The steatotic and cytotoxic effects of cholesterol oxides in cultured L cells (original) (raw)
Related papers
Arteriosclerosis and thrombosis, 1991
Human umbilical vein endothelial cells were cultured in the presence of several oxygenated cholesterol derivatives that are known to affect the viability of other cell lines. 5-Cholestene-3/3,7/J-diol (70-hydroxycholesterol) caused a time-and concentration-dependent perturbation of the endothelial cells. Exposure to 50 /tmol/1 of this compound for 18 hours resulted in marked contraction of the cells, followed by increasing cell detachment from the substrate and Trypan Blue uptake in detached cells. Concomitantly, release of lactate dehydrogenase from the cells reached about 80% at 24 hours. The release of tissue plasminogen activator and tissue plasminogen activator inhibitor-1 antigens decreased at a concentration of 7/3-hydroxycholesterol lower than that required for reducing general protein synthesis. 7/J-Hydroxycholesterol at 50 /i.mol/1 first increased the release and then (at 100 ^mol/l) inhibited the synthesis of von Willebrand factor. Incubation with 100 fimol/l of 5-cholestene-30,7a£2W-triol (7a£2dihydroxycholesterol) and the isomeric 5-cholestene-3ft7/3^2(K >)-triol (7ft22-dihydroxycholesterol) caused formation of intercellular gaps and some detachment of the cells after 24 hours. Cell injury was slightly more pronounced for the 7 a,22-dihydroxycholesterol than for the 70-isomer. Incubations with cholesterol under the same conditions gave no sign of cell injury.
The Role of Cholesterol and Its Biosynthesis in Lymphocyte Proliferation and Differentiation
Annals of the New York Academy of Sciences, 1985
Most membranes of eucaryotes contain sterols with 27 to 30 carbon atoms. In mammalian cells the major membrane sterol is cholesterol (5-cholesten-3P-01). The physiochemical role of this molecule has been investigated intensively and is today fairly well understood, although some details still require further study. This subject has been reviewed and therefore we restrict ourselves to briefly summarizing the present state of knowledge on the role of membrane sterol in lymphocyte function.
Chlorination of Cholesterol in Cell Membranes by Hypochlorous Acid
Archives of Biochemistry and Biophysics, 1996
dative damage associated with neutrophil/monocyte activation. Neutrophils and monocytes produce the highly cyto-Key Words: hypochlorous acid; cholesterol; chlorohytoxic hypochlorous acid (HOCl) via the myeloperoxidrins; myeloperoxidase. ᭧ 1996 Academic Press, Inc. dase (MPO)-catalyzed reaction of H 2 O 2 with Cl 0. We have investigated the reaction of reagent and MPOderived HOCl with cholesterol in a purified liposome system, as well as progressively more complex biological systems. The products were identified by thin-layer Reactive oxygen species produced by stimulated chromatography (TLC) and characterized by mass phagocytes have been implicated in host defense spectrometry (MS). TLC of the HOCl-treated samples against invading pathogens as well as inflammatory gave four major cholesterol products with color develtissue damage (1, 2). Neutrophils and monocytes proopment typical of oxysterols. Two of the products coduce the highly cytotoxic hypochlorous acid (HOCl) eluted with authentic a-and b-chlorohydrin stanvia the myeloperoxidase (MPO 3)-mediated reaction of dards. As was the case with the standards, they were H 2 O 2 with Cl 0. This strong oxidant has been implireadily converted into their respective epoxides durcated in microbial killing (3-7) as well as cytotoxicity ing analysis by MS. Gas chromatography-mass spectoward red blood cells (8-10) and tumor cells (11, 12). trometry analysis of the other major product (band 3) However, the processes involved in cell death are not gave peaks consistent with epoxides as well as a lateyet fully established. HOCl reacts rapidly with many eluting peak with a distinct mass spectrum. Electrobiological compounds, causing the oxidation of thiols, spray MS of this product confirmed its identity as a thioethers, heme groups, and iron-sulfur centers as chlorohydrin based on the presence of the predicted well as the chlorination of amines to form chloramines molecular ion and 3:1 Cl isotope ratios. Lipids ex-(13, 14). tracted from red cells and isolated red cell membranes were exposed to HOCl and gave identical products to HOCl also reacts with the carbon-carbon double the purified cholesterol liposome system as deter-bonds of fatty acyl groups in phospholipids to produce mined by TLC and MS. Higher concentrations of HOCl chlorohydrins (15, 16). Cholesterol has also been shown were required due to competition by other unsatuto react with HOCl. The products detected were, in one rated lipids and protein molecules. When intact red case, chlorohydrins and epoxides (17) and in the other cells, neutrophils, and MCF7 mammary carcinoma epoxides and hydroxy derivatives of cholesterol (16). cells were exposed to HOCl, cholesterol chlorohydrins The latter may have arisen from chlorohydrins via secwere formed, as detected by TLC. The formation of ondary breakdown during analysis. Lipid chlorohycholesterol chlorohydrins could be potentially disrupdrins have a higher polarity than the unmodified lipid tive to cell membranes and result in cell lysis and and thus could potentially disrupt cell membrane indeath. They could also be potential biomarkers for oxitegrity. This could contribute to the antimicrobial and inflammatory tissue damage by peroxidase-containing
Biochemical and Biophysical Research Communications, 2003
Atherosclerosis involves inflammatory processes, as well as cytotoxic and oxidative reactions. In atherosclerotic plaques, these phenomena are revealed by the presence of dead cells, oxidized lipids, and oxidative DNA damage, but the molecules triggering these events are still unknown. As 7b-hydroxycholesterol and 7-ketocholesterol, which are present at elevated concentrations in atherosclerotic lesions, are strongly cytotoxic and pro-oxidative, their effects were determined on cell death, superoxide anion and nitric oxide production, lipid peroxidation, and oxidative DNA damage. 7-Ketocholesterol-and 7b-hydroxycholesterol-induced cell death leads to a loss of mitochondrial potential, to increased permeability to propidium iodide, and to morphological nuclear changes (swelling, fragmentation, and/or condensation of nuclei). These effects are preceded by the formation of cytoplasmic monodansylcadaverine-positive structures and are associated with a rapid enhancement of cells overproducing superoxide anions, a decrease in cells producing nitric oxide, lipid peroxidation (formation of malondialdehyde and 4-hydroxynonenal adducts, low ratio of [unsaturated fatty acids]/[saturated fatty acids]) as well as oxidative DNA damage (8-oxoguanine formation). Noteworthy, none of the cytotoxic features previously observed with 7b-hydroxycholesterol and 7-ketocholesterol were noted with cholesterol, 7bhydroxycholesteryl-3-oleate and 7-ketocholesteryl-3-oleate, with the exception of a slight increase in superoxide anion production with 7b-hydroxycholesteryl-3-oleate. This finding supports the theory that 7b-hydroxycholesterol and 7-ketocholesterol could induce cytotoxic and oxidative processes observed in atherosclerotic lesions and that esterification of these compounds may contribute to reducing atherosclerosis progression.
Cholestan-3gb,5α,6β-triol decreases barrier function of cultured endothelial cell monolayers
Atherosclerosis, 1987
Cholesterol oxidation products (oxysterols) found in foods may be atherogenic, possibly by altering the barrier function of the vascular endothelium. To investigate this hypothesis, endothelial cells were cultured on micropore filters and the effect of cholesterol and the oxysterol cholestan-3fl,5a,6fl-triol (Triol) on albumin transfer across cultured vascular endothelial monolayers (ECM) was studied. Exposure to Triol significantly increased albumin transfer across ECM. The effect of Triol on endothelial cell barrier function was time and concentration dependent, with maximum albumin transfer being reached at 20 #M Triol and after a 24-h exposure. Pure cholesterol, on the other hand, did not affect albumin transfer at concentrations as high as 130/~M. Although an increase in albumin transfer across ECM was observed after a 2-h incubation with Triol-enriched media, a 24-h incubation period was necessary to cause a significant release of cellular lactate dehydrogenase (LDH) into the culture media. Morphological perturbations of the cell monolayers were observed at approx. 14-18 h after cell exposure to Triol-enriched media. Enrichment with cholesterol or vitamin E did not prevent the Triol-induced increase in albumin transfer across ECM. These results suggest that exposure to oxidized cholesterol, but not cholesterol itself, reduces the ability of the endothelium to act as a selectively permeable barrier to plasma components, and that these events may not be prevented by cholesterol or vitamin E.
Comparative atherogenic effects of cholesterol and cholesterol oxides
Atherosclerosis, 1986
Previous findings indicating that the oxidation products of cholesterol are associated with atherogenicity have led to a comparative study of the subchronic effects of feeding rabbits purified cholesterol, oxidized cholesterols free of cholesterol and cholesterol esters, or a mixture of cholesterol and oxidized cholesterols. Macroscopically, the cholesterol-fed animals exhibited 6-fold more arterial lesions than the animals fed cholesterol-free oxidized cholesterols. Microscopically, there was no statistically significant difference from the control in the number of histochemically-defined lesions in any of the groups. However, the lesions in the cholesterol-fed group were more severe, as indicated by a statistically significant increase in the magnitude of the lesions. This increased severity was also characterized by greater frequency and intensity of Azure A/Thionin, VonKossa, and Horseradish Peroxidase-Wheat Germ Agglutinin staining. Electronmicroscopic studies of normal appearing arterial tissues showed an increased density of viable smooth muscle cells and an increase in vacuolar extracellular debris in the cholesterol-fed group. Oxidized cholesterols in the concentrations and relative compositions administered here are markedly less atherogenie to rabbits than highly purified cholesterol.
Cholesterol oxidation: Health hazard and the role of antioxidants in prevention
Biological Research, 2003
BIOLOGICAL IMPORTANCE OF CHOLESTEROL Sterol cholesterol (cholest-5-en-ß-ol) is an essential metabolite required for major biological functions, such as the cell membrane structure where the steroid f o r m s , t o g e t h e r w i t h p h o s p h o l i p i d molecules, the integral part of the lipid b i l a y e r (S p e c t o r & Y o r e k , 1 9 8 5). Cholesterol is inserted into membrane bilayers with its long axis perpendicular to the plane of the membrane, preventing the crystallization of fatty acyl chains by fitting b e t w e e n t h e m (Y e a g l e , 1 9 8 5) a n d modifying the activity of membranebounded enzymes (Valencia et al., 1999). Cholesterol also provides the structural scaffolding for the synthesis of steroids a n d s t e r o i d h o r m o n e s , a n d f o r t h e biosynthesis of bile and bile acid salts. Sterol plays also an important role in embryonic development (Roux et al., 2000).
Interactions of MDL 29,311 and probucol metabolites with cholesteryl esters
Lipids, 1994
The hypothesis that the efficacy of hydrophobic antioxidants in animal models of atherogenesis may, in part, be related to physical effects on cholesteryl esters in cells was probed with analogs and metabolites of probucol. The interactions of an effective bis-thiomethane analog (MDL 29,311) and selected metabolites of probucol with cholesteryl oleate were examined by differential scanning calorimetry and polarized light microscopy. Like probucol, MDL 29,311 and the bisphenol metabolite decrease the liquid-crystalline phase transition enthalpy of cholesteryl oleate with increasing concentrations. At 20 mol%, no transition is detectable. By contrast, the spiroquinone metabolite of probucol and the diphenoquinone metabolite common to both molecules have minimal effects on the liquid-crystalline transitions of cholesteryl oleate. At 20 mol%, neither compound has as great an effect as 1 mol% MDL 29,311. Consistent with their effects on dry cholesteryl oleate, MDL 29,311 and the bisphenol metabolite convert lipid inclusions in cells supplemented with cholesterol to an isotropic physical state similar to that observed with probucol. The number of anisotropic inclusions in the cells decreases with increasing concentration in the medium in the range of 50 to 250 ~g/mL. In cells fed with the spiroquinone or diphenoquinone metabolites, the lipid inclusions are liquid-crystalline and resemble those observed with cholesterol-fed controls. These data are interpreted in terms of a model in which hydrophobic antioxidants closely related to probucol disrupt the packing of cellular cholesteryl esters. MATERIALS AND METHODS Cholesteryl ester loading of cells in culture. The Fu5AH rat hepatoma cell line was graciously provided by J.M. Glick (Philadelphia, PA). Stock cultures were grown in minimal essential medium (MEM) supplemented with basal medium Eagle vitamins, 50 ~g/mL of gentamycin,
Absorption of dietary cholesterol oxidation products and incorporation into rat lymph chylomicrons
Lipids, 1997
Cholesterol oxidation products (oxysterols) induce macrophage lipid loading and accumulate in early arterial fatty streaks. The origin of lesion oxysterols has not been elucidated. The absorption of oxysterols from the diet and transport to the arterial wall by postprandial lipoprotein remnants may be a significant source. This study aimed to investigate the extent of oxysterol absorption and the effect on chylomicron composition. Cholesterol was heat-treated, causing 30% oxidation; the major oxidation products were 7β-hydroxycholesterol, 7-ketocholesterol, 5α,6α-epoxycholesterol, and 5β,6β-epoxycholesterol. Conscious lymph-cannulated rats were given a bolus gastric infusion of 50 mg oxidized cholesterol or 50 mg purified cholesterol in a vehicle of triglyceride. In the rats given the oxidized cholesterol, 6% of the oxysterol load was absorbed and incorporated into lymph chylomicrons. Rats given pure cholesterol had no increase in oxysterols above baseline levels. The incorporation of oxysterols into lymph chylomicrons differed over time with 7β-hydroxycholesterol, having peak absorption at 3 h, followed by 7-ketocholesterol at 4 h and 5α,6α-epoxycholesterol at 5 h. The absorption of oxysterols in animals given the oxidized cholesterol gastric infusate was associated with lymph chylomicron compositional changes at 2-4 h. The oxidized cholesterol-treated group had a twofold increase in the cholesterol (890 ± 84 µg vs. 440 ± 83 µg at 3 h) and triglyceride content (19.76 ± 3.4 µg vs. 8.49 ± 3.8 µg at 3 h). This led to a doubling of chylomicron size over this postprandial period, with particles having a mean diameter of 294 nm in the oxidized cholesterol-treated animals, compared to 179 nm in the purified cholesterol group. In conclusion, dietary oxysterols appear to influence postprandial lipoprotein particle size and composition. These changes may have effects on the clearance of chylomicrons from plasma, arterial delivery of oxysterols, and possible deposition in arterial lesions.
Lipids, 1993
In the present work, several preparatory procedures commonly used for electron microscopy (EM) were evaluated as to their ability to preserve cholesterol (CHO) and CHO derivatives in tissue. We also determined in several rat tissues to what extent the sterols used as tracers are metabolized. Sprague-Dawley rats were injected intraperitoneally with [la,2a(n~3H]cholesterol ([3H]CHO) and cholesterol ([3H]25-OH~HO). Lipids of the fiver, aorta and brain were extracted one and five days after injection, and the distribution of the labeled lipids was followed by thln-layer chromatography. When labeled CHO was injected as tracer, most of the radioactivity re mained associated with the CHO fraction. When 25-hydroxycholesterol {254)H-CHO} was used, we found that it was mostly metabolized to yield more polar compounds. Our results show that the loss of CHO and CHO derivatives from tissues depends not only on the preparatory pro cedure used for EM, but also on the type of tissue studied. Lipids 28, 923-928 (1993).