Dietary administration of sodium arsenite to rats: Relations between dose and urinary concentrations of methylated and thio-metabolites and effects on the rat urinary bladder epithelium

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in drinking water is carcinogenic to humans, inducing skin, urinary bladder and lung tumors. In vivo, inorganic arsenic is metabolized to organic methylated arsenicals including the highly toxic dimethylarsinous acid (DMA^III) and monomethylarsonous acid (MMA^III). Short-term treatment of rats with 100 μg/g trivalent arsenic (As^III) as sodium arsenite in the diet or in drinking water induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation and hyperplasia. The objectives of this study were to determine if these arsenic-induced urothelial effects are dose responsive, the dose of arsenic at which urothelial effects are not detected, and the urinary concentrations of the arsenical metabolites. We treated female F344 rats for 5 weeks with sodium arsenite at dietary doses of 0, 1, 10, 25, 50, and 100 ppm. Cytotoxicity, cell proliferation and hyperplasia of urothelial superficial cells were increased in a dose-responsive manner, with maximum effects found at 50 ppm As^III. There were no effects at 1 ppm As^III. The main urinary arsenical in As^III-treated rats was the organic arsenical dimethylarsinic acid (DMA^V). The thio-metabolites dimethylmonothioarsinic acid (DMMTA^V) and monomethylmonothioarsinic acid (MMMTA^V) were also found in the urine of As^III-treated rats. The LC₅₀ concentrations of DMMTA^V for rat and human urothelial cells in vitro were similar to trivalent oxygen-containing arsenicals. These data suggest that dietary As^III-induced urothelial cytotoxicity and proliferation are dose responsive, and the urothelial effects have a threshold corresponding to the urinary excretion of measurable reactive metabolites.     

Effects of an epidermal growth factor receptor inhibitor on arsenic associated toxicity in the rat bladder epithelium

Arsenite (As^III), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is known to be metabolized to organic methylated arsenicals in vivo. As^III has been reported to have the ability to up-regulate the epidermal growth factor receptor (EGFR)-associated pathway in epithelial cells, including human urothelial cells in vitro. EGFR is a cell-surface receptor belonging to the ErbB family of receptor tyrosine kinases, and the EGFR-associated signaling pathway has been reported to play an important role in carcinogenesis and cancer progression, including in bladder cancer. In this study, we investigated the growth effects of As^III and an organic trivalent arsenical, dimethylarsinous acid (DMA^III), and the effects of co-exposure of gefitinib, an EGFR inhibitor, with As^III to a rat urothelial cell line (MYP3). We also investigated the effects of co-administration of dietary As^III and gefitinib in vivo. In vitro, concentrations of 1.0 μM As^III or 0.5 μM DMA^III induced cytotoxicity. However, lower concentrations of As^III treatment had a slight mitogenic growth effect whereas lower concentrations of DMA^III did not. Gefitinib blocked As^III-induced cell growth in vitro. In vivo, a high dose of gefitinib alone induced slight urothelial cytotoxicity, and did not reduce cytotoxicity and regenerative cell proliferation when co-administered with As^III. The majority of arsenic metabolites present in the urine of As^III-treated rats were organic arsenicals, mainly dimethylarsinic acid (DMA^V). As^III was also present, and its concentration was higher than the concentration required to produce cytotoxicity in vitro. These data suggest that an EGFR inhibitor has the ability to block As^III-induced cell proliferation in vitro but not in vivo in a short-term study.     

Possible role of dimethylarsinous acid in dimethylarsinic acid-induced urothelial toxicity and regeneration in the rat

Dimethylarsinic acid (DMA(V)) is carcinogenic to the rat urinary bladder when administered at high doses in the diet or drinking water. At a dietary dose of 100 ppm (microg/g), it produces cytotoxicity within 6 h and increased proliferation (hyperplasia) by 7 days of administration. We hypothesize that formation of the reactive organic intermediate dimethylarsinous acid (DMA(III)) is involved in the induction of the cytotoxicity. To evaluate the possibility that DMA(V) administration produces urothelial toxicity and regeneration by the formation of trivalent arsenicals, 2,3-dimercaptopropane-1-sulfonic acid (DMPS, 5600 ppm), a chelator of trivalent arsenicals, was co-administered with DMA(V) (100 ppm) for 2 weeks to groups of female Fischer F344 rats. Based on light and scanning electron microscopy, and bromodeoxyuridine labeling index, DMA(V) produced cytotoxicity and regenerative hyperplasia of the urothelium which was inhibited by co-administration with DMPS. The major forms of arsenic in the 24-h urine of rats administered DMA(V) were high concentrations of DMA(V) (66.4 +/- 2.7 microM) itself and the pentavalent organic arsenical trimethylarsine oxide (TMAO) (73.2 +/- 9.5 microM). Co-administration with DMPS led to an increase in DMA(V) (507 +/- 31 microM) with a decrease in TMAO (2.8 +/- 0.4 microM) excretion. The formation of TMAO from DMA(V) mechanistically suggests formation of the intermediate trivalent metabolite, DMA(III). In a second experiment evaluating fresh void urines collected on study days 1, 71, and 175, we detected DMA(III) in the urine of DMA(V) and DMA(V) plus DMPS-treated rats at approximately micromolar concentrations. Using rat (MYP3) and human (1T1) urothelial cells, cytotoxicity for trivalent arsenicals, sodium arsenite, monomethylarsonous acid (MMA(III)), and DMA(III) was demonstrated at 0.4-4.8 microM concentrations, whereas MMA(V), DMA(V), and TMAO were cytotoxic at millimolar concentrations. The presence of DMA(III) at micromolar concentrations in the urine of rats fed 100 ppm DMA(V) suggests that DMA(III) produced in vivo may be involved in the toxic effects in the rat urinary bladder after dietary administration of DMA(V).     

The accumulation and toxicity of methylated arsenicals in endothelial cells: important roles of thiol compounds

Excess intake of arsenic is known to cause vascular diseases as well as skin lesions and cancer in humans. Recent reports suggest that trivalent methylated arsenicals, which are intermediate metabolites in the methylation process of inorganic arsenic, are responsible for the toxicity and carcinogenicity of environmental arsenic. We investigated acute toxicity and accumulation of monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), trimethylarsine oxide (TMAO), and monomethylarsonous acid diglutathione (MMA(III) (GS)(2)) in rat heart microvessel endothelial (RHMVE) cells. MMA(V) (LC(50) = 36.6 mM) and DMA(V) (LC(50) = 2.54 mM) were less toxic than inorganic arsenicals (cf. LC(50) values for inorganic arsenite (iAs(III)), and inorganic arsenate (iAs(V)) was reported to be 36 and 220 microM, respectively, in RHMVE cells. TMAO was essentially not toxic. However, MMA(III) (GS)(2) was highly toxic (LC(50) = 4.1 microM). The order of cellular arsenic accumulation of those four organic arsenic compounds was MMA(III) (GS)(2) > MMA(V) > DMA(V) > TMAO. MMA(III) (GS)(2) was efficiently taken up by the cells and cellular arsenic content increased with the concentration of MMA(III) (GS)(2) in culture medium. N-acetyl-l-cysteine (NAC) reduced cellular arsenic content in DMA(V)-exposed cells and also decreased the cytotoxicity of DMA(V), whereas it changed neither cellular arsenic content nor the viability in MMA(V)-exposed cells. mRNA levels of heme oxygenase-1 (HO-1) were decreased by NAC in DMA(V)-exposed, but MMA(V)-exposed cells. Buthionine sulfoximine (BSO), a cellular glutathione (GSH) depleting agent, enhanced the cytotoxicity of MMA(V). However, BSO reduced, rather than enhanced, the cytotoxicity of DMA(V). These results suggest that intracellular GSH modulated the toxic effects of arsenic in opposite ways for MMA(V) and DMA(V). Even though intracellular GSH decreased the cytotoxicity of MMA(V), extracellularly added GSH enhanced the cytotoxicity of MMA(V). The use of high-performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometric analyses suggested that a small amount of MMA(V) was converted to MMA(III) (GS)(2) in the presence of GSH. These results suggest that MMA(III) (GS)(2) is highly toxic compared to other arsenic compounds because of faster accumulation of this species by cells, in addition to having the toxic nature of methylated trivalent organic arsenics.     

Toxicity of dimethylmonothioarsinic acid toward human epidermoid carcinoma A431 cells

Chronic ingestion of arsenic-contaminated drinking water induces skin lesions and urinary bladder cancer in humans. It is now recognized that thioarsenicals such as dimethylmonothioarsinic acid (DMMTA (V)) are commonly excreted in the urine of humans and animals and that the production of DMMTA (V) may be a risk factor for the development of the diseases caused by arsenic. The toxicity of DMMTA (V) was compared with that of related nonthiolated arsenicals with respect to cell viability, uptake ability, generation of reactive oxygen species (ROS), and cell cycle progression of human epidermoid carcinoma A431 cells, arsenate (iAs (V)), arsenite (iAs (III)), dimethylarsinic acid (DMA (V)), and dimethylarsinous acid (DMA (III)) being used as reference nonthiolated arsenicals. DMMTA (V) (LC 50 = 10.7 microM) was shown to be much more cytotoxic than iAs (V) (LC 50 = 571 microM) and DMA (V) (LC 50 = 843 microM), and its potency was shown to be close to that of trivalent arsenicals iAs (III) (LC 50 = 5.49 microM) and DMA (III) (LC 50 = 2.16 microM). The greater cytotoxicity of DMMTA (V) was associated with greater cellular uptake and distribution, and the level of intracellular ROS remarkably increased in A431 cells upon exposure to DMMTA (V) compared to that after exposure to other trivalent arsenicals at the respective LC 50. Exposure of DMMTA (V) to cells for 24 h induced cell cycle perturbation. Namely, the percentage of cells residing in S and G2/M phases increased from 10.2 and 15.6% to 46.5 and 20.8%, respectively. These results suggest that although DMMTA (V) is a pentavalent arsenical, it is taken up efficiently by cells and causes various levels of toxicity, in a manner different from that of nonthiolated pentavalent arsenicals, demonstrating that DMMTA (V) is one of the most toxic arsenic metabolites. The high cytotoxicity of DMMTA (V) was explained and/or proposed by (1) efficient uptake by cells followed by (2) its transformation to DMA (V), (3) producing ROS in the redox equilibrium between DMA (V) and DMA (III) in the presence of glutathione.     

Arsenic-induced bladder cancer in an animal model

Dimethylarsinic acid (DMA(V)) is carcinogenic to the rat urinary bladder, but not in mice. The carcinogenic mode of action involves cytotoxicity followed by regenerative cell proliferation. Dietary DMA(V) does not produce urinary solids or significant alterations in urinary composition. The cytotoxicity is due to formation of a reactive metabolite, likely dimethylarsinous acid (DMA(III)), concentrated and excreted in the urine. Urinary concentrations of DMA(III) are dose-dependent, and the urinary concentrations are at cytotoxic levels based on in vitro studies. The no observed effect level (NOEL) in these rat dietary studies for detectable levels of DMA(III), cytotoxicity, and proliferation is 2 ppm, with marginal changes at 10 ppm. The tumorigenic dose is 100 ppm. Recent investigations have demonstrated that arsenicals administered to the rat result in binding to a specific cysteine in the hemoglobin alpha chain as DMA(III), regardless of the arsenical being administered. Monomethylarsonic acid (MMA(V)) is not carcinogenic in rats or mice. In short term experiments (< or =10 weeks), sodium arsenate in the drinking water induces significant cytotoxicity and regenerative proliferation. There is little evidence that the cytotoxicity produced following administration of arsenicals is caused by oxidative damage, as antioxidants show little inhibitory activity of the cytotoxicity of the various arsenicals either in vitro or in vivo. In summary, the mode of action for DMA(V)-induced bladder carcinogenesis in the rat involves generation of a reactive metabolite (DMA(III)) leading to cytotoxicity and regenerative proliferation, is a non-linear process, and likely involves a threshold. Extrapolation to human risk needs to take this into account along with the significant differences in toxicokinetics and toxicodynamics that occur between different species.     

Immortalized human urothelial cells as a model of arsenic-induced bladder cancer

Arsenical-induced carcinogenesis in human bladder has been established through epidemiological evidence, and UROtsa cells, a normal, immortalized cell culture model of human urothelium, have proven to be a good model for the bladder epithelium. This cell line does not form tumors when injected into immuno-compromised mice nor does it have anchorage-independent growth. UROtsa can be easily manipulated for acute studies related to arsenical exposure. They have been shown to be sensitive to all arsenicals, in particular, the trivalent species, arsenite and monomethylarsonous acid. UROtsa cells have also opened the area of cellular signaling alterations following subcytotoxic exposure to arsenicals in both the acute and long-term time points. In addition, UROtsa cells were shown to be malignantly transformed following low-level exposure to both As(III) and MMA(III) providing additional models for studying arsenical-induced carcinogenesis of the bladder. These transformed cell lines allow researchers the ability to investigate the process of urothelial tumorigenesis at multiple time points of arsenical exposure. Overall, UROtsa cells are an effective model for cellular insult following arsenical exposure.     

The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.     

Cytotoxic effects of S-(dimethylarsino)-glutathione: a putative intermediate metabolite of inorganic arsenicals

Glutathione (GSH) plays an important role in the metabolism of arsenite and arsenate by generating arsenic-glutathione complexes. Although dimethylarsinic acid (DMA(V)) is the major metabolite of inorganic arsenicals (iAs) in urine, it is not clear how DMA(V) is produced from iAs. In the present study we report that S-(dimethylarsino)-glutathione (DMA(III)(SG)), a putative precursor of dimethylarsinic acid DMA(V), was unstable in the culture medium without excess GSH and generated volatile substances which were highly cytotoxic for both rat heart microvascular endothelial cells and HL60, a human leukemia cell line. Cytotoxicity of DMA(III)(SG) was higher than that of iAs and its LC(50) value was calculated to be 7.8 microM in the endothelial cells. To our surprise DMA(III)(SG) effectively killed cells in the neighbor wells of the same multi-well dish, indicating that volatile toxic compounds generated from DMA(III)(SG) in the culture medium. High performance lipid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) analyses suggested that the freshly generated volatile compounds dissolved into aqueous solution and formed an unstable arsenic compound and the unstable compound was further converted to DMA(V). These results suggested that DMA(III)(SG) exerts its cytotoxicity by generating volatile arsenicals and is implicated in the metabolic conversion of inorganic arsenicals into DMA(V), a major final metabolite of inorganic arsenicals in most mammals.     

Arsenite and monomethylarsonous acid generate oxidative stress response in human bladder cell culture

Arsenicals have commonly been seen to induce reactive oxygen species (ROS) which can lead to DNA damage and oxidative stress. At low levels, arsenicals still induce the formation of ROS, leading to DNA damage and protein alterations. UROtsa cells, an immortalized human urothelial cell line, were used to study the effects of arsenicals on the human bladder, a site of arsenical bioconcentration and carcinogenesis. Biotransformation of As(III) by UROtsa cells has been shown to produce methylated species, namely monomethylarsonous acid [MMA(III)], which has been shown to be 20 times more cytotoxic. Confocal fluorescence images of UROtsa cells treated with arsenicals and the ROS sensing probe, DCFDA, showed an increase of intracellular ROS within five min after 1 microM and 10 microM As(III) treatments. In contrast, 50 and 500 nM MMA(III) required pretreatment for 30 min before inducing ROS. The increase in ROS was ameliorated by preincubation with either SOD or catalase. An interesting aspect of these ROS detection studies is the noticeable difference between concentrations of As(III) and MMA(III) used, further supporting the increased cytotoxicity of MMA(III), as well as the increased amount of time required for MMA(III) to cause oxidative stress. These arsenical-induced ROS produced oxidative DNA damage as evidenced by an increase in 8-hydroxyl-2'-deoxyguanosine (8-oxo-dG) with either 50 nM or 5 microM MMA(III) exposure. These findings provide support that MMA(III) cause a genotoxic response upon generation of ROS. Both As(III) and MMA(III) were also able to induce Hsp70 and MT protein levels above control, showing that the cells recognize the ROS and respond. As(III) rapidly induces the formation of ROS, possibly through it oxidation to As(V) and further metabolism to MMA(III)/(V). These studies provide evidence for a different mechanism of MMA(III) toxicity, one that MMA(III) first interacts with cellular components before an ROS response is generated, taking longer to produce the effect, but with more substantial harm to the cell.     

Monomethylarsonous acid (MMA(III)) is more toxic than arsenite in Chang human hepatocytes

Methylation has been considered to be the primary detoxication pathway of inorganic arsenic. Inorganic arsenic is methylated by many, but not all animal species, to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), and dimethylarsinic acid (DMA(V)). The As(V) derivatives have been assumed to produce low toxicity, but the relative toxicity of MMA(III) remains unknown. In vitro toxicities of arsenate, arsenite, MMA(V), MMA(III), and DMA(V) were determined in Chang human hepatocytes. Leakage of lactate dehydrogenase (LDH) and intracellular potassium (K(+)) and mitochondrial metabolism of the tetrazolium salt XTT were used to assess cytotoxicity due to arsenic exposure. The mean LC50 based on LDH assays in phosphate media was 6 microM for MMA(III) and 68 microM for arsenite. Using the assay for K(+) leakage in phosphate media, the mean LC50 was 6.3 microM for MMA(III) and 19.8 microM for arsenite. The mean LC50 based on the XTT assay in phosphate media was 13.6 microM for MMA(III) and 164 microM for arsenite. The results of the three cytotoxicity assays (LDH, K(+), and XTT) reveal the following order of toxicity in Chang human hepatocytes: MMA(III) > arsenite > arsenate > MMA(V) = DMA(V). Data demonstrate that MMA(III), an intermediate in inorganic arsenic methylation, is highly toxic and again raises the question as to whether methylation of inorganic arsenic is a detoxication process.     

Mitochondria are the main target organelle for trivalent monomethylarsonous acid (MMA(III))-induced cytotoxicity

Excessive generation of reactive oxygen species (ROS) is considered to play an important role in arsenic-induced carcinogenicity in the liver, lungs, and urinary bladder. However, little is known about the mechanism of ROS-based carcinogenicity, including where the ROS are generated, and which arsenic species are the most effective ROS inducers. In order to better understand the mechanism of arsenic toxicity, rat liver RLC-16 cells were exposed to arsenite (iAs(III)) and its intermediate metabolites [i.e., monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III))]. MMA(III) (IC(50) = 1 μM) was found to be the most toxic form, followed by DMA(III) (IC(50) = 2 μM) and iAs(III) (IC(50) = 18 μM). Following exposure to MMA(III), ROS were found to be generated primarily in the mitochondria. DMA(III) exposure resulted in ROS generation in other organelles, while no ROS generation was seen following exposures to low levels of iAs(III). This suggests the mechanisms of induction of ROS are different among the three arsenicals. The effects of iAs(III), MMA(III), and DMA(III) on activities of complexes I-IV in the electron transport chain (ETC) of rat liver submitochondrial particles and on the stimulation of ROS production in intact mitochondria were also studied. Activities of complexes II and IV were significantly inhibited by MMA(III), but only the activity of complexes II was inhibited by DMA(III). Incubation with iAs(III) had no inhibitory effects on any of the four complexes. Generation of ROS in intact mitochondria was significantly increased following incubation with MMA(III), while low levels of ROS generation were observed following incubation with DMA(III). ROS was not produced in mitochondria following exposure to iAs(III). The mechanism underlying cell death is different among As(III), MMA(III), and DMA(III), with mitochondria being one of the primary target organelles for MMA(III)-induced cytotoxicity.     

A comparative study of the sub-chronic toxic effects of three organic arsenical compounds on the urothelium in F344 rats; gender-based differences in response

Epidemiological studies indicated that human arsenic exposure can induce urinary bladder cancer. Methylation of inorganic arsenic can generate more reactive and toxic organic arsenical species. In this regard, it was recently reported that the methylated arsenical metabolite, dimethylarsinic acid [DMA(V)], induced urinary bladder tumors in rats. However, other methylated metabolites, like monomethylarsonic acid [MMA(V)] and trimethylarsine oxide (TMAO) were not carcinogenic to the urinary bladder. In order to compare the early effects of DMA(V), MMA(V), and TMAO on the urinary bladder transitional cell epithelium at the scanning electron microscope (SEM) level, we investigated the sub-chronic (13 weeks) toxicological effects of MMA(V) (187 ppm), DMA(V) (184 ppm), TMAO (182 ppm) given in the drinking water to male and female F344 rats with a focus on the urinary bladder in this study. Obvious pathological changes, including ropy microridges, pitting, increased separation of epithelial cells, exfoliation, and necrosis, were found in the urinary bladders of both sexes, but particularly in females receiving carcinogenic doses of DMA(V). Urine arsenical metabolic differences were found between males and females, with levels of MMA(III), a potential genotoxic form, higher in females treated with DMA(V) than in males. Thus, this study provides clear evidence that DMA(V) is more toxic to the female urinary bladder, in accord with sensitivity to carcinogenesis. Important gender-related metabolic differences including enhanced presentation of MMA(III) to the urothelial cells might possibly account for heightened sensitivity in females. However, the potential carcinogenic effects of MMA(III) need to be further elucidated.     

Monomethylarsonous acid induces transformation of human bladder cells

Arsenic is a human bladder carcinogen. Arsenic is methylated to both monomethyl and dimethyl metabolites which have been detected in human urine. The trivalent methylated arsenicals are more toxic than inorganic arsenic. It is unknown if these trivalent methylated metabolites can directly cause malignant transformation in human cells. The goal of this study is determine if monomethylarsonous acid (MMA(III)) can induce malignant transformation in a human bladder urothelial cell line. To address this goal, a non-tumorigenic human urothelial cell line (UROtsa) was continuously exposed to 0.05 muM MMA(III) for 52 weeks. Hyperproliferation was the first phenotypic change observed in exposed UROtsa (URO-MSC). After 12 weeks of exposure, doubling time had decreased from 42 h in unexposed control cells to 27 h in URO-MSC. Hyperproliferation continued to be a quality possessed by the URO-MSC cells after both 24 and 52 weeks of exposure to MMA(III), which had a 40-50% reduction in doubling time. Throughout the 52-week exposure, URO-MSC cells retained an epithelial morphology with subtle morphological differences from control cells. 24 weeks of MMA(III) exposure was required to induce anchorage-independent growth as detected by colony formation in soft agar, a characteristic not found in UROtsa cells. To further substantiate that malignant transformation had occurred, URO-MSC cells were tested after 24 and 52 weeks of exposure to MMA(III) for the ability to form tumors in SCID mice. Enhanced tumorigenicity in SCID mouse xenografts was observed after 52 weeks of treatment with MMA(III). These observations are the first demonstration of MMA(III)-induced malignant transformation in a human bladder urothelial cell line and provide important evidence that MMA(III) may be carcinogenic in human tissues.     

A major human arsenic metabolite,dimethylarsinic acid,requires reduced glutathione to induce apoptosis

Inorganic arsenicals are important environmental toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenicals often undergo methylation, forming compounds such as dimethyarsinic acid (DMA). Recent evidence indicates DMA is a complete carcinogen in rodents while evidence for inorganic arsenicals as carcinogens in rodents remains equivocal. Thus, we studied the molecular mechanisms of in vitro cytolethality of DMA compared to that of the trivalent inorganic arsenical, sodium arsenite, using a rat liver epithelial cell line (TRL 1215). Arsenite was very cytotoxic in these cells (LC(50) = 35 microM after 48 h of exposure). With arsenite exposure, most dead cells showed histological and biochemical evidence of necrosis. Arsenite cytotoxicity increased markedly when cellular GSH was depleted with the glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO). In contrast, DMA was nearly 3 orders of magnitude less cytotoxic (LC(50) = 1.5 mM) although evidence showed the predominating form of death was apoptosis. Surprisingly, GSH depletion actually decreased DMA-induced apoptosis. A glutathione scavenger, diethyl maleate (DEM), and a glutathione reductase inhibitor, carmustine, also prevented DMA-induced apoptosis. These data indicate that DMA requires intracellular GSH to induce apoptosis. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyzes GSH-substrate conjugation, acivicin, an inhibitor of gamma-glutamyltranspeptidase (GGT) which catalyzes the initial breakdown of GSH-substrate conjugates, and aminooxyacetic acid (AOAA), an inhibitor of beta-lyase which catalyzes the final breakdown of GSH-substrate conjugates, all were effective in suppressing DMA-induced apoptosis. These findings indicate that DMA likely is conjugated in some form with GSH, and that it is this conjugate that induces apoptosis during subsequent metabolic reactions.     

Arsenite uptake and metabolism by rat hepatocyte primary cultures in comparison with kidney- and hepatocyte-derived rat cell lines.

Biotransformation by methylation to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) influences inorganic arsenical toxicity, which is often investigated in cultured cells. Arsenic (III) uptake and methylation was assessed in rat hepatocytes in primary culture and in three established rat cell lines (hepatoma-derived McA-RH 7777 cells and H4-II-EC-3 cells, and kidney epithelium-derived NRK-52E cells) to compare their use as model systems for arsenite metabolism. Incubation of all cell types with 0.27, 0.67, 1.33, 2.67, or 6.67 microM As(III) concentrations resulted in concentration-dependent arsenic uptake and biomethylation. Arsenic uptake by the NRK-52E cells was initially slower than that of the other cells, but by 8 h, total uptake was similar in all cell types. At the lowest arsenite concentration, the percentages of total arsenic methylated to MMA and DMA by the hepatocytes and the McA-RH 7777 cells were similar (67 and 66%); methylation by the H4-II-EC-3 cells was somewhat lower (52%), and methylation by the kidney-derived NRK-52E cells was much lower (15%). Total arsenic methylation was inhibited in the cell lines, but not in the hepatocytes, at the highest arsenite concentrations. In all cases, exposure to increased arsenite concentrations inhibited conversion of MMA to DMA much more than it affected the initial methylation step (inorganic arsenite to MMA). These results indicate that rat hepatocytes in primary culture and established rat hepatoma-derived cell lines are similar in their abilities to accumulate and methylate arsenic to MMA and DMA at environmentally relevant arsenic concentrations in the medium. They differed from the kidney epithelium-derived cells, which exhibited substantially lower biomethylation activity.     

Elevation of 8-hydroxydeoxyguanosine and cell proliferation via generation of oxidative stress by organic arsenicals contributes to their carcinogenicity in the rat liver and bladder

Monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)) and trimethylarsine oxide (TMAO(V)) are well-documented inorganic arsenic (iAs) methylated metabolites. In our previous studies, DMA(V) and TMAO(V) were shown to exert carcinogenicity in the rat bladder and liver, respectively. Furthermore, MMA(V), DMA(V) and TMAO(V) exhibited promoting activity on rat hepatocarcinogenesis. To clarify mechanisms of arsenical carcinogenicity and compare biological responses in the liver and bladder, male F344 rats were sequentially treated for 5, 10, 15, 20 days with MMA(V), DMA(V) and TMAO(V) in their drinking water at a dose of 0.02%. Significant increase of P450 total content and generation of hydroxyl radicals in the liver were observed from 10 and 15 days of treatment with arsenicals, respectively, with the highest levels induced by TMAO(V). Similarly, elevation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation was found in the DNA with significant increase by TMAO(V) treatment in the liver at days 15 and 20, and DMA(V) in the bladder after 20 days treatment. In addition, cell proliferation and apoptosis indices were significantly increased by TMAO(V) in the liver and by DMA(V) in the bladder of rats. These events were accompanied by differential up-regulation of phase I and II metabolizing enzymes, cyclins D1 and E, PCNA, caspase 3 and FasL. The results indicate that early elevation of 8-OHdG and cell proliferation via generation of oxidative stress by TMAO(V) and DMA(V) contributes to their carcinogenicity in the rat liver and bladder.     

Further evidence against a direct genotoxic mode of action for arsenic-induced cancer

Arsenic in drinking water, a mixture of arsenite and arsenate, is associated with increased skin and other cancers in Asia and Latin America, but not the United States. Arsenite alone in drinking water does not cause skin cancers in experimental animals; therefore, it is not a complete carcinogen in skin. We recently showed that low concentrations of arsenite enhanced the tumorigenicity of solar UV irradiation in hairless mice, suggesting arsenic cocarcinogenesis with sunlight in skin cancer and perhaps with different carcinogenic partners for lung and bladder tumors. Cocarcinogenic mechanisms could include blocking DNA repair, stimulating angiogenesis, altering DNA methylation patterns, dysregulating cell cycle control, induction of aneuploidy and blocking apoptosis. Arsenicals are documented clastogens but not strong mutagens, with weak mutagenic activity reported at highly toxic concentrations of inorganic arsenic. Previously, we showed that arsenite, but not monomethylarsonous acid (MMA[III]), induced delayed mutagenesis in HOS cells. Here, we report new data on the mutagenicity of the trivalent methylated arsenic metabolites MMA(III) and dimethylarsinous acid [DMA(III)] at the gpt locus in Chinese hamster G12 cells. Both methylated arsenicals seemed mutagenic with apparent sublinear dose responses. However, significant mutagenesis occurred only at highly toxic concentrations of MMA(III). Most mutants induced by MMA(III) and DMA(III) exhibited transgene deletions. Some non-deletion mutants exhibited altered DNA methylation. A critical discussion of cell survival leads us to conclude that clastogenesis occurs primarily at highly cytotoxic arsenic concentrations, casting further doubt as to whether a genotoxic mode of action (MOA) for arsenicals is supportable.     

Studies on the mechanisms of arsenic-induced self tolerance developed in liver epithelial cells through continuous low-level arsenite exposure.

Arsenic (As) is a human carcinogen. Our prior work showed that chronic (>18 weeks) low level (500 nM) arsenite (As3+) exposure induced malignant transformation in a rat liver epithelial cell line (TRL 1215). In these cells, metallothionein (MT) is hyper-expressible, a trait often linked to metal tolerance. Thus, this study examined whether the adverse effects of arsenicals and other metals were altered in these chronic arsenite-exposed (CAsE) cells. CAsE cells, which had been continuously exposed to 500 nM arsenite for 18 to 20 weeks, and control cells, were exposed to As3+, arsenate (As5+), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), antimony (Sb3+), cadmium (Cd2+), cisplatin (cis-Pt), and nickel (Ni2+) for 24 h and cell viability was determined by metabolic integrity. The lethal concentration for 50% of exposed cells (LC50) for As3+ was 140 microM in CAsE cells as compared to 26 microM in control cells, a 5.4-fold increase in tolerance. CAsE cells were also very tolerant to the acute toxic effects of As5+ (LC50 > 4000 microM) compared to control (LC50 = 180 microM). The LC50 for DMA was 4.4-fold higher in CAsE cells than in control cells, but the LC50 for MMA was unchanged. There was a modest cross-tolerance to Sb3+, Cd2+, and cis-Pt in CAsE cells (LC50 1.5-2.0-fold higher) as compared to control. CAsE cells were very tolerant to Ni2+ (LC50 > 8-fold higher). Culturing CAsE cells in As(3+)-free medium for 5 weeks did not alter As3+ tolerance, implicating an irreversible phenotypic change. Cellular accumulation of As was 87% less in CAsE cells than control and the accumulated As was more readily eliminated. Although accumulating much less As, a greater portion was converted to DMA in CAsE cells. Altered glutathione (GSH) levels were not linked with As tolerance. A maximal induction of MT by Zn produced only a 2.5-fold increase in tolerance to As3+ in control cells. Cell lines derived from MT normal mice (MT+/+) were only slightly more resistant (1.6-fold) to As3+ than cells from MT null mice (MT-/-). These results show that CAsE cells acquire tolerance to As3+, As5+, and DMA. It appears that this self-tolerance is based primarily on reduced cellular disposition of the metalloid and is not accounted for by changes in GSH or MT.     

Metabolic differences between two dimethylthioarsenicals in rats

Thioarsenicals are newly found arsenic metabolites in man and animals, and also in marine organisms. Dimethylmonothioarsinic acid (DMMTA(V)) and dimethyldithioarsinic acid (DMDTA(V)) are the only two thioarsenic metabolites detected in man and/or animals. However, their toxicological and biological significance is not known yet. The present study was performed to gain an insight into the significance of DMMTA(V) and DMDTA(V) in the metabolism of arsenic. The two thioarsenicals were synthesized chemically and injected intravenously into rats at the dose of 0.5 mg As/kg body weight. The distributions of arsenic in organs/tissues and body fluids were determined at 10 min and 12 h after the injection, and arsenic in liver and kidney supernatants, urine, plasma and red blood cell (RBC) lysates was subjected to speciation analysis by HPLC-ICP MS on a gel filtration GS 220 HQ column. Although both thioarsenicals are pentavalent arsenicals, they were distributed in organs/tissues and body fluids differently from the corresponding non-thiolated pentavalent arsenicals, and also from each other. Namely, DMMTA(V) was first found in organs/tissues at 10 min, and then redistributed and retained mostly in RBCs at 12 h, as in the case of trivalent dimethylarsinous acid (DMA(III)). On the other hand, although DMDTA(V) was also found in organs/tissues at 10 min, it had been efficiently excreted in urine in its intact form at 12 h. Thus, DMMTA(V) was unexpectedly distributed in and taken up by organs/tissues in a manner similar to DMA(III) rather than DMA(V), whereas DMDTA(V) was distributed similarly to DMA(V) as expected, but was much more efficiently excreted in urine.