Inorganic and Methylated Arsenic Compounds Induce Cell Death in

arsenate, are strongly toxic to macrophages, and the concentration that decreased the ... In contrast, the cytotoxic effects of methylated arsenic com...
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Chem. Res. Toxicol. 1998, 11, 273-283

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Inorganic and Methylated Arsenic Compounds Induce Cell Death in Murine Macrophages via Different Mechanisms Teruaki Sakurai,* Toshikazu Kaise, and Chiyo Matsubara Laboratory of Environmental Chemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan Received August 8, 1997

We demonstrate in this study the cytotoxic effects of inorganic arsenicals, arsenite and arsenate, and organic arsenic compounds, monomethylarsonic acid (MAA), dimethylarsinic acid (DMAA), and trimethylarsine oxide (TMAO), which are metabolites of inorganic arsenicals in human bodies, using murine macrophages in vitro. Inorganic arsenicals, both arsenite and arsenate, are strongly toxic to macrophages, and the concentration that decreased the number of surviving cells to 50% of that in untreated controls (IC50) was 5 or 500 µM, respectively. These inorganic arsenicals mainly caused necrotic cell death with partially apoptotic cell death; about 80% of dead cells were necrotic, and 20% were apoptotic. The inorganic arsenicals also induced marked release of an inflammatory cytokine, tumor necrosis factor R (TNFR), at cytotoxic doses. This strong cytotoxicity of an inorganic arsenical, arsenite, might be mediated via active oxygen and protease activation because it was inhibited by the addition of some antioxidant reagents, such as superoxide dismutase (SOD), catalase, and GSH, or by a peptide inhibitor of interleukin-1β-converting enzyme (ICE). It is likely that these immunotoxic effects of inorganic arsenicals may evoke both immunosuppression and inflammation, and they may be central factors causing carcinogenesis and severe inflammatory responses, such as hepatomegaly and splenomegaly, in chronic arsenicosis patients who daily ingested arseniccontaminated well water. In contrast, the cytotoxic effects of methylated arsenic compounds were lower than those of inorganic arsenicals. The IC50 value of DMAA was about 5 mM, and MAA and TMAO had no toxicity even at concentrations over 10 mM. Additionally, these methylated chemicals suppressed the TNFR release from macrophages. DMAA induced mainly apoptotic cell death in macrophages as indicated by cellular morphological changes, condensed nuclei, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), and DNA fragmentation. However, the cytotoxicity of DMAA might be induced via a different mechanism from that of inorganic arsenicals because it was not abolished by the additions of SOD, catalase, or ICE inhibitor. Conversely, GSH enhanced the toxicity of DMAA. These data suggest that methylation of inorganic arsenicals in mammals plays an important role in suppression of both severe immunosuppression and inflammatory responses caused by inorganic arsenicals.

Introduction Arsenic is a chemical that evokes many images, most of which are negative. Arsenic is a common constituent of the earth’s crust in its inorganic form, trivalent (arsenite) or pentavalent (arsenate) chemical (1). Humans may encounter inorganic arsenicals in drinking water from wells drilled into arsenic-rich strata. It has been reported that arsenic poisoning has occurred in some countries in Asia and South America (2-5) through the consumption of contaminated well water. For more than a century, various carcinogenic effects of inorganic arsenicals on humans have been documented, mainly involving the skin and lung (6), and recent epidemiological studies have indicated that ingestion of inorganic arsenicals is related to cancer induction in the liver, kidney, urinary bladder, and other internal organs (7, 8). It has been believed that these carcinogenic effects * To whom correspondence should be addressed. Phone: ++81-426/ 76-6792. Fax: ++81-426/76-5354. E-mail: [email protected].

of inorganic arsenicals were due to their direct genotoxicity. Inorganic arsenicals have long been known to cause chromosomal damage, such as chromosome alterations (9-11), and to promote genetic damage in large part by inhibiting DNA repair (12-14). In recent studies, it was also demonstrated that inorganic arsenicals induced abnormal inflammatory-like immunotoxicity, and it is thought possible that this abnormal change in the immune systems presumably contributed to the toxicity and carcinogenicity of inorganic arsenicals. For example, in 1995, Dr. Guha-Mazumder first reported that various severe inflammatory clinical observations, such as hypertrophy of the liver and/or spleen, were found at a higher rate of over 70% in chronic arsenic poisoning patients who ingested inorganic arsenicals from contaminated well water as well as skin lesions in western Bengal, India (15). In 1996, medical observations through ultrasonography and blood tests were done on about 2000 chronic arsenicosis patients in inner Mongolia, China, and many cases of hepatomegaly, hardening of the liver,

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Sakurai et al.

and arsenate, using two populations of macrophages, peritoneal macrophages (PMs) and alveolar macrophages (AMs) from mice.

Experimental Procedures

Figure 1. Metabolism of arsenicals in mammals.

and splenomegaly were found (16). Germolec et al. also showed that growth-promoting cytokines and growth factors were induced in human epidermal keratinocytes following in vitro treatment with inorganic arsenicals and could play a significant role in inorganic arsenicalinduced skin cancer (17). Thus, we concluded that the investigations of the effects of arsenic compounds on immune effector cells would be of great value in enhancing our understanding of the mechanism of the toxic and carcinogenic effects of arsenic compounds in mammals because there is little information on the effects of arsenicals on the immune system. The methylation of inorganic arsenicals is the principal metabolic reaction in humans (18, 19) and most experimental animals (20, 21). In vivo studies suggest that pentavalent inorganic arsenicals in mammals must be reduced to trivalent inorganic arsenicals, arsenites, before methylation can occur (22), and methylarsonic acid (MAA)1 and dimethylarsinic acid (DMAA) have been identified as organic metabolites in human urine after administration of inorganic arsenicals in either the trivalent or pentavalent state (18, 19). DMAA is the ultimate metabolite in humans (19), while DMAA is further methylated to trimethylarsine oxide (TMAO) in hamsters and rats (see Figure 1) (23, 24). It is believed that the methylation of inorganic arsenicals results in the lowering of their general toxicity as indicated by their increased LD50 (25, 26), a far from exact measurement of their toxicity (27). The recent studies of the toxicity of organic arsenic compounds (28-30) increasingly suggest that the methylation of inorganic arsenicals is not universally a detoxification mechanism (27). Macrophages are one of the principal immune effector cells that play essential roles as secretory, phagocytic, and antigen-presenting cells in the immune system. Macrophages release various inflammatory factors when they are activated (31) and are also known to be very sensitive to changes in environmental conditions, including arsenic treatments (32, 33). Taken together, it is suggested that macrophages are very useful to examine the influence of chemical materials upon mammalian immune systems. Hence, we compared the cytotoxic effects of methylated arsenic compounds, MAA, DMAA, and TMAO, with those of inorganic arsenicals, arsenite 1 Abbreviations: MMA, methylarsonic acid; DMAA, dimethylarsinic acid; TMAO, trimethylarsine oxide; PM, peritoneal macrophage; AM, alveolar macrophage; LPS, lipopolysaccharide; SOD, superoxide dismutase; BSO, L-buthionine (S,R)-sulfoximine; L-NMMA, NG-monomethyl-L-arginine acetate; AcYVAD-CMK, acetyl-Tyr-Val-Ala-Aspchloromethyl ketone; AB, AlamarBlue; TdT, terminal deoxynucleotidyltransferase; TUNEL, TdT-mediated dUTP-biotin nick end labeling; TNFR, tumor necrosis factor R; rMu IFNγ, recombinant murine interferon γ; ICE, interleukin-1β-converting enzyme; IC/ICP-MS, ion chromatography with inductively coupled plasma-mass spectrometry.

Reagents. Sodium arsenite, sodium arsenate, and DMAA were purchased from Wako Pure Chemical Co. (Osaka, Japan). MAA was obtained from Trichemical Co. (Yamanashi, Japan). TMAO was synthesized from trimethylarsine using hydrogen peroxide as described elsewhere (34). These arsenic compounds were recrystallized twice, and their purities were >99.9% as determined by GC/MS. Lipopolysaccharide (LPS) contamination of these compounds was not detected ( DMAA (5 mM). These cytotoxic effects of arsenic compounds were parallel to the results of acute toxicity after oral administration in mice as previously described (26). The cytotoxicity of arsenate was 100-fold lower than that of arsenite. It has been reported that the mechanisms of cellular influence of these inorganic arsenicals were different depending on their oxidation state (27, 41, 45, 46). Arsenite, which has a trivalent oxidation state, has a very high affinity for thiol groups; in contrast, arsenate, which has a pentavalent oxidation state, can replace phosphate in many reactions in the mammalian body, and the toxicity of arsenite has been generally higher than that of arsenate. As shown in Table 2, about 7% of arsenate was reduced to arsenite in macrophages during 24-h incubation. Thus, it is suggested that macrophages partially reduced arsenate to arsenite, and the cytotoxicity observed for arsenate might be due to a small amount of it being converted to arsenite. All of the organic compounds, MAA, DMAA, and TMAO, have the pentavalent oxidation state of arsenic in their molecules, and only DMAA showed significant cytotoxicity on macrophages (Figure 2). The toxicity of DMAA was 1000-fold lower than that of arsenite, and MAA and TMAO showed no toxicity even at concentrations over 10 mM. The cytotoxicity of a monomethylated arsenic compound, MAA, was lower than that of another methylated arsenic compound, DMAA (Figure 2). Figure 12 shows the amounts of cellular uptake of methylated arsenicals by macrophages. As a result, similar arsenic uptake was observed when the cells were incubated with DMAA or TMAO, but only 20% as much arsenical was taken up by cells incubated with MAA. It is postulated that the low cytotoxicity of MAA depends, at least in part, on the low uptake of this chemical species. Inorganic arsenicals primarily induced necrotic cell death; in contrast, DMAA induced apoptotic cell death in 100% of both PMs and AMs as indicated by cellular morphological changes (Figure 3), condensed nuclei (figure 4), TUNEL staining (Figure 5), and DNA fragmentation (Figure 6). Inorganic arsenicals also induced apoptosis in 20% of the dead cells. Apoptosis is a process by which organisms eliminate the damaged, precancerous, or redundant cells without invoking an inflammatory response. Thus, the methylation of inorganic arsenicals may not only reduce its acute toxicity but also reduce the inflammatory potency. As shown in Figure 8, stimulation with inorganic arsenicals, either arsenite or arsenate, induced a marked release of an inflammatory cytokine, TNFR, from the PMs at cytotoxic doses. In contrast, highly methylated arsenicals, DMAA and TMAO, reduced TNFR release. In a preliminary experiment, inorganic arsenicals also enhanced the release of another inflammatory cytokine, interleukin-1R (unpublished data). These data suggest that inorganic arsenicals induce necrotic cell death in

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macrophages in concert with the enhanced release of inflammatory cytokines. These cytokines may cause inflammatory responses, such as hepatomegaly and splenomegaly, in chronic arsenicosis patients. Methylation of arsenicals may play an important role in suppressing the inflammation by inhibiting the release of inflammatory mediators from macrophages. Because the culture supernatants of macrophages prestimulated with arsenic compounds showed no cytotoxicity on other fresh macrophages (Table 1), we postulated that the cytotoxic effects of inorganic arsenicals and DMAA on macrophages might be direct rather than due to autocrine mechanisms mediating some factors secreted by arsenite-stimulated cells. An inorganic arsenical, arsenite, has been reported to induce significant cellular oxidative responses in murine macrophages in vitro (32, 33). In addition, DMAA induces potent active oxygen production in vivo after oral administration in a murine model (47). Thus, we examined the effect of some antioxidants on the toxicity of arsenicals to macrophages. Addition of exogenous SOD or catalase, scavenger enzymes for superoxide anion or hydrogen peroxide, respectively, significantly inhibited the cell death of PMs induced by arsenite (Table 3 and Figure 9). About 50% of PMs remained viable in the presence of arsenite and antioxidants. Catalase was more effective than SOD. However, neither addition of SOD nor addition of catalase abolished DMAA-induced apoptotic cell death at all (Table 3 and Figure 9). GSH reduces hydrogen peroxide and binds a variety of essential and nonessential metals, including arsenicals, playing critical roles in the cellular and systemic metabolism of these metals (48). GSH is a critical cofactor for promotion of the in vitro enzymatic methylation of arsenate and arsenite (27, 49). In the present study, exogenous GSH markedly reduced arseniteinduced cell death of PMs; 90% of the cells survived. Conversely, GSH depletion by BSO, a selective inhibitor of γ-glutamylcysteine synthetase, significantly enhanced the cytotoxicity of arsenite on PMs (Figure 10). This strong protection of GSH against arsenite-induced cytotoxicity might depend on both the scavenging of hydrogen peroxide produced by arsenite-stimulated PMs and the chelation of arsenite by GSH. This hypothesis is supported by the results of Bannai and co-workers who found that exposure to arsenite increased intracellular GSH level in murine PMs (33). Compared with these results, exogenous GSH increased the DMAA-induced apoptotic cell death in PMs, and GSH depletion completely abolished the cytotoxicity of DMAA (Figure 10). These findings suggest that the interaction of DMAA and GSH is an important factor in the potent cytotoxicity of DMAA. GSH may reduce the DMAAs5+ to DMAAs3+, resulting in formation of a more toxic chemical (41). However, in our preliminary experiment, dimethylarsine gas, which is well-known to be a very toxic gas, was not detected from a mixture of GSH and DMAA in vitro (unpublished data), and Delnomdedieu et al. recently reported that GSH formed a stable complex with DMAA, DMAAs3+SG (41). On the other hand, Thompson suggested in a previous review that GSH reduced DMAA and that an unstable compound, dimethylarsinous acid, which was thought to be more toxic than DMAA, might be formed (50). Thus, it is possible that this unstable chemical induced cytotoxicity in macrophages, but this hypothesis has not been verified. Further experiments are needed

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to determine the role of GSH in the cytotoxicity of DMAA. ICE is a cysteine protease which is known to regulate apoptosis (41). As shown in Table 4 and Figure 11, the addition of a peptide inhibitor of ICE, AcYVAD-CMK, significantly inhibited the arsenite-induced cell death in macrophages. It has been reported that reactive oxygen intermediators regulate the apoptosis via activation of ICE (51). Hence, the ICE inhibitor may suppress arsenite-induced apoptosis by blocking the action of active oxygen species produced by arsenite-stimulated cells. We also demonstrated in this study that both exogenous antioxidant reagents and the ICE inhibitor equally, but not completely, abrogate arsenite-induced cell death; about 50% of the cells survived. The ICE inhibitor also had a small effect on DMAA-induced cell death (Table 4). Thus, the activation of ICE might play some role in inducing apoptosis in DMAA-stimulated macrophages; however, there must be other unknown signal pathways to regulate apoptosis after exposure to DMAA. These pathways may be mediated via the interaction of DMAA with GSH. Because arsenicals can modulate some enzyme activities by combination with a thiol group (52), it is possible that arsenite- or DMAA-induced apoptosis might be associated with direct modulation of ICE activity. In conclusion, we demonstrate in this study that inorganic arsenicals, both arsenite and arsenate, are potent cytotoxins for the principal immune effector cells, macrophages, and that this effect is partly mediated via cellular oxidative responses and by the activation of a cysteine protease. Inorganic arsenicals were also involved in necrotic cell death and in the enhanced release of an inflammatory cytokine, TNFR, by macrophages. It is likely that these immunotoxic effects of inorganic arsenicals may evoke the breaking up of immune systems, and they may be central causing carcinogenesis and severe inflammatory responses, such as hepatomegaly and splenomegaly, in chronic arsenicosis patients who daily ingested arsenical-contaminated well water. In contrast, the immunotoxic effects of methylated arsenic compounds, MAA, DMAA, and TMAO, which are metabolites of inorganic arsenicals in mammals, were much lower than those of inorganic arsenicals. The IC50 values of these arsenic compounds on macrophages were very high, and they induced apoptosis and released suppression of TNFR in macrophages. Taken together, methylation of inorganic arsenicals by mammals is suggested to play an important role in their detoxification, and chronic arsenicosis may be caused by the accumulation of inorganic arsenicals which could not be methylated (43). Our data suggest an important mechanistic basis for chronic arsenicosis.

Acknowledgment. We express our thanks to Mrs. Masumi H. Sakurai and Noriko N. Miura for their valuable help with the limulus test and to Misses Yukie Takagi and Ayako Yamaura for their excellent technical assistance.

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