Screening of novel nuclear receptor agonists by a convenient reporter gene assay system using green fluorescent protein derivatives.

Nuclear receptors represent a very good family of protein targets for the prevention and treatment of diverse diseases. In this study, we screened natural compounds and their derivatives, and discovered ligands for the retinoic acid receptors (RARs) and the farnesoid X receptor (FXR). In the reporter assay systems of nuclear receptors presented here, two fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), were used for detection of a ligand-based induction and as an internal control, respectively. By optimizing the conditions (e.g., of hormone response elements and promoter genes for reporter plasmids), we established a battery of assay systems for ligands of RARs, retinoid X receptor (RXR) and FXR. The screening using the reporter assay system can be carried out without the addition of co-factors or substrates. As a result of screening of more than 140 compounds, several compounds were detected which activate RARs and/or FXR. Caffeic acid phenylethyl ester (CAPE), known as a component of propolis from honeybee hives, and other derivatives of caffeic acid up-regulated the expression of reporter gene for RARs. Grifolin and ginkgolic acids, which are non-steroidal skeleton compounds purified from mushroom or ginkgo leaves, up-regulated the expression of the reporter gene for FXR.

Perfluorooctanoic acid, a peroxisome-proliferating hypolipidemic agent, dissociates apolipoprotein B48 from lipoprotein particles and decreases secretion of very low density lipoproteins by cultured rat hepatocytes.

The hypolipidemic effect is evoked by various peroxisome proliferators. Modulation of gene transcription via peroxisome proliferator-activated receptor (PPAR) is generally responsible for this effect. In addition, we have found a PPAR-independent mechanism in which fibrates, known peroxisome proliferators, decrease hepatic secretion of very low density lipoproteins (VLDL) through inhibition of phosphatidylcholine synthesis via methylation of phosphatidylethanolamine (PE) (T. Nishimaki-Mogami et al., Biochim. Biophys. Acta 1304 (1996) 21-31). In the present study, we show a novel mechanism by which perfluorooctanoic acid (PFOA), a potent peroxisome proliferator and inhibitor of PE methylation, exerts its hypolipidemic effect. PFOA (100 microM) added to the medium rapidly decreased the secretion of triglyceride by cultured rat hepatocytes, which was independent of the activity of cellular PE methylation. Analysis of the density of apoB secreted into the medium showed that PFOA decreased apoB48 in VLDL, but increased apoB48 in the bottom d>1.21 fraction. This lipid-poor apoB48 was also generated by incubating medium that had been harvested from control cells with PFOA, indicating that PFOA has the ability to dissociate apoB48 from lipoprotein particles. Exposure of cells to PFOA for 2 h prior to the experiment was sufficient to generate lipid-poor apoB48, indicating that PFOA exerted its effect intracellularly. Taken together, the data suggest that a strong interaction of PFOA with apoB48 disturbs the association of apoB48 with lipids in the process of intracellular VLDL assembly, thereby inhibiting VLDL secretion. This study shows that the mechanisms of hypolipidemic effect caused by various classes of peroxisome proliferators are diverse.

Bezafibrate and clofibric acid are novel inhibitors of phosphatidylcholine synthesis via the methylation of phosphatidylethanolamine.

The effects of bezafibrate and clofibric acid, fibrate hypolipidemic agents, on phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway were studied. In cultured rat hepatocytes, bezafibrate and clofibric acid added to the medium rapidly and markedly reduced the conversion of ethanolamine-labeled PE to PC (IC50 30 and 150 microM, respectively). Furthermore, the methylation of PE derived from serine was also blocked by bezafibrate, as was the secretion of PC derived from either serine or ethanolamine. The microsomal activity of PE N-methyltransferase was inhibited by these agents. Perfluorooctanoic acid but not DCQVA, though both are potent peroxisome proliferators comparable to fibrates, produced this inhibition. The inhibitory effects produced by these agents were diminished by dithiothreitol (DTT) added to the assay or alkaline pH assay condition. Inhibition by oleic acid was also attenuated under these conditions, suggesting a common mechanism of inhibition. However, unlike fatty acids, fibrates did not have rapid stimulatory effects on the CDP-choline pathway in hepatocytes. These results suggest that fibrates may mimic fatty acids in regulating PC synthesis from the PE methylation pathway but not the CDP-choline pathway.

The role of phosphatidylethanolamine methylation in the secretion of very low density lipoproteins by cultured rat hepatocytes: rapid inhibition of phosphatidylethanolamine methylation by bezafibrate increases the density of apolipoprotein B48-containing lipoproteins.

The role of phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway in the secretion of very low density lipoproteins (VLDL) by cultured rat hepatocytes has been investigated by determining the effects of inhibitors. We have shown that bezafibrate and clofibric acid, known hypolipidemic agents, are potent inhibitors of PE methylation (see accompanying paper by Nishimaki-Mogami et al. (1996) Biochim. Biophys. Acta 1304). In hepatocytes incubated with ethanolamine, which maintained cellular PE levels and PE methylation activity, bezafibrate (200 microM) decreased the secretion of triacylglycerol (TG), PC, apolipoproteins B48, and E in VLDL by 50-75%. In contrast, bezafibrate at this concentration had marginal effect on VLDL secretion (83-115% of control) by hepatocytes that had been cultured in the absence of ethanolamine. In these cells PE levels and PE methylation activity had decreased by approx. 40%. VLDL secretion was decreased at concentrations similar to those required to inhibit PE methylation, and was accompanied by an increase in cellular TG levels. The same ethanolamine-dependent effects were produced by clofibric acid and also by 3-deazaadenosine (DZA), an inhibitor of cellular methylation reactions. These results indicate that PC synthesis via the PE methylation pathway plays a significant role in VLDL secretion in rat hepatocytes if the pathway is maintained at levels comparable to those in vivo. The reductions of PE methylation by bezafibrate and DZA did not affect the total amount of apolipoprotein B48 secreted into the medium. The decrease in apolipoprotein B48 in VLDL caused by bezafibrate was accompanied by an increase in apolipoprotein B48 in the HDL density range. In contrast, the amount of apolipoprotein B100 in VLDL and density determined by sequential flotation were unaffected. These findings indicate that rapid reduction of PC synthesis via PE methylation does not affect the secretion of apolipoprotein B48- or B100-containing lipoprotein particles, but does impair the lipidation of particles containing apolipoprotein B48, but not apolipoprotein B100.

Activation induces dephosphorylation of cofilin and its translocation to plasma membranes in neutrophil-like differentiated HL-60 cells.

We suggested that a cytosolic 21-kDa phosphoprotein played an important role in opsonized zymosan-trigered activation of superoxide-generating enzyme in neutrophil-like HL-60 cells through dephosphorylation (Suzuki, K., Yamaguchi, T., Oshizawa, T., Yamamoto, Y., Nishimaki-Mogami, T., Hayakawa, T., and Takahashi, A (1995) Biochim. Biophys. Acta 1266, 261-267). In the present study, we characterized the phosphoprotein and studied changes in it localization upon activation of phagocytes. The 21-kDa phosphoprotein was rapidly dephosphorylated upon activation not only wit opsonized zymosan but also with formyl-Met-Leu-Phe and arachidonic acid. The peptide fragments derived from the 21-kDa phosphoprotein were found to have the same amino acid sequences as those of cofilin, an actin-binding protein. The phosphoprotein reacted exclusively with anti-cofilin antibody on two dimensional immunoblots. Accordingly, together with its apparent molecular weight, isoelectric point, and detection of phosphoserine as a phosphoamino acid, we concluded that the 21-kDa phosphoprotein was a phosphorylated form of cofilin. The amount of cofilin in membranous fractions was increased upon activation. Furthermore, confocal laser scanning microscopy showed that cofilin existed diffusely in the cytosol and nuclear region of the resting cells, while in the activated cells, it was accumulated at the plasma membrane area, forming ruffles or endocytic vesicles on which O2.- should be produced. These results suggested that in resting cells cofilin exists as a soluble phosphoprotein in the cytosol and nuclei, while upon stimulation a large portion of cofilin is dephosphorylated and translocated to the plasma membrane regions.

Okadaic acid induces both augmentation and inhibition of opsonized zymosan-stimulated superoxide production by differentiated HL-60 cells. Possible involvement of dephosphorylation of a cytosolic 21K protein in respiratory burst.

We found that okadaic acid (OA), a potent tumor promoter and a phosphatase inhibitor, has a unique opposing effect on opsonized zymosan (Op.-zym.)-elicited O2.- production by differentiated HL-60 cells in a narrow range of concentrations but does not induce any O2.- production by itself. Okadaic acid magnified the O2.- production 2.5-fold at 1.0 microM, while it inhibited it at 2.0 microM or higher concentrations. This effect of OA did not correspond to the changes in the expression of surface receptors (CD11b/CD18, CR3) for Op.-zym., because they were weakly down-regulated by OA at any concentration. Two-dimensional gel electrophoresis revealed that in the absence of OA, Op.-zym. induced rapid dephosphorylation of a cytosolic 21K protein with a very slight increase in phosphorylation of membranous p47phox, which is one of the cytosolic factors required for respiratory burst. In the presence of a stimulatory concentration (1.0 microM) of OA, the Op.-zym.-caused dephosphorylation of the 21K protein was still observed and the phosphorylation of p47phox was enhanced. In the presence of an inhibitory concentration (2.0 or 5.0 microM) of OA, the Op.-zym.-induced dephosphorylation of the 21K protein was strongly inhibited while p47phox was heavily phosphorylated. Acid hydrolysis of the 21K phosphoprotein yielded only phosphoserine as a phosphoamino acid. Furthermore, at least part of the 21K protein seemed to be associated with p67phox and p47phox, because it was co-immunoprecipitated with those cytosolic factors. These results suggest that a cytosolic 21K protein plays an important role in respiratory burst through dephosphorylation by a phosphoserine phosphatase, and that the dephosphorylated 21K protein may work synergistically with the phosphorylated p47phox on the pathway for activation of the respiratory burst oxidase.

Activation of a peroxisome-proliferating catabolite of cholic acid to its CoA ester.

We have shown that a microbial cholic acid catabolite (4R)-4-(2,3,4,6,6a beta,7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-3-oxo- 1H-cyclopenta[f]quinolin-7 beta-yl)valeric acid (DCQVA), is a potent peroxisome proliferator. In this paper a possible key stage in DCQVA metabolism, the activation of DCQVA to its CoA ester, has been investigated in rat liver microsomes and particulate fractions. The microsomal reaction was dependent on CoA, ATP, DCQVA (0.2-1 mM) and protein content. The reaction was decreased by storage at 4 degrees C, preincubation of microsomes at 37 degrees C for 5 min, or inclusion of Triton X-100 in the reaction mixture. Such treatments also enhanced generation of long-chain fatty acyl-CoAs, as determined by h.p.l.c. analysis. The same effect was caused by exposing the microsomes to phospholipase A2, suggesting that endogenous fatty acids may compete with DCQVA for esterification with CoA. Subcellular fractionation of rat liver demonstrated that the activity of DCQVA-CoA synthesis was localized predominantly in the microsomal fraction, in contrast to long-chain fatty acyl-CoA synthetase, which was distributed among all particulate fractions. Administration of clofibrate of rats did not affect the distribution of DCQVA-CoA synthesis activity. In contrast to a 2-fold induction of long-chain fatty acyl-CoA synthetase by clofibrate treatment, the activity of DCQVA-CoA synthesis in the microsomal fraction decreased by 80%. These results suggest that DCQVA is activated by an enzyme distinct from long-chain fatty acyl-CoA synthetase. The resulting perturbation of fatty acid metabolism may be involved in the mechanism whereby DCQVA causes peroxisome proliferation.

Induction of peroxisomal beta-oxidation by a microbial catabolite of cholic acid in rat liver and cultured rat hepatocytes.

The capability of (4R)-4-(2,3,4,6,6a beta,7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-3-oxo-1H-cyclopental[f]quinolin-7 beta-yl)valeric acid (DCQVA), a catabolite of cholic acid produced by enterobacteria, to induce peroxisome proliferation in vivo and in vitro was studied. Rats given 0.3% DCQVA in the diet for 2 weeks showed marked increases in peroxisomal beta-oxidation, mitochondrial 2,4-dienoyl-CoA reductase and microsomal laurate omega-oxidation activities in the liver compared with control rats given the diet without DCQVA. Cultured rat hepatocytes treated with DCQVA for 72 h also exhibited greatly enhanced beta-oxidation activity. The increased activity was concentration-dependent and the effective concentrations were comparable with those of clofibric acid that produced the same degree of induction in the assay. The results demonstrate that DCQVA is a potent peroxisome proliferator that occurs naturally in rat intestine.

Evidence that the rate of phosphatidylcholine catabolism is regulated in cultured rat hepatocytes.

The regulation of phosphatidylcholine (PC) catabolism has been studied in choline-deficient rat hepatocytes. Supplementation of choline-deficient hepatocytes, prelabeled with [3H]choline, with 100 microM choline increased the rate of PC catabolism by approx. 2-fold. The major product of PC degradation was glycerophosphocholine in both choline-deficient and choline-supplemented cells. Choline supplementation decreased the radioactivity recovered in lysoPC by 50%. This effect was accompanied by a 2-fold increase of labeled glycerophosphocholine. Comparable results were obtained when PC of the cells was prelabeled with [3H]methionine or [3H]glycerol. The activity of phospholipase A in cytosol, mitochondria and microsomes isolated from choline-deficient rat liver was similar to the activity in control liver, when determined with [3H]PC vesicles as the substrate. Measurement of the activity of phospholipase A with endogenously [3H]choline-labeled PC showed that the formation of lysoPC in mitochondria isolated form choline-supplemented cells was 40% lower than in choline-deficient cells. Alternatively, the formation of [3H]glycerophosphocholine and [3H]choline in microsomes from choline-supplemented cells was significantly higher (1.4-fold) than in microsomes from choline-deficient cells. These results suggest that the rate of PC catabolism is regulated in rat hepatocytes and that the concentration of PC might be an important regulatory factor.

Effect of sorbic acid feeding on peroxisomes and sorboyl-CoA metabolizing enzymes in mouse liver. Selective induction of 2,4-dienoyl-CoA hydratase.

On the basis of the finding that sorbic acid (SA)-induced hepatoma was correlated with the depletion of reduced glutathione (GSH) in mouse liver (Tsuchiya et al., Mutation Res 130: 267-262, 1984), the possible conversion of SA to a metabolite which is reactive with SH-compounds was studied. Sorboyl-CoA was hydrated and then reduced to 3-keto-4-hexenoyl-CoA by the combined actions of mitochondrial hydratase (crotonase) and L-3-hydroxyacyl-CoA dehydrogenase. Upon the addition of GSH or coenzyme A, 3-keto-4-hexenoyl-CoA was nonenzymatically converted to another 3-ketoacyl-CoA derivative, possibly a Michael type adduct, in a time- and concentration-dependent manner. Alternatively, sorboyl-CoA can be reduced by 2,4-dienoyl-CoA reductase and completely beta-oxidized without the generation of 3-keto-4-hexenoyl-CoA. Two-week feeding of mice of 15% SA caused a 2.0-fold induction of peroxisome beta-oxidation in the liver. SA caused a marked induction (3.6-fold) of hydratase toward sorboyl-CoA but a less pronounced induction (1.3-fold) of 2,4-dienoyl-CoA reductase, leading to about a 3-fold elevation in the hydratase: reductase ratio. The elevated ratio was sustained throughout the period of SA feeding up to 12 weeks. Thus, a large amount of SA could be converted to 3-keto-4-hexenoyl-CoA during this period. Oxidative stress caused by a depleted cellular SH-pool together with the induction of peroxisome proliferation by SA-feeding may implicate the mechanism by which non-mutagenic SA caused hepatoma.

Distribution, metabolism, and excretion of musk xylene in rats.

Distribution, metabolism and excretion of musk xylene (MX) were investigated in male Wistar rats. Urinary and fecal excretion accounted for 10 and 75% of the dose (70 mg/kg), respectively, on day 7 after orally administration of 3H-MX to rats. Total residue of radioactivity in tissues on day 7 was less than 2.0% of the administered dose. The highest concentration was found in adipose tissue and the second was in liver. Some metabolites of MX were identified using GC-MS and NMR after purification by column or thin layer chromatography of feces, bile and urine extracts. MX, 2-NH2-MX, 2-Ac-MX, 2-NH2-3-CH2OH-MX, and 2-NH2-5-tert-BuOH-MX were found in feces, bile and urine. 4-NH2-MX and metabolite X were found in feces and urine. 4-NH2-3-CH2OH-MX was found in urine. HO-MX was found in bile. The major route of excretion for MX was the feces via bile. The reduction of the 2-nitro group of MX to the amino group was a key step in metabolism. Further metabolism of 2-NH2-MX may proceed by decreased steric hindrance of functional group.

Characterization of skin-surface lipids from the monkey (Macaca fascicularis).

Skin-surface lipids from the monkey Macaca fascicularis are composed of sterol esters (38%), cholesterol (4%) and two types of wax diesters, identified as Type II (IIa and IIb, 17% and 40%, respectively). Type IIa contained diesters of 1,2-alkanediols esterified with two molecules of long-chain (C14-C34) fatty acids having straight and branched chains. In the diesters IIa, fatty acids shorter than C19 predominated in position 1, and fatty acids longer than C20 predominated in position 2. Type IIb contained diesters of 1,2-alkanediols esterified with C4 and C5 branched-chain fatty acids (predominantly isovaleric acid) at position 1 and long-chain (C14-C27) acids, having straight and branched chains, at position 2. The short-chain acids were converted to 2-nitrophenylhydrazides and analyzed by high-performance liquid chromatography (HPLC). Ammonia chemical ionization (CI)-gas chromatography (GC)-mass spectrometry (MS) resolved the intact diesters IIb into 12 peaks corresponding to molecular weights ranging from 597 to 748, and showed that the molecular species, such as C21-C16-C5 (diol, fatty acid in position 2, fatty acid in position 1), C22-C16-C5 and C23-C16-C5, were prevalent. The fatty acids from both diesters were mostly (greater than 98%) saturated. The 1,2-alkane-diols from both diesters consisted of C16-C26 saturated straight- and branched-chain components. The acyl groups of sterol esters contained 86% C14-C34 branched-chain acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of rose bengal on serum levels of thyroid hormones and thyroid peroxidase activity in male mice.

The thyrotoxic effect of Rose bengal (RB) (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein disodium salt; Food Red No. 105) was examined in male (C57BL/6N X C3H/N) F1 mice. They were given drinking-water containing RB at levels of 0 (control), 0.125 and 0.250% for 2 weeks. The effect resulted in decreases in serum levels of 3,5,3'-triiodothyronine (T3) and thyroxine (T4), and slight increases in serum 3,3',5'-triiodothyronine (rT3) levels and thyroid weight, but no difference in the values for the body-weight gain, serum thyroid stimulating hormone (TSH) levels and thyroid peroxidase (TPO) activities. However, the in vitro inhibitory effect of RB on TPO activity was observed by addition of RB to the TPO-catalyzed guaiacol oxidation. These results suggest that RB might have weak goitrogenic properties, inhibiting the peripheral conversion of T4 to T3 and/or inhibiting TPO to lead a decrease of T4 and T3 formation.

Isolation and identification of metabolites of 2-ethylhexyl diphenyl phosphate in rats.

The metabolic fate of 2-ethylhexyl diphenyl phosphate (EHDPP) was studied in male rats. Orally administered 14C-EHDPP was rapidly absorbed and about 80% of the radioactivity was excreted in the urine and feces in the first 24 h. By 7 days, 48% and 52% of the radioactivity was recovered in urine and feces, respectively. Since biliary excretion was low (6% for 2 days), urine seems to be the major excretion route of EHDPP. Radioactivity was widely distributed in all tissues examined. At 2 h, the concentration was relatively high in blood, liver kidney and adipose tissue. The elimination of radioactivity from adipose tissue and liver was somewhat delayed, but almost all the radioactivity was eliminated by 7 days. The major metabolites in the urine were diphenyl phosphate (DPP) and phenol. p-Hydroxyphenyl phenyl phosphate (OH-DPP) and monophenyl phosphate (MPP) were also identified as minor metabolites.

Deuterium and tritium labeling of (3-xenyl)cyclohexane by Clemmensen and Wolff-Kishner reduction.

Tritium-labeling of (3-xenyl)cyclohexane was investigated by deuterium-labeling on Clemmensen and Wolff-Kishner reduction of 2-(3-xenyl)cyclohexane. Deuterium-labeling by the Clemmensen reduction was proved to be accompanied by isotope exchange via acid-catalyzed enolyzation of the carbonyl group. Thus, the labeled (3-xenyl)cyclohexane was a mixture of compounds with various numbers of deuterium atoms. Labeling by the modified Wolff-Kishner reduction gave a mixture of compounds labeled predominantly at position 2.

Involvement of the fatty acid oxidation complex in acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli.

The activity of acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli was enhanced when the organism was grown on oleic acid as the sole carbon source, but not detected when grown on glucose. Antibodies raised against fatty acid oxidation complex of E. coli inhibited both the reaction catalyzed by crotonase and the chain elongation in a similar manner, showing that the oxidation complex participates in the chain elongation. The activities of condensation and the activities of NADH- and NADPH-dependent 3-ketoacyl reduction in the cell-free extract were precipitated by antibodies to the complex in parallel with those of 3-ketoacyl-CoA thiolase and crotonase. These results together with the presence of NADPH-dependent trans-2-enoyl-CoA reductase in E. coli (Mizugaki, et al. (1982) Chem. Pharm. Bull. 30, 2503-2511) indicate that the acetyl-CoA-dependent chain elongation of fatty acids in E. coli occurs by the reversal of fatty acid oxidation other than the step of enoyl reduction.