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THE EFFECTS OF ARSENITE ON SELENOPROTEINS BIOSYTHESIS
Title
THE EFFECTS OF ARSENITE ON SELENOPROTEINS BIOSYTHESIS.
Creator
Konate, Fanta, Self, William, University of Central Florida
Abstract / Description
Arsenic contamination of drinking water is a real public health problem in certain areas of South-East Asia where chronic exposure has been correlated to higher rates of lung, skin, bladder, kidney, and liver cancer. Although arsenic carcinogenicity is well established, the mechanism by which it induces cancer is poorly understood. Recent evidence suggests that oxidative stress could be a possible mechanism for the carcinogenic effects of arsenic. Selenium, in the form of selenocysteine, is...
Show moreArsenic contamination of drinking water is a real public health problem in certain areas of South-East Asia where chronic exposure has been correlated to higher rates of lung, skin, bladder, kidney, and liver cancer. Although arsenic carcinogenicity is well established, the mechanism by which it induces cancer is poorly understood. Recent evidence suggests that oxidative stress could be a possible mechanism for the carcinogenic effects of arsenic. Selenium, in the form of selenocysteine, is necessary for the activity of several enzymes with a role in the defense against reactive oxygen species (ROS), primarily thioredoxin reductases (TrxR) and glutathione peroxidases (Gpx). One of the key enzymes in the incorporation of Se into selenoproteins is selenophosphate synthetase (SPS). SPS catalyzes the activation of Se to selenophosphate, and is the first step in the pathway of selenoprotein biosynthesis. SPS contains a conserved vicinal dithiol motif (CXC) within a region of amino acids that have been predicted to be a selenium binding site. Our hypothesis is that arsenite inhibits new selenoprotein synthesis, thus indirectly increasing the level of ROS. In this study we have developed a spectrophotometric assay for SPS. Using this assay, we have determined that arsenite inhibits SPS activity. Kinetic analysis of this inhibition showed that arsenite, a trivalent form of arsenic, acts as a competitive inhibitor with the substrate, sodium selenide. This inhibition of SPS could represent a potential molecular mechanism for oxidative stress induced upon arsenite treatment of human cell lines in culture. To further study the effects of trivalent arsenicals at a cellular level we decided to use a human keratinocyte cell line, HaCaT as a cell culture model. Our study showed that although arsenite does not alter cell proliferation or protein synthesis, it specifically inhibits new selenoprotein synthesis. However, short term or long term exposure of HaCaT cells to arsenite failed to result in changes to Gpx and TrxR levels. Since the radioisotope selenium used in labeling studies is selenite, these results indicate that an alternate source of selenium may bypass the inhibitory effects of arsenite. Future studies will focus on studying the effects of arsenicals on keratinocytes cultured in a defined medium allowing a better control of the selenium source.
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Date Issued
2005
Identifier
CFE0000453, ucf:46406
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000453
THE EFFECTS OF TRIVALENT ARSENICALS AND THIOREDOXIN REDUCTASE INHIBITORS ON SELENIUM METABOLISM IN LUNG CELL CULTURE MODELS
Title
THE EFFECTS OF TRIVALENT ARSENICALS AND THIOREDOXIN REDUCTASE INHIBITORS ON SELENIUM METABOLISM IN LUNG CELL CULTURE MODELS.
Creator
Talbot, Sarah, Self, William, University of Central Florida
Abstract / Description
Arsenic exposure, through various routes, is associated with the development of cancer of the skin, lung, liver, kidney, and bladder. Treatment of cells in culture with trivalent arsenicals has been shown to increase reactive oxygen species (ROS). In particular, monomethylarsonous acid (MMAIII), a trivalent metabolite of arsenite, is highly cytotoxic and possibly carcinogenic. Three trivalent arsenicals; arsenite, arsenic trioxide (ATO), and MMAIII, are also known inhibitors of the...
Show moreArsenic exposure, through various routes, is associated with the development of cancer of the skin, lung, liver, kidney, and bladder. Treatment of cells in culture with trivalent arsenicals has been shown to increase reactive oxygen species (ROS). In particular, monomethylarsonous acid (MMAIII), a trivalent metabolite of arsenite, is highly cytotoxic and possibly carcinogenic. Three trivalent arsenicals; arsenite, arsenic trioxide (ATO), and MMAIII, are also known inhibitors of the selenoprotein thioredoxin reductase (TrxR). Selenium, an essential micronutrient in mammals, is needed in the form of selenocysteine for activity of this enzyme and other selenoproteins. TrxR is part of a key component of the cell's ability to defend against ROS. It has been speculated that TrxR is also involved directly in selenium metabolism, but this has yet to be demonstrated in vivo. The promoter region of the gene encoding the cytosolic TrxR (TrxR1) also contains an antioxidant responsive element (ARE). The ARE is activated by the transcription factor, Nrf2, which is governed by the Nrf2/Keap1 response, and can be triggered by certain oxidants. ATO and arsenite both inhibited incorporation of selenium into selenoproteins. Auranofin, a gold chemotherapeutic inhibitor of TrxR1, also inhibited selenoprotein synthesis. These results seem to support the hypothesis that TrxR1 is needed for selenoprotein synthesis. However, siRNA mediated reduction of TrxR1 did not block incorporation of selenium into selenoproteins. It is likely that ATO and auranofin are forming As-Se and Au-Se complexes, respectively. We also found that exposure of primary lung fibroblasts (WI-38) to MMAIII led to increased synthesis of TrxR1. This increase was dependent on the activation of transcription of the TrxR1 gene, specifically mediated through the ARE element. These results indicate exposure to MMAIII induces the Nrf2 response. The results obtained in these studies aid in both our understanding of the carcinogenic potential of arsenic as well as give new insight into the mechanism of action of emerging cancer drugs.
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Date Issued
2007
Identifier
CFE0001943, ucf:47467
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001943
SELENOTRISULFIDE DERIVATIVE OF ALPHA-LIPOIC ACID: EVALUATION IN A CELL CULTURE MODEL FOR POTENTIAL USE AS A TOPICAL ANTIOXIDANT
Title
SELENOTRISULFIDE DERIVATIVE OF ALPHA-LIPOIC ACID: EVALUATION IN A CELL CULTURE MODEL FOR POTENTIAL USE AS A TOPICAL ANTIOXIDANT.
Creator
Alonis, Melenie, Self, William, University of Central Florida
Abstract / Description
Selenium is a required micronutrient in mammalian cells. It is incorporated in the form of selenocysteine into selenoenzymes such as glutathione peroxidase and thioredoxin reductase, and is absolutely required for activity. Thioredoxin reductase is necessary for reduction of oxidized thioredoxin and therefore plays a major role in maintaining the redox status of the cell. Glutathione peroxidase is responsible for reducing peroxides into their corresponding alcohols and water. Together, these...
Show moreSelenium is a required micronutrient in mammalian cells. It is incorporated in the form of selenocysteine into selenoenzymes such as glutathione peroxidase and thioredoxin reductase, and is absolutely required for activity. Thioredoxin reductase is necessary for reduction of oxidized thioredoxin and therefore plays a major role in maintaining the redox status of the cell. Glutathione peroxidase is responsible for reducing peroxides into their corresponding alcohols and water. Together, these selenoenzymes constitute a significant part of the cell's arsenal to defend itself against oxidative stress. Exogenous sources of oxidative stress, such as UV radiation, are capable of generating reactive oxygen species (ROS). Elevated levels of ROS can lead to covalent modifications of lipids, nucleic acids, and proteins within a cell. This damage has been implicated in the development of cancer and degenerative diseases. As the skin is the first level of defense for UV radiation, skin cancer is an obvious concern. Previous studies have demonstrated a protective effect against UV-induced cytotoxicity when selenium compounds were administered to skin cells in cell culture models. Topical selenium application to mice has also been shown to reduce UV damage to skin. Although a variety of chemical forms of selenium are available in nutritional supplements, the efficiency by which they are used for selenoprotein synthesis varies greatly. It is debated within the selenium research community which form is best for use as a supplement. In this study, we have focused on a selenotrisulfide derivative of alpha-lipoic acid (LASe). We have examined its utilization for selenoprotein synthesis through radiolabeling studies (75Se) in a human keratinocyte cell line (HaCaT). We have determined that is incorporated into selenoproteins with nearly the same efficiency as selenite and L-selenocysteine. We have also determined that LASe is far more efficient as a supplement in cell culture than selenate or L-selenomethionine, two forms of selenium commonly used as supplements. LASe was also found to protect HaCaT keratinocytes from UV- induced cytotoxicity. Cells pretreated with LASe and exposed to 500J/m2 and 750J/m2 of broadband (UVA/UVB) UV radiation showed greater survival than untreated controls in a dose –dependent manner. Cells pre-treated either with lipoic acid or selenium in the form of selenite alone also observed protection. Nonetheless, these finding are significant given that LASe was previously shown to penetrate the skin better than other forms of selenium. These results indicate that LASe has the potential for use as a topical antioxidant upon further testing in animal studies.
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Date Issued
2005
Identifier
CFE0000663, ucf:46531
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000663