Epigenetic drift is hypothesized to occur, in part, through exposure to environmental and dietary compounds that affect the processes responsible for the proper maintenance of the proteome and epigenome [27
]. The recent discovery that significant differences in epigenetic markers exist in monozygotic twins who lived different lifestyles and spent little time together supports this environmentally driven epigenetic drift hypothesis [29
]. Additionally, the finding that methoxychlor, a common DDT substitute used to control mosquitoes, and vinclozolin, a broadly used fungicide applied in the wine industry [30
], caused transgenerational epigenetic effects up to the F4 generation [31
] indicates that humans are likely exposed to epigenetic toxicants causing epigenetic drift on a regular basis. These are intriguing findings that raise the question of whether the diet and environment contain epigenetic toxicants that may also inhibit the sirtuin deacetylases.
We have designed a yeast mating assay to identify environmental chemicals that inhibit heterochromatic repression in S. cerevisiae
to address the possibility that humans are exposed to epigenetic toxicants that inhibit sirtuin deacetylases. After screening more than 100 environmental chemicals to which humans are exposed, including coumarins, bioflavonoids, benzene metabolites, and arsenic, we identified that DHC disrupted heterochromatic repression (see A). Further analyses demonstrated that DHC-mediated heterochromatic derepression caused yeast colony formation in a concentration-dependent manner that was similar to splitomicin (B and C). Splitomicin is an established Sir2p inhibitor [32
] with a structure similar to DHC, thus it was likely that DHC-mediated heterochromatic derepression in the mating assay was due to Sir2p inhibition. Further experiments with an overexpressing SIR2
-induced death phenotype were conducted to identify if Sir2p was the target of DHC. DHC-mediated reversal of the SIR2
overexpressed death phenotype () indicated that DHC is a Sir2p inhibitor and that this inhibition was responsible for the heterochromatic derepression observed in the mating assay. DHC thus joins a short list of established Sir2p inhibitors that includes nicotinamide, splitomicin, and sirtinol [33
The identification that DHC is a Sir2p inhibitor is of particular interest because it is a natural component of M. officinalis
and is synthetically manufactured for use as a common fragrance in perfumes, cosmetics, lotions, soaps, and as a flavoring agent in beverages and chewing gum [23
]. DHC is present at concentrations above 100 ppm (670 μM) in gelatins, puddings, soft candy, frozen dairy products, and baked goods [22
]. Due to the potential DHC exposure to humans, we tested whether it could also inhibit human sirtuin deacetylases and identified that DHC inhibited both SIRT1 and SIRT2 deacetylases in vitro (A and B). We also measured SIRT3 deacetylase activity but did not identify it to be affected by DHC (data not shown). It is only recently that the cellular function of the human sirtuin deacetylases has begun to be revealed. Because there are at least seven human sirtuins [1
], it is expected that the substrates and functions of each will vary. SIRT2 is a cytoplasmic protein identified to be a microtubule deacetylase [26
], an observation that may help explain its requirement for proper progression through mitosis [35
]. SIRT1 has been identified to deacetylate a variety of nuclear substrates [9
], including several that are transcription factors regulating differentiation and development or proteins involved in the apoptotic response, including FOXO [12
], ku70 [11
], p53 [10
], and MEF2 [37
]. Although SIRT1 has been identified to have mostly nonhistone targets, there is growing evidence to indicate that SIRT1 interacts with a number of transcription factors with results that may mimic epigenetic modifications. How SIRT1 inhibitors can affect these processes is just beginning to be understood.
Because SIRT1 has been identified to deactivate p53 through deacetylation, experiments in the human TK6 lymphoblastoid cell line were conducted to identify if DHC could induce a phenotype consistent with SIRT1 inhibition and p53 activation. DHC was identified to increase p53 acetylation, cytotoxicity, and apoptosis levels in vitro (). In addition, DHC enhanced cell killing to etoposide in both the TK6 human lymphoblastoid and human embryonic kidney 293 cell lines (data not shown), data compatible with the observation that SIRT1−/−
cells are more susceptible to killing by genotoxic agents [18
]. Previous in vivo and in vitro studies have demonstrated that DHC is not a mutagen, clastogen, or aneugen [34
], suggesting that it is unlikely that DHC is stabilizing p53 and increasing cytotoxicity and apoptosis through a genotoxic mechanism. Rather, these data are consistent with the phenotypes of SIRT1 compromised cells [10
] and support the hypothesis that DHC-mediated SIRT1 inhibition enhances p53 stabilization and increases apoptosis. Increasing tumor suppression and the susceptibility of a cell to undergo apoptosis, however, cannot solely be viewed in a beneficial chemotherapeutic light, particularly in those cases where exposure to an apoptosis-inducing agent may be widespread. The deleterious effects of a hyperactive p53 have been demonstrated in a transgenic mouse model that displayed a shortened life span and a variety of early aging-associated phenotypes [38
]. It is hypothesized that increased levels of apoptosis cause a rapid depletion of stem cells, leading to premature tissue senescence [20
], possibly decreasing longevity. Increasing p53 tumor suppression through inhibition of SIRT1 may tip the cell-survival balance toward apoptosis in a cell that would otherwise not undergo programmed cell death. It is interesting to speculate whether sirtuin deacetylases, which have a role in regulating longevity in a variety of organisms, may also regulate longevity in mammals through the control of apoptosis. Further work is clearly needed to address the possibility that enhanced apoptosis through chemical-mediated inhibition of SIRT1 can increase tissue senescence and affect longevity.
Our finding that the common flavoring agent DHC inhibits sirtuin deacetylases linked to aging is potentially worrisome. SIRT1 inhibition may lead to epigenetic alterations as well as possible stem cell depletion and early tissue senescence due to increased levels of apoptosis. While no compounds to our knowledge are currently classified as “senescegens,” it is possible that a number of environmental chemicals can increase tissue senescence and aging. The present study demonstrates that a more extensive screening of dietary and environmental chemicals is needed to identify other epigenetic toxicants to which humans are commonly exposed.