Arsenic Toxicology: Five Questions - Chemical Research in

JANUARY 2006 VOLUME 19, NUMBER 1 © Copyright 2006 by the American Chemical Society

ReViews Arsenic Toxicology: Five Questions† H. Vasken Aposhian* and Mary M. Aposhian Department of Molecular and Cellular Biology, The UniVersity of Arizona, Life Sciences South, Room 444, P.O. Box 210106, Tucson, Arizona 85721-0106 ReceiVed April 19, 2005

Contents 1. Preface 2. Introduction 3. Question One: What Enzyme Is Responsible for the Methylation of Arsenic Species in the Human? 3.1. Arsenic Biotransformations 3.2. Dual Enzymes for the Same Step in Arsenic Biotransformation 3.3. Hydrogen Peroxide and Arsenic Biotransformation 3.4. Conclusion One 4. Question Two: How Does Inorganic Arsenic, More Specifically Arsenite, Inhibit the Pyruvic Acid Dehydrogenase Multienzyme Complex? 4.1. Conclusion Two 5. Question Three: What Are the Relationships as Judged by Urinary Arsenic Species between Genetic Polymorphisms and Arsenic Biotransformation in the Human? 5.1. hGSTO 5.2. hGSTO Polymorphisms 5.3. CYT 19 Polymorphisms 5.4. PNP Polymorphisms

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* To whom correspondence should be addressed. Tel: 520-621-7565. Fax: 520-621-3709. † This paper is written in honor of the 90th birthday of Professor John P. Lambooy, the graduate school mentor of one of the authors. His versatility and outspoken love for clear thinking, hard work, and original research have always inspired the authors. The inspiration, respect, and gratitude have remained for over 50 years.

5.5. Other Polymorphisms 5.6. Conclusion Three 6. Question Four: Is There a Useful Treatment for Arsenic Intoxication that Can Replace BAL (Dimercaprol)? 6.1. Treatment of Acute Exposure 6.2. Treatment of Chronic Exposure 6.3. Conclusion Four 7. Question Five: What Is the Role of Protein Binding in Arsenic Metabolism and Toxicity? 7.1. Hemoglobin Binding 7.2. Metallothionein Binding 7.3. Other Pertinent Proteomic Papers 7.4. DIGE 7.5. Conclusion Five 8. Areas of Concern and Conclusions 8.1. Tolerance 8.2. Inappropriate Procedures for Synthesis 8.3. Inappropriate Procedures for Urine Collection 8.4. Compound Identification 8.5. Inadequacy of the Rat as a Model for Arsenic in Humans 8.6. Summary

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1. Preface We have addressed five questions dealing with arsenic toxicology: biotransformation, reactive oxygen species (ROS),1

10.1021/tx050106d CCC: $33.50 © 2006 American Chemical Society Published on Web 12/10/2005

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polymorphism, treatment, and protein binding. The first question, “What enzyme is responsible for the methylation of arsenic species in the human?”, still needs further investigative effort because arsenic methylation is an important biotransformation pathway in the human and some animals. No arsenic methyltransferase has been isolated from surgically removed or biopsied human tissue. For CYT 19 to be accepted as the methylation enzyme, purification of the protein and its activity from human tissue is required. This has not been accomplished. There is little doubt, however, that CYT 19 and the rabbit arsenic methyltransferase have some role in arsenic biotransformation. Second, how does inorganic arsenic, more specifically arsenite, inhibit enzymes, e.g., the pyruvic acid dehydrogenase (PDH) multienzyme complex? The classical mechanism is now in doubt. The conventional belief that arsenite inhibits PDH and perhaps other dithiol-containing enzymes by chelating or complexing the thiol groups now needs further study because experiments have demonstrated that PDH is more sensitive to inhibition by ROS than to arsenic-containing agents that bind vicinal thiols (for example, phenyldichloroarsine). ROS can be generated by arsenicals. Third, what are the relationships as judged by urinary arsenic species between genetic polymorphisms and inorganic arsenic biotransformation? It is well-accepted that there is intraindividual variation in response to arsenic exposure as judged by the urinary arsenic profile. Is interindividual variation due to genetic polymorphisms? A number of polymorphisms in human GST omega (ω), CYT 19, and purine nucleoside phosphorylase (PNP) have been reported. Two studies have linked these polymorphisms with changes in urinary arsenic species in the human. There have been a minimum of investigations combining studies of polymorphisms of human genes known to be involved in arsenic metabolism with determinations of urinary arsenic species. In fact, the genetics of arsenic toxicity is a barren field at present. Fourth, is there an effective treatment for arsenic intoxication that can replace British anti-Lewisite (BAL, dimercaprol)? meso2,3-Dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1 sulfonic acid, Na salt (DMPS) are effective in mobilizing the excretion of arsenic from the human. DMPS seems to be more consistently effective in the clinical improvement of individuals chronically exposed to arsenic. With the millions of people now known to be consuming toxic amounts of arsenic in their drinking water or food, a large-scale clinical trial of arsenic antidotes is needed and recommended. An effective arsenic-mobilizing agent is of little benefit as long as exposure continues but may be of value to decrease the arsenic body burden once the exposure has ceased. Fifth, what is the role of protein binding in arsenic metabolism and toxicity? Are proteins only the site of the toxic action of arsenic species or are they also an arsenic-binding storage reservoir involved in detoxication? A recent study clearly has shown that the differences in the number of cysteine residues in human and mouse hemoglobin are responsible for the greater accumulation of arsenic species in rat blood. With new 1Abbreviations: DMA, generic term including DMA(III) and DMA(V); DMA(III), dimethylarsinous acid; DMA(V), dimethylarsinic acid; DTT, dithiothreitol; MMA, generic term including MMA(III) and MMA(V); MMA(III), monomethylarsonous acid; MMA(V), monomethylarsonic acid; SAM, S-adenosyl-methionine; GST, glutathione-S-transferase; PDH, pyruvate dehydrogenase; PNP, purine nucleoside phosphorylase; ICP-MS, inductively coupled plasma mass spectrometry; BAL, British anti-Lewisite; DMSA, meso-dimericapto succinic acid; DMPS, 2,3-dimercaptopropane1-sulfonic acid, Na; PAO, phenylarsineoxide; ROS, reactive oxygen species; hGSTO, human glutathione-S-transferase ω; EST, expressed sequence tags; DIGE, differential in-gel electrophoresis.

Aposhian and Aposhian Scheme 1

proteomic techniques available, such as differential in-gel electrophoresis (DIGE), research in arsenic toxicology now may be expanded to acquire more specific knowledge as to the exact role of specific proteins in arsenic intoxication and detoxication. Two-dimensional electrophoresis procedures, in which protein extracts from two subjects can be electrophoresed together and simultaneously by using two different fluorescent dyes, is a productive approach to help answer these questions. While a number of recent papers have been emphasized in this review, the need for confirmation of their conclusions by other investigators is needed. The importance of their results at this time, however, should not be minimized. Rather, they should stimulate investigators to reexamine and expand their thinking and investigations and, hopefully, attract new investigators.

2. Introduction According to ancient Greek myths, Hercules killed Hydra and then dipped all of his arrows into the poisonous venom of Hydra’s many arms. Subsequently, Hercules used these poisoned arrows to win his legendary battles to atone for his wrongdoings. The word for arrow in ancient Greek is toxin. Thus, the names toxin and toxicology can be traced back to stories of Hercules and the poison-filled, many-armed Hydra. Figuratively, the toxicology of inorganic arsenic also has many arms (Scheme 1). Many of these will not be addressed in this review because it is not meant to be a summary of the literature. We do not feel compelled to cite every arsenic toxicology paper published during some given time period. Rather, the purpose of this review is to deal with some of these arms by addressing five important questions central to arsenic toxicology with the intent of stimulating investigators, especially new ones, to seek answers. First, what enzyme is responsible for methylation of arsenic species in the human? Second, how does inorganic arsenic, more specifically arsenite, inhibit enzymes, e.g., the PDH multienzyme complex? The classical mechanism is now in doubt. Third, what are the relationships as judged by urinary arsenic species between genetic polymorphisms and inorganic arsenic biotransformation? Is interindividual variation due to polymorphisms? Fourth, is there an effective treatment for arsenic intoxication that can replace BAL (dimercaprol)? Fifth, what is the role of protein binding in arsenic metabolism and toxicity? Are proteins

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Figure 1. Generally accepted pathway for biotransformation of inorganic arsenic. Is it becoming obsolete?

Figure 2. Proposed pathway by Hayakawa et al. (5) for biotransformation of inorganic arsenic. Reprinted with permission from ref 5. Copyright Springer Science and Business Media.

not only the site of the action of toxic arsenic species but also an arsenic-binding storage reservoir involved in detoxication? Only the papers that we consider to be pertinent to these five questions have been cited. Carcinogenicity, signal transduction, and the use of arsenic trioxide to cure human cancer are not reviewed because each could be the single topic of a review and many other investigators are much more competent and knowledgeable in such areas (1-4).

3. Question One: What Enzyme Is Responsible for the Methylation of Arsenic Species in the Human?3.1. Arsenic Biotransformations First, a review of inorganic arsenic biotransformation is pertinent. The usually accepted pathways from inorganic arsenate to dimethylarsinate are outlined in Figure 1. Although a figure very often is interpreted as a final version embedded in stone, the authors wish to make it clear that there are many uncertainties and unknowns about this one. This can be seen in comparing the reactions of Figure 1 with a very interesting and novel pathway for inorganic arsenic biotransformation (Figure 2) suggested by Hayakawa et al. (5). The generally accepted pathway of arsenic biotransformation (Figure 1), usually credited to Challenger (6) and Cullen and Reimer (7), consists of a series of reductions and oxidations coupled with methylations (Figure 1). In the reactions, the +5 oxidative arsenic species is formed before the analogous +3 arsenic species e.g., monomethylarsonic acid [MMA(V)] before monomethylarsonous acid [MMA(III)], dimethylarsinic acid

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[DMA(V)] before dimethylarsinous acid [DMA(III)], and TMAO before TMA(III). Only two putative enzymes, MMA(V) reductase and arsenic methyltransferase, in the pathway have been extensively purified and studied (8-13) as has an alternative one, CYT 19 (14-18). Human MMA(V) reductase and hGST ω are identical proteins (13). Both names are used in this paper to allow a greater relevance and emphasis as needed for the reader. The recent Hayakawa et al. proposal (5) appears to be a reasonable pathway for arsenic biotransformation. The most original part of their proposal is that +3 arsenic species are formed before +5 species, the former being oxidized by hydrogen peroxide or other agents to produce +5 species that are end products of arsenic metabolism. At least one of them, DMA(V), has been the major end product found in the urine of most species and in the past believed to be the end point of arsenic metabolism after exposure. The Hayakawa et al. (5) model (Figure 2) proposes that arsenic triglutathione (ATG) and monomethylarsonic diglutathione [MA(SG)2] are substrates of CYT 19, one possible methylating enzyme. This new pathway also opens the possibility that these glutathione-arsenic compounds, in order to be formed and oxidized, need new enzymes. Once the arsenicglutathione substrates are formed, it is proposed that they are methylated by CYT 19 and S-adenosyl-methionine (SAM) to form MADG and DMAG. The methylated compounds are then oxidized to MMA(V) and DMA(V), the major arsenic metabolites found in the urine. Aposhian et al. (19, 20) in 2004 proposed that the more toxic +3 arsenic species might be oxidized and detoxified by hydrogen peroxide to form the less toxic +5 species. The differences between this newly proposed pathway (Figure 2) and the older one (Figure 1) are important, although the urinary arsenic species, namely, the +5 species, are the same. What is most attractive about the Hayakawa scheme is that MMA(V) and DMA(V) are proposed as the end products, not just intermediates, in arsenic biotransformation. The Cullen and Reimer (7) scheme had the anomaly of the major intermediates appearing as end products in the urine. These experiments of Hayakawa et al. (5) are important and novel enough to require confirmation. One of the major criticisms of its proposed pathways is that the structures of the new intermediates involved, specifically the arsenic-glutathione substrates and products, have not been confirmed by adequate procedures. Chromatography retention times are no longer acceptable as the only proof of structure of new, closely related arsenic compounds. Second, a search of the scientific literature indicated that human CYT 19 has been produced only by DNA recombinant technology (18). It has not been purified and isolated from human tissue. To suggest that human CYT 19 is the arsenite methyltransferase, when CYT 19 does not appear to be expressed in human liver, is at present unwarranted. A search of NCBI (21) indicated that CYT 19 mRNA does not appear to be expressed or is expressed in only small undetectable amounts in human liver (Figure 3). The expression profile suggested by analysis of expressed sequence tags (EST) events in human liver was zero transcripts per million RNA molecules. In mouse liver, it was 76 transcripts per million RNA molecules (21). It does appear to be expressed in human kidneys and a few other tissues. Yet, the major site of arsenic methylation always has been claimed to be the liver (22, 23). However, in a recent letter to us, Dr. David S. Barber of The University of Florida has informed us that he has “observed expression of CYT 19 at the message level using RT-PCR in 8 of 8 human liver samples from male Caucasians” (personal communication).


Aposhian and Aposhian

Figure 3. mRNA transcripts of human CYT 19. Expression profile suggested by analysis of EST counts (21). The National Library of Medicine Web pages are public domain.

While this is definitely a step forward, the enzyme activity responsible for methylating inorganic arsenic in humans still has not been purified from human tissue. Neither have there been reported experiments to determine whether CYT 19 is inducible. Experiments in our laboratory dealing with the inducibility of rabbit methyltransferase have been numerous but inconclusive.

3.2. Dual Enzymes for the Same Step in Arsenic Biotransformation It is pertinent to this review to examine the hypothesis that alternative reactions are available for each step of the biotransformation of inorganic arsenic beginning with the reduction of arsenate to arsenite. For many years, it was proposed that this reduction was accomplished by GSH in a strictly chemical, nonenzymatic reaction. Radabaugh and Aposhian (24) showed that extracts of human liver carried out the reduction of arsenate. In 2002, Radabaugh et al. (10) reported that PNP with inosine as a required constituent of the enzyme reaction had arsenate reductase activity. Simultaneously, the Gregus group (25) reported the same reaction in rat tissue. Subsequently, the latter group reported that PNP was not a factor in arsenate reduction (26) but their studies can be criticized for using the rat, a species generally acknowledged to be a poor model for inorganic arsenic metabolism in the human (see the discussion of question five below) and ignoring the functions of hGSTO (8, 13). The primary reaction for arsenate reduction in humans is catalyzed by human glutathione-S-transferase ω (hGST-01) (8, 13, 27-29) and is probably the explanation for a process described in a paper by other workers dealing with the GSHdependent reduction of arsenate in human erythrocytes (26). The Km and Vmax data for hGSTO1 are given in Table 1. The enzyme requires GSH. Once arsenate is reduced to arsenite, it is methylated. An enzyme from rabbit liver or a human hepatocyte cell line has

Table 1. Kinetics Data for hGSTO1-1 (27, 28) substrate

Km (M)

Vmax (µmol/mg/h)

arsenate MMA(V) DMA(V)

34.8 × 10-3 53.6 × 10-3 30.6 × 10-3

12.8 52.6 18.0

been partially purified that will catalyze the methylation of arsenite or MMA(III) (9, 11, 12, 29). SAM is the methyl donor. Because a reducing agent such as GSH or L-cysteine is needed for methylation, the glutathione-arsenic type structures proposed by Hayakawa et al. (5) as substrates or intermediates are attractive. Certainly, the methylation of arsenite or MMA(III) in vitro can be carried out by either the rabbit type of methyltransferase (11), found in a variety of animals including the hamster (12) and lacking in the chimpanzee, tamarin, and guinea pig, or the rat CYT 19 methyltransferase (14). The CYT 19 relevance has been enormously strengthened by the preparation of human CYT 19 using DNA recombinant technology (18). In addition, it has been suggested recently that the lack of arsenic methylation in the chimpanzee is due to a 275 nucleotide deletion in its CYT 19 gene beginning at nucleotide 612 leading to a premature stop codon (18). In no way does the present discussion mean to minimize the importance of CYT 19, especially in the rat. Its importance and relevance to the methylation of arsenic species in general are established. The rabbit methyltransferases are not without problems. Although extracts have been purified 9000-fold (30), sequence identification has been unsuccessful. Thus, we have two alternative methylation and three reduction pathways for inorganic arsenic metabolism. Scheme 1 uses GSTω, PNP, or perhaps GSH nonenzymatically for arsenate reduction, arsenite/MMA(III) methyltransferase or CYT 19 for methylation, and GSTO for subsequent reduction of methylarsenic(V) and dimethylarsonic(V) species. On the other hand, the Hayakawa et al. (5) scheme uses GSH complexed with +3

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arsenic species as substrates for methylation to +3 arsenic species and perhaps oxidation by H2O2 to resulting +5 species. It should be understood that the biotransformation of inorganic arsenic in many animals and perhaps the human needs only two enzymes, hGSTO and a methyltransferase. The attempts to link other reducing enzymes and systems are unnecessary in our opinion. Perhaps some investigators would be helped by remembering and utilizing Occam’s razor (31).2 On the other hand, as compared to many other complex biological structures, arsenate has a relatively simple structure that is composed primarily of an arsenic atom and oxygen atoms. It should not be surprising that other reducing enzymes might reduce arsenate to arsenite.

3.3. Hydrogen Peroxide and Arsenic Biotransformation The importance of hydrogen peroxide is often underestimated in toxicology. It is usually considered a dangerous oxidant that can damage the structure of the DNA, RNA, and proteins in the cell (32). It is also, however, an important signal that by reacting with thiols of proteins can turn on (or off) signaling pathways. Human peroxiredoxins rid the cell of H2O2 faster than other antioxidant enzymes and are considered to be the controlling switch that determines whether H2O2 is to be a damaging agent or a signaling agent (32). To all of this now must be added the possible role of hydrogen peroxide in the metabolism of inorganic arsenic where its oxidation role is becoming more evident. There also are signals being sent by the H2O2 to other biological processes involving arsenic (33). Before we leave this discussion of arsenic biotransformation, it is pertinent to ask: Why does DMA(V) especially and to some extent MMA(V) appear in human urine in large concentrations? Although water solubility has been cited by some to be the reason, a more reasonable explanation is that DMA(V) and MMA(V) are much less reactive and have much less affinity for tissue components than do the more reactive DMA(III) and MMA(III). Also, there is a stability problem with MMA(III) and DMA(III), which can be easily oxidized. Finally, the Hayakawa et al. (5) scheme has proposed MMA(V) and DMA(V) as the end points of metabolism, not as intermediates (Figure 2).

3.4. Conclusion One As to the question of what enzyme catalyzes the methylation of arsenic in the human, the answer is still unknown. There are the rabbit type methyltransferases and the CYT 19. The relevance and importance of each are established, but questions about both remain, especially for the human. Until an enzyme is purified from surgically removed human tissue, the question remains unanswered. Perhaps more novel approaches in studying arsenic biotransformation are needed. The Hayakawa et al. (5) scheme introduces new reactions especially for the formation as well as the oxidation of arsenic-glutathione compounds. Investigations of enzymes involved in these new pathways are needed.

4. Question Two: How Does Inorganic Arsenic, More Specifically Arsenite, Inhibit the Pyruvic Acid Dehydrogenase Multienzyme Complex? For many years, the usually accepted mechanism for arsenite toxicity has been that it combines with thiols, especially the 2I think the following is the most useful statement of this principle for scientists: “When you have two competing theories which make exactly the same predictions, the one that is simpler is better.”

vicinal thiols of enzymes, and this was believed to result in the inhibition of the catalytic activity (34-36). One of the most studied enzyme models has been the pyruvate dehydrogenase (PDH) complex. It has been considered the target site most sensitive to inhibition by arsenite. More specifically, the site has been believed to be the lipoamide dehydrogenase subunit of the large PDH multienzyme complex (37, 38). PDH catalyzes the oxidative decarboxylation of pyruvate to form acetyl CoA. Inhibition of PDH would be expected to disrupt the energy system of the cell with resulting cell damage and death. Recently, Samikkannu et al. (39) asked the question: Does arsenic trioxide inactivate PDH activity in human cells via binding to the thiols of the enzyme complex or via inhibition by ROS? The results of this provocative and important paper indicated that arsenic trioxide exposure stimulates ROS production that causes PDH inactivation by oxidation. These results have disrupted the dogma as to the mechanism of inorganic arsenic inhibition of enzymes and other metabolic processes. They compared the PDH activity of HL60 cells and purified porcine PDH after exposure to either arsenic trioxide or phenylarsineoxide (PAO). Surprisingly, the intracellular PDH activity, measured in extracts after the cells had been exposed to arsenic trioxide, was more sensitive to inhibition than purified porcine PDH. The IC50 values were 2 and 182 µM, respectively. Arsenic trioxide was approximately 90 times more inhibitory for the intracellular PDH than for the purified PDH. This is quite unusual since purified enzymes are usually more sensitive than cells to inhibitors because the latter has membranes to be penetrated, stimulators, inhibitors, and/or other interfering substances. Phenylarsineoxide reacts specifically with vicinal thiols of proteins to form stable rings. Models for the inhibition of lipoic acid-containing enzymes by PAO have appeared (37, 38). When PAO and arsenic trioxide were compared as to their activity as thiol and vicinal thiol-reacting agents, PAO was more potent (39). The IC50 values for PAO to decrease the thiol and vicinal thiol content of HL60 cells were 2.3 and 1.9 µM, respectively, while for arsenic trioxide they were 82.5 and 81.7 µM, respectively. It is pertinent to point out that the IC50 value for arsenic trioxide to inhibit HL60 PDH activity was 2 µM, a level found in humans after inorganic arsenic exposure, while the IC50 value of arsenic trioxide to decrease thiol and vicinal thiol content of HL60 cells was about 80 µM. Dithiols such as dithiothreitol (DTT), DMSA, or DMPS prevented the PAO inhibition of PDH activity in HL60 cells, but at the level used, they did not reverse the arsenic trioxidecaused inhibition. However, antioxidants such as pyruvate, catalase, and selenite prevented the arsenic trioxide inhibition of PDH but not the PAO inhibition. Arsenic trioxide, but not PAO, increased hydrogen peroxide levels in the HL60 cells. Thus, according to the Samikkannu et al. (39) results, arsenite exposure appears to increase hydrogen peroxide production, producing hydroxyl free radicals through the Fenton reaction resulting in oxidative damage and inactivation of the PDH protein. This process occurs at arsenite concentrations much lower than that for arsenite binding to critical and essential thiols. The production of ROS in biological systems after arsenic exposure is well-known (40, 41). Oxidation of toxic +3 arsenic species by hydrogen peroxide has been proposed as a mechanism for decreasing their toxicity (19, 20). These innovative studies by Samikkannu et al. (39) are very provocative. They dealt, however, primarily with a human leukemia cell line. The extension of these experiments to nonleukemic cells and whole animals might further strengthen their hypothesis. A careful



reading of Samikkannu et al. (39) is recommended for new insights dealing with the inhibitory properties of inorganic arsenic. In addition, the Samikannu et al. (37) proposal is strengthened by the study in humans that showed that oxidative stress could be caused by chronic exposure of humans to drinking water containing inorganic arsenic (42). Elevated serum lipid peroxide levels and decreased nonprotein sulfhydryl were correlated with blood levels of arsenic species (42).

4.1. Conclusion Two “How does inorganic arsenic, more specifically arsenite, inhibit the pyruvic acid dehydrogenase multienzyme complex?” The well-entrenched, usually accepted, mechanism that inhibition by arsenite is the result of its reaction with PDH vicinal thiols is now under question. The generation of ROS by arsenite and the resulting oxidative damage to proteins (PDH) is a reasonable mechanism for the inhibition of PDH by arsenite. This novel and reasonable explanation for arsenite mitochondrial toxicology involving ROS production and inhibition deserves confirmation and further study.

5. Question Three: What Are the Relationships as Judged by Urinary Arsenic Species between Genetic Polymorphisms and Arsenic Biotransformation in the Human? Recently, genetic techniques have been applied to this problem. According to one of the biotransformation pathways, only two genes and enzymes are necessary for the metabolism of inorganic arsenic in the human, hGSTO and an arsenic methyltransferase. It appeared that the most relevant gene and enzyme to study in order to unravel the cause of interindividual variability of arsenic metabolism in the human was hGSTO. Its gene, protein structure, and amino acid sequences were known (12, 13, 43). Its properties and its relationship to the metabolism of arsenic by humans were understood (8, 13, 43).

Figure 4. Speciation of urinary arsenic as a percentage of total inorganic arsenic (52). Also included are the averages of all of the subjects from La Virgen and the average of all 75 subjects in the study population. The analysis did not detect MMA(III) and DMA (III) for reasons unknown to the investigators. Table 2. Concentration of Arsenic Species in the Urine (µg/g Creatinine) (52)a subject no.

As(V) As(III) MMA(V) MMA(III) DMA(V) DMA(III)

44 132.6 6.2 47 4.5 244.9 average for 5.1 14.4 100 µg As/L group average for all 7.91 16.02 groups, n ) 75 a

7.1 9.3 9.7

ND ND 0.18

14.1

0.43

29.7 114.2 54.5 81.17

NDa ND ND ND

ND, not detected.

hGSTO-1 and hGSTO-2, the dehydroascorbate reductase specific activity of hGSTO-2 was 70-100 times greater than that of hGSTO-1 (43, 48, 51).

5.2. hGSTO Polymorphisms 5.1. hGSTO A brief review of this enzyme is appropriate. MMA(V) reductase and hGSTO-1 are identical proteins (13) that can reduce arsenate, MMA(V), and DMA(V) to arsenite, MMA(III), and DMA(III), respectively (27, 28). The products are more toxic than the substrates (44, 45). No other enzyme is necessary for the reduction of these arsenic species. The glutathione-S-transferases (GSTs) make up a superfamily of intensively investigated enzymes that have many different functions. Reviews are available (46, 47). The function most usually associated with them is the conjugation of GSH with xenobiotics. Discovered by the Board group (43), the ω class GSTs have many properties different from other mammalian GSTs such as R, µ, π, and κ. The ω GSTs have thiol transferase, dehydroascorbate reductase, as well as arsenate, MMA(V), and DMA(V) reductase activity. The other members of the GST superfamily essentially lack such activities (43, 48). GSTO1-1 is expressed in many human tissues as indicated by mRNA transcription (49) and in hamster tissue as judged by enzyme activity (50). The GST ωs have a cysteine (Cy 32) in the active site rather than serine or tyrosine, which have been found in other mammalian GSTs (40). GSTO2 has been recently solubilized and characterized (48). Both hGSTO1 and hGSTO2 have six exons and are separated by 7.5 kb on chromosome 10q24.3. There is 64% amino acid identity of hGSTO2 with hGSTO1. Although many substrates are reduced almost equally by

The Aposhian group in collaboration with Professor Gonzalo Garcia-Vargas studied a group of 39 females and 36 males living in the vicinity of Durango and Torreon, Mexico (52). Most of the subjects were chronically exposed to high levels of arsenic in their drinking water. When 11 h overnight urines were collected and analyzed for arsenic species by HPLC-inductively coupled plasma mass spectrometry (ICP-MS), two of the subjects were found to have unusual urine arsenic profiles (Figure 4 and Table 2). Subject 44 had an unusually large amount of arsenate, almost 76% of the total urinary arsenic, and much lower amounts of arsenite, MMA(V), and DMA(V). When his hGSTO1 gene was examined, polymorphisms indicated that the glutamate at 155 had been deleted and the glutamate 208 was replaced by lysine. The other subject, 47, also had an unusual urine arsenic profile and was the mother of subject 44. She had an unusually large percent of arsenite in her urine (Figure 4 and Table 2). While the glutamate 155 deletion and the 208 glutamate replacement by lysine were common to both, the mother also had the 140 alanine replaced by asparagine as well as a GGC deletion. The urine arsenic profile of subject 44 might be explained by a polymorphism resulting in an inhibition of the arsenate reducing activity of hGSTO1. The urine arsenic profile of subject 47 might be explained by the increased activity of hGSTO1 and/or an inhibition of arsenic methyltransferase. Some support for this is that in a variant protein expressed in

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Table 3. Specific Activity of Allelic Variants of GSTO1-1 and GSTO2-2 (48)a monomethylarsonate(V) dimethylarsonate(V) reductase reductase Ala 140/Glu155Glu208 Asp140/Glu155Glu208 Ala140∆Glu155/Glu208 Ala140/Glu155/Lys208 Ala140∆Glu155/Lys208

GSTO1-1 0.33 ( 0.037 0.27 ( 0.039 0.65 ( 0.007 0.39 ( 0.037 0.67 ( 0.066

0.12 ( 0.006 0.15 ( 0.005 0.30 ( 0.014 0.26 ( 0.006 0.37 ( 0.015

Asn142 Asp142

GSTO2-2 0.42 ( 0.071 0.44 ( 0.044

0.03 ( 0.012 0.05 ( 0.019

a All activities are shown as µmol NADPH consumed per min per mg protein at 30 °C. All values are the means of at least three determinations ( standard deviation.

Escherichia coli, a deletion of glutamate 155 doubled the MMA(V) and tripled the DMA(V) reductase activity of hGSTO1-1 (48). Perhaps, the arsenate reductase activity also was increased. Unfortunately, the arsenate reductase activity was not determined. Another variant protein in which glutamate 208 was replaced by lysine had a minimal effect on MMA(V) reductase activity and almost doubled the DMA(V) reductase activity (Table 3). Other polymorphisms and genotypes also have been found (48, 51). The MMA(V) reductase activity of hGSTO2 was slightly different than that of hGSTO1-1 (Table 3). The DMA(V) reductase activity of hGSTO2-2 was about one-fourth that of hGSTO1-1. A polymorphism for hGSTO-2 consisting of the substitution of the 142 asparagine by aspartic acid (N142D) was detected (48), but it was without significant effect on the specific activity of this enzyme with its usual substrates (Table 3). The Klimecki group (53) has studied polymorphisms in hGSTO and PNP in 22 European and 24 indigenous American individuals. A total of 33 hGSTO1-1 polymorphisms were observed. The Europeans had more polymorphisms in the hGSTO gene than did the indigenous Americans. The genetic polymorphisms of each group were essentially exclusive to that group. For the European group, the minor allele frequency was 34% for Ala140Asp, 5% for the glutamic 155 deletion, and 5% for glutamate 208 lysine. These polymorphisms were absent from the indigenous American group. On the other hand, the indigenous American group had a 4% frequency of the Ala236Val, which was absent in the European group. Because the Klimecki study (53) has been the most complete one dealing with genetic variations in hGSTO1-1, including an extensive list of intron polymorphisms, a figure from their paper is included for informational purposes (Figure 5). Tanaka-Kagawa et al. (54) characterized two GSTO-1 recombinant variants. The 140 alanine was replaced by asparagine, and in the second variant, the 217 threonine was replaced by asparagine. The former variant had similar kinetics as the wild when MMA(V) was the substrate. The Km and Vmax values of the Thr217Asn variant were 64 and 56%, respectively, of the wild type, but its relevance has been questioned (48).

5.3. CYT 19 Polymorphisms Polymorphisms for human CYT 19 have been found (55) in a Mexican population (Figure 6). Some of these sites were associated with the ratio of urinary dimethylarsinate:monomethylarsinate in children. Ratios, however, usually exaggerate small differences. The authors neglected to consider and cite the work from Vahter’s group pointing out the unusual decreased amount of MMA in a small number of indigenous American

children (56). It seems appropriate at this time to point out that the consequences of acute and chronic exposure to arsenic in children are often a neglected area of arsenic research. Papers by Concha et al. (56) and Calderon et al. (57) are recommended reading.

5.4. PNP Polymorphisms An investigation of PNP polymorphisms found that glycine 51 was replaced by serine in exon 2 in 14% of European group and 35% of the indigenous American group (53). The latter group had a greater number of PNP polymorphisms. The relevance of PNP for arsenic metabolism has become controversial (10, 26). Although other polymorphisms were found, they do not appear pertinent at the present time.

5.5. Other Polymorphisms There have been other studies as to how polymorphisms in other genes may affect arsenic metabolism, but the genes studied were not proven to be directly involved in arsenic metabolism. In a population from northeastern Taiwan, there was a slightly increased percentage of urinary inorganic arsenic for the null genotype of GSTM1 and an elevated percentage of urinary DMA in null genotype of GSTT1 (58). A genetic polymorphism in p53 involving a change from arg/arg to pro/pro has been suggested to increase the risk of skin cancer in subjects in southwest Taiwan (58). Ironically, arsenic trioxide has been used for the successful treatment of acute promyelocytic leukemia. In some patients, however, there was increased toxicity and even death (59). Polymorphisms in hGSTO or other genes involved in arsenic biotransformation should not be overlooked as a potential cause of these severe reactions.

5.6. Conclusion Three To be able to decipher and ascertain the specific areas of the genes that may be responsible for the interindividual variations found in humans chronically exposed to inorganic arsenic, it is advisable to measure changes in at least three parameters: gene nucleotide sequence, the relevant enzyme activity of the gene product, and changes in the concentrations of various arsenic species in the urine. While it is advisable to study all of these changes in one laboratory, it appears that very few, at present, have all of the capabilities necessary. Polymorphisms of hGSTO1 and CYT 19 have been correlated with some changes in urine arsenic species, but much more research dealing with polymorphisms of hGSTO and CYT 19 and correlating them with the urine profiles of arsenic species is needed.

6. Question Four: Is There a Useful Treatment for Arsenic Intoxication that Can Replace BAL (Dimercaprol)? Because arsenic has been used for many years as a suicidal or homicidal agent, because it is a major contaminant of drinking water in a number of countries (60-62), and because it is present in Lewisite, the chemical warfare agent, it is not surprising that there have been continuing efforts to find antidotes for it. It should be clearly understood, howeVer, that the best way to deal with arsenic toxicity is to preVent exposure. BAL (dimercaprol, 2,3 dimercapto-1-propanol) (Figure 7) was developed during World War II as an antidote against the arsenic-containing chemical warfare agent Lewisite (34, 63). BAL is a fat-soluble, easily oxidizable oil that must be given



Figure 5. Summary of frequency and gene context of polymorphisms discovered in hGSTO1 in European ancestry (Europe) and indigenous American (America) ancestry subjects (53). The ID column indicates the polymorphism identification numbers relative to the location in the consensus sequence with the first base of the consensus numbered 1. The ATG offset column indicates the polymorphism location relative to the first base “A” of the ATG methionine initiation codon. The freq % column is the minor allele frequency graphically displayed in the column to the right (53). Reprinted with permission from EnVironmental Health PerspectiVes.

Figure 6. Summary of frequency and gene context of polymorphisms discovered in CYT 19 in European ancestry (Europe) and indigenous American (America) ancestry subjects (55). The ID column indicates the polymorphism identification number relative to the location in the consensus sequence, with the first base of the consensus numbered 1. The ATG offset column indicates the polymorphism location relative to the first base “A” of the ATG methionine initiation codon. The freq % column is the minor allele frequency, graphically displayed in the column to the right (55). Reprinted with permission from EnVironmental Health PerspectiVes.

by deep intramuscular injection. More than 50% of the patients to whom it is administered suffer side effects. While the side effects usually are not serious and quickly disappear when BAL treatment is stopped, they are annoying and discomforting. In addition, because it is not given by mouth, because of its

instability during storage, and because of concerns that treatment with it resulted in increased brain arsenic levels in rabbits (64), BAL has been replaced by two water-soluble chemical analogues, DMPS and DMSA. The former was synthesized and tested in animals by Petrunkin’s group in Kiev (65). DMSA

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Figure 7. Chemical formulas for chelating agents used to treat arsenic toxicity.

was developed by Ding and Liang (66) in China and Freidheim (67) in Switzerland. The latter agent is approved by the U.S. Food and Drug Administration (FDA) for treating children with blood lead levels equal to or greater than 45 µg/dL. In addition, there have been a number of “off-label” uses as arsenic and mercury antidotes (68). Although no drug is without unwanted side effects, DMPS is a relatively safe chelating and reducing agent approved in Germany for treatment of mercury toxicity. However, there have been reports of serious idiosyncratic reactions to it (69). Even though a new drug application for it has never been submitted to the U.S. FDA, it has been used extensively in the United States by alternative medicine physicians who obtain it from compounding pharmacists. An excellent, extensive, well-written monograph entitled “Dimaval, DMPS” (70) is available from the manufacturer and its U.S. subsidiary Heyl-Tex. The present authors recommend that any physician prescribing DMPS obtain and read it.

6.1. Treatment of Acute Exposure After two brothers ingested 1 and 3 g of pure arsenic trioxide, DMPS was given intravenously at 5 mg/kg every 4 h for 24 h and then 400 mg orally every 4 h for an additional 5 days. There was no prolonged renal or neurological impairment that usually is seen in untreated arsenic poisoning (71). There have been other reports of the successful use of DMPS for recovery from arsenic-induced neuropathy (72). A recent report (69) concerning a young man who tried to commit suicide by ingesting arsenic trioxide is of interest. The concentration of total arsenic in the first urine collected after hospital admission was 215 mg/L. After 8 days of DMPS treatment, it decreased 1000-fold. The chelating agent was administered over 12 days with a total dose of 15.25 g of DMPS, some by iv perfusion and some by mouth. The urinary DMA accounted for less than 5% of total urinary arsenic. It usually accounts for 60-70%. On day eight of therapy, the urine contained arsenite, arsenate, MMA, and DMA, the sum of which amounted to only 64.4% of the total urinary arsenic indicating that the excreted DMPS-arsenic species complex was not being detected or measured by the analytical method employed. This had been noted and suggested previously in studies by the Aposhian group (74-76). The almost complete absence of DMA state may have been due to the inhibition of the second methylation reaction, although other mechanisms are possible (76).

6.2. Treatment of Chronic Exposure Treatment after chronic exposure to toxic levels of inorganic arsenic, usually via drinking water but also by the ingestion of

food contaminated by coal fly ash containing large amounts of arsenic (77), has presented a challenge. To decrease the body’s arsenic burden and then return the patient to the area where arsenic exposure occurred is of little and questionable benefit to the patient. Both DMPS and DMSA have been studied. DMPS increased the urinary excretion of arsenic in such chronic exposures (78, 79), but just because the excretion of a toxic metal has been increased does not mean clinical improvements always occur (80). Guha Mazumder et al. (81), using a randomized placebo-controlled trial of DMPS and a scoring system before and after treatment, concluded that this chelating agent not only increased urinary excretion of arsenic but also improved the clinical score that was used to judge the clinical condition of the arsenosis patients. The number of patients, 11, was small. Although DMSA has had success in increasing the urinary excretion of arsenic after acute exposure (73), it was unsuccessful in reversing the biochemical or histological response in chronic arsenosis (83). While a clinical trial with a much larger number of subjects needs to be done, the immediate usefulness of such a trial for people in India and Bangladesh is doubtful since after chelation treatment most would return to their homes where exposure would continue. Thus, the chelation treatment would appear to have limited, if any, justification. In addition, chronic arsenosis occurs in areas that are among the poorest in the world, for example, Bangladesh, the West Bengal region of India, southwest China, Mongolia, and others. Most people in these areas could not afford chelation therapy, if it were useful and available. We cannot help but wonder in this time of concerns over environmental justice if chronic arsenic toxicity due to drinking contaminated water had occurred in the developed, wealthy, Western countries rather than in the poor, developing countries, that an inexpensive, easy to use water purification procedure as a solution to the greatest public health calamity of the last 30 years would have been achieved by now. The economic middle and upper classes can buy low arsenic bottled water. The poor cannot, and it is their children who suffer the most from such environmental injustice. Excellent and more extensive reviews of chelating agents by Andersen (84) and BAL by Muckter et al. (85) are available and highly recommended for reading. In unexposed individuals, the arsenic concentrations usually found in the blood are 2.5 µg As/L, and in the urine, they are 10-50 µg As/L (82). A recent report concerning the concentrations of arsenic species in human organs after arsenic trioxide poisoning is available and useful (86). It appears reasonable that a cheap, effective, and useful method of purifying highly contaminated drinking water eventually will be found. At that time, the removal of body stores of arsenic needs to be considered. The removal of these arsenic


storage depots may be necessary to eliminate any potential carcinogenic manifestations that had not occurred up to that time. It is at such a point that chelating agents might be helpful.

6.3. Conclusion Four Although the clinical studies are limited, it would appear that DMPS is the best drug available for increasing the excretion of arsenic and improving the conditions of humans exposed to various forms of this metalloid. Idiosyncratic reactions, however, have occurred. A well-designed, large clinical trial of the effectiveness of DMPS in treating chronic arsenosis is needed. It is necessary to plan now for what will be needed once a useful method for purifying drinking water contaminated with high concentrations of arsenic becomes available. At that time, there may be the need to use DMPS or other chelating agents to reduce body stores of arsenic to minimize arsenic cancer risks.

7. Question Five: What Is the Role of Protein Binding in Arsenic Metabolism and Toxicity? A number of early reports appeared dealing with the binding of arsenic species to proteins (87-90). The importance of such binding is at least 2-fold. First, it includes the binding and resulting inhibition of enzymes to cause arsenic toxicity. Second, it may be a mechanism for the modulation of arsenic toxicity by immobilization of the arsenic compound in an arsenic protein reservoir (91). In hindsight, many of the reports used inadequate procedures that subsequently have been replaced by mass spectrometry and other more sophisticated procedures. For years, the mechanism of arsenic species having a +3 oxidation state has been claimed to be their reaction with thiol compounds, e.g., GSH, cysteine, lipoic acid, and/or the thiols of proteins. Very few of these proteins have been identified. Such a broad description of arsenic binding and toxicity, although correct, is no longer adequate with today’s highly sophisticated proteomic techniques such as DIGE and mass spectrometry. Certainly, protein binding of arsenic species is implicated in their metabolism and toxicity. The arsenic species with a +3 oxidation state are chemically more reactive than the +5 species (92). The former species, however, each have a different degree of reactivity. For example, arsenite has three binding sites, MMA(III) has two, and DMA(III) has one. This has been extensively discussed in an excellent review by Carter et al. (93). The review, written mainly by Professor Carter, is highly recommended reading for those who wish to learn about the many facets and needs of arsenic toxicology research from a chemical point of view.

7.1. Hemoglobin Binding A collaborative effort has used chemical and biological techniques to elucidate the binding of +3 arsenic species to rat and human hemoglobin (93). In the past, the prevailing speculation had been that the increased concentration of arsenic in rat blood was due to DMA(V) binding to hemoglobin (93, 94). Because one of the major changes in understanding arsenic metabolism and toxicity has been the result of the recent evidence demonstrating the greater reactivity and toxicity of MMA(III) and DMA(III) (44, 45, 92), the investigators studied the affinity of these +3 arsenic species for rat or human hemoglobin. They used chromatography and nanoelectrospray mass spectrometry. The apparent binding constants (Table 4) showed that arsenic binding to rat hemoglobin was 3-16-fold greater than for binding to human hemoglobin.

Aposhian and Aposhian Table 4. Apparent Binding Constants (nK) for Trivalent Arsenicals Binding to rHB and hHb (93) nK (M-1) arsenic species

rHb

hHb

iAs (III) MMA(III) DMA(III) PhAs(III)O

.0233 × 105 .469 × 105 2.22 × 105 5.35 × 105

.007 × 105 .050 × 105 .136 × 105 .775 × 105

The binding was consistent with the number of reactive cysteine residues in the R- and β-chains of hemoglobin. There are three cysteines, Cys 13, Cys 104, and Cys 111, in each R-chain and two cysteines, Cys 93 and Cys 125, in each β-chain of rat hemoglobin. The rat tetramer contains two R- and two β-units, a total of 10 DMA(III) molecules can be bound to the 10 sulfhydryl groups of rat hemoglobin. Human hemoglobin contains only one cysteine in each R-chain (Cys 104) and two in each β-chain (Cys 93 and Cys 112). It would be expected that six DMA(III) molecules would bind to human hemoglobin. However, the investigators suggest that hydrophobicity may also be a factor. It is unfortunate that they did not incorporate some ROS studies into this excellent paper (93). These studies were extended to in vivo rat experiments (93). Young rats were fed a diet containing 100 mg DMA(V)/kg for 72 days after which they were euthanized and plasma and red cells were collected. The arsenic in red cells was predominantly protein bound. The arsenic concentration in the plasma was 7.3 ( 1.0 µM and that in the red blood cells (RBCs) was 1101 ( 130 µM. Therefore, the arsenic concentration of rat RBCs under these experimental conditions was 150 times greater than that found in the plasma. Hayakawa et al. (5) incubated at pH 7.0 a 20% rat liver homogenate for 20 min at 37 °C with 1 µM various arsenic species. The reaction was stopped by boiling, centrifuging the pellet, and acid digestion, and the arsenic concentration was measured by ICP-MS. The binding of MMA(III) was the greatest. Arsenite had 60% of the MMA(III) binding activity. DMA(III), arsenate, MMA(V), and DMA(V) each had about 15%. Because the DMA(III) was produced by the reduction of DMA(V) using metabisulfite-thiosulfate (5, 94), questions remain about the interpretations of these results. The Reay-Asher procedure not only would produce DMA(III) but also significant amounts of thioarsenicals (95, 96).

7.2. Metallothionein Binding Metallothionein is an unusual polypeptide. It contains 20 cysteine residues. It has been implicated in the detoxication of many toxic metals (97), scavenging of free radicals (98), and metal transport (99). There have been many unconvincing attempts to connect it with arsenic metabolism. A straightforward, careful study of how arsenic compounds having a +3 oxidation state interact with apo-metallothioneins has appeared (100) using commercially available rabbit metallothionein II. Each metallothionein molecule bound up to six As(III), 10 MMA(III), and 20 DMA(III) molecules. Because arsenite has three binding sites and metallothioneins have 20 cysteine residues, the maximum number of arsenite binding to metallothionein should be six. MMA(III) has two binding sites so up to 10 of this arsenic species would be expected to bind to metallothionein. Because DMA(III) has only one binding site, it would be expected that MT would bind up to 20 DMA(III). Their experimental results confirmed the theoretical expectations, and no binding of TMAO(V) was detected. Neither could complexes of MMA(V) nor DMA(V) with metallothionein be

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Figure 9. Example of information obtainable by DIGE. Figure 8. DIGE: Sample preparation.

detected. It would be of interest to do such experiments in vivo to determine its relevance to arsenic modulations in the whole animal. While this study is a revealing one, it would have been improved if experiments or previously reported data were used to estimate the relative affinities of these arsenic species as compared to cadmium, mercury, and lead.

7.3. Other Pertinent Proteomic Papers An excellent, innovative paper from van Houten’s laboratory combining genomics and proteomics of metals and yeast is unusual for its comprehensiveness, clarity, and relevance (101). Kitchen and Wallace (102) have reported arsenite binding to synthetic peptides based on the Zn finger region and estrogenbinding region of the human estrogen receptor. One must not ignore older, classic papers on arsenic toxicology by Vahter. For example, the first indication of some animals not methylating inorganic arsenic but detoxifying by protein binding was presented by her (88, 89).

more specific knowledge as to the exact role of specific proteins in arsenic intoxication and detoxication. Using two-dimensional electrophoresis procedures in which protein extracts from two subjects can be electrophoresed together by using two fluorescent dyes followed by MS of protein spots may quicken the understanding of the role of proteins in arsenic metabolism. A recent study clearly has shown that the differences in the number of cysteine residues in human and mouse hemoglobin are responsible for the greater accumulation of arsenic species in rat blood.

8. Areas of Concern and Conclusions

In the authors’ laboratory, DIGE has been used to study the liver proteins of mice given sodium arsenite in drinking water each day for 14 days. Proteomics (DIGE) has many advantages over microgene array analysis. The latter depends on measuring the gene transcript (mRNA). The former measures protein synthesis including any posttranslational modifications. Not everything that is transcribed is translated. This proteomic procedure (103) is becoming an important exploratory procedure in toxicology research. It can compare the relative amounts of specific proteins in body fluids or tissues of two subjects by placing extracts on the same space of a single gel and performing an electrophoresis first by isoelectric point and second by molecular weight (Figure 8). By having a dye that fluoresces a certain color in one extract and a dye of a different color in the second extract, not only can the proteins be separated, but by using a scanner and specific software, the relative amounts of a specific protein in one sample can be determined to be more, equal to, or less than in the other (Figure 9). The procedure is expensive because of the costs of the dyes, use of a scanner plus appropriate software, and finally mass spectrometry of each protein spot.

There are areas of concern that are related to most of the five questions that we have addressed. Investigators need to keep them in mind while they do research and deal with the enigma of arsenic toxicology. Dose-response is one such area. An attempt to characterize a dose-response relationship between arsenic concentrations in drinking water in West Bengal, India, and arsenic-induced skin keratoses and hyperpigmentation has appeared (104). The average latency for skin lesions was 23 years from the first exposure. The lowest peak arsenic ingested by a confirmed case was 115 µg As/L. Although such values are helpful signposts, they need to be viewed with caution when attempts are made to extend such values to other areas of the world where genes, nutrition, and other factors may be different, even though the drinking water contains similar concentrations of arsenic. The major cause for such concern would be genetics more specifically polymorphisms of the arsenic biotransformation genes in different ethnic groups. Climate differences in other parts of the world also can influence the volume of water consumed and the resulting arsenic exposure. Arsenic antagonists such as selenium salts (28) being present in different concentrations in different countries may influence threshold values. The above-mentioned dose-response study, hopefully, is preceding more extensive studies. The results of a paper (105) studying arsenic exposure and the intellectual function of Bangladesh children need to be of concern to policy makers and to financial organizations that support research on the arsenic catastrophe in Bangladesh and in parts of India. Exposure from drinking water with high levels of arsenic was associated with reduced intellectual function in a dose-related manner in these children. This important, highly relevant paper by the Graziano group is further proof for the need of environmental justice for the children of the poor.

7.5. Conclusion Five

8.1. Tolerance

With new proteomic techniques available, such as DIGE, research in arsenic toxicology now may be expanded to acquire

Ever since the arsenic eaters of Styria were studied by British physicians (106, 107) during the mid-1800s, the question of

7.4. DIGE


the mechanisms for the development of tolerance to inorganic arsenic by humans has been a challenge to many arsenic investigators. After all of these years, the challenge has not been answered successfully. Romach et al. (108) using chronic arsenite-exposed rat liver epithelial cells found that the cells accumulated 87% less As as compared to controls. The tolerance seemed to be acquired without changes in GSH or metallothionein. More studies of these fascinating arsenic tolerance phenomena are needed including whether there is inducibility of the arsenic biotransforming enzymes in human and rabbit cells.

8.2. Inappropriate Procedures for Synthesis The Reay and Asher procedure (94) was designed to reduce arsenate to arsenite using metabisulfate-thiosulfate. Many investigators have used this procedure believing that they were converting MMA(V) to MMA(III) and DMA(V) to DMA(III). Some neglected, however, to confirm the structure of the product and/or to remove unreacted starting materials and unknown products of the reaction. The results of most of these published experiments that used the Reay and Asher procedure to prepare MMA(III) and DMA(III) as enzyme substrates or tissue culture media constituents require, at the minimum, reevaluation and, at the maximum, disbelief. Most of these now questionable studies were performed before the Reay and Asher procedure was shown to produce thioarsenicals (95, 96).

8.3. Inappropriate Procedures for Urine Collection Since the initial finding of MMA(III) in human urine by Aposhian et al. (79), there has been concern about how to collect and store urine in order to minimize oxidation of MMA(III). The Le group has been a major contributor in trying to solve this problem (109, 110, 111). A recent study by Valenzuela et al. (112) found that MMA(III) was 7.4% and DMA(III) 49% of the total urinary arsenic. These are the highest levels ever reported especially for DMA(III), and the authors deserve to be complimented on obtaining such results due to quickly freezing the urine in dry ice and analysis within 6 h after collection. However, because most studies of arsenic-exposed populations take place in countries without adequate analytical laboratories, the need continues for easy, convenient methods of collecting and storing urine samples that cannot be analyzed within a period of 6 h because of transporting time. Because it is generally accepted that the matrix affects the stability of all urinary arsenic species, the Valenzuela et al. results also might have been influenced by reducing substances in urine originating in the diet. This, however, does not minimize the importance of their results. Continued efforts are needed to stabilize arsenic species in the urine.

8.4. Compound Identification Another area of concern has been the use of only one property of a compound to identify it. For example, the use of only retention times after HPLC or Rf after paper or thin-layer chromatography leaves a great deal to be desired as far as identification of new intermediates and metabolites. Mass spectrometry now is available at most research institutions and needs to be used for initial or confirmatory identification of chemical structures.

8.5. Inadequacy of the Rat as a Model for Arsenic in Humans It has been known for many years that DMA will bind rat RBCs to a greater extent than red cells of other species. (The


reader may want to review question five of this review at this point). The extrapolation of arsenic toxicokinetic and metabolic studies from rat experiments can lead to erroneous conclusions since many investigators have ignored these different properties of rat RBCs. In addition, the urinary arsenic species, as percent of total arsenic, of the rat are very different than the human (113). On the basis of urine arsenic species, if an animal must be used, it appears that the hamster and rabbit are the most reasonable and desirable. Rat studies should be viewed with caution and the result should not be extended to or used as a model for the human. Having said this, it needs to be realized that the rat may be one of the few arsenic carcinogenic models available at present. The best model system for studying arsenic toxicology and risk assessment in the human remains the human. Human tissues are available for in vitro studies, and the human body can be used in vivo for ethical and safe excretion and epidemiology studies.

8.6. Summary We have tried to address five questions dealing with five of the arms of arsenic toxicology: biotransformation, ROS, polymorphism, treatment, and protein binding. The first question, “What enzyme is responsible for the methylation of arsenic species in the human?”, still needs further investigative effort to obtain an answer. The dilemma continues. For CYT 19 to be accepted as the methylation enzyme of humans, purification of the protein and its activity from surgically removed human tissue is required. This has not been accomplished for either CYT 19 or the rabbit methyltransferase. Second, the conventional belief that arsenite inhibits PDH and perhaps other dithiol-containing enzymes by chelating the thiol groups now needs to be expanded to include ROS. The latter also can be generated as an inhibitory agent by arsenicals. Third, a number of polymorphisms in human GST ωs, CYT 19, and PNP have been reported. Two studies have linked these polymorphisms with changes in urinary arsenic species. There has been a minimum of investigations dealing with both studies of polymorphisms of human genes known to be involved in arsenic metabolism and the determinations of all of the possible urinary arsenic species, especially MMA(III) and DMA(III). In fact, the genetics of arsenic toxicity is a barren field at present. Fourth, DMSA and DMPS are effective in mobilizing the excretion of arsenic from the human. DMPS seems to be more consistently effective in the clinical improvement of individuals chronically exposed to arsenic. With the millions of people now known to be consuming toxic amounts of arsenic in their drinking water or food, a large-scale clinical trial of arsenic antidotes is needed and recommended so that when remediation of arsenic exposure is finally accomplished, arsenic body burdens of exposed humans can be decreased safely. Fifth, with new proteomic techniques available, such as DIGE, research in arsenic toxicology now may be expanded to acquire more specific knowledge as to the exact role of specific proteins in arsenic intoxication and detoxication. Using two-dimensional electrophoresis procedures in which protein extracts from two subjects can be electrophoresed together by using two fluorescent dyes followed by MS of protein spots may quicken the understanding of the role of proteins in arsenic metabolism. A recent study clearly has shown that the differences in the number of cysteine residues in human and mouse hemoglobin are responsible for the greater accumulation of arsenic species in rat blood. While a number of recent papers have been emphasized in this review, the need for confirmation of their conclusions by

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other investigators is needed. The importance of their results at this time, however, should not be minimized. Rather, they should stimulate investigators to reexamine and expand their thinking and investigations and, hopefully, attract new investigators. Acknowledgment. We are grateful to Dr. Robert A. Zakharyan and Dr. Uttam Chowdhury for reading and critically reviewing this manuscript. This review was written while the research in our laboratory was supported in part by Superfund Basic Research Program NIEHS Grant ES-04940, the Southwest Environmental Health Sciences Center Grant P30-Es-06694 from the National Institute of Environmental Health Sciences, and the Wallace Research Foundation.

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