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PubMed1999

Apoptosis induced by oxysterol in CEM cells is associated with negative regulation of c-myc.

Previously we have demonstrated that treatment of the human lymphoblastic leukemic CEM cells with 25-hydroxycholesterol (25OHC) induces apoptosis. In the present study, we show that both c-myc mRNA and c-Myc protein levels are reduced only in oxysterol-sensitive and not in oxysterol-resistant cells after treatment with concentrations of 25OHC that kill the sensitive CEM cells. The repression of c-Myc protein precedes c-myc mRNA reduction, and both events occur before the onset of cell death. Our data suggest that 25OHC-induced suppression of c-myc gene expression in CEM cells results from posttranscriptional regulation. These results demonstrate the regulation by an oxysterol of a gene/gene product important for cell growth and viability and an association between oxysterol-induced apoptosis of CEM cells and the negative regulation of c-myc.

Apoptosis↗
PubMed1993

Regulation of phenobarbital-inducible cytochrome P450 2B1/2 mRNA by lovastatin and oxysterols in primary cultures of adult rat hepatocytes.

Lovastatin (LOVA) is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase widely used in clinical practice. We treated primary cultures of adult rat hepatocytes, maintained in a minimal, serum-free medium on Matrigel, a reconstituted basement membrane, with this drug, and found that the amounts of P450 2B2 mRNA detected on Northern blots were increased at the same doses (10(-5) to 3 x 10(-5) M) required for induction of HMG-CoA reductase mRNA, a gene known to be under oxysterol regulatory control. LOVA treatment produced selective effects increasing also the mRNA levels for P450s 2C6, 2C7, 3A1, and 4A1 but not for 1A1, 2A1/2, or NADPH-cytochrome P450 oxidoreductase. LOVA treatment increased the induction of 2B1/2 mRNA in cells cotreated with either phenobarbital (PB; 10(-4) M) or clotrimazole (CTZ; 10(-5) M), or of 3A1 mRNA in cells cotreated with PB (2 x 10(-3) M), but not dexamethasone (10(-5) M. LOVA treatment did not potentiate the induction of 1A1 or 4A1 mRNA in cells cotreated with beta-naphthoflavone (10(-5) M) or ciprofibrate (10(-4) M), respectively. In contrast to the potentiation of 2B1/2 mRNA induction produced by treatments with LOVA in combination with PB or CTZ, cotreatment of hepatocytes with PB and CTZ did not result in increased induction relative to that seen in cells treated with either agent alone. Treatment of hepatocyte cultures with either mevalonate (3 x 10(-4) to 3 x 10(-3) M), the immediate product of HMG-CoA reductase, or 25-hydroxycholesterol (10(-6) to 10(-5) M), a model oxysterol, resulted in dose-dependent suppression of 2B1/2 mRNA induction in cells treated with PB-like inducers. Taken together, our results demonstrate that LOVA is a unique inducer of P450 mRNA in cultured rat hepatocytes and implicate oxysterols as potential intracellular modulators of 2B1/2 induction. We conclude that endogenous metabolic factors including those related to cholesterol biosynthesis are critical in induction of liver cytochromes P450 2B1 and 2B2 by PB and "PB-like" agents.

Animals↗
PubMed1998

Effect of a dopamine agonist on luteinizing hormone receptors, cyclic AMP production and steroidogenesis in rat Leydig cells.

Dopamine agonists are known to increase the incidence of Leydig cell hyperplasia/adenomas when administered to rats over periods of 1-2 years. We have examined the early changes in factors affecting luteinizing hormone (LH)-controlled signal transduction pathways and steroidogenesis in Leydig cells in vitro after chronic oral administration of one of these dopamine agonists, Mesulergine (CU327-085) (N-(1-6,dimethylergolin-8a-yl)-N',N'-dimethylsulphamide hydrochloride) to Sprague-Dawley (SD) rats. Eight-week-old rats were given this dopamine agonist (2 mg/kg body wt/day) in food for 1, 5, or 12 weeks. The Leydig cells from control and treated rats were purified by elutriation and density gradient centrifugation. The dopamine agonist treatment was found to decrease the specific binding of 125I-human chorionic gonadotrophin (hCG) binding to the Leydig cells: a decrease was detected as early as 1 week after treatment and was more pronounced after 5 and 12 weeks. This was found to be due to a decrease in the LH/hCG receptor numbers and not to a decrease in LH/hCG-receptor binding affinity. Both basal and LH-stimulated cAMP and testosterone production were also decreased; cAMP production was decreased by approximately 50% by all concentrations of LH added whereas testosterone production was only decreased with submaximum stimulating concentrations of LH. The formation of testosterone in response to dibutyryl cAMP was also decreased by approximately 50%, indicating additional lesions in the signal transduction pathway. The addition of the cell permeant 22R-hydroxycholesterol (22R) demonstrated that testosterone but not pregnenolone production was decreased by treatment with the dopamine agonist, thus indicating that the 17 alpha-hydroxylase/C17-20 lyase may have been inhibited. Supporting evidence for this was found because the dopamine agonist also increased aromatase activity in the Leydig cells and thus the potential to produce estrogens; previous studies have shown that estradiol is an inhibitor of the 17-20 lyase enzyme. The addition of the dopamine agonist directly to the Leydig cells did not inhibit cAMP production or testosterone production except at high concentrations. It is concluded that treatment of rats with the dopamine agonist indirectly (i.e., via the pituitary) affects Leydig cell function resulting in a rapid decrease in LH receptors and cAMP and testosterone production. Aromatase activity is increased and thus the capacity to produce estrogens. These early changes in the signal transduction pathways and steroidogenesis may be involved in the Leydig cell hyperplasia/adenoma formation that subsequently occurs.

Administration, Oral↗
PubMed1976

The role of cytochrome P-450 in the regulation of steroid biosynthesis.

A cytochrome P-450 from bovine adrenocortical mitochondria has been purified to near homogeneity. The protein catalyzes side-chain cleavage of cholesterol (cholesterol leads to pregnenolone) but neither 11beta- nor 18-hydroxylation. It consists of 16 subunits of two species (MW 52,000) and contains 8 heme groups. The enzyme has been used to determine the stoichiometry of side-chain cleavage with the following results: (TPNH and O2 consumed/mole of cleavage), cholesterol 3:3:1, 20S-hydroxycholesterol 2:2:1 and 20S,22R-dihydroxycholesterol 1:1:1. These findings support the occurrence of the proposed pathway for the side-chain cleavage of cholesterol. Cleavage of the diol is inhibited by CO and shows a characteristic P-450 photochemical action spectrum. Evidently the diol is cleaved in a typical monoxygenase reaction. The active form of the enzyme contains 16 subunits (protein 16); forms consisting of 8 (protein 8) and 4 (protein 4) subunits can be isolated and are enzymatically active only by prior conversion to protein 16.

Adrenal Cortex↗
PubMed1991

Characterization of the LDL receptor in rat promegakaryoblasts in culture.

Rat promegakaryoblasts (RPM, a precursor platelet cell line) in culture exhibited a capacity to bind, take up and degrade 125I-LDL. The low density lipoprotein (LDL) binding showed the following characteristics: (a) high affinity, (b) saturability, (c) specificity, (d) down-regulation, after exposure to 25 hydroxycholesterol. Furthermore the proteolytic degradation of 125I-LDL by RPMs was inhibited by chloroquine which interferes with the lysosomal degradation processes. These findings show LDL receptor cell biology of RPM to be of the classical type and to differ from that of platelets.

Animals↗
PubMed1988

Further characterization of a somatic cell mutant defective in regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Two enzymes of mammalian cellular mevalonate biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase, have been shown to be regulated by exogenous sterols. It has been demonstrated that these enzymes are regulated, at least in part, by transcriptional control of their synthesis. We have previously described a somatic cell mutant (CR1) of the CHO-K1 cell line that is defective in regulation of the activity of these enzymes in response to exogenous sterols. In this report, we demonstrate that this mutant is defective in regulation of the mRNA levels for HMG-CoA reductase and HMG-CoA synthase by 25-hydroxycholesterol and mevinolin. In the case of HMG-CoA reductase, this loss of apparent transcriptional control is not accompanied by a comparable loss in regulation of synthesis of this enzyme. This observation is consistent with prior studies suggesting that HMG-CoA reductase can be regulated translationally. We also show that CR1 cells exhibit a constitutively rapid rate of degradation of HMG-CoA reductase.

Animals↗
PubMed1996

Microscale preparation of natural and labeled oxysterols.

The preparation of cholesterol and radiocholesterol oxidation products on a microscale is difficult. Cholesterol generally resists oxidation unless it is well dispersed under controlled conditions. A method was developed to reliably produce 7 alpha- and 7 beta-hydroxycholesterol and 7-ketocholesterol. Small changes in pH, metal ions present, or in the colloidal dispersion, resulted in production of completely different oxysterols. Attempts to oxidize aged radiocholesterol were not successful even after purification by several thin-layer chromatographic steps, and this appeared to be due to a time-related change in the radioactive material. Fresh radiocholesterol oxidized readily.

Carbon Radioisotopes↗
PubMed1996

Application of 2-hydroxypropyl-beta-cyclodextrin in the assay of acyl-CoA:cholesterol acyltransferase and neutral and acid cholesterol ester hydrolases.

The utility of 2-hydroxypropyl-beta-cyclodextrin for increasing the sensitivity of assays for the microsomal acylCoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-beta-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-beta-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-beta-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-beta-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1981

Regulation of 3-hydroxy-3-methylglutaryl CoA reductase by analogs of cholesterol and bile acids in cultured intestinal mucosa.

Sodium fusidate and its glycine conjugate, which have the same detergent properties as bile acids, significantly (p less than 0.05) stimulate HMG-CoA reductase of cultured intestine below the critical micellar concentration (CMC) without affecting brush border enzymes. Above CMC, both amphiphiles are cytotoxic. At concentrations between 1 and 5 mM, sodium fusidate decreased cholesterol contents of cultured mucosa (P less than 0.05), the increase in synthesis only partially compensating for the sterol loss. Oxygenated sterols, 7-keto- and 25-hydroxycholesterol, also depleted mucosal cholesterol at 0.5 mM, exerting their effect differently by inhibiting HMG-CoA reductase (p less than 0.01). In contrast to their marked effect on total mucosal cholesterol contents, brush border cholesterol was unaffected by both cholesterol and bile acid analogs.

Animals↗
PubMed1981

Lethal effect of cis- but not trans-22-dehydrocholesterol on mouse fibroblast cells.

cis- and trans-22-dehydrocholesterol were incorporated into the culture medium for mouse fibroblasts cells. Although neither isomer had an effect on sterol biosynthesis, the cis isomer inhibited cell growth and viability and increased Rb+ efflux from the cells. The trans isomer had no effect on growth and could replace exogenous cholesterol for growth of cells for which sterol biosynthesis was blocked by 25-hydroxycholesterol.

Cell Division↗
PubMed2006

Short-term hepatic effects of depleted uranium on xenobiotic and bile acid metabolizing cytochrome P450 enzymes in the rat.

The toxicity of uranium has been demonstrated in different organs, including the kidneys, skeleton, central nervous system, and liver. However, few works have investigated the biological effects of uranium contamination on important metabolic function in the liver. In vivo studies were conducted to evaluate its effects on cytochrome P450 (CYP) enzymes involved in the metabolism of cholesterol and xenobiotics in the rat liver. The effects of depleted uranium (DU) contamination on Sprague-Dawley were measured at 1 and 3 days after exposure. Biochemical indicators characterizing liver and kidney functions were measured in the plasma. The DU affected bile acid CYP activity: 7alpha-hydroxycholesterol plasma level decreased by 52% at day 3 whereas microsomal CYP7A1 activity in the liver did not change significantly and mitochondrial CYP27A1 activity quintupled at day 1. Gene expression of the nuclear receptors related to lipid metabolism (FXR and LXR) also changed, while PPARalpha mRNA levels did not. The increased mRNA levels of the xenobiotic-metabolizing CYP3A enzyme at day 3 may be caused by feedback up-regulation due to the decreased CYP3A activity at day 1. CAR mRNA levels, which tripled on day 1, may be involved in this up-regulation, while mRNA levels of PXR did not change. These results indicate that high levels of depleted uranium, acting through modulation of the CYP enzymes and some of their nuclear receptors, affect the hepatic metabolism of bile acids and xenobiotics.

Animals↗
PubMed2006

Biosynthetic capacity of rainbow trout (Oncorhynchus mykiss) interrenal tissue after cadmium exposure.

The disruption of endocrine system function in wildlife species, including teleosts, by contaminants such as metals is presently of major environmental concern. Recently, it has been shown that cadmium (Cd) exposure results in significant reductions in corticosteroid secretion by fish interrenal steroidogenic cells, likely through an inhibition of intracellular cortisol synthesis. In the present study, the effects of CdCl(2) on unstimulated and stimulated interrenal steroidogenesis in rainbow trout were examined with the intention of furthering an understanding of the site(s) of Cd toxic action. CdCl(2) alone reduced cortisol secretion in minced interrenal tissues to 59% and 55% of control values when exposed to 10 and 100 microM, respectively. Incubation of interrenal tissues with 0.01 IU/mL adrenocorticotropic hormone (ACTH), which activates rate-limiting steps in steroid synthesis, resulted in significant stimulation of steroidogenesis in controls. However, ACTH-stimulated steroidogenesis was reduced when tissues were previously incubated with Cd. Maximal rates of unstimulated cortisol secretion were achieved by augmentation using 5 microM 25-hydroxycholesterol (25-OHC) or 0.8 microL/mL synthetic cholesterol [SyntheChol(SC)]. Steroidogenesis augmentation by 25-OHC was significantly reduced in tissues incubated with Cd. Interestingly, cortisol secretion was significantly higher in SC-augmented tissue exposed to 1 and 10 microM Cd when compared to augmented control tissues. The results of this study show that Cd affects both stimulated and unstimulated steroidogenesis in rainbow trout, and that one major site(s) of action of Cd in the cortisol synthesis pathway is likely prior to cytochrome P450 side chain cleavage.

Adrenocorticotropic Hormone↗
PubMed1999

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

BACKGROUND AND AIMS: Cholesterol oxidation products (oxysterols) are commonly found in foods of animal origin and are also produced endogenously in the body. Oxysterols are cytotoxic to certain cell lines and in some cases have been shown to induce apoptosis. The aim of this study was to investigate the effects of 7beta-hydroxy-cholesterol (7beta-OHC) and 25-hydroxycholesterol (25-OHC) on cytotoxicity and induction of apoptosis in U937 and HepG2 cells, treated in media containing either 2.5% foetal calf serum (FCS) or 10% FCS to examine the effect of increasing the cholesterol level. METHODS: The cells were incubated for 24 and 48 h with 30 microM oxysterol. Viability was assessed by fluorescein diacetate/ethidium bromide staining and cell proliferation was determined by haemocytometer counting. Apoptosis was monitored by detection of DNA fragments (laddering) in 1.5% agarose gels. Cells with condensed or fragmented nuclei were identified by Hoechst 33342 staining. The percentage of cells with sub-G1 levels of DNA was measured by flow cytometry. RESULTS: Treatment of U937 cells with 7beta-OHC, in contrast to 25-OHC, resulted in a decrease in cell viability and proliferation at 24 and 48 h (P <.01). 25-OHC and 7beta-OHC were both equally cytotoxic to the HepG2 cell line. 7beta-OHC induced DNA laddering and an increase in the percentage of condensed or fragmented nuclei at both time points and at both serum concentrations in the U937 cell line. 25-OHC induced faint laddering in the U937 cells after 48 h in reduced serum media and resulted in a small increase in percentage condensed or fragmented nuclei which was independent of time of oxysterol exposure and serum concentration. The percentage of condensed or fragmented nuclei was low in the HepG2 cell line and no laddering was observed under any of the conditions studied. Flow cytometric analysis showed that only 7beta-OHC treated U937 cells had an increased level of hypodiploid cells. CONCLUSION: Both oxysterols appear to be equally cytotoxic to the HepG2 cell line. In U937 cells, 25-OHC is much less cytotoxic than 7beta-OHC. In addition, we have shown that 7beta-OHC induces apoptosis in U937 cells. 10% FCS displays a protective effect on cytotoxicity (as well as on 7beta-OHC induced apoptosis in U937 cells), although the data did not reach statistical significance.

Apoptosis↗
PubMed2006

RhoA and p38 MAPK mediate apoptosis induced by cellular cholesterol depletion.

Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.

Animals↗
PubMed2001

Growth modulation of low density lipoprotein receptor sterol sensitivity in cultured human fibroblasts.

Sterols regulate low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene expressions by end product repression. Studies on cultured cells have shown that growing cells have a higher LDL uptake than quiescent cells and that incubation of cells with growth factors or mitogenic compounds leads to sterol-resistant upregulation of LDL receptor gene expression. The recent finding that elevated LDL receptor activity in acute myelogenous leukemia cells was characterized by a decreased sensitivity to downregulation by sterols raises the possibility that the mechanism behind this is related to the cellular growth rate. By using cultured human fibroblasts as a model system we therefore studied whether growth modulation of sterol sensitivity takes place in normal actively growing cells. Judging from the ability of sterols (25-hydroxycholesterol + cholesterol) to inhibit 125I-LDL degradation, we found that the sensitivity to sterols varied markedly between cells of different densities. The lowest sensitivity to sterols and highest 125I-LDL degradation rate were found in subconfluent cells, whereas sparse and confluent cells were the most sensitive ones. In contrast to the LDL receptor, HMG-CoA reductase sterol sensitivity did not appear to be growth regulated. We conclude that growth-dependent modulation of sterol sensitivity and LDL receptor activity takes place in normal human fibroblasts. Modulation of sterol sensitivity may be an important mechanism to ensure an adequate cholesterol supply in growing cells.

Cell Division↗
PubMed2005

Mitochondrial cholesterol transport: a possible target in the management of hyperlipidemia.

Sterol 27-hydroxylase (CYP27A1) may defend cells against accumulation of excess cholesterol, making this enzyme a possible target in the management of hyperlipidemia. The study objective was to analyze cholesterol homeostatic responses to increases in CYP27A1 activity in HepG2 cells and primary human hepatocytes. Increasing CYP27A1 activity by increasing enzyme expression led to significant increases in bile acid synthesis with compensatory increases in HMG-CoA reductase (HMGR) activity/protein, LDL receptor (LDLR) mRNA, and LDLR-mediated cholesterol uptake. Under these conditions, only a small increase in cellular 27-hydroxycholesterol (27OH-Chol) concentration was observed. No changes were detected in mature sterol regulatory element-binding proteins (SREBP) 1 or 2. Increasing CYP27A1 activity by increasing mitochondrial cholesterol transport (i.e., substrate availability) led to greater increases in bile acid synthesis with significant increases in cellular 27OH-Chol concentration. Mature SREBP 2 protein decreased significantly with compensatory decreases in HMGR protein. No change was detected in mature SREBP 1 protein. Despite increasing 27OH-Chol and lowering SREBP 2 protein concentrations, LDLR mRNA increased significantly, suggesting alternative mechanisms of LDLR transcriptional regulation. These findings suggest that regulation of liver mitochondrial cholesterol transport represents a potential therapeutic strategy in the treatment of hyperlipidemia and atherosclerosis.

Adenoviridae↗
PubMed1998

Cholesterol oxidation in meat from chickens fed alpha-tocopherol- and beta-carotene-supplemented diets with different unsaturation grades.

The production of B-ring and side-chain oxysterols was evaluated in meat from chickens fed diets differing by the kind of oil or fat added. The effect of supplementary levels of natural antioxidants, as alpha-tocopherol and beta-carotene, on the meat cholesterol oxidative stability was also studied. Lard, sunflower and olive oil were used as dietary fat. Raw and cooked meats were analyzed for oxysterols, and cholesterol was also quantified. Oxysterol analyses were carried out by combining the use of solid-phase extraction, thin-layer chromatography, capillary gas chromatography, and capillary gas chromatography-mass spectrometry. Oxysterols were detected within the 0.1-0.5 microg/g range in raw meat. Cooking increased the oxysterol content of the meat, and levels as high as 5 microg/g muscle tissue were observed. B-Ring oxysterols were mainly produced: the alpha- and the beta-epoxycholesterols, the 7alpha- and 7beta-hydroxycholesterols, and the 7-ketocholesterol. The results showed that the meat from the chickens fed the olive oil-based diet containing alpha-tocopherol at 200 mg/kg of diet presented the best cholesterol oxidative stability. A positive effect could not be found for dietary beta-carotene administered at levels of 15 and 50 mg/kg of diet. Furthermore, a significant decrease in the tissue cholesterol content was observed with the olive and the sunflower oil-based diets.

Animal Feed↗
PubMed1988

Assay of cholesterol 7 alpha-hydroxylase activity in rat hepatocytes in primary monolayer culture.

A sensitive and precise method is described to assay cholesterol 7 alpha-hydroxylase activity in homogenates of rat hepatocytes cultured in monolayers for up to 76 h. The assay is based on measurement of the amount of radioactive cholesterol converted into 7 alpha-[14C]-hydroxycholesterol. Since no subcellular fractionation was applied to measure enzyme activity, this method is rapid and can be performed with cell protein, corresponding to as little as 1 to 2 million hepatocytes. Optimal assay conditions were determined and the reproducibility of this cholesterol 7 alpha-hydroxylase determination was established. Exogenous cholesterol (105 microM), solubilized in Tween 80, was added to saturate the enzyme, giving an apparent Km of 56 microM. Under these conditions, 70% of the cholesterol present in the homogenates is directly accessible to the cholesterol 7 alpha-hydroxylase. The detection limit of the assay was found to be about 10 pmol per incubation. A time course of the cholesterol 7 alpha-hydroxylase activity in cultured hepatocytes revealed that after an initial loss of approximately 60% of the activity as compared with 287 pmol/h/mg for freshly isolated cells, the enzyme activity was increased to the initial level in hepatocytes cultured for 52 h. This result and the finding that the cholesterol 7 alpha-hydroxylase activity was diminished by 94% after a 24-h incubation with 5 microM cycloheximide suggest that the enzyme activity is associated with de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗