Methylated Organic Metabolites of Arsenic and their Cardiovascular Toxicities.

Recently, arsenic-toxicity has become the major focus of strenuous assessment and dynamic research from the academy and regulatory agency. To elucidate the cause and the mechanism underlying the serious adverse health effects from chronic ingestion of arsenic-contaminated drinking water, numerous studies have been directed on the investigation of arsenic-toxicity using various in vitro as well as in vivo systems. Neverthless, some questions for arsenic effects remain unexplained, reflecting the contribution of unknown factors to the manifestation of arsenic-toxicity. Interestingly, very recent studies on arsenic metabolites have discovered that trivalent methylated arsenicals show stronger cytotoxic and genotoxic potentials than inorganic arsenic or pentavalent metabolites, arguing that these metabolites could play a key role in arsenic-associated disorders. In this review, recent progress and literatures are summarized on the metabolism of trivalent methylated metabolites and their toxicity on body systems including cardiovascular system in an effort to provide an insight into the future research on arsenic-associated disorders.

human is contaminated drinking water and food (Gebel, 1999). Exposure via the contaminated food is mainly from arsenobetaine, arsenocholine and arsenosugars in sea foods (Edmonds et aI., 1997). However, due to their low toxicities and rapid urinary excretion, their effects on human are known to be minor (Buchet et aI., 1994). On the contrary, chronic arsenic exposure via contaminated drinking water is drawing an enormous public attention worldwide. (Abemathy et aI., 2003;Mead, 2005). US Environmental Protection Agency (EPA) and World Health Organization (WHO) are regulating the arsenic level in drinking water up to 10 fl9/1 (ppb) in an effort to prevent chronic arsenic exposure but the reports of arsenic exposure through contaminated drinking water are continuing throughout world. In a certain region of China, 0.09-1.86 mg/I level of arsenic was detected in the drinking water from a natural well (Xia and Liu, 2004) and in Bengal Delta Plain surrounded by India, West Bengal and Bangladesh, extremely high levels of arsenic ranging up to 3.2 mg/I have been reported (Bhattacharyya et a/., 2003). 3.05 mg/I arsenic level was detected in the underground water from a region of Vietnam (Berg et a/., 2001), and 0.01-1.82 mg/I of arsenic level was reported in some regions of Taiwan (Bates et aI., 1992). These epidemic arsenic contaminations have been also reported in other parts of world such as Latin America including Chile, Argentina and Mexico Hopenhayn-Rich et al., 1996;Caceres et al., 2005,), America and Europe (Lewis et al., 1999;Lamm et al., 2004).
Distribution, metabolism and excretion of arsenic. Arsenic in drinking water is mainly inorganic form and it can be easily absorbed through gastrointestinal tract to systemic blood circulation. Orally administered arsenic is found to be rapidly absorbed and cleared from body in human volunteer through urine for 3-4 days. However, some remain in body and are slowly excreted through hair, nail and sweat (Buchet et al., 1981).
Arsenic distribution in blood has been investigated through the bioanalysis of arsenic concentration in plasma, serum and erythrocytes of arsenic exposed human (Heydorn, 1970;Concha et al., 1998). Normal healthy people without arsenic exposure have the blood arsenic concentration less than 5.1 f.Lg/1 while arsenicexposed group showed arsenic concentration of around 32.7 f.Lg/1 in erythrocytes and 15.4 f.Lg/1 in plasma (Heydorn, 1970). In addition, female population exposed to approximately 200 f.Lg/1 of arsenic contaminated drinking water showed average blood arsenic level of 11 ~lg/I (Concha et al., 1998). Even after 2 years of wash-out period from arsenic exposure, plasma and blood concentration of arsenic were determined to be 7.48 ~lg/I and 26.3 f.Lg/l, respectively (Mandai et al., 2004). Other study in the population exposed to 5-410 f.Lg/1 of arsenic contaminated drinking water showed blood arsenic concentration of 2-42.1 f.Lg/1 (Olguin et al., 1983;Pi et al., 2000).
Major arsenic metabolite in urine is known to be DMA v but trivalent state was recently identified to be another major metabolite of arsenic in urine Aposhian et al., 2000b;Mandai et al., 2004). In human urine, arsenic metabolite contents were measured to be in the order of DMA'II > DMA v > ASIII > Asv > MMA'II > MMA v (Valenzuela et al., 2005). Although the actual level of trivalent methylated metabolites in environment and human tissue is still poorly investigated, several reports have dealt with the exposure level using in vitro and in vivo models. Upto now, MMA'II and DMA'II are known to be present in contaminated water (Hasegawa, 1994), hamster liver tissue (Sampayo-Reyes et al., 2000), cultured human hepatocyte (Del , and human urine (Le et al., 2000a, b;Del Razo et al., 2001). In addition, the presence of MMA'II in bile acid were reported in rats . Tissue distribution of radioactive DMA'II was also examined in mouse model (Hughes et al.,2008).
Toxicity of trivalent arsenic metabolites. Biological activities of MMA v and DMA v are known to be inferior to those of inorganic arsenic and therefore, methylation pathways have been regarded as the detoxification of inorganic arsenic (Vahter and Marafante, 1983;Eguchi et a/., 1997;. Although trivalent methylated metabolites, MMA'II and DMA'II had been known to be chemically reactive (Cullen et a/., 1984), their toxicological roles were questioned due to the absence of knowledge about their biological concentrations. With the recent discovery of trivalent methylated metabolites, MMA'II and DMA'II as major human metabolites of inorganic arsenic and their strong toxicities, however, a renewed view of arsenic methylated metabolites as toxic metabolites is gaining more and more strength (Petrick et a/., 2000(Petrick et a/., , 2001Styblo et a/., 2000;Sakurai et a/., 2002). Petrick et a/. (2000) showed that in Chang human hepatocyte, MMA'II showed 10 times stronger toxicities than ASIII and potent toxicites of MMA'II and DMA'II could be also observed in other cell types such as normal human hepatocyte, epidermal keratinocyte, bronchial epithelial cell and urinary bladder UROtsa cell (Styblo et a/., 2000). In addition, toxicities of MMA'II and DMA'II were shown in various cancer cell lines including human hepatocellular carcinoma, human bladder transient carcinoma, human acute promyelocytic leukemia and human osteocarcinoma (Styblo et a/., 2002) and inhibition potency of MMA'II against glutathione reductase, glutathione peroxidase and thioredoxin reductase was also the strongest among arsenic species (Chouchane and Snow, 2001;Lin et a/., 2001). DMAIII is known to have neurotoxic potential in contrast to DMA v (Kruger et a/., 2007). Furthermore, trivalent methylated arsenic species are known to directly react with DNA to induce genotoxicity (Mass et a/., 2001) and they have been identified as clastogen in human lymphocyte and mouse lymphoma cell (Kligerman et a/., 2003). Indeed, after intraperitoneal injection, MMA'II showed strong acute toxicities in hamsters. LD50 level of MMA'II was estimated to be around 29.3 mmol/kg which is 5 times lower than that of inorganic arsenic (Petrick et a/., 2001).
The reason for the potent toxicities of trivalent methylated metabolites over inorganic or pentavalent methylated metabolites has not been fully elaborated, but several explanations such the higher affinity to free thiol (Shiobara et a/., 2001) and the strong potential of oxidative stress (Nesnow et a/., 2002) of trivalent methylated metabolite have been suggested. Some reports on the thiol-bound arsenicals suggested that thiol-bound form, which is referred as arsenothiol, could be responsible for the toxicity of methylated metabolites (Styblo et a/., 1997). Supporting this view, a recent paper has shown that low-concentration of dithiol compounds potentiate the toxicity of trivalent methylated metabolites (Jan et a/., 2006). In addition, higher intracellular accumulation of MMA'II over ASIII might be suggested as well for the strong toxicity of MMA'II (Hirano et a/., 2004).
Effects of trivalent arsenic metabolites on cardiovascular system. Based on the epidemiological reports on the link between chronic ingestion of arsenic-contaminated drinking water and diverse cardiovasular diseases inculding peripheral vascular disease, ischemic heart disease, atherosclerosis and hypertension (Yu et a/., 2002;Abernathy et a/., 2003;Tseng et a/., 2003;Simeonova and Luster, 2004;Yoshida et a/., 2004;Kwok et a/., 2007), numerous studies have been directed to elucidate the effects of arsenic on cardiovascular system using in vitro as well as in vivo assays. It is now well-established by substantial number of evidences that the inorganic arsenic including arsenite and arsenate has deleterious effects on cardiovascular system. Exposure to arsenite and arsenate could lead to the development of atherosclerosis, increase of reactive nitrogen species, thrombus formation, and decrease of nitric oxide and cyclic GMP in vivo Simeonova et a/., 2003;Bunderson et a/., 2004). In isolated vascular tissues, alteration of vascular tone was induced by arsenite-induced excessive contraction as well as impaired relaxation (Lee et a/., 2003(Lee et a/., , 2005. In vascular cells like vascular smooth muscle cells and endothelial cells, inorganic arsenic could induce oxidative stress, DNA damage and apoptosis with the expression of several proteins by activation of transcription factors (Hirano et a/., 2003;Tsou et a/., 2003;Kumagai and Pi, 2004;Soucy et a/., 2004). Inorganic arsenic also enhances the activation of platelets, potentiating agonist-induced platelet aggregation and procogulant activity through phosphatidylserine exposure and microparticle generation Bae et a/., 2007).
Although a wide range of cardiovascular effects of inorganic arsenic is well-characterized, information on the role of trivalent methylated metabolites in arsenicassociated diseases is extremely limited. Hirano et a/. (2004) showed that MMA'II has higher toxicity than other arsenic species in rat heart endothelial cells, and Li et a/. (2007) have reported that MMA'II exposure leads to the inhibition of eNOS activity and alteration of eNOS phosphorylation in endothelial cells. Recently, we investigated the effects of trivalent methylated metabolites on vascular tone using isolated blood vessel, and found that MMA'II and DMA'II could induce smooth muscle dysfunction through the impairment of agonist-induced blood vessel contraction, which is mediated by the disturbance of calcium regulation (Fig. 2). Dysfunction of smooth muscle cells is well-correlated with the alteration of blood pressure after exposure to MMA'II in in vivo animal model. Besides vascular cells like smooth muscle cells or endothelial cells, trivalent methylated arsenic metabolites could affect platelet function. In our recent study, MMA'II and DMA'II were shown to induce apoptotic-like events in platelets, accompanied by phosphatidylserine exposure, resulting in the promotion of procoagulant activity which could be also confirmed in in vivo thrombosis model (unpublished data). Notably, procoagulant activity could be increased directly by MMA'II and DMA'II in the absence of activating agonists, in contrast to the case of inorganic arsenic where agonist was required for the inorganic arsenic-induced procoagulant activity (Bae et al., 2007).
Concluding remarks. Due to the potent toxicity and/or high reactivity of trivalent methylated arsenics in vitro, increasing efforts are currently being made to explain the role of these metabolites for the arsenicassociated adverse health effects. Although their actual level in environment and body after the chronic ingestion of arsenic need to be addressed further to confirm their roles, it is a matter of fact that trivalent methylated arsenic metabolites have deleterious effects on the diverse body systems, especially cardiovascular system. Upto now, most of the toxicological studies for the regulatory purposes have been focused on the effects of inorganic arsenic, but now the bottom line for assessment of arsenic-toxicity should be changed to explore the possible involvement of trivalent methylated metabolites. The potent effects of trivalent methylated metabo-lites with distinct toxic mechanisms from that of inorganic arsenic could give a novel clue for the explanation of the unsolved questions for arsenic-associated health effects.