25400-23-1

A unique enzyme, MMA(v) reductase, has been partially purified from rabbit liver by using DEAE-cellulose, carboxymethylcellulose, and red dye ligand chromatography. The enzyme is unique since it is the rate-limiting enzyme in the biotransformation of inorganic arsenite in rabbit liver. The K(m) and V(max) values were 2.16 x 10-3 M and 10.3 μmol h-1 (mg of protein)-1. When DMA(v) or arsenate was tested as a substrate, the K(m) was 20.9 x 10-3 or 109 x 10-3 M, respectively. The enzyme has an absolute requirement for GSH. Other thiols such as DTT or L-cysteine were inactive alone. At a pH below the physiological pH, GSH carried out this reduction, but this GSH reduction in the absence of the enzyme had little if any value at pH 7.4. When the K(m) values of rabbit liver arsenite methyltransferase (5.5 x 10-6 M) and MMA(III) methyltransferase (9.2 x 10-6) were compared to that of MMA(v) reductase (2.16 x 10-3 M), it can be concluded that MMA(v) reductase was the rate-limiting enzyme of inorganic arsenite biotransformation. MMA(v) reductase was also present in surgically removed human liver.

The drinking of water containing large amounts of inorganic arsenic is a worldwide major public health problem because of arsenic carcinogenicity. Yet an understanding of the specific mechanism(s) of inorganic arsenic toxicity has been elusive. We have now partially purified the rate-limiting enzyme of inorganic arsenic metabolism, human liver MMAv reductase, using ion exchange, molecular exclusion, and hydroxyapatite chromatography. When SDS-βmercaptoethanol-PAGE was performed on the most purified fraction, seven protein bands were obtained. Each band was excised from the gel, sequenced by LC-MS/MS and identified according to the SWISS-PROT and TrEMBL Protein Sequence databases. Human liver MMAv reductase is 100% identical, over 92% of sequence that we analyzed, with the recently discovered human glutathione-S-transferase Omega class hGSTO 1-1. Recombinant human GSTO1-1 had MMAv reductase activity with Km and Vmax values comparable to those of human liver MMAv reductase. The partially purified human liver MMAv reductase had glutathione S-transferase (GST) activity. MMAv reductase activity was competitively inhibited by the GST substrate, 1-chloro 2,4-dinitrobenzene and also by the GST inhibitor, deoxycholate. Western blot analysis of the most purified human liver MMAv reductase showed one band when probed with hGSTO1-1 antiserum. We propose that MMAv reductase and hGSTO 1-1 are identical proteins.

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 (iAsIII) 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). iAsIII is methylated to mono (MMA)- and dimethylated (DMA) arsenics in the liver during circulation in the conjugated form [iAsIII(GS)3], and a part of MMA is excreted into the bile in the forms of MMAIII and MMAv, the former being mostly in the conjugated form [CH3AsIII(GS)2], and the latter being in the nonconjugated free form. DMAIII and DMAv were not detected in the bile. In the urine, arsenic was detected in the forms of iAsIII, arsenate, MMAv, and DMAv, iAsIII being the major arsenic in the first 6-h-urine, and DMAv being increased in the second 6-h-urine. The present metabolic balance and speciation study suggests that iAsIII is methylated in the liver during its hepato-enteric circulation through the formation of the GSH-cojugated form [iAsIII(GS)3], and MMAIII and MMAv are partly excreted into the bile, the former being in the conjugated form [CH3AsIII(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 DMAv in rats.

Speciation of arsenic in urine from rats treated with dimethylarsinic acid (DMAV) alone or in combination with dimercaptopropane sulfonate (DMPS) were studied. Methods were developed for the determination of the methylarsenic metabolites, especially trace levels of dimethylarsinous acid (DMAIII) and trimethylarsine oxide (TMAO), in the presence of a large excess of DMAV. Success was achieved by using improved ion-exchange chromatographic separation combined with hydride generation atomic fluorescence detection. Micromolar concentrations of DMAIII were detected in urine of rats fed with a diet supplemented with either 100 μg/g of DMAV or a mixture of 100 μg/g of DMAV and 5600 μg/g of DMPS. No significant difference in the DMAIII concentration was observed between the two groups; however, there was a significant difference in TMAO concentrations. Urine from rats fed with the diet supplemented with DMAV alone contained 73 ± 30 μM TMAO, whereas urine from rats fed with the diet supplemented with both DMA V and DMPS contained only 2.8 ± 1.4 μM TMAO. Solutions containing mixtures of 100 μg/L DMAV or TMAO and 5600 μg/L DMPS did not show reduction of DMAV and TMAO. The significant decrease (p V and DMPS suggests that DMPS inhibits the biomethylation of arsenic.