Genotoxic potential of methyleugenol and selected methyleugenol metabolites in cultured Chinese hamster V79 cells

Abstract

Methyleugenol is a substituted alkenylbenzene classified by the European Union's Scientific Committee on Food as a genotoxic carcinogen. We addressed cytotoxicity, genotoxicity and mutagenicity caused by methyleugenol and selected oxidative methyleugenol metabolites in Chinese hamster lung fibroblasts V79 cells. Cytotoxicity was measured by two cell proliferation assays, water soluble tetrazolium salt (WST) 1 and sulforhodamine B (SRB) assays. Genotoxicity was determined by using single cell gel electrophoreses (comet assay) and the in vitro micronuclei test, while mutagenicity was investigated with the hypoxanthinephosphoribosyl transferase (hprt) assay. Methyleugenol and 1′-hydroxymethyleugenol showed no or marginal cytotoxic effects, but caused DNA strand breaks at concentrations ≥10 μM. The metabolites methyleugenol-2′,3′-epoxide and 3′-oxomethylisoeugenol exhibited growth inhibitory properties with IC₅₀-values of 70–90 μM after 48 h or 72 h of incubation. These metabolites significantly enhanced cytotoxicity and DNA damage after 1 h of incubation. Overall, no increase in formamidopyrimidine DNA glycosylase sensitive sites were detected with the comet assay. The metabolites 1′-hydroxymethyleugenol and methyleugenol-2′,3′-epoxide exceeded the DNA strand breaking properties of the parent compound methyleugenol. However, only 3′-oxomethylisoeugenol and methyleugenol-2′,3′-epoxide induced the formation of micronucleated cells in comparison to the negative control. These compounds were found to be not or rather weakly mutagenic at the hprt locus. In summary, phase I metabolites exceeded the cytotoxic and genotoxic properties of the parent compound methyleugenol.