Yeh S L, Hu M L
Department of Food Science, National Chung-Hsing University, 250 Kuo-Kuang Road, Taichung, Taiwan ROC.
Free Radic Res. 2001 Aug;35(2):203-13. doi: 10.1080/10715760100300751.
Two recent clinical trials suggest that beta-carotene may be harmful to smokers. In this study we examined the hypothesis that beta-carotene may become toxic when degradation occurs. beta-Carotene (BC) and lycopene (LP) with or without prior heat treatment (60 degrees C for 1 h in open air) were incubated at 20 and 40 microM with calf thymus DNA or human fibroblasts Hs68 cells. The heat treatment resulted in ca. 80% and 35% bleaching of BC and LP, respectively. When Hs68 cells were incubated with the oxidized beta-carotene (OBC) or oxidized lycopene (OLP) at 37 degrees C for 20 h, cell viability was significantly and dose-dependently decreased whereas cell viability was not affected by BC or LP. Cell death, which was already evident at 4 h after incubation with OBC or OLP, was possibly attributable to apoptosis, as shown by the increased histone-associated DNA fragmentation. However, cell lysis, measured as release of lactate dehydrogenase, also occurred at 4 h after incubation with OBC and OLP, although the extent was relatively small and was greater for OLP than for OBC. When calf thymus DNA was incubated with OBC or OLP at 37 degrees C for 20 h, the 8-hydroxy-2-deoxyguanosine (8-OH-dG) level was significantly and dose-dependently increased by OLP whereas the increase by OBC was only significant at 40 microM. When Hs68 cells were incubated with OBC and OLP for 20 h, both compounds increased the 8-OH-dG level, but the effect was only significant for 40 microM OLP. Comet (single-cell gel electrophoresis) assay of DNA damage in Hs68 cells was determined at 2 h after incubation with OBC or OLP because of its high sensitivity. Both OBC and OLP significantly and dose-dependently increased DNA breakage while BC and LP had no effect. Inclusion of BHT during incubation of cells with 40 microM OBC or OLP partially inhibited (ca. 40%, p < .05) the extent of comet formation. Intriguingly, OBC and OLP neither induce lipid peroxidation in Hs68 cells (measured as thiobarbituric acid-reactive substances released into the medium) nor increased the intracellular level of reactive oxygen species. Although it is presently unclear about what degradation products are formed, this study has demonstrated that, when oxidized, BC and LP lead to oxidative damage to both purified DNA and cellular DNA. The results suggest that such damage may contribute to the adverse effects of beta-carotene reported in recent clinical studies and caution that it is important to prevent oxidation of BC and LP for human uses such as in supplemental studies.
两项近期的临床试验表明，β-胡萝卜素可能对吸烟者有害。在本研究中，我们检验了这样一个假设：当β-胡萝卜素发生降解时可能会产生毒性。将经过或未经预先热处理（在空气中60摄氏度处理1小时）的β-胡萝卜素（BC）和番茄红素（LP），分别以20和40微摩尔的浓度与小牛胸腺DNA或人成纤维细胞Hs68细胞一起孵育。热处理分别导致BC和LP约80%和35%的褪色。当Hs68细胞与氧化型β-胡萝卜素（OBC）或氧化型番茄红素（OLP）在37摄氏度孵育20小时时，细胞活力显著且呈剂量依赖性降低，而细胞活力不受BC或LP的影响。与OBC或OLP孵育4小时后就已明显出现的细胞死亡，可能归因于细胞凋亡，这可通过组蛋白相关的DNA片段增加来表明。然而，以乳酸脱氢酶释放量衡量的细胞裂解，在与OBC和OLP孵育4小时后也会发生，尽管程度相对较小，且OLP比OBC更明显。当小牛胸腺DNA与OBC或OLP在37摄氏度孵育20小时时，OLP会显著且呈剂量依赖性地增加8-羟基-2'-脱氧鸟苷（8-OH-dG）水平，而OBC仅在40微摩尔时增加才显著。当Hs68细胞与OBC和OLP孵育20小时时，两种化合物都会增加8-OH-dG水平，但仅40微摩尔的OLP这种作用才显著。由于其高灵敏度，在与OBC或OLP孵育2小时后对Hs68细胞中的DNA损伤进行彗星（单细胞凝胶电泳）分析。OBC和OLP都显著且呈剂量依赖性地增加DNA断裂，而BC和LP则没有影响。在细胞与40微摩尔OBC或OLP孵育期间加入丁基羟基甲苯（BHT）可部分抑制（约40%，p < 0.05）彗星形成的程度。有趣的是，OBC和OLP既不会在Hs68细胞中诱导脂质过氧化（以释放到培养基中的硫代巴比妥酸反应性物质来衡量），也不会增加细胞内活性氧的水平。虽然目前尚不清楚会形成哪些降解产物，但本研究已证明，氧化时BC和LP会导致对纯化DNA和细胞DNA的氧化损伤。结果表明，这种损伤可能是近期临床研究中报道的β-胡萝卜素产生不良反应的原因，并警示在诸如补充研究等人类用途中防止BC和LP氧化很重要。
