Chem. Res. Toxicol. 2001, 14, 371-378
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Identification of Dimethylarsinous and Monomethylarsonous Acids in Human Urine of the Arsenic-Affected Areas in West Bengal, India Badal Kumar Mandal, Yasumitsu Ogra, and Kazuo T. Suzuki* Faculty of Pharmaceutical Sciences, Chiba University, Inage, Chiba 263-8522, Japan Received December 4, 2000
A speciation technique for arsenic has been developed using an anion-exchange highperformance liquid chromatography/inductively coupled argon plasma mass spectrometer (HPLC/ICP MS). Under optimized conditions, eight arsenic species [arsenocholine, arsenobetaine, dimethylarsinic acid (DMAV), dimethylarsinous acid (DMAIII), monomethylarsonic acid (MMAV), monomethylarsonous acid (MMAIII), arsenite (AsIII), and arsenate (AsV)] can be separated with isocratic elution within 10 min. The detection limit of arsenic compounds was 0.14-0.33 µg/L. To validate the method, Standard Reference Material in freeze-dried urine, SRM-2670, containing both normal and elevated levels of arsenic was analyzed. The method was applied to determine arsenic species in urine samples from three arsenic-affected districts of West Bengal, India. Both DMAIII and MMAIII were detected directly (i.e., without any prechemical treatment) for the first time in the urine of some humans exposed to inorganic arsenic through their drinking water. Of 428 subjects, MMAIII was found in 48% and DMAIII in 72%. Our results indicate the following. (1) Since MMAIII and DMAIII are more toxic than inorganic arsenic, it is essential to re-evaluate the hypothesis that methylation is the detoxification pathway for inorganic arsenic. (2) Since MMAV reductase with glutathione (GSH) is responsible for conversion of MMAV to MMAIII in vivo, is DMAV reductase with GSH responsible for conversion of DMAV to DMAIII in vivo? (3) Since DMAIII forms iron-dependent reactive oxygen species (ROS) which causes DNA damage in vivo, DMAIII may be responsible for arsenic carcinogenesis in human.
Introduction The toxicological and biological properties of metals depend on their chemical forms. Chemical speciation analysis aims to qualitatively and quantitatively determine the compounds of an element present in a given material. The toxicity and bioavailability of arsenic depend on its chemical form (1, 2). Inorganic arsenic compounds are more toxic than organic arsenic compounds and have been established as human carcinogens (2-5). Ingestion of inorganic arsenic may cause cancer of the skin, urinary bladder, kidney, lung, and liver as well as with disorders of the circulatory and nervous system (2, 4, 6-11). So, knowledge of speciation is especially necessary for As. In humans, like many mammalian species, inorganic arsenic undergoes metabolic conversion from pentavalent to trivalent arsenic with subsequent methylation (12, 13). In the case of inorganic arsenic ingestion, it has been reported that approximately 60-75% of the dose was excreted into urine (4, 12, 14, 15) within a few days and the distribution of arsenic species in urine was 10-15% inorganic arsenic, 10-15% monomethylarsonic acid (MMAV), and 60-80% dimethylarsinic acid (DMAV) (5, 14, 16). The methylation of arsenite (AsIII) is catalyzed by a specific methyltransferase with S-adenosylmethion* To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences, Chiba University, Inage, Chiba 263-8522, Japan. Fax or phone: 81-43-290-2891. E-mail:
[email protected].
ine (SAM)1 as a methyl group donor (17). The first methylation step forms MMAV, a pentavalent monomethylated arsenical. According to the metabolic scheme postulated by Cullen et al. (18), MMAV is then reduced to MMAIII and further methylated to yield DMAV. Similarly, reduction of DMAV to DMAIII is a pathway (19) for the third methylation step, forming trimethylarsenic oxide (TMAVO). DMAV is a major urinary metabolite in humans exposed to inorganic arsenic (5, 20). Methylation is considered to be the principal pathway for detoxification of inorganic arsenic since pentavalent methylated metabolites are less acutely toxic than either AsIII or arsenate (AsV) (21). However, DMAV induces DNA damage via formation of dimethylarsenic peroxyl radical, superoxide anion, or hydroxyl radicals in vivo and may induce arsenic-mediated cellular toxicity and/or carcinogenesis in humans (22-24). However, little is known about the toxicity of trivalent methylated metabolites, MMAIII and DMAIII. MMAIII and DMAIII have high affinity for thiols such as AsIII and also for some specific cellular proteins (25, 26). MMAIII inhibits glutathione reductase (27) whereas reduced GSH can reduce AsV to AsIII (28, 29) and donates electrons for reduction steps 1 Abbreviations: SAM, S-adenosylmethionine; TMAVO, trimethylarsine oxide; DMAIII, dimethylarsinous acid; MMAIII, monomethylarsonous acid; NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; ROS, reactive oxygen species; DMPS, sodium 2,3dimercapto-1-propanesulfonate; GSH, glutathione; SAHC, S-adenosylhomocysteine.
10.1021/tx000246h CCC: $20.00 © 2001 American Chemical Society Published on Web 03/17/2001
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Table 1. Demographic Details of the Studied Groups in West Bengal, India location no. of subjects no. of men no. of women no. of patientsb durationc (years) no. of water samples analyzed AsV in watere AsIII in watere sum of AsV and AsIII in watere AsIII/AsV ratio
control
group A
group B
group C
group D
Jhilimili, Ranibandh, Bankura 46 23 23 10 NDf ND ND -
Bibipur, Baduria, 24 PGS (N)a 105 44 61
