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PubMed1980

Differences in the characteristics of inhibition of lymphocyte stimulation by 25-hydroxycholesterol and by the immunoregulatory serum lipoprotein LDL-In.

Suppression of peripheral blood lymphocyte phytohemagglutinin-stimulated 3H-thymidine uptake in vitro by serum lipoprotein fractions enriched in the immunoregulatory low density lipoprotein (LDL-In) and by 25-hydroxycholesterol was compared to determine whether the biologically active constituent of LDL-In might be an oxygenated sterol. Dose titrations indicated that 25-hydroxycholesterol was 100 times more potent than LDL-In on the basis of total sterol. Both were similar in that maximal inhibitory activity was evident when they were present in the cultures for at least 24 hr before mitogen stimulation. However, the suppression of 3H-thymidine uptake by 25-hydroxycholesterol required the continuous presence of the sterol, whereas equivalent suppression by LDL-In was irreversible and did not require the presence of the lipoprotein beyond the preincubation period. 25-Hydroxycholesterol-mediated suppression of 3H-thymidine uptake was mitigated by the addition of cholesterol or mevalonic acid to the lymphocyte cultures, consistent with an effect on sterol biosynthesis, whereas, LDL-In mediated suppression was not. Finally, when chloroquine was used to inhibit lymphocyte lysosomal hydrolysis, which is required for the intracellular degradation and release of cholesterol esters from internalized low density lipoprotein, the phenotypic expression of LDL-In mediated suppression was not altered, indicating that this known pathway of regulation of cellular function by low density lipoproteins is not the primary mode of action of LDL-In. These data indicated that the mode of suppression of mitogen-stimulated thymidine uptake by serum lipoprotein fractions enriched in LDL-In differs from that produced by 25-hydroxycholesterol, and that LDL-In suppression does not appear to result from inhibition of intracellular cholesterol biosynthesis.

Cell Division↗
PubMed1995

Inhibition of cellular cholesterol efflux by 25-hydroxycholesterol.

The effect of oxysterols on efflux of cholesterol from mouse L-cell fibroblasts, rat Fu5AH hepatoma cells, J774 macrophages, and human EA.hy 926 endothelial cells was studied. Cells were preincubated with 25-hydroxycholesterol (25-OHC) either during labeling of the cells with [3H]cholesterol or during equilibration after labeling. Subsequently, the release of [3H]cholesterol into medium containing 0.2 mg HDL3/ml was measured and the fractional release of cellular [3H]cholesterol was calculated. Pretreatment with 25-OHC (1 microgram/ml) caused a 30% reduction in [3H]cholesterol efflux from L-cells during 8 h of incubation with HDL3. 25-OHC also inhibited cholesterol efflux from Fu5AH and J774 cells, but the effect was less marked. There was only a small, nonsignificant reduction of efflux from EA.hy 926 cells. The mechanisms of 25-OHC-induced inhibition of cellular cholesterol efflux was further studied in L-cells, because of their sensitivity to 25-OHC treatment. The effect of 25-OHC on cholesterol efflux was dose-dependent, with significant effects seen at 25-OHC concentrations as low as 50 ng/ml. The half-time for cholesterol efflux from 25-OHC-treated cells (5 micrograms/ml) was 13.0 +/- 3.3 h compared to 5.7 +/- 1.0 in control cells, corresponding to a 55% reduction in the rate of cholesterol release. Other oxysterols, including 7-ketocholesterol, 7 alpha- and 7 beta-hydroxycholesterol, and 22(S)-hydroxycholesterol also inhibited [3H]cholesterol efflux from L-cells significantly, but to a lesser degree. 25-Hydroxycholesterol (5 micrograms/ml) reduced efflux from both normal and cholesterol-enriched cells by 31 and 14%, respectively. Inhibition of efflux was similar when reconstituted HDL3-apolipoprotein/phosphatidylcholine particles or small unilamellar phosphatidylcholine vesicles were used as cholesterol acceptors instead of HDL3. The content of phospholipids, cholesterol and the FC/PL ratio of intact cells and from isolated plasma membrane vesicles were the same for control and 25-OHC-treated cells. Efflux of [3H]cholesterol from plasma membranes isolated from 25-OHC-treated cells was 20% less than efflux from membranes from control cells. The difference in efflux observed in intact cells is partially explained by the reduction in efflux from the plasma membrane. In conclusion, our studies suggest that oxysterols, especially 25-hydroxycholesterol, can reduce cellular cholesterol efflux in vitro. Therefore oxysterols, either endogenous or derived from the diet, may influence cellular cholesterol efflux in vivo, the first step in reverse cholesterol transport.

Animals↗
PubMed1997

High levels of (24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide in the serum and urine of children with severe cholestatic liver disease.

Extracts of urine and serum from children with cholestatic liver disease were analyzed by fast atom bombardment (FAB) mass spectrometry. About half of all spectra showed a peak at m/z 657, compatible with the presence of a glucuronidated cholestenediol sulfate. Separation by ion exchange chromatography before and after solvolysis and treatment with beta-glucuronidase, combined with analyses by gas chromatography-mass spectrometry and FAB mass spectrometry with collision-induced dissociation, showed that the major compound responsible for the peak at m/z 657 was (24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide. The double conjugate of 27-hydroxycholesterol was also identified and double conjugates of cholestene- and cholestanetriols were also present. Semiquantitative analyses of the double conjugate of 24-hydroxycholesterol in patients whose FAB spectra showed a peak at m/z 657 indicated serum levels of 2-18 microM and a daily urinary excretion of 0.1-2.7 mumol/24 h. Eleven of 13 studied patients with a prominent peak at m/z 657 in the FAB spectra of their serum or urine either underwent liver transplantation or died. It is concluded that double conjugation of hydroxysterols with sulfuric and glucuronic acids can be an important metabolic pathway, particularly for (24S)-24-hydroxycholesterol. It is speculated that serious cholestatic liver disease may induce an increased formation and release of (24S)-24-hydroxycholesterol from brain (Lütjohann et al. 1996, Proc. Nutl. Acad. Sci. USA. 93: 9799-9804) with subsequent extracerebral conjugation with sulfuric and glucuronic acids.

Biomarkers↗
PubMed1997

Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells.

We have characterized the death of human aortic smooth muscle cells induced by 25-hydroxycholesterol, an oxidation product of cholesterol. Chromatin condensation characteristic of apoptosis was observed by enzymatic (TUNEL) staining of chromatin, and by electron microscopy. Fourteen percent of cells treated with 5 microg/ml of 25-hydroxycholesterol for 24 h displayed chromatin degradation as determined by positive TUNEL staining. Addition of TNF alpha (10 ng/ml) and IFN gamma (20 ng/ml) increased the proportion of TUNEL positive cells to 30%, whereas the cytokines alone were without effect. After 48 h, 40% of the cells treated with 5 microg/ml of 25-hydroxycholesterol were TUNEL positive, and 21% of the cells displayed chromatin condensation. Oligonucleosomal DNA fragmentation typical of apoptosis was demonstrated by agarose gel electrophoresis. Furthermore, activation of the ICE-like protease caspase 3 (CPP32) was observed in cells treated with 25-hydroxycholesterol. Addition of the Ca2+ entry blockers verapamil or nifedipine to the culture medium inhibited apoptosis by more than 70% and reduced cytotoxicity, while removal of Ca2+ from culture medium reduced apoptosis by 42%. Within a few minutes after addition, 25-hydroxycholesterol induced intracellular Ca2+ oscillations with a frequency of approximately 0.3-0.4 min(-1). Thus it appears that Ca2+ influx through plasma membrane channels is an important signal in oxysterol-induced apoptosis. Addition of TNF alpha and IFN gamma enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cytokines can increase the cytotoxicity of lipid oxidation products.

Aorta↗
PubMed2002

Human sterol 27-hydroxylase (CYP27) overexpressor transgenic mouse model. Evidence against 27-hydroxycholesterol as a critical regulator of cholesterol homeostasis.

CYP27-overexpressed transgenic mice were generated with the use of a human full-length CYP27 coding region cloned into a ubiquitous expression vector. Positive transgenic mice were identified by tail DNA genotyping and high fecal 27-hydroxycholesterol content. The levels of 27-hydroxycholesterol were found to be 3-5 times higher in the circulation and the tissues of the overexpressed mice when compared with littermate controls. There were no gross morphological differences between the overexpressed mice and their controls. Total cholesterol and triglyceride levels were not affected by overexpression of CYP27. Serum lathosterol was also normal, suggesting a normal rate of cholesterol synthesis. Serum levels of 7alpha-hydroxycholesterol were unaffected, suggesting a normal rate of bile acid formation in the pathway involving cholesterol 7alpha-hydroxylase. Biliary bile acid composition was slightly affected by CYP27 overexpression in female but not in male mice. Fecal levels of neutral steroids were slightly but significantly increased in overexpressor female mice but not in male mice. Levels of 24-hydroxycholesterol in the circulation were significantly reduced in the overexpressed mice, probably as a consequence of a recently described catabolic pathway involving CYP27. Combined with the results of our previous work on mice with a disruption of the CYP27 gene, the present results suggest that the levels of 27-hydroxycholesterol are not of critical importance for cholesterol homeostasis in mice.

Animals↗
PubMed1985

25-Hydroxycholesterol-induced elevations in 45Ca uptake: permeability changes in P815 cells.

Certain oxysterols are capable of suppressing the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. We have previously demonstrated that treatment of P815 cells with 1 microgram 25-hydroxycholesterol/ml culture results in a rapid influx of 45Ca, and supplemental cholesterol prevents this from occurring. In this paper, we report on investigations into the means whereby this influx of calcium takes place. Through the use of respiratory inhibitors which prevent mitochondrial retention of calcium it was determined that the large increase in slow phase (intracellular) calcium uptake caused by 25-hydroxycholesterol treatment was related to mitochondrial uptake. The effects of various inhibitors of calcium uptake into cells, including verapamil, diltiazem, quinidine, ruthenium red, Co++, Mn++, were tested. Of these only Co++ and ruthenium red had any effect on 45Ca uptake. 25-Hydroxycholesterol has been shown to be capable of membrane insertion and this could result in plasma membrane permeability changes. To test this hypothesis P815 cells were treated with 1 microgram 25-hydroxycholesterol/ml or 5 micrograms mevinolin/ml culture. Mevinolin, being a water soluble competitive inhibitor of HMG-CoA reductase, should be unable to disrupt membrane architecture in a manner analogous to 25-hydroxycholesterol. While both inhibitors rapidly suppressed the synthesis of digitonin-precipitable sterols, only 25-hydroxycholesterol was able to increase 45Ca influx. The implications of these findings are discussed.

Animals↗
PubMed1982

Effect of detergents on in vitro 7 alpha-hydroxycholesterol formation by rat liver microsomes.

Formation of 7 alpha-hydroxycholesterol by rat liver microsomes was quantitated using a gas chromatograph-mass spectrometer (GC/MS) operated in selected ion monitoring (SIM) mode. Microsomes from normal rat livers incubated for different periods were found to yield increased 7 alpha-hydroxycholesterol with time. This was also true when incubations contained Tween-80, but in this instance, the rate of 7 alpha-hydroxycholesterol production was lower and dependent on the concentration of Tween used. Similarly, Triton X-100, Renex-30, Kyro EOB, Cutscum, and Emulgen 911 all lowered the formation of 7 alpha-hydroxycholesterol by rat liver microsomes, whereas Triton WR-1339 stimulated its production. Analysis of data obtained from following the enzyme reaction over an extended period using an integrated Michaelis-Menten equation indicated the enzyme possesses a very significant affinity for the product (Ks greater than Kp). Similar analysis shows that Tween-80 is a noncompetitive inhibitor of the enzyme.

Animals↗
PubMed1997

Sex difference in serum 7 alpha-hydroxycholesterol levels in the rat reflect hepatic activity of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase and cholesterol 7 alpha-hydroxylase.

Factors that affect serum levels of 7 alpha-hydroxycholesterol were studied in the rat. Serum levels of 7 alpha-hydroxycholesterol differed in male and female rats fed regular chow (male; 0.2 +/- 0.1 nmol/ml (mean +/- SD); n = 8; female; 0.4 +/- 0.1 nmol/ml; n = 8). When rats were fed with chow to which 3% cholestyramine had been added, the level increased significantly, particularly in female rats (male; 0.6 +/- 0.3 nmol/ml; n = 8; female; 2.4 +/- 1.5 nmol/ml; n = 8). The liver activity of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme for degradation of cholesterol, did not show any sex differences, irrespective of whether the animals were fed with regular chow (male; 51 +/- 15 pmol/min per mg protein; n = 8; female; 58 +/- 21 pmol/min per mg protein; n = 8), or the cholestyramine-supplemented chow (male; 162 +/- 33 pmol/min per mg protein; n = 8; female; 172 +/- 33 pmol/min per mg protein; n = 8). In contrast, the activity of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase, which acts after cholesterol 7 alpha-hydroxylase in the catabolism of cholesterol, showed a marked difference between the sexes. In both sexes this enzyme activity was higher in cholestyramine-treated rats (male; 963 +/- 78 pmol/min per mg protein; n = 8; female; 708 +/- 106 pmol/min per mg protein, n = 8) compared to that in that rats received regular chow (male; 622 +/- 83 pmol/min per mg protein; n = 8; female; 469 +/- 41 pmol/min per mg protein; n = 8). If the serum level of 7 alpha-hydroxycholesterol depended solely on the enzyme activity of cholesterol 7 alpha-hydroxylase, it would be difficult to explain these sex differences, since there were no sex differences in levels of cholesterol 7 alpha-hydroxylase. These results clearly indicate that, in the rat, the serum level of 7 alpha-hydroxycholesterol depends not only on cholesterol 7 alpha-hydroxylase activity but also on 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

7 alpha-hydroxylation of 27-hydroxycholesterol in human liver microsomes.

Human liver microsomes were found to catalyze 7 alpha-hydroxylation of 27-hydroxycholesterol at a rate of up to about 0.2 nmol/mg protein per min. The product of the reaction, 5-cholestene-3 beta, 7 alpha, 27-triol, was identified by means of combined gas chromatography-mass spectrometry. Liver microsomes from two patients with an upregulated cholesterol 7 alpha-hydroxylase, did not have higher 7 alpha-hydroxylase activity towards 27-hydroxycholesterol than those from untreated patients, suggesting that the 7 alpha-hydroxylase active towards 27-hydroxycholesterol is not the same as that active towards cholesterol. The mitochondrial fraction of liver from untreated patients and patients treated with cholestyramine, had negligible 7 alpha-hydroxylase activity towards 27-hydroxycholesterol less than 0.01 nmol/mg protein per min). The results are in accord with the possibility that there is a pathway to bile acids in human liver in which the first step is a 27-hydroxylation of cholesterol.

Adult↗
PubMed1993

Hepatic uptake and metabolism of ingested 24-hydroxycholesterol and 24(S),25-epoxycholesterol.

Although two hepatic sterol metabolites, 24(S)-hydroxycholesterol and 24(S),25-epoxycholesterol, are thought to be important regulators of cholesterol biosynthesis, nothing is known of their degradation and disposal in liver, nor of the mechanisms that regulate their levels. As an initial approach to these questions the two sterols were administered intragastrically, as a bolus, to mice and their hepatic accumulation and conversion to more polar compounds were examined as a function of time. These results were compared to those obtained for cholesterol and for the unnatural epimer of one of the oxysterols, 24(R)-hydroxycholesterol. Maximum concentrations of the three oxysterols in liver were reached by approx. 4 h and then declined to control levels by 8 h. More polar neutral and acidic metabolites were found in the liver extracts. Radiolabeled oxysterols and their metabolites were found in bile glands. In comparison, the amounts of hepatic free and esterified cholesterol and of acidic products formed from it increased gradually over the measured period of time. Rates of conversion of the two 24-hydroxycholesterol epimers into acidic compounds by a liver mitochondrial fraction in vitro exceeded those of 24(S),25-epoxycholesterol and cholesterol. 24(S)-Hydroxycholesterol did not lower the level of hepatic HMG-CoA reductase activity, consistent with the absence of any significant accumulation of the free sterol. Accumulation of appreciable amounts of free 24(S),25-epoxycholesterol was associated with lowered levels of reductase. The existence of hepatic systems for the rapid inactivation and degradation of the oxysterols is consistent with their postulated role in the regulation of cholesterol synthesis.

Animals↗
PubMed1992

7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid by cytochrome P-450 in pig liver microsomes.

Pig liver microsomes were found to catalyze the 7 alpha-hydroxylation of several potential bile acid precursors besides cholesterol. 26-Hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid were all efficiently converted into the 7 alpha-hydroxylated products. Two cytochrome P-450 fractions showing 7 alpha-hydroxylase activity could be isolated. One fraction catalyzed 7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid but was inactive towards cholesterol. The other fraction catalyzed 7 alpha-hydroxylation of cholesterol in addition to the other substrates. 26-Hydroxycholesterol in equimolar concentration did not inhibit the cholesterol 7 alpha-hydroxylase activity of this fraction. It is concluded that liver microsomes contain a cytochrome P-450 catalyzing 7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid. The results indicate that this cytochrome P-450 is different from that catalyzing 7 alpha-hydroxylation of cholesterol.

Animals↗
PubMed1993

27-Hydroxycholesterol modulation of low density lipoprotein metabolism in cultured human hepatic and extrahepatic cells.

27-Hydroxycholesterol**, 25-hydroxycholesterol and cholesterol suppressed LDL uptake and degradation in human extrahepatic and hepatic cell lines in a concentration-dependent manner. Cholesterol was the least potent, and the inhibitory effect of oxysterols was more pronounced in skin fibroblasts and in endothelial cell line EAhy 926 than in hepatoma HepG2 cells. Shorter incubations were required for oxysterols to achieve 50% inhibition of LDL uptake and degradation in all cultured cells. The inhibition of LDL catabolism in extrahepatic cells by 27-hydroxycholesterol occurred at concentrations close to those observed in human plasma (0.2-0.6 microM). The results support a possible role of 27-hydroxycholesterol, a physiological oxysterol, in the regulation of cellular cholesterol homeostasis in non-hepatic tissues.

Cells, Cultured↗
PubMed1982

Distribution of 25-hydroxycholesterol in plasma lipoproteins and its role in atherogenesis. A study in squirrel monkeys.

Oxidation products of cholesterol have been shown to be potent inhibitors of cholesterol biosynthesis and also highly toxic to cultured aortic smooth muscle cells. In rabbit experiments, these compounds produced arterial injury resulting in arteriosclerosis. Purified cholesterol only minimally inhibited cholesterol biosynthesis and had no effect on cultured aortic smooth muscle cells. This raises the possibility that plasma lipoproteins containing beta-apoprotein (i.e. LDL and VLDL), which are considered to be atherogenic, may carry more oxidation products than HDL which is not atherogenic [3H]25-hydroxycholesterol and [14C]cholesterol were given only orally to 10 squirrel monkeys (Saimiri sciureus) and blood samples were collected via femoral puncture 24 h after administration. Lipoproteins were separated by ultracentrifugation and the radioactivity in each fraction was counted. Results show that the distribution of labeled cholesterol in VLDL, LDL, and HDL was almost identical to that of unlabeled cholesterol. Most of the radio-activity of 25-hydroxycholesterol was located in LDL & VLDL (55.1% and 34.7%, respectively), only 10.2% was present in HDL. If the radioactivity of 25-hydroxycholesterol were calculated on the basis of the apoprotein content of the lipoprotein micelle, the relative capacity of VLDL and LDL to carry 25-hydroxycholesterol was even greater and more significant than that of HDL (90 X and 42 X, respectively).

Animals↗
PubMed1986

26-Hydroxycholesterol disulfate: metabolism and excretion in the normal neonate.

Deuterated 26-hydroxycholesterol disulfate has been given in a tracer amount to neonates to evaluate the pool size, metabolism and excretion of the endogenously occurring compound in meconium. In a group of 5 normal neonates excretion of endogenous 26-hydroxycholesterol during the initial 72 h of life ranged from 327 to 1096 micrograms. Recovery of administered isotope during the same period was 66-98%. Only trace amounts of 26-hydroxycholesterol were recovered in urine. The findings indicate that relative to bile acid pool size, the normal neonate has a small intestinal pool of 26-hydroxycholesterol which, for the most part, is rapidly excreted and does not contribute significantly to bile acid synthesis. The techniques developed during the course of the study provide an approach to non invasive metabolic studies that give insights on the normal transition from fetal to neonatal life.

Bile Acids and Salts↗
PubMed1991

The effect of 25-hydroxycholesterol on accumulation of intracellular calcium.

Liposomes prepared with 25-hydroxycholesterol and egg phosphatidylcholine (PC) were incubated with bovine arterial smooth muscle cells for 8 h at 37 degrees C. Cells incubated in the absence of liposomes or with liposomes containing cholesterol and PC were used as controls. The results indicated that calcium accumulated in the smooth muscle cells incubated in the presence of 25-hydroxycholesterol containing liposomes in an amount proportional to the time of incubation. The calcium accumulation, as indicated by kinetic analysis, resulted from an increased compartment size. (Ca(2+)+Mg2+)-ATPase exhibited decreased activity after pretreatment with 25-hydroxycholesterol containing liposomes and the increased intracellular calcium content was directly proportional to the decreased (Ca(2+) + Mg2+)-ATPase activity. When lipids in the cell membrane were examined, a failure to change the cholesterol/phospholipids ratio in the membrane was noted. The 25-hydroxycholesterol content in the membrane determined by HPLC did not increase. An increase in sphingomyelin and a decrease in phosphatidylethanolamine and acidic phospholipids in the membrane was noted. We suggest that the accumulation of intracellular calcium comes from both an increase of calcium influx and a decrease of (Ca(2+) + Mg2+)-ATPase activity, which may be the consequence of changes in membrane phospholipid composition.

Animals↗
PubMed1979

Effects of cholesterol, hydroxycholesterols and calcium on pregnenolone production rates in mitochondrial fractions from rat testes.

The in vitro regulation of the mitochondrial conversion of cholesterol to pregnenolone in rat testis tissue has been further investigated. Pregnenolone production rates by isolated mitochondrial fractions could be stimulated by the addition of cholesterol. The stimulation was always highest in mitochondria isolated from lutropin-treated testes relative to control and cycloheximide treated testes. Addition 20- or 25-hydroxycholesterol resulted in a greater stimulation of pregnenolone production rates and these rates were unaffected by prior treatment with cycloheximide. When both cholesterol and 20- or 25-hydroxycholesterol were present in the incubation medium, pregnenolone production rates were mainly influenced by the hydroxycholesterol, even in the presence of a ten-fold excess of cholesterol. Ca2+ in vitro stimulated pregnenolone production rates from endogenous cholesterol as well as from added cholesterol. However, pregnenolone production rates in the presence of hydroxycholesterol were not influence by the addition of Ca2+ in vitro.

Animals↗
PubMed2006

7beta-hydroxycholesterol reduces the extent of reactive gliosis caused by iron deposition in the hippocampus but does not attenuate the iron-induced seizures in rats.

7beta-Hydroxycholesterol has been previously demonstrated to inhibit astrocytosis in injured cortex or spinal cord of rats. In this study, we explored the inhibitory effects of the liposome containing 7beta-hydroxycholesterol on the reactive astrocytosis caused by the injection of iron into the hippocampus of rats and furthermore evaluated the involvement of reactive astrocytosis in iron-induced epilepsy. Injection of ferric chloride solution unilaterally into the hippocampus of rats induced spontaneous spiking activity ipsilaterally then developed into bilateral hippocampi and generalized convulsive seizures within the first week post-operation, and spontaneous epileptiform activity and generalized seizures lasted as long as 2 weeks post-operation, whereas none of the rats injected with sodium chloride solution unilaterally into the hippocampus developed generalized seizures. With immunohistochemistry and Western blot analyses, apparent reactive astrocytosis in bilateral hippocampi was detected using antibody against glial fibrillary acidic protein 14 days after the injection of ferric chloride solution, but no significant differences were found in the amount of synaptophysin protein, a presynaptic vesicle protein, as compared with the rats injected with sodium chloride solution. Infusion of liposome suspension containing 7beta-hydroxycholesterol into the same site immediately after the injection of ferric chloride solution reduced the extent of the reactive astrocytosis by 50%-55% of the amount of glial fibrillary acidic protein in the hippocampi of both hemispheres, and non-significantly elevated the amount of synaptophysin protein in both sides of hippocampus. However, these effects did not significantly modify the seizure latency and the incidence of generalized seizures in the rats. These findings demonstrate the effects of 7beta-hydroxycholesterol on the inhibition of reactive astrocytosis caused by iron deposition in the hippocampus of rats, and suggest that the reactive astrocytosis may not play a causal role in the development of iron-induced seizures.

Action Potentials↗
PubMed2003

Impairment of the cytotoxic and oxidative activities of 7 beta-hydroxycholesterol and 7-ketocholesterol by esterification with oleate.

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 7 beta-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 7 beta-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 7 beta-hydroxycholesterol and 7-ketocholesterol were noted with cholesterol, 7 beta-hydroxycholesteryl-3-oleate and 7-ketocholesteryl-3-oleate, with the exception of a slight increase in superoxide anion production with 7 beta-hydroxycholesteryl-3-oleate. This finding supports the theory that 7 beta-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.

Arteriosclerosis↗