Abstract
Cholesterol oxidation products or oxysterols are of interest due to their hypothesized role in the development of atherosclerosis. The objective of the present study was to assess the cytotoxic effects of mixtures of oxysterols: 25-hydroxycholesterol (25-OHC), 7β-hydroxycholesterol (7β-OHC), and cholesterol-5β,6β-epoxide (β-epox) on two cell types associated with the atherosclerotic process, bovine aortic endothelial (BAE) cells and human monocytic U937 cells. Cells were exposed to 25-OHC, 7β-OHC, or β-epox, or equimolar mixtures (30 μM) of 25-OHC and 7β-OHC, 25-OHC and β-epox, or 7β-OHC and β-epox for 48 h. Cell viability was assessed using the fluorescein diacetate/ethidium bromide (FDA/ EtBr) assay and nuclear morphology following staining with Hoechst 33342. 25-OHC was the least toxic of the oxysterols and did not induce apoptosis in either cell line. Both 7β-OHC and β-epox treatments were cytotoxic and induced apoptosis in the cells. Cotreatment with 25-OHC did not alter the toxicity of 7β-OHC and β-epox in U937 cells but did decrease the percentage apoptotic cell death. In contrast, in the BAE cells cotreatment with 25-OHC had a slight protective effect on 7β-OHC and β-epox–induced toxicities and a marked decrease in apoptotic cell death. The 7β-OHC and β-epox mixture induced a significant increase in apoptotic cell death in U937 cells but decreased this mode of cell death in the BAE cells. The effects of oxysterols on glutathione levels also differed between the cells with changes noted in U937 and not in BAE cells. Results demonstrate interactive effects when oxysterols are studied as mixtures rather than single compounds in vitro.
Cholesterol oxidation products (oxysterols) comprise a large family of biologically active compounds derived from the oxidation of cholesterol both in vivo and in vitro. Plasma oxysterols arise from either absorption from dietary sources such as processed foods of animal origin (Bascoul, Domergue, and Crastes de Paulet 1985; Bascoul et al. 1986; Emanuel et al. 1991; Osada, Sasaki, and Sugano 1994) or may be produced endogenously. Endogenous production occurs during the enzymatic oxidation of cholesterol to bile acids and steroid hormones (Bosinger, Luf, and Brandl 1993). Nonenzymatic oxidation or auto-oxidation of cholesterol also occurs in vivo.
Oxysterols are a diverse group of compounds with various biological activities. In cell culture, oxysterols have been shown to be toxic to a variety of human and animal cell types including smooth muscle cells, fibroblasts, vascular endothelial cells, macrophages, and lymphocytes. In a number of these studies the mechanism of this toxicity was identified as apoptosis (Nishio and Watanbe 1996; Aupeix et al. 1995; Lizard et al. 1998, 2000; O’Callaghan, Woods, and O’Brien 2002; Ryan, O’Callaghan, and O’Brien 2004a). Many factors that induce apoptosis have also been shown to elicit an oxidative stress (Ghibelli et al. 1999). Lizard et al. (1998) demonstrated the involvement of reactive oxygen species (ROS) during oxysterol-induced apoptosis in vitro. Ryan, O’Callaghan, and O’Brien (2004a) reported decreased glutathione levels coupled with an increase in super-oxide dismutase (SOD) activity in U937 cells treated with the oxysterol 7β-hydroxycholesterol (7β-OHC). Glutathione forms the most abundant and important cellular antioxidant protecting the cell against ROS. Cellular glutathione depletion may be involved in the early stages of and may preceed the final commitment of cells to apoptotic death (Ghibelli et al. 1998, 1999).
In normal human plasma, average oxysterol concentrations are approximately 1 μM. In disease states, however, such as in hypercholesterolemia, concentrations may be elevated to as high as 20 to 30 μM (Schroepfer 2000). Accumulating evidence suggests certain oxysterols may play a role in the pathogenesis and progression of atherosclerosis (Leonarduzzi et al. 2004; Brown and Jessup 1999). Oxysterols have been implicated in the formation of lesions on the vascular cell wall, which initiate the atherosclerotic process (Berliner and Heinecke 1996). Lizard et al. (1999) examined the cytotoxicity of oxysterols to cells of the vascular cell wall in vitro and reported that the oxysterols 7β-OHC and 7-ketocholesterol (7-keto) induced apoptosis in endothelial and smooth muscle cells and necrosis in fibroblast cell lines. Both oxysterols have been shown to be present at high concentrations in atherosclerotic plaques (Brown et al. 1997). A human epidemiological study has indicated that raised plasma levels of 7β-OHC may be associated with an increased risk of atherosclerosis (Zieden et al. 1999). Oxidation of low-density lipoprotein (LDL) is considered as a key event in the pathogenesis of atherosclerosis. The cytotoxic effects of oxidised LDL appear to be associated with the presence of oxysterols. Yuan et al. (2000) demonstrated that oxysterols were the most potent component of oxidized LDL, capable of inducing cholesterol accumulation and eventual death to vascular cells.
Individual oxysterols have received considerable attention in cell culture models; however, little is known about the way in which oxysterols act collectively, as might occur in the in vivo environment. Equimolar amounts of 7-ketocholesterol and a mixture of oxysterols representative of that found in oxidized LDL were compared in terms of their apoptotic effects on cultured macrophages. Only the oxysterol alone strongly induced apoptosis (Leonarduzzi et al. 2001). More recently, Biasi et al. (2004) demonstrated that 7-ketocholesterol–induced apoptosis in U937 cells was largely attenuated in the presence of equimolar concentrations of 7β-OHC. The authors of these studies propose that mixtures of oxysterols like those occurring in oxidized LDL may be less toxic to vascular cells than single purified oxysterols.
The objective of the present study was to explore the potential antagonistic/synergistic effects of oxysterol mixtures. The cell lines chosen included a vascular cell, bovine aortic endothelial (BAE) cells, and a blood-derived cell, human U937 cells. The BAE cell line has previously been used to study the effects of oxysterols in vitro and has been shown to respond in a similar way to human aortic cells (Lizard et al. 1997). The oxysterols chosen were 7β-OHC, cholesterol-5β,6β-epoxide (β-epox), and 25-hydroxycholesterol (25-OHC). All three oxysterols are commonly found in foods. 7β-OHC is one of the more potent oxysterols in vitro and has previously been shown to induce apoptosis in U937 cells (Lizard et al. 1998; O’Callaghan, Woods, and O’Brien 1999, 2002; Lyons, Woods, and O’Brien 2001). Lizard et al. (1997) have previously shown that, in BAE cells, oxysterols oxidized at the C-7 position are more potent inducers of apoptosis than those oxidized on the side chain. The epoxide, β-epox, has also been shown to induce apoptosis in the U937 cell line (O’Callaghan, Woods, and O’Brien 2001; Ryan, O’Callaghan, and O’Brien 2004b). On the other hand, 25-OHC, a known inhibitor of hydroxymethylglutamyl coenzyme A (HMG-CoA) reductase, does not induce apoptosis in U937 cells but has previously been shown to have a cytostatic effect on these cells (O’Callaghan, Woods, and O’Brien 1999). In this study we report on the differential effects of mixtures of these oxysterols on BAE and U937 cells.
MATERIALS AND METHODS
All chemicals and tissue culture reagents were purchased from the Sigma Chemical Co. (Poole, UK) unless otherwise stated. Tissue culture plastics were supplied by Costar (Cambridge, UK). Information on the purity of oxysterols (purity >95%) was obtained from Sigma. Cell lines were obtained from European Collection of Animal Cell Cultures (Salisbury, UK).
Maintenance of Cell Lines
Human monocytic U937 cells were grown in suspension in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS). BAE cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM
Treatment of Cells with Oxysterols
BAE cells were adjusted to a density of 1 × 105 cells/ml and grown in complete medium (10% FBS) for 2 days in Costar 10-cm2 dishes prior to treatment. U937 cells were also adjusted to a density of 1 × 105 cells/ml. Oxysterols were added to give a final concentration of 30 or 60 μM of 25-OHC, 7β-OHC, or β-epox. The oxysterol mixtures consisted of equimolar amounts (30 μM each) of both 25-OHC and 7β-OHC (25-OHC + 7β-OHC), 25-OHC and β-epox (25-OHC + β-epox), and 7β-OHC and β-epox (7β-OHC + β-epox). The cells were exposed to the oxysterols or oxysterol mixtures for periods of 12 h and 48 h. Each of the oxysterols was dissolved in ethanol as a delivery vehicle and the final concentration of ethanol in the cultures did not exceed 0.25% (v/v). Equivalent quantities of ethanol were added to control cells. All treatments were carried out in reduced serum medium (2.5% FBS) and incubated at 37°C/5% CO2. In the case of BAE cells both floating and attached cells were collected for analysis.
Cell Viability
Following treatment, 25 μl of cells were removed for assessment of cell viability. Viability was monitored using a modification of the fluorochromemediated viability assay as described by Strauss (1991). Briefly, cells were mixed 1:1 (v/v) with a solution of fluorescein diacetate (FDA) and ethidium bromide (EtBr), then incubated at 37°C for 2 to 5 min before being layered onto a microscope slide. Under the fluorescence microscope, live cells fluoresce green, whereas dead cells fluoresce red. Samples were examined at 200× magnification on a Nikon fluorescence microscope using blue light (450 to 490 nm). Cells (200) were scored from each slide and cell viability was expressed as a percentage of viable (green) cells relative to the control.
Morphological Analysis
The nuclear morphology of the cells was assessed by fluorescence microscopy after staining with Hoechst 33342. Approximately 4 × 105 cells were centrifuged at 200 × g for 10 min to form a pellet. Hoechst 33342 stain (200 μl, 5 μg/ml) was added and the samples were incubated at 37°C/5% CO2 for 1 h. Stained samples (25 μl) were placed on a microscope slide and examined under ultraviolet (UV) light (Nikon Labophot fluorescence microscope 400× magnification). A total of 300 cells per sample were analyzed and the percentage of fragmented and condensed nuclei was calculated. Apoptotic cells were characterized by nuclear condensation of chromatin and/or nuclear fragmentation (Dubrez et al. 1996).
Determination of Cellular Glutathione Levels
The cellular level of glutathione was measured according to the method of Hissin and Hilf (1976). Briefly, 4× 106 cells were centrifuged at 100,000 × g for 20 min. The supernatant (100 μl) was diluted in 1.8 ml phosphate-EDTA buffer (0.1 mol/L sodium phosphate, 0.005 mol/L EDTA, pH 8) and mixed with 100 μl o-phthalaldehyde (1 μg/ml). Following incubation at 25°C for 15 min, the fluorescence at 420 nm was detected after activation at 350 nm. Results were expressed in nmols reduced glutathione (GSH)/mg protein. Protein was determined by the bicinchoninic acid (BCA) method (Smith et al. 1985).
Statistics
All data points are mean values (±SE) of at least three independent experiments. Where appropriate, data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (Prism 3.0). Data described as different in this report are significantly different p <.05.
RESULTS
Cytotoxicity of Oxysterols to U937 and BAE Cells
The viability of control samples was found to be 97.5% in the U937 cell line and 82% in the BAE cell line. At 30 μM concentration, all oxysterols were found to be more toxic to the BAE cell line than U937 cells (Figure 1). However, the trend observed was the same for both cell lines, with 25-OHC as the least cytotoxic, followed by 7β-OHC and β-epox. At the higher concentration (60 μM), the toxicity of 25-OHC was increased in both cell lines with a more apparent effect in the BAE cell line. The toxicity of 7β-OHC was also increased at the higher concentration; however, the toxicity of β-epox appeared to be unaffected by increasing concentration (Figure 1).
Incubation of BAE or U937 cells with equimolar concentrations (30 μM each) of 25-OHC and 7β-OHC produced similar results to those obtained for cells incubated with 30 μM of 7β-OHC alone. Similarly, treatment of both cell types with the 25-OHC + β-epox mixture resulted in a decrease in viability similar to that observed in cells treated with 30 and 60 μM β-epox only. Therefore, cotreatment with 25-OHC did not appear to affect the toxicity of either 7β-OHC or β-epox in either cell line. In the U937 cell line, treatment with the 7β-OHC + β-epox mixture was found to be more toxic (p < .05) than treatment with 30 μM 7β-OHC alone (15.6% and 38.4% viability, respectively). In the BAE cell line, a similar decrease in viability was observed in cells treated with 7β-OHC alone (18.6%), β -epox alone (11.5%), and in those cells incubated with the 7 β-OHC + β-epox mixture (14.6%). Therefore, the 7β-OHC + β-epox mixture exacerbated the toxic effects in U937 cells but not in the BAE cells.
Morphological Analysis of Cell Nuclei
Condensed fragmented nuclei were determined by morphological assessment after staining with Hoechst 33342, following 48 h of incubation with the various treatments. The percentage of apoptotic cells in controls did not exceed 3% (Figure 2). In both U937 and BAE cells, 25-OHC did not induce a significant (p < .05) level of apoptosis above control at either 30 or 60 μM concentrations. On the other hand, percentage apoptosis were significantly higher (p < .05) than control in both U937 and BAE cells exposed to 7β-OHC (30 μM) and β-epox (30 μM). The percentage apoptosis induced by 7β-OHC and β-epox was greater in the BAE cells compared to U937 cells. In BAE cells, the level of apoptosis induced by 7β-OHC and β-epox did not increase significantly with increasing concentration. Similarly in U937 cells, the level of apoptosis induced by 7β-OHC was not concentration dependent, whereas it appears to be so with β-epox (Figure 2).
The percentage apoptosis in U937 cells treated with 25-OHC + 7β-OHC and 25-OHC + β-epox was half that seen in cells exposed to 7β-OHC and β-epox individually (Figure 2). Thus, cotreatment with 25-OHC appears to decrease the apop-totic potency of these two oxysterols in U937 cells. On the other hand, percentage apoptosis induced in the U937 cell treated with the 7β-OHC + β-epox mixture was significantly greater (p < .05) than that of either of the oxysterols alone. In the BAE cell line, cotreatment with 25-OHC decreased the apoptotic potency of both 7β-OHC and β-epox. However, treatment with the 7β-OHC + β-epox mixture resulted in less apoptosis compared to the compounds individually.
Cellular Glutathione Content after 12 Hours
Changes in cellular glutathione levels may be an early event in oxysterol-induced apoptosis. In the present study cellular glutathione content was measured after 12 h incubation with the various treatments (Figure 3). In U937 cells, treatment with 7β-OHC and the 7β-OHC + β-epox mixture resulted in significant (p < .001) reduction in cellular glutathione content. Thus, apoptosis induced in U937 in response to 7β-OHC and the 7β-OHC + β-epox mixture was preceded by a significant reduction in cellular glutathione levels. In contrast, apoptosis induced by β-epox treatment was not preceded by a decrease in cellular glutathione content in U937 cells. In BAE cells oxysterol-induced apoptosis did not appear to be preceded by a reduction in cellular glutathione content.
DISCUSSION
To date, the majority of cellular in vitro studies involving oxysterols have administered the compounds individually whereas few studies have examined the potential synergistic/antagonistic effects of these compounds. Thus, in this study we used a mixture of oxysterols, which are found both in the diet and in human plasma. There is strong evidence implicating 7β-OHC in the development of atherosclerotic plaques (Brown and Jessup 1999), and it has previously been shown to induce apoptosis in U937 cells at a concentration of 30 μM (O’Callaghan, Woods, and O’Brien 1999). β-Epox has also been found to be highly cytotoxic to U937 cells; however, the mechanism of toxicity appears to differ from that of 7β-OHC (Ryan, O’Callaghan, and O’Brien 2004b). 25-OHC was found not to be cytotoxic to U937 cells (O’Callaghan, Woods, and O’Brien 1999) and was investigated in the present study to determine if it has an ameliorating effect on the toxicity induced by either 7β-OHC or β-epox. We employed two cell lines, an aortic cell line and a macrophage cell line, BAE and U937 cells, respectively.
In the present study, when U937 cells were treated simultaneously with 7β-OHC and β-epox the percentage cell viability was 15.6% and death was 40% apoptotic, whereas when the BAE cells were subjected to the same treatment the majority of the cells died by necrosis (Figures 1 and 2). Therefore, although both 7β-OHC and β-epox induced apoptosis when administered alone to BAE cells, it seems when these apoptotic agents are given together as an equimolar mixture the cells respond with passive or necrotic cell death. Leonarduzzi et al. (2001) compared the effects of equimolar amounts (30 μM) of 7-keto with a mixture of oxysterols, representative of that found in oxidized LDL, on cultured murine macrophages. When 7-keto was administered to the cells alone, cell death was largely by apoptosis. However, when 7-keto was coincubated with the oxysterol mixture, cell viability was reduced by 10% to 15% and the mode of cell death was not characterized as apoptosis. In the present study we found a differential response in the two cell lines employed. In U937 cells, 25-OHC, a nonapoptotic oxysterol, did not alleviate the toxicity of 7β-OHC and β-epox. However, it decreased the percentage apoptotic cell death induced by 7β-OHC and β-epox and hence its presence increased death by necrosis. In the BAE cells, 25-OHC may have had a very slight protective effect on the cytotoxicity of 7β-OHC and β-epox and had a dramatic effect on decreasing the percentage apoptotic cell death induced by these oxysterols. When the two proapoptotic oxysterols were incubated together, apoptosis was strongly induced in the U937 cells (40.6%), whereas in the BAE cell line the mixture was much less apoptotic compared to the individual oxysterols. Therefore, simultaneous administration of two proapoptotic oxysterols to BAE cells induced a switch from apoptotic to necrotic cell death. There have been very few reports on the possible quenching effects of oxysterols in a mixture. Aupeix et al. (1995) described that the cotreatment of U937 cells with 7β-OHC and 25-OHC led to a decrease in cytotoxicity induced by these oxysterols when added singly. In an earlier study we reported that simultaneous addition of equal concentrations of 7β-OHC and 25-OHC (30 μM) to U937 cells resulted in a slight but significant increase in the percentage of viable cells relative to cells treated with 7β-OHC only. This corresponded with a decrease in the percentage of apoptotic nuclei, relative to cells treated with 7β-OHC alone (Lyons, Woods, and O’Brien 2003). Biasi et al. (2004) reported that coincubation of U937 cells with equimolar concentrations (20 μM) of two strongly apoptotic oxysterols, 7-keto and 7β-OHC, resulted in the attenuation of apoptosis. Both these oxysterols induce apoptosis by similar signalling pathways in U937 cells (Miguet-Alfonsi et al. 2002). Interestingly, more recent work from our laboratory suggests that the proapoptotic oxysterols 7β-OHC and β-epox induce apoptosis by different signaling pathways in U937 cells (Ryan, O’Callaghan, and O’Brien 2004b). For example, apoptosis induced by 7β-OHC is accompanied by changes in cellular glutathione levels status whereas this is not seen with β-epox–induced apoptosis. On the other hand, Biasi et al. (2004) suggest the proapoptotic effects of 7-ketocholesterol appeared to rely on its ability to up-regulate intracellular ROS levels, and cotreat-ment with 7β-OHC may competitively inhibit production of ROS resulting in protection against apoptosis.
It has been proposed that the generation of an oxidative stress (mediated by ROS production) is a key governing factor in oxysterol-induced apoptosis and/or cytotoxicity (Lizard et al. 1998). Moreover, it was found that the addition of an endogenous antioxidant such as glutathione (GSH) or an exogenous antioxidant such as a-tocopherol prevented oxysterol activation of the apoptotic pathway (Lizard et al. 1998; Ghibelli et al. 1998; Lyons, Woods, and O’Brien 2001). It has previously been suggested that loss of GSH may be the earliest cellular change associated with apoptosis triggered by an oxysterol, because none of the morphological changes consistent with apoptosis precede GSH depletion (Lizard et al. 1998). In our study, apopto-sis induced in U937 in response to 7β-OHC and the 7β-OHC + β-epox mixture was preceded by a significant reduction in cellular glutathione levels. However, there was no observed reduction the cellular glutathione content of BAE cells following oxysterol treatment. Therefore, oxysterol-induced cytotoxicity in the BAE cells does not appear to be related to a decrease in glutathione and apoptosis may occur by an alternative pathway in this cell line. A number of apoptotic pathways which do not involve a depletion of glutathione have previously been described (Ghibelli et al. 1999) however the signalling pathway initiated by oxysterols in the BAE cells has yet to be elucidated.
The present study demonstrates how two cell types may respond differently to the same oxysterol mixture. Damage to these cell types in vivo would assist in the development of atherosclerosis. An increased concentration of proapoptotic or necrotic agents in vivo could have a deleterious effect on the vascular endothelium and oxysterol-induced macrophage cell death may contribute to plaque formation. Further work is needed with oxysterol mixtures representative of normal plasma concentrations in order to elucidate the mode of cell death and gain a better understanding of the likely interactive effects of these compounds. A full understanding of the mechanisms involved in oxysterol-induced apoptosis would facilitate the development of improved therapies against the development of atherosclerosis.