the mouse and rat, values in the PO treated mice were 50% higher than expected. The dosage of DMSA had no effect on this change In contrast to the earlier findings by Paternain et al. [18], fetal Zn concentrations tended to be higher, rather than lower, in the DMSA treated groups, while Cu concentrations tended to be lower. These findings support the idea that DMSA administration can alter the balance between Zn and Cu in the products of conception. The observed increase in fetal Zn concentrations may have been secondary to the Cu deficiency rather than due to a direct effect of the drug Similar to our results (Table IX), Paternain et al. [18] observed a dose-dependent decline in fetal liver Cu with increasing DMSA doses, up to 1000 mg/kg per day.

38

The low fetal liver Cu concentrations associated with gestational DMSA exposure may pose complications for the neonate Copper is accumulated by the fetal liver m the normal course of events and it is thought that this store of Cu is mobilized for use during the early postnatal growth spurt [33-36]. Possible comphcatlons of poor Cu stores at birth are numerous, with neutropenla, hypochromic anemia, decreased skin pigmentation, poor superoxxde radical defense, elastin and collagen crosshnklng defects, and osteoporosls and bone fragility potentially resulting [36,37]. In summary, the increased incidence of skeletal variations and the dose-dependent decrease in fetal liver Cu show that DMSA administration during gestation influences the normal development of the fetal and neonatal mouse. These findings agree with results from studies m the rat conducted by Domlngo et al. [17,38], who observed subtle changes in skeletal ossification and a dramatic dose-dependent decrease in fetal liver Cu with increasing DMSA doses up to 1000 mg/kg per day. A previous study by these investigators of the impact of SC DMSA on the pregnant mouse did not assess mineral status, but revealed an increased incidence of numerous skeletal variations with increasing DMSA doses of up to 1640 mg/kg per day [16]. Given the effects of DMSA on fetal hver Cu, we suggest that the developmental toxicity of DMSA may be mediated, in part, through altered fetal Cu metabolism If D M S A is to be used during pregnancy, the drug should be further evaluated for its full effects on both maternal and fetal Cu status.

Acknowledgements Supported in part by N . I . H . - H D 0 1 7 4 3 ( C . L . K ) and a U S.D.A Fellowship (M W.T.).

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