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Intracellular, time-resolved speciation and quantification of arsenic compounds in human urothelial and hepatoma cells

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Abstract

In contrast to the temporary administration of arsenic in cancer therapy (e.g. in the form of arsenic trioxide), chronic exposure to low doses can cause bladder cancer and other cancers. Especially the trivalent arsenic species MMA(III) (monomethylarsonous acid) and DMA(III) (dimethylarsinous acid) are known to be highly toxic. In the present study we analysed the soluble, intracellular biotransformation products of MMA(III) in methylating HepG2 (hepatocytes) and non-methylating UROtsa cells (urothelial cells) after various times of exposure. As most of the intracellulararsenic is bound to cellular structures and proteins the soluble arsenicmetabolites can hardly be speciated and even less quantified. Using an improved isolation procedure and HPLC-ICP/MS, we investigated the time-resolved biotransformation of MMA(III) and detected and quantified MMA(V) (monomethylarsonic acid) as an oxidation product of MMA(III) and, to a minor degree, DMA(V) (dimethylarsenic acid) as a methylation and oxidation product of MMA(III) in the lysates of HepG2 cells. In contrast, only MMA(V) but no DMA(V) was detected in the lysates of UROtsa cells. We conclude from our study that MMA(III) is taken up by HepG2 and UROtsa cells and immediately oxidized to MMA(V). Only in HepG2 cellsMMA(V) is finally methylated to DMA(V) over time. The new method might help to advance the analysis of metabolic pathways of arsenic in mammalian cells.

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... 40 However, pentavalent arsenic species cannot be considered completely nontoxic, as they may interfere with the phosphate uptake and transport phenomena of the body by acting as a phosphate analog, 22 furthermore it has also been suggested that arsenate (iAs V ) and phosphate can share the same transportation process, as addition of phosphate can reduce the absorption of arsenic. 41 The mechanisms by which arsenic causes toxicities including cancer are still undefined, [42][43][44] as there is much complexity in inducing toxicity within animal models using the same arsenic concentrations that can be deleterious to humans, making it much more difficult to investigate the exact mode of arsenic toxicity. 42,43,45,46 However, it is reported that arsenic may act as an efficient co-carcinogen in experimental rodents by promoting the effects of other carcinogens. ...
... Furthermore, most of the administered arsenic (approximately 70-80% of dose) was recovered in urine and feces at 12 h or 24 h after dosing, in an unmodified form. Hippler et al. 44 also portrayed a precise mechanism of MMA III metabolic pathway using the Hepatoma cell line (HepG2), signifying that when MMA III was exposed to HepG2, it was rapidly taken up by the cells and followed the binding to cellular proteins, and the liberated MMA V and DMA V were excreted. However, some of these pentavalent species may further undergo reduction by GSH instead of being excreted. ...
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... The following methodology (Hippler et al., 2011) was used for intracellular arsenic speciation and quantification: Cells were seeded into 150 cm² flasks and grown to confluence before experiments were performed. Both cell lines (UROtsa and HepG2) were incubated for five minutes to 24 hours in fresh growth medium containing 5 µM MMA(III) (exposure medium). ...
... To study the intracellular arsenic biotransformation of MMA(III) we incubated UROtsa and HepG2 cells with 5 µM MMA(III) for 5 min up to 24 hours, followed by a newly developed sample preparation process (Hippler et al., 2011). Using HPLC-ICP/MS analysis we were able to detect more than 99.99% of the total arsenic in the non-soluble fraction of both cell lines and only 0.003% in the soluble fraction of UROtsa cells and 0.01% of HepG2 cells, respectively. ...
... The reducing intracellular environment might suggest that DMA III is the predominant form of dimethylated arsenic within the cell; however, this has been difficult to prove and DMA V has been detected in human cell lines and mouse liver homogenate (Currier et al., 2011). The highly reactive DMA III is highly protein bound and unlikely to be available for cellular export (Hippler et al., 2011;Shen et al., 2013). An equilibrium between DMA III and DMA V will exist within the cell, and the highaffinity high-capacity export of DMA V by MRP1 would provide a good mechanism for cellular detoxification. ...
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... For better comparability, results are reported in ng Se taken up per 10 6 cells normalized to μM of initially applied selenium. The medium was removed after 24 h and the cells were successively rinsed with Dulbecco's Phosphate Buffered Saline (DPBS, GIBCO), Ampuwa, and 0.1 mM 2,3-dimercapto-1-propanesulfonic acid sodium salt monohydrate (DMPS, Alfa Aesar, Karlsruhe, Germany, purity 95 %) according to a published procedure (Hippler et al. 2011) to ensure the absence of extracellular selenium. Cells were trypsinized, collected in 2 mL DPBS, and mechanically lysed using glass beads (Retsch, Haan, Germany). ...
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... Despite the highly reducing environment of the cell, DMA V is detected in murine liver homogenates and human cell lines analyzed with oxidation state-specific hydride generationcryotrapping-atomic absorption spectroscopy (Currier et al., 2011). Furthermore, the highly reactive nature of DMA III combined with the lack of evidence for physiologic formation of dimethylarsenic glutathione [DMA(GS)] (Leslie, 2012) mean that DMA III is highly bound to protein and not available for transport (Hippler et al., 2011;Shen et al., 2013). MRP4 transport of DMA V was with high apparent affinity (K 0.5 , 0.22 mM) and would allow for the efficient efflux of DMA V at low cellular concentrations. ...
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... After 24 h of exposure, the medium was collected. To ensure complete removal of extracellular arsenic, the cell layer was rinsed four times using DPBS, Ampuwa, 0.1 mM DMPS, and DPBS according to a published procedure (Hippler et al. 2011). Subsequently, the cells were trypsinized (trypsin-EDTA was used for HepG2 cells, trypsin was used for UROtsa cells), collected in 2 mL DPBS, counted with the CASY Model TT (Roche Applied Sciences), and mechanically lysed using glass beads (Retsch GmbH, Germany). ...
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4-hydroxynonenal (HNE), a highly reactive lipid peroxidation product, may adversely modify proteins. Accumulation of HNE-modified proteins may be responsible for pathological lesions associated with oxidative stress. The objective of this work was to determine how HNE-modified proteins are removed from cells. The data showed that alphaB-crystallin modified by HNE was ubiquitinated at a faster rate than that of native alphaB-crystallin in a cell-free system. However, its susceptibility to proteasome-dependent degradation in the cell-free system did not increase. When delivered into cultured lens epithelial cells, HNE-modified alphaB-crystallin was degraded at a faster rate than that of unmodified alphaB-crystallin. Inhibition of the lysosomal activity stabilized HNE-modified alphaB-crystallin, but inhibition of the proteasome activity alone had little effect. To determine if other HNE-modified proteins are also degraded in a ubiquitin-dependent lysosomal pathway, lens epithelial cells were treated with HNE and the removal of HNE-modified proteins in the cells was monitored. The levels of HNE-modified proteins in the cell decreased rapidly upon removal of HNE from the medium. Depletion of ATP or the presence of MG132, a proteasome/lysosome inhibitor, resulted in stabilization of HNE-modified proteins. However, proteasome-specific inhibitors, lactacystin-beta-lactone and epoxomicin, could not stabilize HNE-modified proteins in the cells. In contrast, chloroquine, a lysosome inhibitor, stabilized HNE-modified proteins. The enrichment of HNE-modified proteins in the fraction of ubiquitin conjugates suggests that HNE-modified proteins are preferentially ubiquitinated. Taken together, these findings show that HNE-modified proteins are degraded via a novel ubiquitin and lysosomal-dependent but proteasome-independent pathway.
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Arsenic is a prominent environmental toxicant and carcinogen; however, its molecular mechanism of toxicity and carcinogenicity remains poorly understood. In this study, we performed microarray-based expression profiling on liver of zebrafish exposed to 15 parts/million (ppm) arsenic [As(V)] for 8-96 h to identify global transcriptional changes and biological networks involved in arsenic-induced adaptive responses in vivo. We found that there was an increase of transcriptional activity associated with metabolism, especially for biosyntheses, membrane transporter activities, cytoplasm, and endoplasmic reticulum in the 96 h of arsenic treatment, while transcriptional programs for proteins in catabolism, energy derivation, and stress response remained active throughout the arsenic treatment. Many differentially expressed genes encoding proteins involved in heat shock proteins, DNA damage/repair, antioxidant activity, hypoxia induction, iron homeostasis, arsenic metabolism, and ubiquitin-dependent protein degradation were identified, suggesting strongly that DNA and protein damage as a result of arsenic metabolism and oxidative stress caused major cellular injury. These findings were comparable with those reported in mammalian systems, suggesting that the zebrafish liver coupled with the available microarray technology present an excellent in vivo toxicogenomic model for investigating arsenic toxicity. We proposed an in vivo, acute arsenic-induced adaptive response model of the zebrafish liver illustrating the relevance of many transcriptional activities that provide both global and specific information of a coordinated adaptive response to arsenic in the liver.
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Agonist-stimulated β2-adrenergic receptor (β2AR) ubiquitination is a major factor that governs both lysosomal trafficking and degradation of internalized receptors, but the identity of the E3 ubiquitin ligase regulating this process was unknown. Among the various catalytically inactive E3 ubiquitin ligase mutants that we tested, a dominant negative Nedd4 specifically inhibited isoproterenol-induced ubiquitination and degradation of the β2AR in HEK-293 cells. Moreover, siRNA that down-regulates Nedd4 expression inhibited β2AR ubiquitination and lysosomal degradation, whereas siRNA targeting the closely related E3 ligases Nedd4-2 or AIP4 did not. Interestingly, β2AR as well as β-arrestin2, the endocytic and signaling adaptor for the β2AR, interact robustly with Nedd4 upon agonist stimulation. However, β2AR-Nedd4 interaction is ablated when β-arrestin2 expression is knocked down by siRNA transfection, implicating an essential E3 ubiquitin ligase adaptor role for β-arrestin2 in mediating β2AR ubiquitination. Notably, β-arrestin2 interacts with two different E3 ubiquitin ligases, namely, Mdm2 and Nedd4 to regulate distinct steps in β2AR trafficking. Collectively, our findings indicate that the degradative fate of the β2AR in the lysosomal compartments is dependent upon β-arrestin2-mediated recruitment of Nedd4 to the activated receptor and Nedd4-catalyzed ubiquitination.
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This review deals with environmental origin, occurrence, episodes, and impact on human health of arsenic. Arsenic, a metalloid occurs naturally, being the 20th most abundant element in the earth's crust, and is a component of more than 245 minerals. These are mostly ores containing sulfide, along with copper, nickel, lead, cobalt, or other metals. Arsenic and its compounds are mobile in the environment. Weathering of rocks converts arsenic sulfides to arsenic trioxide, which enters the arsenic cycle as dust or by dissolution in rain, rivers, or groundwater. So, groundwater contamination by arsenic is a serious threat to mankind all over the world. It can also enter food chain causing wide spread distribution throughout the plant and animal kingdoms. However, fish, fruits, and vegetables primarily contain organic arsenic, less than 10% of the arsenic in these foods exists in the inorganic form, although the arsenic content of many foods (i.e. milk and dairy products, beef and pork, poultry, and cereals) is mainly inorganic, typically 65-75%. A few recent studies report 85-95% inorganic arsenic in rice and vegetables, which suggest more studies for standardisation. Humans are exposed to this toxic arsenic primarily from air, food, and water. Thousands and thousands of people are suffering from the toxic effects of arsenicals in many countries all over the world due to natural groundwater contamination as well as industrial effluent and drainage problems. Arsenic, being a normal component of human body is transported by the blood to different organs in the body, mainly in the form of MMA after ingestion. It causes a variety of adverse health effects to humans after acute and chronic exposures such as dermal changes (pigmentation, hyperkeratoses, and ulceration), respiratory, pulmonary, cardiovascular, gastrointestinal, hematological, hepatic, renal, neurological, developmental, reproductive, immunologic, genotoxic, mutagenetic, and carcinogenic effects. Key research studies are needed for improving arsenic risk assessment at low exposure levels urgently among all the arsenic research groups.
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The presence of arsenic in marine samples was first reported over 100 years ago, and shortly thereafter it was shown that common seafood such as fish, crustaceans, and molluscs contained arsenic at exceedingly high concentrations. It was noted at the time that this seafood arsenic was probably present as an organically bound species because the concentrations were so high that if the arsenic had been present as an inorganic species it would certainly have been toxic to the humans consuming seafood. Investigations in the late 1970s identified the major form of seafood arsenic as arsenobetaine [(CH(3))(3)As(+)CH(2)COO(-)], a harmless organoarsenic compound which, following ingestion by humans, is rapidly excreted in the urine. Since that work, however, over 50 additional arsenic species have been identified in marine organisms, including many important food products. For most of these arsenic compounds, the human toxicology remains unknown. The current status of arsenic in seafood will be discussed in terms of the possible origin of these compounds and the implications of their presence in our foods.
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The major route of human exposure to arsenic is via ingestion. Seafoods contain large amounts of various arsenic compounds. Recently, people have been advised not to eat Hijiki seaweed (Hijikia fusiforme) in the UK because of its high content of inorganic arsenic (iAs). To clarify the risks of Hijiki ingestion, a 42-year-old male volunteer ingested 825 µg of iAs compounds contained in eight servings of commercial Hijiki food, after refraining from eating seafood for 3 months. In order to determine the distribution of arsenic species in his urine, arsenic compounds were analyzed using HPLC-ICP-MS. The maximum concentrations of arsenate (AsV), arsenite (AsIII), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) were found at 4, 6.5, 13 and 17.5 h after ingestion, respectively. Arsenobetaine concentration was very low, and almost constant throughout the observation period. A total of 28% of ingested arsenic was excreted in urine. The total amounts of AsV, AsIII, MMA and DMA excreted in urine over 50 h were 11.2, 31.8, 40.9 and 104.0 µg, respectively. After eating one serving of Hijiki, arsenic intake and urinary excretion were at levels similar to those in individuals affected by arsenic poisoning. Long-term ingestion of Hijiki might thus have the potential to cause arsenic poisoning. Copyright © 2006 John Wiley & Sons, Ltd.
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The growing importance of arsenic speciation analysis has led to the development of a wide range of high-performance liquid chromatographic based hyphenated techniques. During the present study a method was developed for the high-speed separation of several biologically and environmentally important arsenic compounds. The method is based on the use of an octadecyldimethylsilyl reversed-phase narrow-bore HPLC column. Separation of anionic arsenic species [arsenite (Aite), dimethylarsinic acid (DMAA), monomethylarsonic acid (MMAA), and arsenate (Aate)] can be achieved using a mobile phase containing 5 mM tetrabutylammonium hydroxide as the ion-pairing reagent, at pH 6.0, in less than 2 min, when employing a ¯ow rate of 0.7 ml min 21 . Adding 4-hydroxyphenylarsonic acid as the internal standard prolongs the total separation time by 30 s. On-line coupling with inductively coupled plasma mass spectrometry affords high sensitivity, as well as low limits of detection (low ppb or pg of arsenic). The in¯uence of mobile phase pH and ion-pairing reagent concentration on the separation ef®ciency was studied. A loss of resolution occurs with increasing ion-pairing reagent concentration; the optimum pH is between 6.0 and 6.2. The ion-pair reversed-phase narrow-bore HPLC-ICP-MS method was subsequently applied to the speciation of arsenic in wine and kelp samples. Aite at trace levels was found to be the only arsenic species present in several wines. Average spike recoveries for Aite, Aate, MMAA and DMAA were 95¡3, 94¡5, 98¡1 and 92¡1%, respectively, for all wines examined. The method was also used for the speciation of four arsenosugars and DMAA in a kelp powder extract.
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The dynamics of interactions between rat liver cytosolic proteins and arsenicals were examined in anin vitromethylation system that contained cytosol, glutathione,S-adenosylmethionine, and 1 μm[73As]arsenite. After incubation at 37°C for up to 90 min, low-molecular-weight components of the assay system (<10 kDa) were removed by ultrafiltration and cytosolic proteins were separated by size-exclusion chromatography on Sephacryl S-300 gel. Five73As-labeled protein peaks were found in chromatographic profiles. The estimated molecular masses of73As-labeled proteins eluting in the three earliest peaks were as follows:Vo, ≥1000 kDa; A, 135 kDa; and B, 38 kDa. Peak C eluted immediately before the total volume (VT) of the chromatographic column; peak D eluted after theVT.73As bound to proteins was released by CuCl treatment and speciated by thin-layer chromatography. Amounts and ratios of inorganic As, methyl As, and dimethyl As associated with cytosolic proteins depended upon the incubation interval. Inorganic As was present in all protein peaks. Methyl As was primarily associated with peaks A and C; dimethyl As was associated with peaks B and C. To examine the effect of valence on the binding of methylarsenicals to cytosolic proteins, trivalent or pentavalent14C-labeled methyl As or dimethyl As was incubated in anin vitrosystem designed to minimize the enzymatically catalyzed production of methylated arsenicals. Proteins in peaks A, B, and C bound preferentially trivalent methyl and dimethyl As. Peak D bound either trivalent or pentavalent methyl and dimethyl As. Protein-bound inorganic and methyl As were substrates for the production of dimethyl As in anin vitromethylation system, suggesting a role for protein-bound arsenicals in the biomethylation of this metalloid.
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Ubiquitin is a common demoninator in the targeting of substrates to all three major protein degradation pathways in mammalian cells: the proteasome, the lysosome, and the autophagosome. The factors that direct a substrate toward a particular route of degradation likely include ubiquitin chain length and linkage type, which may favor interaction with particular receptors or confer differential susceptibility to deubiquitinase activities associated with each pathway.
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While the metal(loid)s arsenic, bismuth, and selenium (probably also tellurium) have been shown to be enzymatically methylated in the human body, this has not yet been demonstrated for antimony, cadmium, germanium, indium, lead, mercury, thallium, and tin, although the latter elements can be biomethylated in the environment. Methylated metal(loid)s exhibit increased mobility, thus leading to a more efficient metal(loid) transport within the body and, in particular, opening chances for passing membrane barriers (blood-brain barrier, placental barrier). As a consequence human health may be affected. In this review, relevant data from the literature are compiled, and are discussed with respect to the evaluation of assumed and proven health effects caused by alkylated metal(loid) species.
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Arsenobetaine has always been referred to as a non-toxic but readily bioavailable compound and the available data would suggest that it is neither metabolised by nor accumulated in humans. Here this study investigates the urine of five volunteers on an arsenobetaine exclusive diet for twelve days and shows that arsenobetaine was consistently excreted by three of the five volunteers. From the expected elimination pattern of arsenobetaine in rodents, no significant amount of arsenobetaine should have been detectable after 5 days of the trial period. The arsenobetaine concentration found in the urine was constant after 5 days and varied between 0.2 and 12.2 microg As per L for three of the volunteers. Contrary to the established belief that arsenobetaine is neither accumulated nor generated by humans, the presented results would suggest that either accumulated arsenobetaine in the tissues is slowly released over time or that arsenobetaine is a human metabolite of dimethylarsinic acid or inorganic arsenic from the trial food, or both. Either possibility is intriguing and raises fundamental questions about human arsenic metabolism and the toxicological and environmental inertness of arsenobetaine.
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Exposure to high levels of arsenic can cause a wide range of health effects, including cancers of the bladder, lung, skin, and kidney. However, the mechanism(s) of action underlying these deleterious effects of arsenic remains unclear. Arsenic binding to cellular proteins is a possible mechanism of toxicity, and identifying such binding is analytically challenging because of the large concentration range and variety of proteins. We describe here an affinity selection technique, coupled with mass spectrometry, to select and identify specific arsenic-binding proteins from a large pool of cellular proteins. Controlled experiments using proteins either containing free cysteine(s) or having cysteine blocked showed that the arsenic affinity column specifically captured the proteins containing free cysteine(s) available to bind to arsenic. The technique was able to capture and identify trace amounts of bovine biliverdin reductase B present as a minor impurity in the commercial preparation of carbonic anhydrase II, demonstrating the ability to identify arsenic-binding proteins in the presence of a large excess of non-specific proteins. Application of the technique to the analysis of subcellular fractions of A549 human lung carcinoma cells identified 50 proteins in the nuclear fraction, and 24 proteins in the membrane/organelle fraction that could bind to arsenic, adding to the current list of only a few known arsenic-binding proteins.
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Glutathione (GSH; gamma-glutamylcysteinylglycine) is ubiquitous in mammalian and other living cells. It has several important functions, including protection against oxidative stress. It is synthesized from its constituent amino acids by the consecutive actions of gamma-glutamylcysteine synthetase and GSH synthetase. gamma-Glutamylcysteine synthetase activity is modulated by its light subunit and by feedback inhibition of the end product, GSH. Treatment with an inhibitor, buthionine sulfoximine (BSO), of gamma-glutamylcysteine synthetase leads to decreased cellular GSH levels, and its application can provide a useful experimental model of GSH deficiency. Cellular levels of GSH may be increased by supplying substrates and GSH delivery compounds. Increasing cellular GSH may be therapeutically useful.
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Biomethylation is the major human metabolic pathway for inorganic arsenic, and the speciation of arsenic metabolites is essential to a better understanding of arsenic metabolism and health effects. Here we describe a technique for the speciation of arsenic in human urine and demonstrate its application to the discovery of key arsenic metabolic intermediates, monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), in human urine. The study provides a direct evidence in support of the proposed arsenic methylation pathway in the human. The finding of MMAIII and DMAIII in human urine, along with recent studies showing the high toxicity of these arsenicals, suggests that the usual belief of arsenic detoxification by methylation needs to be reconsidered. The arsenic speciation technique is based on ion pair chromatographic separation of arsenic species on a 3-micron particle size column at 50 degrees C followed by hydride generation atomic fluorescence detection. Speciation of MMAIII, DMAIII, arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV) in urine samples is complete in 6 min with detection limits of 0.5-2 micrograms/L. There is no need for any sample pretreatment. The capability of rapid analysis of trace levels of arsenic species, which resulted in the findings of the key metabolic intermediates, makes the technique useful for routine arsenic speciation analysis required for toxicological and epidemiological studies.
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Although arsenic can be poisonous, and chronic arsenic exposure from industrial or natural sources can cause serious toxicity, arsenic has been used therapeutically for more than 2,400 years. Thomas Fowler's potassium bicarbonate-based solution of arsenic trioxide (As(2)O(3)) was used empirically to treat a variety of disorders, and in 1878, was reported to reduce white blood cell counts in two normal individuals and one with "leucocythemia." Salvarsan, an organic arsenical for treating syphilis and trypanosomiasis, was developed in 1910 by Paul EHRLICH: In the 1930s, arsenic was reported to be effective in chronic myelogenous leukemia. After a decline in the use of arsenic during the mid-20th century, reports from China described a high proportion of hematologic responses in patients with acute promyelocytic leukemia (APL) who were treated with arsenic trioxide. Randomized clinical trials in the U.S. led to FDA approval of arsenic trioxide for relapsed or refractory APL in September 2000.
Article
Chronic arsenic exposure increases risk for the development of diabetes, vascular disease, and cancers of the skin, lung, kidney, and bladder. This study investigates the effects of arsenite [As(III)] on human urothelial cells (UROtsa). As(III) toxicity was determined by exposing confluent UROtsa cells to As(III) (0.5-200 microM). Depleting cellular glutathione levels with buthionine sulfoximine (BSO) potentiated the toxicity of As(III). Cell viability was assessed with the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. UROtsa cell ability to biotransform As(III) was determined by dosing cells with environmentally relevant concentrations of As(III) followed by HPLC/ICP-MS analysis of cell media and lysate. Both pentavalent and trivalent monomethylated products were detected. Although cytotoxicity was observed at high doses of As(III) (approximately 100 microM) in UROtsa cells, perturbations of a variety of molecular processes occurred at much lower doses. Exposure to low-level As(III) (0.5-25 microM) causes an accumulation of ubiquitin (Ub)-conjugated proteins. This effect is enhanced when cellular glutathione levels have been reduced with BSO treatment. Because As(III) has many effects on UROtsa cells, a greater understanding of how As(III) is affecting cellular proteins in a target tissue will lead to a better understanding of the mechanism of toxicity and pathogenesis for low-level As(III).
Article
Inorganic arsenic is converted to methylated metabolites, and most is excreted in urine as dimethylarsinic acid in humans and animals. The present study was conducted to investigate the metabolism of arsenic and identify hepatic and renal metabolites of arsenic after an intravenous injection of arsenite (0.5 mg As/kg body weight) in rats. Similar levels of arsenic were found in the soluble (SUP) and nonsoluble sediment (SED) fractions of both organs after 1 h. More than 80% of the SUP arsenic was bound to high molecular weight (HMW) proteins in both organs. Arsenic bound to the HMW and SED proteins were oxidized with H(2)O(2) and released in the pentavalent forms (arsenate, monomethylarsonic, and dimethylarsinic acids). The relative ratios of the three arsenicals changed depending on organ, fraction (HMW and SED), and time. Since the arsenic metabolites/intermediates were liberated from proteins by oxidation with H(2)O(2) and recovered in the pentavalent forms, and only tri- but not pentavalent arsenicals were bound to proteins in vitro, it was deduced that arsenic metabolites bound to proteins during the successive methylation pathway are in the trivalent forms; that is, successive methylation reaction takes place with simultaneous reductive rather than stepwise oxidative methylation. Thus, on the basis of the present observations, it was proposed that inorganic arsenic was successively methylated reductively in the presence of glutathione, rather than a stepwise oxidative methylation, and pentavalent arsenicals (MMA(V) and DMA(V)) were present as end products of metabolism, rather than intermediates. We also discussed the in vitro formation of dimethylthioarsenicals after incubating dimethylarsinous acid with liver homogenate.
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The lysosomal compartment is the place for cellular degradation of endocytosed and autophagocytosed material and a center for normal turnover of organelles as well as most long-lived proteins. Lysosomes were long considered stable structures that broke and released their many hydrolytic enzymes only following necrotic cell death. It is now realized that lysosomes instead are quite vulnerable, although in a heterogeneous way. Their exposure to a number of events, such as oxidative stress, lysosomotropic detergents and aldhydes, as well as overexpression of the p53 protein, causes time-and-dose-dependent lysosomal rupture that is followed by apoptosis or necrosis. Partial lysosomal rupture has often been found to be an early upstream event in apoptosis, while necrosis results from fulminant lysosomal rupture. Consequently, factors influencing the stability of lysosomes, for instance their content of labile and redox-active iron, seem to be essential for the survival of cells.
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Cells from patients with t(15;17) acute promyelocytic leukemia (APL) express the fusion protein between the promyelocytic leukemia protein and retinoic acid receptor alpha (PML/RAR alpha). Patients with APL respond to differentiation therapy with all-trans-retinoic acid, which induces PML/RAR alpha degradation. When resistance to all-trans-retinoic acid develops, an effective treatment is arsenic trioxide (arsenite), which also induces this degradation. We investigated the mechanism of arsenite-induced PML/RAR alpha degradation. NB4-S1 APL cells were treated with clinically relevant concentrations of arsenite. Lysosomes were visualized with a lysosome-specific dye. Lysosomal protein esterase was measured by immunoblot analysis. Lysosomal cathepsin L was detected by immunogold labeling and transmission electron microscopy, and its activity was measured in cytosolic cellular fractions. In vitro degradation assays of PML/RAR alpha in cell lysates were performed with and without protease inhibitors and assessed by immunoblot analysis. Only nonparametric two-sided statistical analyses were used. The nonparametric Wilcoxon test was used for group comparison, and the nonlinear regression technique was used for analysis of dose-response relationship as a function of arsenite concentration. Arsenite treatment destabilized lysosomes in APL cells. Lysosomal proteases, including cathepsin L, were released from lysosomes 5 minutes to 6 hours after arsenite treatment. PML/RAR alpha was degraded by lysate from arsenite-treated APL cells, and the degradation was inhibited by protease inhibitors. At both 6 and 24 hours, substantially fewer arsenite-treated APL cells, than untreated cells, contained cathepsin L clusters, a reflection of cathepsin L delocalization. Cells with cathepsin L clusters decreased as a function of arsenite concentration at rates of -2.03% (95% confidence interval [CI] = -4.01 to -.045; P = .045) and -2.39% (95% CI = -4.54 to -.024; P = .029) in 6- and 24-hour treatment groups, respectively, per 1.0 microM increase in arsenite concentration. Statistically significantly higher cytosolic cathepsin L activity was detected in lysates of arsenite-treated APL cells than in control lysates. For example, the mean increase in cathepsin activity at 6 hours and 1.0 microM arsenite was 26.3% (95% CI = 3.3% to 33%; P < .001), compared with untreated cells. In APL cells, arsenite may cause rapid destabilization of lysosomes.
Article
Traditional Chinese medicines (TCMs) often contain significant levels of potentially toxic elements, including arsenic. Niu Huang Jie Du Pian pills were analyzed to determine the concentration, bioaccessibility (arsenic fraction soluble in the human gastrointestinal system) and chemical form (speciation) of arsenic. Arsenic excretion in urine (including speciation) and facial hair were studied after a one-time ingestion. The pills contained arsenic in the form of realgar, and although the total arsenic that was present in a single pill was high (28 mg), the low bioaccessibility of this form of arsenic predicted that only 4% of it was available for absorption into the bloodstream (1 mg of arsenic per pill). The species of arsenic that were solubilized were inorganic arsenate (As(V)) and arsenite (As(III)) but DMAA and MMAA were detected in urine. Two urinary arsenic excretion peaks were observed: an initial peak several (4-8) hours after ingestion corresponding to the excretion of predominantly As(III), and a larger peak at 14 h corresponding predominantly to DMAA and MMAA. No methylated As(III) species were observed. Facial hair analysis revealed that arsenic concentrations did not increase significantly as a result of the ingestion. Arsenic is incompletely soluble under human gastrointestinal conditions, and is metabolized from the inorganic to organic forms found in urine. Bioaccessible arsenic is comparable to the quantity excreted. Facial hair as a bio-indicator should be further tested.
Article
The tissue distribution and chemical forms of arsenic were compared in two animal species with different metabolic capacity and toxicity to arsenic. Hamsters and rats were given a single oral dose of arsenite (iAsIII) at 5.0 mg As/kg body weight, and then the concentrations of arsenic were determined; more than 75% of the dose accumulated in rat red blood cells (RBCs) in the form of dimethylarsinous acid (DMAIII), whereas less than 0.8% of the dose accumulated in hamster RBCs, mostly in the form of monomethylarsonous acid (MMAIII). Reflecting the low accumulation in RBCs, more than 63% of the dose was recovered in hamster urine within one week (7.8-fold higher than that in rat urine). The quantity of arsenic distributed in the liver and kidneys was significantly higher in hamsters than in rats, and arsenic in livers stayed much longer in hamsters than in rats. Arsenic accumulated more and was retained longer in the kidneys than in the livers in both animals, and in hamster kidneys, it accumulated at levels higher than those in rat kidneys in the form of MMAIII bound to proteins. In the first 24 h urine, dimethylmonothioarsinic (DMMTAV) and dimethyldithioarsinic (DMDTAV) acids were detected in hamsters, but only DMMTAV was found in rats, together with an unknown arsenic metabolite in both animals. The unknown urinary arsenic metabolite was identified as monomethylmonothioarsonic acid (MMMTAV; CH3As(=S)(OH)2). The present results indicate that in hamsters, arsenic does not accumulate in RBCs, and therefore, hamsters exhibit a more uniform tissue distribution and faster urinary excretion of arsenic than rats. In addition, arsenic was thiolated more in hamsters than in rats excreting mono and dimethylated thioarsenicals in urine.
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Autophagy is a process by which cytoplasmic components are sequestered in double membrane vesicles and degraded upon fusion with lysosomal compartments. In yeast, autophagy is activated in response to changes in the extracellular milieu. Depending upon the stimulus, autophagy can degrade cytoplasmic contents nonspecifically or can target the degradation of specific cellular components. Both of these have been adopted in higher eukaryotes and account for the expanding role of autophagy in various cellular processes, as well as contribute to the variation in cellular outcomes after induction of autophagy. In some cases, autophagy appears to be an adaptive response, whereas under other circumstances it is involved in cell death. In mammals, autophagy has been implicated in either the pathogenesis or response to a wide variety of diseases, including neurodegenerative disease, chronic bacterial and viral infections, atherosclerosis, and cancer. As the basic molecular pathways that regulate autophagy are elucidated, the relationship of autophagy to the pathogenesis of various disease states emerges.
Article
The bladder and skin are the primary targets for arsenic-induced carcinogenicity in mammals. Thioarsenicals dimethylmonothioarsinic (DMMTA(V)) and dimethyldithioarsinic (DMDTA(V)) acids are common urinary metabolites, the former being much more toxic than non-thiolated dimethylarsinic acid (DMA(V)) and comparable to dimethylarsinous acid (DMAIII) in epidermoid cells, suggesting that the metabolic production of thioarsenicals may be a risk factor for the development of cancer in these organs. To reveal their production sites (tissues/body fluids), we examined the uptake and transformation of the four dimethylated arsenicals by incubation with rat and human red blood cells (RBCs). Although DMA(V) and DMDTA(V) were not taken up by either type of RBCs, DMAIII and DMMTA(V) were taken up by both (more efficiently by rat ones), though DMMTA(V) was taken up slowly, and then the arsenic transformed into DMDTA(V) was excreted from both types of animal RBCs. On the other hand, although DMA(III) taken up rapidly by rat RBCs was retained in the RBCs, that taken up by human RBCs was immediately transformed into DMMTA(V) and then excreted into the incubation medium without being retained in the RBCs. In a separate experiment, arsenic remaining in primary rat hepatocytes after incubation with 1.5 microM DMAIII was recovered from the incubation medium in the forms of DMA(V) and DMMTA(V) in the presence of human RBCs, but not in the presence of rat RBCs (in which the arsenic was bound to hemoglobin). Thus, DMMTA(V) was detected in the medium only in the presence of human RBCs and increased with incubation time. It was proposed that arsenic is excreted from hepatocytes into the bloodstream in the form of DMAIII and then taken up by RBCs in humans, where it is transformed into DMMTA(V) and then excreted again into the bloodstream.
Article
To evaluate the contribution of an autophagic mechanism to the As2O3- induced death of human acute myeloid leukaemia cell line HL60 cells. The growth inhibition of HL60 cells induced by As2O3 was assessed with 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. The activation of autophagy was determined with monodansylcadaverine labeling and transmission electron microscope. The role of autophagy in the As2O3-induced death of HL60 cells was assessed using autophagic and lysosomal inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. After treatment with As2O3, the proliferation of HL60 cells was significantly inhibited and the formation of autophagosomes increased. The blockade of autophagy maturation with the autophagy-specific inhibitor 3-methyladenine (3-MA) or the lysosome-neutralizing agent NH4Cl 1 h before As2O3 potentiated the As2O3-induced death of HL60 cells. In contrast, 3-MA attenuated As2O3-induced death when administered 30 min after As2O3. 3-MA and NH4Cl also inhibited As2O3-induced upregulation of microtubule-associated protein 1 light chain 3, the protein required for autophagy in mammalian cells. Following As2O3, lysosomes were activated as indicated by increased levels of cathepsins B and L. The apoptotic response of HL60 cells to As2O3 was suggested by the collapse of mitochondrial membrane potential, release of cytochrome c from mitochondria, and the activation of caspase-3. Pretreatment with 3-MA prior to As2O3 amplified these apoptotic signals, while posttreatment with 3-MA 30 min after As2O3 attenuated the apoptotic pathways. Autophagy plays complex roles in the As2O3-induced death of HL60 cells; it inhibits As2O3-induced apoptosis in the initiation stage, but amplifies the As2O3-mediated apoptotic program if it is persistently activated.
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