ABSTRACT
Methyleugenol is a substituted alkenylbenzene found in several herbs and spices. It is classified by the European Union's Scientific Committee on Food as a genotoxic carcinogen. We addressed the biological mechanism of the genotoxic properties of methyleugenol and its oxidative metabolites. Methyleugenol and the oxidative metabolites significantly enhanced the DNA damage in human colon carcinoma cells (HT29). Methyleugenol did not affect the protein status of γH2AX, a biomarker of DNA double-strand breaks, whereas its metabolites methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol significantly increased the cellular phosphorylated H2AX level. Both of these metabolites also showed a significant induction of micronuclei in HT29 cells. Furthermore, we investigated whether topoisomerase interaction contribute to the observed effect on DNA integrity. Methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol inhibited the activity of recombinant topoisomerase I. In HT29 cells, neither methyleugenol nor the metabolites affected the level of topoisomerase protein bound to DNA, excluding a topoisomerase poisoning mode of action. In addition, 3'-oxomethylisoeugenol potently diminished the level of camptothecin-stabilized topoisomerase I/DNA intermediates and camptothecin-induced DNA strand breaks. In conclusion, it could be suggested that 3'-oxomethylisoeugenol may also interact with classical or food-borne topoisomerase I poisons, diminishing their poisoning effectiveness.
Subject(s)
Carcinogens, Environmental/toxicity , Colonic Neoplasms/chemically induced , DNA Damage , DNA Topoisomerases, Type I/metabolism , Eugenol/analogs & derivatives , Mutagens/toxicity , Topoisomerase I Inhibitors/toxicity , Biomarkers, Tumor/agonists , Biomarkers, Tumor/metabolism , Biotransformation , Carcinogens, Environmental/analysis , Carcinogens, Environmental/metabolism , Carcinoma/chemically induced , Carcinoma/enzymology , Colonic Neoplasms/enzymology , Colonic Neoplasms/metabolism , Comet Assay , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/genetics , Epoxy Compounds/analysis , Epoxy Compounds/metabolism , Epoxy Compounds/toxicity , Eugenol/analysis , Eugenol/metabolism , Eugenol/toxicity , Food Contamination , HT29 Cells , Histones/agonists , Histones/metabolism , Humans , Micronucleus Tests , Mutagens/analysis , Mutagens/metabolism , Neoplasm Proteins/agonists , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Oxidation-Reduction , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spices/adverse effects , Spices/analysis , Topoisomerase I Inhibitors/analysis , Topoisomerase I Inhibitors/metabolismABSTRACT
Various ursanic, oleanic and lupanic pentacyclic triterpenoids found in apple peel were studied for anti-inflammatory effects in vitro using T84 colon carcinoma cells. After pretreatment with single triterpenoids, cells were stimulated with pro-inflammatory cytokines (TNF-α, INF-γ, IL-1ß). Regulation of mRNA expression was analysed for three specific inflammation-associated marker genes (TNF-α, IL-8, IP-10) using qRT-PCR. Furthermore, the effects of ursolic acid (UA) and oleanolic acid (OA) on the synthesis of certain pro-inflammatory proteins were examined. IP-10 expression was inhibited in a dose-dependent manner by all the tested compounds at concentrations ≥25 µM. The mRNA expression of TNF-α was slightly affected and the IL-8 level was increased. At the protein level, UA and OA (25 µM) reduced the synthesis of IP-10; sICAM-1, IL-23 and GROα were slightly repressed. The TNF-α level was not modulated, whereas induction of IL-8 was increased. UA also enhanced the synthesis of IL-1ra, while OA suppressed the level of I-TAC. The present study confirms that triterpenoids present in apple peel and ß-damascone may be implicated in the anti-inflammatory properties of apple constituents, suggesting that these substances might be helpful in the treatment of IBD as nutrient supplements.
