Corey W Speers and Dr. Kim L O’Neill, Microbiology
Abstract
One of the most effective strategies in cancer control is chemoprevention. Previous studies have shown naturally occurring organosulfur compounds from garlic and onion to be effective against carcinogenesis. The effects of two of these organosulfur compounds, diallyl sulfide (DAS) and diallyl disulfide (DADS), are thought to be of especial worth. Their anti-carcinogenic effects in the human cell line HepG2 are, however, less understood. HepG2 cells are human hepatoblastoma cells that have maintained their xenobiotic metabolizing competence. Because they are similar in function to cells obtained from human liver tissue, they are ideal for research in vitro. Benzo[a]pyrene (B[a]P) is an especially potent carcinogen found naturally in incompletely combusted organic compounds. The extent and mechanisms of its carcinogenesis have been well documented. The comet (single cell gel electrophoresis) assay is a rapid and sensitive technique for quantifying DNA damage. In this assay, cells are suspended in low melting point agarose, lysed, and electrophoresed at a high pH in order to separate the damaged DNA from the nucleus, forming a comet tail. The resulting comet tail is then compared against a control and an anti-carcinogenic effect of compounds can be quantified.
Introduction
One of the most effective strategies in cancer control is chemoprevention, the prevention of cancer by ingestion of chemical agents that reduce the risk of carcinogenesis (1). Extrinsic factors, including nutrition, play a major role in the development of most human malignancies (2). Foods contain not only numerous mutagens, but also a variety of chemicals that can block carcinogenesis (3-4). Chemopreventative agents inhibit or reverse cellular events associated with tumor initiation, promotion, and progression. It has recently been proposed that the mechanisms of chemoprotective activities might correlate to a balance between phases I and phase II enzyme activity. It has been proven that a diet rich in fruits and vegetables will reduce the incidence of cancer (5). A number of naturally occurring products from vegetables exert chemopreventative effects against carcinogenesis. It has been shown that naturally occurring organosulfur compounds (OSCs) isolated from garlic and onion have chemopreventative and anti-tumorogenic effects in mice, and that this prevention correlates to induction of phase II enzyme activity (6). Phase II enzymes are biologically important because they allow for the safe detoxification of xenobiotic substances found in the body. It is an upregulation of these phase II enzymes that may indeed correlate to the compound’s chemopreventative effects.
Materials and Methods
HepG2 cells were seeded at 1,000,000 cells/ml and allowed to attach to T25 flasks for 24 hours. They were then treated with varying concentrations of the garlic extracts (25-200 μM), with positive and negative controls, for 24 hours. Cells were then treated with 25μM benzo (a) pyrene (enough to cause significant DNA damage) and allowed to incubate for 24 hours. Evaluation of benzo (a) pyrene induced DNA damage, or the inhibition thereof, was then done utilizing the single cell gel electrophoresis (comet) assay.
Results
In this study, the effects of DAS and DADS on anti-carcinogenesis in HepG2 cells were evaluated using the comet assay. HepG2 cells were pre-treated with 25, 50, 100, and 200 μM DAS respectively and allowed to incubate for 24 hours. Cells were then treated with 25 μM B[a]P in the presence of fresh DAS and again allowed to incubate for 24 hours. DNA damage was then assessed using the comet assay. HepG2 cells treated with 200 μM DAS showed a 48% decrease in DNA damage as compared with the positive control. In HepG2 cells pre-treated with similar concentrations of DADS, those pre-treated with 200 μM DADS showed a 53% decrease in DNA damage. In both experiments, a dramatic, dose-dependent response was noted (see Figure 1). My study establishes not only the efficacy with which the comet assay can detect DNA damage in HepG2 cells, it indicates that DAS and DADS have significant chemopreventative potential in human models.
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