Chem. Res. Toxicol. 2004, 17, 243-249
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Formation and Urinary Excretion of Arsenic Triglutathione and Methylarsenic Diglutathione Subbarao V. Kala,† Geeta Kala,† Christopher I. Prater,† Alan C. Sartorelli,‡ and Michael W. Lieberman*,†,§ Departments of Pathology and Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, and Department of Pharmacology and Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520 Received October 6, 2003
Taking advantage of mice deficient in γ-glutamyl transpeptidase that are unable to metabolize glutathione (GSH), we have identified two previously unrecognized urinary metabolites of arsenite: arsenic triglutathione and methylarsenic diglutathione. Following administration of sodium arsenite to these mice, ∼60-70% of urinary arsenic is present as one of these GSH conjugates. We did not detect the dimethyl derivative, dimethyl arsenic GSH; however, dimethyl arsenic (DMAV) represented approximately 30% of urinary arsenic. Administration of buthionine sulfoximine, an inhibitor of GSH synthesis, to wild-type mice reduced urinary arsenic excretion by more than 50%, indicating the GSH dependence of arsenic metabolism, transport, or both. Rodents deficient in three known ABC family transporters (MRP1, MRP2, and MDR1a/1b) exhibited urinary arsenic levels similar or greater than those in wild-type rodents; however, administration of MK571, an MRP inhibitor, reduced urinary arsenic excretion by almost 50%. MK571-treated mice showed ∼50% reduction of AsIII, MMAV, and AsV as compared to untreated wild-type controls, while DMAV levels were unchanged. These findings suggest that arsenic excretion is in part dependent on GSH and on an MRP transporter other than MRP1 or 2.
Introduction Arsenic is a well-documented human bladder, lung, and skin carcinogen; yet, millions of people in Bangladesh, India, Taiwan, and Latin America are exposed to arsenic levels in drinking water that exceed the World Health Organization and U.S. standard of 10 µg/L by as much as 100-fold (1-5). Urinary arsenic excretion accounts for 60-80% of total arsenic ingested; in most mammals, including humans, much of the ingested arsenic is rapidly metabolized to its methylated derivatives and is excreted as AsIII or methylated products (69). A majority of xenobiotics and toxins are cleared from the body either by conjugation to GSH or by glucuronide formation (see review, 10). It is not known if urinary arsenic excretion is similarly dependent on GSH conjugate formation. These conjugates have not been detected in urine to date either because of the methodology used in previous studies or because of their degradation by GGT,1 which cleaves GSH and most GSH conjugates and is abundantly expressed on the microvilli of the proximal tubules of the kidney (11, 12). However, the development of mice deficient in GGT in our laboratory (13) has * To whom correspondence should be addressed. Tel: 713-798-6501. Fax: 713-798-6001. E-mail:
[email protected]. † Department of Pathology, Baylor College of Medicine. ‡ Department of Pharmacology and Cancer Center, Yale University School of Medicine. § Department of Molecular and Cellular Biology, Baylor College of Medicine. 1 Abbreviations: GGT, γ-glutamyl transpeptidase; ATG, arsenic triglutathione; MADG, methylarsenic diglutathione; DMAG, dimethylarsenic glutathione; MMAV, monomethylarsonic acid; DMAV, dimethyl arsenic; MRP, multidrug resistance associated protein; MDR, multidrug resistance; ABC, ATP binding cassette; cMOAT, canalicular multispecific organic anion transporter; BSO, buthionine sulfoximine; ICP, inductively coupled plasma; IC, ion exchange chromatography.
allowed us to investigate the role of GSH conjugation of arsenic in the urinary excretion of this element. The ABC transporter superfamily contains the P-glycoprotein (pgp)/MDR protein and the MRPs gene family. MRPs are known to transport GSH conjugates of drugs and xenobiotics and to encode for GS-X pumps (14, 15). MRP1 is ubiquitously expressed throughout the body, and its levels in the lung, testes, and kidney are relatively high (16-18); it has been shown to transport cysteinyl leukotrienes as well as other GSH and glucuronide conjugates (19-22). MRP1 deficient mice exhibit higher tissue levels of GSH and increased sensitivity to etoposide toxicity (22-24). The MRP1 transporter is expressed basolaterally at relatively lower levels. In contrast, the MRP2/cMOAT transporter localizes to the apical side of the bile canalicular membrane of hepatocytes. MRP2 has been implicated in the transport of several GSHconjugated substrates including cisplatin (25-27). We have recently shown that the MRP2 pump is responsible for biliary arsenic excretion and that GSH plays an obligatory role in this process (28). We have also demonstrated the presence of two arsenic-GSH conjugates, ATG and MADG, in the bile of rats in vivo. The role of other MRP transporters (MRP3 to MRP9) in the transport of GSH conjugates has not been elucidated yet. With respect to MRP3, there is conflicting evidence as to whether it functions to transport GSH conjugates (29, 30). The MDR1a and MDR1b genes in mice have also been shown to participate in the excretion of xenobiotics (31-35). In the mouse, the MDR1a gene is predominantly expressed in the intestine, liver, and blood capillaries of brain and testes, whereas the MDR1b gene is mainly expressed in the adrenal glands, placenta, and ovaries and the expression of MDR1a and MDR1b is similar in the kidney (34).
10.1021/tx0342060 CCC: $27.50 © 2004 American Chemical Society Published on Web 01/17/2004
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Kala et al.
In the present study, we have taken advantage of GGT deficient mice to identify two arsenic-GSH conjugates, ATG and MADG, not previously recognized in urine. We have also used mice deficient in MRP1 and MDR1a/1b and rats deficient in MRP2 to examine their role in the transport of arsenic into the urine.
Experimental Procedures Animals. GGT deficient mice of the C57BL/6/129SvEv hybrid strain developed in our laboratory (13) and age-matched controls were used. GGT deficient mice were supplemented with Nacetylcysteine (10 mg/mL) in the drinking water from birth as described previously to maintain normal growth, and 6-8 week old mice were used for all experiments. MRP1 deficient mice developed by Lorcio et al. (24) were bred, and 6-8 week old mice were used for arsenic studies. C57BL6/129 mice served as controls. MDR1a/1b deficient mice and FVB (controls) used in this study were purchased from Taconic Engineering (Germantown, NY). Rats (Wistar; Harlan, Houston, TX) and TR- rats (on a Wistar background originally obtained from Dr. Oude Elferink of the Academic Medical Center, Department of Gastrointestinal and Liver Diseases, Amsterdam, The Netherlands) 6-8 weeks of age were used for bile and urinary arsenic excretion experiments. Arsenic Exposure. Mice or rats were injected with sodium arsenite (0.5-5 mg/kg) subcutaneously, and urine and bile samples were collected for the analysis of total arsenic, arsenic speciation, and arsenic-glutathione conjugates. Animals were anesthetized prior to arsenic administration, and bile and urine samples were collected after 1 h. Bile samples were collected as described previously (28). Urine samples were collected directly from the bladder. All procedures followed guidelines established by the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Baylor College of Medicine Animal Care and Use Committee. LC/MS Analysis of Arsenic-Glutathione Conjugates. ATG, MADG, and DMAG were synthesized as described earlier (28), and standard chromatograms were generated using LC/ MS to identify arsenic-GSH conjugates in the urine. ArsenicGSH conjugates in urine samples of wild-type and mutant mice were separated on reversed phase HPLC column (Aqua C18 75 mm × 2 mm column width with a particle size of 3 µm; Phenomenex, Torrance, CA) using 0.1% formic acid (pH 2.65) and an acetonitrile gradient (0-40%). The column effluent was analyzed by monitoring negative ions by ESI coupled to a mass spectrometer. Selective ion monitoring (SIM) was used to identify ATG (m/z 992), MADG (m/z 701), and DMAG (m/z 410). The recovery of ATG, MADG, and DMAG on this column was found to be approximately 98-100% under the conditions used. The urine samples were fractionated on an LC column to determine the arsenic content of the arsenic-GSH conjugates by ICP-MS. ICP-MS and IC Coupled ICP-MS Analysis of Arsenic Species. Urine samples from arsenic-treated rats and mice were diluted with 1% nitric acid and analyzed for arsenic content by ICP-MS (HP4500). Arsenic content in the urine was quantified by using a standard calibration curve of inorganic arsenic (SPEX, Metchen, NJ) with terbium as an internal standard. The creatinine content in urine samples was measured by using a Sigma Kit (no. 555-A; Sigma, St.Louis, MO). Total arsenic content in urine samples was expressed as µg/mg of creatinine. A portion of the urine was used for the arsenic speciation using IC/ICP-MS. Arsenic species (DMAV, AsIII, MMAV, and AsV) were separated on a Dionex anionic ion exchange column (AS7, Dionex, Houston, TX), with a gradient containing 30 mM ammonium acetate (pH 9.0), 30 mM ammonium phosphate (pH 4.5), 200 mM ammonium hydroxide, and water. The flow rate of the column was maintained at 1.5 mL/min, and the arsenic content in the effluent was continuously monitored by ICP-MS.
Figure 1. Analysis of ATG and MADG in urine samples of arsenic-treated GGT deficient mice. (A) LC/MS profile of ATG, MADG, and DMAG. (B) Analysis of arsenic-GSH conjugates in urine samples of GGT deficient mice. (C) ICP-MS analysis of arsenic in urine fractionated by LC. Mixtures of synthetic ATG, MADG, and DMAG were separated on a reversed phase HPLC column (Aqua C18 75 mm × 2 mm column with a particle size of 3 µm, Phenomenex) using 0.1% formic acid (pH 2.6) and an acetonitrile gradient (0-40%) as described previously (28). The column effluent was analyzed by ESI-MS (HP1100) for negative ions using SIM. Extracted ion chromatograms of m/z 992 (ATG), m/z 701 (MADG), and m/z 410 (DMAG) are presented (A). GGT deficient mice were treated with 1 mg/kg of sodium arsenite, and urine samples collected after 1 h were analyzed using LC/ MS (B). A portion of the urine sample was also fractionated by LC and was used for the analysis of total arsenic by ICP-MS (C). DMAG was not detected in urine samples of arsenic-treated GGT deficient mice (B). See Table 1 for the details of the quantitative analysis of ATG and MADG at different doses of sodium arsenite. Standard chromatograms were generated using commercially available DMAV, As III (sodium arsenite), MMAV, and AsV (sodium arsenate) (Sigma) to identify and quantify arsenic species. Statistical Analysis. Student’s t-test was used for statistical analyses. Results were expressed as mean ( SEM. A p value < 0.05 was considered statistically significant.
Results We have used GGT deficient mice that are incapable of degrading glutathione to identify arsenic-glutathione conjugates in urine (13). We identified two arsenic-GSH conjugates, ATG and MADG, in urine samples of GGT deficient mice treated with sodium arsenite (1.0 mg/kg, s.c.) (Figure 1A,B). We were unable to detect DMAG in
Arsenic-Glutathione Conjugates in Urine
Chem. Res. Toxicol., Vol. 17, No. 2, 2004 245 Table 1. Arsenic-Glutathione Conjugates in Urine of GGT Deficient Micea dose (mg/kg)
arsenic in ATG (µg/mL)
arsenic in MADG (µg/mL)
0.5 1.0 5.0
2.3 ( 0.5 6.2 ( 0.6 24.5 ( 6.2
3.8 ( 0.3 4.2 ( 0.3 1.6 ( 0.3
a Groups of four GGT deficient mice were injected subcutaneously with different doses of sodium arsenite (0.5, 1.0, and 5.0 mg/ kg), and the urine was collected after 1 h. Arsenic-GSH conjugates in the urine samples were analyzed using LC/MS. Values are the mean ( SE of four individual experiments.
Figure 2. Arsenic speciation in wild-type and GGT deficient mice. (A) Levels of arsenic species in urine samples of wild-type and GGT deficient mice. (B) IC/ICP-MS chromatogram of arsenic speciation in a urine sample of GGT deficient mice. (C) IC/ICP-MS chromatogram of arsenic speciation in a urine sample of GGT deficient mice after treatment with 1% H2O2. Sodium arsenite (1 mg/kg, s.c.) was injected into wild-type and GGT deficient mice, and urine samples were collected for 1 h for the determination of arsenic speciation. Values are expressed as the mean ( SEM of data obtained from six mice (A). Urine samples were treated with 1% H2O2 for 15 min prior to ion exchange separation (C).
these samples (Figure 1B). To quantify the contribution of these arsenic-GSH conjugates to total urinary arsenic excretion, we fractionated urine samples on a C18 reversed phase column and collected samples at 20 s intervals. Arsenic content was determined by ICP-MS (Figure 1C). We found that 60-70% of the arsenic was present as either ATG or MADG. The recoveries of ATG, MADG, and DMAG on the C18 column under the experimental conditions used were ∼98-100%. The recoveries of DMAV and MMAV were found to be 90% of arsenic was found in fractions between 240 and 360 s corresponding to elution times of ATG and
MADG. This value corresponds to ∼60-70% of the total arsenic in urine; 30% of the arsenic was present as DMAV, which is not eluted under these conditions (see Figure 2A for DMAV levels in urine samples of WT and GGT deficient mice measured using IC/ICP-MS). We treated GGT deficient mice with three different doses of arsenic (0.5, 1.0, and 5.0 mg/kg) and quantified the levels of ATG and MADG in urine (Table 1). The level of ATG increases almost linearly with dose while that of MADG remains constant and falls at the 5 mg/kg dose. The most likely explanation for this behavior is that the ability of mice to methylate arsenic is saturated at the lower doses used in the experiments and may even be inhibited by very high levels of arsenic (36). Other studies have also shown that high doses of As III inhibit MMAsIII methyl transferase activity (37) and formation of methylated AsIII metabolites (38). Urine samples from arsenic-treated GGT deficient mice were also analyzed by IC/ICP-MS. In contrast to the soft ESI used for the analysis of arsenic-GSH conjugates, the ion exchange chromatographic separation is harsh with respect to the stability of the arsenic-GSH conjugates (especially for ATG) and is used here mainly to identify various arsenic species (DMAV, AsIII, MMAV, and AsV). Other investigators (39, 40) have also used similar methods for urinary arsenic determinations and did not identify urinary arsenic-GSH conjugates. Although methods have been developed recently to identify arsenicGSH conjugates using IC/ICP-MS (41), more satisfactory methodology for identification of these compounds relies on LC/MS (28). Urine samples from wild-type and GGT deficient mice showed more or less the same level of arsenic species (Figure 2A). However, an unidentified peak of arsenic was observed in the urine samples of GGT deficient mice (peak 1 in Figure 2A,B) as opposed to wildtype mice where no such peak was found. Because ATG is unstable during the ion exchange separation conditions, we hypothesized that the unretained peak should be MADG, as we did not detect DMAG in GGT deficient mice using LC/MS (Figure 1B). When we injected standard MADG or DMAG separately on to an ion exchange column and analyzed the eluent by ICP-MS, we found that both of these compounds elute at the same retention time (1.1 min). To rule out the possibility that peak 1 is DMAG and to positively identify peak 1 as MADG, we treated urine samples of GGT deficient mice with H2O2 prior to ion exchange separation and analyzed samples by IC/ICP-MS. As expected, pretreatment of urine samples of GGT deficient mice with H2O2 resulted in the disappearance of the first peak with the simultaneous appearance of MMAV (Figure 2C), confirming that peak 1 is MADG. Hydrogen peroxide treatment of urine samples also resulted in the conversion of AsIII into AsV without affecting DMAV.
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Figure 3. Time-dependent breakdown of MADG by GGT. Synthetic MADG (3 mM) was incubated with GGT (Sigma) in 50 mM Tris-HCl (pH 8.0) buffer. Aliquots were removed at different time intervals, and the reaction was stopped by the addition of 1% formic acid containing bathophenanthroline disulfonic acid (1 mM) and analyzed by LC/MS. The breakdown products of MADG were monitored using SIM mode. MACG was measured at m/z 572, whereas m/z 701 was used for MADG.
Because we were not able to detect arsenic-GSH conjugates in urine samples from wild-type mice because of the abundant presence of GGT in kidney proximal tubules, we have determined the role of GGT in the breakdown of arsenic-GSH conjugates. We used commercially available GGT (Sigma) to study the breakdown of MADG by this enzyme. We found that GGT was capable of cleaving the γ-glutamyl group of one of the GSH molecules in MADG to yield the methylarsenic cysteinylglycine glutathione (MACG) complex (m/z 572) (Figure 3). However, we could not detect the final breakdown product of MADG (loss of two glutamyl groups from two GSH molecules in MADG), i.e., methylarsenic dicysteinylglycine conjugate using LC/MS. This finding is probably the result of the instability of this conjugate. This interpretation is supported by our inability to make synthetic methyl dicysteinylglycine arsenic conjugate, as opposed to ATG and MADG in vitro (data not shown). These results indicate that GGT may be involved in the metabolism and/or processing of arsenic-GSH conjugates in vivo. To further confirm the role of GSH in urinary arsenic excretion, we used BSO, an inhibitor of GSH synthesis (42). Pretreatment of wild-type mice with 10 mmol/kg BSO reduced the urinary arsenic excretion to
