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PubMed1997

25-Hydroxycholesterol enhances eicosanoid production in cultured bovine coronary artery endothelial cells by increasing prostaglandin G/H synthase-2.

Oxidized derivatives of cholesterol, the oxysterols, have been implicated in the development of atherosclerosis. We have found that the oxysterol, 25-hydroxycholesterol (25OHC), is a potent stimulator of eicosanoid production in endothelial cells. Confluent monolayers of bovine coronary artery endothelial cells (BCAECs) were treated with 25OHC (10 micrograms/ml) or interleukin-1 beta (IL-1 beta, 1 ng/ml), a known prostaglandin G/H synthase-2 (PGHS-2) inducer, for 48 h at 37 degrees C. Following incubation with [14C]arachidonic acid, the 14C-labeled metabolites of arachidonic acid were extracted and resolved using both normal and reverse phase high-performance liquid chromatography (HPLC) systems. 25OHC-treated cells had a five-fold increase in their prostaglandin production when compared to untreated cells. Eicosanoid production in IL-1 beta treated cells was not as pronounced. The major component in both sets of cells comigrated with 6-ketoPGF1 alpha. Other PGHS metabolites, 15- and 11-hydroxyeicosatetraenoic acids (HETEs) and 12-hydroxyheptadecatrienoic acid (HHT) were also identified and increased following 25OHC treatment. Epoxyeicosatrienoic acids, metabolites of cytochrome P450, were not effected. Enhanced production of 6-ketoPGF1 alpha, 15-HETE, 11-HETE and HHT were inhibited by indomethacin. Thus, all eicosanoids induced by 25OHC treatment were PGHS products. Western immunoblot analysis of lysates from 25OHC, IL-1 beta, or vehicle treated cells using anti-PGHS-1 and -2 antibodies revealed a significant increase in PGHS-2, but not PGHS-1, in both 25OHC and IL-1 beta treated cells. The notion that oxysterols can modulate vascular eicosanoid production through enzyme induction may be important in ultimately understanding their role in the pathogenesis of atherosclerosis.

Animals↗
PubMed2001

Limitations of the single-cell gel electrophoresis assay to monitor apoptosis in U937 and HepG2 cells exposed to 7beta-hydroxycholesterol.

The single-cell gel electrophoresis (comet) assay is a method which allows the detection of DNA strand breaks in individual cells. It has been suggested that the single cell gel electrophoresis assay, as an index of DNA fragmentation during cell death, may be applied to monitor apoptosis. The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to discriminate between the mode of cell death in two cell lines (U937, a human monocytic blood cell line and HepG2, a human hepatocarcinoma cell line) which were treated with 30 microM 7beta-hydroxycholesterol (7betaOHC) over a 48 hr period. The single cell gel electrophoresis assay was compared with more established methods for the determination of apoptosis such as morphological examination, flow cytometry and DNA laddering. The percentage of maximally damaged nuclei as measured by the single cell gel electrophoresis assay was found to be similar at 48 hr in both U937 and HepG2 cells when treated with 7betaOHC. However, morphological examination, flow cytometry and DNA laddering techniques showed that 7betaOHC induced apoptosis in U937 cells but not in HepG2 cells. Thus, although the alkaline single cell gel electrophoresis assay detected DNA strand breaks occurring during cell death, these breaks were observed only when the process was fairly well advanced and a major part of the cells had lost membrane permeability. Therefore the present report demonstrates that the single cell gel electrophoresis assay, used in isolation, cannot accurately be used to distinguish between the mode of cell death induced by 7betaOHC in U937 cells (apoptosis), or HepG2 cells (cell lysis).

Apoptosis↗
PubMed1999

The neurotoxic effect of 24-hydroxycholesterol on SH-SY5Y human neuroblastoma cells.

Neurodegenerative disorders are characterized by a massive loss of nerve cells. The neuronal cell death is accompanied by an increased cholesterol release and conversion of cholesterol into the polar metabolite, 24-hydroxycholesterol (24-OH-Chol), appears to be an important mechanism in the central nervous system for eliminating cholesterol from the brain. We tested the influence of 24-OH-Chol on SH-SY5Y human neuroblastoma cells by recording cell morphology, Trypan blue exclusion, LDH-release into the culture medium, intracellular calcium and reactive oxygen species (ROS). The exposure of SH-SY5Y human neuroblastoma cells to 50 microM 24-OH-Chol led to a 90% loss in cell viability within 30 h, the LDH-release into the medium increased rapidly after 24 h, and after 24 to 30 h we found an elevation in intracellular calcium. These results show that, in a physiological concentration range, 24-OH-Chol damages neuronal cells, thus we speculate that this oxysterol may be involved in the etiology of neurodegenerative disease.

Analysis of Variance↗
PubMed2003

Membrane incorporation of 22-hydroxycholesterol inhibits chemokine receptor activity.

Cell membrane exposure to oxysterols, such as 22-hydroxycholesterol (22-OHC), has previously been shown to induce a suppressive effect on lymphocyte activation. Based on our previous findings that chemokine binding was significantly inhibited by the extraction of membrane cholesterol, we sought to assess the effects of 22-OHC treatment on chemokine ligand-binding and receptor activity. Our results revealed that 22-OHC, but not nonoxidized cholesterol, significantly reduced the binding of both SDF-1alpha and MIP-1beta to human T-cell lines and PBMCs within 1 h of treatment. Incubating the treated cells at 37 degrees C for 1 h reversed a majority of the inhibitory effects on chemokine binding. 22-OHC also inhibited intracellular calcium mobilization and cell migration in response to SDF-1alpha treatment. Interestingly, while the presence of oxysterols in cell membranes significantly inhibits chemokine receptor function, this inhibitory effect does not involve alterations in receptor conformation, expression, or a direct antagonism of chemokine binding. We propose here a novel mechanism for oxysterol-mediated inhibition of chemokine receptor function and the implications for the presence of oxysterols on immune cells.

Antibodies, Monoclonal↗
PubMed1999

Distribution of exogenous 25-hydroxycholesterol in Hep G2 cells between two different pools.

Binding of [26,27-(3)H]25-hydroxycholesterol (25HC) to human hepatoma Hep G2 cells was saturated within 120 min. Two intracellular pools of 25HC were identified in a pulse-chase experiment: (i) an exchangeable pool which was in dynamic equilibrium with 25HC in the medium (t(1/2) of reversible exchange 15 min) and (ii) an unexchangeable pool which remained in cells during incubation in medium containing LPDS. 25HC from the exchangeable pool inhibits cholesterol biosynthesis, decreases the HMG CoA reductase mRNA level and stimulates cholesterol acylation. 25HC from the unexchangeable pool was partially bound to cytosolic proteins and apparently utilized for metabolic transformation. Incubation of Hep G2 cells with [26,27-(3)H]25HC in the presence of a 30-fold molar excess of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one was found to cause (i) 2-fold decrease in the binding of [26,27-(3)H]25HC to cytosolic proteins (sedimentation constant of radioactive complex was 4-5 S) and (ii) the 35% inhibition of 25HC transformation to polar metabolites.

Acylation↗
PubMed2003

Hypoxia increases 25-hydroxycholesterol-induced interleukin-8 protein secretion in human macrophages.

Interleukin-8 (IL-8) is a chemotactic factor for T-lymphocytes and smooth muscle cells and may therefore have an important effect in atherogenesis. It is secreted from oxysterol-containing foam cells which have been found in hypoxic zones in atherosclerotic plaques. The aim of this study was to investigate the effect of hypoxia on the secretion of IL-8 by oxysterol-stimulated macrophages. Hypoxia enhances 25-hydroxycholesterol (25-OH-chol)-induced IL-8 secretion in human monocyte-derived macrophages. The effect is most pronounced when macrophages are incubated with low concentrations of 25-OH-chol. Both 25-OH-chol and hypoxia increases the intracellular level of the signalling molecule hydrogen peroxide (H(2)O(2)). This event coincided with an enhanced binding of the transcription factor c-jun to the IL-8 gene promoter and an increased IL-8 mRNA expression in hypoxic macrophages. These observations suggest that similar intracellular signalling pathways are used for both 25-OH-chol-induced IL-8 expression and hypoxia-induced IL-8 expression. Thus, hypoxia in atherosclerotic plaques may increase the secretion of IL-8 from oxysterol-containing foam cells, which subsequently may accelerate the progression of atherosclerosis.

ATP Synthetase Complexes↗
PubMed2002

Synthesis of (25R)-26-hydroxycholesterol.

We describe the synthesis of (25R)-cholest-5-en-3beta,26-diol ((25R)-26-hydroxycholesterol) from diosgenin in four steps in 58% overall, yield via a modified Clemmensen reduction followed by a Barton deoxygenation reaction.

Hydroxycholesterols↗
PubMed2003

Synthesis of deuterated isotopomers of 7alpha- and (25R,S)-26-hydroxycholesterol, internal standards for in vivo determination of the two biosynthetic pathways of bile acids.

Deuterated isotopomers of 7alpha- and (25R,S)-26-hydroxycholesterol, internal standards for in vivo determination of the two biosynthetic pathways of bile acids formation from cholesterol, were prepared from [2,2,3,4,4,6-2H(6)]-cholesterol and (20S)-[7,7,21,21-2H(4)]-3beta-(tert-butyldimethylsilyl)oxy-20-methylpregna-5-en-21-ol, respectively.

Bile Acids and Salts↗
PubMed2002

Characteristics of 7 beta-hydroxycholesterol-induced cell death in a human monocytic blood cell line, U937, and a human hepatoma cell line, HepG2.

Oxysterols have been shown in a number of cell lines to induce apoptosis by a mechanism as yet unclear. The induction of apoptosis by certain agents has been associated with the generation of oxidative stress and the depletion of the endogenous antioxidant, glutathione, which may result in cytochrome c release and caspase activation. The aim of the present study was to determine whether 7 beta-hydroxycholesterol (7 beta-OH) alters glutathione levels or the activities of catalase, superoxide dismutase (SOD) or caspase-3 in association with cell death in either the U937 or the HepG2 cell lines. 7 beta-OH, which induced significant apoptosis at 12 h in the U937 cell line, was shown to cause a significant decrease in glutathione levels and an increase in the activity of SOD at this time point. An increase in caspase-3 activity was also observed in the U937 cell line following a 24-h incubation with 7 beta-OH. Glutathione concentration, SOD activity and caspase-3 activity were unchanged in the HepG2 cell line, which underwent necrosis following incubation with 7 beta-OH. The activity of the enzyme catalase remained unchanged in both cell lines. These results provide evidence that the generation of an oxidative stress may be a significant event occurring during 7 beta-OH-induced apoptosis.

Apoptosis↗
PubMed2002

Changes in human plasma levels of the brain specific oxysterol 24S-hydroxycholesterol during progression of multiple sclerosis.

24S-Hydroxycholesterol (24S-OH-chol) is almost exclusively derived from the brain and the levels of this oxysterol in the circulation may be used as a marker for changes in the turnover of brain cholesterol. In the present study we assayed plasma and cerebrospinal fluid 24S-OH-chol in patients with multiple sclerosis (n = 118) at different stages of the disease. In the oldest groups of patients the levels of 24S-OH-chol were significantly lower than in the controls, possibly reflecting loss of neuronal cells responsible for the synthesis. There was a significant inverse relation between the expanded disability status scale-grade of the disease and the plasma cholesterol related levels of 24S-OH-chol. There was a tendency to increased plasma levels of 24S-OH-chol in the younger patients and almost all of the high levels were found in the patients in the 3rd and 4th decades of life. Increased levels were only found in patients with positive cranial magnetic resonance imaging. The possibility is discussed that plasma levels of 24S-OH-chol may add significantly to existing methods used for evaluation of the different phases of multiple sclerosis.

Adult↗
PubMedDate not supplied

Metabolism in vitro of cholesterol and 25-hydroxycholesterol by the larval prothoracic glands of Manduca sexta.

The prothoracic glands in vitro convert 25-hydroxycholesterol (25C) to 25-hydroxy-7-dehydrocholesterol (7d25C) and to ecdysteroids at a greater rate than cholesterol (C) is converted to ecdysteroids via 7-dehydrocholesterol (7dC). Mediated via a cytochrome P450 most probably located in the endoplasmic reticulum (ER), both intact and extensively homogenized prothoracic glands, as well as crude subcellular fractions, were able to 7,8-dehydrogenate 25C to 7d25C eight-fold more efficiently than they could convert C to 7dC. However, less than a two-fold difference was observed in the subsequent monooxygenase mediated conversion of these two intermediates formed in situ into ecdysteroids, mainly ecdysone (E) and 2-deoxyecdysone (2dE) and/or their 3-dehydroderivatives. When 7dC, and particularly 7d25C, were made directly available to these tissue preparations, their conversion to ecdysteroids greatly exceeded that of the in situ conversion of either C or 25C, via 7dC or 7d25C, respectively. Indeed, there was an eight-fold increase in the VMAX for 25C dehydrogenation by homogenized glands relative to the dehydrogenation of C. Most important, however, was the 1000-fold increase in the VMAX observed for the direct production of E from emulsified 7d25C by gland homogenates relative to E production from 25C via 7d25C synthesized in situ. Thus, it is apparent that even after the rapid and efficient conversion of 25C to 7d25C within the ER, the subsequent rate of conversion of this intermediate to E is greatly retarded relative to that observed following the direct incubation of emulsified 7d25C with gland homogenates. These differential kinetics of direct and indirect 7d25C incorporation into E are interpreted as evidence for the existence of a barrier to the efficient translocation of the delta 5,7-sterol intermediates from the ER to another site where the subsequent, uncharacterized initial conversions leading to ecdysteroids take place. On the basis of studies on mammalian adrenal cortical steroidogenesis, this site is postulated to be the inner membrane/matrix of the mitochondria. The present data support the hypothesis that the translocation of both 7dC and 7d25C, first from the site of their probable synthesis within the ER membranes, next through the cytosol to the outer mitochondrial membrane, and then across the intramitochondrial aqueous space to the inner membrane/matrix compartment, may be analogous to the translocation in the adrenal cortex of ER-derived C, first to the plasma membrane and/or to the outer mitochondrial membrane and then to the inner mitochondrial membrane/matrix for P450scc-mediated conversion into pregnenolone.

Animals↗
PubMed2000

Kinetics and plasma concentrations of 26-hydroxycholesterol in baboons.

26-Hydroxycholesterol (26OHC), a major oxysterol in human blood, is believed to play an important role in reverse cholesterol transport, bile acid formation, and regulation of various cellular processes. Using isotope dilution mass spectrometry, we measured plasma 26OHC concentrations in baboons fed either a high cholesterol/saturated fat (HC-SF) or normal chow diet. Plasma 26OHC levels in baboons were comparable to those reported for humans and were positively correlated with plasma cholesterol concentrations. Animals on the HC-SF diet had significantly higher 26OHC levels (0.274+/-0.058 microM, mean+/-S.D.) than those on the chow diet (0.156+/-0.046 microM). In separate experiments, [(3)H]26OHC was injected into four tethered baboons, and multiple blood samples drawn over a 1-h period were analyzed for [(3)H]26OHC and 26OHC. Fitting the specific radioactivity data to a two-pool compartmental model indicated a rapidly turning over plasma compartment (t(1/2) 2.9-6.0 min) and a second compartment with slow turnover (t(1/2) 76-333 min). The calculated 26OHC production rate was 2.5 micromol/kg body weight/day. Assuming all 26OHC is converted to bile acids, the 26OHC production rate corresponds to about 10% of total bile acid production in adult baboons. These results indicate that rapid turnover of plasma 26OHC at submicromolar concentrations could significantly contribute to bile acid synthesis.

Animals↗
PubMed2000

Biologic role(s) of the 25(R),26-hydroxycholesterol metabolic pathway.

Expression of the gene coding for the synthesis of 25(R), 26-hydroxycholesterol in many tissues and the finding that this sterol can be the sole pathway for the production of bile acids have led to a renewed interest in this metabolic pathway. A further impetus for exploring the normal biologic roles that are served by expression of the CYP27A1 gene is the knowledge that mutations in humans are associated with accelerated atherosclerosis and with severe neurologic impairment. The molecular mechanisms governing these phenotypic expressions are not known but in light of the traditional role of steroids as ligands for receptors that regulate gene expression it seems likely that the intermediates in this pathway modulate a number of enzymatic activities that remain to be elucidated.

Animals↗
PubMed1990

Monophosphoric acid diesters of 7 beta-hydroxycholesterol and of pyrimidine nucleosides as potential antitumor agents: synthesis and preliminary evaluation of antitumor activity.

7 beta-Hydroxycholesterol, which has been shown to be selectively cytotoxic toward tumor cells cultured in vitro, was converted into the corresponding water-soluble phosphoric acid ester and linked to a pyrimidine nucleoside such as 5-fluoro-2'-deoxyuridine or 2'-deoxyuridine. 2-Chlorophenyl phosphorodichloridate (3), without activation, was used directly to phosphorylate the protected oxygenated sterol. The intermediate phosphorylated the 5'-OH group of nucleoside selectively, leading to compounds 1a and 1b after deprotection. These compounds were screened for their antiproliferative activity toward EL-4 murine leukemia cells in vitro and for their antitumor activity against the mice bearing Krebs II ascitic carcinoma in vivo.

Animals↗
PubMed2005

Different apoptotic mechanisms are involved in the antiproliferative effects of 7beta-hydroxysitosterol and 7beta-hydroxycholesterol in human colon cancer cells.

Plant sterols are found in fruits and vegetables. Their cholesterol-lowering effect is well documented. Our study aimed at comparing antiproliferative effects of 7beta-hydroxysitosterol (7beta-OHsito) versus 7beta-hydroxycholesterol (7beta-OHchol) on the human colon cancer cell line Caco-2. When cells were exposed for 32 h to 60 microM 7beta-OHsito or to 30 microM 7beta-OHchol, both compounds caused 50% growth inhibition. Cells treated with 7beta-OHsito showed enhanced caspase-9 and -3 activities followed by DNA fragmentation. In contrast, 7beta-OHchol did not activate caspase-3 and activation of caspase-9 and DNA fragmentation were delayed. The treatment of cells with the caspase inhibitor Z-VAD.fmk retarded the 7beta-OHsito-induced apoptotic process but not that triggered by 7beta-OHchol. Our data suggest that the two compounds in spite of their structural similarities target different cellular pathways, which lead to cell death.

Amino Acid Chloromethyl Ketones↗
PubMed1988

Cholesterol metabolism. Effect of 26-thiacholesterol and 26-aminocholesterol, analogues of 26-hydroxycholesterol, on cholesterol synthesis and low-density-lipoprotein-receptor binding.

1. The effects of 26-aminocholesterol and 26-thiacholesterol on cholesterol synthesis and LDL (low-density lipoprotein)-receptor activity were compared with naturally occurring 26-hydroxycholesterol utilizing both human fibroblasts and hepatoma (Hep G2) cells. 2. At equimolar concentrations (0.625 microM), down-regulation of LDL-receptor activity and cholesterol synthesis was greater with human fibroblasts than with Hep G2 cells. 3. At much higher concentrations (5-20 microM) the 26-thia analogue had little effect on either cholesterol synthesis or LDL-receptor activity.

Animals↗
PubMed1989

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.

Apolipoprotein A-I↗
PubMed1996

Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.

We have investigated whether side chain-hydroxylated cholesterol species are important for elimination of cholesterol from the brain. Plasma concentrations of 24-hydroxycholesterol (24-OH-Chol) in the internal jugular vein and the brachial artery in healthy volunteers were consistent with a net flux of this steroid from the brain into the circulation, corresponding to elimination of approximately 4 mg cholesterol during a 24-h period in adults. Results of experiments with rats exposed to 18O2 were also consistent with a flux of 24-OH-Chol from the brain into the circulation. No other oxysterol measured showed a similar behavior as 24-OH-Chol. These results and the finding that the concentration of 24-OH-Chol was 30- to 1500-fold higher in the brain than in any other organ except the adrenals indicate that the major part of 24-OH-Chol present in the circulation originates from the brain. Both the 24-OH-Chol present in the brain and in the circulation were the 24S-stereoisomer. In contrast to other oxysterols, levels of plasma 24-OH-Chol were found to be markedly dependent upon age. The ratio between 24-OH-Chol and cholesterol in plasma was approximately 5 times higher during the first decade of life than during the sixth decade. There was a high correlation between levels of 24-OH-Chol in plasma and cerebrospinal fluid. It is suggested that the flux of 24-OH-Chol from the brain is important for cholesterol homeostasis in this organ.

Adolescent↗