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

Xing Cui 1 Yayoi Kobayashi 1 Toru Hayakawa 0 Seishiro Hirano 1 0 Graduate School of Pharmaceutical Sciences, Chiba University , Inage, Chiba 263-8522 , Japan 1 Environmental Health Sciences Division, National Institute for Environmental Studies , 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 , Japan - 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 SpragueDawley 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 chromatographyinductively 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 methylarsenicdiglutathione (MADG) and/or dimethylarsenic acid (DMAV) forms in iAsIII- or iAsV-po rats, but that MADG and arsenictriglutathione (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. 1 To whom correspondence should be addressed. Fax: 181-29-850 2892. E-mail: . Toxicological Sciences vol. 82 no. 2 # Society of Toxicology 2004; all rights reserved. Inorganic arsenic (iAs) is known to cause multiorgan dysfunction and human cancers (Bates et al., 1992; Chiou et al., 1995; Kitchin, 2001). 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., 1998; Pi et al., 2000; Shraim et al., 2003). 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., 2000; Thomas et al., 2001; Vahter, 2002). 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., 1997; Hopenhayn-Rich et al., 1996). 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., 2000; Le et al., 2000; Mandal et al., 2001). 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, 2001; Schuliga et al., 2002). 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 resistanceassociated protein 2 (MRP2) (Gyurasics et al., 1991; Kala et al., 2000). 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., 2001). Pretreatment of the rats with GSH depletors, diethyl maleate or buthionine sulfoximine (BSO), abolished the excretion of the arsenic into bile (Borst et al., 2000; Dietrich et al., 2001; Ramos et al., 1995). 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 These arsenic compounds are toxic and should be handled 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 6 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)-p (...truncated)