Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair

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Abstract

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimutagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1) HCT116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1–3 weeks) treatment of HCT116 cells with VAN at minimally toxic concentrations (0.5–2.5 mM) reduced the spontaneous HPRT mutant fraction (MF, mutants/106 survivors) in a concentration-related manner by 19–73%. A similar treatment with CIN at 2.5–7.5 μM yielded a 13–56% reduction of the spontaneous MF. Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimutagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip®) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116 + chr3) and deficient (HCT116) cells at concentrations that were antimutagenic in HCT116 cells. There were 64 genes whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for four selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN are antimutagenic against spontaneous mutations in mammalian (human) cells. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair, which reduces spontaneous mutations.

Introduction

Epidemiological studies have demonstrated that certain components of the diet can have beneficial effects to health, such as reducing risks for various types of cancers [1], [2], [3], [4]. A wide array of dietary agents have been studied or are considered for chemoprevention of cancer [5], and many have antimutagenic activity in laboratory studies [6]. Vanillin (VAN) and cinnamaldehyde (CIN) are food flavorings that inhibit induced mutations in bacteria [7], [8], [9], [10] and mammalian cells [11], [12], [13], [14]. Although VAN and CIN inhibit spontaneous mutation in bacteria [15], [16], [17], [18], [19], this effect has not been reported in mammalian cells. VAN and CIN have been classified as bioantimutagens because they can modulate cellular processes such as DNA replication and repair [18]. Studies in bacteria led to the hypothesis that VAN and CIN inhibited mutagenesis by increasing repair of DNA damage through the recombinational repair pathway [10]. Later studies in Drosophila supported this view [20], [21].

We showed previously that VAN and CIN reduced spontaneous mutations in Salmonella TA104 at GC sites but not at AT sites and that this inhibition required the pKM101 plasmid [17]. Recently, we demonstrated that VAN and CIN required RecA recombinational repair, but not mismatch, SOS, or nucleotide excision repair, in order to reduce spontaneous mutations in Escherichia coli [19]. In addition, VAN, but not CIN, was actually mutagenic in a mismatch repair (MMR)-defective strain of E. coli [19]. Thus, we proposed that VAN and CIN produced DNA damage that elicited recombinational repair, but not other types of repair, resulting in a reduction of spontaneous mutation in bacteria [19].

To examine whether these compounds exhibited antimutagenic effects in mammalian (human) cells, we evaluated the ability of VAN and CIN to reduce spontaneous mutagenesis in mismatch repair-deficient (MMR) human colon cancer HCT116 cells. These cells have an elevated spontaneous HPRT mutation rate of ∼86 × 10−6 mutations/cell/generation, which is ∼19× higher than in the mismatch repair-corrected cell line HCT116 + chr3 (4.55 × 10−6) [22]. The spontaneous mutant fraction (MF) for the MMR HCT116 cells is ∼12.5× higher (150 × 10−6) than in MMR+ HCT116 + chr3 cells (12 × 10−6) [23]. Cells with a high spontaneous mutation rate are needed to effectively detect treatment-induced reductions in spontaneous mutagenesis. HCT116 cells were used to demonstrate the antimutagenicity of dietary antioxidants such as ascorbate, α-tocopherol, (−) epigallocatechin gallate (EGCG), and lycopene against spontaneous mutation [22], [24]. Lycopene was the most effective of these antimutagens, reducing the spontaneous mutation rate at HPRT by 70% [22].

Because VAN was mutagenic in mismatch repair-deficient E. coli [19] and both VAN and CIN required recombinational repair in order to be antimutagenic, we evaluated the reduction of the spontaneous MF at the HPRT locus in HCT116 cells as well as the induction of DNA damage (comet assay) by VAN and CIN in both HCT116 and HCT116 + chr3 cells. In addition, we examined the influence of VAN and CIN on global gene expression in these cells in order to identify genes and pathways that might play a role in the antimutagenic effects of these agents.

Section snippets

Chemicals

VAN (>98% pure, Sigma, St. Louis, MO) and CIN (trans-cinnamaldehyde, >99% pure, Aldrich, Milwaukee, WI) were dissolved in dimethyl sulfoxide (DMSO, Burdick and Jackson, Muskegon, MI), and stored at 4 °C as stock solutions at 5 M for VAN and 0.5 M for CIN. Fresh dilutions were made in medium immediately before each experiment. The final concentration of DMSO in the culture medium was 0.1%.

Cell culture

HCT116 cells are MMR-deficient (hMLH1) human colon cancer cells derived from a hereditary non-polyposis colon

Survival and antimutagenesis for VAN and CIN

Before antimutagenicity testing, the cytotoxicities of VAN and CIN were evaluated in 7-day “in situ” clonal survival assays and in 1–3-week exposure assays. In the 7-day clonal survival assays, survival curves were generated for HCT116 cells with 5–50-μM CIN and 0.5–5.0-mM VAN (data not shown). At the highest concentrations tested for both CIN and VAN, cell survival was 10% or less. We chose concentrations that produced ∼70% survival, 7.5 μM CIN and 2.5-mM, as the maximum concentration of each

Discussion

Antimutagenic effects of VAN and CIN against a variety of chemical and physical mutagens have been reported in numerous studies in bacteria [6], [7], [8], [9], mammalian cells [11], [12], [13], [14], [29], [30], and in vivo systems [11], [31], [32], [33]. Only a few studies have reported on the antimutagenic effects of VAN and CIN against endogenous mutations in bacteria [15], [16], [17] and for VAN in Drosophila [34]. In the present study, we found that VAN and CIN inhibited spontaneous

Acknowledgments

This research was supported in part by NIH R03 CA89732 (to C.B.K.), by the NYU/NIEHS Center ES000260, by the NYU Cancer Center CA016087, and by an NIEHS T32 ES007324 training grant (A.K.). This research was also supported in part by the Intramural Research Program of the National Institute of Environmental Health Sciences, NIH, DHHS. D.T.S. acknowledges support from an NIEHS Intramural Research Training Award. We thank the NIEHS Microarray Center for assistance with the gene expression results

References (48)

  • D.T. Shaughnessy et al.

    The antimutagenic effect of vanillin and cinnamaldehyde on spontaneous mutation in Salmonella TA104 is due to a reduction in mutations at GC but not AT sites

    Mutat. Res.

    (2001)
  • D.T. Shaughnessy et al.

    Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: dependence on recombinational repair

    Mutat. Res.

    (2006)
  • J.H. Santos et al.

    The synergistic effects of vanillin on recombination predominate over its antimutagenic action in relation to MMC-induced lesions in somatic cells of Drosophila melanogaster

    Mutat. Res.

    (1999)
  • K. Mure et al.

    Reduction of spontaneous mutagenesis in mismatch repair-deficient and proficient cells by dietary antioxidants

    Mutat. Res.

    (2001)
  • T.G. Rossman et al.

    Modeling and measurement of the spontaneous mutation rate in mammalian cells

    Mutat. Res.

    (1995)
  • N.P. Singh et al.

    A simple technique for quantitation of low levels of DNA damage in individual cells

    Exp. Cell Res.

    (1988)
  • Y.F. Sasaki et al.

    Effects of vanillin on sister-chromatid exchanges and chromosome aberrations induced by mitomycin C in cultured Chinese hamster ovary cells

    Mutat. Res.

    (1987)
  • K. Tamai et al.

    Different modifications by vanillin in cytotoxicity and genetic changes induced by EMS and H2O2 in cultured Chinese hamster cells

    Mutat. Res.

    (1992)
  • T. Inouye et al.

    Suppression of mitomycin C-induced micronuclei in mouse bone marrow cells by post-treatment with vanillin

    Mutat. Res.

    (1988)
  • Y.F. Sasaki et al.

    Suppressing effects of vanillin, cinnamaldehyde, and anisaldehyde on chromosome aberrations induced by X-rays in mice

    Mutat. Res.

    (1990)
  • T. Imai et al.

    Inhibitory effects of cinnamaldehyde on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung carcinogenesis in rasH2 mice

    Cancer Lett.

    (2002)
  • H.H. de Andrade et al.

    Suppressing effect of vanillin on chromosome aberrations that occur spontaneously or are induced by mitomycin C in the germ cell line of Drosophila melanogaster

    Mutat. Res.

    (1992)
  • H. Ka et al.

    Cinnamaldehyde induces apoptosis by ROS-mediated mitochondrial permeability transition in human promyelocytic leukemia HL-60 cells

    Cancer Lett.

    (2003)
  • L. Foucaud et al.

    Oxidative stress induction by short time exposure to ozone on THP-1 cells

    Toxicol. In Vitro

    (2006)
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