The Flavoring Agent Dihydrocoumarin Reverses Epigenetic Silencing and Inhibits Sirtuin Deacetylases

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Abstract
Sirtuins are a family of phylogenetically conserved nicotinamide adenine dinucleotide-dependent deacetylases that have a firmly established role in aging. Using a simple Saccharomyces cerevisiae yeast heterochromatic derepression assay, we tested a number of environmental chemicals to address the possibility that humans are exposed to sirtuin inhibitors. Here we show that dihydrocoumarin (DHC), a compound found in Melilotus officinalis (sweet clover) that is commonly added to food and cosmetics, disrupted heterochromatic silencing and inhibited yeast Sir2p as well as human SIRT1 deacetylase activity. DHC exposure in the human TK6 lymphoblastoid cell line also caused concentration-dependent increases in p53 acetylation and cytotoxicity. Flow cytometric analysis to detect annexin V binding to phosphatidylserine demonstrated that DHC increased apoptosis more than 3-fold over controls. Thus, DHC inhibits both yeast Sir2p and human SIRT1 deacetylases and increases p53 acetylation and apoptosis, a phenotype associated with senescence and aging. These findings demonstrate that humans are potentially exposed to epigenetic toxicants that inhibit sirtuin deacetylases. The effects of chronic low-dose human exposure to environmental chemicals are difficult to study and poorly understood. Chemicals are routinely tested for the ability to induce DNA mutations, cause chromosome damage, or produce cell death, but are rarely tested for their ability to cause epigenetic changes, which can influence the behavior of a cell without directly changing the DNA sequence. Epigenetic changes have become the focus of intense research in an attempt to understand the mechanisms by which they function. The Sir2 family of deacetylases is one class of proteins that controls some epigenetic processes and, interestingly, has been implicated in extending the longevity of several organisms. Here the authors describe a novel assay based upon yeast Sir2p function to screen environmental chemicals for their ability to alter epigenetic silencing. From screening a relatively small number of agents, the authors found that dihydrocoumarin, a natural compound found in Melilotus officinalis (sweet clover) that is synthetically manufactured and frequently added to both food and cosmetics, disrupted epigenetic processes in the yeast Saccharomyces cerevisiae. Dihydrocoumarin also inhibited several human Sir2 family deacetylases (SIRT1 and SIRT2) and, when added to cells in culture, increased p53 tumor suppressor protein acetylation and caused elevated levels of apoptosis. The present study suggests that humans are exposed to a number of environmental chemicals that may be classified as epigenetic toxicants.