Monomethylated arsenic was the Major methylated arsenic in Red blood cells of acute promyelocytic leukemia patients treated with arsenic trioxide.

BACKGROUND: Arsenic trioxide (ATO) has been successfully applied in the treatment of acute promyelocytic leukemia (APL). Arsenic metabolites including inorganic arsenic and methylated arsenic could lead to different toxicity and curative effect. This study aims to establish a method to determine arsenic species in red blood cells (RBCs), clarify the distribution characteristics of arsenic species in RBCs. METHODS: Steady state blood samples were collected from 97 APL patients. H2O2 and HClO4 were used to release the hemoglobin bounding arsenic and precipitate protein. Arsenite (iAsIII), arsenate (iAsV), monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) in plasma and RBCs were detected by HPLC-HG-AFS. Free and bound arsenic species in RBCs were separated by 30 kDa molecular mass cutoff filters and determined to evaluate hemoglobin binding capacity of different arsenic species. RESULTS: The method was validated with accuracy ranged from 84.75% to 104.13%. Arsenic species in RBCs followed the trend iAs > MMA > DMA (p < 0.01), while the concentration of DMA was significantly higher than iAs and MMA in plasma (p < 0.01). The correlation between iAs concentration in plasma and corresponding RBCs arsenic level was weak. And the concentrations of DMA and MMA in plasma were moderately positive correlated with those in RBCs. Hemoglobin-binding ratios of iAs, MMA and DMA were all over 70 %. CONCLUSIONS: In this study, we provided a reliable method to determine arsenic species in RBCs of APL patients treated with ATO by HPLC-HG-AFS. It was confirmed that the concentration of DMA is the highest in plasma, while MMA is the most predominant methylated arsenic in RBCs. High affinity of MMA with human Hb was responsible for the accumulation of arsenic in RBCs of APL patients.

Time course of arsenic species in red blood cells of acute promyelocytic leukemia (APL) patients treated with single agent arsenic trioxide.

BACKGROUND: Arsenic trioxide (ATO) is widely applied to treat acute promyelocytic leukemia (APL). To elucidate metabolism and toxicity of arsenic, we analyzed time course of arsenic species in red blood cells (RBCs) of APL patients. METHODS: Nine APL patients received ATO (0.16 mg/kg/day) through 18-h infusion. Blood was collected before daily administration (days 2 to 9), and at different time points on day 8. Inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were detected by HPLC-ICP-MS. RESULTS: Arsenic species reached Cmax at 18 h on day 8. Arsenicals gradually accumulated during days 2 to 9, whereas their percentages remained almost constant. The general trend in red blood cells (RBCs) was iAs > MMA > DMA. MMA was consistently the predominant methylated arsenic metabolite in RBCs. iAs, MMA, and tAs (tAs = iAs + DMA + MMA) concentrations (P < 0.0001), MMA/DMA ratios (P = 0.0016) and iAs% (P = 0.0013) were higher in RBCs than in plasma. CONCLUSIONS: Time course of arsenic species reveal kinetic characteristic of ATO metabolites in RBCs. Arsenic species accumulated with administration frequency. Arsenic species in RBCs were remarkably different from those in plasma. Time course of arsenic species in RBCs is important in ATO clinical application.

Pharmacokinetic Properties of Arsenic Species after Intravenous and Intragastrical Administration of Arsenic Trioxide Solution in Cynomolgus Macaques Using HPLC-ICP-MS.

A rapid and sensitive method was established for arsenic (As) speciation based on high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). This method was validated for the quantification of four arsenic species, including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) in cynomolgus macaque plasma. Separation was achieved in just 3.7 min with an alkyl reverse phase column and highly aqueous mobile phase containing 20 mM citric acid and 5 mM sodium hexanesulfonate (pH = 4.3). The calibration curves were linear over the range of 5⁻500 ng·mL-1 (measured as As), with r > 0.99. The above method was validated for selectivity, precision, accuracy, matrix effect, recovery, carryover effect and stability, and applied in a comparative pharmacokinetic study of arsenic species in cynomolgus macaque samples following intravenous and intragastrical administration of arsenic trioxide solution (0.80 mg·kg-1; 0.61 mg·kg-1 of arsenic); in addition, the absolute oral bioavailability of the active ingredient AsIII of arsenic trioxide in cynomolgus macaque samples was derived as 60.9 ± 16.1%.

Association between arsenic exposure and thyroid function: data from NHANES 2007-2010.

The association of arsenic variables in urine, total arsenic (UAS), arsenobetaine (UAB), dimethylarsinic acid (UDMA), and arsenic adjusted for arsenobetaine (UAAS) with thyroid-stimulating hormone (TSH), free and total serum thyroxine (FT4, TT4), free and total triiodothyronine (FT3, TT3), and thyroglobulin (TGN) was evaluated by analyzing data from 2007-2010 National Health and Nutrition Examination Survey. For iodine deficient males, there was a positive association between TSH and UDMA (p < 0.01) and a negative association between the levels of TT4 and UDMA (p < 0.01). Levels of UAAS were inversely associated with the levels of TT4 for both iodine-deficient (p = 0.054) and iodine-replete females (p < 0.01). For iodine-replete females, levels of both TSH and TGN increased with decrease in the levels of both UAB (p < 0.01) and UAS (p < 0.01). There was also a negative association between TSH and UAB as well as UAS (p < 0.01). For iodine-replete males, increased levels of UDMA were associated with decreasing levels of FT4 (p = 0.03).

[Arsenic speciation in rat plasma after oral administration of realgar and Niu Huang Jie Du Pian].

The arsenic species in rat plasma were studied after oral administration of realgar and Niu Huang Jie Du Pian (NHJDP) and the possible compatible effects of realgar was evaluated by comparing the pharmacokinetics of arsenic species after administration of realgar and NHJDP. The separation of the arsenicals was performed by a high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) technique. Dimethylarsinic acid (DMA) was found to be the main species in rats' plasma after dosing. No traces of arsenite [As(Ⅲ)], monomethylarsonic acid (MMA) or arsenate [As(Ⅴ)] were detected at any sampling time points. Compared with realgar administration alone, dose-normalized peak concentration(C(max)) and AUC(0-t) of DMA were significantly decreased by NHJDP administration, while the t(max) was significantly delayed with the clearance and apparent volume of distribution significantly increased, indicating that the pharmacokinetics of As from realgar was affected by other ingredients in the compound prescription of NHJDP.

Renal function is associated with indicators of arsenic methylation capacity in Bangladeshi adults.

BACKGROUND: Arsenic (As) methylation capacity in epidemiologic studies is typically indicated by the proportions of inorganic As (%InAs), monomethylarsonic acid (%MMA), and dimethylarsinic acid (%DMA) in urine as a fraction of total urinary As. The relationship between renal function and indicators of As methylation capacity has not been thoroughly investigated. OBJECTIVES: Our two aims were to examine (1) associations between estimated glomerular filtration rate (eGFR) and %As metabolites in blood and urine, and (2) whether renal function modifies the relationship of blood %As metabolites with respective urinary %As metabolites. METHODS: In a cross-sectional study of 375 As-exposed Bangladeshi adults, we measured blood and urinary As metabolites, and calculated eGFR from plasma cystatin C. RESULTS: In covariate-adjusted linear models, a 1 ml/min/1.73 m(2) increase in eGFR was associated with a 0.39% increase in urinary %InAs (p<0.0001) and a mean decrease in urinary %DMA of 0.07 (p=0.0005). In the 292 participants with measurable blood As metabolites, the associations of eGFR with increased blood %InAs and decreased blood %DMA did not reach statistical significance. eGFR was not associated with urinary or blood %MMA in covariate-adjusted models. For a given increase in blood %InAs, the increase in urinary %InAs was smaller in those with reduced eGFR, compared to those with normal eGFR (p=0.06); this effect modification was not observed for %MMA or %DMA. CONCLUSIONS: Urinary excretion of InAs may be impaired in individuals with reduced renal function. Alternatively, increased As methylation capacity (as indicated by decreased urinary %InAs) may be detrimental to renal function.

Proposal for novel metabolic pathway of highly toxic dimethylated arsenics accompanied by enzymatic sulfuration, desulfuration and oxidation.

The International Agency for Research on Cancer (IARC) has concluded that dimethylarsinic acid [(CH3)2AsO(OH), DMA(V)], a main metabolite of inorganic arsenic, is responsible for carcinogenesis in urinary bladder and lung in rodents, and various modes of carcinogenic action have been proposed. One theory concerning the mode of action is that the biotransformation of dimethylarsinous acid [(CH3)2AsOH, DMA(III)] from DMA(V) plays an important role in the carcinogenesis by way of reactive oxygen species (ROS) production. Furthermore, dimethylmonothioarsinic acid [(CH3)2AsS(OH), DMMTA(V)], a metabolite of DMA(V), has also been noted because of its higher toxicity. However, the metabolic mechanisms of formation and disappearance of DMA(III) and DMMTA(V), and their toxicity are not fully understood. Thus, the purpose of the present study was to clarify the mechanism of metabolic formation of DMMTA(V) and DMA(V) from DMA(III). The in vitro transformation of arsenicals by treatment with liver homogenate from rodents and sulfur transferase was detected by HPLC-ICP-MS and HPLC-tandem MS. DMMTA(V) is produced from DMA(III) but not DMA(V) by cellular fractions from mouse liver homogenates and by rhodanese from bovine liver in the presence of thiosulfate, a sulfur donor. Not only DMMTA(V) thus produced but also DMA(III) are re-converted into DMA(V) by an in vitro addition of S9 mix. These findings indicate that the metabolic process not only of DMA(III) to DMA(V) or DMMTA(V) but also of DMMTA(V) to DMA(V) consists of a complicated mode of interaction between monooxygenase including cytochrome P450 (CYP) and/or sulfur transferase.

Effects of exogenous methionine on arsenic burden and NO metabolism in brain of mice exposed to arsenite through drinking water.

The aim of this study was to explore the effects of exogenous methionine (Met) on arsenic burden and metabolism of nitric oxide (NO) in the brain of mice exposed to arsenite via drinking water. Mice were exposed to sodium arsenite through drinking water contaminated with 50 mg/L arsenic for four consecutive weeks, and treated intraperitoneally with saline solution, 100 mg/kg body weight (b.w), 200 mg/kg b.w or 400 mg/kg b.w of Met, respectively at the fourth week. Levels of inorganic arsenic (iAs), monomethylarsenic acid (MMAs), and dimethylarsenic acid (DMAs) in the liver, blood and brain were determined by method of hydride generation coupled with atomic absorption spectrophotometry. Nitric oxide synthase (NOS) activities and NO levels in the brain were determined by colorimetric method. Compared with mice exposed to arsenite alone, administration of Met increased significantly the primary methylation ratio in the liver, which resulted in decrease of percent iAs and increase of percent DMAs in the liver, and decrease of iAs, MMAs and total arsenic levels (TAs) in the blood and DMAs and TAs in the brain. NOS activities and NO levels in the brain of mice exposed to arsenite alone were significantly lower than those in control, however administration of Met could increase significantly NO levels. Findings from this study suggested that exogenous Met could benefit the primary arsenic methylation in the liver, which might increase the production of methylated arsenicals and facilitate arsenic excretion. As a consequence, arsenic burden in both blood and brain was reduced, and toxic effects on NO metabolism in the brain were ameliorated.

Arsenic methylation capacity and its correlation with skin lesions induced by contaminated drinking water consumption in residents of chronic arsenicosis area.

Chronic exposure to excess level of arsenic through contaminated drinking water is associated with many injuries, among which skin lesions are the most prominent. In this study, we measured the concentrations of inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in the blood of the residents of arsenicosis area, who demonstrated different skin lesion grade from mild, moderate to advanced. We evaluated the individual methylation capacity by two indices of the first and secondary methylation ratio (FMR and SMR). We found that SMR of moderate and advanced groups were markedly lower than that of mild group. Significant negative correlation was found between SMR of all the subjects and the grade of skin lesion, with Spearman's correlation coefficient of -0.429 (P = 0.016). Moreover, blood MMA proportion of moderate and advanced groups was found to be significantly higher than that of the mild group. These results suggest that low secondary arsenic methylation capacity and high MMA proportion are associated with the severity of arsenic-related skin lesions. Our findings evaluated by blood speciation is consistent with that evaluated by the generally accepted urinary arsenic speciation in the relationship between arsenic methylation capacity and arsenic-related lesions.

Distribution and metabolism of four different dimethylated arsenicals in hamsters.

Arsenic toxicity and distribution are highly dependent on animal species and its chemical species. Recently, thioarsenical has been recognized in highly toxic arsenic metabolites, which was commonly found in human and animal urine. In the present study, we revealed the mechanism underlying the distribution and metabolism of non-thiolated and thiolated dimethylarsenic compounds such as dimethylarsinic acid (DMA(V)), dimethylarsinous acid (DMA(III)), dimethylmonothioarsinic acid (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) after the administration of them into femoral vein of hamsters. DMA(V) and DMDTA(V) distributed in organs and body fluids were in their unmodified form, while DMA(III) and DMMTA(V) were bound to proteins and transformed to DMA(V) in organs. On the other hand, DMA(V) and DMDTA(V) were mostly excreted into urine as their intact form 1 h after post-injection, and more than 70% of the doses were recovered in urine as their intact form. By contrast, less than 8-14% of doses were recovered in urine as DMA(V), while more than 60% of doses were distributed in muscles and target organs (liver, kidney, and lung) of hamsters after the injection of DMMTA(V) and DMA(III). However, in red blood cells (RBCs), only a small amount of the arsenicals was distributed (less than 4% of the doses) after the injection of DMA(III) and DMMTA(V), suggesting that the DMA(III) and DMMTA(V) were hardly accumulated in hamster RBCs. Based on these observations, we suggest that although DMMTA(V) and DMDTA(V) are thioarsenicals, DMMTA(V) is taken up efficiently by organs, in a manner different from that of DMDTA(V). In addition, the distribution and metabolism of DMMTA(V) are like in manner similar to DMA(III) in hamsters, while DMDTA(V) is in a manner similar to DMA(V).

Identification of the major arsenic-binding protein in rat plasma as the ternary dimethylarsinous-hemoglobin-haptoglobin complex.

Chronic exposure to arsenic causes a wide range of diseases such as hyperkeratosis, cardiovascular diseases, and skin, lung, and bladder cancers, and millions of people are chronically exposed to arsenic worldwide. However, little is known about the mechanisms underlying these toxic actions. The metabolism of arsenic is essential for understanding the toxic actions. Here, we identified the major arsenic-binding protein (As-BP) in the plasma of rats after oral administration of arsenite by the use of two different HPLC columns, gel filtration and anion exchange ones, coupled with an inductively coupled argon plasma mass spectrometer (ICP MS). The molecular mass of the As-BP was estimated to be 90 kDa based on results using the former column, and arsenic bound to this protein only in the form of dimethylarsinous acid (DMA (III)) in the plasma in vivo. In addition, the purified As-BP was shown to consist of two different proteins, haptoglobin (Hp) of 37 kDa (three bands) and the hemoglobin (Hb) alpha chain of 14 kDa (single band), using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), respectively, suggesting that the As-BP was the ternary DMA (III)-Hb-Hp complex. To confirm the present observations, an arsenic-binding assay was carried out in vitro . Although DMA (III) bound directly to fresh rat plasma proteins, they were different from that identified in vivo. However, when a DMA (III)-exposed rat RBC lysate (DMA (III) binds to Hb in rat RBCs) was added to control rat plasma, a new arsenic peak increased at the expense of the arsenic-Hb one. Furthermore, this new arsenic peak was consistent with the As-BP identified in the plasma in vivo, suggesting that arsenic bound to Hb further binds to haptoglobin (Hp), forming the ternary As-Hb-Hp complex.

Impact of arsenic in foodstuffs on the people living in the arsenic-affected areas of West Bengal, India.

Although the accumulation of arsenic (As) in human blood is linked with some diseases and with occupational exposure, there are few reports on speciation of As in blood. On the basis of our earlier article, elevated level of arsenicals in human urine and blood were found in the ex-exposed population via As-containing drinking water. The aim of the present study was to get an insight on impact of As in foodstuffs on the people living in the As-affected areas. Moreover, speciation of arsenicals in urine, and water-samples found in arsenobetaine (AsB). Since sampling population (n=25) was not taking any seafood, As in foodstuffs was thought to be the prime source for this discrepancy. So, speciation of methanol extract of freeze-dried red blood cells (RBCs) and foodstuffs, and trichloro acetic acid (TCA) treated plasma by high performance liquid chromatography-inductively coupled argon plasma mass spectrometer (HPLC-ICP MS) collected from the study population (n=33) was carried out to support our hypothesis. Results showed that urine contained AsB (1.7%), arsenite (iAs(III)) (14.3), arsenate (iAs(V)) (4.9), monomethylarsonous acid (MMA(III)) (0.64), monomethylarsonic acid (MMA(V)) (13.6), dimethylarsinous acid (DMA(III)) (7.7), and dimethylarsinic acid (DMA(V)) (65.4). Blood contained 21.3 microg L(- 1) (mean) As and of which 27.3% was in plasma and 72.7% in RBCs. RBCs contained AsB (21.6%) and DMA(V) (78.4) and blood plasma contained AsB (12.4%), iAs(III) (25.9), MMA(V) (30.3), and DMA(V) (31.4). Furthermore, speciation of As in foodstuffs showed that most of them contained AsB (3.54-25.81 microg kg(- 1)) (25.81-312.44 microg kg(- 1)) along with iAs(III) (9.62-194.93), iAs(V) (17.63-78.33), MMA(V) (9.47-73.22) and DMA(V) (13.43-101.15) that supported the presence of AsB and elevated As in urine and blood samples of the present study group. Inorganic As (iAs) predominates in rice (67.17-86.62%) and in spices (40-90.35%), respectively over organic As. So, As in the food chain is a real threat to human health.

In vivo assessment of arsenic bioavailability in rice and its significance for human health risk assessment.

BACKGROUND: Millions of people worldwide consume arsenic-contaminated rice; however, little is known about the uptake and bioavailability of arsenic species after arsenic-contaminated rice ingestion. OBJECTIVES: In this study, we assessed arsenic speciation in greenhouse-grown and supermarket-bought rice, and determined arsenic bioavailability in cooked rice using an in vivo swine model. RESULTS: In supermarket-bought rice, arsenic was present entirely in the inorganic form compared to greenhouse-grown rice (using irrigation water contaminated with sodium arsenate), where most (approximately 86%) arsenic was present as dimethylarsinic acid (organic arsenic). Because of the low absolute bioavailability of dimethylarsinic acid and the high proportion of dimethylarsinic acid in greenhouse-grown rice, only 33 +/- 3% (mean +/- SD) of the total rice-bound arsenic was bioavailable. Conversely, in supermarket-bought rice cooked in water contaminated with sodium arsenate, arsenic was present entirely in the inorganic form, and bioavailability was high (89 +/- 9%). CONCLUSIONS: These results indicate that arsenic bioavailability in rice is highly dependent on arsenic speciation, which in turn can vary depending on rice cultivar, arsenic in irrigation water, and the presence and nature of arsenic speciation in cooking water. Arsenic speciation and bioavailability are therefore critical parameters for reducing uncertainties when estimating exposure from the consumption of rice grown and cooked using arsenic-contaminated water.

Effect of selenite on the disposition of arsenate and arsenite in rats.

Selenite (SeIV) and inorganic arsenicals counter the toxicity of each other. SeIV inhibits arsenic methylation in hepatocytes, however, it is unknown whether it decreases the formation of the highly toxic monomethylarsonous acid (MMAsIII). Therefore, we examined, in comparison with the methylation inhibitor periodate-oxidised adenosine (PAD), the effect of SeIV (10 micromol/kg, i.v.) on the appearance of arsenic metabolites in blood, bile and urine as well as the distribution of arsenic metabolites in the liver and kidneys in rats injected i.v. with 50 micromol/kg arsenite (AsIII) or arsenate (AsV). Arsenic metabolites were analysed by HPLC-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). In rats given either arsenical, PAD decreased the excretion and tissue concentrations of methylated arsenic metabolites (MMAsIII, monomethylarsonic acid [MMAsV], and dimethylarsinic acid [DMAsV]), while increasing the tissue retention of AsV and AsIII. The effect of SeIV on arsenic disposition differed significantly from that of PAD. For example, both in AsIII- and AsV-injected animals, SeIV lowered the tissue levels of MMAsIII and MMAsV, but increased the levels of DMAsV. SeIV almost abolished the biliary excretion of MMAsIII in AsV-exposed rats, but barely influenced it in AsIII-dosed rats. The SeIV-induced changes in arsenic disposition may largely be ascribable to formation of the known complex containing trivalent arsenic and selenide (SeII), which not only depends on but also influences the availability and effects of these metalloid species in tissues. By such complexation SeII compromises monomethylation of arsenic when trivalent arsenic availability is limited (e.g. in AsV-exposed rats), but affects it less when the presence of AsIII is overwhelming (e.g. in AsIII-dosed rats). As an auxiliary finding, it is shown that DMAsV occurs in the blood of rats not injected with arsenic and that DMAsV formation in rats can be followed by measuring the build-up of blood-borne DMAsV.

Dose-dependent biotransformation of arsenite in rats--not S-adenosylmethionine depletion impairs arsenic methylation at high dose.

Arsenite (AsIII) is eliminated via excretion and methylation. Monomethylarsonous acid (MMAsIII) is a super toxic metabolite of AsIII, while dimethylarsinic acid produced in the next metabolic step is relatively atoxic. Since the role of methylation in the acute toxicity and elimination of AsIII in vivo is unclear, we have examined the excretion and tissue retention of AsIII and its metabolites in rats exposed to increasing AsIII doses. Rats were injected i.v. with 20, 50 and 125 micromol/kg AsIII and arsenic metabolites in bile, urine and tissues were analysed. The excretion of AsIII increased almost proportionately to the dose, while its concentration in tissues rose more than proportionately. In contrast, the excretion and tissue concentrations of methylated metabolites increased less than the dosage, or they even decreased after injection of the largest dose of AsIII. To elucidate the mechanism of the dose-dependent decrease of methylation, we quantified S-adenosylmethionine (SAME), glutathione (GSH), and adenine nucleotides in the liver of AsIII-injected rats. AsIII decreased the hepatic concentrations of GSH and adenosine 5'-triphosphate (ATP) and the energy charge in a dose-dependent manner, but increased the level of SAME. Thus, impaired methylation after AsIII overdose is not due to SAME shortage, but probably to methyltransferase inhibition. It appears that exhausted elimination capacity of AsIII, rather than MMAsIII produced from AsIII, contributes significantly to the acute toxicity of AsIII. After GSH depletion the retained AsIII can increasingly inhibit SH-enzymes, thus causing ATP depletion and energetic disorder.

Chemical speciation of arsenic in serum of uraemic patients.

Chemical speciation of arsenic was carried out in serum of a total of 51 uraemic patients: 19 non-dialysis (ND), 18 haemodialysis (HD) and 14 continuous ambulatory peritoneal dialysis (CAPD) patients. The low molecular mass As species were separated by ion-exchange liquid chromatography and measured on-line by hydride generation atomic absorption spectrometry (HGAAS). The high molecular mass As species were separated by fast protein liquid chromatography, either size-exclusion, ion-exchange or affinity chromatography, and the fractions were digested and measured off-line with HGAAS. The mean total As concentrations in the serum of the three groups of the uraemic patients were significantly higher than the reference value (6.47 +/- 4.28, 5.12 +/- 5.58 and 4.67 +/- 5.41 micrograms l-1 for HD, ND and CAPD patients, respectively, versus the reference value of 0.96 +/- 1.52 micrograms l-1. The major As species in serum of the patients were dimethylarsinic acid (DMA) and arsenobetaine. The HD patients showed a significantly higher mean DMA level than ND and CAPD patients. No selective removal of different As species in serum of HD patients was observed after 4 h of haemodialysis. The inorganic As species in serum were bound to proteins, mainly transferrin (about 5-6% of total As in serum). This binding may play an important role in arsenic detoxification.

Metabolism of arsenic after acute occupational arsine intoxication.

Among the elements of toxicological relevance, inorganic arsenic (As) probably exhibits the most complex metabolism, and we deemed it interesting to identify and quantify the different As species excreted after an occupational acute intoxication with arsine. For this purpose total As and five As species were determined using an hybrid analytical method coupling liquid chromatography with inductively coupled plasma mass spectrometry. The highest urinary elimination of total As was observed in the first 5 d after admission. The As species mostly excreted were monomethylarsonate (MMA), dimethylarsinate (DMA), As3+, arsenobetaine (AsB), and to a lesser extent As5+. The amount of AsB excreted in urine by the subject does not appear to be completely justified by AsB intake through food. Arsenic is excreted mainly via the urine with a clearance of 7.8 ml/h/kg and follows a triphasic model with periods of 28 h, 59 h, and 9 d, respectively. The evidence that DMA excretion culminates after a few days, when the excretion of the inorganic form is substantially reduced (while that of MMA is still elevated), seems to confirm the existence of two successive methylating enzyme activities. Furthermore, the elimination rate of As from blood follows a three-phase model and the half-lives of different species vary from about 27 to 86 h with the following gradient As5+ < MMA < As3+ < DMA < AsB.

Accumulation of arsenic species in serum of patients with chronic renal disease.

Speciation of arsenic was determined in serum of 19 non-hemodialysis (non-HD) and 18 HD patients. The respective mean values of serum creatinine in these groups were 410 +/- 250 and 914 +/- 173 mumol/L (reference range for healthy subjects: females 50-80; males 57-93 mumol/L). The mean total arsenic concentrations were 5.12 +/-5.58 and 6.47 +/- 4.28 micrograms/L, respectively (reference value: 0.958 +/-1.52 micrograms/L). Dimethylarsinic acid (DMA) and arsenobetaine (AsB) were the major As species in serum of the non-HD and HD patients, with mean values of 0.82 +/- 1.05 and 1.93 +/- 1.51 micrograms/L for DMA and 3.55 +/- 4.58 and 3.47 +/- 2.89 micrograms/L for AsB, respectively. Serum concentrations of inorganic As and monomethylarsonic acid in both groups were below the detection limits for these compounds. Measurement of As concentration before vs after 4 h of HD treatment indicated that 68% of total As in serum was removed, as was 16% of the total As in packed cells. The efficiency of DMA and AsB removal during dialysis corresponded to that of total As.

A unique metabolism of inorganic arsenic in native Andean women.

The metabolism of inorganic arsenic (As) in native women in four Andean villages in north-western Argentina with elevated levels of As in the drinking water (2.5, 14, 31, and 200 micrograms/1, respectively) has been investigated. Collected foods contained 9-427 micrograms As/kg wet weight, with the highest concentrations in soup. Total As concentrations in blood were markedly elevated (median 7.6 micrograms/1) only in the village with the highest concentration in the drinking water. Group median concentrations of metabolites of inorganic As (inorganic As, methylarsonic acid (MMA) and dimethylarsinic acid (DMA)) in the urine varied between 14 and 256 micrograms/1. Urinary concentrations of total As were only slightly higher (18-258 micrograms/1), indicating that inorganic As was the main form of As ingested. In contrast to all other populations studied so far, arsenic was excreted in the urine mainly as inorganic As and DMA. There was very little MMA in the urine (overall median 2.2%, range 0.0-11%), which should be compared to 10-20% of the urinary arsenic in all other populations studied. This may indicate the existence of genetic polymorphism in the control of the methyltransferase activity involved in the methylation of As. Furthermore, the percentage of DMA in the urine was significantly higher in the village with 200 micrograms As/1 in the water, indicating an induction of the formation of DMA. Such an effect has not been observed in other studies on human subjects with elevated exposure to arsenic.

Time dependence of accumulation and binding of inorganic and organic arsenic species in rabbit erythrocytes.

The uptake by rabbit erythrocytes of 0.4 mM arsenate, As(V), monomethylarsinate, MMA(V) and dimethylarsonate, DMA(V) were compared over 24 h. In membrane-free hemolysate, the distribution of As between proteins (10 kDa) and ultrafiltrate was determined by ultrafiltration and arsenic species in the ultrafiltrate were identified by thin layer chromatography methods. 1H spin-echo Fourier transform NMR was used to follow the binding of these arsenic species to glutathione (GSH). 31P-NMR was used to observe their effects on high-energy adenine nucleotide levels (ATP, ADP). These results demonstrate that As(III) readily accumulates in cells, reaches a quasi-plateau at 78% of the total As in the incubation after 1 h and 88% of the total As after 24 h. On average, 20% of the total erythrocyte As(III) burden is associated with the protein fraction, particularly with hemoglobin (Hb). About 68% of the erythrocyte As(III) burden is bound to GSH. As(III) has no effect on ATP levels during a 5-h incubation. By comparison, As(V) enters erythrocytes more slowly (53% of the total As after 5 h). Erythrocytes take up 81% of the As(V) in the reaction system after a 24 h incubation. Of the total As burden in As(V)-exposed erythrocytes, 22% was associated with the proteins (10 kDa) and possibly reduced to As(III) and 59% was in the ultrafiltrate (8% as As(III) and 51% as As(V)). This finding indicates that, over a 24 h incubation period, the reduction of As(V) to As(III) may account for 30% of the total As in rabbit erythrocytes. As(V) present in the erythrocytes enters the phosphate pool and depletes ATP. In comparison, about 65% of the total MMA(V) or about 44% of the total DMA(V) in the incubation system is taken up by rabbit erythrocytes during a 24 h incubation. Neither organoAs species perturbed the Hb signals observed by spin-echo Fourier transform NMR and the binding to GSH was minimal. Unlike As(V), MMA(V) and DMA(V) do not perturb phosphate metabolism, showing that, despite their pentavalent oxidation state, these arsenic species are not analogs for phosphate.