Peroxisome proliferator-activated receptor alpha (PPARα) activation by fibrates controls expression of several genes involved in hepatic cholesterol metabolism. Other genes could be indirectly controlled in response to changes in cellular cholesterol availability. To further understand how fibrates may affect cholesterol synthesis, we investigated in parallel the changes in the metabolic pathways contributing to cholesterol homeostasis in liver. Ciprofibrate increased HMG-CoA reductase and FPP synthase mRNA levels in rat hepatocytes, together with cholesterogenesis from [14C] acetate and [3H] mevalonate. The up-regulation observed in fenofibrate- and WY-14,643-treated mice was abolished in PPARα-null mice, showing an essential role of PPARα. Among the three sterol regulatory element-binding protein (SREBP) mRNA species, only SREBP-1c level was significantly increased. In ciprofibrate-treated hepatocytes, cholesterol efflux was decreased, in parallel with cholesteryl ester storage and bile acids synthesis. As expected, AOX expression was strongly induced, supporting evidence of the peroxisome proliferation. Taken together, these results show that fibrates can cause cholesterol depletion in hepatocytes, possibly in part as a consequence of an important requirement of cholesterol for peroxisome proliferation, and increase cholesterogenesis by a compensatory phenomenon afterwards. Such cholesterogenesis regulation could occur in vivo, in species responsive to the peroxisome proliferative effect of PPARα ligands.

Fibrates are hypolipidemic drugs that exert multiple effects on lipid metabolism by activating peroxisome proliferator-activated receptor alpha (PPARα) and modulating the expression of many target genes. In order to investigate the link between PPARα and cholesterol synthesis, we analysed the effect of fibrates on expression of the farnesyl diphosphate synthase (FPP synthase) gene, known to be regulated by sterol regulatory element-binding proteins (SREBPs), in conjunction with HMG-CoA reductase. In wild-type mice, both fenofibrate and WY 14,643 induced FPP synthase gene expression, an effect impaired in PPARα-null mice. A three-fold induction was observed in ciprofibrate-treated rat hepatocytes, in primary culture. This effect was decreased in presence of 5,6-dichlorobenzimidazole riboside (DRB) and cycloheximide (CHX), transcription and translation inhibitors, respectively. Acyl-CoA oxidase (AOX), a bona fide PPARα target gene, was induced by ciprofibrate but slower and more strongly than FPP synthase. In addition, induction of FPP synthase gene expression was abolished in the presence of 25-hydroxycholesterol (25-OH Chol). Thus, activation of PPARα by fibrates induced FPP synthase gene expression in both hepatocytes in culture and in mouse liver. This effect is likely to be dependent on cellular sterol level, possibly through SREBP-mediated transcriptional activation.

Human HepG2, rat Fao and MH1C1 hepatoma cell lines have been examined for their response to ciprofibrate, a potent peroxisome proliferator. Changes in the morphological characteristics of peroxisomes, the inductibility of their proliferation and of their beta-oxidation enzymes, palmitoyl-CoA oxidase and bifunctional enzyme, were studied in control and treated cells. In Fao cells, peroxisomes are less numerous and smaller than in rat liver, but they increase in size and number under the effect of ciprofibrate, similarly to those of treated rat liver. The high peroxisome proliferation is accompanied by a strong induction of beta-oxidation enzymes as in vivo. In MH1C1 cells, peroxisomes are seen in irregular clusters in the cytoplasm, small with rounded to tubular forms, suggesting rapid peroxisomal growth. A striking observation is the particularly elongated, worm-like form of many of the peroxisomes. Under the effect of ciprofibrate, the proliferation is low, as is the induction of beta-oxidation enzymes. HepG2 cells contain few, small peroxisomes with a heterogeneity of forms, from spherical to elongated. The only peroxisomal response to ciprofibrate in these cells seemed to be a morphological reorganization. There is little or no induction of beta-oxidation enzymes by ciprofibrate in HepG2 cells, as in cultured human hepatocytes. Therefore, on the one hand, Fao and MH1C1 cells are complementary tools in the investigation of the regulation of the hepatic response to peroxisome proliferators in the rat, on the other hand, HepG2 and Fao cells are useful in the study of the species specificity of the response.

The Y-1 adrenal cell line was shown to produce 20α-dihydroaldosterone from deoxycorticosterone. This compound was identified by GC-MS by comparison with the previously synthesized reference compound. Two other 18-hydroxylated metabolites were identified as 11β,18-dihydroxy-20α-dihydroprogesterone from endogenous cholesterol and 18-hydroxy-20α-dihydro-11-dehydrocorti-costerone from DOC. The conditions necessary for the synthesis of these compounds are culturing in 20% serum-supplemented medium and repeated incubations with the substrate. The production of 11β-hydroxylated steroids and that of 18-oxygenated steroids is stimulated differently by ACTH and angiotensin II suggesting the expression of two different enzymes, cytochrome P-45011β and cytochrome P-450aldo The Y-1 cell line can secrete either 11β-hydroxylated steroids characteristic of the glucocorticoid pathway or 18-oxygenated steroids characteristic of the mineralocorticoid pathway, which in vivo are generally produced in two different zones of the adrenal cortex. This cell line should be an interesting model for the study of the molecular mechanisms regulating the expression of these two enzymes involved in the final steps of the steroidogenic pathways.

Summary— The response of two rat cell lines, Fao and MH 1 C 1 , and one human cell line, HepG2, to the peroxisome proliferator ciprofibrate, was studied. Using a fluorometric assay for palmitoyl‐CoA oxidase, the dose‐ and time‐dependent increase of this enzymatic activity was determined. From the lowest concentration (100 μM) stimulation is evident in the two rat cell lines. In the Fao line, the activity was stimulated reaching a seven‐fold increase over the control level at 250 μM after 72 h of treatment. In the MH 1 C 1 line, the maximum stimulation, four‐ to five‐fold, was obtained at 250 and 500 μM after 72 h. In the HepG2 cell line, activity increased two‐fold at 250 μM after 72 h reaching a three‐fold increase at 1000 μM after 48 h. Ciprofibrate was more toxic to Fao cells than to MH 1 C 1 and HepG2 cells which is also the order of the acyl‐CoA oxidase stimulation by ciprofibrate. These preliminary results suggest that the two rat cell lines are appropriate for investigating the induction of peroxisomal β‐oxidation enzymes and the expression of their genes. The HepG2 cell line is a complementary model for the study of interspecies differences in the response to peroxisomal proliferators and of the peroxisomal functions implied in the lipid metabolism of human liver.

The 20alpha-reduced derivative of aldosterone, 20alpha-dihydroaldosterone, was needed as reference compound in order to continue the studies on 18-hydroxylation in the Y-1 adrenal cell line. It was obtained by reduction of aldosterone with sodium borohydride. Analysis of the products of the reaction as methoxime trimethylsilyl (MO-TMS) derivatives by gas chromatography (GC) and GC-mass spectrometry (GC-MS) showed three possible forms of the compound. Their identification was confirmed by comparison with the products obtained by stereospecific reduction of aldosterone using 3alpha,20beta-hydroxysteroid dehydrogenase. Chromatographic behavior and mass spectra are given for the three forms of 20alpha-dihydroaldosterone as the MO-TMS derivatives; that is, the 18-aldehyde, the 18,11beta-hemiacetal, and the 11beta: 18,18:20alpha-acetal. The possible origin of these different forms is discussed as a function of these results and of the results obtained by complementary analysis on high-performance liquid chromatography.

The 18-hydroxylation of deoxycorticosterone in the Y-1 adrenal cell line was studied under various incubation and cell culture conditions and compared to 11β-hydroxylation. Repeated incubation of the substrate increased both 18- and 11β-hydroxylation in the Y-1 cells. Furthermore, both 18- and 11β-hydroxylation were increased with increased serum concentration and prolonged incubation time. While the increase in 11β-hydroxylation seemed to be independent of the type of serum, 18-hydroxylation was much more important in cells cultured in fetal or newborn calf serum supplemented medium than in those cultured in horse serum supplemented medium. As expected, ACTH treatment increased 11β-hydroxylation; however, it decreased 18-hydroxylation. The different regulation of these two hydroxylating pathways by ACTH, point to a heterogeneity of the cytochrome P-45011β of the Y-1 cell line.

The expression of 19-hydroxylase activity in the Y1 adrenal cell line is reported here for the first time. Two new metabolites from the incubation of deoxycorticosterone (DOC) with these cells, 19-hydroxy-20 alpha-dihydroDOC and 19-hydroxy-20 alpha-dihydrocorticosterone, have been identified. The most important of the two is the 11 beta,19-dihydroxylated metabolite, which is produced in smaller amounts than 18-hydroxy-20 alpha-dihydrocorticosterone. A third 19-hydroxylated metabolite was identified as 19-hydroxy-20 alpha-dihydroprogesterone, produced from the cholesterol in the serum supplemented medium. These results show that the cytochrome P-450(11)beta of this cell line expresses 19-hydroxylase activity in addition to 11 beta- and 18-hydroxylase activities, as do those of other species.

During the course of a study to produce reference compounds, the metabolism of tetrahydrogenated derivatives (ring A reduced) of progesterone, 6 alpha-hydroxyprogesterone, 11-deoxycorticosterone and corticosterone in newborn rat adrenal cells in primary culture was studied. Analysis of the metabolites was made by gas chromatography-mass spectrometry. Most products resulted from the enzymatic reactions of 11 beta-, 18- and 21-hydroxylation, reduction of the 20-oxo group and oxidoreduction of the 3-hydroxyl group. However, unexpected metabolites were produced from the incubation of 3 beta, 5 alpha-tetrahydroprogesterone and 6 alpha-hydroxy-3 alpha, 5 beta-tetrahydroprogesterone. They resulted from the 16 alpha-hydroxylation of the precursors and probably from the 15 alpha-, 16 beta- and 17 alpha-hydroxylation of 6 alpha-hydroxy-3 alpha, 5 beta-tetrahydroprogesterone. These hydroxylating activities are weak and were not detected from the endogenous steroidogenesis. They were not detected either from the incubation of exogenous steroids with a 3-oxo-4-ene structure or from steroids with a 21-hydroxyl substituent. They result only from substrates showing diminished or no affinity towards the 11 beta/18- and 21-steroid hydroxylase systems. These unusual hydroxylations could be catalyzed by monooxygenase systems in the endoplasmic reticulum similar to those present in the liver or by the monooxygenase systems specific to steroidogenesis. In particular, the reaction specificity of cytochrome P-450(11) beta could be altered by the presence of a 6 alpha-hydroxyl group in a tetrahydrogenated steroid.

The formation and gas Chromatographic behavior of syn-and anti- isomers in position 20 of the methoxime-trimethylsilyl (MO-TMS) derivatives of many 20-oxo and 3,20-dioxo-21-hy-droxysteroids is reported. The existence of such isomers was established from the gas Chromatographic (GC) and mass spectrometric analysis of the MO-TMS derivatives of 3α,21-dihydroxy-5β-pregnan-20-one and its 17α-epimer. The degree of separation during GC analysis of the syn-and anti-isomers in position 20, as well as those in position 3, is associated to the position of additional hydroxyl groups on the steroid ring. These data are very important for the location of oxygenated substituents such as 2α/2β, 6α/6β, 11β, 16α, 17α, 18, 19 or 21-hydroxyl groups during structural studies of 20-oxo and 3,20-dioxosteroids.

18-Hydroxylase activity, reported here for the first time in the mouse adrenal tumor cell line (Y1), was expressed in the metabolism of 11-deoxycorticosterone (DOC) and corticosterone (B). Detected after 24 h of incubation, it was more evident after 48 h and produced mostly 18-hydroxy-20 alpha-DHB from these exogenous substrates. However, 18-hydroxylation was quantitatively less significant than the metabolism of 20 alpha-reduction and 11 beta-hydroxylation (of DOC). The latter is also the predominant metabolism of progesterone in this cell line, during the conversion of cholesterol from the serum-supplemented culture media. The cytochrome P-450 11 beta activity of the Y1 cells is similar to that of the mouse in vivo which catalyzes the production of an 11 beta 18-dihydroxylated metabolite as the principal 18-hydroxylated steroid. It is different from that of other species, such as the rat and the bovine, both in terms of the ratio of 11 beta- to 18-hydroxylated metabolites and of the structure of these metabolites.

Consacree essentiellement a l'analyse des phenomenes survenant lors de l'interaction entre un faisceau laser co#2 continu et des particules metalliques de quelques dizaines de micrometres, cette etude experimentale est une approche complementaire pour interpreter et controler la formation d'alliage ou le depot de surface par laser avec apport de poudre. Elle comporte une phase experimentale initiale de caracterisation des ecoulements a particules qui se concretise par le developpement d'un dispositif de visualisation des trajectoires des particules et de mesure de leurs vitesses. La determination des vitesses et des concentrations volumiques des particules constitue une etape indispensable pour evaluer l'energie qu'elles absorbent pendant leur temps d'illumination dans le faisceau laser ainsi que l'attenuation du faisceau par le nuage de particules. La realisation d'un dispositif pour caracteriser ces deux grandeurs (absorption et attenuation), en presence du faisceau laser, constitue la partie experimentale de l'etude. L'exploitation du bilan energetique relatif au nuage de particules, devrait simplifier la recherche systematique des parametres de traitement de surface. Le dispositif de qualification des ecoulements mis au point, permet d'optimiser la geometrie des injecteurs de poudre en vue d'une industrialisation du procede

The metabolism of deoxycorticosterone (DOC) by newborn rat adrenal cells in primary culture at various times after culture, with and without ACTH, was studied. After 5 days in culture before addition of ACTH, the main products of the metabolism of DOC were corticosterone and 18-hydroxy-11-deoxycorticosterone in a 2:1 ratio. Smaller amounts of 20 alpha-dihydrocorticosterone and 18-hydroxycorticosterone were also found. No reduced metabolites of DOC were detected. Without ACTH the conversion of DOC to corticosterone and 18-hydroxyDOC declined rapidly. After 13 days in culture, this conversion accounted for only half the metabolites. The reductive metabolism of DOC which yields products reduced at 20 alpha and/or 3 alpha/beta and 5 alpha accounted for the other half. When ACTH (22 mU/ml) was added to the culture daily for several weeks, the primary metabolism of DOC remained that of 11 beta- and 18-hydroxylation yielding corticosterone and 18-hydroxyDOC. A minor reductive metabolism was found. Both cultures produced 6 beta-hydroxyDOC. These results demonstrate that ACTH is needed to maintain the efficiency of the 11 beta/18-hydroxylating system. They also show that ACTH controls the type of metabolism predominant in the rat adrenal cell and may be responsible for the balance between the biosynthesis of glucocorticoids and their reductive catabolism in the fasciculata zone of the adrenal gland.

Analysis of urinary steroids excreted by a 7-year old girl with low renin hypertension following ACTH treatment revealed several unknown steroids, which have been analysed by gas chromatography-mass spectrometry. It is proposed that these steroids are monohydroxylated derivatives of cortisol, cortisone, either or both tetrahydro and allo-tetrahydrocortisol and either or both tetrahydro and allo-tetrahydro-11-deoxycortisol. Further analysis indicated that there are two likely positions for the additional hydroxyl group, either on the A or B ring.

Stereospecific or nonstereospecific reductions of corticosteroids with an 18-hydroxyl group or an 18-oxo group were carried out either with sodium borohydride or 20β-hydroxysteroid dehydrogenase of Streptomyces hydrogenous to unambiguously characterize the two possible 20α- and 20β-epimers. The resulting products were analyzed as methyloxime trimethylsilyl ethers by gas chromatography-mass spectrometry. Characteristic ions are described and a mechanism of a specific fragmentation of the 18,20,21-trihydroxystcroids is proposed.

Unconjugated and conjugated androgens were determined in rat plasma by a method combining mass fragmentography and isotopic dilution. Two internal standards were used: [4-14C]-testosterone added to plasma and 5α-cholestane added to the reagent mixture for gas phase analysis. The testosterone concentration in plasma can be computed directly from the ratio between the peaks at m/e 389 (M+) and 391 (M + 2), the number of d.p.m. added to the plasma sample and the volume of the plasma sample. This method is compared to previously established radioimmunoassay techniques. In plasma from 5-month-old male rats, this method showed the presence of testosterone: 3,55 ± 0,33 ng/ml and androsterone: 3.56 ±0,44 ng/ml. In addition four conjugated isomers of androstane-3, 17β-diol were identified.

Extensive studies of parameters conditioning high plating efficiency of epithelial liver cells at primary seeding allowed us to set up a technique for the routine culture of liver cells from rats of various ages (18 day-old pc to 7 month-old) in Ham F10 medium supplemented with 10 p. cent fetal calf serum and 10 p. cent human serum. Cultures, after several passages, or sometimes at primary seeding were free of fibroblasts. Cell lines were kept for over one year with a subculture splitting ratio of 1 to 10 each week. The generation time of the cells was 16–18 hours. Caryotype analysis showed a high majority of normal diploid and tetraploid cells. Various enzymes and metabolic pathways have been studied in primary culture and in cell lines: enzymes of the anaerobic metabolism of hexoses and metabolism of steroid hormones. Activity glucose-6-phosphatase was nearly lost in all cultures. Aldolase showed a specific liver activity with a cleavage ratio of phosphofructoses (F-1,6-diP/-1-P) equal to 1 or about 1 in several primary cultures and cell lines. Many metabolites arising from incubation of cell lines with 14C-labelled corticosterone, corticosterone-21-sulfate, testosterone and progesterone have been isolated and quantitated by gas liquid chromatography (GC) and mass fragmentography coupled to GC, using 14C/12C isotope ratio measurements. These metabolites indicate the presence in cultured cells of 3α/β-steroid-reductases, 4-enesteroid-reductases, 17β-steroid-oxido-reductase, 11β-steroid-oxido-reductase and ring hydroxylases.

In order to study the metabolism of 18-OH-11-deoxycorticosterone, the mineralocorticoid hormone responsible for hypertension in rats and humans, we have synthesized the following dihydrogenated and tetrahydrogenated reference derivatives: 18,21-dihydroxy-5alpha-pregnane-3,20-dione, 18,21-dihydroxy-5beta-pregnane-3,20-dione, 3alpha,18,21-trihydroxy-5alpha-pregnan-20-one (I), 3beta,18,21-trihydroxy-5alpha-pregnan-20-one (II), 3alpha,18,21-trihydroxy-5beta-pregnan-20-one (III) and 3beta,18,21-trihydroxy-5beta-pregnan-20-one (IV). A complete separation of these compounds from each other and from tetrahydrocorticosterone isomers has been realized only by the association of thin-layer chromatography and gas-liquid chromatography on high-efficiency glass capillary columns. Characterization by gas-liquid chromatography-mas spectrometry is described. The stereoisomer distribution in rats is: adrenals of adult males and females (compounds I and II in the ratio 4:1); adrenals of 23-day-old males and females (compounds I, II and III, 11:11:3); liver of females (compound I and traces of III); and the liver of adult male (compounds II, III and IV, 4.5:4.5:1).