O'Callaghan Y C, Woods J A, O'Brien N M
Department of Food Science and Technology, University College, Cork, Ireland.
Eur J Nutr. 1999 Dec;38(6):255-62. doi: 10.1007/s003940050075.
Cholesterol oxidation products (oxysterols) are commonly found in foods of animal origin and are also produced endogenously in the body. Oxysterols are cytotoxic to certain cell lines and in some cases have been shown to induce apoptosis. The aim of this study was to investigate the effects of 7beta-hydroxy-cholesterol (7beta-OHC) and 25-hydroxycholesterol (25-OHC) on cytotoxicity and induction of apoptosis in U937 and HepG2 cells, treated in media containing either 2.5% foetal calf serum (FCS) or 10% FCS to examine the effect of increasing the cholesterol level.
The cells were incubated for 24 and 48 h with 30 microM oxysterol. Viability was assessed by fluorescein diacetate/ethidium bromide staining and cell proliferation was determined by haemocytometer counting. Apoptosis was monitored by detection of DNA fragments (laddering) in 1.5% agarose gels. Cells with condensed or fragmented nuclei were identified by Hoechst 33342 staining. The percentage of cells with sub-G1 levels of DNA was measured by flow cytometry.
Treatment of U937 cells with 7beta-OHC, in contrast to 25-OHC, resulted in a decrease in cell viability and proliferation at 24 and 48 h (P <.01). 25-OHC and 7beta-OHC were both equally cytotoxic to the HepG2 cell line. 7beta-OHC induced DNA laddering and an increase in the percentage of condensed or fragmented nuclei at both time points and at both serum concentrations in the U937 cell line. 25-OHC induced faint laddering in the U937 cells after 48 h in reduced serum media and resulted in a small increase in percentage condensed or fragmented nuclei which was independent of time of oxysterol exposure and serum concentration. The percentage of condensed or fragmented nuclei was low in the HepG2 cell line and no laddering was observed under any of the conditions studied. Flow cytometric analysis showed that only 7beta-OHC treated U937 cells had an increased level of hypodiploid cells.
Both oxysterols appear to be equally cytotoxic to the HepG2 cell line. In U937 cells, 25-OHC is much less cytotoxic than 7beta-OHC. In addition, we have shown that 7beta-OHC induces apoptosis in U937 cells. 10% FCS displays a protective effect on cytotoxicity (as well as on 7beta-OHC induced apoptosis in U937 cells), although the data did not reach statistical significance.
胆固醇氧化产物（氧化甾醇）常见于动物源性食品中，也可在体内内源性产生。氧化甾醇对某些细胞系具有细胞毒性，且在某些情况下已显示可诱导细胞凋亡。本研究的目的是调查7β-羟基胆固醇（7β-OHC）和25-羟基胆固醇（25-OHC）在含有2.5%胎牛血清（FCS）或10% FCS的培养基中处理时，对U937和HepG2细胞的细胞毒性及凋亡诱导作用，以研究增加胆固醇水平的影响。
将细胞与30μM氧化甾醇孵育24小时和48小时。通过荧光素二乙酸酯/溴化乙锭染色评估细胞活力，通过血细胞计数器计数确定细胞增殖。通过检测1.5%琼脂糖凝胶中的DNA片段（梯状条带）监测细胞凋亡。用Hoechst 33342染色鉴定细胞核浓缩或碎片化的细胞。通过流式细胞术测量DNA亚G1水平细胞的百分比。
与25-OHC相比，用7β-OHC处理U937细胞在24小时和48小时时导致细胞活力和增殖下降（P<.01）。25-OHC和7β-OHC对HepG2细胞系的细胞毒性相同。在U937细胞系中，7β-OHC在两个时间点和两种血清浓度下均诱导DNA梯状条带形成以及浓缩或碎片化细胞核百分比增加。在低血清培养基中，25-OHC在48小时后诱导U937细胞出现微弱的梯状条带，并导致浓缩或碎片化细胞核百分比略有增加，这与氧化甾醇暴露时间和血清浓度无关。HepG2细胞系中浓缩或碎片化细胞核的百分比很低，在所研究的任何条件下均未观察到梯状条带。流式细胞术分析表明，只有用7β-OHC处理的U937细胞亚二倍体细胞水平增加。
两种氧化甾醇对HepG2细胞系的细胞毒性似乎相同。在U937细胞中，25-OHC的细胞毒性远低于7β-OHC。此外，我们已表明7β-OHC可诱导U937细胞凋亡。10% FCS对细胞毒性（以及对U937细胞中7β-OHC诱导的凋亡）具有保护作用，尽管数据未达到统计学显著性。
