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In this study, we observed the in vivo acute immunotoxicity of a trimethyl arsenic compound, arsenobetaine (AsBe), which is present in large quantities in various marine animals that are daily ingested as seafood in many countries. The synthetic pure AsBe was orally administered to CDF(1) mice at a dose of 1.625 g/kg mouse weight once a day on days -6, -4, -2 and 0 (four times, total 6.5 g/kg mouse weight), and its effect on the immune organs and immune effector cells were assessed until day 8. Orally administered AsBe was temporally distributed to the immune organs, such as the spleen and thymus, but was not very toxic both quantitatively and qualitatively on these immune organs and immune effector cells, splenocytes, thymocytes, Peyer's patch lymphocytes and peritoneal macrophages. This finding suggests that the ingestion of AsBe contained in marine animals is relatively safe to the health of people who often consume marine animals in their daily diet.

Abstract: Inorganic arsenite has caused severe inflammatory chronic poisoning in humans through the consumption of contaminated well water. In this study, we examined the effects of arsenite at nanomolar concentrations on the in vitro differentiation of human macrophages from peripheral blood monocytes. While arsenite was found to induce cell death in a culture system containing macrophage colony stimulating factor (M-CSF), macrophages induced by granulocyte-macrophage CSF (GM-CSF) survived the treatment, but were morphologically, phenotypically, and functionally altered. In particular, arsenite-induced cells expressed higher levels of a major histocompatibility complex (MHC) class II antigen, HLA-DR, and CD14. They were more effective at inducing allogeneic or autologous T cell responses and responded more strongly to bacterial lipopolysaccharide (LPS) by inflammatory cytokine release as compared to cells induced by GM-CSF alone. On the other hand, arsenite-induced cells expressed lower levels of CD11b and CD54 and phagocytosed latex beads or zymosan particles less efficiently. We also demonstrated that the optimum amount of cellular reactive oxygen species (ROS) induced by nM arsenite might play an important role in this abnormal monocyte differentiation. This work may have implications in chronic arsenic poisoning because the total peripheral blood arsenic concentrations of these patients are at nM levels. [Copyright &y& Elsevier]

Abstract: A trivalent inorganic arsenic, arsenite, has been causing chronic inflammation in humans through the consumption of contaminated well water. The total peripheral blood arsenic concentrations of chronic arsenic-exposed patients, who had inflammatory-like immune responses, are less than 1 μM, thus, nM concentrations may be very important regarding the chronic inflammatory effects by arsenite. However, there are few reports about the biological effects of low concentrations of arsenite in mammalian cells, especially in normal immune effector cells. In this study, we examined whether arsenite has any biological and/or toxicological effects on the differentiation of human peripheral blood monocytes into macrophages using the colony-stimulating factor (CSF) in vitro compared with that of other metallic compounds, and found that arsenite sensitively inhibited the CSF-induced in vitro maturation of monocytes into macrophages at nM levels, and it also induced small, nonadhesive and CD14-positive abnormal macrophage generation from monocytes with granulocyte-macrophage CSF (GM-CSF) at 50–500 nM without cell death. The addition of other metallic compounds, including chromium, selenium, mercury, cadmium, nickel, copper, zinc, cobalt, manganese and other human pentavalent arsenic metabolites, such as inorganic arsenate, monomethylarsonic acid and dimethylarsinic acid, could not induce the same abnormal cell generation from monocytes with CSFs at any concentration and any additional time schedules; they showed only simple cytolethality in monocytes and macrophages at any concentration and any additional time schedules; they showed only simple cytolethality in monocytes and macrophages at n-mM levels accompanied by cell death. This work may have implications in the arsenic-induced chronic inflammation in humans. [Copyright &y& Elsevier]

Abstract: Human pentavalent arsenic metabolic intermediate, monomethylarsonic acid (MMAsV), is a major arsenic type found in the blood in chronic arsenic poisoning patients, but little information is available on its toxicity potential or mechanisms of action. In this study, we investigated the molecular mechanisms of in vitro cytolethality of MMAsV using rat liver TRL 1215 cells. Cellular arsenic concentrations reached the nanomolar range in TRL 1215 cells when cells were exposed to millimolar levels of MMAsV, and most of the MMAsV was not metabolized during the 48-h incubation. Under these conditions, MMAsV showed significant cytolethality when cellular reserves of reduced glutathione (GSH) were depleted. Morphological and biochemical evidence confirmed that MMAsV induced both necrosis and apoptosis in the cellular GSH-depleted cells. MMAsV significantly enhanced cellular caspase 3 activity in the cellular GSH-depleted cells, and a caspase 3 inhibitor blocked MMAsV-induced apoptosis. MMAsV also enhanced the production of cellular reactive oxygen species (ROS) in the cellular GSH-depleted cells, and addition of a membrane-permeable radical trapping reagent completely prevented both MMAsV-induced cellular caspase 3 activation and cytolethality in these cells. These observations suggest that MMAsV typically generates harmful ROS in cells, and cellular GSH prevents cytolethality by scavenging these toxic ROS. However, when cellular GSH levels are decreased, MMAsV induces oxidative stress in the cells, and this leads to apoptosis and/or necrosis depending on the cellular ROS/GSH ratio. [Copyright &y& Elsevier]

A trivalent inorganic arsenic, arsenite, has been causing chronic inflammation in humans through the consumption of contaminated well water. The total peripheral blood arsenic concentrations of chronic arsenic-exposed patients, who had inflammatory-like immune responses, are less than 1 μM, thus, nM concentrations may be very important regarding the chronic inflammatory effects by arsenite. However, there are few reports about the biological effects of low concentrations of arsenite in mammalian cells, especially in normal immune effector cells. In this study, we examined whether arsenite has any biological and/or toxicological effects on the differentiation of human peripheral blood monocytes into macrophages using the colony-stimulating factor (CSF) in vitro compared with that of other metallic compounds, and found that arsenite sensitively inhibited the CSF-induced in vitro maturation of monocytes into macrophages at nM levels, and it also induced small, nonadhesive and CD14-positive abnormal macrophage generation from monocytes with granulocyte-macrophage CSF (GM-CSF) at 50–500 nM without cell death. The addition of other metallic compounds, including chromium, selenium, mercury, cadmium, nickel, copper, zinc, cobalt, manganese and other human pentavalent arsenic metabolites, such as inorganic arsenate, monomethylarsonic acid and dimethylarsinic acid, could not induce the same abnormal cell generation from monocytes with CSFs at any concentration and any additional time schedules; they showed only simple cytolethality in monocytes and macrophages at n-mM levels accompanied by cell death. This work may have implications in the arsenic-induced chronic inflammation in humans. [Copyright &y& Elsevier]

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenicals often undergo methylation, forming compounds such as dimethylarsinic acid (DMAsV). Recent evidence indicates that DMAsV is a complete carcinogen in rodents although evidence for inorganic arsenicals as carcinogens in rodents remains equivocal. Thus, we studied the molecular mechanisms of in vitro cytolethality of DMAsV using a rat liver epithelial cell line (TRL 1215). DMAsV selectively induced apoptosis in TRL 1215 cells; its LC50 value after 48 h exposure was 4.5 mM. The addition of a glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO), actually decreased DMAsV-induced apoptosis. DMAsV exposure temporarily decreased cellular reduced glutathione (GSH) levels and enhanced cellular glutathione S-transferase (GST) activity from 6 h after the exposure when the cells were still alive. Also, DMAsV exposure activated cellular caspase 3 activity with a peak at 18 h after the exposure when apoptosis began, and BSO treatment completely inhibited this enzyme activity. The additions of inhibitors of caspase 3, caspase 8, and caspase 9 significantly reduced DMAsV-induced apoptosis. Taken together, these data indicate that cellular GSH was required for DMAsV-induced apoptosis to occur, and activation of cellular caspases after conjugation of DMAsV with cellular GSH appears to be of mechanistic significance. Further research will be required to determine the role of intracellular GSH and methylation in the toxicity of arsenicals in chronic arsenic poisoning or in cases where arsenicals are used as chemotherapeutics. [Copyright &y& Elsevier]

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenic often undergoes methylation, forming compounds such as monomethylarsonic acid (MMAsV) and dimethylarsinic acid (DMAsV). However, much less information is available on the in vitro toxic potential or mechanisms of these methylated arsenicals, especially MMAsV. We studied the molecular mechanisms of in vitro cytolethality of MMAsV using a rat liver epithelial cell line (TRL 1215). MMAsV was not cytotoxic in TRL 1215 cells even at concentrations exceeding 10 mM, but it became weakly cytotoxic and induced both necrotic and apoptotic cell death when cellular reduced glutathione (GSH) was depleted with the glutathione synthase inhibitor, l-buthionine-[S,R]-sulfoximine (BSO), or the glutathione reductase inhibitor, carmustine. Similar results were observed in the other mammalian cells, such as human skin TIG-112 cells, chimpanzee skin CRT-1609 cells, and mouse metallothionein (MT) positive and MT negative embryonic cells. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyses GSH–substrate conjugation, also enhanced the cytolethality of MMAsV, but aminooxyacetic acid (AOAA), an inhibitor of β-lyase that catalyses the final breakdown of GSH–substrate conjugates, had no effect. Both the cellular GSH levels and the cellular GST activity were increased by the exposure to MMAsV in TRL 1215 cells. On the other hand, the addition of exogenous extracellular GSH enhanced the cytolethality of MMAsV, although cellular GSH levels actually prevented the cytolethality of combined MMAsV and exogenous GSH. These findings indicate that human arsenic metabolite MMAsV is not a highly toxic compound in mammalian cells, and the level of cellular GSH is critical to its eventual toxic effects. [Copyright &y& Elsevier]