Biliary and Urinary Excretion of Inorganic Arsenic ...

56, 18 –25 (2000) Copyright © 2000 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Biliary and Urinary Excretion of Inorganic Arsenic: Monomethylarsonous Acid as a Major Biliary Metabolite in Rats ´ gnes Gyurasics, and Ivań Csanaky Zoltań Gregus, 1 A Department of Pharmacology, University Medical School of Pećs, Szigeti u´t 12, H-7643 Pećs, Hungary Received December 17, 1999; accepted February 28, 2000

Huff, 1997; Iffland, 1994; Ishinishi et al., 1986). AsV is the predominant form in arsenic-contaminated drinking water. The considerably more toxic AsIII is a notorious poison and is also a metabolite of AsV. Based on its selective toxicity to specific leukemic cells, arsenic trioxide, the dehydrated form of AsIII, is now used in the treatment of patients with acute promyelocytic leukemia with curative result (Chen et al., 1996; Soignet et al., 1998). The fate of AsIII and AsV in the body involves biotransformation by reduction and methylation and excretion of the parent compound and metabolites into urine and bile (Healy et al., 1999; Styblo et al., 1995a; Vahter, 1983, 1994). Studies with radioactively labeled AsIII and AsV in rats indicate that significant fractions of the intravenous dose of these arsenicals appear in bile (Gregus and Klaassen, 1986, 1995; Gyurasics et al., 1991a,b; Klaassen, 1974). We have shown that the biliary excretion of arsenic in rats injected with either AsIII or AsV depends completely on the availability of hepatic glutathione, because pretreatment of the rats with the glutathione depletor diethyl maleate abolished the excretion of arsenic into bile (Gyurasics et al., 1991b). Furthermore, simultaneously with the biliary excretion of arsenic, large amounts of glutathione appeared in the bile in rats given AsIII or AsV (Gyurasics et al., 1991a). These findings were attributed to the hepatobiliary transport of arsenic as unstable glutathione complexes that decompose in the bile-releasing glutathione. Formation of such complexes in vitro has since been demonstrated (Delnomdedieu et al., 1994; Gailer and Lindner, 1998; Scott et al., 1993; Styblo et al., 1997). It was also hypothesized that both AsIII and AsV are excreted into bile in trivalent form(s) (Gyurasics et al., 1991b) because only the trivalent arsenic can react covalently with thiols such as glutathione. However, the chemical form of arsenic in bile has not been determined, unlike the urinary metabolites of AsIII and AsV, which are well known in humans and experimental animals (Concha et al., 1998; Hughes et al., 1994; Vahter, 1983). Therefore, this study was carried out to specify the forms of arsenic in the bile and to determine the rates of excretion of these arsenicals and their metabolites. For this purpose, the bile and urine of anesthetized rats injected with AsIII or AsV was analyzed for arsenic compounds using HPLC-hydride genera-

In rats exposed to arsenite (AsIII) or arsenate (AsV), the biliary excretion of arsenic depends completely on availability of hepatic glutathione, suggesting that both AsIII and AsV are transported into bile in thiol-reactive trivalent forms (Gyurasics et al. [1991], Biochem. Pharmacol. 42, 465– 468). To test this hypothesis, the bile and urine of bile duct-cannulated rats injected with AsIII or AsV (50 ␮mol/kg, iv) were collected periodically for 2 h and analyzed for arsenic metabolites by HPLC-hydride generation-atomic fluorescence spectrometry. Arsenic was excreted predominantly into bile in AsIII-injected rats, but the urine was the main route of excretion in AsV-exposed rats. Injected AsIII was excreted in urine practically unchanged, whereas both AsV and AsIII appeared in urine after administration of AsV. Irrespective of the arsenical administered, the bile contained 2 main arsenic species, namely AsIII and a hitherto unidentified metabolite. Formation of this metabolite could be prevented by pretreatment of the rats with the methylation inhibitor periodate-oxidized adenosine, indicating that it is a methylated arsenic compound. This metabolite could be converted in vitro into monomethylarsonic acid (MMAsV) by oxidation, whereas synthetic MMAsV could be converted into the unknown metabolite by reduction. Consequently, this biliary metabolite of both AsIII and AsV is monomethylarsonous acid (MMAsIII), a long-hypothesized, but never identified, intermediate in the biotransformation of AsIII and AsV. Although MMAsIII is thought to be formed from an oxidized precursor, rats injected with MMAsV did not excrete MMAsIII. In summary, the inorganic arsenicals investigated are transported into bile exclusively in trivalent forms, namely as AsIII and MMAsIII, but are excreted in urine in both tri- and pentavalent forms. Identification of MMAsIII is signified by the fact that this metabolite is more toxic than AsIII and AsV and thus formation of MMAsIII represents toxification of inorganic arsenic. Key Words: arsenic; monomethylarsonous acid; biliary excretion; methylation; urinary excretion.

Pentavalent arsenate (AsV) and the trivalent arsenite (AsIII), the major forms of inorganic arsenic, are of great toxicological relevance because of their considerable acute toxicity and human carcinogenicity upon prolonged exposure (Chan and 1

To whom correspondence should be addressed. Fax: 36 –72–211–761. E-mail: [email protected]. 18

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BILIARY ARSENIC METABOLITES

tion-atomic fluorescence spectrometry as a speciation technique. This paper reports not only on verification of the hypothesis put forward earlier as to the valence state of biliary arsenic (Gyurasics et al., 1991b), but also on identification of a significant arsenic metabolite, monomethylarsonous acid (MMAsIII), whose existence has been proposed, but not demonstrated. MATERIALS AND METHODS Chemicals. Sodium arsenite (AsIII) was purchased from Carlo Erba (Milan, Italy), disodium hydrogen arsenate (AsV), high purity hydrochloric acid, potassium dihydrogen phosphate, hydrogen peroxide and the chemicals used for reduction of pentavalent arsenic (see below) were from Reanal (Budapest, Hungary); disodium methylarsenate (MMAsV) was obtained from Chem Service (West Chester, PA), cacodylic acid sodium salt (DMAsV) from Sigma, di-potassium hydrogen phosphate from Merck and sodium borohydride from Aldrich. Periodate-oxidized adenosine (PAD) was prepared by incubation of adenosine with sodium periodate according to the method of Hoffman (1980). Immediately before use, a saturated aqueous solution of PAD was prepared and its PAD concentration was determined spectrophotometrically as described by Tandon et al. (1986). Animal experiments. Male Wistar rats (LATI, Go¨do¨llo˝, Hungary) weighing 230 –270 g were used. The animals were kept at 22–25° temperature, at 55– 65% relative air humidity, and on a 12-h light/dark cycle and provided with tap water and lab chow (Altromin, LATI, Go¨do¨llo˝, Hungary) ad libitum. The biliary and urinary excretion studies were performed largely as described (Gregus et al., 1998). To induce urine production, the rats were hydrated by gavage of 30 mL/kg of saline containing 10 mM potassium chloride, anesthetized by ip injection of a mixture of fentanyl, midazolam, and droperidol (0.045, 4.5, and 5.5 mg/kg, respectively), and their body temperature was maintained at 37° by means of heating radiators. Subsequently, the urinary bladder was exteriorized through a lower abdominal incision in the midline, the common bile duct was cannulated with the shaft of a 23-gauge needle attached to PE-50 tubing (Clay Adams, Parsippany, NY) through a higher abdominal incision, and the right carotid artery was cannulated with PE-50 tubing. The rats thus prepared were administered 6 mL/kg 10% mannitol in saline via the carotid cannula to promote urine flow, and were subsequently injected with sodium arsenite or arsenate (50 ␮mol/kg) in saline (3 mL/kg) into the left saphenous vein. To inhibit methylation, some rats were pretreated ip with PAD (50 ␮mol/kg, in 5–10 mL/kg of saline, depending on the actual concentration of PAD in the solution) 30 min before administration of arsenite or arsenate. Bile and urine samples were then collected in 20-min periods into pre-weighed 1.5-mL microcentrifuge tubes. The tubes into which the bile was collected were immersed in ice. To obtain urine, the urinary bladder was gently compressed manually when full and at the end of each collection period. To maintain urine flow at rates of 130 –180 ␮L/kg 䡠 min, 3 mL/kg 10% mannitol in saline was injected via the carotid cannula every 20-min. The volumes of bile and urine samples were measured gravimetrically, taking 1.0 as specific gravity. Biliary and urinary excretion rates of arsenic compounds were calculated as the products of their concentration in bile or urine and the biliary or urinary flow, respectively. Analysis. Arsenic in bile and urine was speciated and quantified by HPLChydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) based on the description of Gomez-Ariza et al. (1998). The eluents containing 10 mM (A) or 60 mM (B) K 2HPO 4-KH 2PO 4 (pH 5.75) were pumped at a combined flow rate of 1 mL/min with 2 HPLC pumps (Model 501, Waters, Milford, MA) through an injector (Rheodyne 7125) equipped with a 20-␮L sample loop onto a strong anion-exchange guard column linked to an analytical column (both Hamilton PRP X-100 with dimensions of 20 ⫻ 4.1 mm and 250 ⫻ 4.1 mm, respectively). To the effluent exiting from the analytical column, first 1.5 M HCl, then 1.5%-m/v sodium borohydride in 0.1 M sodium

hydroxide were admixed using 2 tees and a mixing coil. Both solutions were pumped with a peristaltic pump (Type 313S, Watson-Marlow Ltd., Falmouth, UK) at flow rates of 1 mL/min. These reagents convert the arsenic compounds eluted from the HPLC column into gaseous arsenic hydrides, which are subsequently separated from the combined liquids by a gas-liquid separator (A type, PS Analytical, Kent, UK). The hydrides were conveyed with a mixed stream of argon (300 mL/min) and hydrogen gas (60 mL/min) through a hygroscopic-membrane drying tube (Perma Pure Products, Farmingdale, NJ) into a hydrogen-argon diffusion flame inside the atomic fluorescence spectrometer (PSA Excalibur, PS Analytical), which was equipped with an arsenic boosted-discharge hollow cathode lamp. The fluorescence signal of the detector was recorded by computer using Millennium Chromatography Manager (Waters), which also controlled the HPLC pumps. Immediately after collection, the bile and urine samples were deproteinized by mixing with 9 volumes of 80% methanol in water and centrifuged for 2 min in a Beckman Microfuge E. The resultant supernatant was subsequently diluted with water 10 –25-fold. Both diluents were purged with argon to minimize oxidation of trivalent arsenic in the samples. After injecting the appropriately diluted samples into the HPLC column, the arsenic compounds were eluted by pumping 100% eluent A for 2.1 min, then 100% eluent B till 8.6 min, after which the eluent was changed to 100% A again till minute 12. Quantification of arsenic compounds was based on peak areas of samples and authentic standards. Because pure MMAsIII is not available, the biliary arsenic compound identified in this work as MMAsIII was quantified based on the MMAsIII peak area in the bile sample and the AsIII peak area in the standard. Reduction and oxidation of arsenic compounds. In order to identify the unknown biliary arsenic metabolite (see later), bile samples of rats injected with arsenite or arsenate and authentic MMAsV were subjected to oxidation and reduction, respectively. To oxidize trivalent biliary arsenic, bile was mixed with 9 volumes of a mixture of concentrated hydrogen peroxide and methanol (2:8) and centrifuged for 2 min. The resultant supernatant was diluted 25-fold with water and analyzed by HPLC-HG-AFS as described above. Reduction of MMAsV was carried out with the metabisulfite-thiosulfate reagent as described by Reay and Asher (1977). Two volumes of aqueous MMAsV (5 mM) was mixed with one volume of the reagent and incubated at room temperature for 60 min. This reaction mixture was subsequently diluted with water to a final arsenic concentration of 5 ␮M and analyzed by HPLC-HG-AFS.

RESULTS

Biliary Metabolites of AsIII and AsV In order to test the hypothesis that AsIII and AsV are excreted into bile in trivalent form, bile samples from rats injected with these arsenicals were speciated with HPLC-HGAFS. Representative chromatograms of these bile samples and the standard arsenicals are presented in Figure 1. The HPLC-HG-AFS analysis revealed the presence of two arsenic compounds in the bile of AsIII-injected rats (Fig. 1, middle). One of these (labeled 1) coeluted with the authentic AsIII (Fig. 1, top), whereas the other did not coelute with any of the 3 other arsenic compounds, i.e., DMAsV, MMAsV, and AsV, that have been shown to be present in the urine of arsenic-exposed humans and experimental animals. Apparently, this metabolite of AsIII has not been identified and was thus labeled X. The bile from the AsV-injected rats also contained compound X, moreover, it was quantitatively the most significant biliary metabolite of AsV (Fig. 1, bottom). In addition, lower amounts of AsIII and even less MMAsV were

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GREGUS, GYURASICS, AND CSANAKY

experiment gave qualitatively similar results. Thus, only AsIII and the unknown arsenic metabolite were invariably detected in the bile of AsIII- and AsV-injected rats, whereas MMAsV was found occasionally, but AsV and DMAsV never. MMAsIII as a Biliary Arsenic Metabolite

FIG. 1. HPLC-HG-AFS analysis of arsenic compounds in the bile of rats injected with AsIII or AsV. The figure demonstrates analyses of authentic standard compounds (top), of a representative bile sample collected from a rat 20 – 40 min after iv injection of 50 ␮mol/kg AsIII (middle), and of a representative bile sample collected from a rat 20 – 40 min after iv injection of 50 ␮mol/kg AsV (bottom). Peak identification is given in the top panel. Peak X represents an unknown arsenic metabolite to be identified by investigations presented in Figure 2.

detected in the bile of AsV-injected rats. The latter metabolite, however, was not found consistently; moreover MMAsV was typically undetectable as a biliary metabolite of AsIII or AsV. Figure 1 presents the analysis of bile collected 20 – 40 min after administration of AsIII or AsV; however, analysis of bile samples collected earlier and later until the end of the 2-h-long

Several approaches were taken to determine the chemical identity of metabolite X. First, it was examined to see whether this metabolite is a tri- or pentavalent arsenical, based on the fact that trivalent arsenicals can be converted into pentavalent form by oxidizing agents such as hydrogen peroxide. For this purpose, the bile samples whose analyses are presented in Figure 1 were exposed to hydrogen peroxide and reanalyzed. Comparison of the appropriate chromatograms in Figure 1 with those in Figure 2 reveals that upon exposure to hydrogen peroxide, AsIII disappeared. However, AsV appeared in the bile sample obtained from both the AsIII-injected rat (Fig. 2, middle left) and the AsV-injected animal (Fig. 2, bottom left), indicating that hydrogen peroxide did oxidize AsIII to AsV. More importantly, hydrogen peroxide also eliminated the unknown arsenic metabolite from the bile, and caused appearance of MMAsV (Fig. 2, middle and bottom left). Because this observation suggested that metabolite X is the reduced form of MMAsV, we examined whether reduction of the authentic MMAsV would give rise to a compound with chromatographic properties identical to those of metabolite X. Indeed, incubation of MMAsV with the metabisulfite-thiosulfate reagent partially converted MMAsV into a metabolite that coeluted with the unknown biliary metabolite of AsIII and AsV (Fig. 2, upper right). Finally, we examined whether formation of metabolite X from AsIII and AsV requires methylation. For this purpose, we analyzed the arsenic metabolites in the bile of rats that had been pretreated with the methylation inhibitor PAD before administration of AsIII or AsV. Comparison of the respective chromatograms in Figure 1 with those in Figure 2 (middle and bottom left) indicates that while appearance of AsIII in bile was barely affected, biliary excretion of metabolite X was almost abolished in the PAD-treated rats. These observations indicate that formation and biliary excretion of the unknown arsenic metabolite depends on the activity of methyltransferases, and suggest that metabolite X is a methylated arsenic compound. Thus, the collective evidence provided by the investigations presented in Figure 2 indicates that the hitherto unknown biliary metabolite of both AsIII and AsV is MMAsIII. Biliary and Urinary Excretion Rates of Arsenicals and Their Metabolites Quantification of the concentrations of the injected arsenicals and their metabolites in bile and urine HPLC-HG-AFS analysis permitted calculation of the biliary and urinary excretion rate of each arsenic compound as well as the cumulative excretion of total arsenic into bile and urine. The time courses


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FIG. 2. Identification by HPLC-HG-AFS of the unknown biliary metabolite, X, of AsIII and AsV by treatment of bile from AsIII- and AsV-injected rats with the oxidant hydrogen peroxide, of MMAsV with a reducing agent, and of rats injected with AsIII or AsV with the methylation inhibitor PAD. The figure demonstrates analyses of authentic standards (top left), of H 2O 2-treated bile samples collected from rats given AsIII (middle left) or AsV (bottom left), of MMAsV incubated with a reducing agent (top right), and of bile samples collected from PAD-pretreated rats given AsIII (middle right) or AsV (bottom right). The bile samples were collected from rats 20 – 40 min after injection of AsIII or AsV (50 ␮mol/kg, iv). PAD (50 ␮mol/kg, ip) was given 30 min before administration of AsIII or AsV. The chromatograms of all bile samples shown above and in Figure 1 are directly comparable because all bile samples were diluted 250-fold. Treatment of bile with H 2O 2 and of MMAsV with the reductant was described in Materials and Methods.

of excretion of the major biliary and urinary arsenic species following injection of AsIII and AsV are demonstrated in Figures 3 and 4, respectively. In bile duct-cannulated rats, AsIII and its metabolites were excreted preferentially into bile, as 22% of the dose was delivered into bile, but only 8% into urine in 2 h. AsIII appeared in bile rapidly and constituted the vast majority of biliary arsenic in the first 20 min (Fig. 3, left). Thereafter, the rate of AsIII excretion declined precipitously, while the rate of

MMAsIII output increased gradually. Thus, from 40 min after AsIII administration till the end of the experiment, MMAsIII became the dominant form of biliary arsenic, and by 120 min, 9.2% of the AsIII dose was delivered into bile as MMAsIII. Injected AsIII was also excreted into urine and in fact was the main form of urinary arsenic throughout the experiment (Fig. 3, right). AsV appeared in the urine of only half of the AsIIIinjected rats, and its average excretion rate remained below 3 nmol/kg 䡠 min. Even lower were the average excretion rates of

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FIG. 3. Biliary and urinary excretion of AsIII and its metabolites in rats. Bile and urine were collected at 20-min intervals after injection of AsIII (50 ␮mol/kg, iv). At the fourth collection interval, some rats excreted minute amounts of MMAsV and DMAsV (less than 2 and 5 nmol/ kg/min, respectively) (not shown). Symbols represent means ⫾ SEM of 6 rats.

MMAsV and DMAsV, as they were found in urine only occasionally (not shown). Following iv injection of AsV, arsenic was excreted preferentially by the kidneys, with as much as 46% of the dose being cleared into urine but only 8% into bile within 2 h. Unchanged AsV was the major urinary form, although AsIII was also excreted at significant rates (Fig. 4, right). Some rats excreted minor amounts of MMAsV and DMAsV in urine at later times after AsV administration (not shown). The newly identified MMAsIII was the main biliary metabolite in AsV-injected rats (Fig. 4, left). Its rate of excretion gradually increased then slowly declined and its cumulative 2-h biliary excretion amounted to 7.3% of the AsV dose. The AsV-exposed rats also excreted AsIII into bile; however, the biliary excretion rate of this trivalent arsenical varied between 5 and 10 percent of the rate of MMAsIII excretion (Fig. 4, left). In order to determine whether exogenous MMAsV can be converted into MMAsIII in vivo, the bile and urine of rats injected with MMAsV was analyzed for arsenic compounds. As shown in Figure 5, MMAsV was rapidly cleared from the body by the kidneys and 60% of dose was excreted into urine by 2 h. The biliary excretion rate and cumulative biliary excretion was below 1 nmol/kg 䡠 min and 0.1% of dose, respectively. No metabolite of MMAsV was detected either in bile or urine.

FIG. 4. Biliary and urinary excretion of AsV and its metabolites in rats. Bile and urine was collected in 20-min periods after injection of AsV (50 ␮mol/kg, iv). From the third period, some rats excreted DMAsV at rates below 5 nmol/kg/min (not shown). Symbols represent means ⫾ SEM of 5 rats.

DISCUSSION

In mammals, inorganic arsenic undergoes extensive biotransformation (Aposhian, 1997; Healy et al., 1999; Vahter,

FIG. 5. Biliary and urinary excretion of MMAsV in rats. Bile and urine were collected at 20-min intervals after injection of MMAsV (50 ⫾ ␮mol/kg, iv). Symbols represent means ⫾ SEM of 5 rats.


1994), which is thought to involve the following alternating steps of reductions and methylations: AsV 3 AsIII 3 MMAsV 3 MMAsIII 3 DMAsV. Although most investigators have proposed that methylation of AsIII is oxidative, i.e., results in formation of MMAsV (as above) (Cullen et al., 1984; Thompson, 1993; Styblo et al., 1995a; Vahter, 1994), Braman (1983) has suggested that the product of AsIII methylation is MMAsIII, which is subsequently oxidised to MMAsV. Nevertheless, AsIII, MMAsV and DMAsV have been shown to be present in the urine and tissues of humans and animals (Benramdane et al., 1999; Vahter, 1994), whereas MMAsIII has not been identified as an in vivo metabolite of AsIII or AsV. The present study has revealed the presence of an unknown arsenic compound in the bile of AsIII- and AsV-exposed rats, which was differentiated chromatographically from AsIII, AsV, MMAsV, and DMAsV. Subsequent investigations clarified that this unique compound is a methylated arsenic metabolite, because its excretion into bile was practically abolished in rats pretreated with PAD prior to administration of AsIII or AsV. As an inhibitor of S-adenosylhomocysteine hydrolase, PAD causes accumulation of S-adenosylhomocysteine, which, in turn, inhibits methyltransferases (Hoffman, 1980). It has been shown that PAD effectively impairs formation of MMAsV and DMAsV in mice and rabbits (Marafante and Vahter, 1984; Vahter and Marafante, 1988) and of methylated selenium metabolites in rats (Gregus et al., 1998; Tandon et al., 1986). Thus, the new methylated arsenic compound must have been formed in an S-adenosylmethionine-dependent, methyltransferase-catalyzed reaction, rather than in the recently reported methylcobalamin-dependent non-enzymatic reaction (Zakharyan and Aposhian, 1999). In addition to being methylated, the unknown biliary metabolite was also proved to be a trivalent arsenical because, just like AsIII, it disappeared upon exposure to hydrogen peroxide. More importantly, this oxidizing agent converted the unknown biliary metabolite into an arsenic compound that was indistinguishable chromatographically from MMAsV. In addition, a reducing agent converted the synthetic MMAsV into an arsenic compound that was indistinguishable chromatographically from the unidentified biliary arsenic metabolite. These latter findings especially represent compelling evidence that the biliary metabolite of both AsIII and AsV is MMAsIII, the long hypothesized intermediate in the biotransformation of arsenic. Because as much as 9.2% and 7.3% of the dose of arsenic was recovered in bile as MMAsIII within 2 h after injection of AsIII and AsV, respectively, MMAsIII can be considered to be a quantitatively significant arsenic metabolite. Yet it could have been expected that AsIII-exposed rats would excrete considerably more of this metabolite than the AsV-injected rats, because AsIII is a closer precursor of MMAsIII than AsV

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(see above). The observation that this is not the case suggests that formation and/or hepatobiliary transport of MMAsIII is comparatively less facile in the AsIII-exposed rats than the AsV-injected animals. It may be hypothesized that AsIII, which initially should be present at a much higher concentration in the liver of rats given AsIII than in the liver of rats receiving AsV, inhibits the methyltransferase that forms MMAsIII and/or competes for the hepatobiliary transport with MMAsIII. The finding that the biliary excretion rate of MMAsIII peaked at 20 – 40 min in rats given AsV, but only at 40 – 80 min in rats injected with AsIII is compatible with both hypotheses. As to the first possible mechanism, it has been shown that AsIII inhibits the formation of DMAsV from MMAsV in vitro (Buchet and Lauwerys, 1988; Styblo et al., 1996) and that formation of MMAsIII from AsIII decreases when the concentration of the latter is increased above a certain value (Styblo et al., 1996; Zakharyan et al., 1995). Because, in the generally accepted metabolic scheme of inorganic arsenic, MMAsIII is formed from MMAsV (see above), we investigated whether MMAsIII is produced and excreted into bile in rats injected with MMAsV. These studies indicated that the exogenous MMAsV is probably not reduced to any significant extent to MMAsIII, because this metabolite was not detectable in the bile. They also confirmed in vitro investigations using rat liver cytosol (Styblo et al., 1995b) and experiments in humans (Buchet et al., 1981) as well as hamsters (Yamauchi et al., 1988), indicating that biotransformation of MMAsV is negligible. Thus, if this proposed metabolic scheme is valid, these findings imply that the exogenous MMAsV cannot enter the biochemical compartment in which the endogenously formed MMAsV is converted into MMAsIII. Such a compartmentalization has been proposed by Thompson (1993), who assumes that AsIII is bound to a dithiol “cofactor” in vivo and that the subsequent oxidative methylation and reduction take place in this dithiol-bound form. The present investigations verified our hypothesis that both AsIII and AsV are transported into bile in trivalent forms, i.e., AsIII and MMAsIII. Although we occasionally found small amounts of MMAsV in bile, it is unlikely that MMAsV undergoes significant hepatobiliary transport, as only a negligible amount of MMAsV was excreted in the bile of rats injected with a large dose of this arsenical. Rather, MMAsV is likely formed by intrabiliary oxidation of the excreted MMAsIII. This conclusion is supported by the observation that MMAsIII is readily oxidized and that, upon a short storage of bile samples on air, their MMAsIII content decreased with concomitant appearance of MMAsV (not shown). Both AsIII and MMAsIII form glutathione complexes in vitro (Delnomdedieu et al., 1994; Gailer and Lindner, 1998; Scott et al., 1993; Styblo et al., 1997). Because glutathione conjugates are readily excreted into bile, it is likely that AsIII and MMAsIII are transported from the liver into the bile canaliculi as glutathione conjugates. These conjugates, however, are unstable at the pH of bile (Delnomdedieu et al., 1994) and therefore they are

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expected to decompose after being transported into the bile canaliculi. This decomposition accounts probably for the presence of unconjugated AsIII and MMAsIII together with large amounts of glutathione (Gyurasics et al., 1991a) in the bile of AsIII- and AsV-exposed rats. The present study also confirmed some findings reported earlier, such as extensive reduction of arsenate to arsenite (Vahter, 1994), but minimal oxidation of arsenite to arsenate (Vahter and Envall, 1983). The results obtained in this work with speciation of biliary arsenic corroborate our previous data gained by quantification of total radioactive arsenic in bile. Both works indicated larger participation of biliary excretion in the fate of AsIII than AsV and the extremely fast, but rapidly subsiding hepatobiliary transport of arsenic in rats exposed to AsIII (Gyurasics et al., 1991b). In contrast, unlike others (Buchet and Lauwerys, 1987), we found that the urinary excretion of MMAsV and DMAsV is negligible following administration of inorganic arsenic. This discrepancy may be caused, at least in part, by the intactness of the enterohepatic circulation in other experiments, which permitted the biliary MMAsIII to be reabsorbed from the intestine and excreted into urine after conversion to MMAsV and DMAsV. Studies with radioactive arsenic did indicate that arsenic undergoes enterohepatic circulation in rats (Klaassen, 1974). Because the present investigations were extensions of our earlier studies in rats, they were carried out on this species. The metabolic fate of arsenic in the rat is qualitatively similar to that in many other animal species and humans (Vahter, 1994). Nevertheless, because arsenic is strongly retained in the red blood cells of rats, but not in the erythrocytes of other species, the rat may not be a good model for arsenic disposition in humans (Aposhian, 1997). Therefore, formation of MMAsIII from AsIII and AsV in other species, including humans, awaits confirmation. In conclusion, this study verifies the hypothesis that inorganic arsenic is transported into bile exclusively in trivalent forms, one of which is MMAsIII, a quantitatively significant metabolite of both AsIII and AsV. The significance of identification of MMAsIII is related not only to the quantity in which this metabolite is produced in the body, but also to its chemical reactivity and toxicity in relation to the reactivity and toxicity of its parent compounds, AsV and AsIII. Chemical reactivity and toxicity of arsenicals are markedly different. The pentavalent AsV imitates phosphate (Dixon, 1997), whereas the trivalent arsenicals form compounds of different stability with thiols (Knowles and Benson, 1983). While it is widely recognized that AsIII is considerably more acutely toxic than AsV, it is not generally appreciated that of MMAsIII has a larger toxic potential than AsIII, although this has been demonstrated in microorganisms (Cullen, 1989) and more recently in rat hepatocytes (Styblo et al., 1999). MMAsIII is over 100 times more potent than AsIII as an in vitro inhibitor of thioredoxin reductase (Lin et al., 1999). Thus, formation of MMAsIII appears to represent toxification for both AsV and AsIII. Fur-

ther studies should investigate whether MMAsIII, which is now known to exist in vivo, is important in the acute toxicity, carcinogenicity and the therapeutically exploited selective toxicity of inorganic arsenic. ACKNOWLEDGMENTS This publication is based on a work supported by the Hungarian National Scientific Research Fund, the Ministry of Health, and the Hungarian Academy of Sciences. The authors thank Istvań Schweibert for the excellent assistance in the analytical works as well as Mrs. Katalin Gyulai for typing the manuscript.

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