Grantee Research Project Results

2000 Progress Report: Arsenicals, Glutathione Reductase and Cellular Redox Status

EPA Grant Number: R826136
Title: Arsenicals, Glutathione Reductase and Cellular Redox Status
Investigators: Stýblo, Miroslav , Thomas, David J. , Del Razo, Luz M. , Cullen, William R. , Lin, Shan
Current 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
Current 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 Period Covered by this Report: September 1, 1999 through August 31, 2000
Project Amount: $322,936
RFA: Arsenic Health Effects Research (1997) RFA Text |  Recipients Lists
Research Category: Drinking Water , Human Health , Water

Objective:

The major goal of this project is to study effects of inorganic arsenic on a major cellular redox enzyme, glutathione reductase (GR), and on the redox status of cells exposed to arsenic. To better understand the role of metabolism in the toxicity of arsenic, the correlation between effects induced by arsenic and the patterns of its metabolic conversions are systematically examined in live cells. Cells from tissues that metabolize arsenic (liver) and tissues that are targets for carcinogenic effects of arsenic (skin, lung, and bladder) are used in the study.

Progress Summary:

The following is a summary of results obtained during the 3 years (1997-2000) of the study:

1. An inorganic form of trivalent arsenic, arsenite (iAs^III), is toxic for most cell types examined only at relatively high concentrations. It is a weak inhibitor of GR and of another major oxidoreductase, thioredoxin reductase (TR), in cultured cells. iAs^III is effectively metabolized to methylarsenic (MAs) and dimethylarsenic (DMAs) species in primary human hepatocytes. However, the capacity of human hepatocytes to methylate iAs^III varies significantly among individuals. Human epidermal keratinocytes and human bronchial epithelial cells are much less effective methylators of iAs^III than are hepatocytes. Epithelial cells derived from human bladder do not methylate iAs^III. There is no apparent correlation between the capacity of cells to methylate iAs and their susceptibility to acute toxicity of iAs^III. Trivalent methylated metabolites of iAs^III, methylarsonous acid (MAs^III) and dimethylarsinous acid (DMAs^III), are significantly more toxic for most cells types than is iAs^III. In addition, MAs^III is an order of magnitude more potent inhibitor of GR and TR than is iAs^III. An acute exposure of cells to MAs^III results in a significant reduction of intracellular levels of a major cellular antioxidant, glutathione (GSH). Pentavalent methylated metabolites of iAs^III, methylarsonic acid (MAs^V) and dimethylarsinic acid (DMAs^V), do not inhibit either GR or TR and are not cytotoxic. In cells exposed to iAs^III, inhibition of TR activity strongly correlates with intracellular MAs, suggesting that the trivalent form of MAs^III is a product of the metabolism of iAs^III in cells.

2. Treatments with antioxidants (catalase, N-acetylcysteine or glutathione-ethyl ester) partially protect cultured cells against toxicity of iAs^III, MAs^III, or DMAs^III, indicating that generation of reactive oxygen species may be in part responsible for cytotoxicity of trivalent arsenicals. On the other hand, concurrent exposure to selenite increases cytotoxicity of all three species.

3. Using an optimized hydride generation atomic absorption spectrometric technique, we found that trivalent methylated arsenicals, MAs^III and DMAs^III, are indeed products of the methylation of iAs^III in human hepatic (HepG2) cells (Figure 1). Both MAs^III and DMAs^III were also found in urine of individuals chronically exposed to iAs from drinking water (Table 1). The amounts of MAs^III and DMAs^III positively correlated with total urinary arsenic and thus, with the extent of the exposure to iAs.

In conclusion, trivalent methylated metabolites, MAs^III and DMAs^III, are more potent cytotoxins and enzyme inhibitors than is iAs^III. Trivalent methylated arsenicals are natural products of the metabolism of iAs in humans, suggesting that methylation of iAs is not necessarily a detoxification mechanism. In fact, these species may significantly contribute to the adverse effects associated with the exposure to iAs. Because of their pronounced biological effects, MAs^III and DMAs^III may become sensitive biomarkers of arsenic toxicity in humans.

Table 1. Arsenic metabolites in urine (ng/ml) 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
19/m	25.2	16.8	2.3	37.7	18.2	40.1	140.3
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
20/f	14.7	10.1	1.2	19.3	5.7	90.5	141.5
11/f	11.2	12.2	1	11.2	18.3	52.7	106.6

Future Activities:

Continue to prepare publications for peer reviewed journals and prepare and submit the final report for the grant.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Publications Views

Other project views:	All 43 publications	12 publications in selected types	All 12 journal articles

Publications

Type	Citation	Project	Document Sources
Journal Article	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)	Abstract from PubMed Abstract: ACS-Abstract Exit
Journal Article	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)	Abstract from PubMed Full-text: ScienceDirect-Full Text PDF Exit Abstract: ScienceDirect-Abstract Exit
Journal Article	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)	Abstract from PubMed Full-text: ScienceDirect-Full Text PDF Exit Abstract: ScienceDirect-Abstract Exit

Supplemental Keywords:

drinking water, exposure, health effects, human health, metabolism, methylation, carcinogen, cellular, cell culture, cell viability, cytotoxicity, enzymes, inhibitor, inhibition, susceptibility, chemicals, metals, metalloids, arsenic, glutathione, antioxidants, oxidative stress, ROS, risk assessment, animal, mammalian, uptake, efflux, detoxification, arsenothiols, mechanism, protein binding, mouse., 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 binding

Progress and Final Reports:

Original Abstract

1998

1999 Progress Report

2001

Final Report