Lead Detoxification Activities of a Class of Novel ... - ACS Publications

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Lead Detoxification Activities of a Class of Novel DMSA
Amino Acid Conjugates Yanxia Xu, Yuji Wang, Ling Wang, Ming Zhao,* Xiaoyi Zhang, Xiaomin Hu, Baoguang Hou, Li Peng, Meiqing Zheng, Jianhui Wu, and Shiqi Peng* College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, P. R. China

bS Supporting Information ABSTRACT: The coupling of the 1-carboxyl of DMSA with L-amino acids led to a class of novel 1-(carbonyl-L-amino-acid)2,3-dimercaptosuccinic acids (DMSA
amino acid conjugates, DMSA-Gly, -Ser, -Val, -Leu, -Ile, -Asn, -Asp, -Gln, -Glu, -Met, Phe, and -Trp). In the in vivo evaluation of Pb-loaded mice, 0.4 mmol/kg of the conjugates effectively decreased the Pb levels of the femur, brain, kidney, liver, and blood, greatly enhanced urination, and increased the Pb levels of both urine and feces, while causing no redistributions of Pb to the other organs, especially to the brain. With respect to lowering the bone and brain Pb, DMSA-Ile, -Asn, -Gln, and -Met were more effective than DMSA. This benefit was attributed to their high transmembrane ability. In contrast to Pb, the essential metals such as Fe, Cu, Zn, and Ca of the treated mice were not affected by the administration of the conjugates. Silico molecular modeling predicted that the conjugates had little hepatotoxicity, except possibly for DMSA-Phe.

’ INTRODUCTION During the past two decades, the industrialization and agriculture modernization in developing countries have significantly increased the levels of lead (Pb) in air, water, and soil. For the developed countries, Pb poisoning remains a major concern despite the declining use of Pb-based products.1
5 As a widespread toxic metal, in the body the half-life time of Pb is 6 to 10 years.4,6,7 In addition to hypertension, hyperthyroidism, osteoporosis, and skeletal disorders,3
5,8 the exposure of Pb in the environment has been associated with developmental, neuropsychological, and behavioral deficits in particular.9 In the foreseeable future, Pb exposure will continue to be a major public health issue around the world, and the discovery of a novel therapy for the treatment of Pb poisoning is an urgent need.10
14 It has been well documented that chelation therapy is one of the most common treatments of Pb intoxication, while meso-2,3-dimercaptosuccinic acid (DMSA) is the most acceptable antidote in lowering Pb levels in the bone and brain, causing no redistributions to other organs, especially to the brain.9,15
20 In a previous paper, by preparing a class of novel 5-(1-carbonyl-L-amino-acid)2,2-dimethyl[1,3]dithi-olane-4-carboxylic acids, it was explored that the linking of the two mercapto groups with an isopropyl group and the coupling of its 5-carboxyl group with L-amino acids may significantly increase the efficacy of DMSA in lowering brain Pb.21 In the discovery of a novel therapy of Pb poisoning, it was also explored that with respect to enhancing Pb detoxification activity of the pharmacophore Gly, L-Ser, L-Val, L-Leu, L-Ile, LAsn, L-Asp, L-Gln, L-Glu, L-Met, L-Phe, L-Trp, and L-Cys were more effective than the other amino acids.22 In this context, we introduced these amino acids into the 1-carboxyl of DMSA to r 2011 American Chemical Society

form a class of novel 1-(carbonyl-L-amino-acid)-2,3-dimercaptosuccinic acids (DMSA-amino acid conjugates, 4a
l) to increase the efficacy of DMSA in lowering brain and bone Pb.

’ MATERIALS AND METHODS Chemicals. All chemicals were purchased from Sigma Aldrich Co. (Milwaukee, WI, USA). 1-(Carbonyl-L-amino-acid)-2,3-dimercaptosuccinic acids (4a
l) and their intermediates were prepared via a four-stepreaction procedure (Scheme 1), and the procedures as well as the physical chemical data are given as Supporting Information. Animals. Male imprinting control region (ICR) mice (weighing 22 ( 2 g) were purchased from Experimental Animal Center of Peking University. The chemicals used in the in vivo assays were purchased from Sigma Aldrich Chemical Co. (Milwaukee, WI, USA). Following a protocol reviewed and approved by an ethics committee of Capital Medical University, the investigations were performed. The committee assures that the welfare of the animals was maintained in accordance with the requirements of the Animal Welfare Act and according to the Guide for Care and Use of Laboratory Animals. Lead Detoxification Assay. Fifteen groups (12 each) of mice were injected (i.p.) with a solution of 8.2 mg/kg of Pb(C2H3O2)2 3 3H2O in 0.5 mL of water per day for seven consecutive days to load Pb. After a 2-day interval, one group (blank control) was given a daily injection (i.p.) of 0.2 mL of normal saline (NS), 14 groups were given a daily injection (i.p.) of 0.4 mmol/kg of DL-penicillamine (positive controls), sodium meso-2,3-dimer captosuccinate (NaDMS, lead compound control), or 4a
l for five consecutive days. On each day, 2 h after the Received: April 13, 2011 Published: May 17, 2011 979

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acid (4a
l), and 1 and 2 as well as 3a
l were the intermediates of 4a
l. The yields of 1, 2, 3a-l, and 4a
l were 78%, 91%, 22
56%, and 40
87%, respectively, and the overall yields of 4a
l were 6
35%. The synthetic conditions and the chemical physical properties of 1, 2, 3a-l, and 4a
l are given as Supporting Information. The data imply that using this four-step-reaction procedure 4a
l can be smoothly gained. Pb Levels in the Livers, Kidneys, Femurs, Brains, and Blood of Mice. In the Pb detoxification assay, the Pb-loaded mice were administered with NS (blank control) or 0.4 mmol/kg of DL-penicillamine (a known heavy metal chelating agent,25 positive control), or 0.4 mmol/kg of NaDMS (the lead compound of 4a
l, positive control) or 0.4 mmol/kg of 4a
l. The Pb levels of the livers, kidneys, brains, femurs, and blood of mice were measured, and the data are shown in Table 1. As seen, the Pb levels of the livers, kidneys, femurs, brains, and blood of the mice receiving DL-penicillamine, NaDMS, and 4a
l are significantly lower than that of the mice receiving NS. This suggests that 0.4 mmol/kg of DL-penicillamine, NaDMS, and 4a
l are effective in lowering Pb levels of the livers, kidneys, femurs, brains, and blood of mice. With respect to bone Pb, though the Pb levels of the femurs of the mice receiving 4a
d,g,i,k,l are not significantly different from that of the femurs of the mice receiving NaDMS, which suggests that the efficacy of DMSA in lowering bone Pb is not affected by the conjugates DMSA-Gly, -Ser, -Val, -Leu, -Asp, Glu, -Phe, and -Trp, the Pb levels of the femurs of the mice receiving 4e,f,h,j are significantly lower than that of the femur of the mice receiving NaDMS. Therefore, the conjugate DMSAAsn, -Gln, -Met, and -Ile are more effective than DMSA in bone Pb detoxification. With respect to brain Pb, the Pb levels of the brains of the mice receiving 4a,b,e,f,h,i,j are significantly lower than that of the brains of the mice receiving NaDMS. This suggests that the efficacy of DMSA in lowering brain Pb is greatly enhanced by the coupling of the 1-carboxyl of DMSA with Gly, L-Ser, L-Ile, L-Asn, L-Gln, L-Glu and L-Met. Besides, the Pb levels of the kidneys and the livers of the mice receiving 4a
l are not significantly different from that of the kidneys and the livers of the mice receiving NaDMS. This suggests that the coupling of the 1-carboxyl of DMSA with Lamino acids does not decrease the efficacy of DMSA in lowering kidney and liver Pb. Further more, the Pb levels of the blood of the mice receiving conjugates 4a,b,e
j are significantly lower than that of the blood of the mice receiving NaDMS. This suggests that the efficacy of DMSA in lowering blood Pb is enhanced by the coupling of the 1-carboxyl of DMSA with Gly, L-Ser, L-Ile, L-Asn, L-Asp, L-Gln, LGlu, and L-Met. This reduced the probability of the redistributions of Pb. To clarify the advance of the present modification in both bone and brain Pb, the detoxification shown in Table 5 of Supporting Information, the Pb levels of the bone and brain of Pb-loaded mice receiving 0.4 mmol/kg of 4a
l were compared with that of the bone and brain of the Pb-loaded mice receiving 0.4 mmol/kg of 5-(1-carbonyl-L-amino-acid)-2,2-dimethyl-[1,3]dithiolane-4carboxylic acids (CDDCAs, 3a
l of Scheme 1), which were capable of enhancing the activity of brain Pb detoxification.21 The comparison indicated that except for 4g,i, in which the femur Pb detoxification activity was lower than that of CDDCA, and 4a,j,k, in which the femur Pb detoxification activity was not significantly different from that of CDDCA, the femur Pb of

Scheme 1. Synthetic Route of 1-(Carbonyl-L-amino-acid)2,3-dimercaptosuccinic Acida

a

(i) Hydrogen chloride and acetone; (ii) acetyl chloride; (iii) amino acid; (iv) mercuric acetate and H2S. In 3a and 4a, AA = Gly; 3b and 4b, AA = L-Ser; 3c and 4c, AA = L-Val; 3d and 4d, AA = L-Leu; 3e and 4e, AA = L-Ile; 3f and 4f, AA = L-Asn; 3g and 4g, AA = L-Asp; 3h and 4h, AA = L-Gln; 3i and 4i, AA = L-Glu; 3j and 4j, AA = L-Met; 3k and 4k, AA = L-Phe; and 3l and 4l, AA = L-Trp. injection, the urine samples of each group were continually collected for 24 h, and the fecal samples of each group were continually collected for 24 h. Twenty-four hours after the last injection, all the mice were weighed, sacrificed by diethyl ether anesthesia, and dissected to immediately get the livers, kidneys, brains, and left femurs. On a heating block, the collected urine, fecal, liver, kidney, brain, and left femur were digested in a 1/3 mixture of HClO4/HNO3, dried at 80 °C, and dissolved in nitric acid (1%) to measure their lead contents as well as the contents of essential metals on a Varian Spectra AA-40 atomic absorption spectrometer in the graphite furnace. All data are expressed as the means ( SD. The statistical analysis of the data was carried out by using an ANOVA test. p < 0.05 was considered significant. Apparent Permeability Evaluation. The permeability of the compounds across Caco-2 monolayers has been correlated with human oral absorption in vivo.23 The permeability was also correlated with the lead decorporation activity of the chelating agents such as pentahydroxylhexyl-L-cysteine and its dimer.24 In general, the i.p. administration is close to oral administration. On the basis of these, here the apparent permeability assay was selected to understand the different Pb detoxification activities of the present chelants. Caco-2 cells were cultivated on polycarbonate filters (traswell cell culture inserts, 12 mm in diameter, and 3.0 μm in mean pore size). After growth on filter supports for 21 days, Caco-2 cells were used for all transport tests, and the integrity of the monolayer was routinely checked with the measured transepithelial electrical resistance (∼700 Ω 3 cm2). All absorptions were tested in Hank’s balanced salt solution (HBSS). DLPenicillamine, NaDMS, and 4e,f,h,j were dissolved in HBSS, and the final concentration of the formed solution was 4 mM. In the apical to the basolateral direction, the transport was initiated by adding the test solution (total AP volume, 0.5 mL) to the apical compartment of the inserts held in the transwell containing 1.5 mL of HBSS (basolateral compartment). In the basolateral to the apical direction, the transport was initiated by the addition of 1.5 mL of the test solution to the basolateral compartment and 0.5 mL of HBSS as receiving solution to the apical side of the monolayer. The monolayer was incubated in air at 37 °C and 95% relative humidity. At 30, 60, 90, and 120 min, the samples were withdrawn from the receiving side, and the concentration of the sample was measured with HPLC. The resistance of the monolayer was checked at the end of each experiment. The apparent permeability coefficient (Papp) was calculated following Papp = dQ/dt 3 1/(A 3 C0), wherein dQ/dt represents the permeability rate, C0 represents the initial concentration in the donor chamber, and A represents the surface area of the monolayer (1 cm2).

’ RESULTS AND DISCUSSION Preparation of Conjugates 4a
l. Using a four-step reaction procedure and in the corresponding reaction conditions (Scheme 1), we prepared 1-(carbonyl-L-amino-acid)-2,3-dimercaptosuccinic 980

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Table 1. Lead Levels of the Femur, Brain, Liver, Kidney and Blood of Micea compd

femur

brain

kidney

liver

blood

NS

23.70 ( 3.08

2.04 ( 0.40

8.92 ( 1.46

7.22 ( 1.64

0.54 ( 0.05

DL-PA

14.99 ( 2.05 b

1.61 ( 0.42 b

7.24 ( 2.08 b

3.52 ( 0.86 c

0.40 ( 0.03 b

NaDMS

14.31 ( 2.07 b

1.72 ( 0.34 b

6.23 ( 1.89 c

4.75 ( 1.35 c

0.39 ( 0.04 b

DMSA-Gly, 4a

13.54 ( 1.77 c

0.93 ( 0.45 c

5.90 ( 1.53 c

3.46 ( 1.56 c

0.30 ( 0.02 c

DMSA-Ser, 4b

13.22 ( 1.93 c

1.02 ( 0.49 c

7.30 ( 1.54 b

5.29 ( 1.09 c

0.36 ( 0.04 d

DMSA-Val, 4c

14.14 ( 1.06 b

1.42 ( 0.53 b

7.00 ( 1.95 b

5.65 ( 0.74 b

0.37 ( 0.03 e

DMSA-Leu, 4d

14.73 ( 1.70 b

1.44 ( 0.58 b

6.62 ( 1.82 c

5.64 ( 1.02 b

0.34 ( 0.02 c

DMSA-Ile, 4e DMSA-Asn, 4f

12.70 ( 1.41 d 12.56 ( 1.30 d

1.08 ( 0.52 d 0.98 ( 0.41 c

7.40 ( 1.36 b 7.14 ( 1.79 b

5.62 ( 1.36 b 5.18 ( 1.24 c

0.33 ( 0.04 c 0.35 ( 0.05 f

DMSA-Asp, 4g

14.00 ( 1.15 c

1.46 ( 0.45 b

7.07 ( 1.86 b

4.89 ( 1.68 c

0.36 ( 0.02 d

DMSA-Gln, 4h

12.79 ( 1.23 d

1.16 ( 0.46 d

5.65 ( 1.78 c

4.78 ( 1.56 c

0.31 ( 0.03 c 0.30 ( 0.04 c

DMSA-Glu, 4i

16.05 ( 1.53 b

1.06 ( 0.46 c

5.31 ( 2.64 c

4.78 ( 1.40 c

DMSA-Met, 4j

12.80 ( 1.30 d

0.99 ( 0.43 c

6.69 ( 1.80 c

5.52 ( 1.39 b

0.34 ( 0.02 c

DMSA-Phe, 4k

15.58 ( 1.98 b

1.41 ( 0.70 b

7.01 ( 1.83 b

4.69 ( 1.68 c

0.37 ( 0.03 e

DMSA-Trp, 4l

15.64 ( 1.20 b

1.40 ( 0.74 b

7.41 ( 1.62 b

5.66 ( 1.34 b

0.38 ( 0.03 b

Data are represented with the mean ( SD μg of Pb/g of organ or excrement. NS = normal saline = vehicle, NaDMS = sodium meso-2,3dimercaptosuccinate, DL-PA = DL-penicillamine, dose of DL-PA, NaDMS, and DMSA-amino acid conjugates = 0.4 mmol/kg; n = 12. The normal mice were also used as an absolute blank control, and no Pb was measured in their femurs, brains, kidneys, livers, and blood. For femurs lead: b, compared to NS p < 0.01; c, compared to NS p < 0.01, to DL-PA p < 0.05; d, compared to NS p < 0.01, to NaDMS and DL-PA p < 0.05. For brain lead: b, compared to NS p < 0.01; c, compared to NS, DL-PA and NaDMS p < 0.01; d, compared to NS p < 0.01, to NaDMS p < 0.05. For kidney lead: b, compared to NS p < 0.05; c, compared to NS p < 0.01. For liver lead: b, compared to NS p < 0.05; c, compared to NS p < 0.01; d, compared to NS; and 4b
f,j,l, p < 0.01, to NaDMS and 4g
i,k p < 0.05. For blood lead: b, compared to NS p < 0.01; c, compared to NS, DL-PA and NaDMS p < 0.01; d, compared to NS and DLPA p < 0.01, to NaDMS p < 0.05; e, compared to NS p < 0.01, to DL-PA p < 0.05; f, compared to NS p < 0.01, to DL-PA and NaDMS p < 0.05. a

Table 2. Lead Levels of Urine and Feces of the Micea compd.

urinary volume

fecal weight

fecal Pb

urinary Pb

NS

9.62 ( 0.86

3.50 ( 0.40

1.27 ( 1.15

0.93 ( 0.11

DL-PA

16.85 ( 1.84 b

3.56 ( 0.29

3.26 ( 1.40 b

1.94 ( 0.67 b

NaDMS

19.98 ( 1.90 c

3.54 ( 0.31

4.59 ( 0.84 c

1.79 ( 1.46 b

DMSA-Gly, 4a

20.11 ( 2.01 c

3.87 ( 0.32 b

5.01 ( 1.56 c

2.73 ( 1.37 b

DMSA-Ser, 4b

23.30 ( 1.94 d

3.83 ( 0.30 b

4.98 ( 0.64 c

2.89 ( 1.02 c

DMSA-Val, 4c

20.24 ( 1.98 c

3.55 ( 0.28

6.48 ( 2.01 e

1.92 ( 1.08 b

DMSA-Leu, 4d DMSA-Ile, 4e

20.33 ( 1.99 c 23.29 ( 1.98 d

3.84 ( 0.26 b 3.87 ( 0.33 b

4.93 ( 1.79 c 4.87 ( 1.73 c

2.63 ( 1.17 b 2.28 ( 1.10 b

DMSA-Asn, 4f

23.32 ( 1.92 d

3.82 ( 0.30 b

4.96 ( 0.65 c

2.22 ( 0.99 b

DMSA-Asp, 4g

19.80 ( 1.85 c

3.51 ( 0.27

6.80 ( 1.04 e

1.86 ( 0.85 b

DMSA-Gln, 4h

23.41 ( 2.00 d

3.57 ( 0.34

6.95 ( 1.41 e

2.88 ( 0.96 c

DMSA-Glu, 4i

23.11 ( 1.93 d

3.90 ( 0.41 b

4.64 ( 1.69 c

2.50 ( 1.04 b

DMSA-Met, 4j

23.30 ( 1.99 d

3.84 ( 0.35 b

5.20 ( 1.42 d

2.86 ( 0.90 c

DMSA-Phe, 4k

20.63 ( 1.88 c

3.86 ( 0.30 b

4.98 ( 1.38 c

2.55 ( 0.96 b

DMSA-Trp, 4l

20.57 ( 1.91 c

3.80 ( 0.26 b

4.44 ( 0.95 c

2.41 ( 0.99 b

Fecal Pb is represented with the mean ( SD μg of Pb/g of feces, urinary Pb is represented with the mean ( SD μg of Pb/mL of urine, fecal weight is represented with the mean ( SD g/24 h, and urinary volume is represented with the mean ( SD mL/24 h. NS (normal saline) = vehicle, NaDMS = sodium meso-2,3-dimercaptosuccinate, DL-PA = DL-penicillamine, dose of DL-PA, NaDMS and 4a
l = 0.4 mmol/kg, n = 12. The normal mice were also used as an absolute blank control, and no Pb was measured in their urine and feces. For urinary volume: b, compared to NS p < 0.01; c, compared to NS and DL-PA p < 0.01; and d, compared to NS and DL-PA p < 0.01, to NaDMS p < 0.05. For fecal weight: b, compared to NS p < 0.05. For fecal lead: b, compared to NS p < 0.01; c, compared to NS p < 0.01, to DL-PA p < 0.05; d, compared to NS and DL-PA p < 0.01; and e, compared to NS and DL-PA p < 0.01, to NaDMS p < 0.05. For urinary lead: b, compared to NS p < 0.01; c, compared to NS p < 0.01, to NaDMS, DL-PA p < 0.05. a

4b
f,h,l treated mice is significantly lower than that of CDDCA treated mice. This means that to increase bone Pb detoxification efficacy 4a
l are more preferable than CDDCA. The comparison also indicated that except for 4a, in which the brain Pb detoxification activity was higher than that of CDDCA, and 4f, in which the brain Pb detoxification activity was lower than that of

CDDCA, the brain Pb of 4b
e,g
l treated mice is not significantly different from that of CDDCA treated mice. This means that there is no significant difference between brain Pb detoxification activities of 4a
l and CDDCA. Therefore, the present modification of DMSA is superior to the previous modification.21 981

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Table 3. Dose-Dependent Pb Detoxification Activities of 4ha compd

dose (mmol/kg)

femur

brain

kidney

liver

NS

0.2 mL

23.70 ( 3.08

2.04 ( 0.40

8.92 ( 1.46

7.22 ( 1.64

DMSA-Gln (4h)

0.6

10.21 ( 1.15 b

0.79 ( 0.30 b

4.33 ( 1.21 b

3.34 ( 1.10 b

0.4

12.79 ( 1.23 c

1.16 ( 0.46 c

5.65 ( 1.78 c

4.78 ( 1.56 c

0.2

16.10 ( 1.66 d

1.70 ( 0.37 d

7.40 ( 1.55 d

6.10 ( 1.48 d

Data are represented with the mean ( SD μg of Pb/g of organ. NS = normal saline = vehicle, n = 12. For femur lead: b, compared to NS, 0.2 mmol/kg of 4h and 0.4 mmol/kg of 4h, p < 0.01; c, compared to NS and 0.2 mmol/kg of 4h, p < 0.01; and d, compared to NS, p < 0.01. For brain lead: b, compared to NS and 0.2 mmol/kg of 4h, p < 0.01, to 0.4 mmol/kg of 4h, p < 0.05; c, compared to NS, p < 0.01, to 0.2 mmol/kg of 4h, p < 0.01; and d, compared to NS, p < 0.05. For kidney lead: b, compared to NS and 0.2 mmol/kg of 4h, p < 0.01, to 0.4 mmol/kg of 4h, p < 0.05; c, compared to NS, p < 0.01, to 0.2 mmol/ kg of 4h, p < 0.05; and d, compared to NS, p < 0.05. For liver lead: b, compared to NS and 0.2 mmol/kg of 4h, p < 0.01, to 0.4 mmol/kg of 4h, p < 0.05; c, compared to NS, p < 0.01, to 0.2 mmol/kg of 4h, p < 0.05; d, compared to NS, p > 0.05. a

Table 4. Apparent Permeability Coefficients of 4e,f,h,j

Table 5. ADMET Hepatotoxicity Probability of 4a
l

Papp  10
6 (cm/s) compd

AfB

BfA

compd AfB/BfA

ADMET score

compd

ADMET score

NaDMS

0.033

DMSA-Asp, 4g

0.105

DMSA-Gly, 4a

0.006

DMSA-Gln, 4h

0.033

DL-PA

13.12

11.50

1.14

DMSA-Ser, 4b

0.086

DMSA-Glu, 4i

0.033

NaDMS

11.01

10.00

1.10

DMSA-Ile, 4e

17.23

5.22

3.30

DMSA-Val, 4c DMSA-Leu, 4d

0.066 0.092

DMSA-Met, 4j DMSA-Phe, 4k

0.125 0.443

DMSA-Trp, 4l

0.238

DMSA-Asn, 4f

15.93

5.14

3.10

DMSA-Ile, 4e

0.099

DMSA-Gln, 4h DMSA-Met, 4j

15.86 16.60

5.10 5.17

3.11 3.21

DMSA-Asn, 4f

0.132

Therefore, conjugates 4a
l show their Pb detoxification efficacy mainly via enhancing the urination of the treated mice. Effect of 4a
l on Essential Metals of Mice. The levels of essential metals, Fe, Cu, Zn, Mn, and Ca, in the kidneys, livers, and brains of the mice receiving NS or 0.4 mmol/kg of 4a
l were measured and the data are given in Supporting Information (Tables 1
3). As seen, there is no difference between the essential metals of the organs of the mice receiving NS and the essential metals of the organs of the mice receiving 4a
l. This suggests that the essential metal levels of the treated mice are not affected by conjugates 4a
l. Effect of 4a
l on the Growth of Mice. The effect of the treatment of 4a
l on the growth of Pb-loaded mice was examined by comparing the body weights of the mice receiving NS or 0.4 mmol/kg of 4a
l, and the data are given in Supporting Information (Table 4). As seen, there is no difference between the body weights of the mice receiving NS and 4a
l. This suggests that the growth of Pb-loaded mice is not affected by the treatment of 4a
l. Dose-Dependent Pb Detoxification Activity of 4h. The dose-dependent response of Pb-loaded mice to the treatment DMSA-Gln (4h) was selected as a representative of 4a
l, and three doses (0.2 mmol/kg, 0.4 mmol/kg, and 0.6 mmol/kg) were used. The Pb levels of the livers, kidneys, femurs, and brains of the treated mice were measured, and the data are listed in Table 3. As seen, the Pb levels progressively decreased with the increase of the dose. Therefore, DMSA-Gln dose-dependently removes the Pb accumulated in the livers, kidneys, femurs, and brains of the treated mice. Membrane Permeability of Conjugates 4e,f,h,j. The correlation of the membrane permeability of 4a
l with their Pb detoxification activities was explored with the Caco-2 cell monolayer experiments of DL-PA, NaDMS, and conjugates 4e,f,h,j, the representatives of 4a
l. Their membrane permeability is listed in Table 4. The experiments gave DL-PA, NaDMS, and 4e,

Pb Levels of Feces and Urine of the Mice. The Pb levels of

the feces and the urine, as well as the quantity of the feces and the urine of the mice receiving NS or 0.4 mmol/kg of DLpenicillamine, or 0.4 mmol/kg of NaDMS, or 0.4 mmol/kg of 4a
l are shown in Table 2. The Pb levels of the feces and urine of the mice receiving DL-penicillamine, NaDMS, and 4a
l are significantly higher than that of the feces and urine of the mice receiving NS. This suggests that DL-penicillamine, NaDMS, and 4a
l are able to enhance Pb excretion. The Pb levels of the feces of the mice receiving of 4c,g,h,j are significantly higher than that of the feces of the mice receiving NaDMS. This suggests that DMSA-Val, -Asp, and -Gln benefit Pb excretion via feces. The urinary volume of the mice receiving NaDMS and 4a
l is not only signifycantly higher than that of the mice receiving NS but also significantly higher than that of the mice receiving DLpenicillamine. This suggests that NaDMS and 4a
l are able to more effectively increase the urination of the mice. However, the urinary volume of the mice receiving 4b,e,f,h,i,j are significantly higher than that of the mice receiving NaDMS. This suggests that for enhancing the urination 4b,e,f,h,i,j are the most potent conjugates among 4a
l. In contrast to urination, the feces weights per 24 h of the mice receiving NS, DL-penicillamine, NaDMS, and 4a
l are not significantly different from each other. This means that 4a
l do not enhance fecal excretion. From Table 2, we also noticed that the feces weight of 4a
l treated mice ranged from 3.51 to 3.90 g, and the Pb levels of the feces ranged from 4.44 μg to 6.95 μg Pb per g of feces; thus, the Pb of the feces totally ranged from 15.58 μg to 27.11 μg. While the urinary volume of 4a
l treated mice ranged from 19.80 to 23.41 mL, and the Pb levels of the urine ranged from 1.86 μg to 2.89 μg Pb per mL of urine, the Pb of the urine totally ranged from 36.83 μg to 67.65 μg. This means that for the treated mice the Pb of the urine is much higher than that of the feces. 982

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Chemical Research in Toxicology f,h,j the 11.01  10
6 cm/s to 17.23  10
6 cm/s of Papp values from the apical side to the basolateral side (Papp,AfB), the 4.68  10
6 cm/s to 11.50  10
6 cm/s of Papp values from the basolateral side to the apical side (Papp,BfA). The Papp,AfB values are 1.10-fold to 3.30-fold higher than the Papp,BfA values. An actively absorbed compound generally shows much faster transport from the apical to the basolateral direction, and the Papp,AfB value is more than 10  10
6 cm/s.26 As seen, the Papp, AfB values of DL-PA, NaDMS, and 4e,f,h,j are more than 10  10
6 cm/s. The Papp,AfB values of 4e,f,h,j are 3.10-fold to 3.30fold higher than their Papp,BfA values, while the Papp,AfB value of NaDMS is only 1.10-fold higher than its Papp,BfA value. This means that the coupling of 1-carboxyl of DMSA with amino acids significantly increases the membrane permeability, and their higher membrane permeability should be responsible for the higher activities of 4e,f,h,j in lowering bone Pb and brain Pb. ADMET Hepatotoxicity of 4a
l. The hepatotoxicities of 4a
l were predicted with the absorption, distribution, metabolism, elimination, and toxicity (ADMET) program developed from available data of a number of compounds that were known to possess liver toxicity or trigger dose-related elevated aminotransferase levels in more than 10% of the human population.27,28 Two scores (0 represents nonhepatotoxin and 1 represents hepatotoxin) are defined. The calculated scores (0.006
0.443) of NaDMS and 4a
l are listed in Table 5. As seen, all scores are much less than 1. This suggests that NaDMS and 4a
l possess little or no hepatotoxicity, except possibly for 4k. The calculated hepatotoxicity of 4k has a value of 0.443 and is much higher than any of the others and close to 0.5. Considering that 0 represents nonhepatotoxin and 1 represents hepatotoxin 0.443 should be a critical value. This suggests that introducing L-Phe into DMSA could induce hepatotoxicity.

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’ AUTHOR INFORMATION Corresponding Author

*(M.Z.) Tel: þ86-10-8391-1535. Fax: þ86-10-83911535. E-mail: [email protected]. (S.P.) Tel: 86-10-8391-1528. Fax: 86-10-83911528. E-mail: [email protected]. Funding Sources

This work was supported by the following research funds: PHR (IHLB, KZ200910025004, and KM200910025-009), the National Natural Scientific Foundation of China (30801426), Special Project (2011ZX09302-007-01) of China, and Innovation Platform Project of Education Committee of Beijing.

’ ACKNOWLEDGMENT We acknowledge Beijing Area Major Laboratory of Peptide and Small Molecular Drugs. ’ REFERENCES (1) Nriagu, J., Afeiche, M., Linder, A., Arowolo, T., Ana, G., Sridhar, M. K. C., Oloruntoba, E. O., Obi, E., Ebenebe, J. C., Orisakwe, O. E., and Adesina, A. (2008) Lead poisoning associated with malaria in children of urban areas of Nigeria. Int. J. Hyg. Environ. Health 211, 591–605. (2) Liu, Z. P. (2003) Lead poisoning combined with cadmium in sheep and horses in the vicinity of non-ferrous metal smelters. Sci. Total Environ. 309, 117–126. (3) Kachur, A. N., Arzhanova, V. S., Yelpatyevsky, P. V., von Braun, M. C., and von Lindern, I. H. (2003) Environmental conditions in the Rudnaya River watershed: a compilation of Soviet and post-Soviet era sampling around a lead smelter in the Russian Far East. Sci. Total Environ. 303, 171–185. (4) Franco-Uria, A., Lopez-Mateo, C., Roca, E., and FernandezMarcos, M. L. (2009) Source identification of heavy metals in pastureland by multivariate analysis in NW Spain. J. Hazard. Mater. 165, 1008– 1015. (5) Cheng, H., and Hu, Y. (2010) Lead (Pb) isotopic fingerprinting and its applications in lead pollution studies in China: A review. Environ. Pollut. 158, 1134–1146. (6) Takser, L., Mergler, D., and Lafond, J. (2005) Very low level environmental exposure to lead and prolactin levels during pregnancy. Neurotoxicol. Teratol. 27, 505–508. (7) Patra, M., Bhowmik, N., Bandopadhyay, B., and Sharma, A. (2004) Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant systems and the development of genetic tolerance. Environ. Exp. Bot. 52, 199–223. (8) Osterode, W., Winker, R., Bieglmayer, C., and Vierhapper, H. (2004) Effects of parathyroidectomy on lead mobilization from bone in patients with primary hyperparathyroidism. Bone 35, 942–947. (9) Rademachera, D. J., Steinpreisa, R. E., and Weber, D. N. (2001) Short-term exposure to dietary Pb and/or DMSA affects dopamine and dopamine metabolite levels in the medulla, optic tectum, and cerebellum of rainbow trout (Oncorhynchus mykiss). Pharmacol. Biochem. Behav. 70, 199–207. (10) Gurer, H., and Ercal, N. (2000) Can antioxidants be beneficial in the treatment of lead poisoning? Free Radical Biol. Med. 29, 927–945. (11) Pachauri, V., Saxena, G., Mehta, A., Mishra, D., and Flora, S. J. S. (2009) Combinational chelation therapy abrogates lead-induced neurodegeneration in rats. Toxicol. Appl. Pharmacol. 240, 255–264. (12) Flora, S. J. S., Kannan, G. M., and Pant, B. P. (2002) Combined administration of oxalic acid, succimer and its analogue for the reversal of gallium arsenide-induced oxidative stress in rats. Arch. Toxicol. 76, 269– 276. (13) Shaban, E. M., and Said, E. Y. (2011) Influence of vitamin C supplementation on lead-induced histopathological alterations in male rats. Exp. Toxicol. Pathol. 63, 221–227.

’ CONCLUSIONS In conclusion, the coupling of the 1-carboxyl of DMSA with Lamino acid leads to DMSA
amino acid conjugates, a series of novel antidotes for organ and blood Pb detoxification, in particular for bone and brain Pb detoxification. The order of bone Pb detoxification activities of these conjugates is DMSA-Ile, -Asn, Gln, and -Met > DMSA-Gly, -Ser, and -Asp > DMSA-Val, -Leu, Glu, -Phe, and -Trp. The order of brain Pb detoxification activities of these conjugates is DMSA-Gly, -Ser, -Asn, -Glu, and -Met > DMSA-Ile and -Gln > DMSA-Val, -Leu, -Asp, -Phe, and -Trp. Taking the bone Pb and brain Pb detoxifycation activities together into account, DMSA-Ile, -Asn, -Gln, and Met have the highest efficacy, and this could be partly correlated with their high membrane permeability. Comparing with DMSA, the most acceptable antidote in lowering the Pb levels of bone and brain causing no redistributions of Pb to other organs, conjugates DMSA-Ile, -Asn, -Gln, and -Met are more efficacious in lowering bone and brain Pb levels. The administration of DMSA
amino acid conjugates does not induce hepatotoxity and does not lower the levels of essential metals. ’ ASSOCIATED CONTENT

bS

Supporting Information. Experimental procedures, biological evaluation methods, synthetic data, analytical data, physical chemical constants, and spectral data. This material is available free of charge via the Internet at http://pubs.acs.org. 983

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