Oxysterol-induced cell death in U937 and HepG2 cells at reduced and normal serum concentrations

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.