Binding of dimethylarsinous acid to cys-13alpha of rat hemoglobin is responsible for the retention of arsenic in rat blood

The metabolism, disposition, and carcinogenicity of arsenic differ dramatically between humans and rats. To understand the molecular basis of these differences, we have characterized arsenic species in rats that were treated with inorganic arsenate (iAsV), monomethylarsonic acid (MMAV), or dimethylarsinic acid (DMAV) for up to 15 weeks. Arsenic significantly accumulated in the red blood cells (RBCs) of rats in the form of hemoglobin (Hb) complexed with dimethylarsinous acid (DMAIII), regardless of whether the rats were treated with iAsV, MMAV, or DMAV, suggesting rapid methylation of arsenic species followed by strong binding of DMAIII to rat Hb. The binding site for DMAIII was identified to be cysteine 13 in the alpha-chain of rat Hb with a stoichiometry of 1:1. Over 99% of the total arsenic (maximum 2.5-3.5 mM) in rat RBCs was bound to Hb for all rats examined (n = 138). In contrast, only 40-49% of the total arsenic (maximum approximately 10 muM) in rat plasma was bound to proteins. The ratios of the total arsenic in RBCs to that in plasma ranged from 88-423 for rats that were fed iAsV, 100-680 for rats that were fed MMAV, and 185-1393 for rats that were fed DMAV, when samples were obtained over the 15-week exposure duration. Previous studies have shown an increase in urothelial hyperplasia in rats fed DMAV. This is the first article reporting that treatment with iAsV in the drinking water also produces urothelial hyperplasia and at an even earlier time point than dietary DMAV. Dietary MMAV produced only a slight urothelial response. A correlation between the Hb-DMAIII complex and urothelial lesion severity in rats was observed. The lack of cysteine 13alpha in human Hb may be responsible for the shorter retention of arsenic in human blood. These differences in the disposition of arsenicals may contribute to the observed differences between humans and rats in susceptibility to arsenic carcinogenicity.     

Glutathione-conjugated arsenics in the potential hepato-enteric circulation in rats.

The metabolic pathways for arsenic were precisely studied by determining the metabolic balance and chemical species of arsenic to gain an insight into the mechanisms underlying the animal species difference in the metabolism and preferential accumulation of arsenic in red blood cells (RBCs) in rats. Male Wistar rats were injected intravenously with a single dose of arsenite (iAs(III)) at 2.0 mg of As/kg of body weight, and then the time-dependent changes in the concentrations of arsenic in organs and body fluids were determined. Furthermore, arsenic in the bile was analyzed on anion and cation exchange columns by high-performance liquid chromatography-inductively coupled argon plasma mass spectrometry (HPLC-ICP MS). The metabolic balance and speciation studies revealed that arsenic is potentially transferred to the hepato-enteric circulation through excretion from the liver in a form conjugated with glutathione (GSH). iAs(III) is methylated to mono (MMA)- and dimethylated (DMA) arsenics in the liver during circulation in the conjugated form [iAs(III)(GS)(3)], and a part of MMA is excreted into the bile in the forms of MMA(III) and MMA(V), the former being mostly in the conjugated form [CH(3)As(III)(GS)(2)], and the latter being in the nonconjugated free form. DMA(III) and DMA(V) were not detected in the bile. In the urine, arsenic was detected in the forms of iAs(III), arsenate, MMA(V), and DMA(V), iAs(III) being the major arsenic in the first 6-h-urine, and DMA(V) being increased in the second 6-h-urine. The present metabolic balance and speciation study suggests that iAs(III) is methylated in the liver during its hepato-enteric circulation through the formation of the GSH-cojugated form [iAs(III)(GS)(3)], and MMA(III) and MMA(V) are partly excreted into the bile, the former being in the conjugated form [CH(3)As(III)(GS)(2)]. DMA is not excreted into the bile but into the bloodstream, accumulating in RBCs, and then excreted into the urine mostly in the form of DMA(V) in rats.     

A new metabolic pathway of arsenite: arsenic–glutathione complexes are substrates for human arsenic methyltransferase Cyt19

The metabolism of arsenic is generally accepted to proceed by repetitive reduction and oxidative methylation; the latter is mediated by arsenic methyltransferase (Cyt19). In human urine, the major metabolites of inorganic arsenicals such as arsenite (iAs^III) and arsenate (iAs^V) are monomethylarsonic acid (MMA^V) and dimethylarsinic acid (DMA^V). On the other hand, in rat bile, the major metabolites of iAs^III have been reported to be arsenic–glutathione (As-GSH) complexes. In the present study we investigate whether these As-GSH complexes are substrates for arsenic methyltransferase by using human recombinant Cyt19. Analyses by high-performance liquid chromatography–inductively coupled plasma mass spectrometry suggested that arsenic triglutathione (ATG) was generated nonenzymatically from iAs^III when GSH was present at concentrations 2 mM or higher. Human recombinant Cyt19 catalyzed transfer of a methyl group from S-adenosyl-l-methionine to arsenic and produced monomethyl and dimethyl arsenicals. The methylation of arsenic was catalyzed by Cyt19 only when ATG was present in the reaction mixture. Moreover, monomethylarsonic diglutathione (MADG) was a substrate of Cyt19 for further methylation to dimethylarsinic glutathione (DMAG). On the other hand, monomethylarsonous acid (MMA^III), a hydrolysis product of MADG, was not methylated to dimethyl arsenical by Cyt19. These results suggest that As-GSH complexes such as ATG and MADG were converted by Cyt19 to MADG and DMAG, respectively. Both MADG and DMAG were unstable in solution when the GSH concentration was lower than 1 mM, and were hydrolyzed and oxidized to MMA^V and DMA^V, respectively. Metabolism of iAs^III to methylated arsenicals by Cyt19 was via ATG and MADG rather than by oxidative methylation of iAs^III and MMA^III.     

Stability of arsenic metabolites, arsenic triglutathione [As(GS)3] and methylarsenic diglutathione [CH3As(GS)2], in rat bile

Inorganic arsenicals such as arsenite (iAs(III)) and arsenate (iAs(V)) are well-known human carcinogens. Arsenic is metabolized by repetitive reduction and oxidative methylation, and is excreted mainly in urine as monomethylated arsenicals (MMAs) and dimethylated arsenicals (DMAs). Recently, it has been shown that iAs(III) administered intravenously or orally is excreted into bile as arsenic-glutathione (As-GSH) complexes such as arsenic triglutathione [As(GS)(3)] and methylarsenic diglutathione [CH(3)As(GS)(2)]. In order to carry out the speciation of As-GSH complexes, it is important to understand their stability. The present study was designed to clarify the stability of As-GSH complexes in rat bile, and the role of GSH in stabilizing these complexes. Arsenic species were separated on an anion-exchange column and were analyzed by high-performance liquid chromatography-inductively coupled argon plasma mass spectrometry (HPLC-ICP MS). As(GS)(3) and CH(3)As(GS)(2) were unstable in bile and were hydrolyzed to iAs(III) and monomethylarsonous acid (MMA(III)) in the absence of GSH. As(GS)(3) appeared to be stable in the presence of 10mM GSH. Exogenously added GSH also stabilized CH(3)As(GS)(2) in bile at the concentrations of 5mM or higher. It has been suggested that trivalent arsenicals, especially MMA(III), are more toxic than corresponding pentavalent ones. These results suggest that GSH plays an important role in preventing hydrolysis of As-GSH complexes and the generation of well-known toxic trivalent arsenicals.     

Formation and urinary excretion of arsenic triglutathione and methylarsenic diglutathione

Taking advantage of mice deficient in gamma-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, approximately 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 approximately 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.     

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water

The relationship of exposure and tissue concentration of parent chemical and metabolites over prolonged exposure is a critical issue for chronic toxicities mediated by metabolite(s) rather than parent chemical alone. This is an issue for AsV because its trivalent metabolites have unique toxicities and relatively greater potency compared to their pentavalent counterparts for many endpoints. In this study, dose-dependency in tissue distribution and urinary excretion for inorganic arsenic and its methylated metabolites was assessed in female C57Bl/6 mice exposed to 0, 0.5, 2, 10 or 50 ppm arsenic (as arsenate, AsV) in their drinking water for 12 weeks. No adverse effects were observed and body weight gain did not differ significantly among groups. Urinary excretion of arsenite monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), dimethylarsinic acid (DMAV), and trimethylarsine oxide (TMAO) increased linearly with dose, whereas AsV and monomethylarsonic acid (MMAV) excretion was non-linear with respect to dose. Total tissue arsenic accumulation was greatest in kidney > lung > urinary bladder > skin > blood > liver. Monomethyl arsenic (MMA, i.e. MMA(III)+MMAV) was the predominant metabolite in kidney, whereas dimethylarsenic (DMA, i.e., DMA(III)+DMAV) was the predominant metabolite in lung. Urinary bladder tissue had roughly equivalent levels of inorganic arsenic and dimethylarsenic, as did skin. These data indicate that pharmacokinetic models for arsenic metabolism and disposition need to include mechanisms for organ-specific accumulation of some arsenicals and that urinary metabolite profiles are not necessarily reflective of target tissue dosimetry.     

Decreased enzyme activity of hepatic thioredoxin reductase and glutathione reductase in rabbits by prolonged exposure to inorganic arsenate

Chronic exposure of humans to inorganic arsenic, mainly pentavalent arsenate (iAsV), results in drinking water-induced oxidative stress (Pi et al., 2002). Thioredoxin reductase (TR) and glutathione reductase (GR) are the two critical enzymes in the response to oxidative stress in vivo. In the present study we examined alterations in enzyme activities of hepatic TR and GR from prolonged exposure of male New Zealand white rabbits to iAsV. Exposure of rabbits to iAsV in drinking water (5 mg/L) for 18 weeks caused a significant suppression of hepatic TR and GR activities, of approximately 30% and 20%, respectively, below controls. In vitro experiments suggested that trivalent inorganic arsenic (iAsIII) but not pentavalent arsenicals including iAsV, monomethylarsonic acid (MMAsV), and dimethylarsinic acid (DMAsV) affected the hepatic TR activity of rabbit. So it was suggested that in the present study iAsV ingested via drinking water was metabolized to reactive trivalent arsenicals, such as iAsIII, which may play an important role in the decreased TR and GR activities from prolonged exposure to iAsV observed in vivo.     

Distributions and chemical forms of arsenic after intravenous administration of dimethylarsinic and monomethylarsonic acids to rats

The observed toxicity of arsenic is highly dependent on animal species and differences in metabolism. Rats are one of the most tolerant species, and the metabolic pathway is quite different in some aspects from those of other mammals. The distinct metabolic pathway including the preferential accumulation in red blood cells (RBCs) has been explained, whereby allowing an effective use of rats as an animal model for the arsenic metabolism. In the present study, distributions of arsenic among organs/tissues/body fluids and their chemical forms were studied after intravenous injection of arsenic in the forms of dimethylarsinic (DMA(V)) and monomethylarsonic acids (MMA(V)) to rats. DMA(V) and MMA(V) were mostly excreted into urine immediately after the injection as the intact forms, and both forms were taken up less effectively by organs/tissues than arsenite. The methylated arsenics distributed in organs/tissues were excreted directly into urine and excreted before being redistributed in RBCs. DMA(V) and MMA(V) taken up by the liver were transformed to metabolites not yet identified, accumulated transiently in the liver, and then they disappeared from the liver. The unidentified metabolites were assumed to be transformed from dimethylarsinic acid (DMA(III)) following the consecutive metabolic reactions [MMA(V) --> monomethylarsonous acid (MMA(III)) --> DMA(V) --> DMA(III)]. The unidentified metabolites were excreted not into the bile but into the bloodstream. Injections of DMA(V) and MMA(V) induced a biliary excretion of arsenic but only at 0.2-0.3% of the dose, the arsenic in the bile being their intact free forms.     

Mechanism of arsenic-induced neurotoxicity may be explained through cleavage of p35 to p25 by calpain.

In recent studies we have demonstrated that arsenic (As) metabolites change the composition of neuronal cytoskeletal proteins in vivo and in vitro. To further examine the mechanism of arsenic-induced neurotoxicity with various arsenate metabolites (iAsV, MMAV and DMAV) and arsenite metabolites (iAsIII, MMAIII and DMAIII), we investigated the role of the proteolytic enzyme calpain and its involvement in the cleavage of p35 protein to p25, and also mRNA expression levels of calpain, cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3 beta (gsk3ss). A HeLa cell line transfected with a p35 construct (HeLa-p35) was used as a model, since all other proteins such as calpain, CDK5 and GSK3beta are already present in HeLa cells as they are in neuronal cells. HeLa-p35 cells were incubated with various As metabolites and concentrations of 0, 10 and 30 microM for duration of 4 h. Subsequently the cells were either lysed to study their relative quantification levels of these genes or to be examined on their p35-protein expression. P35-RNA expression levels were significantly (p<0.01) increased by arsenite metabolites, while p35 protein was cleaved to p25 (and p10) after incubation with these metabolites. The cleavage of p35 is caused by calcium (Ca2+) induced activation of calpain. Inhibition of calpain activity by calpeptin prevents cleavage of p35 to p25. These results suggest that cleavage of p35 to p25 by calpain, probably As-induced Ca2+-influx, may explain the mechanism by which arsenic induces its neurotoxic effects.     

Biliary and urinary excretion of inorganic arsenic: monomethylarsonous acid as a major biliary metabolite in rats.

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 micromol/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.     

Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells

Biomethylation is considered a major detoxification pathway for inorganic arsenicals (iAs). According to the postulated metabolic scheme, the methylation of iAs yields methylated metabolites in which arsenic is present in both pentavalent and trivalent forms. Pentavalent mono- and dimethylated arsenicals are less acutely toxic than iAs. However, little is known about the toxicity of trivalent methylated species. In the work reported here the toxicities of iAs and trivalent and pentavalent methylated arsenicals were examined in cultured human cells derived from tissues that are considered a major site for iAs methylation (liver) or targets for carcinogenic effects associated with exposure to iAs (skin, urinary bladder, and lung). To characterize the role of methylation in the protection against toxicity of arsenicals, the capacities of cells to produce methylated metabolites were also examined. In addition to human cells, primary rat hepatocytes were used as methylating controls. Among the arsenicals examined, trivalent monomethylated species were the most cytotoxic in all cell types. Trivalent dimethylated arsenicals were at least as cytotoxic as trivalent iAs (arsenite) for most cell types. Pentavalent arsenicals were significantly less cytotoxic than their trivalent analogs. Among the cell types examined, primary rat hepatocytes exhibited the greatest methylation capacity for iAs followed by primary human hepatocytes, epidermal keratinocytes, and bronchial epithelial cells. Cells derived from human bladder did not methylate iAs. There was no apparent correlation between susceptibility of cells to arsenic toxicity and their capacity to methylate iAs. These results suggest that (1) trivalent methylated arsenicals, intermediary products of arsenic methylation, may significantly contribute to the adverse effects associated with exposure to iAs, and (2) high methylation capacity does not protect cells from the acute toxicity of trivalent arsenicals.     

Biliary excretion of arsenic in rats,rabbits, and dogs

The disappearance of ⁷⁴As from blood and plasma of rats and its excretion into bile was measured for 2 hr after the iv administration of 0.01, 0.46, 1.0, 2.1, and 4.6 mg/kg of arsenic given as the trichloride. Arsenic disappearance from plasma was biphasic; the half-life during the late phase was greater than 2 hr. Even though the arsenic was injected iv, the concentration in the blood increased through the first 2 hr. Arsenic was rapidly excreted into the bile, reaching its highest rate of excretion 6 min after administration, after which it rapidly decreased. This rapid decrease in excretion is due to redistribution of arsenic from the liver to the blood. Arsenic enters bile against an apparent bile/plasma concentration gradient of 630, 8 min after 1 mg/kg of arsenic. At this time the liver/plasma gradient is 17 and the liver/bile gradient is 37. Twenty-five percent of the arsenic administered to bile duct-cannulated rats is excreted into the bile within 2 hr. However, less than 10% of the administered dose is excreted into the feces of intact rats over a 7-day period. In the rabbit and dog, arsenic is excreted into the bile at a much slower rate. These data demonstrate that arsenic is excreted into the bile, and this occurs against a large bile/plasma concentration gradient in rats, suggesting excretion by an active transport mechanism. However, the overall importance of bile as a route of elimination for arsenic is minimized due to enterohepatic circulation and species variations in its biliary excretion rate.     

Arsenic metabolism and thioarsenicals in hamsters and rats

The tissue distribution and chemical forms of arsenic were compared in two animal species with different metabolic capacity and toxicity to arsenic. Hamsters and rats were given a single oral dose of arsenite (iAsIII) at 5.0 mg As/kg body weight, and then the concentrations of arsenic were determined; more than 75% of the dose accumulated in rat red blood cells (RBCs) in the form of dimethylarsinous acid (DMAIII), whereas less than 0.8% of the dose accumulated in hamster RBCs, mostly in the form of monomethylarsonous acid (MMAIII). Reflecting the low accumulation in RBCs, more than 63% of the dose was recovered in hamster urine within one week (7.8-fold higher than that in rat urine). The quantity of arsenic distributed in the liver and kidneys was significantly higher in hamsters than in rats, and arsenic in livers stayed much longer in hamsters than in rats. Arsenic accumulated more and was retained longer in the kidneys than in the livers in both animals, and in hamster kidneys, it accumulated at levels higher than those in rat kidneys in the form of MMAIII bound to proteins. In the first 24 h urine, dimethylmonothioarsinic (DMMTAV) and dimethyldithioarsinic (DMDTAV) acids were detected in hamsters, but only DMMTAV was found in rats, together with an unknown arsenic metabolite in both animals. The unknown urinary arsenic metabolite was identified as monomethylmonothioarsonic acid (MMMTAV; CH3As(=S)(OH)2). The present results indicate that in hamsters, arsenic does not accumulate in RBCs, and therefore, hamsters exhibit a more uniform tissue distribution and faster urinary excretion of arsenic than rats. In addition, arsenic was thiolated more in hamsters than in rats excreting mono and dimethylated thioarsenicals in urine.     

Effects of exogenous GSH and methionine on methylation of inorganic arsenic in mice exposed to arsenite through drinking water

We hypothesized that chronic exposure to arsenic would deplete the reduced glutathione (GSH) and methionine in vivo, thereby impair the methylation capacity of inorganic arsenic (iAs) ingested. Our experiment was designed to explore the effects of exogenous GSH and methionine on arsenic methylation in mice exposed to arsenite via drinking water. Levels of iAs, monomethylarsenic acid (MMAs), and dimethylarsenic acid (DMAs) in the liver and blood were determined by the method of hydride generation coupled with atomic absorption spectrophotometry. Compared with mice exposed to arsenite alone, administration of GSH or methionine increased the secondary methylation index in the liver and primary methylation index in the blood, which resulted in the consequent increase of DMAs percent and decrease of iAs percent in the blood. Moreover, administration of GSH resulted in the increase of DMAs percent in the liver and total arsenic in the blood. Increase of total arsenic in the blood was mainly due to the increase of DMAs. Findings from the present study suggested that administration of GSH or methionine might potentiate the methylation capacity of arsenic in both liver and extrahepatic tissues, which may facilitate the excretion of arsenic and decrease arsenic related toxicities in the body. © 2009 Wiley Periodicals, Inc. Environ Toxicol 25: 361–366, 2010.     

Role of glutathione in the biliary excretion of the arsenical drugs trimelarsan and melarsoprol

After administration of the inorganic sodium arsenite or arsenate to rats, the biliary excretion of arsenic is rapid, is accompanied by the biliary output of large amounts of GSH, and is completely arrested by the GSH depletor diethyl maleate (DEM). We studied the biliary excretion of trimelarsan (TMA) and melarsoprol (MAP) in rats in order to determine whether biliary excretion is also significant in the disposition of these trivalent organic arsenicals that are used as therapeutic agents and whether GSH is also involved in their hepatobiliary transport. After injection of either drug (100 micromol/kg, i.v.), arsenic was rapidly excreted in bile (up to 1 micromol/kg. min, approximately 55% of dose/100 min). Concurrently, TMA and MAP increased the biliary output of GSH 3- and 6 fold, and lowered the hepatic GSH content by 24% and 27%, respectively. In TMA-injected rats, pretreatment with DEM or buthionine sulfoximine decreased the initial biliary excretion of arsenic by 75% and 40%, respectively, whereas in MAP-injected rats these GSH depletors diminished arsenic output by 45% and 20%. Both arsenicals reacted with GSH in vitro, giving rise to the same product, which was also shown by HPLC analysis to be a major biliary metabolite of both TMA and MAP. This metabolite was sensitive to gamma-glutamyltranspeptidase in vitro and its biliary excretion was virtually prevented by the GSH depletors, confirming that it is a GSH conjugate (purportedly melarsen-diglutathione). Some TMA was excreted in the bile unchanged, whereas a significant amount of MAP also appeared there as two glucuronides. The biliary excretion of unchanged TMA and MAP glucuronides was increased by experimental depletion of GSH. These studies indicate that the biliary excretion of TMA and MAP (1) is very significant in their disposition, (2) is partially dependent on the hepatic availability of GSH, as these arsenicals are excreted in part as a GSH conjugate, and (3) is concomitant with the increased appearance of GSH in bile, probably originating from dissociation of the unstable GSH conjugate of these arsenicals. Thus, conjugation with GSH is important in the elimination of both TMA and MAP, although glucuronidation is also involved in the fate of MAP.     

Metabolism and excretion of orally administrated arsenic trioxide in the hamster

It was shown that a single dose of arsenic trioxide administered to hamsters was chiefly methylated in vivo into methylarsonic acid (MAA) and dimethylarsinic acid (DMAA), and that inorganic arsenic accounted for the major portion of total arsenic that deposited in organs and tissues, followed by MAA and DMAA in decreasing sequence of significance. The single oral dose of arsenic trioxide was followed by a very small amount of trimethylarsenic compounds (TMA) occurring only in the liver but not in any other organs, tissues, blood or feces. The distribution pattern of arsenic in the blood following the single oral dose of arsenic trioxide was such that inorganic arsenic and MAA occurred chiefly in the blood cells; DMAA, chiefly in the plasma; and the arsenic compounds disappeared rapidly from blood. The single oral dose of arsenic trioxide was further followed by excretion of an amount of arsenic equivalent to about 60% of the administered dose: 49% in the urine and 11% in the feces. In other words, more arsenic tended to be excreted in the urine. DMAA accounted for the major portion of arsenic excreted in the urine and feces, and this finding re-confirmed that DMAA is the major metabolite of arsenic trioxide. Although it is believed that arsenic trioxide is not converted into TMA, the results of the present study suggest that a very small amount of arsenic trioxide is converted into TMA in the liver.     

Arsenic speciation in urine from acute promyelocytic leukemia patients undergoing arsenic trioxide treatment

Arsenic has been used successfully in clinical trials for treating acute promyelocytic leukemia (APL). Although sublethal doses of inorganic arsenic are used, little is known about the pharmacokinetics and metabolism of the high levels of arsenic in APL patients. To fill this important gap, this study describes the speciation of arsenic in urine from four APL patients treated with arsenic. Each patient was injected daily with an arsenite (As(III)) solution that contained 10 mg of As(2)O(3) precursor. Speciation analysis of the patient urine samples collected consecutively for 48 h, encompassing two intravenous injections of arsenic, revealed the presence of monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), and dimethylarsinic acid (DMA(V)). The intermediate methyl arsenic metabolites, MMA(III) and DMA(III), were detected in most urine samples from all of the patients when a preservative, diethyldithiocarbomate, was added to the urine samples to stabilize these trivalent arsenic species. The major arsenic species detected in the urine samples from the patients were As(III), MMA(V), and DMA(V), accounting for >95% of the total arsenic excreted. The relative proportions of As(III), As(V), MMA(V), and DMA(V) in urine samples collected 24 h after the injections of As(III) were 27.6 +/- 6.1, 2.8 +/- 2.0, 22.8 +/- 8.1, and 43.7 +/- 13.3%, respectively. The relatively lower fraction of the methylated arsenic species in these APL patients under arsenic treatment as compared with that from the general population exposed to much lower levels of arsenic suggests that the high levels of As(III) inhibit the methylation of arsenic (inhibits the formation of methyl arsenic metabolites). The arsenic species excreted into the urine accounted for 32-65% of the total arsenic injected. These results suggest that other pathways of excretion, such as through the bile, may play an important role in eliminating (removing) arsenic from the human body when challenged by high levels of As(III).     

URINARY EXCRETION OF ARSENIC METABOLITES AFTER LONG-TERM ORAL ADMINISTRATION OF VARIOUS ARSENIC COMPOUNDS TO RATS

The metabolism of arsenic compounds in rats was studied by comparing urinary metabolites of arsenic compounds administered for 1 wk or 7 mo. Male F344/DuCrj rats were given 100 mg As/L as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), or arsenobetaine (AsBe), or 10 mg As/L as arsenite \[As(III)] via drinking water for 7 mo. Urine was collected by forced urination after 1 wk or 7 mo. Arsenic metabolites in urine were analyzed by ion chromatography with inductively coupled plasma mass spectrometry. In the case of As(III) ingestion, a small portion of all arsenic excreted in urine (about 6% ) was excreted in inorganic form, while most arsenic was excreted as methylated arsenic metabolites. Following MMA treatments for 1 wk or 7 mo, the predominant products excreted were unchanged MMA and DMA accompanied by small amounts of TMAO and tetramethylarsonium (TeMA). In the case of DMA treatment the urinary compounds found were mainly the parent DMA and TMAO with minute amounts of TeMA. TMAO was methylated to TeMA to a slight extent after 1 wk and 7 mo of administration, although most TMAO was excreted in the form of unchanged TMAO. AsBe was predominantly eliminated in urine without any transformation. Two unidentified metabolites were detected in urine after 7 mo of arsenic species exposure; the amounts of these metabolites increased in the order DMA &gt; MMA &gt; TMAO with only small quantities of these detected in the As(III)-treated group. These results suggest that these unidentified metabolites are formed during a demethylation process, and not during methylation. Our findings indicate that long-term exposure to As(III), MMA, or DMA decreases the proportion of TMAO elimination in urine and increases that of DMA, M-1, and M-2, and that further methylation to TMAO to TeMA does occur to a slight extent following long-term exposure to arsenical compounds in rats.     

Increased biliary excretion of glutathione is generated by the glutathione-dependent hepatobiliary transport of antimony and bismuth.

We have recently demonstrated that the hepatobiliary transport of arsenic is glutathione-dependent and is associated with a profound increase in biliary excretion of glutathione (GSH), hepatic GSH depletion and diminished GSH conjugation (Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 41: 937-944 and Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 42: 465-468, 1991). The present studies in rats aimed to determine whether antimony and bismuth, other metalloids in group Va of the periodic table, also possess similar properties. Antimony potassium tartrate (25-100 mumol/kg, i.v.) and bismuth ammonium citrate (50-200 mumol/kg, i.v.) increased up to 50- and 4-fold, respectively, the biliary excretion of non-protein thiols (NPSH). This resulted mainly from increased hepatobiliary transport of GSH as suggested by a close parallelism in the biliary excretion of NPSH and GSH after antimony or bismuth administration. Within 2 hr, rats excreted into bile 55 and 3% of the dose of antimony (50 mumol/kg, i.v.) and bismuth (150 mumol/kg, i.v.), respectively. The time courses of the biliary excretion of these metalloids and NPSH or GSH were strikingly similar suggesting co-ordinate hepatobiliary transport of the metalloids and GSH. However, at the peak of their excretion, each molecule of antimony or bismuth resulted in a co-transport of approximately three molecules of GSH. Diethyl maleate, indocyanine green and sulfobromophthalein (BSP), which decreased biliary excretion of GSH, significantly diminished excretion of antimony and bismuth into bile indicating that hepatobiliary transport of these metalloids is GSH-dependent. Administration of antimony, but not bismuth, decreased hepatic GSH level by 30% and reduced the GSH conjugation and biliary excretion of BSP. These studies demonstrate that the hepatobiliary transport of trivalent antimony and bismuth is GSH-dependent similarly to the hepatobiliary transport of trivalent arsenic. Proportionally to their biliary excretion rates, these metalloids generate increased biliary excretion of GSH probably because they are transported from liver to bile as unstable GSH complexes. The significant loss of hepatic GSH into bile as induced by arsenic or antimony may compromise conjugation of xenobiotics with GSH.