Arsenic Speciation in Bile and Urine Following Oral and Intravenous Exposure to Inorganic and Organic Arsenics in Rats

Xing Cui*,1, Yayoi Kobayashi*, Toru Hayakawa{dagger} and Seishiro Hirano*

* Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan; and {dagger} Graduate School of Pharmaceutical Sciences, Chiba University, Inage, Chiba 263-8522, Japan

Received July 5, 2004; accepted August 23, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although inorganic arsenate (iAsV) and arsenite (iAsIII) are metabolized in liver and excreted into bile and urine, the metabolites in the bile after the oral intake of iAs remain unclear. Male Sprague-Dawley rats were orally (po) or intravenously (iv) exposed to iAs and methylated arsenics, and the arsenic speciation in the urine and bile was analyzed by high performance liquid chromatography-inductively coupled argon plasma mass spectrometry. Arsenic caused induction of multidrug resistance-associated protein 2 (MRP2), and changes of glutathione (GSH) levels in the liver and bile were also determined. The metabolic speciation studies revealed that arsenic was excreted into bile in the methylarsenic-diglutathione (MADG) and/or dimethylarsenic acid (DMAV) forms in iAsIII- or iAsV-po rats, but that MADG and arsenic-triglutathione (ATG) are the main forms excreted into bile both in iAsIII- and iAsV-iv rats. In MADG-po rats, the MADG was excreted into bile in the MADG and DMAV forms. Monomethylarsonic acid (MMAV)- and DMAV-iv rats did not excrete significant amounts of either MMAV or DMAV into bile and mostly excreted into urine in the unchanged chemical forms. Taken together, the DMAV detected in the bile is mostly supposed to be the dissociation of dimethylarsenic-glutathione (DMAG). Urinary arsenic speciation showed that arsenic metabolized to 43% methylated DMAV, 47% unmethylated iAsIII, and 10% iAsV in iAsIII-iv rats, whereas only 3% methylated DMAV, 87% unmethylated iAsV, and 10% iAsIII were detected in iAsV-iv rats. Arsenic was accumulated dose dependently, and arsenic concentration was significantly higher in the iAsIII-po rat liver than in the iAsV-po rat liver. GSH levels in the bile were decreased by relatively higher doses of iAsV-po, but significantly increased by iAsIII- or iAsV-iv. iAs-exposure increased the expression of MRP2 in the liver. Pretreatment with buthionine sulfoximine predominantly inhibited arsenic excretion into bile in iAs-iv rats. In conclusion, our data demonstrated that biliary and urinary arsenic excretion and speciation are affected by the route, dose, and chemical forms of arsenical administration, and GSH plays a key role in arsenic metabolism. We are also first to show that DMAV that probably originated from DMAG is excreted into the bile in iAs-po rats.

Key Words: arsenic; glutathione; metabolite; rat; bile; urine.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inorganic arsenic (iAs) is known to cause multiorgan dysfunction and human cancers (Bates et al., 1992Go; Chiou et al., 1995Go; Kitchin, 2001Go). Both pentavalent arsenate (iAsV) and trivalent arsenite (iAsIII) exposure via drinking water or burning of coal have been reported in many countries of the world (Mazumder et al., 1998Go; Pi et al., 2000Go; Shraim et al., 2003Go). Soluble arsenic compounds are rapidly absorbed from the gastrointestinal tract, and then the parent compound and metabolites are excreted into bile and urine through biotransformation by reduction and methylation in the liver (Gregus et al., 2000Go; Thomas et al., 2001Go; Vahter, 2002Go). Urinary excretion of iAs is mainly nonmethylated arsenic and methylated arsenic in the forms of monomethylarsonic acid (MMAV) and dimethylarsenic acid (DMAV) (Del Razo et al., 1997Go; Hopenhayn-Rich et al., 1996Go). Recently, methylated trivalent arsenicals, MMAIII and DMAIII, formed in the course of iAs methylation in the liver have also been detected in urine from men who were chronically exposed to iAs in drinking water (Aposhian et al., 2000Go; Le et al., 2000Go; Mandal et al., 2001Go).

Glutathione (GSH) is important as an intracellular reductant for arsenic methylation and is critical as a cellular antioxidant. One of the basic mechanisms that underlie the toxicity of iAs is the interaction of iAs with thiol-containing residues of peptides and proteins (Maiti and Chatterjee, 2001Go; Schuliga et al., 2002Go). The biliary excretion of arsenic in iAsIII- or iAsV-iv rats is in methylated trivalent arsenic-GSH conjugated forms and depends on the availability of hepatic GSH and multidrug resistance-associated protein 2 (MRP2) (Gyurasics et al., 1991Go; Kala et al., 2000Go). MRP2 is a member of the ATP-binding cassette family of transporter proteins, localized in the canalicular membrane of hepatocytes and involved in the transport of organic anions, various GSH, and sulfate conjugates (Vernhet et al., 2001Go). Pretreatment of the rats with GSH depletors, diethyl maleate or buthionine sulfoximine (BSO), abolished the excretion of the arsenic into bile (Borst et al., 2000Go; Dietrich et al., 2001Go; Ramos et al., 1995Go). Hepatobiliary transport of GSH via MRP2 is thought to play a key role in the biliary excretion of physiologically important copper and zinc as well as toxic arsenics.

iAs is known to be excreted into bile and urine in the forms of methylated intermediates and products that are more reactive and toxic than iAs, but the molecular basis of the arsenic metabolic process is still unclear. Because human exposure to iAs is largely oral, it is important to clarify the excretion products and patterns in the bile after oral intake of iAs. We designed the present study to examine the speciation of arsenic metabolites in bile fluids and urine in rats orally or intravenously exposed to iAs and methylated arsenics by using a high performance liquid chromatography-inductively coupled argon plasma mass spectrometry (HPLC-ICP MS) system.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Caution. These arsenic compounds are toxic and should be handled with care.

Animals. Six-week old male Sprague-Dawley (SD) rats (CLEA Japan Inc., Tokyo Japan) were acclimated to laboratory conditions in a temperature controlled room of 24 ± 2°C under a 12 h (light)/12 h (dark) illumination cycle for 1 week prior to the start of the study. The animals were randomly assigned to iAs- and methylated arsenic-exposed groups. The iAs-exposed groups were divided into orally (po) and intravenously (iv) exposed subgroups. The orally exposed rats were allowed free access to distilled water containing 0, 1, 10, and 100 ppm sodium arsenate (iAsV, Na2HAsO4•7H2O; Sigma, St. Louis, MO) or arsenite (iAsIII, NaAsO2; Sigma, St. Louis, MO) for one week. The intravenously exposed rats were administered 0.25 mg/kg body weight iAsV or iAsIII in sodium chloride solutions via the tail vein. The methylated arsenic-exposed groups were divided into MMAV-iv (0.25 mg/kg body weight, TRI Chemical Laboratory Inc., Yamanashi-Japan), DMAV-iv (0.25 mg/kg body weight, Wako Pure Chemical Industries, Ltd, Japan), and methylarsenic diglutathione (MADG)-po subgroups (0.1 or 0.5 mg/kg body weight; because MADG is unstable and decomposes easily, we prepared it just before the experiment and administered it through gastric tube). All procedures were approved by Animal Care and Use Committee of National Institute for Environmental Studies.

Synthesis of arsenic-glutathione conjugates. The synthesis of arsenic triglutathione (ATG), MADG, and dimethylarsenic glutathione (DMAG) was carried out according to the literature (Kala et al., 2000Go; Scott et al., 1993Go). Stoichiometric amounts of GSH and the respective arsenic species were mixed, and the reaction was carried out for 12 h under a nitrogen atmosphere at room temperature. The experimental conditions for each of the arsenic-GSH complexes were as follows: for ATG, react 0.129 g NaAsO2 and 0.921 g GSH with 10 ml degassed ultrapure water; for MADG, react 0.056 g MMAV and 0.491 g GSH with 2 ml degassed ultrapure water; for DMAG, react 0.138 g DMAV and 0.921 g GSH with 10 ml degassed ultrapure water. All the synthesized compounds were precipitated with 40 ml methanol and evaporated to dryness without heating.

Bile and urine collection from iAs-exposed rats. The animals were anesthetized with pentobarbital (40 mg/kg ip); then the common bile duct was cannulated with polyethylene tubing (PE-10), the distal end of which flowed into an Eppendorf tube resting on a small pad of ice. Bile was collected at 15-min intervals for the analysis of arsenic speciation. BSO (2.5 mmol/kg) (Wako Pure Chemical Industries, Ltd, Japan) was administered intraperitoneally 4 h prior to bile duct cannulation. Urine samples were taken from the bladder by needle puncture at the end of bile collection. Urine and bile samples were examined immediately by the HPLC-ICP MS system.

Reagents and solutions used in HPLC-ICP MS analysis. The HPLC-ICP MS operating condition was the same as we previously reported (Shraim et al., 2003Go). In brief, the mobile phase used for a reversed-phase C18 column (Inertsil ODS-3, 150 x 3 mm, 3 µm particle size, GL Sciences Inc., Tokyo, Japan) was as follows: 5 mM tetrabutylammonium hydroxide (TBAH, 0.5 M, HPLC grade) + 3 mM malonic acid (98%, 104.06 g/mol) + 5% methanol (99.7%, HPLC grade). We used 20 mM oxalic acid (pH adjusted to 2.3 with ammonia) as a mobile phase for the anion-exchange column (Shodex RSpak JJ-50 4D, 4.6 mm x 150 mm id, Showa Denko, Tokyo, Japan). All chemicals used were obtained from Wako Pure Chemicals (Osaka, Japan) and were of analytical-reagent grade. Arsenobetaine (AsB) was obtained from TRI Chemical Laboratory Inc. (Yamanashi, Japan). All mobile phases were passed through 0.22 µm membrane filters before use. MMAIII solution used as a standard was prepared by using MMAV reacted with the reducing solution (0.28 g of sodum metabisulfite, 15 ml of water, 2 ml of 1% sodium thiosulfate, and 0.1 ml of concentrated sulfuric acid) in the ratio 1:1 (v/v) at room temperature for 1 h (Reay and Asher, 1977Go).

Analyses of bile and urine samples by HPLC-ICP MS. Arsenic speciation was carried out using the HPLC-ICP MS system. The HPLC is composed of an LC-10AD solvent delivery unit (Shimadzu, Kyoto, Japan), a CTO-10A column oven (Shimadzu, Kyoto, Japan) and a Rheodyne (California-USA) 7125 six-port injection valve with a 20 µl injection loop. The mobile phase for speciation analyses was isocratically delivered to the HPLC at a flow rate of 1.0 ml/min (reversed-phase C18 column) or 0.6 ml/min (anion-exchange column). A shield torch ICP MS system (HP 4500 series, Yokogawa Analytical Systems Inc., Tokyo, Japan) was used as a detector. The flow from the HPLC column was directly introduced to the ICP MS spray chamber via a 30-cm PEEK tube (0.175 mm id). The injection loop and syringe were properly flushed with water or acid blank after switching from standards to samples and between samples to eliminate any carryover from the previous run. All bile and urine samples from arsenic-exposed rats were diluted with mobile phase and passed through 0.22 µm membrane filters. The detection limits of ICP MS for arsenic ranged between 100 and 200 ppt. Arsenic quantification in the bile and urine was carried out using external calibration curves (up to 20 ppb As) based on the peak area and calculated by ICP MS Chemstation software (Aglient 7500, Yokogawa analytical system). Arsenic levels were expressed as ppb or ppm.

Arsenic concentration in liver. All animals orally exposed to iAs were sacrificed, and the livers were removed and weighed. The experimental animal livers were digested with a mixture of HNO3–HClO4 solution (conc. HClO4:conc. HNO3 = 1:3) for 2 days at 130°C. After the HNO3 was removed by evaporation, the digested samples were diluted with deionized water, and the arsenic concentration was measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) (ICAP-61E-Trace, Thermo Jarrell-Ash) (Cui et al., 2004Go).

GSH concentration in liver and bile. GSH levels in the liver and bile were estimated by using a total glutathione quantification kit (Dojindo Molecular Technologies Inc., Japan). According to the manufacture's protocol, first, the bile (20 µl in 10 µl of 5% 5-sulfosalicylic acid) or homogenized liver tissue (100 mg in 1 ml of 5% 5-sulfosalicylic acid) was centrifuged at 8000 x g for 10 min, and then the supernatant was transferred to a new tube. To each well, enzyme working solution (20 µl), coenzyme working solution (140 µl), and one of either the GSH standard solutions or the sample solution (20 µl) were added. The concentration of 5-sulfosalicylic acid in the sample was adjusted to 0.5–1% with distilled water, and the samples were incubated at 37°C for 10 min. A 20-µl aliquot of DTNB working solution was added. Finally, the absorbance was measured using a microplate reader (InterMed immuno mini NJ-2201), and concentrations of GSH in the sample solutions determined using a calibration curve.

Analysis of MRP2 mRNA level in iAs-exposed rat liver. Both control and arsenic-exposed rat livers were homogenized in TRIZOL (Gibco BRL, Gaithersburg, MD) to obtain total RNA. cDNA was synthesized from 1 µg of RNA with a Thermoscript RT-PCR System (Gibco BRL, Gaithersburg, MD), and gene expression levels were quantitatively measured with a quantitative real-time RT-PCR system (iCycler iQ, Bio-Rad, Hercules, CA) using SYBR® Green I (ABI, Foster, CA) and corresponding primers: for rat MRP2, sense 5'-GGG ATA AAT CTC AGT GGT-3', antisense 5'-ATA TGC TCC ACA GAG TTG-3'; for ß-actin, sense 5'-CGA GGC CCC TCT GAA CCC TA-3', antisense 5'-GGG GCA TCG GAA CCG CTC AT-3'. The mRNA level for the MRP2 in the liver was normalized against ß-actin. For a visual evaluation of mRNA expression of the MRP2, a common RT-PCR was also performed. In brief, specific ß-actin primers were used for the internal control to normalize the sample amounts. PCR was performed in the thermal cycler (GeneAmp® PCR System 9700, ABI, Foster, CA) for 29–40 cycles consisting of denaturation at 94°C for 1 min, annealing at 55°C (ß-actin) for 30 s, or 63°C (MRP2 primer sets) for 30 s and extension at 72°C for 1 min, followed by a final 7 min extension at 72°C. The reaction products were loaded onto 1.8% agarose gels containing ethidium bromide, and visualized under UV illumination.

Statistical analysis. One-way ANOVA and Bonferroni multiple comparison tests were used when differences between the groups were evaluated. For all comparisons, p values less than 0.05 were defined as significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Speciation of Authentic Arsenic Solution by HPLC-ICP MS
Figures 1A and 1E show a standard chromatogram for the speciation of the four arsenic species (iAsIII, iAsV, MMAV, and DMAV) by reverse-phase C18 column and anion-exchange column, respectively. Arsenic-GSH complexes have recently been identified as intermediates in arsenic transport in vivo and appear to be unstable. We synthesized three arsenic-GSH conjugates, ATG, MADG, and DMAG, and developed a method for their separation and identification by HPLC-ICP MS using these two different columns. ATG and MADG were eluted at the same retention time as those of iAsIII (96 s) and MMAIII (120 s) on the reverse-phase C18 column (Figs. 1A and 1D, 1B and 1C). It is revealed that ATG and MADG were degraded to iAsIII and MMAIII in the mobile phase for the reverse-phase C18 column, respectively. However, we successfully separated ATG from iAsIII (retention time 110 s vs. 180 s) and MADG from MMAIII (retention time 120 s vs. 180 s) by using an anion-exchange column (Figs. 1E vs. 1H, 1F vs. 1G). In the present study, because biliary speciation by anion exchange column revealed that only arsenic-GSH conjugates, ATG and MADG were detected, we regarded the chromatograms of iAsIII and MMAIII as ATG and MADG, respectively, in the case of biliary speciation by reverse-phase C18 column. The synthesized DMAG was unstable in the mobile phase and could not detected as a single peak in the chromatogram by HPLC-ICP MS on reverse-phase C18 column or on an anion-exchange column.



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FIG. 1. HPLC-ICP MS chromatogram of authentic standard compounds by reversed-phase C18 column (A–D) and anion-exchange column (E–H). Aliquots of 20 µl of standard solution were applied to a reverse-phase C18 column at 50°C with 5 mM TBAH + 3 mM malonic acid with 5% methanol (pH 5.6, 25°C) at a flow rate of 1.0 ml/min. Aliquots of 20 µl of standard solution were applied to the anion-exchange column at room temperature with 20 mM (pH 2.3, 25°C) at a flow rate of 0.6 ml/min. (A and E) Standard solution of arsenic mixture containing iAsV, iAsIII, DMAV, and MMAV (20 ppb). (B and F) Standard solution of MADG (20 ppb), a small part of MMAV was also detected in the chromatogram because of highly instability of MADG in mobile phase. (C and G) Standard solution of MMAIII, a small part of MMAIII was oxidized to MMAV. (D and H) Standard solution of ATG (20 ppb). The vertical bar indicates the counts per second (cps) for arsenic.

 
Biliary and Urinary Arsenic Speciation in iAs- and MADG-po Rats
Because human exposure to iAs is largely oral, we administered iAs-containing drinking water to SD rats and collected bile and urine for analysis of arsenic speciation with HPLC-ICP MS. We found that most of the arsenic was present as MADG and/or DMAV in the bile. At the lower doses (1 ppm) of iAs that are relevant to human exposure, we were unable to detect either MADG or DMAV excreted into the bile (Fig. 2A). In the 10-ppm iAsV- or iAsIII-po rats, we detected 20.8 ppb DMAV in the bile, and 2190 ppb DMAV, 320 ppb iAsIII, 12.8 ppb iAsV in the urine (Figs. 2B, 2D, 2G, 3A, and 3C). However, in the high dose (100 ppm) exposure group, we were able to detect 297 ppb MADG and 62 ppb DMAV excreted into the bile (Figs. 2C and 3B) and 53700 ppb DMAV, 3100 ppb iAsIII, 400 ppb iAsV, and 910 ppb MMAV in the urine (Figs. 2F and 3D). Urinary arsenic speciation showed that DMAV is the main metabolite both in iAsV and iAsIII-po rats and that the DMAV increased dose dependently (Figs. 2D–2F, 3C, and 3D). In MADG-po rats, the MADG was excreted into bile in the MADG and DMAV forms and was detected in iAsIII, DMAV and DMAIII forms in urine (Fig. 4).



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FIG. 2. HPLC-ICP MS analyses of biliary and urinary arsenic speciation (by reversed-phase C18 column) in rats orally exposed to iAsV or iAsIII at different concentrations. The samples were diluted with mobile phase. A, B, and C show biliary arsenic speciation of the to 1, 10, and 100 ppm iAsV-po rats for 1 week, respectively. DMAV was detectable in the 10, 100 ppm iAs-po rat bile; D, E, and F show urinary arsenic speciation of the 1, 10, and 100 ppm iAsV-po for 1 week rats, respectively. G shows biliary arsenic speciation of the 10 ppm iAsIII-po for 1 week rats. H shows urinary arsenic speciation of the 10 ppm iAsIII-po for 1 week rats. DMAV was the main metabolite in the urine.

 


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FIG. 3. Arsenic speciation (by reversed-phase C18 column) in iAsV-po rats. (A) Biliary arsenic metabolite in the 10 ppm iAsV-po rats. (B) Biliary arsenic metabolites in the 100 ppm iAsV-po rats. (C) Urinary arsenic metabolites in the 10 ppm iAsV-po rats. (D) Urinary arsenic metabolites in the 100 ppm iAsV-po rats.

 


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FIG. 4. Arsenic speciation (by reversed-phase C18 column) in MADG-po rats. A shows a biliary arsenic speciation exposed to 0.1 mg/kg MADG. B shows a biliary arsenic speciation exposed to 0.5 mg/kg MADG. C shows a urinary arsenic speciation exposed to 0.5 mg/kg MADG. DMAV was detectable in the MADG-po rat bile.

 
Biliary and Urinary Arsenic Speciation in iAs-iv Rats
To further clarify the arsenic metabolic mechanism, we administered iAs intravenously to SD rats. Speciation of biliary arsenic metabolites in both iAsIII- and iAsV-iv rats indicates that MADG and ATG are the main forms and that DMAV is not detectable. Arsenic was excreted rapidly into bile mainly in MADG and partly in ATG forms in iAsIII-iv rats. When rats were injected with 0.25 mg/kg iAsIII, we detected 115 ppm MADG and 17 ppm ATG in the first 15 min, but after 45 min MADG and ATG significantly decreased to 7.67 ppm and 0.17 ppm, respectively (Fig. 5A). We found only 2.8 ppm iAsIII (47%), 0.6 ppm iAsV (10%), and 2.6 ppm DMAV (43%) in the urine (Fig. 5B). In contrast, when rats were injected with 0.25 mg/kg iAsV, 1.8 to 4.1 ppm MADG was slowly excreted into bile within 60 min, and 156 ppm iAsV (87%), 18 ppm iAsIII (10%), and 4.5 ppm DMAV (3%) were detected in the urine (Figs. 5C and 5D). Pretreatment with 2.5 mmol/kg BSO almost completely inhibited the excretion of arsenic into bile both in 0.25 mg/kg iAsV- and 0.25 mg/kg iAsIII -iv rats (Figs. 6A, 6B, 6D, and 6E). However, arsenics were still predominantly excreted into urine (Figs. 6C and 6F).



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FIG. 5. Time course of arsenic excretion in bile and urine. A and B show arsenic metabolites excreted into bile and urine in the 0.25 mg/kg iAsIII-iv rats, respectively. C and D show arsenic metabolites excreted into bile and urine in the 0.25 mg/kg iAsV-iv rats, respectively. Bile was collected at 15 min intervals for 60 min and analyzed by HPLC-ICP MS. Urine was collected at the end of bile collection. Values represent the means ± SE of four rats.

 


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FIG. 6. Biliary arsenic speciation (by reversed-phase C18 column) in rats with 0.25 mg/kg iAsV-iv or 0.25 mg/kg iAsIII-iv alone (A, D) or pretreated with 2.5 mmol/kg BSO-ip (B, E). C and F show urinary speciation in iAsV- or iAsIII-iv rats with BSO pretreatment. Arsenic excretion into bile was predominantly abolished by BSO. However, a large amount of arsenic was still excreted into urine.

 
Biliary and Urinary Arsenic Speciation in MMAV- and DMAV-iv Rats
To examine the transport of MMAV and DMAV into bile, we also administered these methylated arsenic compounds (0.25 mg/kg) intravenously to SD rats. Interestingly, a negligible amount of either MMAV or DMAV was excreted into bile in MMAV- or DMAV-iv rats, and they were mostly excreted into urine in the unchanged chemical forms (Fig. 7). A very small part of the MMAV was further methylated to DMAV and excreted into urine (Fig. 7C). A small amount of iAsIII could be found in urine from both MMAV- and DMAV-iv rats (Figs. 7C and 7F).



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FIG. 7. Arsenic speciation in MMAV- and DMAV-iv rats. Biliary (A) and urinary (B, C) arsenic speciation in 0.25 mg/kg MMAV-iv rats; Biliary (D) and urinary (E, F) arsenic speciation in 0.25 mg/kg DMAV-iv rats. C is the magnification of B, and F is the magnification of E. A very small part of MMAV was metabolized to DMAV.

 
Arsenic Concentration and GSH Levels in Liver and Bile
Arsenic concentrations in the liver were increased dose dependently, and the arsenic accumulation in the liver of the 10 ppm iAsIII group was more than two times that of the 10 ppm iAsV group (Table 1). GSH levels in the liver and bile of the 10 and 100 ppm iAsV-po rats were decreased but were unchanged in the 10 ppm iAs III-po rats (Table 1). However, intravenous exposure to both iAsIII and iAsV significantly increased GSH levels in the bile. In addition, pretreatment of rats with BSO (2.5 mmol/kg) significantly reduced the GSH level and subsequently decreased the biliary excretion of arsenic compared to the control (Table 1). These data provide direct evidence for the essential role of GSH transport in the biliary excretion of arsenic.


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TABLE 1 Arsenic Concentrations in the Liver and GSH Levels in the Liver and Bile of Rats Exposed to iAs

 
MRP2 mRNA Levels in Liver of Rats Exposed to iAs
The MRP2 transporter is known to transport organic anions including GSH. The transport of GSH and the transport of arsenic are two separate processes that both depend on the MRP2 pump. Both oral and intravenous exposure to either iAsIII or iAsV significantly increased MRP2 mRNA expression levels in the rat liver (Fig. 8). However, the MRP2 mRNA level was suppressed by BSO pretreatment.



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FIG. 8. Relative mRNA levels of MRP2 mRNA expression in iAs-exposed rat liver were determined by real-time quantitative RT-PCR (upper panels). Total RNA was extracted from the livers of rats with 100 ppm iAsIII-po or 100 ppm iAsV-po for 1 week, or 0.25 mg/kg iAsIII-iv, 0.25 mg/kg iAsV-iv alone or pretreated with 2.5 mmpl/kg BSO-ip. cDNA was synthesized from 1 µg of RNA with a Thermoscript RT-PCR System, and gene expression levels were quantitatively measured with a real-time RT-PCR system. For a visual evaluation, representative photographs of the agarose gels for each RT-PCR product stained with ethidium bromide were displayed (Lower panels). *p < 0.01.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we found that arsenic was excreted into bile in MADG and/or DMAV forms in iAs-po rats, but mainly in MADG and ATG forms into bile in iAsV- or iAsIII-iv rats. It was hard to detect either MADG or DMAV in the bile of rats at lower doses (1 ppm) of iAs-po. However, when iAsV- or iAsIII-po at 10 or 100 ppm, both MADG and DMAV were detectable in the bile. We speculated that after hepato-enteric circulation a part of the MADG is further metabolized to DMAG and finally excreted into bile and urine in the DMAV form. To confirm this hypothesis, we orally administered MADG to the rats and found that the MADG was excreted into bile in the MADG and DMAV forms. This implies that after hepatic-enteric circulation a part of the MADG must be further methylated into DMAG, and the DMAG swiftly is oxidized to DMAV in the bile. In fact, the presence of DMAG in the bile and mobile phase is unstable and undetectable, so probably the dissociation of DMAG occurred (data not shown). Because oral intake of arsenic is the main route for human exposure, and both arsenic metabolic intermediates, MADG and DMAG are reported to be much more toxic than any iAs (Styblo et al., 2002Go), this finding is important for us in understanding the arsenic toxicity and arsenic-induced diseases.

Comparatively, MADG and ATG are the main forms of arsenic excreted into bile both in iAsIII and iAsV-iv rats, but the output rate of iAsV-iv is much slower than iAsIII-iv. This finding is consistent with recent reports that MADG and ATG have been identified as in vivo metabolites of iAsIII or iAsV in the bile of Wistar rats (Maiti and Chatterjee et al., 2001Go; Suzuki et al., 2001Go). iAsIII-iv rats excrete considerably more MADG than the iAsV-iv rats. Urinary arsenic speciation showed that arsenic metabolized to 43% methylated DMAV and 47% unmethylated iAsIII in iAsIII-iv rats. In contrast, only 3% methylated DMAV and 87% unmethylated iAsV were detected in iAsV-iv rats. These data indicate that iAsIII metabolism largely depends on biliary excretion and is extremely fast, but elimination from the body through urine is limited. However, urinary excretion of arsenic is the main route for iAsV, and a small part is metabolized by hepatobiliary transport. The cause of the different features of biliary arsenic speciation of oral, compared to intravenous, exposure to iAs is the existence of hepato-enteric circulation.

Because MMAV was able to react with GSH and produce MADG in vitro, we examined whether MADG is produced and excreted into bile in vivo, in MMAV-iv rats. Interestingly, we did not detect any significant amount of MADG in the bile. This implies that the endogenously formed MMAV can be converted into MADG, but exogenous MMAV cannot enter this biotransformation. Similarly, we only detected a negligible amount of DMAV excreted into bile in DMAV-iv rats. Following oral exposure to MADG, an arsenic-GSH conjugate, the MADG could be metabolized into bile in MADG and DMAV forms, but MMAV- and DMAV-iv rats excrete an imperceptible amount of either MMAV or DMAV into bile, suggesting that, for MMAV or DMAV to be excreted into bile, they must be formed in vivo within the liver and conjugated with GSH. Thus, we speculate that DMAV detected in the bile of iAs-po rats mainly originates from DMAG.

It has been reported that arsenic dose-dependently accumulated in various organs including the liver (Cui et al., 2004Go). In this study, the data showed that arsenic was accumulated significantly more in the liver of the iAsIII-po rats than that of the iAsV-po rats. GSH is known to be an important intracellular reductant and cellular antioxidant and plays a key role in arsenic methylation and metabolism (Maiti and Chatterjee, 2001Go; Schuliga et al., 2002Go). In the present study, the GSH levels in the bile were decreased by relatively higher doses of iAsV-po for 1 week, but significantly increased by both iAsIII- and iAsV-iv. That acute intravenous exposure to higher doses of iAs elevated the GSH levels in the bile suggests that a large amount of arsenic-GSH conjugates was excreted. Pretreatment with BSO, a GSH depletor, predominantly inhibited arsenic excretion into bile in both iAsIII- and iAsV-iv rats. Failure of BSO-pretreated rats to excrete any arsenic after loading with iAs suggests that both arsenic and its methylated products are transported as conjugates with GSH. It has been shown that MRP2 transports GSH and GSH conjugates (Borst et al., 2000Go; Dietrich et al., 2001Go; Wielandt et al., 1999Go); our data demonstrate that expression of MRP2 was significantly increased by either iAsIII or iAsV-exposure. Taken together, these data suggest that GSH and MRP2 play a key role in the transport of arsenic into bile (Gyurasics et al., 1991Go; Leslie et al., 2004Go; Liu et al., 2001Go).

In summary, our data demonstrated that arsenic was excreted into bile in MADG and/or DMAV forms in iAs-po rats, with the existence of hepato-enteric circulation, whereas MADG and ATG but not DMAV were excreted into the bile in iAs-iv rats. We speculate that DMAV detected in the bile of iAs-po rats mainly originates from DMAG.


    NOTES
 

1 To whom correspondence should be addressed. Fax: +81-29-850-2892. E-mail: xing.cui{at}nies.go.jp.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aposhian, H. V., Gurzau, E. S., Le, X. C., Gurzau, A., Healy, S. M., Lu, X., Ma, M., Yip, L., Zakharyan, R. A., Maiorino, R. M., et al. (2000). Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic. Chem. Res. Toxicol. 13, 693–697.[CrossRef][ISI][Medline]

Bates, M. N., Smith, A. H., and Hopenhayn-Rich, C. (1992). Arsenic ingestion and internal cancers: A review. Am. J. Epidemiol. 135, 462–476.[Abstract]

Borst, P., Evers, R., Kool, M., and Wijnholds, J. (2000). A family of drug transporters: The multidrug resistance-associated proteins. J. Natl. Cancer Inst. 92, 1295–1302.[Abstract/Free Full Text]

Chiou, H. Y., Hsueh, Y. M., Liaw, K. F., Horng, S. F., Chiang, M. H., Pu, Y. S., Lin, J. S., Huang, C. H., and Chen, C. J. (1995). Incidence of internal cancers and ingested inorganic arsenic: A seven-year follow-up study in Taiwan. Cancer Res. 55, 1296–1300.[Abstract]

Cui, X., Li, S., Shraim, A., Kobayashi, Y., Hayakawa, T., Kanno, S., Yamamoto, M., and Hirano, S. (2004). Subchronic exposure to arsenic through drinking water alters expression of cancer-related genes in rat liver. Toxicol. Pathol. 32, 64–72.[CrossRef][ISI][Medline]

Del Razo, L. M., Garcia-Vargas, G. G., Vargas, H., Albores, A., Gonsebatt, M. E., Montero, R., Ostrosky-Wegman, P., Kelsh, M., and Cebrian, M. E. (1997). Altered profile of urinary arsenic metabolites in adults with chronic arsenicism. A pilot study. Arch. Toxicol. 71, 211–217.[CrossRef][ISI][Medline]

Dietrich, C. G., Ottenhoff, R., de Waart, D. R., and Oude Elferink, R. P. (2001). Role of MRP2 and GSH in intrahepatic cycling of toxins. Toxicology 167, 73–81.[CrossRef][ISI][Medline]

Gregus, Z., Gyurasics, A., and Csanaky, I. (2000). Biliary and urinary excretion of inorganic arsenic: Monomethylarsonous acid as a major biliary metabolite in rats. Toxicol. Sci 56, 18–25.[Abstract/Free Full Text]

Gyurasics, A., Varga, F., and Gregus, Z. (1991). Glutathione-dependent biliary excretion of arsenic. Biochem. Pharmacol. 42, 465–468.[CrossRef][ISI][Medline]

Hopenhayn-Rich, C., Biggs, M. L., Kalman, D. A., Moore, L. E., and Smith, A. H. (1996). Arsenic methylation patterns before and after changing from high to lower concentrations of arsenic in drinking water. Environ. Health Perspect. 104, 1200–1207.[ISI][Medline]

Kala, S. V., Neely, M. W., Kala, G., Prater, C. I., Atwood, D. W., Rice, J. S., and Lieberman, M. W. (2000). The MRP2/cMOAT transporter and arsenic-glutathione complex formation are required for biliary excretion of arsenic. J. Biol. Chem. 275, 33404–33408.[Abstract/Free Full Text]

Kitchin, K. T. (2001). Recent advances in arsenic carcinogenesis: Modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol. 172, 249–261.[CrossRef][ISI][Medline]

Le, X. C., Ma, M., Cullen, W. R., Aposhian, H. V., Lu, X., and Zheng, B. (2000). Determination of monomethylarsonous acid, a key arsenic methylation intermediate, in human urine. Environ. Health. Perspect. 108, 1015–1018.[ISI][Medline]

Leslie, E. M., Haimeur, A., and Waalkes, M. P. (2004). Arsenic transport by the human multidrug resistance protein 1 (MRP1/ABCC1). Evidence that a tri-glutathione conjugate is required. J. Biol. Chem. 279, 32700–32708.[Abstract/Free Full Text]

Liu, J., Chen, H., Miller, D. S., Saavedra, J. E., Keefer, L. K., Johnson, D. R., Klaassen, C. D., and Waalkes, M. P. (2001). Overexpression of glutathione S-transferase II and multidrug resistance transport proteins is associated with acquired tolerance to inorganic arsenic. Mol. Pharmacol. 60, 302–309.[Abstract/Free Full Text]

Maiti, S. and Chatterjee, A. K. (2001). Effects on levels of glutathione and some related enzymes in tissues after an acute arsenic exposure in rats and their relationship to dietary protein deficiency. Arch. Toxicol. 75, 531–537.[CrossRef][ISI][Medline]

Mandal, B. K., Ogra, Y., and Suzuki, K. T. (2001). Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal, India. Chem. Res. Toxicol. 14, 371–378.[CrossRef][ISI][Medline]

Mazumder, D. N., Das Gupta, J., Santra, A., Pal, A., Ghose, A., and Sarkar, S. (1998). Chronic arsenic toxicity in west Bengal–the worst calamity in the world. J. Indian Med. Assoc. 96, 4–7, 18.

Pi, J., Kumagai, Y., Sun, G., Yamauchi, H., Yoshida, T., Iso, H., Endo, A., Yu, L., Yuki, K., Miyauchi, T., et al. (2000). Decreased serum concentrations of nitric oxide metabolites among Chinese in an endemic area of chronic arsenic poisoning in inner Mongolia. Free Radic. Biol. Med. 28, 1137–1142.[CrossRef][ISI][Medline]

Ramos, O., Carrizales, L., Yanez, L., Mejia, J., Batres, L., Ortiz, D., and Diaz-Barriga, F. (1995). Arsenic increased lipid peroxidation in rat tissues by a mechanism independent of glutathione levels. Environ. Health Perspect. 103 (Suppl. 1), 85–88.[ISI][Medline]

Reay, P. F. and Asher, C. J. (1977). Preparation and purification of 74As-labeled arsenate and arsenite for use in biological experiments. Anal. Biochem. 78, 557–560.[ISI][Medline]

Schuliga, M., Chouchane, S., and Snow, E. T. (2002). Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethal concentrations of inorganic arsenic. Toxicol. Sci. 70, 183–192.[Abstract/Free Full Text]

Scott, N., Hatlelid, K. M., MacKenzie, N. E., and Carter, D. E. (1993). Reactions of arsenic(III) and arsenic(V) species with glutathione. Chem. Res. Toxicol. 6, 102–106.[ISI]

Shraim, A., Cui, X., Li, S., Ng, J. C., Wang, J., Jin, Y., Liu, Y., Guo, L., Li, D., Wang, S., et al. (2003). Arsenic speciation in the urine and hair of individuals exposed to airborne arsenic through coal-burning in Guizhou, PR China. Toxicol. Lett. 137, 35–48.[CrossRef][ISI][Medline]

Styblo, M., Drobna, Z., Jaspers, I., Lin, S., and Thomas, D. J. (2002). The role of biomethylation in toxicity and carcinogenicity of arsenic: A research update. Environ. Health Perspect. 110 (Suppl. 5), 767–771.[ISI][Medline]

Suzuki, K. T., Tomita, T., Ogra, Y., and Ohmichi, M. (2001). Glutathione-conjugated arsenics in the potential hepato-enteric circulation in rats. Chem. Res. Toxicol. 14, 1604–1611.[CrossRef][ISI][Medline]

Thomas, D. J., Styblo, M., and Lin, S. (2001). The cellular metabolism and systemic toxicity of arsenic. Toxicol. Appl. Pharmacol. 176, 127–144.[CrossRef][ISI][Medline]

Vahter, M. (2002). Mechanisms of arsenic biotransformation. Toxicology 181–182, 211–217.[CrossRef][ISI]

Vernhet, L., Seite M,P., Allain N,, Guillouzo, A., and Fardel, O. (2001). Arsenic induces expression of the multidrug resistance-associated protein 2 (MRP2) gene in primary rat and human hepatocytes. J. Pharmacol. Exp. Ther. 298, 234–239.[Abstract/Free Full Text]

Wielandt, A. M., Vollrath, V., Manzano, M., Miranda, S., Accatino, L., and Chianale, J. (1999). Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver. Biochem. J. 341, 105–111.[CrossRef][ISI][Medline]