Grantee Research Project Results
Final Report: Arsenicals, Glutathione Reductase and Cellular Redox Status
EPA Grant Number: R826136Title: Arsenicals, Glutathione Reductase and Cellular Redox Status
Investigators: Stýblo, Miroslav , Beck, Melinda A. , Del Razo, Luz M. , Cullen, William R. , Walton, Felecia , Lin, Shan
Institution: University of North Carolina at Chapel Hill , University of British Columbia
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 1997 through August 31, 2000 (Extended to September 14, 2001)
Project Amount: $322,936
RFA: Arsenic Health Effects Research (1997) RFA Text | Recipients Lists
Research Category: Drinking Water , Human Health , Water
Objective:
One of the basic mechanisms that underlie toxicity of inorganic arsenic (iAs) is the interaction of trivalent iAs, arsenite (iAsIII), with thiol-containing residues of peptides and proteins. This interaction is responsible for the inhibition of various enzymes and for the inactivation of critical cellular receptors. Glutathione reductase (GR, NADPH:oxidized glutathione oxidoreductase, E.C. 1.6.4.2) has been implicated among enzymes that are sensitive to the inhibition by iAsIII. GR is the key enzyme in the redox metabolism of glutathione (GSH), a major intracellular antioxidant. GR maintains a constant intracellular ratio of GSH to glutathione disulfide (GSSG), playing an important role in the regulation of the cellular redox status. Methylated arsenicals that contain trivalent arsenic (AsIII) are orders of magnitude more potent GR inhibitors than iAsIII. Thus, production of methylated trivalent arsenicals in the course of iAs metabolism may result in GR inhibition, shifting the GSH:GSSG ratio and the cellular redox balance. Changes in the cellular redox status have been linked to modulations of gene transcription that underlie an uncontrolled cell proliferation, the main attribute of cancerous growth. This project was originally designed to study the relationship between metabolism of iAs, GR activity, and cellular redox status. The main focus of the project was on:
(1) Effects of arsenicals on GR activity, GSH:GSSG ratio, and the production of reactive oxygen species (ROS) in human cells as compared with patterns of arsenic metabolism and toxicity in these cells. Cells derived from tissues that are targets for carcinogenic effects of iAs (skin, lung, and urinary bladder) or are the major sites of iAs metabolism (liver) were of particular interest.
(2) Interactions of iAs and methylated arsenicals with GR purified from human cells.
(3) GR activity and GSH:GSSG ratio in tissues of laboratory animals in vivo exposed to arsenicals.
Inorganic arsenicals, iAsIII, and arsenate (iAsV), were used in this study along with trivalent and pentavalent methylated arsenicals that are chemically consistent with methylated metabolites of iAs, methylarsonic acid (MAsV), methylarsonous acid (MAsIII), dimethylarsinic acid (DMAsV), and dimethylarsinous acid (DMAsIII). The main goal of this research was to establish whether GR activity and the GSH:GSSG ratio can serve as sensitive markers of arsenic toxicity in individuals exposed to iAs from the environment. The original objectives were modified considerably as the work proceeded to reflect new findings and to focus on more sensitive markers of arsenic toxicity in human cells.
Summary/Accomplishments (Outputs/Outcomes):
Metabolism and Toxicity of Arsenicals in Cultured Cells. In cultured human cells, including primary hepatocytes, normal epidermal keratinocytes (NHEK), bronchial epithelial cells (HBEC), and UROtsa (SV40-immortalized bladder epithelial cells), trivalent arsenicals were significantly more cytotoxic than their pentavalent counterparts. Table 1 shows LC50 values for inorganic and methylated trivalent arsenicals, including iAsIII, methylarsine oxide (MAsIIIO), methylarsonous diiodide (MAsIIII2), dimethylarsinous iodide (DMAsIIII), and dimethylarsinous glutathione complex (DMAsIIIGS). The methylation rate for iAsIII in every cell type examined also is shown. Regardless of the cell type, trivalent monomethylated arsenicals, MAsIIIO and MAsIIII2, were the most potent cytotoxins with LC50 values ranging from 0.8 to 5.5 µM. The DMAsIII derivatives, DMAsIIIGS, and DMAsIIII, were as cytotoxic as MAsIII species and more cytotoxic than iAsIII in some cell types. Similar cytotoxic patterns were found in primary rat and primary guinea pig hepatocytes used throughout the study as positive and negative metabolic controls, respectively. Primary rat hepatocytes were superior methylators of iAsIII followed by primary human hepatocytes. The other cell types had little or no methylation capacity for iAsIII. Notably, there was no apparent correlation between the capacity of cells to methylate iAs and their sensitivity to the cytotoxic effects of trivalent arsenicals, indicating that the capacity to methylate has little to do with the resistance of cells to acute toxicity of AsIII.
Table 1. Methylation Capacities and Susceptibility of Cells to Toxic Effects of Trivalent Arsenicals
| Cell Type | Estimated LC50 Values a (µM) |
Methylation Rateb
pmol As/106cells/h |
||||
| iAsIII | MAsIIIO | MAsIIII2 | DMAsIIIGS | DMAsIIII | ||
| Primary human hepatocytes | >20 | 5.5 | >20 | 3.3 | ||
| NHEK | 10- >20 | 2.6 | 8.5 | 0.12 | ||
| HBEC | 3.2 | 2.7 | 6.8 | 0.05 | ||
| UROtsa | 17.8 | 2.0 | 0.8 | 14.2 | >20 | NDc |
| Primary rathepatocytes | 10- >20 | 2.8 | 1.8 | 14.5 | 2.7 | 19 |
| Primary guinea pig hepatocytes | >50 | 3.2 | 5.0 | ND | ||
a LC50 is defined as the concentration of an arsenical that resulted in a 50 percent decrease in cell viability over a 24-hour incubation period. The MTT assay was used to examine cell viability in all cell types.
b Methylation rates were determined in cultures exposed to 0.05 or 0.1 µM iAsIII for 24 or 48 hours.
c Methylation activity not detected.
Effects of Arsenicals on GR Activity and Redox Status in Cultured Cells. Short-time exposures to cytotoxic concentrations of iAsIII or MAsIIIO resulted in a concentration-dependent inhibition of GR activity and a decrease in GSH concentration in cultured cells. No changes in GSSG concentration were detected. Figure 1 shows the inhibition of GR activity and the partial GSH depletion in primary rat hepatocytes exposed to iAsIII (4, 10, and 50 µM) or MAsIIIO (1, 5, and 10 µM). The GSH:GSSG ratio, a marker of the cellular redox status, decreased significantly in exposed cells as a result of GSH depletion. MAsIIIO was considerably more potent than iAsIII inhibiting GR and decreasing GSH concentration. Exposures to either arsenical did not change activity of glutathione peroxidase (GPx), another enzyme involved in GSH turnover in the cell. Similar effects of iAsIII and MAsIIIO on GR activity and GSH concentration were observed in
Figure 1. Effects of iAsIII (4, 10, or 50 µM) and MAsIIIO (1, 5, or 10 µM) on activities of GR and GPx (a) and concentrations of GSH and GSSG (b) in primary rat hepatocytes (mean and SD, n = 3). *Values are statistically different (p<0.05) from those in untreated (control) cells (C).
primary human hepatocytes, NHEK, and UROtsa cells. In all cell types, the inhibition of GR activity and/or depletion of GSH were associated only with exposures to cytotoxic concentrations of iAsIII and MAsIIIO. These data suggest that GR activity and GSH:GSSG ratio are not sensitive markers of arsenic toxicity at low exposure levels. Notably, DMAsIII derivatives (up to 10 µM) did not modify GR activity or GSH concentrations in cultured cells. Exposures to iAsIII or MAsIII were associated with an increased production of peroxides as monitored in intact cells by dichlorofluorescein fluorescence. The increased peroxide levels were detected in cells exposed to both high (cytotoxic) and to low (noncytotoxic) concentrations of iAsIII or MAsIII. Thus, induction of ROS in human cells may be an early sign of the toxicity of trivalent arsenicals.
Effects of Trivalent Arsenicals on Thioredoxin Reductase Activity in Cultured Cells. Unlike GR activity, the activity of thioredoxin reductase (TR, NADPH: oxidized-thioredoxin oxidoreductase, E.C. 1.6.4.5) was very sensitive to inhibition by trivalent arsenicals, particularly by MAsIII species. TR catalyzes the reduction of dithiol bonds in molecules of various peptides and proteins, including thioredoxin (TRx), a 12 kDa protein with redox-active cysteinyl residues. Through its capacity to reduce dithiols in proteins, Trx is an important component of the machinery that regulates the response of cells to oxidative stress. In primary rat and human hepatocytes, exposure to 1 µM MAsIIIO inhibited TR activity by about 30 percent (Figure 2). Fifty times higher concentration of iAsIII was required to attain a comparable degree of inhibition. MAsIIIO was a more potent TR inhibitor than was aurothioglucose (ATG), a specific
Figure 2. TR activity in primary rat and human hepatocytes exposed to iAsIII, MAsIIIO, or aurothioglucose (ATG) for 30 minutes (mean ± SD, n = 3). *Values are significantly different (p < 0.05) from those in control (untreated) cells.
Figure 3. TR activity and intracellular concentrations of arsenic metabolites in primary rat hepatocytes exposed to 10 µM iAsIII for 24 hours (mean ± SD, n = 3). *TR activity in treated cells is significantly different (p < 0.05) from that in control (untreated) cells.
inhibitor of the enzyme. In methylating rat hepatocytes exposed to iAsIII, TR inhibition correlated with production and intracellular concentration of MAs, but not iAs (Figure 3), indicating that trivalent form of MAs, MAsIII, accumulated in cells and was responsible for the inhibitory effect. These data suggest that inhibition of TR activity may be a sensitive marker of the production and accumulation of MAsIII in tissues of individuals exposed to iAs. Inhibition of TR by MAsIII may have profound effects on various cellular mechanisms that require TR activity (e.g., redox metabolism of TRx and ascorbate, or redox sensitive mechanisms involved in the signal transduction pathway). Hence, MAsIII produced in the course of iAs methylation may significantly contribute to adverse effects associated with exposures to this arsenical.
Figure 4. Tri- and pentavalent metabolites in HepG2 cell cultures exposed to 10 µM iAsIII for 24 hours. Averages of duplicates (cells + medium) are shown.
Analysis of Methylated Trivalent Metabolites in Biological Samples. To provide direct information about formation of MAsIII in the course of iAs metabolism in humans, a hydride generation atomic absorption spectrophotometrical (HG-AAS) technique was optimized for analysis of oxidation states of methylated metabolites in biological matrices. Using this technique, methylated trivalent arsenicals were analyzed in human cells exposed to iAs and in urine from individuals chronically exposed to iAs in drinking water. Human hepatocellular carcinoma (HepG2) cells exposed to iAsIII (0.1, 1, or 10 µM) synthesized DMAsIII regardless of the exposure levels (Figure 4). In contrast, MAsIII was detected only in cultures exposed to 1 or 10 µM iAsIII. Notably, significant portions of either MAsIII or DMAsIII were found in culture medium, indicating that these toxic metabolites can be released in vivo from hepatic cells and translocated via the blood circulation to nonmethylating tissues or cells. Both MAsIII or DMAsIII also were found in urines collected from residents of Zimapan region (Mexico) who drink water contaminated with iAs (Table 2). In urine from these individuals, MAsIII accounted for up to 9 percent of the total urinary MAs; DMAsIII accounted for up to 31 percent of the total urinary DMAs. The amounts of MAsIII and DMAsIII in urine positively correlated with total urinary arsenic. These results show that toxic trivalent methylated arsenicals, MAsIII and DMAsIII, are natural products of the metabolism of iAs in humans.
Table 2. Arsenic metabolites in urine (mg/l) collected from residents of Zimapan region, Mexico.
| age/sex | iAsIII | iAsV | MAsIII | MAsV | DMAsIII | DMAsV | Total As |
| 39/f | 8.1 | 4.5 | ND* | 7.1 | ND* | 18.6 | 38.3 |
| 11/f | 11.2 | 12.2 | 1 | 11.2 | 18.3 | 52.7 | 106.6 |
| 19/m | 25.2 | 16.8 | 2.3 | 37.7 | 18.2 | 40.1 | 140.3 |
| 20/f | 4.7 | 10.1 | 1.2 | 19.3 | 5.7 | 90.5 | 141.5 |
| 21/m | 59.4 | 72.1 | 5.2 | 119.8 | 59.8 | 408.2 | 724.5 |
| 12/m | 104.2 | 122.8 | 12.3 | 276.7 | 114.3 | 467.2 | 1097.5 |
* ND, not detected
Conclusions:
Methylated trivalent arsenicals, particularly MAsIII derivatives, are more toxic for cultured human and animal cells than is iAsIII. iAsIII and MAsIII derivatives inhibit GR activity, decreasing GSH concentration in cells. These effects are likely to contribute to the cytotoxicity of trivalent arsenicals. However, neither GR activity nor GSH concentration is a sensitive marker of toxicity of trivalent arsenicals in mammalian cells.
Trivalent arsenicals induce ROS production in mammalian cells at low exposure levels. These effects that are not associated with acute toxicity may be early indicators of arsenic exposure. MAsIII derivatives are potent inhibitors of TR in mammalian cells. There is a direct link between production of MAs metabolites in cells exposed to iAs and inhibition of TR activity. Thus, TR activity may be a sensitive marker of MAsIII toxicity at low exposure levels. Methylated trivalent arsenicals, MAsIII and DMAsIII, are formed in the course of iAs methylation in human cells and are urinary metabolites in humans chronically exposed to iAs in drinking water. Urinary levels of MAsIII and DMAsIII may be used as indicators of health risks associated with exposures to iAs.
Taken together, results of this project provide further evidence about biomethylation as a process that activates rather than detoxifies iAs in humans.
Journal Articles on this Report : 12 Displayed | Download in RIS Format
| Other project views: | All 43 publications | 12 publications in selected types | All 12 journal articles |
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Del Razo LM, Styblo M, Cullen WR, Thomas DJ. Determination of trivalent methylated arsenicals in biological matrices. Toxicology and Applied Pharmacology 2001;174(3):282-293. |
R826136 (Final) |
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Easterling MR, Styblo M, Evans MV, Kenyon EM. Pharmacokinetic modeling of arsenite uptake and metabolism in hepatocytes–mechanistic insights and implications for further experiments. Journal of Pharmacokinetics and Pharmacodynamics 2002;29(3):207-234. |
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Lin S, Del Razo LM, Styblo M, Wang C, Cullen WR, Thomas DJ. Arsenicals inhibit thioredoxin reductase in cultured rat hepatocytes. Chemical Research in Toxicology 2001;14(3):305-311. |
R826136 (2000) R826136 (Final) |
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Lin S, Shi Q, Nix FB, Styblo M, Beck MA, Herbin-Davis KM, Hall LL, Simeonsson JB, Thomas DJ. A novel S-adenosyl-L-methionine: arsenic(III) methyltransferase from rat liver cytosol. Journal of Biological Chemistry 2002;277(13):10795-10803. |
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Mass MJ, Tennant A, Roop BC, Cullen WR, Styblo M, Thomas DJ, Kligerman AD. Methylated trivalent arsenic species are genotoxic. Chemical Research in Toxicology 2001;14(4):355-361. |
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Styblo M, Del Razo LM, LeCluyse EL, Hamilton GA, Wang C, Cullen WR, Thomas DJ. Metabolism of arsenic in primary cultures of human and rat hepatocytes. Chemical Research in Toxicology 1999;12(7):560-565. |
R826136 (1999) R826136 (Final) |
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Styblo M, Del Razo LM, Vega L, Germolic DR, LeCluyse EL, Hamilton GA, Reed W, Wang C, Cullen WR, Thomas DJ. Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Archives of Toxicology 2000;74(6):289-299. |
R826136 (1999) R826136 (Final) |
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Styblo M, Thomas DJ. Selenium modifies the metabolism and toxicity of arsenic in primary rat hepatocytes. Toxicology and Applied Pharmacology 2001;172(1):52-61. |
R826136 (2000) R826136 (Final) |
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Styblo M, Drobna Z, Jaspers I, Lin S, Thomas DJ. The role of biomethylation in toxicity and carcinogenicity of arsenic: a research update. Environmental Health Perspectives 2002;110(Suppl 5):767-771. |
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Thomas DJ, Styblo M, Lin S. The cellular metabolism and systemic toxicity of arsenic. Toxicology and Applied Pharmacology 2001;176(2):127-144. |
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Thomas DJ, Waters SB, Styblo M. Elucidating the pathway for arsenic methylation. Toxicology and Applied Pharmacology 2004;198(3):319-326. |
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Vega L, Styblo M, Patterson R, Cullen W, Wang C, Germolec D. Differential effects of trivalent and pentavalent arsenicals on cell proliferation and cytokine secretion in normal human epidermal keratinocytes. Toxicology and Applied Pharmacology 2001;172(3):225-232. |
R826136 (2000) R826136 (Final) |
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Supplemental Keywords:
arsenic, drinking water, exposure, health effects, human health, metabolism, methylation, carcinogen, mammalian, organism, cell, cellular, cell viability, toxicity, enzyme, inhibitor, susceptibility, chemical, metal, metalloid, glutathione, antioxidants, oxidative stress, ROS, glutathione reductase, thioredoxin reductase, atomic absorption spectrophotometry, analysis, toxicology, biochemistry, analytical chemistry, environmental chemistry., RFA, Health, Scientific Discipline, Toxics, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, POLLUTANTS/TOXICS, National Recommended Water Quality, Arsenic, Fate & Transport, Risk Assessments, Disease & Cumulative Effects, Water Pollutants, Biology, Drinking Water, cancer risk, Pathology, fate and transport, health effects, carcinogenesis, human health effects, redox metabolism, cellular metabolism, arsenothiols, trivalent methylated arsenicals, detoxification, cellular biology, exposure and effects, cell biology, exposure, inorganic arsenic, cellular physiology, effects, human exposure, GSH, methylation, carcinogens, toxicity, metabolism, water quality, protein bindingProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.