The potential biological mechanisms of arsenic-induced diabetes mellitus

Toxicology and Applied Pharmacology 197 (2004) 67 – 83 www.elsevier.com/locate/ytaap

The potential biological mechanisms of arsenic-induced diabetes mellitus

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Chin-Hsiao Tseng * Division of Endocrinology and Metabolism, Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan Division of Environmental Health and Occupational Medicine of the National Health Research Institutes, Taipei, Taiwan Received 10 December 2003; accepted 13 February 2004 Available online 20 April 2004

Abstract Although epidemiologic studies carried out in Taiwan, Bangladesh, and Sweden have demonstrated a diabetogenic effect of arsenic, the mechanisms remain unclear and require further investigation. This paper reviewed the potential biological mechanisms of arsenic-induced diabetes mellitus based on the current knowledge of the biochemical properties of arsenic. Arsenate can substitute phosphate in the formation of adenosine triphosphate (ATP) and other phosphate intermediates involved in glucose metabolism, which could theoretically slow down the normal metabolism of glucose, interrupt the production of energy, and interfere with the ATP-dependent insulin secretion. However, the concentration of arsenate required for such reaction is high and not physiologically relevant, and these effects may only happen in acute intoxication and may not be effective in subjects chronically exposed to low-dose arsenic. On the other hand, arsenite has high affinity for sulfhydryl groups and thus can form covalent bonds with the disulfide bridges in the molecules of insulin, insulin receptors, glucose transporters (GLUTs), and enzymes involved in glucose metabolism (e.g., pyruvate dehydrogenase and a-ketoglutarate dehydrogenase). As a result, the normal functions of these molecules can be hampered. However, a direct effect on these molecules caused by arsenite at physiologically relevant concentrations seems unlikely. Recent evidence has shown that treatment of arsenite at lower and physiologically relevant concentrations can stimulate glucose transport, in contrary to an inhibitory effect exerted by phenylarsine oxide (PAO) or by higher doses of arsenite. Induction of oxidative stress and interferences in signal transduction or gene expression by arsenic or by its methylated metabolites are the most possible causes to arsenic-induced diabetes mellitus through mechanisms of induction of insulin resistance and h cell dysfunction. Recent studies have shown that, in subjects with chronic arsenic exposure, oxidative stress is increased and the expression of tumor necrosis factor a (TNFa) and interleukin-6 (IL-6) is upregulated. Both of these two cytokines have been well known for their effect on the induction of insulin resistance. Arsenite at physiologically relevant concentration also shows inhibitory effect on the expression of peroxisome proliferator-activated receptor g (PPARg), a nuclear hormone receptor important for activating insulin action. Oxidative stress has been suggested as a major pathogenic link to both insulin resistance and h cell dysfunction through mechanisms involving activation of nuclear factor-nB (NF-nB), which is also activated by low levels of arsenic. Although without supportive data, superoxide production induced by arsenic exposure can theoretically impair insulin secretion by interaction with uncoupling protein 2 (UCP2), and oxidative stress can also cause amyloid formation in the pancreas, which could progressively destroy the insulin-secreting h cells. Individual susceptibility with respect to genetics, nutritional status, health status, detoxification capability, interactions with other trace elements, and the existence of other well-recognized risk factors of diabetes mellitus can influence the toxicity of arsenic on organs involved in glucose metabolism and determine the progression of insulin resistance and impaired insulin secretion to a status of persistent hyperglycemia or diabetes mellitus. In conclusions, insulin resistance and h cell

Abbreviations: ADP, adenosine diphosphate; AP-1, activating protein-1; ATP, adenosine triphosphate; DNA, deoxyribonucleic acid; ERK, extracellular signal-regulated kinase; GLUT, glucose transporter; GR, glucocorticoid receptor; GSH, glutathione; IL-6, interleukin-6; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; NADPH, nicotinamide adenine dinucleotide phosphate; NF-nB, nuclear factor-nB; PAO, phenylarsine oxide; PEPCK, phosphoenolpyruvate carboxykinase; PI-3 kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PPARg, peroxisome proliferator-activated receptor g; ROS, reactive oxygen species; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus; TNFa, tumor necrosis factor a; UCP2, uncoupling protein 2. $ The scientific content of this manuscript has been reviewed and approved for publication by the Division of Environmental Health and Occupational Medicine of the National Health Research Institutes. Approval for publication does not necessarily signify that the content reflects the view and policies of the DEHOM/NHRI, or condemnation or endorsement and recommendation for use on this issue presented. * Division of Endocrinology and Metabolism, Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan. Fax: +886-2-23883578. E-mail address: [email protected]. 0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.02.009

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dysfunction can be induced by chronic arsenic exposure. These defects may be responsible for arsenic-induced diabetes mellitus, but investigations are required to test this hypothesis. D 2004 Elsevier Inc. All rights reserved. Keywords: Inorganic arsenic; Diabetes mellitus; Glucose metabolism; Reactive oxygen species; Individual susceptibility; Environmental pollutants; Trace elements

Introduction Arsenic is a Class A human carcinogen, which causes an increased risk of cancers originating from the lung, liver, kidney, bladder, and skin (Kitchin, 2001). Arsenic is also atherogenic and neuropathogenic. Exposure to arsenic from drinking artesian well water in the arseniasis-hyperendemic areas in Taiwan can induce an endemic peripheral vascular disease known as blackfoot disease (Tseng, 2002). Recent studies also demonstrated that arsenic is closely related to ischemic heart disease (Tseng et al., 2003), stroke (Chiou et al., 1997), hypertension (Chen et al., 1995a, 1995b), subclinical sensory neuropathy (Tseng, 2003), and neurobehavioral dysfunction (Tsai et al., 2003). According to the classification recommended by the American Diabetes Association (2004), diabetes mellitus can be classified as type 1, type 2, other specific types, and gestational diabetes mellitus. Type 1 diabetes mellitus (T1DM) always occurs in childhood and adolescence with abrupt clinical manifestation of diabetic ketoacidosis resulting from absolute deficiency of insulin (a minimal amount of insulin is required to suppress lipolysis with end products of ketone body, which causes diabetic ketoacidosis) caused by autoimmune or idiopathic destruction of pancreatic h cells (American Diabetes Association, 2004). Insulin injection is always necessary for survival in T1DM because of the complete lack of endogenous insulin (American Diabetes Association, 2004). Type 2 diabetes mellitus (T2DM) accounts for more than 90 –95% of all diabetes with unknown specific etiology, but hereditary factors, aging, and obesity are important risk factors (American Diabetes Association, 2004). Insulin resistance (a term refers to impaired tissue response to insulin) occurs during the early phase of T2DM, but the disease frequently goes undiagnosed for many years because hyperglycemia during the earlier stages is not severe enough to cause symptoms (American Diabetes Association, 2004). Insulin resistance can be resulted from mutations or posttranslational modifications of the insulin receptor itself or any of its downstream effector molecules. However, postreceptor defects are the most commonly seen (Le Roith and Zick, 2001). Initially, insulin resistance is compensated for by hyperinsulinemia, through which normal glucose level is maintained, but at the time of diagnosis of T2DM, h cell dysfunction is always present (Kahn, 2003). Ketoacidosis is not common at the time of diagnosis of T2DM because the pancreas can still secrete the minimal

concentration of insulin required for the suppression of lipolysis (Mahler and Adler, 1999). Most patients do not require insulin injection and oral antidiabetic agents can be given during the earlier stages of T2DM. With the progression of T2DM, especially when long-term glycemic control is not adequate, pancreatic h cell dysfunction can be so severe that insulin injection is necessary (Mahler and Adler, 1999). If the underlying cause of diabetes mellitus is well characterized, it can be classified under the term of ‘‘other specific types’’, which includes known genetic defects of h cells and insulin action, diseases of the exocrine pancreas, some endocrinopathies, drug- or chemical-induced, infections, and other uncommon forms associated with immune disorders or genetic syndromes (American Diabetes Association, 2004). The term ‘‘gestational diabetes mellitus’’ only refers to the diagnosis of diabetes in women during pregnancy. The relationship between arsenic exposure and diabetes mellitus is a relatively novel finding. This link has been observed in people drinking contaminated well water in Taiwan (Lai et al., 1994; Tseng et al., 2000, 2002) and Bangladesh (Rahman et al., 1998, 1999), and in people working in copper smelters (Rahman and Axelson, 1995) and art glass industry (Rahman et al., 1996) in Sweden. If the cause of diabetes mellitus can be confirmed as ascribed to arsenic exposure, the diabetes can be classified as ‘‘arsenic-induced’’ fitting the classification of ‘‘drug- or chemical-induced’’ under ‘‘the other specific types’’ according to the recommendation of the American Diabetes Association (2004). The features of diabetes mellitus observed in arsenic-exposed subjects in epidemiologic studies are actually similar to T2DM. This similarity is clearly demonstrated by the prospective study carried out in the blackfoot disease-hyperendemic villages in Taiwan by Tseng et al. (2000), in which all of the incident cases were diagnosed by an oral glucose tolerance test, and none of them developed diabetic ketoacidosis or required insulin treatment during the period of follow-up. Therefore, with the knowledge learned from the epidemiologic studies, the pathophysiology involved in the development of diabetes mellitus after long-term and sublethal dosage of arsenic exposure in human beings is not due to a quick, massive, and complete destruction of the pancreatic h cells, which would clinically mimics T1DM rather than T2DM. Because of the slow and progressive clinical course similar to T2DM, it is believed that the pathophysiology associated with

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arsenic-induced diabetes mellitus should be more likely to that of T2DM, which is characterized by a dual defect of both insulin resistance and a relative deficiency in insulin secretion (Kahn, 2003). Although epidemiologic data can provide the information for the generation of hypotheses, they are not able to evaluate the cellular and molecular mechanisms of disease pathogenesis. Therefore, it is necessary to have a further look into the potential biological effects of arsenic on glucose metabolism, which could provide evidence for the biological plausibility in arsenic-induced diabetes mellitus. In this paper, the author reviewed the glucose homeostasis under normal physiological conditions, the pathophysiology of T2DM, and the biochemical properties of arsenic first, and then discussed and summarized the most possible mechanisms responsible for arsenic-induced diabetes mellitus.

An overview on glucose homeostasis The balance between the rates of glucose entry into the circulation (mainly from the liver in the fasting state and the gut after meals) and of its uptake into the peripheral tissues (mainly skeletal muscles, and to a lesser extent, the adipocytes) maintains blood glucose levels within tight limits in normal conditions. Insulin, a hormone secreted by the islet h cells of the pancreas, is the principal hormone to lower blood glucose by suppressing gluconeogenesis and glycogenolysis in the liver and by stimulating the uptake of glucose into skeletal muscle and fat. Insulin molecule contains 2 peptide chains called the A chain and B chain. Two disulfide bonds link these two chains together and insulin exerts its physiological actions by binding to its receptor on cell membrane (Massague et al., 1980). The insulin receptor complex contains two a- and two h-subunits, linked together by interchain disulfide bridges (Massague et al., 1980). Insulin binding to the a-subunits causes a cascade of signaling events including autophosphorylation of tyrosine residues on the h-subunits, tyrosine phosphorylation of the insulin receptor substrates (IRS), and activation of the phosphatidylinositol 3-kinase (PI-3 kinase) pathway, which triggers a series of downstream events involving protein kinase C (PKC), leading to insulin-stimulated translocation of glucose transporters (GLUTs) to the plasma membrane and glucose transport via the transporters (Bandyopadhyay et al., 1997; Gammeltoft and Van Obberghen, 1986; Kotani et al., 1998; Le Roith and Zick, 2001; Rosen, 1987; Standaert et al., 1997; White and Kahn, 1994). This PI-3 kinase dependent pathway is responsible primarily for the metabolic response to insulin (Le Roith and Zick, 2001). However, alternative pathways exist for the stimulation of GLUTs translocation. The mitogen-activated protein kinases (MAPK) are serine/threonine protein kinases, which are activated in response to a variety of external stimuli. There are four known families of MAPK at present: extracellular

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signal-regulated kinase (ERK), c-jun-NH2-terminal kinase, p38, and big MAPK-1 (Harris and Shi, 2003). Glucose transport induced by muscle contraction is mediated by the MAPK pathway (Widegren et al., 2001). Full stimulation of glucose uptake following translocation of GLUTs by insulin may also involve a p38 of the MAPK pathway (Somwar et al., 2001). Activating protein-1 (AP-1), a class of transcription factors that can be activated by the MAPK pathway, is associated with GLUT1 expression in cardiac myocytes (Santalucia et al., 2003) and formation of GLUT4-containing vesicles in rat adipocytes (Gillingham et al., 1999). Metabolic stress can induce glucose transport via the 5Vadenosine monophosphate-activated protein kinase pathway, which is also independent of insulin-activated PI-3 kinase (Hayashi et al., 2000). Hyperglycemia per se can stimulate translocation of GLUTs through a pathway involving PKC and dependent on the proline-rich tyrosine kinase-2/ERK/phospholipase D, but independent of PI-3 kinase (Bandyopadhyay et al., 2001a, 2001b). The disulfide bonds are essential for both insulin binding and autophosphorylation (Pike et al., 1986). Specific GLUTs carry extracellular glucose across cell membrane into the cell (Olson and Pessin, 1996). The widely distributed GLUT1 mediates much of the body’s basal glucose transport and non-insulin-mediated glucose uptake. Insulinsensitive tissues express GLUT4, which is responsible for the large increase in glucose uptake into skeletal muscle, cardiac muscle, and fat. Pancreatic h cells express GLUT2, which plays important role in the regulation of insulin secretion (Olson and Pessin, 1996). The exofacial sulfhydryl groups of the GLUTs play an important role in the maximal activity of the transporters and are crucial for the regulation of transport rates by insulin (May, 1985). Thus, the sulfhydryl groups have important structural and functional roles in insulin, insulin receptors, and GLUTs. In addition to the effect of transporting glucose into target cells, insulin also influences glucose synthesis and metabolism, through the phosphorylation and dephosphorylation of enzymes, possibly by affecting their expression at the level of gene transcription (Le Roith and Zick, 2001). Therefore, the effect of insulin on gene transcription may explain some of its effects on glucose uptake, synthesis, and metabolism.

Pathophysiology of type 2 diabetes mellitus The pathophysiology leading to hyperglycemia in T2DM is very complicated and has not been completely understood. Any gene mutation or metabolic disturbance leading to a defect in insulin secretion, insulin transport, insulin action, glucose transport, or enzymes associated with glucose metabolism can theoretically result in hyperglycemia or clinical diabetes. An example for genetic subtypes of T2DM involves mutations in glucokinase, which phosphorylates glucose to glucose-6-phosphate,

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leading to impaired glycolysis (Fajans et al., 2001). The impairment of glucose transport into skeletal muscle and adipocytes, which can be due to a variety of mechanisms involving insulin receptor defects or postreceptor defects, is the main cause of T2DM (Le Roith and Zick, 2001). Overexpression of tumor necrosis factor a (TNFa) in fat and muscle cells has been implicated as an inducer of insulin resistance by increasing the serine phosphorylation of IRS-1 and IRS-2, resulting in a reduction in the ability of the IRS molecules to dock with receptor and interact with downstream pathways (Le Roith and Zick, 2001). Interleukin-6 (IL-6) has also been found to play an important role in the induction of insulin resistance in adipocytes (Lagathu et al., 2003; Rotter et al., 2003). Chronic treatment with IL-6 to adipocytes can diminish expression of the h-subunit of insulin receptor, IRS-1, and GLUT4, resulting in reduced glucose transport (Lagathu et al., 2003). Insulin-induced activation of h-subunit of insulin receptor, ERK-1, and ERK-2 are also inhibited by IL-6 (Lagathu et al., 2003). Although the expression of p38 MAPK in patients with T2DM and in nondiabetic subjects is similar and basal p38 MAPK phosphorylation is increased in skeletal muscle in patients with T2DM, the insulin-stimulated p38 MAPK phosphorylation is only noted in nondiabetic subjects, but not in patients with T2DM (Koistinen et al., 2003). On the other hand, peroxisome proliferator-activated receptor g (PPARg) is an adipose-selective nuclear hormone receptor that plays a key role in the control of adipocyte differentiation by complexing with a retinoid X receptor (Umek et al., 1991). Activation of PPARg also plays an important role in glucose homeostasis by increasing the sensitivity of insulin (Lehmann et al., 1995). One of the results of activating PPARg is a reduction of TNFa expression (Jiang et al., 1998). PPARg is also downregulated in adipocytes with the treatment of IL-6, and the effects of IL-6 can be prevented by rosiglitazone, a PPARg agonist (Lagathu et al., 2003). The expression of TNFa and IL-6 are regulated by nuclear factornB (NF-nB) (Chen et al., 1999). Recent studies have pointed out that reactive oxygen species (ROS) resulting from hyperglycemia or other stress stimuli can lead to insulin resistance through its interactions with cytokines and other mediators involving the activation of NF-nB pathway (Haber et al., 2003; Hitsumoto et al., 2003; Katsuki et al., 2004; Rudich et al., 1997; Shimosawa et al., 2003; Shinozaki et al., 2003; Urakawa et al., 2003). Another mechanism leading to hyperglycemia in patients with T2DM involves the inability of insulin to inhibit hepatic glucose production. Enhanced phosphoenolpyruvate carboxykinase (PEPCK, an enzyme catalyzing the rate-limiting step in gluconeogenesis) activity leading to increased gluconeogenesis is a major source of increased hepatic glucose production in patients with T2DM (Consoli et al., 1990). Decreased glycogen synthesis has also been reported in patients with T2DM, but it is probably secondary to a reduction in glucose transport (Le Roith and Zick, 2001).

Pancreatic h cell dysfunction has also been demonstrated in patients with T2DM (Kahn, 2003). Progressive formation of amyloidosis with loss of h cells is always a major pathological change found in patients with T2DM (Marzban et al., 2003). The severity of amyloidosis is highly associated with h cell dysfunction during the development of T2DM in animal and human studies (Clark and Nilsson, 2004). Pancreatic h cells are most vulnerable to damages caused by oxidative stress because they are low in free radical quenching enzymes such as catalase, glutathione peroxidase, and superoxide dismutase (Tiedge et al., 1997). ROS can induce rapid polymerization of monomeric pancreatic islet amyloid polypeptide into amylin-derived islet amyloid, which is extremely resistant to proteolysis (for reviews, see Hayden, 2002; Hayden and Tyagi, 2002). The aggregation and deposition of amyloid not only results in a space-occupying lesion preventing the release of insulin into the circulation, it can also cause destruction to the insulinsecreting islet cells (Hayden, 2002; Hayden and Tyagi, 2002). Evidence has shown that the injury of pancreatic h cells incurred by ROS is also activated by the NFnB pathway (Ho and Bray, 1999; Ho et al., 1999).

Biochemical properties of arsenic Substitution of arsenate for phosphate Arsenic, being in Group VA of the periodic table, can occur in the +5, +3, 0, and 3 states and can form alloys with metals and covalent bonds with carbon, hydrogen, oxygen, and sulfur (Ferguson and Gavis, 1972). Because of its similar biochemical properties to phosphate, arsenate can replace phosphate in energy transfer phosphorylation reactions, resulting in the formation of adenosine diphosphate (ADP)-arsenate instead of adenosine triphosphate (ATP) (Gresser, 1981). However, the concentration required for half-maximal stimulation of the formation of ADP-arsenate is high, at approximately 0.8 mM arsenate (Moore et al., 1983). Nonenzymatic hydrolysis of ATP is negligible, whereas the arsenate ester of ADP-arsenate is unstable in aqueous media at neutral pH and undergoes rapid nonenzymatic hydrolysis at a rate constant estimated to be between 5 and 70 min 1 (Moore et al., 1983). This process is probably the major mechanism by which arsenate uncouples oxidative phosphorylation (Gresser, 1981; Moore et al., 1983). ADP-arsenate can also serve as a substrate for hexokinase resulting in the formation of glucose-6-arsenate instead of glucose-6-phosphate (Gresser, 1981). At high concentrations of arsenate, the activity of hexokinase is also inhibited (Moore et al., 1983). Unlike the inorganic forms of arsenate, neither the two pentavalent forms of methylated metabolites as monomethylarsinate and dimethylarsonate will perturb phosphate metabolism or bind significantly to sulfhydryl groups (Delnomdedieu et al., 1995).

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Fig. 1. Formation of cyclic thioarsenite complex by arsenite and dihydrolipoamide.

Arsenite reaction with sulfhydryl groups Arsenite readily enters the cells and has always been demonstrated to be more toxic than arsenate (Delnomdedieu et al., 1995), but it does not show similar substitution for phosphate as described previously for arsenate (Delnomdedieu et al., 1994, 1995). On the other hand, arsenite has high affinity for sulfhydryl groups of proteins and can form stable cyclic thioarsenite complexes with vicinal or paired sulfhydryl groups of cellular proteins (Delnomdedieu et al., 1994,

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1995; Peters, 1955). Similar reactions with sulfhydryl groups do not occur in the form of arsenate (Delnomdedieu et al., 1994). One example of such reaction is the complex formation of arsenite with dihydrolipoamide (Fig. 1), a cofactor of the enzymes pyruvate dehydrogenase and aketoglutarate dehydrogenase (Peters, 1955). This chemical change can cause aberration in structures of proteins and inactivate many enzymes and receptors. Inhibition of pyruvate dehydrogenase can impair the production of ATP by blocking the processing of citric acid cycle, which is critical for providing reducing equivalents to the mitochondria needed for electron transport (Fig. 2). However, a high concentration of arsenite (IC50 > 100 AM) is required for the inhibition of pyruvate dehydrogenase (Petrick et al., 2001). Recent studies have shown that the trivalent methylated metabolites of inorganic arsenic can be more toxic than arsenite. Monomethylarsonous acid can be detected in the liver of hamsters exposed to arsenate (Sampayo-Reyes et al., 2000), identified in bile of rats injected with either arsenite or arsenate (Gregus et al., 2000), and

Fig. 2. The potential sites of interference by arsenic on biochemical pathways of glucose metabolism. Known metabolic disturbances in patients with diabetes mellitus include impaired glucose transport, impaired glycolysis, increased gluconeogenesis, and decreased glycogen synthesis (see text for detailed discussion). The blockade of these metabolic pathways by arsenic as indicated is theoretically possible, but are not necessarily implicated as etiologic causes to arsenic-induced diabetes mellitus in subjects chronically exposed to arsenic. Glucose transport into target cells may be affected by interaction of arsenite and disulfide bridges in the molecules of insulin, insulin receptor, and glucose transporters, or by the inhibitory effect of arsenic on the synthesis and translocation of glucose transporters (site 1). Because of the substitution of phosphate by arsenate, glucose-6-arsenate brings to a slowdown or even a halt in the metabolic pathways that use glucose-6-phosphate as an intermediate or initiator, such as the glycolysis (site 2), gluconeogenesis (site 6), pentose phosphate pathway (site 7), glycogenesis, and glycogenolysis (site 8). By forming thioarsenite complexes with paired sulfhydryl groups of the enzymes pyruvate dehydrogenase (site 3) and a-ketoglutarate dehydrogenase (site 4), citric acid cycle cannot be processed, leading to insufficient energy production. Arsenic can also interfere with the respiratory chain on mitochondrial membrane by acting as an uncoupler of oxidative phosphorylation (site 5).

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detected in the urine of humans chronically exposed to arsenic (Aposhian et al., 2000), indicating that it can be a stable toxic metabolite of inorganic arsenic. Petrick et al. (2001) demonstrated that the LD50 of monomethylarsonous acid administered intraperitoneally to hamsters is many folds lower than that for sodium arsenite (29.3 vs. 112.0 Amol/kg of body weight). In addition, trivalent methylated metabolites can exhibit inhibitory effect on pyruvate dehydrogenase with a higher potency than arsenite. Petrick et al. (2001) showed that the concentrations of sodium arsenite required to inhibit the activity of pyruvate dehydrogenase in hamster kidney or porcine heart by 50% are 2- to 6-fold higher (>100 AM) than the concentrations of trivalent methylated metabolites required to exert similar effects. For hamster kidney, the IC50 for methylarsine oxide, diiodomethylarsine, and arsenite are 59.9, 62.0, and 115.7 AM, respectively; and for porcine heart, the IC50 for methylarsine oxide and arsenite are 17.6 and 106.1 AM, respectively. The median concentration of arsenic in well water in blackfoot disease-hyperendemic villages in Taiwan has been estimated to range from 0.7 to 0.93 mg/l (Tseng et al., 2000). If we use 1 mg/l for calculation and assume a 70-kg human taking 5 l of water per day, then the total amount of arsenic taken per day should be 5 mg. If we assume that absorption of arsenic is complete, the blood volume is 5 l and there is no redistribution to other components of body fluid and no metabolism or excretion of arsenic during the day, then the blood concentration of arsenic will be 1 mg/l (or 13.3 AM) at most. Actually, in the study by Wu et al. (2003), the highest blood level of arsenic in a group of arsenic-exposed subjects is 46.5 Ag/l or 0.6 AM. Thus, it seems impossible for arsenite or arsenate to exhibit direct effect on the energy production or enzyme activity involving glycolysis in such conditions of low concentrations. It is also doubtful that the methylated metabolites would exert such effect at physiologically relevant doses. However, in the liver where methylation of arsenic takes place, it is not known whether the endogenously produced methylated trivalent metabolites could accumulate to a high enough concentration to exert an effect locally. Taken together, trivalent arsenite, pentavalent arsenate, or the pentavalent methylated metabolites probably play an insignificant role in the inhibition of pyruvate dehydrogenase or the production of ADP-arsenate in humans with chronic arsenic exposure. On the other hand, the trivalent methylated metabolites produced endogenously in the liver at high concentrations might probably exert a local effect on slowing down the citric acid cycle resulting in insufficient energy production by interaction with sulfhydryl groups on enzymes such as the pyruvate dehydrogenase. However, a complete blockade of the metabolic pathways associated with energy production seems to be not the scenario in human body with chronic arsenic exposure as observed in most epidemiologic studies because this is not compatible with survival. Up to now, such inhibitory effects on glycolysis or energy production have not been demonstrated in humans.

Oxidative stress Oxidative stress is an imbalance between the production of highly reactive molecular species and the antioxidant defenses, which can lead to tissue damage. During the metabolism of arsenic, oxidative stress can be generated by the production of ROS and free radicals like hydrogen peroxide (Barchowsky et al., 1996, 1999; Chen et al., 1998; Wang et al., 1996), hydroxyl radical species (Wang et al., 1996), nitric oxide (Gurr et al., 1998; Lynn et al., 1998), superoxide anion (Barchowsky et al., 1999; Lynn et al., 2000), dimethylarsinic peroxyl radical (Yamanaka et al., 1991, 1997), and dimethylarsinic radical (Yamanaka et al., 1997, 2001). A variety of normal physiological functions can be disrupted through the production of ROS and induction of oxidative stress. For example, arsenite increases nicotinamide adenine dinucleotide oxidase activity and induces superoxide, which then causes oxidative deoxyribonucleic acid (DNA) damage in human vascular smooth muscle cells (Lynn et al., 2000). Arsenite also increases the levels of superoxide-driven hydroxyl radicals, which play an important causal role in the genotoxicity induced by arsenical compounds (Liu et al., 2001). The oxidative stress induced by arsenic is also one of the possible mechanisms leading to apoptosis (Nakagawa et al., 2002). Oxidative stress can also be induced by methylated metabolites of inorganic arsenic and is possibly responsible for the carcinogenesis induced by arsenic. Yamanaka et al. (1989, 1990) observed that, in mice treated with dimethylarsinic acid orally, DNA single-strand breaks can be induced by a further metabolite (dimethylarsine) specifically in the lung involving peroxyl radical. The same group later reported that dimethylarsinic acid also promotes and causes the progression of skin tumorigenesis in mice, probably via the formation of dimethylarsenic peroxyradicals during the metabolism of dimethylarsinic acid (Yamanaka et al., 2001). The production of hydroxyradicals found in the early phase of metabolism of methylated arsenicals is also a possible cause to the induction of liver cancer in rats (Nishikawa et al., 2001). The trivalent methylated metabolites of arsenic are found to cause chromosomal mutations, probably via a mechanism involving ROS (Kligerman et al., 2003). Oxidative DNA damage in cultured human cells can also be induced by very low physiologically relevant doses (nM or AM) of arsenite and the trivalent and pentavalent methylated metabolites (Schwerdtle et al., 2003a, 2003b). Significant amount of inorganic arsenicals and their trivalent and pentavalent methylated metabolites can be identified from the urine of arsenic-exposed subjects in West Bengal, India (Mandal et al., 2001), suggesting that the oxidative damages caused by ROS during the metabolism of arsenic can be one of the factors responsible for human diseases associated with arsenic exposure. Oxidative stress induced by arsenic was not only demonstrated in animal or cell biology studies, recent studies in human beings disclosed an increased oxidative stress in

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subjects with chronic arsenic exposure. Pi et al. (2000) showed a reduced serum concentration of nitric oxide metabolites in arsenic-exposed subjects in China while compared to a group of control. In total samples, the nitric oxide metabolites were also correlated negatively with blood inorganic arsenic and its methylated metabolites, and positively with nonprotein sulfhydryl in whole blood (Pi et al., 2000). In a later study, the same group demonstrated a higher oxidative stress (indicated by an increased serum level of lipid peroxide and a decreased level of whole blood nonprotein sulfhydryl) in a high-arsenic-exposed group than a low-exposed group, and a significant correlation between oxidative stress and inorganic arsenic and its methylated metabolites (Pi et al., 2002). Thus, abundant evidence has shown that oxidative stress can play etiologic roles in arsenic-induced carcinogenesis, and that excess oxidative stress can be demonstrated in humans chronically exposed to arsenic. It is justified to believe that the injury of oxidative stress caused by chronic arsenic exposure is not restricted to carcinogenesis.

Table 1 Major biochemical properties of arsenic on glucose homeostasis and the potential link to arsenic-induced diabetes mellitus Biochemical property

Effects on glucose homeostasis

Comments on potential link to arsenic-induced diabetes mellitus

Similarity with phosphorus

Substituting phosphate and forming ADParsenate and glucose6-arsenate, leading to impaired glucose metabolism and inefficient energy production.

High affinity for sulfhydryl groups

Formation of cyclic thioarsenite complex with paired sulfhydryl groups in proteins (insulin, insulin receptor, glucose transporters), and enzymes (pyruvate dehydrogenase and a-ketoglutarate dehydrogenase) could lead to impaired glucose transport and metabolism. Oxidative stress can lead to formation of amyloid in pancreatic islet cells, leading to progressive h cell dysfunction. Superoxide may impair insulin secretion by interaction with uncoupling protein 2. Insulin resistance can also be induced by oxidative stress.

May be associated with acute intoxication, but possibly not the major cause of arsenic-induced diabetes mellitus with chronic exposure, because high concentration of arsenate is required for the reactions. Probably effective when increased methylated metabolites are produced locally. Lack of directly supportive data for interferences on metabolic pathways involving these proteins by arsenite. These effects may be counterregulated in conditions with chronic exposure to arsenic.

Gene expression Arsenic can influence the expression of a variety of proteins involving signal transduction and gene transcription. For example, in a recently published study evaluating the gene expression of 708 transcripts of known human genes by microarray in circulating lymphocytes from arsenic-exposed subjects in Taiwan, a variety of cytokines and growth factors associated with inflammation including IL-6 was found to be upregulated in persons with increased arsenic exposure (Wu et al., 2003). Arsenite may also interfere with the glucocorticoid-dependent expression of PEPCK by interaction directly with glucocorticoid receptor (GR) complexes and selectively inhibit GR-mediated transcription at nontoxic doses (0.3 – 3.3 AM) (Kaltreider et al., 2001). It has been shown that, in bladder epithelial cells, arsenite may stimulate MAPK cascade with a consequent increase in the expression or phosphorylation of the two major AP-1 constituents, c-Jun and c-Fos (Simeonova et al., 2000), and the methylated metabolites may be more potent activators than inorganic arsenite (Drobna et al., 2003). Arsenic has also been shown to upregulate TNFa (Yu et al., 2002) and to inhibit the expression of PPARg (Wauson et al., 2002) (to be discussed later). Furthermore, arsenite can compete with zinc in metalbinding proteins, displaying vicinal dithiols contained in zinc fingers of DNA binding and repair proteins and transcription factors (Asmuss et al., 2000). However, arsenite could not exert this effect at concentrations of V1 mM (Asmuss et al., 2000). A recent study demonstrated that both trivalent and pentavalent methylated metabolites of arsenic are more potent than arsenite in releasing zinc from its binding sites in the nanomolar or micromolar concentration range (Schwerdtle et al., 2003a).

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Increased oxidative stress

Interference with gene expression

Induction of insulin resistance by enhancing the expression of NF-nB, TNFa, and IL-6 and by inhibiting the expression of PPARg.

Oxidative stress has been implicated as a major cause to both insulin resistance and h cell dysfunction. Arsenic is well known for its capability to produce reactive oxygen species and free radicals. Increased oxidative stress can be demonstrated in arsenic-exposed subjects. The oxidative stress induced by arsenic may lead to diabetes mellitus. NF-nB, TNFa, and IL-6 have direct link to insulin resistance and activation of PPARg can improve insulin sensitivity. Subjects with chronic arsenic exposure have been shown to have higher expression of IL-6. Physiologically relevant concentrations of arsenite can induce NF-nB and TNFa, and inhibit PPARg. These effects are potential cause of arsenic-induced diabetes mellitus.

ADP, adenosine diphosphate; NF-nB, nuclear factor-nB; TNFa, tumor necrosis factor a; IL-6, interleukin-6; PPARg, peroxisome proliferatoractivated receptor g.

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The above biochemical properties of arsenic and their potential association with diabetes mellitus are summarized in Table 1 and will be discussed as follows.

Arsenic effects on glucose transport and potential link with insulin resistance Exofacial sulfhydryl groups in GLUTs in rat adipocytes are crucial for the maximal activity of the transporters and for the transport rates of glucose regulated by insulin (May, 1985). Phenylarsine oxide (PAO), a trivalent arsenical that forms stable cyclic thioarsenite complexes with vicinal or paired sulfhydryl groups of cellular proteins, has been shown to inhibit glucose transport in adipocytes (Douen and Jones, 1988; Frost and Lane, 1985). Although the study by Douen and Jones (1988) suggested a direct inhibitory effect of PAO on both the receptor and the transporter systems possibly by reacting with sulfhydryl groups at or near the receptor or transporter sites, Frost and Lane (1985) showed that PAO inhibits insulin-stimulated glucose transport with targets distal to the receptor tyrosine kinase activity and does not interfere directly with insulin binding to its receptor. Wheeler (1989) also demonstrated that PAO does not inactivate transporters directly. The phosphorylation of the two endogenous phosphoproteins (pp24 and pp240) blocked by PAO are serine-specific (Frost et al., 1987). Jhun et al. (1991) later demonstrated that PAO causes insulin-dependent GLUT4 degradation in rat adipocytes rather than inhibits the insulin-induced transporter recruitment. Begum’s study (1994) disclosed that PAO may exert its inhibitory effect on insulin-stimulated glucose transport by abolishing insulin’s ability to activate protein phosphatase 1 and dephosphorylate GLUT4. The effect of PAO on glucose transport seems to be insulin-dependent and specific to GLUT4, which are present on adipocytes and muscle cells, and not effective on GLUT2 or GLUT 1 (Jhun et al., 1991). Similar inhibition of glucose transport occurs in the skeletal muscle and kidney cells. For example, PAO exhibits an inhibitory effect on insulin-stimulated or hypoxia-stimulated glucose transport in rat skeletal muscle (Henriksen and Holloszy, 1990). Denervation-induced postreceptor resistance of glucose transport to insulin and insulin-like growth factor I also involves primarily a PAO-sensitive pathway in rat skeletal muscle (Sowell et al., 1988). In cell cultures, PAO inhibits glucose uptake in Madin– Darby canine kidney cells (Liebl et al., 1995). The site of inhibition is probably at the membrane transport or phosphorylation, and PAO exhibits a far more potent inhibitory activity on glucose uptake than arsenite does (Liebl et al., 1992). PAO is membrane-permeable and highly toxic, and has always been used as a probe for functional thiol groups and as a tyrosine phosphatase inhibitor in experimental studies. Although the above studies using PAO as an arsenical in investigating the effects of arsenic on glucose transport

showed an inhibitory effect, the results might only suggest that sulfhydryl groups are critical in glucose transport and should not be used to explain what would happen in human body with chronic arsenic exposure because PAO is a synthetic compound and may not behave similarly as other naturally occurring inorganic arsenicals. There remains great concern on the use of possibly high PAO concentration, leading to severe impairment in cellular viability and nonspecific inhibition of cellular function. Although arsenite also exhibits inhibitory effect on glucose transport in muscle cells, the concentration for halfmaximum inhibition by arsenite is much greater than that by PAO (0.5 – 1 mM vs. 5 – 30 AM) (Liebl et al., 1992). Interestingly, an opposite effect on glucose transport by arsenite has been observed with lower doses. A recent study well demonstrated that arsenite at concentrations of 0.01– 0.5 mM may stimulate glucose transport in a dose-responsive pattern in 3T3-L1 adipocytes involving GLUT4 translocation in the presence of PKC-E (Bazuine et al., 2003). The concentration of 0.5 mM of arsenite is actually the concentration that maximally stimulate glucose uptake. At higher concentrations, glucose uptake will decrease (Bazuine et al., 2003). Arsenite does not induce PKC-E activation over basal levels, but requires basal levels of PKC-E to induce glucose uptake (Bazuine et al., 2003). The response is not dependent on PI-3 kinase or ERK-1 or -2, but involves the p38 MAPK pathway (Bazuine et al., 2003). The total amount of GLUT4 does not change (Bazuine et al., 2003), indicating that arsenite does not have an effect on the regulation of the gene expression of GLUT4. For the study by Liebl et al. (1992), the used concentration of 0.5 mM is extraordinarily higher than the concentration that might occur in humans. If we do not consider the different cell types used by Liebl et al. (1992) and by Bazuine et al. (2003), it may be justified to conclude that lower concentrations of arsenite in the range of human exposure doses encountered in epidemiologic studies may probably stimulate, rather than inhibit, glucose uptake. Taken together, arsenite-induced inhibition of glucose uptake will probably occur in acute intoxication but not in chronically exposed subjects as observed in epidemiologic studies, and it is not reasonable to ascribe the increased prevalence or incidence of diabetes mellitus associated with chronic arsenic exposure to a direct inhibitory effect of arsenic on glucose transport taking into account the total amount of arsenic taken daily. Because methylated metabolites of arsenic can be more toxic than the inorganic forms, whether an inhibitory effect of arsenic on glucose transport can be amplified by these methylated metabolites in humans with chronic arsenic exposure awaits further investigation. On the other hand, the stimulatory effect of low concentrations of arsenite on GLUT translocation might not be effective in conditions with chronic arsenic exposure if the expression of GLUT or the signal transduction pathways leading to glucose transport are inhibited by the other effects of arsenic as described below.

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Recent studies have pointed to another possible mechanism for arsenic-induced diabetes mellitus. Arsenic may induce a status of insulin resistance through its molecular effects on gene expression of some cytokines. Arsenic has been demonstrated to induce TNFa release from mononuclear cells at a concentration of 1 AM (Yu et al., 2002) and IL-6 expression is upregulated in peripheral lymphocytes in subjects with high arsenic exposure from drinking water in Taiwan (Wu et al., 2003). The concentration of arsenite used in the experiments by Yu et al. is physiologically relevant, and the study by Wu et al. was done in biospecimens obtained from humans. It is remarkable that expression of IL-6 in subjects with blood levels of arsenic in the intermediate (4.64 – 9.00 Ag/l) and high (9.60 – 46.5 Ag/l) range is significantly higher than that in the low (0 –4.32 Ag/l) range of exposure (Wu et al., 2003). Abundant evidence has confirmed TNFa and IL-6 as mediators of insulin resistance as discussed earlier. These cytokines might persistently exert their effects on insulin-responsive cells even if they are not produced endogenously because evidence has shown that chronic treatment with IL-6 can induce insulin resistance in adipocytes (Lagathu et al., 2003). Because the increased expression of IL-6 can be observed directly in subjects chronically exposed to arsenic and it is possible for this increased level of IL-6 to exert its effect in adipocytes, it is highly possible that cytokines can play an important role on the development of arsenic-induced diabetes mellitus. It is also possible, though without evidence, that the expression of TNFa and IL-6 can be upregulated by chronic arsenic exposure inside insulin-sensitive cell types such as skeletal muscle and adipocytes. The study by Wauson et al. (2002) provided another piece of evidence for an effect of arsenite on the induction of insulin resistance in the adipocytes. They have observed that sodium arsenite at physiologically relevant concentration of 6 AM prevents adipocyte differentiation of C3H 10T1/2 cells through a mechanism of inhibiting PPARg expression and that the differentiating effect on adipocytes induced by pioglitazone (a PPARg agonist that has been marketed for the treatment of T2DM by improving insulin sensitivity) can be inhibited by arsenite (Wauson et al., 2002). This inhibitory effect of arsenite on the expression of PPARg in adipocytes and the induction of TNFa and IL-6 expression associated with arsenic exposure are supportive for the hypothesis that insulin resistance can be induced by chronic arsenic exposure via the induction of cytokines and the regulation of related genes, leading to diabetes mellitus. A growing number of reports in recent years have suggested a link between increased ROS production or oxidative stress and the development of insulin resistance and h cell dysfunction in humans as discussed earlier. This stresssensitive pathway may involve the transcription factor NFnB. It is notable that NF-nB can also be responsible for ROS damages induced by low levels of arsenic at micromolar ranges (Barchowsky et al., 1996; Wijeweera et al., 2001). A recent study has also shown that lower concentrations (V1

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AM) of arsenite will induce NF-nB in keratinocytes, but higher concentrations (z5 AM) will not induce NF-nB and will actually cause apoptosis (Laio et al., 2004). Because NFnB could be linked to insulin resistance by regulating the expression of TNFa and IL-6 and activated by oxidative stress as discussed earlier, it is also justified to speculate a role of NF-nB in the induction of arsenic-induced diabetes mellitus. In addition to cancer and diabetes mellitus, ROS damages can also be easily used to explain the commonly seen diseases associated with arsenic exposure. For example, ROS has also been implicated as potential pathogenic factors for the development of hypertension (Taniyama and Griendling, 2003), atherosclerosis (Taniyama and Griendling, 2003), and neuropathy (Lelkes et al., 2001). All of these health problems have also been reported to be associated with chronic arsenic exposure. Therefore, ROS, cytokines, PPARg, and NF-nB can work together in the induction of insulin resistance and diabetes mellitus associated with chronic exposure to arsenic. However, this hypothesis requires further confirmation.

Arsenic effects on glucose metabolism and energy production Because of its biochemical properties, arsenic may theoretically impair glucose metabolism by acting as an uncoupler of oxidative phosphorylation, as an inhibitor of sulfhydryl containing enzymes such as a-ketoglutarate dehydrogenase and pyruvate dehydrogenase, and as a competitor for phosphate-binding sites on glycolytic enzymes (Brazy et al., 1980; Liebl et al., 1995). The formation of ADP-arsenate instead of ATP can cause an inefficient production of energy and results in generalized inhibition of the metabolic pathways that require ATP. Glucose-6phosphate is not only important as a mediator for glycolysis, gluconeogenesis, glycogenesis, and glycogenolysis, it is also important as an initiator for the pentose phosphate pathway (Fig. 2), which generates nicotinamide adenine dinucleotide phosphate [NADPH, an important cofactor in the reduction of glutathione (GSH)] and provides the cell with ribose-5-phosphate for the synthesis of nucleotides and nucleic acids. Substitution of phosphate in the formation of glucose-6-phosphate by yielding glucose-6-arsenate may lead to an inefficient metabolism of glucose. Insufficient production of NADPH from the pentose phosphate pathway further disrupts the ability of the cells to deal with oxidative stress. Although, theoretically, arsenic in the forms of arsenite or arsenate can affect glucose metabolism and energy production as described previously, a complete blockade of these metabolic pathways is unlikely in subjects with chronic arsenic exposure as observed in most epidemiologic studies because this is not compatible with survival. However, a slowdown of the metabolic pathways induced by arsenic or its metabolites is possible, but its contribution to

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the development of arsenic-induced diabetes mellitus is also in doubt because an increased intracellular glucose and a resulting hyperglycemia due to a slowdown of glucose metabolism would probably be counteracted by other mechanisms. For example, gluconeogenesis may also be inhibited by arsenite through its inhibition on PEPCK expression by downregulating glucocorticoid receptor at nontoxic doses (Kaltreider et al., 2001).

Arsenic effects on insulin biosynthesis and secretion Physiologically, glucose transport into the h cells can initiate insulin secretion by the following mechanism. The ATP produced as a result of glycolysis leads to the closure of the ATP-sensitive potassium channel, which in turn depolarizes and opens the voltage-dependent calcium channel. The calcium flux through the opened channel releases insulin from the h cells (MacDonald and Wheeler, 2003). Insulin forms hexamer with zinc before its secretion (Olsen et al., 2003). Up to now, there has not been any study evaluating the pancreatic function associated with environmental or occupational arsenic exposure in humans, but arsenic may theoretically cause impairment in insulin secretion by mechanisms involving functional or structural changes. Based on the observation that arsenic might not interfere with glucose transport through GLUT2 (Jhun et al., 1991) or at lower concentrations of arsenite (Bazuine et al., 2003), if arsenic would functionally interfere with insulin secretion, the mechanisms should involve events occurring after the transport of glucose into the cells. Because arsenic may cause formation of ADP-arsenate instead of ATP, it is possible that the ATP-dependent insulin secretion could be impaired in the absence of sufficient energy supply. However, because a relatively high concentration of arsenate is also required for the formation of ADP-arsenate and it is not known whether the slowdown of energy production via the citric acid cycle by arsenite or its methylated metabolites is in play in the pancreas of subjects chronically exposed to arsenic, impairment of insulin secretion via this shortage of energy supply is in doubt. Interaction between arsenite or its methylated metabolites and the sulfhydryl groups of insulin or proinsulin, and competition with zinc in the formation of hexamers during the synthetic stages are also theoretically possible, but all of these are speculative without supportive data. Arsenic may also indirectly cause functional impairment in insulin secretion through the generation of free radicals. Uncoupling protein 2 (UCP2) is a negative regulator of insulin secretion. It mediates proton leak across the inner mitochondrial membrane (Jaburek et al., 1999). A superoxide-UCP2 pathway has been suggested to cause impairment in insulin secretion in pancreatic h cells observed in association with obesity and hyperglycemia resulting from endogenously produced mitochondrial superoxide, which

activates UCP2-mediated proton leak, leading to decreased level of ATP and impaired glucose-stimulated insulin secretion (Krauss et al., 2003). Arsenic is well known for its ability to induce the production of superoxide (Barchowsky et al., 1999; Lynn et al., 2000). If excess superoxide is produced in the pancreatic h cells, an impairment of insulin secretion is expected. Although without supportive data, the increased oxidative stress induced by arsenic as previously discussed could theoretically cause structural damages to the pancreatic islets with the formation of amyloidosis, which not only prevents the release of insulin into the circulation, but also destroys the insulin-secreting h cells insidiously after prolonged exposure to arsenic. ERK1 and ERK2 are important regulators for glucosestimulated expression of insulin gene in rat h cells (Khoo et al., 2003; Arnette et al., 2003). Whether chronic arsenic exposure could have an impact on the expression of these genes and how it could be related to arsenic-induced diabetes mellitus awaits further investigations.

Individual susceptibility to arsenic-induced diabetes mellitus It is true that not all subjects exposed to arsenic develop diabetes mellitus. Individual susceptibility is likely to vary based on duration and cumulative dosage of exposure, genetics, metabolism, nutritional status, health status, and other possible factors. The variation in individual susceptibility may influence the toxic effect of arsenic on target organs and determine the clinical development of diabetes mellitus (Fig. 3). Currently, studies evaluating the association between these susceptibility factors and arsenic-induced diabetes mellitus are still lacking. Some published data on the association between nutritional status in arsenic-exposed subjects and disease occurrence might have implications for a link to arsenic-induced diabetes mellitus. Nutritional status has been found to be an important factor determining the chronic toxicity associated with arsenic exposure in epidemiologic studies (Hsueh et al., 1995, 1998). Studies carried out in residents of the blackfoot disease-hyperendemic areas in Taiwan demonstrated that undernourishment, as indicated by high consumption of dried sweet potato, is associated with increased prevalence of skin cancer (Hsueh et al., 1995) and that serum levels of antioxidants such as a- and h-carotene have significant influence on the development of atherosclerotic disease (Hsueh et al., 1998). Although there is no direct supportive data for a link between deficiency of antioxidants and arsenic-induced diabetes mellitus, it is highly possible that this link does exist because recent studies have suggested that perturbations to the antioxidant defense mechanism within skeletal muscle can lead to T2DM (Bruce et al., 2003) and that individuals taking less antioxidant would have increased risk of diabetes mellitus and cardiovascular

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Fig. 3. The interactions between individual susceptibility and arsenic effects on target organs leading to the development of diabetes mellitus. Individual susceptibility (including the genetic factors, nutritional status, health status, detoxification capability, interactions with other trace elements, and the presence of other risk factors for diabetes mellitus, etc.) acts like a check valve that determines the flow of toxic effects to the organs and the clinical manifestation of diabetes mellitus. TNFa, tumor necrosis factor a; IL-6, interleukin-6; PPARg, peroxisome proliferator-activated receptor g.

disease (Ford et al., 2003). Thus, good nutritional status with sufficient intake of antioxidants can reduce oxidative stress induced by arsenic and can possibly prevent the onset of arsenic-induced diseases including diabetes mellitus. In animals, protein-restricted diets can lead to a lower level of GSH in the liver (Carrillo et al., 1989). GSH is essential for insulin action in hepatic cells in rats (Guarino et al., 2003). Diabetic patients would have a lower level of GSH (Dincer et al., 2002) and GSH infusion has been shown to improve insulin action and increase glucose uptake in patients with T2DM (De Mattia et al., 1998). Because residents of the blackfoot disease-hyperendemic villages in Taiwan (Chen et al., 1988) and the people exposed to arsenic in Bangladesh (Mitra et al., 2002) may have poorer nutritional conditions, selenium (required in the biosynthesis of GSH) in residents of the blackfoot diseaseendemic area in Taiwan is significantly lower than normal controls (Horng and Lin, 1997; Lin and Yang, 1988), and GSH is obligatory for the excretion of arsenic (Kala et al., 2000), it is possible that a poor nutritional status as frequently observed in most arsenic-exposed individuals will also lead to a less formation of arsenic-GSH complexes and less excretion of arsenic. All of these will aggravate the hyperglycemia induced by arsenic. Arsenic has been shown to inhibit the absorption of water, sodium, glucose, and leucine from the intestine of male Sprague– Dawley rats in a dose-responsive pattern (Hunder et al., 1993). Whether arsenic can inhibit the absorption of antioxidants, such as a-tocopherol, ascorbic acid, and h-carotene is an issue that remains to be seen. General risk factors of diabetes mellitus are also important in the determination of progression of hyperglycemia and clinical onset of diabetes mellitus in the arsenic-exposed

subjects. As demonstrated in the epidemiologic follow-up study in the blackfoot disease-hyperendemic areas in Taiwan, in addition to arsenic exposure, the general risk factors of diabetes mellitus including age and body mass index are also important for the incidence of diabetes mellitus (Tseng et al., 2000). It is worth mentioning that the impact of body build on the development of diabetes mellitus is interesting. On the one hand, obesity, which is often a result of excess calorie intake, is a risk factor for diabetes mellitus; on the other hand, undernourishment with insufficient intake of antioxidants could probably aggravate the development of diabetes mellitus in subjects chronically exposed to arsenic. Most of the trace elements do not work in isolation and the interactions between arsenic and other trace elements from environmental co-contamination and oral intake could modulate the chronic toxicity and clinical manifestations associated with arsenic exposure. These interactions could also occur in the progression and development of diabetes mellitus in subjects exposed to arsenic. In patients with blackfoot disease in Taiwan, the concentrations of zinc and selenium in urine and serum are significantly lower than normal controls (Horng and Lin, 1997; Lin and Yang, 1988). Diabetic patients are often deficient in zinc (Anetor et al., 2002; Car et al., 1992; Chen et al., 1995a, 1995b; Ekin et al., 2003), magnesium (Anetor et al., 2002; Chen et al., 1995a, 1995b; Ekin et al., 2003), and chromium (Ekmekcioglu et al., 2001; Ghosh et al., 2002). Feeding ob/ob mice (an animal model for T2DM) with zinc for 2 weeks (Hwang et al., 2002) or intraperitoneal injection with zinc for 13 days in KK-Ay mice (Kojima et al., 2003) can significantly improve glycemic control. Although supplementation of zinc for 6 months in patients with T2DM does not improve glycemic control, oxidative

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stress does improve significantly (Roussel et al., 2003). In a clinical study, patients with T2DM have significantly lower level of chromium, and chromium supplementation for 12 weeks can significantly improve glycemic control by increasing insulin action (Ghosh et al., 2002). The improvement of insulin action by chromium can also be demonstrated in type 2 diabetic rats (but not in type 1 diabetic rats) (Sun et al., 2002). A variety of other studies in humans or animals also agree with an insulin-enhancing effect of chromium (Appleton et al., 2002; Bahijri and Mufti, 2002; Cefalu et al., 2002). Increased magnesium intake has been demonstrated to reduce the risk of T2DM through a mechanism of improving insulin action in two independent papers published recently. One followed 85 060 women and 42 872 men without history of diabetes for more than 10 years (Lopez-Ridaura et al., 2004) and the other followed 39 345 healthy women for an average of 6 years (Song et al., 2004). Selenium possesses antioxidant and insulin-mimetic effects (Stapleton, 2000). In streptozotocin-induced diabetic rats, dietary supplement of selenium can significantly improve oxidative stress (Douillet et al., 1998) and intraperitoneal injection of selenium significantly reduced blood glucose by improving insulin action (Battell et al., 1998). In 3T3-L1 adipocytes, selenium has been demonstrated to stimulate glucose transport through a PI-3 kinase activity and increase GLUT1 content in the plasma membrane, but this effect is independent of insulin receptor activation (Heart and Sung, 2003). Whether the risk of T2DM could be reduced by supplementation with selenium in humans requires further investigation. Arsenic can interact with most of these trace elements. For examples, low and noncytotoxic levels of arsenite and its methylated metabolites may compete with zinc (Asmuss et al., 2000; Schwerdtle et al., 2003a) and zinc pretreatment can protect the mice against arsenite toxicity (Kreppel et al., 1994). Arsenic can counteract the glucose lowering effect of chromium in growing rats (Aguilar et al., 1997) and the hypoglycemia induced by experimental selenium toxicity in guinea pigs (Das et al., 1989). Selenium is also the most efficient antioxidant found at the subcellular level in the glutathione peroxidase enzyme system and can attenuate the effects of arsenic on cytotoxicity, viability, and cell cycle in porcine endothelial cells (Yeh et al., 2003). The contents of selenium in drinking water are low and diet is the main source of this element (Gebel, 2000). In case that selenium is deficient, as known in the residents of the blackfoot disease-endemic areas in Taiwan (Horng and Lin, 1997; Lin and Yang, 1988), arsenic can cause an increased oxidative injury. It is now known that both arsenite and arsenate are actively transported into cells by aquaglyceroporins and by phosphate transporters, respectively (Rosen, 2002). In low phosphate conditions, arsenate uptake will be accelerated and its toxicity will probably be promoted. Although there is not much published research, the interactions between arsenic and other trace elements that have an effect on glucose homeostasis, such as lithium and

vanadium, can also contribute to the hyperglycemic effect of arsenic.

Limitations of current evidence It should be admitted that there has not been any successful animal model for arsenic-induced diabetes mellitus and extensive research on arsenic-induced diabetes mellitus is still lacking. Because most of the biological effects of arsenic on glucose homeostasis are derived from in vitro studies or from animals treated with high dosage of arsenic compounds, the appropriateness to extrapolate these findings to human beings is limited. The dosages, routes of administration, and arsenic species used in most studies could be different from the environmental or occupational exposure in humans and should be interpreted with caution. Currently, we can only obtain most of the information from these observations because it is not ethical to carry out experiments in humans. The extensive contamination of drinking water with arsenic in Bangladesh and West Bengal, India, nowadays can provide quasi-experimental observations, but it is mandatory to exclude the co-contamination of other trace elements and to consider the effect of other risk factors of diabetes mellitus. The complexity of human physiology may make the clinical manifestations of arsenic exposure more diversified in different ethnicities and individuals. This complexity may include many facets of individual susceptibility as described previously. The growing number of papers showing methylated metabolites more toxic than inorganic arsenic makes the problems more complicated. Pathological observations in the target organs such as the pancreas or the liver are lacking. According to current knowledge, most of the effects of arsenic are not specific and may involve different organs and systems, and it is not easy to weight the relative importance of each organ in the development of arsenic-induced diabetes mellitus. In addition to insulin, a variety of counter-regulatory hormones and the nervous system may also participate in the tight regulation of glucose levels in normal conditions. However, studies on these issues are still lacking and further investigations are necessary.

Conclusions Studies from experimental animals and cell biology can provide pathophysiological mechanisms for arsenic-induced diabetes mellitus. With current knowledge and evidence, arsenic-induced diabetes mellitus in chronically exposed subjects may not be directly related to the exposed arsenic species and may involve the methylated metabolites of arsenic. Insulin resistance and h cell dysfunction can be induced by chronic arsenic exposure through the activation of ROS, NF-nB, and cytokines (TNFa and IL-6), and the inhibition of PPARg. The pathogenic role of direct interfer-

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ences on glucose metabolic pathways and energy production as always observed in acute intoxication might not be responsible for the development of arsenic-induced diabetes mellitus in chronically exposed subjects. Individual variability in detoxification capability, nutritional status, and interactions with other trace elements could influence the susceptibility of arsenic-exposed subjects to develop diabetes mellitus. The potential link among oxidative stress, cytokines and PPARg, and arsenic-induced diabetes mellitus can yield new insights for the development of strategies on the prevention and treatment of arsenic-induced diabetes mellitus.

Acknowledgments The author thanks the Department of Health (DOH89TD-1035) and the National Science Council (NSC-89-2320B-002-125, NSC-90-2320-B-002-197, and NSC-92-2320B-002-156), Executive Yuan, Republic of China, for supporting a series of epidemiologic studies on diabetes mellitus and blackfoot disease.

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