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Experimental STudies This chapter discusses the effects of arsenic that have been observed in experimental studies. It begins with a summary of the experimental studies described in the 1999 report. Following that summary, toxicokinetic, animal toxicity, and mechanistic studies of arsenic, published since the previous NRC report was released, are discussed. This chapter does not provide a compre- hensive discussion of all the toxicologic mechanisms of arsenic. SUMMARY OF EXPERIMENTAL STUDIES DISCUSSED IN THE 1999 REPORT The previous NBC Subcommittee on Arsenic in Drinking Water reviewed the data on the toxicokinetics, animal toxicity studies, and mode of action of arsenic, focusing on the modes of action that might underlie the carcinogenic effects of arsenic (NRC 1999~. It concluded that inorganic arsenic is readily absorbed from the gastrointestinal tract in humans and it is mainly transported in the blood bound to sulfhydryl groups. At low-to-moderate doses, inorganic arsenic has a half-life in the body of about 4 days and is excreted primarily in the urine (NRC 19994. Humans and some animals methylate inorganic arsenic compounds to pentavalent monomethylarsonic acid (MMAV) and pentavalent dimethylarsinic acid (DMAV), which are less acutely toxic and readily ex- creted. At the time of the NRC 1999 report, there was little information on the 75
76 ARSENIC INDR1rNKING WA TER: 2001 UPDA TE distribution and toxicity ofthe trivalent methylated metabolites (monomethyI- arsonous acid AMMAN and dimethylarsinous acid (DMA~. It was noted that the fractions of the various metabolites of arsenic in urine (inorganic arsenic, MMA, and DMA) vary markedly among humans, and the toxicoki- netics of arsenic varies considerably among animal species. It is not known in which animal species the toxicokinetics more closely resembles that in humans, and this uncertainty makes it difficult to extrapolate from animals to humans. The subcommittee concluded that the mechanisms or modes of action by which inorganic arsenic causes toxicity, including cancer, is not well estab- lished (NRC 1999~. The data available on the ability of inorganic arsenic to act as a cocarcinogen or a tumor promoter in rats and mice are conflicting, but studies conducted at very high doses indicate that DMA~ is not a tumor initia- tor but might act as a tumor promoter. Furthermore, inorganic arsenic and its metabolites have been shown to induce chromosomal alterations (aberrations, aneuploidy, and sister chromatic exchange) and large deletion mutations, but not point mutations. Data on other genotoxic responses that might indicate mode of action for arsenic were not sufficient for conclusions to be drawn. Therefore, the 1999 subcommittee concluded that "the most plausible and generalized mode of action for arsenic carcinogenicity is that it induces struc- tural and numerical chromosomal abnormalities without acting directly with DNA." The subcommittee also discussed other mechanisms, such as cell proliferation and oxidative stress. An indirect mechanism of mutagenicity suggests that the mostplausible shape ofthe carcinogenic dose-response curve is sublinear "at some point below the level at which a significant increase in tumors is observed tin the available epidemiological studies]." There was insufficient scientific evidence to identify the dose at which sublinearity might occur. Therefore, the subcommittee concluded that "because a specific mode (or modes) of action has not been identified at this time, it is prudent not to rule out the possibility of a linear response." The subcommittee further concluded that arsenicals inhibit some types of mitochondrial-respiratory function, leading to decreased cellular ATE produc- tion and increased production of hydrogen peroxide (H2O2) (NRC 1999~. Those effects could cause the formation of reactive oxygen species, resulting in oxidative stress. Oxidative stress can have numerous effects, including inhibition of heme-biosynthetic pathways and induction of major stress pro- teins. Although the role of arsenic-induced oxidative stress in mediating DNA damage is not clear, the intracellular production of reactive oxygen species
EXPERIMENTAL STUDIES 77 might play an initiating role in the carcinogenic process by producing DNA damage. In the remainder ofthis chapter, more recent studies on the toxicoki- netics of arsenic will be discussed, followed by in vitro and in vivo studies that provide additional information on the mode of action of arsenic. TOXICOKINETICS Methylation of Arsenic . Arsenic can exist in methylated and inorganic forms as well as in different valence states (NRC 1999~. The form and valence state can affect the toxicity of arsenic; therefore, it is important to understand the metabolism and toxicokinetics of arsenic. As discussed in the 1999 NRC report, inorganic arsenic is believed to be methylated via sequential reduction of pentavalent arsenic to trivalent arsenic, followed by oxidative addition of a methyl group from S-adenosy~methionine (SAM) to the trivalent form (Figure 3-11. The main products of that methyla- tion, MMAV and DMAV, are readily excreted in the urine. More recent exper- iments have detected the presence of the reduced methylated forms EMMA and DMA~) in human urine.) The development ofthe analytical methods for the speciation of arsenic metabolites, as well as the advantages and disadvan- tages ofthese methods, were thoroughly discussed in the previous NRC report NRC 1999~. The reduced methylated forms and their toxicity are discussed later in this chapter. Further methylation of DMA to trimethylarsine is frequently seen in mi- croorganisms exposed to arsenite ARC 1999~. A smallpercentage of urinary arsenic as trimethylarsine oxide (TMAO) has been detected in mice, hamsters, and humans following exposure to DMA (for review, see Kenyon and Hughes 2001), but TMAO or demethylated products of DMA were not detected in the blood or tissues of mice exposed intravenously to DMA at a dose of 1 or 100 mg/kg (Hughes et al. 2000~. By contrast, TMAO has not been reported to be present in the urine of mammals exposed to inorganic arsenic. DMA foe by methylation of inorganic arsenic (Ash) and MMAi~has been shown to clear 'Because trivalent methylated forms of arsenic have been detected only recently, they often have not been specifically assayed for or discussed in most experiments. In those cases, the abbreviations MMA and DMA are used without indicating the valence state.
78 ARSENIC IN DRINKING WA TER: 2001 UPDA TE rapidly from cells (Styblo et al. ~999a; Lin et al. 200~). That rapid clearance might prevent the accumulation of intracellular concentrations of DMA re- quired for further methylation to TMAO, explaining why TMAO is not de- tected following exposure to Astir. Nonenzyrnatic methylation of arsenic has been seen in vitro. Studies have demonstrated that methy~cobalam~n (a form of vitamin By) can mediate the nonenzymatic methylation of arsenite (Zakharyan and Aposhian 1999a), but whether that occurs in viva is not known. Glutathione (GSH), end possibly other thiols, can act es reducing agents in the methylation process (NRC 1999~. Recent in vitro studies indicate that dithiols (e.g., reduced lipoic acid) might be more active than GSH in providing the reduc- ing environment required for methylation by MA methyTtransferase (Zakharyan et al. 1999~. Thiol binding might also be involved in arsenic biotransformation. Trivalent arsenic metabolites are highly bound to cytosolic proteins in the cells (Styblo and Thomas 1997~. In in vitro studies, protein-bound inorganic arsenic and MMA were methylated to MMA and DMA, respectively. It has been proposed that arsenic is bound to a protein dithiol cofactor before the sequential methylation of arsenic (Thompson 1993; DeKimpe et al. 1999a). The exact sequence of events in arsenic biotransformation remains urdmown, but it seems clear that S-adenosy~me~ionine (SAM) is the main source of methyl H2AsO4- CH3+ 2e~ CH3+ 2e~ AsO33- CH3AsO32- Reductase Methyl- MMAV transferase 2e~ (CH3~2AsO2----------------> (CH3~2AsO Methyl- DMAV Reductase DMA~i transferase CH3ASo2 Reductase MMA~ FIGURE 3-1 Proposed chemical pathway for the methylation of inorganic arsenic in humans. The enzymes that have been proposed for the reduction and methylation reactions are indicated. Uncertainties regarding this pathway are discussed in the text.
EXPERIMENTAL STUDIES 79 groups for the methylation of arsenic and that the methylation is dependent upon methyTtransferases. Studies have shown that SAM is required for arsenic methylation in various in vitro systems and that arsenic methylation in viva is decreased by specific inhibitors of SAM-dependent methylation and by low methionine intake ARC 1999~. Because of the importance of SAM in the methylation of arsenic, much research has been aimed at characterizing the methyTtransferases involved in SAM-dependent arsenic methylation. During recent years, arsenite and MMAi~ methyTtransferases from the liver of rabbits, hamsters, and rhesus monkeys have been purified and partially characterized (Wilt/fang et al. 1998; Za~aryan et al. 1999~. The Kin,, determined using Michaelis-Menten kinetics, for arsenite methyTtransferase for hamsters was ~ .79 x ~o-6 M and for MMA methy~transferase was 7.98 x 10- M. The MMA methy~transferase was higher than the arsenite methyTtransferase. Similar values were reported for rabbit methy~transferases, but the rhesus monkey had Knot values for MMA methyTtransferase of 3.5 X 10-6 M and for arsenite methy~transferase of S.5 x 10-6 M. Zakharyan et al. (1999) reported that the rabbit liver MMA methyTtransferase had higher affinity for MA than for MMAV. Furthermore, the Km for MMA~i methyTtransferase from Chang hu- man hepatocytes was not very different from that of rabbit liver. The exact structures ofthe arsenic methy~transferases have not been deter- mined (NRC ~ 999), but recent in vitro studies using rabbit liver cytoso! show- ed that the two steps in arsenic methylation to DMA are markedly inhibited by pyrogalloT (0.3 to 9 millimolar) (mM), a specific inhibitor of catechol-O- methyTtransferase (DeKimpe et al. ~ 999a). Those data suggest that the active sites of arsenite methyTtransferase and MMA methyTtransferase are similar to that of catechol-O-methy~transferase. Furthermore, trichIoromethiazide (TCM), an inhibitor of human microsomal thiopurine methyTtransferase, did not inhibit the formation of MMA but did inhibit the formation of DMA. in contrast, neither ofthose arsenic methylation steps was inhibited by an inhibi- tor of cytosolic thiopurine methy~transferase (p-anisic acid) or by an inhibitor of cytosine DMA methyTtransferase. ADMA methy~transferase, which would further methylate DMA forming TMAO, has not been reported to be present in mammalian cells. Styblo et al. (1 999b) reported that DMA was the only metabolite detected in rat or human hepatocytes incubated with DMAV or a glutathione complex of DMA~ (DMAi~-GS).
80 ARSENIC IN DRINKING WA TER: 2001 UPDA TE Species Differences in the Methylation of Arsenic As discussed in the previous report, there is considerable variation in the methylation of inorganic arsenic among mammalian species (NRC 1999~. Rats, mice, and dogs show a very efficient methylation of arsenic to DMA. Rabbits and hamsters also methylate arsenic relatively efficiently. In most animals, the DMA that is formed is rapidly excreted in urine. However, in rats, most of the DMA that is formed accumulates in the red blood cells and tissues. Rats also appear to methylate administered DMA to TMAO more efficiently than other species (Kitchin et al. 1999; NRC 1999~. Following exposure of mate rats to high concentrations of DMA in drinking water (100 mg/L, equivalent to 100,000 )lg/~), about 10% of the urinary arsenic was present as TMAO (Yoshida et al. 1998~. Consistent with that marked variation in arsenic methylation efficiency seen among animal species (Vahter 1999b), the activity of methy~transferases differs markedly among the animals studied (Healy et al. ~ 999~. The variation in the activity of those enzymes probably underlies most of the cross-species variability in methylation ability (for review, see Healy et al. 1999; Vahter 1 999a). Guinea pigs and several types of non-human primates, including the chimpanzee, seem to be unable to methylate inorganic arsenic (Healy et al. 1999; Vahter ~ 999b), and no methyTtransferase activity was detected in those species (Healy et al. 1999~. Human arsenic methy~transferases were Tong thought to be very unstable, because activity could not be detected in human liver preparations (NRC ~ 999~. Recently, however, arsenic-methylation activity was detected in hu- man hepatocytes; MMA~imethy~transferase activity was detected in cultured Chang human hepatocytes (Zakharyan et al. 1999~. Incubation of primary human hepatocytes with arsenite yielded MMA and DMA, and incubation with EMMA' produced mainly DMA (Styblo et al. 1999a). Rat hepatocytes, however, methylated arsenic considerably faster than did human hepatocytes (Styblo et al. 1999a; Styblo et al. 2000~. In contrast to humans, most mammals do not excrete appreciable amounts of MMA in the urine. Recently, however, the Flemish Giant rabbit was found to excrete substantial amounts of MMA in urine (DeKimpe et al. ~ 999b), and MMA was formed in vitro after incubation of inorganic arsenic with rabbit- liver cytosol (DeKimpe et al. 1999a). No differences, however, were seen in the urinary pattern of arsenic me- tabolites in three strains of mice 24 hours (fur) after administration of an oral
EXPERIMENTAL STUDIES 81 dose of arsenite (Hughes et al. 1999~. More than 95°/O of the arsenic in urine (corresponding to 60%, 68%, and 69% ofthe administered dose in each ofthe three mouse strains) was in the form of DMA in all three mouse strains. As indicated above, the ability to excrete MMA and DMA in the urine varies among species. Because ofthis variation and other species differences in arsenic metabolism discussed in this section, extrapolation of data from studies in animals and animal cells to humans is difficult. Tissue Differences in the Methylation of Arsenic The liver appears to play the central role in arsenic methylation (NRC 1999), but in vitro studies using mate mouse cytosoT demonstrated that most tissues appear to be capable of methylating arsenic (Healy et al. ~998~. The highest activity of arsenite methyTtransferase was observed in the cytoso! from the testis, followed by cytosol from the kidney, liver, and lung. More recent data also point to the liver's important role. Styblo et al. (2000) reported a much higher rate of arsenic methylation in primary human hepatocytes com- pared with human keratinocytes and bronchial cells, and no methylation activ- ity was detected in human urinary-bladder cells. When proliferating human keratinocytes and bronchial epithelial cells were cultured in the presence of the relatively Tow arsenite concentration of 0.05 micromolar (EM) (approxi- mately 3.7 high), more than two-thirds of the cell-associated methylated arsenic consisted of MMA, most of which was retained intracellularly throughout a 24-hr incubation. Human keratinocytes cultured in the presence of ~ EM of methylarsine oxide, a putative substrate for MA methyI- transferase, did not produce any DMA. Recent studies indicate that EMMA' might be the most toxic intracellular form of arsenic in terms of oxidative stress, enzyme inhibition, and DNA `damage (see Mechanistic Data later in this chapter). It is noteworthy that cells from two tissues that are targets of arsenic-induced cancer (skin and lung) seem to have less efficient conversion of MMA to DMA at relevant concentrations of arsenite in culture that is, at concentrations similar to those that might occur in blood and possibly tissue following chronic ingestion of low-to-moderate concentrations of arsenic. However, more studies on this topic are needed before firm conclusions can be reached. The situation is even more complex in viva, where the extent of methyla- tion of inorganic arsenic to MMA and DMA is also influenced by the rate of
82 ARSENIC IN DRINKING WATER: 2001 UPDATE cellular uptake of the inorganic arsenic in the various tissues. Tatum and Hood (1999) reported that the uptake and methylation of Asset by a kidney- epithelium-derived cell line, the NKR-52E cell line, were lower than those of primary rat hepatocytes or hepatoma-derived cell lines. Those data confirm previous findings that Astir is the main form of arsenic taken up by the liver (NRC 1999~. In the presence of phosphate-free media, the uptake and cytotoxicity of AsV in KB oral epidermoid carcinoma cells was greatly en- hanced (Huang and Lee ~996~. Cellular uptake and efflux of arsenic vary considerably among the differ- ent arsenic metabolites, and the variation affects the distribution of metabo- lites formed in the liver following absorption of arsenic compounds. In com- parison to the trivalent methylated forms of arsenic, exposure to the pentava- lent forms (MMAV or DMAV) results in very low tissue concentrations of MMA and DMA (Hughes and Kenyon 1998; NRC 1999~. That difference is probably because of a lower cellular uptake and accumulation of the pentava- lent forms than the trivalent forms. A lower uptake was confirmed in studies in rat and human hepatocytes that showed a several-fold higher cellular uptake of Astir and MA than of the corresponding pentavalent forms (Styblo et al. 1999a). In the presence of phosphate-free media, the uptake and cytotoxicity of AsV in KB oral carcinoma cells were greatly enhanced (Huang and Lee 1996~. Cellular efflux also appears to vary among the different forms of arsenic. Styblo et al. (1999a) demonstrated that DMA is the main excretory product in human and rat hepatocytes. Inhibition of the methylation of MMA to DMA by increasing arsenite concentrations in the medium resulted in the accumula- tion of MMA in the cells, indicating that MMA is not excreted as readily from liver cells as is DMA. Whether that same effect occurs in cells from other tissues is unknown. Induction of Arsenic Methy~transferases The inducibility of arsenic methyTtransferase has been investigated in mice. Arsenic methyTtransferase activity did not appear to be induced in mice exposed subchronicly to arsenic in the drinking water (25 or 2,500 Age) (Hughes and Thompson 1996; Healy et al. 1998~.
EXPERIMENTAL STUDIES 83 Trivalent Methylated Arsenic Metabolites It is obvious from the proposed scheme of arsenic methylation (Figure 3- 1) that pentavalent methylated arsenic compounds can be reduced to trivalent methylated arsenic compounds EMMA and DMAi~. Although pentavalent arsenicals can be reduced directly, for example by glutathione (NRC ~ 999), recent studies indicate the involvement of arsenic reductases. Arsenate reduc- tase activity has been detected in human liver (Radabaugh and Aposhian 2000~. The enzyme has a molecular weight of about 72 kilodaltons (kI:)a), and it requires a thiol and a heat-stable cofactor for activity. It did not reduce MMAV, indicating the presence of two enzymes, arsenate reductase and MMAVreductase. MMAV reductase activity has been detected in rabbit liver (Zakharyan and Aposhian 1999b), hamster tissues (Sampayo-Reyes et al. 2000), and human liver (Zakharyan et al. 2001~. There is evidence that the human MMAV reduc- tase is identical to glutathione-s-transferase omega class A. The rabbit liver MMAV reductase was shown to reduce both DMAV and arsenate (AsV) (Zakharyan and Aposhian 1999b). The Kn~ values were 2.16 x i0-3 M with MMAV as the substrate, 20.9 x 10~3 M with DMAV as the substrate, and 109 X i0-3 M with arsenate as the substrate. ~en the Km for the rabbit liver MMAV reductase was compared with that ofthe As~ methyTtransferase (i.e., 5.5 x lo-6) and that of MMA~ methyTtransferase (i.e., 9.2 x lo-6), the authors concluded that MMAV reductase was the rate-limiting enzyme for arsenite metabolism in the rabbit liver. However, it should be emphasized that, be- cause of the species differences in arsenic methylation (see above), it is not known whether the rate-limiting step in the rabbit liver equates to the rate- limiting step in any particular human organ. It is not clear to what extent the DMA formed following exposure to inorganic arsenic is reduced to DMA~'~ by a specific DMAV reductase. The latter has not been well studied, but DMA~ was detected in the liver of ham- sters given arsenate (Sampayo-Reyes et al. 2000~. Tn addition, as mentioned previously, a small percentage of TMAO is found in the urine following expo- sure to DMA. The formation of TMAO would require the reduction of DMAV to DMAi~ before the addition of the third methyl group, indicating DMAV reductase activity. The activities of the arsenic reductases appear to vary markedly among tissues. In the male hamster, the highest activity was found in the brain, fol- lowed by the bladder, spleen, and liver; the Towest activity was found in the
84 ARSENIC IN DRINKING WA TER. 2001 UPDA TE testis (Sampayo-Reyes et al. 2000~. Therefore, the tissues with high arsenic reductase activity seem to be different from those with high arsenic methyTtransferase activity (testis > kidney > liver > lung, as discussed previ- ously). Considering the marked species differences in the metabolism of arsenic, more data are needed on the tissue variation in arsenic metabolizing enzymes in various species. In particular, more data are needed on tissue variations in enzyme activities in humans. More data are also needed on sex differences, because most experiments have been performed in male animals. There is increasing evidence that the trivalent methylated arsenic metabo- lites (especially EMMA are released from the site of arsenic methylation. Aposhian et al. (2000a) reported that people in Romania exposed to arsenic in drinking water (28, 84, or 161 PAL) had MA in their urine at concentra- tions of 5 -7 ,ug/L, irrespective of their exposure. Mandal et al. (2001) re- ported the presence of both MMAi~ (2-5% of urinary arsenic) and DMA~ (5- 20% of urinary arsenic) in the urine of subjects chronically exposed to inor- ganic arsenic via drinking water (33-250 ,ug/~) in four villages in West Ben- gal, India. The concentrations of MMAi~i and DMAi~ in the four villages ranged from 3-30 ,ug/L and 8-64 vigil, respectively. The concentrations of both EMMA and DMA~ increased with increasing concentration of total arsenic in urine (i.e., the sum of metabolites). It should be noted that the concentrations oftrivaTent metabolites in the urine might have been underesti- mated, because the trivalent metabolites are easily oxidized (Le et al. 2000~. On the other hand, the trivalent metabolites are reported to be highly reactive; therefore, it is unlikely that urinary concentrations of the trivalent metabolites would be as high as MMAV and DMAV, which are much less reactive and readily excreted in urine. There are very few data on the tissue distribution of trivalent methylated arsenic metabolites following exposure to inorganic arsenic, and no data in humans. Bile-duct-canulated rats were injectedintravenously with arsenite or arsenate. Almost 10% ofthe injected dose (50 ~mol/kg) was excreted in the bile as MMAii~ and Astir (Gregus et al. 2000~. Rats excrete much more arsenic in the bile than other species; therefore, it is difficult to generalize those re- sults to other species. However, further support for the formation of trivalent methylated arsenic compounds in vivo following exposure to inorganic arsenic comes from experiments in hamsters. Both MMAiii and DMA~i were detected in the livers of hamsters treated with arsenite (Sampayo-Reyes et al. 2000~. Some ofthe trivaTentmethylated arsenic metabolites found in urine might be, in part, the result of reduction occurring in the kidneys and urinary blad-
EXPERIMENTAL STUDIES 85 den It has been shown that arsenate is reabsorbed and reduced in the proximal renal tubuTi, after which Astir is excreted in the urine ARC 1999~. Also, as mentioned above, high MMAV reductase activity was found in the bladder of the hamster (Sampayo-Reyes et al.2000), indicating that reduction of MMA, and possibly also DMA, might occur in the urinary bladder. Administration of 300 mg ofthe chelating agent sodium 2,3-dimercapto- 1 - propane sulfonate (DMPS) to people exposed to arsenic in drinking water (568 ~ 58 vigil) in Inner Mongolia, China, markedly increased the urinary concentrations of inorganic arsenic (50-125,ug/L, on average) and MMA (50- 325 )lg/~), while the concentration of DMA decreased (240-125 ,ug/~) (Aposhian et al. 2000a; Le et al. 2000~. This study was the first to identify MMAi~ in urine as one of the arsenic metabolites. In vitro studies using par- tially purified rabbit liver MMAi~ methy~transferase showed that the MMAi~- DMPS complex did not serve as a substrate for the enzyme (Aposhian et al. 2000a). Therefore, the authors suggested that DMPS forms a stable complex with MA, which is excreted in urine. It should be noted that the amount of EMMA formed in tissues following exposure to inorganic arsenic is dependent upon the activity of arsenite methy~transferase, the enzyme that forms MMAV, and the presence of MMAV reductase, the enzyme that reduces MMAV to MA. It is also dependent on the presence and activity of MA methyTtransferase, the enzyme that further methylates EMMA to DMAV, which is readily excreted from cells (Styblo et al. 2000~. Inorganic Asiii and the reduced foes of the methylated arsenic metabo- lites EMMA and DMA~) are highly reactive and might contribute to the toxicity observed following exposure to inorganic arsenic (see Mechanisms of Toxicity). As discussed in the previous NBC report (NRC ~ 999) and by Styblo et al. (1997), trivalent inorganic and methylated arsenic metabolites have been shown to complex with GSH. Also, trivalent arsenicals are be- lieved to forte highly stable complexes with molecules containing vicinal thiols. In studies investigating the binding of Astir to proteins following expo- sure to arsenite in human lymphoblastoid cells, at least four 20-50 kDa pro- teins with arsenic affinity were isolated (Menzel et al. 1999~. Two of the proteins identified were tubulin and actin. This ability of arsenic to bind to functional groups (e.g., thiols) can result in the inhibition of certain enzymes (tin et al.2001) and is one possible mechanism underlying arsenic's toxicity. Reactions with sulfhydry] groups are also the basis for arsenic detoxification therapy (e.g., use of DMPS) (Aposhian et al. 2000a).
86 ARSENIC IN DRINKING WA TER: 2001 UPDA TE ANIMAL TOXICITY STUDIES Animal Bioassays Although arsenic is not typically positive in experimental animal carcino- genicity bioassays (NRC ~ 999), several recent experiments have demonstrated that, under certain conditions, some forms of arsenic can induce tumors in animals. For a recent doctoral dissertation, which was also published as a confer- ence proceeding, Ng (1999) administered AsV at 500 ~g/L in drinking water to 90 female C57BL/61 and 140 female metallothionein (MT) knock-out transgenic mice for 26 months. Control groups of 60 mice received tap water containing arsenic at less than 0.l ,ug/L. Weekly water consumption and weight gain were reported, and survival was high (i.e., survival rates of 81% for C57BL/6] exposed mice, 74% for MT knock-out exposed mice, 98% for C57BL/61 control mice, and 97% for MT knock-out control mice after 2 years). The data indicate that the exposed mice developed more tumors than the controls by study termination. The subcommittee's understanding is that the results of this study are undergoing further examination and should be regarded as preliminary. If final analyses confirm the preliminary report, those results will be important because they would be the first data to demon- strate tumors in animals following ingestion of inorganic arsenic. Waalkes et al. (2000) injected rats intravenously (i.v.) with sodium arse- nate at 0.5 mg/kg once weekly for 20 weeks; the study was completed after 96 weeks. Significant skin changes (hyperkeratotic lesions) and renal lesions occurred in arsenate-treated females. Although these repeated arsenate expo- sures were not tumorigenic outright, there was clear evidence of proliferative, preneoplastic lesions of the uterus, testes, and liver. The authors suggested that because estrogen treatment has been associated with proliferative lesions and tumors of the uterus, female liver, and testes in other studies, arsenate might somehow act through an estrogenic mode of action. That hypothesis is supported by the observation that arsenate-induced uterine hyperplastic le- sions showed a strong up-regulation of cyclic D1, an estrogen-associated gene product, and an up-regulation of estrogen receptor (ER) immunoreactive protein in the early lesions of uterine luminal and glandular hyperplasia. Simeonova et al. (2000) administered sodium arsenite in drinking water to mice at doses of either 0, 0.002%, or 0.01% (20,000 or 100,000 ppb) for up to 16 weeks. After 4 weeks of exposure to the high dose (100,000 ppb), mouse urothelium exhibited hyperplasia, accompanied by accumulation of
EXPERIMENTAL STUDIES 3 7 inorganic trivalent arsenic and, to a lesser extent, DMA. The authors found a persistent increase in DNA binding ofthe nuclear transcription factor, AP-1, in bladder epithelium of arsenite-exposed mice at both doses. Using a trans- genic strain of mice possessing a luciferase reporter gene containing an AP-l activation site, they were able to demonstrate that arsenite induces AP- 1 -medi- ated transcriptional activation in the urothelium in viva. Using several tools to determine alterations in gene transcription, including cDNA expression arrays, the authors also demonstrated that arsenite alters the expression of a number of genes associated with cell growth, including the oncogenes c-foe, cjun, and EGRET. The expression of genes involved in cell-cycle arrest, including GADDl 53 and GADD45, were enhanced by arsenite. The authors concluded that "the proliferation-enhancing effect of arsenic on uroepithelial cells likely contributes to its ability to cause cancer." In a follow-up study (Simeonova et al. 2001) in which groups of mice were exposed to a range of arsenite concentrations in drinking water, an increase in AP-l DNA binding in bladder epithelial tissue was detected after ~ weeks of exposure to brining water containing 50,000 ,ug/L and 100,000 ,ug/L, but not after exposure to water containing arsenite at SOO ~g/L and 20,000 Go/. Santra et al. (2000) examined the hepatic effects of chronic ingestion (up to ~ 5 months) of Winking water containing arsenic hi: ~ arsenite to arsenate) at 3.2 mg/L (3,200 ,ug/~) in male BALB/c mice. Groups of arsenic-exposed mice and unexposed controls were sacnficed at 3, 6, 9, 12, and 1 S months for examination of hepatic histology and certain biochemical parameters of oxida- tive stress. Statistically significant decrements in body weight appeared in the exposed animals at 12 months and IS months, without significant differences between exposed and control groups in the amount of food or water consump- tion. No abnormal hepatic morphology was observed by light microscopy during the first 9 months of arsenic exposure, but at 12 months, ~ ~ of 14 mice in the experimental group exhibited hepatocellular degeneration and focal mononuclear cell collection. After 15 months, exposed mice displayed evi- dence of hepatocellular necrosis, intralobular mononuclear cell infiltration, Kupffer cell proliferation, and portal fibrosis. Hepatic morphology was nor- mal in all control mice. Biochemical changes consistent with oxidative stress preceded the overt histological pathology: hepatic glutathione was signifi- cantly reduced after 6 months, hepatic catalase was significantly reduced at 9 months, and hepatic glutathione S-transferase and glutathione reductase activities were significantly reduced at 12 and ~ 5 months. There was a pro- gressive, time-dependent increase in lipid peroxidation, as evidenced by in- creased production of malondialdehyde, and concomitant time-dependent
88 ARSENIC IN DRINKING WA TER. 2001 UPDA TE damage to hepatocellular plasma membranes, as evidenced by decreases in membrane Na+/K+ ATPase activity. The findings of Santra et al. (2000) repre- sent the first animal model to demonstrate hepatic fibrosis following chronic arsenic ingestion. Periportal fibrosis, sometimes with noncirrhotic portal hypertension, is a recognized sequela of chronic arsenic ingestion in humans (Santra et al. 1999; NRC 1999~. Biochemical changes observed in this long- term in vivo animal-feeding experiment suggest that these adverse effects of arsenic may be mediated through oxidative stress. In the past, DMAV has been considered primarily a detoxification product in arsenic metabolism that is rapidly eliminated from cells and excreted in the urine. However, over the past several years, there has been mounting evi- dence that DMA might possess biological activity that is relevant to arsenic carcinogenesis. Wei et al. (1999) found that chronic administration of DMA in drinking water, albeit at very high doses, to mate Fischer 344 (F344) rats induced bladder cancer. The rats were given DMA at 0,12.5,50, or 200 parts per million (ppm) (0, 12,500, 50,000, 200,000,ug/~) in drinking wafer for 104 weeks. From weeks 97 to 104, urinary bladder tumors were seen in ~ of 3 ~ rats (26%) at 50 ppm, and 12 of 3 ~ rats (39%) at 200 ppm. No bladder tumors were seen in any ofthe control animals (36 rats) or in the 12.5-ppm group (33 rats). In a study of lung tumors in mice, Hayashi et al. (1998) found that admin- istration of DMA at 400 ppm (400,000 ~g/~) in drinking water for 50 weeks to A/] mice produced an increase in the number of pulmonary tumors per mouse (~ .36 and 0.5 tumors per mouse in exposed and control groups, respec- tively), and the authors concluded that DMA alone can act as a carcinogen in mice. Neither of the studies by Wei et al. (1999) and Hayashi et al. (1998) pro- vices dose-response data, but the types of tumors observed (bladder and lung tumors) are highly relevant to what has been seen in human epidemiological studies. Yamanaka et al. (2000) administered DMA at either 400 or 1,000 ppm (400,000 or 1,000,000 ,ug/L) in drinking water to hairless mice that were concomitantly exposed to ultraviolet B radiation (2 kilojoules per square meter (kI/m2) twice weekly) for 25 weeks. The number of maTignant skin tumors per mouse was slightly, but significantly, increased in the I,000-ppm treatment group, but no differences from the control were seen in the 400-ppm group. There were no differences in the percentage of tumor-bearing mice over the 25-week period. Therefore, in this study, there was only a modest
EXPERIMENTA ~ STUDIES 89 cocarcinogenic effect seen at a very high dose of exogenously administered DMA. In addition to these bioassays of DMA, another study found that adminis- tration of DMA in drinking water (100 ppm, or ~ 00,000 ~g/L, for 36 weeks) can act as a bladder tumor promoter in Lewis x F344 strain of rats initiated with nitrosamine in drinking water (Chen et al. 1999~. Cohen et al. (2001) showed that DMA at 100 ppm (100,000 ,ug/L) in the diet of female F344 rats produced cytotoxic changes in the rat urothelium as early as 6 hr after expo- sure was begun. Necrosis was evident by 3 days, followed by regenerative hyperplasia of the bladder epithelium. Morikawa et al. (2000) found that 3.6 mg of DMA applied topically to a skin-tumor sensitive strain of mice twice a week for 18 weeks significantly accelerated skin tumor development. The authors concluded that DMA has a promoting effect on skin tumorigenesis in this strain of mice. The cancer bioassays of DMA utilized extremely high doses administered to rats. Their interpretation is limited by the lack of any studies relating the relative intracellular concentrations of DMA achieved by direct administration of DMA in drinking water to the intracellular concentrations derived from biomethylation of arsenic to DMA following chronic exposure to inorganic arsenic in drinking water. The relative uptake of exogenously administered DMA into cells has not been determined directly, but Males et al. (1998) found that the transmembrane permeability coefficient for DMA was two orders of magnitude greater than that for MMA when measured in unilameliar vesicles, suggesting that DMA may readily diffuse across cell membranes. Furthermore, trimethylarsine oxide has been identified in human urine follow- ing administration of DMAV, indicating that some uptake and metabolism of DMA does occur in humans (for review, see Kenyon and Hughes 2001~. In summary, several new animal bioassays of various forms of arsenic have been reported since the previous NBC report (NRC 1999~. Although these studies have demonstrated "positive" carcinogenic responses to arsenic in rodent species under certain conditions, the doses used were very high relative to human exposures to arsenic via drinking water. Relatively few doses were used, and information on the shape of the in vivo dose-response curve is limited at best. Although these studies are of qualitative interest in demonstrating the carcinogenic potential of arsenic in rodents, the studies provide little useful quantitative information relevant to human risk assess- ment of arsenic following exposure via drinking water and cannot replace the current body of epidemiological data used for risk assessment.
90 ARSENIC IN DRINKING WA TER. 2001 UPDA TE Species Differences in Toxicity As noted in the 1999 NRC report, there are differences in susceptibility to arsenic among animals. As discussed earlier in the Toxicokinetics section, species differences have also been noted in more recent studies. Mice were found to be less susceptible than rats to DMA toxicity (Ahmad et al. ~ 999; Brown et al. 1997~. Mice dosed orally with DMA at 720 mg/kg showed no DNA damage in the lung, no induction of cytochrome P~50 in the liver, and no reduction in serum alanine aminotransferase activity. In contrast, all of those changes were observed in rats dosed with DMA at 387 mg/kg. Both species had reduced liver GSH and reduced lung ornithine decarboxylase activity at the doses administered. Rats and guinea pigs, but not mice, showed an accumulation of copper in the kidneys following administration of sodium arsenite at levels of 0, ~ 0, 30 or 60 mg/kg of diet for I, 2, or 3 weeks (Hunder et al. 1999~. The authors attributed the species difference to more efficient methylation and elimination of arsenic by mice than by rats and guinea pigs. The copper content of the renal cortex increased from 10 Gig of wet weight to as high as 65 Gig of wet weight in a time- and concentration-dependent manner. Developmental Toxicity Studies One recent animal study has been published on the developmental toxicity resulting from repeated oral dosing with arsenic compounds. Data on the developmental effects of arsenic in humans is discussed in Chapter2. Arsenic trioxide was repeatedly administered orally to CrI:CDO(SD)BR rats at doses of 0, I, 2.5, 5, or ~ O mg/kg/day (Holson et al. 2000~. No evidence of develop- mental toxicity was found at doses that did not cause maternal toxicity. Another study examined the effect of genotype in mice on arsenite-in- duced congenital malformations in the neural tube (Machado et al. 1999~. Mice that were bred to include the "splotch" allele were more sensitive to the teratogenic effects of arsenite, illustrating that genotype can influence arsenic toxicity. It should be noted that doses of arsenicals required to elicit teratogen- esis acutely in outbred strains of mice are orders of magnitude higher than doses to which humans are exposed environmentally.
EXPERIMENTAL STUDIES 91 MECHANISMS OF TOXICITY Numerous studies have been completed in the past 3 years that address the potential modes of action and specific mechanisms by which arsenic exerts its toxic effects, including cancer. Four major, and overlapping, areas have. re- ceived much attention: (l ~ induction of mutations and chromosomal abbera- tions; (2) alterations in signal transduction, cell-cycle control, differentiation, and apoptosis; (3) induction of oxidative stress; and (4) alterations in gene expression. It is important to recognize that these categories of potential modes of action are not distinct, and the contribution of a specific mechanism (e.g., particular type of mutation, inhibition of a specific enzyme or pathway, or induction of a specific form of reactive oxygen species) within each cate- gory is difficult to determine. The sensitivity of those targets would be ex- pected to be dose- and time-related and might also be species and tissue spe- ClilC. Because various forms of arsenic have different toxic potencies or modes of action, some discussion of the new findings on the relative differences is warranted before discussion of the new data on each potential mode of action listed above. Resistance and tolerance to the cytotoxicity of arsenic and the implications ofthe experimental data on the carcinogenicity of arsenic are also discussed. Table 3-1 provides an overview of some of the mechanistic studies that have been completed since 1998. The subcommittee focused on studies that appear to induce biochemical effects at moderate to relatively low concentra- tions of arsenic in vitro (e.g., less than 10 AM), although a few studies that used higher concentrations are included for comparative purposes. Studies that require arsenic concentrations greater than ~ O ,uM to produce a biological response in vitro are less likely to be relevant to the health effects related to chronic ingestion of arsenic in drinking water and have not been exhaustively reviewed. Relative Toxicity of Different Forms of Arsenic In the previous NRC report, it was stated that the pentavaTent methylated arsenic metabolites are much less mutagenic than the inorganic arsenicals (NRC 1999; p. 199~. At that time, few studies had directly assessed the relative toxicity and mutagenicity of the trivalent methylated forms of arsenic.
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98 ARSENIC IN DRINKING WA TER: 2001 UPDA TE Aposhian and colleagues (Petrick et al. 2001) recently evaluated the relative toxicities of MA and Astir, in the hamster. Single LDsoS (lethal dose to 50°/O of animals) in hamsters were calculated to be 29 and 113 micromoles (pmol)/kg for MMAii~ and Astir, respectively. Those data represent the first in viva acute toxicity data for ALA and show, somewhat surprisingly, that it is about 4 times more acutely toxic than inorganic arsenite. Earlier LDso studies showed that the pentavaTent species MMAV is less toxic than arsenite by more than one order of magnitude in some species (for a tabular compari- son, see ATSDR 2000~. Petrick et al. (2001) also found that EMMA was a much more potent inhibitor of hamster-kidney and porcine-heart pyruvate dehydrogenase (PDH) than inorganic arsenite. Previous studies demonstrated that Asset was a potent inhibitor of PDH via covalent binding ofthe arsenite to critical vicinal dithiols on the enzyme. Because MMAi~ is in the same oxida- tive state as Astir, the authors suggested that the mechanism for MA is likely to be the same. Using nuclear magnetic resonance (NMR) spectros- copy, they also showed that methylarsine oxide EMMA oxide, (CH3AsO)n) and diiodomethylarsine EMMA iodide, CH3AsT2), the chemical forms of EMMA administered to the animals and used in the in vitro studies, hydrolyze to EMMA (CH3As(OH)2) in aqueous solution. Several other new studies investigated the toxicity of different forms of arsenic and its methylated derivatives in cultured cells. Moderate amounts of Asi~iinhibited the synthesis of DMA, resulting in an accumulation of Ashland monomethy! arsenic species in the cells. The same investigators (Petrick et al. 2001 ) found that, in viva, MMA was more toxic than arsenite when given to hamsters intraperitoneally (LDso = 29.3 ~mol/kg and 112 ,umol/kg, respec- tively). Using primary cells from human and rat liver, human neonatal fore- skin, and human cervix as well as a simian virus (SV)-40-transformed epithe- lial cell line derived from normal human urinary bladder cells, the investiga- tors found that the trivalent methylated metabolites of arsenic were more cytotoxic than arsenite in all cell types (Styblo et al. ~ 999b). The same group further reported that EMMA was the most cytotoxic in all cell types (including primary human bronchial epithelial cells) and that DMA~ was at least as toxic as Astir (Styblo et al. 2000~. Pentavalent arsenicals were much less cytotoxic than the trivalent forms in all cell lines. The transformed cell line derived from human bladder cells had no capacity to methylate the various forms of arsenic. It is uncertain whether primary epithelial cells lack the methylation capacity or whether the transformation process causes the lack of capacity. There was no apparent correlation, however, between susceptibility of cells to arsenic toxicity and their capacity to methylate Asiii. Similar findings were reported by Petrick et al. (2000), who tested the relative toxicities of different
EXPERIMENTAL STUDIES 99 forms of arsenic in Chang human hepatocytes and found that the order of toxicity was monomethylarsonous acid > arsenite ~ arsenate ~ monomethylarsonic acid = `dimethyl arsinic acid. A major conclusion of all of these studies is that, contrary to what was thought earlier, methylation of arsenicals is not entirely a detoxification process, and cells with a high capac- ity to methylate arsenic compounds are not necessarily protected from arsenic toxicity. Induction of Mutations and Chromosomal Abberations The 999 NRC report provided a detailed review of many studies docu- menting that arsenic is not a particularly effective direct-acting mutagen, but it is quite effective in altering chromosomal integrity. Several recent in vitro and in viva studies have examined the impact of arsenic on chromosomal aberrations and gene mutations. Mass et al. (2001) utilized a DNA-nicking assay of phage DNA and the single-cell "comet" assay in cultured human lymphocytes to assess the genotoxic properties of inorganic and various methylated arsenicals. The nicking assay examines the ability of arsenic compounds to induce breaks in naked DNA in a cell-free system without added enzymes or chemical-activa- t~on systems. MA and DMA~ were the only forms of arsenic able to directly damage naked DNA in this assay. As noted by the authors, EMMA' was effective at nicking DNA at 30 mM; however, at 150 `1M of DMA~, nicking could be observed. Although the mechanism ofthe DNA damage was not determined, the authors referred to preliminary studies in which the nick- ing activity was not inhibited by the addition of glutathione or by the use of argon purge (i.e., under reduced oxygen conditions), suggesting that oxidative stress might not have been responsible. In the comet assay, which examined the ability of arsenicals to induce DNA damage in cultured human Tympho- cytes, MA and DMA~i were 77 and 386 times more potent, respectively, than arsenite. The relative potency was determined from the slope of the dose-response curves for MA (from 1.25 to 80 ~M), DMA~ (from 1.4 to 91 EM), and Astir and AsV (from 1 to 1,000 EM). The study by Mass et al. (2001 ) is noteworthy because it provides additional evidence that methylation of arsenic may not be a detoxification pathway and suggests that the trivalent methylated arsenicals might be capable of causing genotoxic damage by acting directly on DNA. Human fibroblasts exposed to arsenite from 1.25 to 10 EM for 24 hr in- duced micro nuclei formation in human fibroblasts in culture. Micro nuclei
1 00 ARSENIC IN DRINKING WA TER: 2001 UPDA TE formation was blocked by both catalase end N-acetyl cysteine, suggesting that the induction of micro nuclei was mediated via arsenic-induced oxidative stress (Yih and Lee ~999~. As noted in the ~999 NRC report, several in viva studies have associated arsenic exposure with the appearance of micronuclei in human cells, including a report of micronuclei in the exfoliated bladder cells of men in Chile who consumed drinking wafer with en arsenic concentra- tion of 54 to 137 ,ug/L (Moore et al. 1997; Biggs et al. 1997~. Liou et al. (1999) investigated the utility of chromosomal aberrations in lymphocytes as a biomarker of cancer risk. In a nested, case-control study of cancer incidence in a cohort of 686 subjects from the area of southwestern Taiwan where blackfoot-disease is endemic, 31 cases of cancer developed during a 4-year follow-up (11 skin, 4 bladder, 3 Jung, and 3 uterine and cervi- cal cancers). Lymphocyte cytogenetics could be examined in 22 cases and 22 matched controls for whom the duration of consumption of artesian well water was approximately 30 years. No significant differences were seen in the frequencies of sister-chromatic-exchange (SCE) and chromatic-type aberra- tions in the cases and the controls. The frequency of chromosomal-type aber- rations, such as gaps, breaks, and breaks plus exchanges, and the total fre- quency of chromosomal-type aberrations were significantly higher (p < 0.05) in cases than in controls. The odds ratio for cancer risk in subjects with at least one chromosomal-type break was 5.0 (95% confidence interval = 1.09 to 22.82~. The odds ratio for cancer risk in subjects with at least one chromo- somal break plus exchanges and a frequency of total chromosomal-type aber- rations greater than 1.007% are 11.0 and 12.0, respectively (p < 0.05~. Sub- jects with a frequency of total chromosomal-type aberrations greater than 4.023% had a 9-fold increase in cancer risk. Those results suggest that chromosomal-type aberrations in lymphocytes might be useful biomarkers for the prediction of cancer development in arsenic-exposed populations. In a small study of subjects exposed to arsenic in drinking water in Fin- land, Maki-Paakkanen etal. (1998) examined the association between urinary arsenic concentration and chromosomal aberrations (gaps, isogaps, breaks, and rearrangements) in human lymphocytes. The total urinary arsenic concentra- tion (sum of inorganic arsenic, MMA, and DMA) was 1 SO ,ug/L (from 7 to 500 ,ug/L) in 27 exposed subjects who were consuming well water, 17 ,ug/I (from 3 to 104 ,ug/L) in nine individuals who had previously consumed the water, and 7 ,ug/L (from 4 to 44 ,ug/L) in eight controls. In multiple regression mociels adjusting for age, gender, and smoking, total urinary arsenic was positively associated with the number of chromosomal aberrations (r2 = 0.23, p = 0.02 when chromosomal gaps were excluded; r2 = 0.15, p = 0.07 when
EXPERIMENTS ~ STUDIES ~ 01 chromosomal gaps were included). Among current users ofthe well water, the ratio of MMA-to-total arsenic in urine was positively correlated with the number of chromosomal aberrations (including gaps), and the ratio of DMA- to-total arsenic in urine was negatively correlated with the number of chromo- somal aberrations (including gaps). One recent study showed that Asset can induce DNA-protein cross-links (DPCs) in vitro in a human liver cell line at concentrations as low as 1 nano- molar (nM) (Ramirez et al. 2000~. The extent of DNA-protein cross-links increased linearly with arsenite concentration. Detectable DPCs decreased within a few hours of substituting arsenite with an arsenite-free culture me- dium, possibly indicating their elimination through DNA repair. Presumably, because of their high cysteine (and hence thiol) content, several cytokeratins were identified by immunoblotting techniques to be among the proteins that cross-linked with DNA. The total cellular content of cytokeratin CK18 was increased following arsenite exposure. Ramirez et al. (2000) suggested that the formation of arsenite-induced DPCs might be involved in arsenite-related chromosomal aberrations. They also suggested that arsenite's effect on cytokeratins might interfere with the important role of these proteins in cellu- lar differentiation. Alterations in Signal Transduction, Cell-Cycle Control, Differentiation, and Apoptosis Although in vitro studies can provide useful inflation on putative mechanisms of arsenic toxicity and carcinogenicity, it is important to recog- nize the many limitations associated with such studies. Quantitative and qualitative responses of an in vitro system to arsenic might be influenced by factors such as species of arsenic used, dose and time frame of exposure, period and nature of observations, experimental and culture conditions (e.g., state of confluence, presence or absence of serum, nature and concentration of other nutrient supplements), and cell type and tissue origin. As discussed in Chapters 2 and 4, there are species differences in response to the toxic and carcinogenic actions of arsenic, and it is also likely that there is interindividual variability in response. Mechanistic studies are helpful in identifying poten- tial reasons for such variability in responses observed in viva, but great cau- tion should be used in inferring relevance of a specific response to arsenic observed in vitro to humans exposed chronically at relatively low doses in drinking water.
102 ARSENICINDRINKING WATER: 2001 UPDATE Cell Signaling Trouba et al. (2000b) found that long-term exposure of epithelial-growth- factor (EGF)-stimulated murine f~broblasts to arsenite increased DNA synthe- sis and the proportion of cells entering S phase. Expression of the positive growth regulators c-myc and E2F-1 were both increased, although extracell- ular signal-regulated protein kineses (ERK-2s) and EGF-receptor expression were unchanged. The negative regulators of proliferation, mitogen-activated protein (MAP) kinase phosphatase-l and p27 (Kipl), were lower in As~i'- treated cells compared with control cells. The authors concluded that long- term exposure to high levels of arsenite might make cells more susceptible to mitogenic stimulation and that alterations in mitogenic signaling proteins might contribute to the carcinogenic actions of arsenite. Porter et al. (1999) evaluated the role of different mitogen-activated pro- tein kinases/extracellular signal-regulated kinase kinase (MEKK) kineses that are involved in arsenate and arsenite-induced activation of c-dun N-terminal kineses (]NKs). Interestingly, both arsenate and arsenite activated INK but, apparently, by slightly different pathways. Arsenite-mediated activation of INK requires MEKK2, MEKK3, and MEKK4, whereas arsenate required only MEKK3 and MEKK4. In addition, arsenite, but not arsenate, activation of INK requires p2 1 -activated kinase, whereas both forms of arsenic require the guanosine triphosphatases (GTPases) Rac and Rho. Chen et al. (2000a) found that Asi" induced the transiocation of several protein kinase C (PKC) proteins from cytosol to membranes and activated activator protein-l (APED. Using selective PKC inhibitors and dominant negative mutants, the authors demonstrated that PKC~, PKC£, and PKCa medi- ate arsenic-induced AP-1 activation through different MAP kinase pathways (e.g., ERKs, JNKs, and p38 kineses). Arsenite has been shown to inhibit the NF-KB signal transduction pathway that is important in transcriptional regulation of a variety of cellular pathways, including the inflammatory cytokines TNF-a and TL-8 (Rousse! and Barchowsky 20001. Kapahi et al. (2000) identified what might be the first specific molecular target for arsenite. They found that arsenite is an effective inhibitor of T-KB kinase (IKK) (ICE, 9.1 ,uM), which is required for the integ- rity and function of the NF-KB signaling pathway. Asia' was shown to bind to Cys- 179 in the activation loop of one of the INK catalytic subunits. Replace- ment of the Cys-179 with alanine provides a fully functional IKK catalytic subunit but abbrogates Astir binding. Overexpression of the CI79A mutant protects NF-KB from inhibition by arsenite, thereby demonstrating that direct
EXPERIMENTAL STUDIES 1 03 binding of arsenite to the Cys-179 residue of IKK subunit is responsible for arsenite-induced inhibition of the NF-KB signaling pathway. Vogt and Rossman (2001) examined the effect of arsenite on cell signal- ing in a cell culture of WI38 normal human lung fibroblasts. Treatment of cells with 0. ~ EM arsenite for 14 days caused a modest (3-fold) increase in the cellular levels of pS3 protein without any increase in p21 protein, a major downstream protein involved in cell-cycle arrest that is often increased by signals associated with increases in pS3. Arsenite exposure blunted the in- crease in pS3 and prevented any increase in p2 ~ when the cells were exposed to irradiation (6 Gy), an insult that alone markedly increased levels of both proteins. Arsenite at 0.1 EM for 14 days heightened expression of cycTin D1, a protein that facilitates progression through the G! phase of the cell cycle. Vogt and Rossman (2001) suggested that the action of arsenite in preventing the p5 3 -dependent p2 ~ -protein increase in cells that experience DNA damage, together with arsenite-induced up-regulation of cyclic D1, might promote replication of a DNA-damaged template. This action might be a mechanism for some ofthe observed comutagenic and cocarcinogenic properties of arsen- ite. Collectively, the studies described above demonstrate that arsenic can interfere with cell signaling pathways (e.g., the pS3 signaling pathway) that are frequently implicated in the promotion and progression of a variety of tumor types in experimental animal models and of some human tumors. How- ever, which specific alterations in signal transduction pathways are actual targets that contribute to the development of arsenic-induced tumors in hu- mans following chronic consumption of arsenic in drinking water remains uncertain. Apoptosis, Cytotoxicity, and Anticancer Activity Recent clinical trials have found that arsenite has therapeutic value in the treatment of acute promyelocytic leukemia, and there is interest in exploring its effectiveness in the treatment of a variety of other cancers (Soignet et al. 1999; Murgo 2001~. It is of historical note that a therapeutic role for arsenic in the treatment of leukemia was suggested nearly a century ago (OsTer ~ 894; Forlmer and Scott 193 ~ ). A number of studies have examined the biochemical basis for this potential therapeutic effect (for reviews, see Chen et al. 2000b; Fenaux et al. 2001~. Detailed descriptions of those studies are beyond the scope ofthis report; however, the potential mode of action of arsenic revealed
~ 04 ARSENIC IN DRINKING WA TER: 2001 UPDA TE from those studies might be relevant to the biological actions of arsenic in other human cell types. In acute promyelocytic leukemia, the specific molecular event critical to the formation of malignant cells is known. Malignant Tyrnphoblasts exhibit a t(15; ~ 7~-chromosomal transIocation that produces a product that regulates expression of the tyrosine kinase oncogene BCR-ABL. Puccetti et al. (2000) demonstrated that 2 ,uM of As2O3 induces apoptosis in human lymphoblasts that carry the t(15; 17) transIocation, but not in wild-type cells, and also found that forced overexpression of BCR-ABI~ susceptibility in these cells resulted in greatly enhanced sensitivity to arsenic-induced apoptosis. From this obser- vation, they concluded that As2O3 is a tumor-specific agent capable of induc- ing apoptosis selectively in acute promyelocytic leukemia cells. Several recent studies showed that As2O3 can induce apoptosis through alterations in other cell signaling pathways. For example, Seo] et al. (1999) found that 2 EM of As2O3 efficiently induced G2/M arrest in PCCI-1 cells, apparently in association with induction of p2 ~ and reduction of cdc2 kinase activity. Numerous other studies found that Tow doses of As2O3 selectively induced apoptosis of NB4 promyelocytic leukemia cells (see review in Alemany and Levin 2000~. In addition to acute promyelocytic leukemia, As2O3 is thought to have therapeutic potential for myeloma. There are several recent studies on the ability of this agent to kill myeloma cells or T cells infected with leukemia virus. Studies on human myeloma-like cell lines indicated that the compound acted as an adjuvant in increasing cell killing by lymphocytes (Deaglio et al. 2001~. Two cell-surface markers, CD38 and CD54m, were up-regulated, as were their correspondingligands (CD31 and CD] la). Those data suggest that increased adhesion was responsible for the improved killing. Park et al. (2000) examined the effect of As2O3 on proliferation, cell-cycle regulation, and apoptosis in human myeloma cell lines. They found that cell proliferation in eight different cell lines were inhibited by As2O3, with ICsoS (the concentration that inhibits the response by 50%) of I-2 ~M. Arsenic induced G! and G2-M phase arrest. As2O3 markedly enhanced the binding of p21 to several cycTins and cycTin-dependent Anise 6. They concluded that As2O3 inhibits proliferation of myeloma tumor cells via cell-cycle arrest in association with induction of p21 and apoptosis. The apoptosis was associ- ated with down-regulation of BcI-2, loss of mitochondrial transmembrane potential, and an increase of caspase-3 activity. Earlier, Zhang et al. (1998) reported the induction of apoptosis and cell-cycle arrest in cell lines from lymphoid neoplasms by As2O3 at micromolar concentrations. Zhang et al.
EXPERIMENTAL STUDIES 105 (1999) also examined the sensitivity of six human cancer cell lines to As2O3- induced inhibition of cell growth and induction of apoptosis. They found that a gastric carcinoma cell line was particularly sensitive to As2O3-induced apop- tosis, with concentrations as low as 0.01 ,uM causing significant growth inhi- bition and apoptosis. Several studies reported that As203 induces cell-cycle arrest and apoptosis in malignant cells. Ishitsuka et al. (2000), using HTEV- ~ infected T-cell lines, showed that As203 at concentrations that might be seen when it is used cTini- cally inhibited cell growth by cell-cycle arrest and induced apoptosis. The induced apoptosis could be prevented by caspase inhibitors. They also ob- served destruction of the BcI-2 protein and enhancement of the teak protein production. The arsenic compound increased expression of p53, Cipl/p21, and Kipl/p27 and dephosphorylation of retinoblastoma protein. Rousselot et al. (1999) found that As2O3 as well as the organic arsenical melarsoprol (2-~4- [~4,6-diamino- ~ ,3 ,5 -triazin-2-yI)amino~phenyI] - ~ ,3 ,2-dithiarsolane~-metha- nol) at pharmacological concentrations ~ ~ O nM to ~ EM) inhibited growth and induced apoptosis in myeloma cell lines. In bone-marrow samples, the com- pounds were able to induce apoptosis in myeloma cells while sparing the normal myeloid cells. As2O3 induced apoptosis in PCI-l head and neck cells after treatment for 3 days with 2 EM of arsenite (Seo! et al. 2001~. The mechanism appeared to be through up-regulation of BcI-2; capase 9 was activated and the mitochon- drial membranes of the cells were depolarized. To test the efficacy of the arsenic in viva, C3H mice inoculated with syngenic SCC7 cells were treated by intratumoral injections of As203 (300 Age daily for 4 days. The tumor was reduced in size and increased apoptosis was seen. Other tumor cell lines have also been reported to be sensitive to arsenite trioxide. UsTu et al. (2000) reported the induction of apoptosis in prostate and ovarian carcinoma cell lines treated with ~ ,uM of As2O3. Jiang et al. (2001) reported the induction of apoptosis in human gastric cancer cells at concentra- tions as Tow as 0. ~ ~M. The treatment resulted in a marked increase in p53 protein levels and increased the activity of capase 3. Shen et al. (1999) studied the concentration of arsenic trioxide required to inhibit growth and induce apoptosis in oesophageal cancer cells in vitro. The ECso for induction of apoptosis was ~ EM. In summary, numerous cancer chemotherapy studies in cell cultures and in patients with acute promyelocytic leukemia demonstrate that arsenic tr~ox- ide can lead to cell-cycle arrest and apoptosis in malignant cells.
~ 06 ARSENIC IN DRINKING WA TER: 2001 UPDA TE
EXPERIMENTAL STUDIES ~ O 7 strafe that activation of ERK, but not INK, by arsenite is required to induce cell transformation. In another study, these same authors (Huang et al. ~ 999b) found that 200 ,uM of arsenite induces both apoptosis and activates INK in ~TB6 cells but did not induce pS3-dependent transactivation. Furthermore, there was no difference in the induction of apoptosis by arsenite in pS3+'+ versus pS3~'~ cells, suggesting that induction of apoptosis at relatively high concentrations of arsenite proceeds via a pS3-independent pathway. Larochette et al. (1999) provided further evidence that arsenite acts on mitochondria to induce apoptosis, as mitochonidria were required for arsenite to induce nuclear apoptosis in a cell-free system. Arsenite caused the release of an apoptosis-inducing factor from the mitochondrial inner membrane space and also altered the permeability transition (PT) pore, suggesting that arsenite (30 ~M) can induce apoptosis via a direct effect on the mitochondrial PT pore. In that study, arsenite induced apoptosis in a myelomonocytic leukemia cell line with an EDSo (the effective dose in inducing apoptosis in 50% ofthe cells) of 20 ,uM. Li and Broome (l 999) found that O.S-S EM of As2O3 induces the expres- sion of myeloid maturation and apoptotic markers, and markedly inhibits GTP-induced polymerization and microtubule foundation. (However, only very high concentrations, 0. I-1 mM, were used.) The authors suggested that Astir binds to cysteine residues in tubulin, blocking the binding of GTP, thus disrupting the normal dynamic of microtubules during mitosis. They further suggested that that might be the mechanism by which As203 induced myeloid- cell maturation and arrest in metaphase, leading to apoptosis. Aside is clearly cytotoxic to a variety of tumor cells in the 1-10 ,uM range; its toxicity is substantially influenced by the levels of intracellular glutathione (Yang et al. ~ 999~. Using a variety of specific modulators of oxidative stress and cell signaling, Chen et al. (1998) found that arsenite-mediated apoptosis involved the formation of reactive oxygen species, activation of CPP32 (a protease) activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from the mitochondria to the cytosol. From those and other data, the authors hypothesized that arsenite-induced apoptosis is triggered by the generation of H202 through activation of flavoprotein-dependent superox- ide, producing enzymes such as NADH oxidase, which then might play a role as a mediator to induce apoptosis through activation of CPP32 protease, re- lease of cytochrome c to cytosol, and PARP degradation. Cell cultures were used to determine the effect of arsenicals on the prolif- eration and cytokine secretion of normal human epiclermal keratinocytes (Vega et al. 20011. Trivalent arsenicals stimulated proliferation of the cells
~ 08 ARSENIC IN DRINKING WA TER: 2001 UPDA TE at concentrations of 0.001 to 0.01 ,uM, and concentrations greater than 0.5 EM inhibited proliferation. Pentavalent arsenicals did not stimulate proliferation. The trivalent arsenicals also stimulated secretion of the growth-promoting cytokines, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor. The study indicated possible mechanisms by which arsenicals can cause damage to human skin or contribute to neoplasia. Those observa- tions are consistent with earlier data showing a stimulatory effect on prolifera- tion of keratinocytes at low doses (Germolec et al. 1997, 1998), as discussed in the ~ 999 NRC report. Sodium arsenite also reduced the proliferation of PHA-stimulated T lym- phocytes from human peripheral blood due to inhibition of secretion of ~-2 (Vega et al. 1999~. A preliminary report presented at a meeting by Chiang et al. (2001) found that continuous exposure of immortalized human keratinocytes (HaCaT) to noncytoxic doses of arsenite (0.5 and 1.0 EM) for 5 months resulted in in- creased cell-culture growth rate, cell density, and colony-forming efficiency. The arsenite-exposed HaCaT cells formed tumors when injected into nude mice, in contrast to the absence of tumor formation following injection of unexposed cells into nude mice. Induction of Oxidative Stress Prior to the 1999 NRC report, a great deal of research was conducted on the genotoxic and chromosomal effects of arsenic. Recently, several studies have provided additional evidence that arsenic can induce DNA damage indi- rectly by promoting the formation of oxygen free radicals. Barchowsky et al. (1999a) found that 5-200 ,uM of arsenite caused cell proliferation and increased superoxide and H2O2 accumulation, increased activity of the cytoplasmic tyrosine kinase cSRC, increased H2O2-dependent tyrosine phosphorylation, and increased NF-KB-dependent transcription. At higher concentrations that caused cell death, As~activated MAP kineses and p3 8. Another study by this group (Barchowsky et al. ~ 999b) demonstrated via electron spin resonance that these same concentrations of As ~~ rapidly in- creased oxygen consumption and superoxide formation, which was abolished by superoxide dismutase (SOD) but not nitric oxide radicals, confirming that superoxide is a predominant form of arsenic-induced reactive oxygen species. The authors suggested from this finding that arsenite-induced oxidant accumu- lation, in the form of superoxide and H2O2, activates tyrosine phosphorylation and might represent a MAP-kinase-independent pathway for proliferation of
EXPERIMENTA ~ STUDIES ~ 09 vascular cells. Lynn et al. (1998) found that arsenite-induced poly(ADP)- ribosylation, NAD depletion, DNA-strand breaks, and micro-nuclei formation in CHO-Ki cells. However, in contrast to the findings of Barchowsky et al. (1999a), Lynn et al. (1998) noted that the DNA damage could be suppressed by pretreatment with inhibitors of nitric oxide synthetase and also found in- creased nitrite levels in cells following Asset treatment. From those results, they concluded that the induction of nitric oxide might be important to the etiology of arsenic-induced vascular disorders in humans. The mechanisms by which arsenic causes oxidant stress were further elucidated in several in vitro studies. The effects of four species of arsenic (arsenite, arsenate, MMA, and DMA) in mobilizing iron from horse-spleen ferritin were investigated (Ahmad et al. 2000~. The results indicate that exog- enous methylated arsenic species administered alone or synergistically in the presence of endogenous ascorbic acid can cause the release of iron from ferrit- in and an iron-dependent formation of reactive oxygen species. In rats, Kitchin et al. (1999) found a linear relationship between the tissue concentration of arsenite and the induction of heme oxygenase (HO), a meta- bolic enzyme known to be induced by stress. Doses resulting in 30 Cool of arsenite per kilogram of liver induced HO, while a dose resulting in 10 ~mol/kg of liver did not induce HO. The tissue concentration of arsenite needed to induce kidney HO was 100 ~mol/kg of kidney; 30 ,umol/kg did not induce HO. The ability of different forms of arsenic to inhibit thioredoxin reductase, a nicotinamide adenine dinucieotide phosphate (NADPH)-`Jependent flavoen- zyme that catalyzes the reduction of many disulf~de-containing substrates and plays an important role in the cellular response to oxidative stress, was investi- gated by Lin et al. (2001~. Of the arsenicals tested (Asia, MMA'i~, and DMA~), the monomethyl form of trivalent arsenic was found to be the most potent inhibitor of thioredoxin reductase, having an ICso of 3 ~M. S.X. Liu et al. (200 ~ ~ used a fluorescent probe sensitive to oxygen radicals and a confocal scanning microscope to show that arsenite is capable of rapidly increasing intracellular levels of oxygen radicals. The arsenic-induced in- crease in oxygen radicals could be reduced by the free radical scavenger, dimethyl sulfoxide (DMSO). Using electron spin resonance spectroscopy and selective spin-trap agents, the authors also showed that arsenite can increase the levels of superoxide-derived hydroxy] radical production in a Chinese hamster ovary cell line that contains a single copy of human chromosome 1 1. Finally, they demonstrated that arsenite could induce mutations in the CD59 gene on chromosome 11 in these cells. The mean lethal dose of arsenite to these cells was I.7 ,ug/mL (23 EM) but was 0.l ~g/mL (2.3 EM) following
~ ~ O ARSENIC IN DRINKING WA TER: 2001 UPDA TE glutathione depletion with buthionine sulfoximine, indicating that cell death was mediated via oxidative stress. These results suggest that arsenite can produce intracellular oxygen free radicals capable of damaging DNA. Matsui et al. (1999) examined the DNA in human skin biopsies obtained from cases of Bowen's disease (a type of skin cancer), invasive squamous-cell carcinoma (SCC), and arsenical keratoses for the frequency of 8-hydroxy-2- deoxyguanosine, a marker of oxidative damage. Cases were obtained from unspecified areas of Taiwan, Japan, and Thailand where the authors indicated "chronic arsenicism was endemic." Control biopsies consisted of 10 Bowen's disease lesions and one SCC obtained from 11 Japanese subjects without known arsenic exposure. The authors found that the frequency of oxidative damage to DNA was significantly higher (22 of 28; 78°/O) in the arsenic-re- lated lesions when compared with those from subjects not exposed to arsenic (l of 1 1; 9%~. For a subset ofthese samples, arsenic exposure was determined by neutron activation analysis of deparaffined skin-tumor samples. Arsenic was readily identified in four of five tumors classified as arsenic-related, but not in any of three cases of non-arsenic-related tumors. Those data provide further evidence that arsenic is capable of causing oxidative damage to DNA and suggest that this mechanism could contribute to the development of arsenic-related skin tumors in humans exposed to arsenic through drinking water. Lynn et al. (2000) found that treatment of human vascular smooth-muscle cells in culture with concentrations of arsenite greater than 1 remold increased superoxide concentrations and induced oxidative damage to DNA. The au- thors also provided evidence suggesting that low concentrations of arsenite can activate NADH oxidase, which could contribute to the formation of super- oxide. Studies by Snow and colleagues (Snow et al. 1999, in press; Chouchane and Snow 2001) in human keratinocytes indicated the different effects of Astir at different concentrations. Ligase function, a process involved in DNA re- pair, was increased in SV-40 transformed cells (AG06) after a 3-fur exposure with approximately ~ O EM of arsenite and was decreased in a dose-dependent manner after 24-hr exposures with arsenite concentrations up to 100 ,uM (Snow et al. 1999~. However, the purified ligases were only inhibited at very high (millimolar) concentrations of arsenite. The authors suggest that the inhibition of DNA repair by arsenite is by an indirect mechanism, such as transcriptional or post-transTational regulation of gene products. At micromolar concentrations of arsenite (maximal effect at 3 ,uM), there was an increase in cellular GSH due to an increase in the activity of the GSH-
EXPERIMENTA ~ STUDIES ~ ~ ~ synthesizing enzyme gamma-glutamyl cysteinyl synthase. GSH andN-acetyI- cysteine were found to protect against arsenite toxicity. However, the various enzymes that use GSH as a substrate (e.g., glutathione transferases, glutathi- one reductase, and glutathione peroxidase) were not inhibited by relevant concentrations of arsenite (Snow et al. ~ 999; Chouchane and Snow 200 ~ ). By contrast, the enzyme pyruvate dehydrogenase, an enzyme involved in energy production, was inhibited with an ICso of 5.6 ,uM of arsenite, and this inhibi- tion is likely to be involved in the cytotoxicity of arsenic in the cells. In addition to the effects on ligase function and cellular redox enzymes, arsenite was found to interact with DNA alkylating agents. The activity of Tow concentrations of arsenite was opposite to that of high concentrations. Cells pre-exposed for 24 hr with 0.2 EM of arsenite before exposure to less than 4 EM of N-methyI-N-nitro-N-nitrosoguanidine (MUNG) showed an en- hanced uptake of neutral red dye compared with that of either exposure alone. (Dye uptake increased rather than decreased, a sign of either increased lysosomal activity or cell proliferation.) In contrast, pre-exposure with 4 or 20 ,uM of arsenite resulted in decreased uptake of neutral red dye compared with that seen with 20 to 35 ,uM of MUNG. (Neutraired dye decreased, a sign of decreased cell viability or cytotoxicity.) Alterations in Gene Expression In the 1999 NRC report, alterations in gene expression via hypo- or hypermethylation of CpG sites in DNA were reviewed. Recently, Zhong and Mass (2001) utilized a sensitive PCR technique to determine whether inor- ganic Asset can alter DNA methylation at relatively low concentrations in immortalized human kidney tumor cells in culture. The kidney cell line was considerably more susceptible to cytotoxic effects of arsenic (ICE = 20 nM) than the human lung cell line, A549 cells (TCso = 400 nM). Those relatively Tow concentrations of arsenic in cell culture produced six differentially hypermethylated regions and two differentially hypomethylated regions of genomic DNA, lending further support to the hypothesis that some forms of arsenic can alter gene regulation through hyper- or hypomethylation of DNA. Although this group of investigators had shown that hypermethylation of CpG sites can inactivate the transcriptional activity of human pS3 tumor suppressor gene (Schroeder and Mass ~997) and that arsenic can alter methylation pat- terns in the promoter region of the p53 gene (Mass and Wang ~ 997), Zhong and Mass (2001) failed to find arsenic-induced alterations in methylation of
~12 ARSENIC INDRINK[NG WATER. 2001 UPDATE the CpG-sensitive promoter of the von Hippel-Lindau tumor suppressor gene in their recent study. Recent studies have examined pS3 gene expression and mutation in tu- mors obtained from subjects with a history of arsenic ingestion. Kuo et al. (1997) compared pS3 overexpression in 26 cases of Bowen's disease lesions from residents in the area of southwestern Taiwan where blackfoot-disease is endemic presumably exposed to arsenic and in 22 cases of Bowen's disease lesions from unexposed control subjects. ~nunohistochemical staining for p53 occurred at a positive rate in biopsies from 42.3% (l ~ of 26) arsenic- exposed patients compared with 9.~°/0 (2 of 22) from nonexposed patients (p = 0.0 ~ ). In another study from the region where blackfoot-disease is endemic, Chang et al. (1998) reported positive immunohistochemical staining for pS3 in 100% of 30 Bowen's disease lesions, 12 basal-cell carcinomas (BCCs), and 10 SCCs. Normal skin adjacent to the lesions also consistently overexpressed p53. ~ a study by Hsu et al. (1999) of skin cancer in subjects from the region where blackfoot-disease is endemic, immunohistochemical staining found p5 3 overexpression in 43.5% (10 of 23) of lesions of Bowen's disease, in 14% (1 of 7) of the BCCs, and in 44% (4 of 9) of the SCCs. Polymerase chain-reac- tion single-strand conformation polymorphism (PCR-SSCP) analysis detected pS3 gene mutations in 39% (9 of 23) of the cases with Bowen's disease, 28.6% (2 of 7) of cases with BCC, and 55.6% of cases with SCC. The two techniques did not have complete concordance: PCR-SSCP analysis did not detect mutations in4 of 15 cases with positive immunohistochemical staining and did detect mutations in 5 of 15 cases that stained negative. In a recent study of BCC from Queensland, Australia, Boonchai et al. (2000) found a Tower percentage of pS3 expression in biopsies obtained from patients with a history of prior chronic ingestion of an arsenic-containing elixir compared with those with no known arsenic exposure and presumed UV-induced basal- cell carcinoma (52.9% of 121 biopsies). Hamadeh et al. (l 999) found that treatment of human keratinocytes to Tow concentrations of Asii~ (1 -l ,000 nM) produced a dose-dependent increase in mUm2 protein (involved in pS3 regulation) and a corresponding decrease in pS3 protein. Effects were seen at all time points from 2 to 14 days of incuba- tion, but peaked at about 4-6 days. The authors proposed that disruption ofthe p53-m~m2 Toop that regulates cell-cycle arrest might be an important event in the development of arsenic-related skin carcinogenesis. Several other recent studies provide additional support for the hypothesis that arsenic can modulate gene expression. Kaltreider et al. (1999) found that relatively low (1 ,uM), noncytotoxic doses of arsenite alter nuclear-binding
EXPERIMENTAL STUDIES 1 1 3 levels of several transcription factors, including AP-l, NF-kB, SP1, end Yb-l, to their cis-acting elements in a human breast cancer cell line and in rat hepa- toma cells. A subsequent study demonstrated that 3.3 ,uM of arsenite altered the function of the nuclear glucocorticoid receptor, apparently by interacting directly with the glucocorticoid receptor complex and interfering with GR- mediated transcriptional activation of genes in dexamethasone-treated, hormone-responsive H4BE rat hepatoma cells (Kaltreider et al. 2001~. This type of toxicity might influence not only the induction of neoplasia but could also influence other corticosteroid-dependent activities. Parrish et al. (1999) investigated the impact of submicromolar concentra- tions of arsenite and arsenate on transcription-factor DNA binding and gene expression in precision-cut rabbit renal cortical slices. Arsenite and arsenate appeared equipotentin several effects, including increasing AP-1 DNA-bind- ing activity and increasing c-foe, c dun, and c-myc gene expression. Peak effects for both arsenic species occurred in the range of 0.1 to ~ .0 ,uM. Arsen- ite, but not arsenate, increased expression of heat-shock protein HSP 32 at submicromolar concentrations. This study extends earlier observations ofthe impact of Tow-dose arsenite on gene expression for example, the finding by Germolec et al. (1997) that ~ EM of arsenite increased expression ofthe c-myc protooncogene in normal human keratinocytes in vitro. Tully et al. (2000) used a battery of recombinant HepG2 cells (a human liver tumor-derived cell line) to evaluate the effects of metals, including AsV, on transcriptional activation of any of 13 signal transduction pathways. At high doses (100 EM and greater), they found that AsV induced transcriptional activation of several genes, including glutathione S-transferase Al, metallothionein lIA, NF-KB, FOS, and HSP70. However, because effects were not generally seen at the lowest concentration tested (50 EM), the rele- Vance of these results to risk assessment of Tow concentrations of arsenic in drinking water is limited. Lu et al. (2001) used c DNA microarrays to evaluate whether gene expres- sion in six needle biopsies of human liver tissue from individuals exposed to arsenic in their environment from the Guizhou region of China was different from normal human liver. Chronic exposure to arsenic apparently occurs from the use of coal containing very high concentrations of arsenic (up to 900 ppm) for the purposes of drying food. All cases were selected from subjects with a history of arsenic-related skin lesions (e.g., keratoses and hyperpigmen- tation) and gastrointestinal disturbances. Furthermore, all of the Chinese patients had histopathological evidence of chronic liver damage that was presumed to be related to their arsenic exposure. Patients did not have a
~14 ARSENIC IN DRINKING WATER: 2001 UPDATE history of hepatitis, and serology for hepatitis B and C viruses was negative. When compared with normal liver obtained from six surgical resections or transplantations, approximately 60 genes of about 600 examined appeared to be differentially expressed. Of particular interest were changes in expression of genes involved in cell-cycle regulation, cell proliferation, apoptosis, and DNA-damage repair. Although there are many uncertainties associated with the design and interpretation of this human study, the authors noted that the observed changes in gene expression were consistent with the result of array analysis of chronically arsenic-exposed mouse livers and chronically arsenic- transformed rat liver cells seen in other studies (Chen et al. 2001; Liu, I. et al. 2000). in another global gene expression study, Liu et al. (2001 a) found that mice exposed to high doses of inorganic arsenicals Astir at 100 ,umol/kg or AsV at 300 ,umol/kg exhibited significant changes in expression of genes related to stress (e.g., HSP60, heme oxygenase 1), DNA damage (e.g., GADD45 and the DNA excision repair protein ERCC ~ ), and xenobiotic biotransformation (e.g., various cytochrome Palms). They also found that arsenic exposure activated the c-Jun/AP-1 transcription complex, which could explain some ofthe wide- spread changes in gene expression. Collectively, these studies provide further evidence that various forms of arsenic can alter gene expression and that such changes could contribute substantially to the toxic and carcinogenic actions of arsenic in humanpopula- tions. Resistance and Tolerance to Arsenic Cytotox~city Romach et al. (2000) investigated the mechanism underlying the develop- ment oftolerance to arsenic in a ret liver epithelial cell line (TRL 1215~. That cell line over-expresses metallothionein (MT), a protein that binds metals and is often associated with the development of tolerance to metals. The cells were chronically ~~ 8-20 weeks) exposed to 500 nM of arsenite. Control cells (not previously exposed to arsenite) and the chronically exposed cells were subsequently exposed to arsenite. The LCso in the chronically exposed cells was 140 ,uM compared with 26 ,uM in the control cells. The LCsOs for AsV and DMA, but not MMA, were also increased. The control cells, however, were extremely tolerant to MMA to begin with (LCso > 60,000 EM). Cultur- ing the chronically exposed cells in arsenite-free media did not alter their tolerance, suggesting an irreversible phenotypic change. The development of tolerance was not linked to GSH levels nor metallothionein levels in the cells.
EXPERIMENTAL STUDIES 115 The tolerant cells accumulated less arsenite than the control cells, had an increased methylation of arsenic to DMA, and eliminated the arsenite more readily. The increased methylation could not fully account for the increased tolerance, and the mechanism underlying the development oftolerance is still unclear. It should be noted that the observed tolerance is to the cytotoxic effects of arsenic, but that does not necessarily imply tolerance to the carcino- genic effects of arsenic. Rossman and Wang (1999) identified two cDNAs, asrl and asr2, which confer As~resistance to As-sensitive cells lines. In a second study, Rossman and Wang (1999) used arsenite-sensitive and arsenite-resistant strains of Chinese hamster V79 cells and found that overexpression of the tumor sup- pressor genefau contributed to arsenic resistance (Rossman and Wang ~ 999~. They hypothesized that becausefau contains a ubiquitin-like region, arsenic might interfere with the ubiquitin system that targets certain proteins for rapid destruction. Because pS3 and several other proteins involved in DNA repair and cell-cycle regulation also require ubiquitin signal sequences for proper function, Rossman and Wang (1999) suggested that the carcinogenic effects of arsenite might, in part, be mediated indirectly by effects on cellular control of DNArepairprocesses, secondary to interference with the ubiquitin system. Liu et al. (200Ib), who earlier found that Tong-term exposure to low con- centrations of arsenite-induced malignant transformation in a rat-liver epithe- lial cell line, reported that these chronically exposed cells developed a toler- ance to later acute exposures. Microarray analysis of the cells showed in- creased expression of the genes encoding for glutathione S-transferase, multi- drug resistance-associated protein genes, and multidrug resistance genes. The tolerant cells were found to be resistant to several anticancer drugs. Implications of Mode-of-Action Studies of Arsenic in Drinking Water to lIuman Carcinogenesis As described in this chapter, numerous recent studies have identified potential modes of action by which arsenic could increase cancer risk in hu- man populations. Several recent in vivo animal studies have been completed, including one preliminary study that potentially demonstrated a significant carcinogenic response in mice to arsenic at 500 ppb (500 Egg) in drinking water. However, the lack of dose-response information and other uncertain- ties in extrapolating Tom rodents to humans makes rodent bioassays unsuit- able for direct application to human risk assessment for arsenic, particularly
~ ~ 6 ARSENIC IN DRINKING WA TER: 2001 UPDA TE in view of the extensive human epidemiological data that are available. Since the ~ 999 NRC report, several studies have reported the presence of trivalent methylated arsenic metabolites in tissues or urine. Because these forms are reactive and highly toxic, they have been considered in the evaluation of possible modes of action of arsenic carcinogenicity. Recent studies have used in vitro experiments to evaluate several putative modes of action for arsenic-induced cancer, including (1) induction of muta- tions and chromosomal abberations, (2) secondary induction of DNA damage via generation of oxygen free radicals (oxidative stress) and altered fidelity of DNA repair, (3) alterations in signal transduction, cell-cycle regulation, and apoptosis, and (4) alterations in gene expression. The newly reported studies on mode of action of arsenic provide insight into the importance of exposure level, exposure duration, and time frame of observation in the assessment of cellular responses to arsenic. Within the same cell system, different levels of exposure appeared to induce different modes of action (Table 3-~. To assess the potential relevance and implications of these in vitro studies to the risk assessment of arsenic in drinking water, it would be useful to deter- mine whether the studies used experimental concentrations of arsenic that are plausible in human target tissues following exposure via drinking water. Human urinary bladder epithelium is one of the primary target tissues for arsenic carcinogenesis. Because this tissue is essentially bathed in urine that contains arsenic following ingestion of arsenic in the diet and drinking water, levels of biologically active forms of arsenic that appear in the urine following exposure to Tow-to-moderate doses in humans are particularly relevant to in vitro studies. Numerous studies have speciated arsenic in urine following ingestion via drinking water. The mechanistic studies reviewed herein and those reviewed previously in the 1999 NRC report suggest that trivalent ar- senic species (primarily Asiii, MMA~', and, possibly, DMAiii) are the forms of arsenic of greatest toxicological concern. Considering a concentration of arsenic in drinking water of 50 ,ug/L (i.e., 50 ppb) for illustrative purposes, the corresponding concentration of As"' in urine can be estimated from previous studies. In general, arsenic at 50 ~g/L of drinking water would be expected to yield a total urinary arsenic concentration in the range of 30-100 ,ug/L (NRC 1999~. Most studies have found that approximately 10-30% of total urinary arsenic is inorganic (As'i' and AsV) (NRC 1999), yielding urinary concentrations of inorganic arsenic in the range of 3-30 ,ug/L. Approximately 50-75% of the inorganic arsenic concentration is Ash (Smith et al. 1977; Yamamura and Yamauchi 1980; Farmer and Johnson 1990; Hakala and Pyy 1995; Crecelius and Yager 1997), yielding a concentration of Ash at approxi- mately 2-24 ,ug/L, equivalent to 0.04-0.3 ~M. Because MMAi" and DMAi'~
EXPERIMENTS ~ STUDIES 1 1 7 might also be toxicologically relevant and have been found as metabolites in urine, some adjustment upward ofthis concentration would be appropriate for comparative purposes. It should tee nosed that background exposure to arsenic commonly results in urinary concentrations of inorganic arsenic of around 1-2 ,ug/L (Foe et al. 1984; Kalman et al. 1990; Lin and Huang 1995), yielding a baseline concentration of approximately 0.02-0.04 EM of Astir. Therefore, in assessing the relevance of in vitro studies to arsenic-induced cancer, the fol- Towing guidelines seem appropriate: 1. Arsenite concentrations of 0.01-0.5 EM approximate the concentra- tions that might exist in human urine following ingestion of drinking water containing arsenic concentrations of 3-50 ppb, a range that is currently the focus of Tow- dose risk assessment. 2. Arsenite concentrations of O.S-10 ,uM have been encountered in the urine of human populations chronically exposed to higher concentrations (more than 100 ~g/~) of arsenic in drinking water and might be relevant to understanding pathological effects observed in human epidemiological stud- ies. 3. Arsenite concentrations in excess of ~ O ,uM generally exceed concen- trations that can occur in the urine of individuals chronically exposed to ar- senic in drinking water and have less direct relevance to understanding the modes of action responsible for human cancer induced by this route of expo- sure. As discussed in this chapter, several recent studies have identifiedbiologi- cal effects of arsenic at in vitro concentrations of less than 1 ,uM; although for many others, high concentrations (greater than 10 EM) were required to pro- duce biological effects. The following biochemical effects have been seen in in vitro studies at arsenic concentrations of 1 ,uM or less, with a few showing effects at concentrations less than 0.1 EM (see Table 3-1~: 1. Induction of oxidative damage to DATA. 2. Altered DNA methylation and gene expression.2 Changes in intracellular levels of mUm2 protein and pS3 protein.2 4. Inhibition of thioredoxin reductase AMMAN, but not As~. 5. Inhibition of pyruvate dehydrogenase. 6. Altered colony-forming efficiency.2 2These biological effects were observed at concentrations of less than 0.1 AM of arsenic.
11 8 ARSENIC IN DRINKING WA TER. 2001 UPDA TE 7. Formation of protein-DNA cross-links.2 8. Induction of apoptosis.2 9. Altered regulation of DNA repair genes, thioredoxin, glutathione reductase, and other stress-response pathways. 10. Stimulation (0.001-0.01 ~M) and inhibition (greater than O.S EM) of normal human keratinocyte cell proliferation.2 ~ ~ . Altered function of the glucocorticoid receptor. Some of the effects described above would act to promote carcinogenic activity (e.g., stimulation of cell proliferation), while others would act to prevent cancer formation (e.g., apoptosis). The results ofthe mode-of-action studies do not provide a clear picture of the shape of the dose-response curve at low doses. At concentrations between ~ and JO ~M, a variety of additional biochemi- cal effects have been observed. For example, induction of apoptosis has been seen in numerous studies and might be an important, if not essential, mecha- nism for the chemotherapeutic effects of arsenic toward acute promyelocytic leukemia and perhaps certain other types of cancer cell lines studied in vitro. Cytogenetic studies of malignant end nonmalignant tissues obtained from humans exposed to arsenic by ingestion have found evidence of genotoxic effects, including DNA fragmentation, chromosomal aberrations andmicronu- clei, alterations in gene expression, and specific gene mutations (notably in pow. The relatively few studies conducted to date have examined populations exposed to concentrations of ingested arsenic associated with urinary inor- ganic arsenic concentrations in the range of 0.1-5 ~M. Thus, evidence is accumulating that relatively low concentrations of ar- senic, potentially achievable through consumption of drinking water contain- ing 10-50 ,ug/L of arsenic, can alter biochemical pathways relevant to carcinogenesis (gene expression, cell-cycle regulation, signal transduction, apoptosis, oxidative stress, and others). Before drawing conclusions on the relevance of these effects, however, one must be aware of the many caveats present in extrapolating from in vitro studies. For example, effects seen in transformed epithelial cell lines might or might not be relevant to normal epithelial-cell responses because ofthe many biochemical differences that are present in transformed cells. This is especially true for tumor cell lines de- rived from different primary tumors. Effects seen in one cell type might or might not be relevant to other cell types because of differences among cell types in uptake andmetabolismofarsenic, differences in gene expression, and differences in redox pathways. For example, effects seen in human keratino-
EXPERIMENTAL STUDIES ~ ~ 9 cytes might or might not be relevant to effects in human bladder or liver epi- thelial cells and vice versa. It is important to note that such differences might make the in vitro model either less or more sensitive to arsenic, relative to in viva exposures. There is no way of knowing a priori whether an effect of arsenic at a given concentration seen in one cell type in vitro is directly rele- vant to target-tissue effects in vivo following arsenic exposure via drinking water. In addition, the uptake and efflux of arsenic and its metabolites might be different in in vitro experiments. Of course, all of the uncertainties associ- ated with cross-species comparisons are also relevant to in vitro studies using cell lines or tissues from different species. All of the above modes of action (i.e., chromosomal alterations, alter- ations in DNA caused by oxidative stress or by altered methylation, and inhi- bition of DNA-repair enzymes) could enhance the carcinogenic actions of other direct tumorigens by loss of efficient repair processes or Toss of control elements in the genetic material. Thus, the mode-of-action studies suggest that the arsenic might be acting as a cocarcinogen, a promoter, or a progressor. The aforementioned biological effects that might occur following expo- sure to arsenic are likely to exhibit a typical sigmoid dose-response relation- ship, characterized by a sublinear, linear, or supralinear shape, depending on where the dose range under consideration falls along the dose-response curve. An important and controversial issue is whether the existing mode-of-action data provide suitable evidence to demonstrate that arsenic acts as a threshold- type carcinogen in humans exposed to arsenic in drinking water. A sublinear response might imply the existence of a threshold below which there is no biologically relevant response. It should be recognized that the actual shape ofthe dose-response curve seen in viva following chronic exposure to arsenic in drinking water will be a composite of many individual dose-response curves for different specific biochemical end points. It is likely that multiple differ- ent cellular perturbations are necessary before a pathological end point, such as tumor development, occurs. Thus, inferring the shape of the in viva dose- response curve from mechanistic studies cannot be done with any confidence. Despite the publication of important new findings since 1999, it is the subcommittee's consensus that the existing research database cannot establish with confidence the precise modes of action involved in arsenic-induced cancer in humans. The shape of the dose-response relationship observed for a candidate biochemical effect in a specific in vitro model should not imply that the overall dose-response relationship for the appearance of cancer in exposed human populations is likely to have the same or similar shape. At the present time, the multiplicity of potential modes of action and the apparent
120 ARSENIC IN DRINKING WA TER. 2001 UPDA TE heterogeneity of susceptibility and responsiveness that characterizes human populations does not allow identification of a specific range of arsenic concen- trations in drinking water for which the occurrence of cancer would be ex- pected to exhibit a nonlinear as opposed to a linear dose-response relationship. SUMMARY AND CONCLUSIONS · inorganic arsenic is methylated via alternating reduction of pentava- lent arsenic to trivalent arsenic and addition of a methyl group from S- adenosy~methionine. The main metabolic products MMAV and DMAV are readily excreted in urine. There is increasing evidence for the presence of reactive amounts of trivalent methylated arsenic metabolites, especially MA, in tissues and urine following exposure to inorganic arsenic. There- fore, the methylation of inorganic arsenic is not entirely a detoxification pro- cess; the formation and distribution of the reduced metabolites might be asso- ciated with increased toxicity. Those metabolites can be released and excreted in urine, especially when complexed with dithiols like DMPS. The exact mechanisms of arsenic methylation are not known. In particular, the arsenic methyTtransferases and reductases are only partially characterized. Arsenic methylation seems to vary considerably between tissues. DMA is the main metabolite excreted from the cells. There are major differences in the methylation of inorganic arsenic among mammalian species; therefore, the results of studies on animals or animal cells are difficult to extrapolate to humans. · Some recent experiments in animals have demonstrated an increase in cancer incidence following exposure to inorganic arsenic or dimethylarsen- ic acid either alone or in the presence of an initiator. However, in view of the extensive human epidemiological data that are available, those studies, al- though qualitatively relevant, are not appropriate for use in a quantitative human-health risk assessment for arsenic. · Experiments in animals and in vitro have demonstrated that arsenic has many biochemical and cytotoxic effects at low doses and concentrations that are potentially attainable in human tissues following ingestion of arsenic in drinking water. Those effects include induction of oxidative damage to DNA; altered DNA methylation and gene expression; changes in intracellular levels of mdrn2 protein and p53 protein; inhibition of thioredoxin reductase (MMAIII but not AsIII); inhibition of pyruvate dehydrogenase; altered colony- forming efficiency; induction of protein-DNA cross-links; induction of
EXPERIMENTAL STUDIES 121 apoptosis; altered regulation of DNA- repair genes, thioredoxin, glutathione reductase, and other stress-response pathways; stimulation or inhibition of normal human keratinocyte cell proliferation, depending on the concentration; and altered function of the glucocorticoid receptor. - Despite the extensive research investigating the modes of action of arsenic, the experimental evidence does not allow confidence in distinguishing between various shapes (sublinear, linear, or supralinear) ofthe dose-response curve for tumorigenesis at low doses. Therefore, the choice of model to ex- trapolate human epidemiological data from the observed range (100-2,000 ~g/~) to the range of regulatory interest (3-50 ,ug/L) cannot be made solely on a mechanistic basis. · Although a threshold for carcinogenic activity for arsenic might con- ceivably exist in a given individual, interindividual variation in response and the variability in background exposure to arsenic via the diet and other sources within the human population complicates the determination of a no-effect level in a diverse human population, if such a level were to exist. RECOMMENDATIONS · Research should be conducted to determine in viva target-tissue con- centrations of the various arsenic metabolites following ingestion of arsenic in drinking water. Of particular interest are the trivalent arsenic metabolites. Additional information is needed on arsenic reduction and methyla- tion reactions and on reactions of the various metabolites with critical tissues and proteins. Studies on the metabolism of arsenic should preferably be con- ducted in humans or in human cells. · Future research into the various potential biochemical and molecular modes of action of arsenic should focus on identifying the dose-response relationship and time course for effects of arsenic at the submicromolar level in human-derived cells or tissues. The inclusion of potential biomarkers of cytogenetic effect in epide- miological studies of arsenic-induced cancer help to elucidate the mechanisms of arsenic carcinogenesis and ultimately increase the power of investigations conducted in populations with low-dose exposure. · Research should be conducted to determine the extent to which ar- senic acts as a cocarcinogen with known carcinogens.
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