Grantee Research Project Results

1999 Progress Report: Arsenicals, Glutathione Reductase and Cellular Redox Status

EPA Grant Number: R826136
Title: Arsenicals, Glutathione Reductase and Cellular Redox Status
Investigators: Stýblo, Miroslav
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, 1998 through August 31, 1999
Project Amount: $322,936
RFA: Arsenic Health Effects Research (1997) RFA Text | Recipients Lists
Research Category: Drinking Water , Human Health , Water

Objective:

The goal of this project is to examine two related aspects on the mode of action of inorganic arsenic (iAs) as a carcinogen and toxin, including: (1) interactions between metabolites of iAs and glutathione reductase (GR), a key enzyme in redox metabolism of glutathione (GSH); and (2) effects of the arsenic-GR interactions on the ratio of GSH to glutathione disulfide (GSSG) that is an indicator of cellular redox status. This project integrates studies at three levels of biological complexity: purified enzyme, cultured cells, and intact animals. The experimental approach includes: (1) examination of the effects of arsenicals on GR activity, the GSH/GSSG ratio and intracellular peroxides in cultured human cells as compared with patterns of arsenic metabolism in cells; (2) examination of the interactions of iAs and its metabolites with GR purified from human erythrocytes; and (3) examination of the in vivo effects of arsenicals on GR activity and GSH/GSSG ratio in tissues of mice.

Progress Summary:

During the first year of the project (October 1997 to September 1998), the work focused predominantly on characterization of the metabolism and toxicity of iAs and methylated arsenicals in those types of cells that were to be used in further experiments. The cells used in this project are derived from normal human liver, a major site of arsenic metabolism, and from tissues that are targets for carcinogenic effects of arsenic: skin, lung, and urinary bladder. Primary rat hepatocytes are being used throughout the project as methylating controls. The relationship between the capacity of cells to metabolize (methylate) iAs and their susceptibility to the toxic effects of arsenicals has been studied in primary human and rat hepatocytes, normal human epidermal keratinocytes, and Urotsa, human epithelial bladder cells. The effects of selected antioxidants (GSH, selenium, N-acetylcysteine, and catalase) on arsenic metabolism and toxicity in cultured cells have been described. During the current funding period (October 1998 to September 1999), additional experiments completed the characterization of arsenic metabolism and toxicity in human cells of interest. Primary bronchial epithelial cells obtained from the lungs of healthy human donors were used in these experiments. The major part of the experimental work during this period focused on the examination of the effects of iAs and methylated arsenicals on GR activity, the concentration of GSH, and induction of oxidative stress in cultured cells. In addition, effects of arsenicals on the activity of another NADPH-dependent oxidoreductase, thioredoxin reductase (TR), have been examined. TR is involved in the reduction of protein disulfides and therefore plays an important role in the maintenance of the cellular redox status. It recently has been reported that trivalent methylarsenic (MAs^III), a putative metabolite of iAs, is a potent inhibitor of TR. The following is a brief summary of results of the 2 years of the project:

Among arsenicals examined, MAs^III was the most toxic for human cell types used in this study, followed by trivalent dimethylarsenic (DMAs^III), and arsenite (iAs^III) (Table 1).
There is no apparent correlation between the capacity of cells to methylate iAs and their susceptibility to acute toxicity of trivalent arsenicals (Table 1).
Trivalent arsenicals, iAs^III and MAs^III, are inhibitors of GR and especially TR in cultured cells. In addition, the exposure to either iAs^III or MAs^III results in a significant decrease of the intracellular concentration of GSH.
MAs^III is at least by an order of magnitude more potent of an enzyme inhibitor than is iAs^III. Similarly, the effect of MAs^III on GSH concentration is more profound than that of iAs^III.
In cells with high methylation capacity, enzymatic methylation of MAs^III to yield DMAs results in the restoration of TR activity. Thus, under these conditions methylation functions as a detoxification process. On the other hand, the production and accumulation of MAs in cells exposed to high concentrations of iAs^III may contribute to the inhibition of the enzyme.
Exposure to iAs results in an increased production of ROS in cultured cells. The induction of oxidative stress may underlie the mechanism of arsenic cytotoxicity.
Treatment with antioxidants protects cells against toxicity of trivalent arsenicals.

Table 1: Methylation Capacities and Susceptibility of Cells to Toxic Effects of Trivalent Arsenicals

Methylation Capacity Estimated IC₅₀Values¹ (M) for Arsenicals

Cell Line (pmol iAs^III/10⁶ cells/h) iAs^III MAs^III DMAs^III

Rat Hepatocytes 387 5.1 - >20 1.6 - 2.8 2.6 - 14.5

Human Hepatocytes 3.1 - 101 >20 5.9 >20

Human Keratinocytes 0.2 9.6 - >10 2.3 - 3.3 9.2 - 10.2

Urotsa ~0 5.5 - 17.7 0.8 - 1.9 3.5 - 14.8

Human bronchial cells 0.05 3.2 2.7 6.8
1 IC50 is defined as a concentration of an arsenical that results in 50% decrease in viability of cells over 24-hr incubation period.

Future Activities:

The experimental work scheduled for the subsequent funding period includes: (1) purification of GR from human erythrocytes and examination of the stability and kinetic characteristics of the enzyme (purification procedures will be optimized using rat or rabbit erythrocytes); (2) examination of the effects of arsenicals on activity of purified GR; (3) binding of radiolabeled arsenicals to the purified enzyme and its subunits; and (4) examination of the effects of arsenicals on GR and TR activity and cellular redox status in tissues of mice. Preliminary experiments will be performed in a limited number of mice to determine optimal levels of exposure to arsenicals and timing for sample collection that will be used in acute and chronic in vivo experiments

Journal Articles on this Report : 2 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	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)	Abstract from PubMed Abstract: ACS-Abstract Exit
Journal Article	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)	Abstract from PubMed Abstract: Springer-Abstract Exit

Supplemental Keywords:

drinking water, exposure, health effects, human health, metabolism, methylation, carcinogen, cellular, cell culture, cell viability, cytotoxicity, enzymes, inhibitor, susceptibility, chemicals, metals, metalloids, arsenic, glutathione, antioxidants, oxidative stress, ROS., 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

The 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.