Glaab V, Collins A R, Eisenbrand G, Janzowski C
University Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, 67663, Kaiserslautern, Germany.
Mutat Res. 2001 Oct 18;497(1-2):185-97. doi: 10.1016/s1383-5718(01)00260-1.
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 1h incubation in V79 cells (LC(50): 4.75mM). The 2-alkenals ((E)-2-octenal (OCTE), (2E,4Z)-2,4-hexadienal (HEXDI), (E)-2-nonenal (NONE), (2E,6Z)-2,6-nonadienal (NONDI)) exhibited a distinctly higher cytotoxicity, except for (E)-2-hexenal (HEX) (LC(50): 3.67mM) and cinnamaldehyde (CA) (LC(50): 4.45mM). If the incubation time was prolonged to 24h, an IC(50) of 15microM was obtained for CHX which is well within the range obtained for the 2-alkenals (4 and 17microM). Concentration-dependent DNA damage was observed after 1h incubation with CHX. The respective DC(50) values (concentration inducing DNA damage in 50% of cells) were 272microM (V79) and 455microM (Caco-2). All 2-alkenals were more active under these conditions, except for CA. In primary human colon cells, CHX (800microM, 30min) exhibited a weak, but still significant DNA-damaging potential. Glutathione levels in V79 cells were effectively depleted (down to approximately 20%) by CHX concentrations not yet inducing DNA damage (c < or = 50microM). Incubation with CHX or 2-alkenals (50 and 100microM, 1h), followed by H2O2 treatment (5min, 25microM) 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.
2-环己烯-1-酮（CHX）作为天然成分存在于一些热带水果中，并且在某些人工甜味软饮料中被检测为污染物。为了阐明其细胞毒性/遗传毒性作用，将CHX与结构相关的2-烯醛相比，在哺乳动物细胞系（V79和Caco-2）以及原代人结肠细胞中进行测试。分别通过用磺酰罗丹明B（SRB）进行蛋白质染色和台盼蓝排斥法测定细胞生长抑制（IC(50)）和细胞毒性（LC(50)）。通过彗星试验对DNA损伤（双链断裂和氧化嘌呤）进行定量。基于5-硫代-2-硝基苯甲酸（TNB）的形成，在动力学试验中测量谷胱甘肽的消耗。对于CHX，在V79细胞中孵育1小时后观察到中等细胞毒性（LC(50)：4.75mM）。除了（E）-2-己烯醛（HEX）（LC(50)：3.67mM）和肉桂醛（CA）（LC(50)：4.45mM）外，2-烯醛（（E）-2-辛烯醛（OCTE）、（2E,4Z）-2,4-己二烯醛（HEXDI）、（E）-2-壬烯醛（NONE）、（2E,6Z）-2,6-壬二烯醛（NONDI））表现出明显更高的细胞毒性。如果孵育时间延长至24小时，CHX的IC(50)为15μM，这完全在2-烯醛所获得的范围内（4至17μM）。与CHX孵育1小时后观察到浓度依赖性DNA损伤。各自的DC(50)值（在50%的细胞中诱导DNA损伤的浓度）为272μM（V79）和455μM（Caco-2）。在这些条件下，除了CA外，所有2-烯醛都更具活性。在原代人结肠细胞中，CHX（800μM，30分钟）表现出微弱但仍显著的DNA损伤潜力。尚未诱导DNA损伤的CHX浓度（c≤50μM）可有效消耗V79细胞中的谷胱甘肽水平（降至约20%）。用CHX或2-烯醛（50和100μM，1小时）孵育，随后进行H2O2处理（5分钟，25μM），在改良彗星试验中导致氧化嘌呤水平升高。在原代人结肠细胞中额外测试的CHX和HEX消耗了谷胱甘肽并增加了对氧化应激的敏感性。
