DNA-damaging potential and glutathione depletion of 2-cyclohexene-1-one in mammalian cells, compared to food relevant 2-alkenals

Abstract 2-Cyclohexene-1-one (CHX) occurs as a natural ingredient in some tropical fruits and has been detected as a contaminant in certain artificially sweetened soft drinks. To elucidate its cytotoxic/genotoxic effectiveness, CHX was tested in mammalian cell lines (V79 and Caco-2) and in primary human colon cells in comparison to structurally related 2-alkenals. Inhibition of cell growth (IC 50 ) and cytotoxicity (LC 50 ) were determined by protein staining with sulforhodamin B (SRB) and by trypan blue exclusion, respectively. DNA damage — both strand breaks and oxidised purines — was quantified by comet assay. Depletion of glutathione was measured in a kinetic assay, based on 5-thio-2-nitrobenzoate (TNB) formation. For CHX, a moderate cytotoxicity was observed after 1 h incubation in V79 cells (LC 50 : 4.75 mM). The 2-alkenals (( E )-2-octenal (OCTE), (2 E ,4 Z )-2,4-hexadienal (HEXDI), ( E )-2-nonenal (NONE), (2 E ,6 Z )-2,6-nonadienal (NONDI)) exhibited a distinctly higher cytotoxicity, except for ( E )-2-hexenal (HEX) (LC 50 : 3.67 mM) and cinnamaldehyde (CA) (LC 50 : 4.45 mM). If the incubation time was prolonged to 24 h, an IC 50 of 15 μM was obtained for CHX which is well within the range obtained for the 2-alkenals (4 and 17 μM). Concentration-dependent DNA damage was observed after 1 h incubation with CHX. The respective DC 50 values (concentration inducing DNA damage in 50% of cells) were 272 μM (V79) and 455 μM (Caco-2). All 2-alkenals were more active under these conditions, except for CA. In primary human colon cells, CHX (800 μM, 30 min) exhibited a weak, but still significant DNA-damaging potential. Glutathione levels in V79 cells were effectively depleted (down to ≈20%) by CHX concentrations not yet inducing DNA damage ( c ≤50 μM). Incubation with CHX or 2-alkenals (50 and 100 μM, 1 h), followed by H 2 O 2 treatment (5 min, 25 μM) resulted in increased levels of oxidised purines in the modified comet assay. CHX and HEX, additionally tested in primary human colon cells, depleted glutathione and increased the sensitivity towards oxidative stress.