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The study, to be published Friday, Dec. 16, in the online journal Public Library of Science (PLoS) Genetics, found that in lab tests, dihydrocoumarin (DHC), a compound found naturally in sweet clover and synthetically manufactured for use in foods and cosmetics, inhibited the activity of Sir2p and SIRT1, forms of sirtuin found in yeast and humans, respectively.

Increased sirtuin activity is known to increase longevity in yeast, roundworms and fruit flies. The opposite effect in yeast has also been shown when the sirtuin enzyme is either deleted or reduced - the lifespan decreases by as much as 30 percent.

"Although studies on sirtuins and longevity have not been done in humans, there is reason to suspect that sirtuins play a role in the aging process of human cells, so identifying sirtuin inhibitors becomes important," said Martyn Smith, professor of toxicology at UC Berkeley's School of Public Health and principal investigator of the study, which was funded by grants from the National Institute of Environmental Health Sciences and the National Institute of General Medical Sciences.

DHC is added as a flavoring agent to a wide variety of foods, including soft drinks, yogurt and muffins, and is used as a common fragrance in cosmetics, lotions and soaps. It can be found in concentrations as high as 100 parts per million in such foods as gelatins, puddings and frozen dairy products.

"It's still a long jump from test tube to person, but our findings may imply that humans are exposed to chemicals in the diet and environment that block an important enzyme involved in longevity," said Smith.

Notably, there are five forms of sirtuins found in yeast, and seven forms of sirtuin in humans. "There is good reason to think that DHC would inhibit at least some of the functions of some of the other forms of sirtuins," said Jasper Rine, professor of genetics at UC Berkeley's Department of Molecular and Cell Biology and co-author of the paper.

The UC Berkeley researchers collaborated with Dr. Eric Verdin, associate director and senior investigator at the Gladstone Institute of Virology and Immunology, which is affiliated with UC San Francisco. Verdin, who is also a professor of medicine at UCSF, is an expert on mammalian sirtuin enzymes.

"It's important to note that the role of sirtuins in aging is conserved in distantly related organisms such as yeast, drosophila and the small roundworm, C. elegans," said Verdin. "So, it is not too much of a stretch to expect that sirtuins also control the aging process in mammals, including humans. In addition, sirtuins control many important cellular functions besides aging, and an agent that modulates sirtuins, such as DHC, could not only affect aging but also other critical functions, such as metabolism, neurodegeneration and cancer."

Sirtuin enzymes are histone deacetylases, which modify the structure of chromatins, complex substances made up of DNA and protein. Histone deacetylases keep chromatins packed and tightly coiled in such a way that enzymes cannot activate the DNA, keeping the gene from being expressed. This type of gene "silencing" is considered epigenetic, which means that it effectively controls the expression of a gene without altering the actual DNA molecules.

"Epigenetic mechanisms are crucial for nearly all aspects of development and have been implicated in a variety of human diseases," said Rine. "Hence, agents which disrupt epigenetic processes can have profound consequences."

Andrew Olaharski, UC Berkeley post-doctoral researcher in environmental health sciences and lead author of the paper, noted that toxicology testing routinely focuses on an agent's ability to cause DNA mutations, chromosome alterations, or cell death, but does not currently test for chemical-induced epigenetic changes. While epigenetic changes do not alter the DNA structure, they can impact gene expression that may lead to disease.

"Identical twins with the exact same DNA may grow up to have different diseases because of epigenetic changes," said Olaharski. "The identification that a common additive to foods and cosmetics may interfere with these epigenetic processes is simply one more piece of evidence supporting the important role environmental agents may play in human health."

To screen for possible sirtuin inhibitors, the researchers used haploid strains of Saccharomyces cerevisiae yeast. The haploid yeast strains, like gametes or sex cells, contained only one set of chromosomes, and would not normally grow on the medium prepared in the lab.

However, if the haploid yeast strains were exposed to an agent that disrupted sirtuin activity, epigenetic silencing would be reversed, the strains would mate to become diploid cells, and the yeast would grow on the medium. Of the compounds tested thus far, DHC was the first environmental agent to have shown the ability to inhibit sirtuin enzymes.

The researchers further tested the role of DHC in apoptosis, a process that causes a cell to die. They exposed DHC to human white blood cells and found that increasing concentrations of the compound led to increases in cell toxicity, apoptosis, and the stability of p53, a tumor-suppressing protein associated with apoptosis.

"Our study raises some concern about the potential effects of DHC on humans," said Olaharski. "However, I would not be alarmed at this point, since we currently do not have any evidence that exposure to DHC in the diet accelerates the human aging process."

Olaharski said the findings highlight an emerging hypothesis regarding the delicate balance a cell must strike between tumor growth and aging. "Cancers and tumors are essentially cells that have uncontrolled or accelerated growth," he said. "In contrast, aging and apoptosis are processes involved in the halting of cell growth and eventually cell death. If there is too much tumor suppression, there may be no cancer, but the tissue may age more rapidly. It is interesting to speculate whether DHC present in the diet tips this balance towards enhanced tumor suppression, and on the potential ramifications this may have on human health."

The trick, researchers say, is to suppress tumor growth without triggering an acceleration of the aging process. The study suggests that properly functioning sirtuin enzymes - and the agents that inhibit or activate them - play a key role in that balance.

Contact: Sarah Yang scyang@berkeley.edu 510-643-7741 University of California - Berkeley

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