A Speciation Study focused on the Identification of Proximate Toxic Arsenic Metabolites. Kazuo T. Suzuki

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1 A Speciation Study focused on the Identification of Proximate Toxic Arsenic Metabolites Kazuo T. Suzuki Graduate School of Pharmaceutical Sciences, Chiba University Inage, Chiba , JAPAN Arsenic Contamination in Groundwater - Technical and Policy Dimensions 45

2 Chemical species of arsenic taken by humans and excreted into urine Ingested forms Inorganic Organic arsenite arsenate arsenosugars arsenocholine arsenobetaines Excreted forms (urinary metabolites) dimethylated arsenic (DMA) (dimethylarsinic acid ( )) Most probable pattern in the toxification of arsenic in humans Ingested forms Urinary metabolites Arsenite or DMA ( ) Arsenate Reduction and methylation reactions in the metabolism of arsenic O As O As CH 3 O As CH 3 CH 3 reduction As As CH 3 CH 3 As CH 3 methylation arsenate arsenite monomethylarsonic acid monomethylarsonous acid dimethylarsinic acid dimethylarsinous acid 46 Arsenic Contamination in Groundwater - Technical and Policy Dimensions

3 Reduction and methylation reactions in the metabolism of arsenic Detoxification pathway or Toxification pathway? Which arsenic metabolites do cause the arsenic toxicity? What are the proximate toxic arsenic metabolites? Tri- and pentavalent forms of arsenic Trivalent Pentavalent More reactive less reactive Arsenic Contamination in Groundwater - Technical and Policy Dimensions 47

4 Possible reactions for trivalent forms of arsenic As III RSH As V 2e - reactive oxygen species (ROS), radicals RS-As (RS-As(CH 3 ) 2, (RS) 2 -AsCH 3, (RS) 3 -As, RS-As=O) Separation and subsequent analytical procedures for arsenic in the body Soluble Free Pentavalent arsenic A (,, ) Trivalent arsenic B (,, ) Conjugated Trivalent arsenic GSH C ( (GS) 3, CH 3 As III (GS) 2, (CH 3 ) 2 As III (GS)) Trivalent arsenic - soluble proteins Non-soluble Conjugated Trivalent arsenic - non-soluble proteins D E Separation and subsequent analytical procedures for arsenic in the body Soluble Free Pentavalent arsenic A Trivalent arsenic B Conjugated Trivalent arsenic GSH C Trivalent arsenic - soluble proteins D Non-soluble Conjugated Trivalent arsenic - non-soluble proteins E A, B: Free arsenic can be speciated simultaneously on an anion or cation exchange column by HPLC-ICPMS. C: Trivalent arsenic conjugated with GSH can be speciated simultaneously on an anion exchange column by HPLC-ICPMS. D: Trivalent arsenic conjugated with thiol groups of soluble proteins can be characterized on a gel filtration column by HPLC-ICP MS. However, the arsenic species and the corresponding protein have to be identified separately. E: Trivalent arsenic conjugated with non-soluble proteins is not applicable to HPLC separation. The arsenic species can be identified after separation to free arsenic. 48 Arsenic Contamination in Groundwater - Technical and Policy Dimensions

5 Why rats are less toxic to arsenic than other mammals? What is the significant difference in the metabolism of arsenic in rats compared with other animals? What are the difference in the toxicity and hence metabolism of arsenic between sensitive and tolerant animal species? Characteristics of arsenic metabolism in rats Longer biological half time in rats than in humans. Why arsenic is more toxic in humans despite of its shorter biological half time? Specific accumulation of arsenic in red blood cells in rats. Why does arsenic accumulate in RBCs in rats? The major urinary metabolite in rats and humans is believed to be DMA( ). Is it true? Speciation of arsenic in the bile of rats after an intravenous injection of arsenite on a cation exchange column by HPLC-ICP MS (A) 30 min 60 min 90 min 120 min 150 min 180 min 210 min 240 min 270 min 300 min X 1/6 30 min (B) iasiii i) Authentic AsB ii) Reduced MMA iii) Reduced DMA iv) As(GS) 3 v) CH 3As(GS) Arsenic Contamination in Groundwater - Technical and Policy Dimensions 49

6 Speciation of arsenic in the bile of rats after an intravenous injection of arsenite on an anion exchange column by HPLC-ICP MS (A) CH 3As(GS) 2 As(GS) min 60 min 90 min 120 min 150 min 180 min 210 min 240 min 270 min 300 min (B) AsB As(GS) 3 i) Authentic ii) Reduced MMA iii) Reduced DMA iv) As(GS) 3 v) CH 3As(GS) Proposed metabolic pathway for arsenic after an intravenous injection of arsenite in rats bloodstream urine RBCs bile duct (SG) 2 (SG) 2 (SG) 3 liver (SG) 3 main route bile excretion intestine Speciation of arsenic in the urine of rats after an intravenous injection of arsenite cation exchange AsB Arsenic Contamination in Groundwater - Technical and Policy Dimensions

7 Selective uptake of by red blood cells (RBCs) chemical species difference animal species difference Plasma As (µg/ml) A) Reduction (-) m/z 75 Reduction (+) B) Incubation time (hr) As ( µg/ml medium) As (µg/ml medium) µg/ml 13.3 µg/ml A) rat 13.3 µg/ml B) hamster 13.4 µg/ml C) mouse D) human Incubation time (hr) Co-authors Y. Shiobara, Y. Ogra and K. T. Suzuki Animal species difference in the uptake of dimethylarsinous acid ( ) by red blood cells. Chem. Res. Toxicol. 14, (2001). B.K. Mandal, Yasumi. Ogra and K.T. Suzuki Identification of dimethylarsinous and momomethylarsonous acids in human urine of the arsenic affected areas in West Bengal, India. Chem. Res. Toxicol. 14, (2001). K.T. Suzuki, T. Tomita, Y. Ogra and M. Ohmichi Glutathione-conjugated arsenics in the potential hepato-enteric circulation in rats Chem. Res. Toxicol. 14, (2001) Arsenic Contamination in Groundwater - Technical and Policy Dimensions 51

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