Brain mitochondrial cytochrome P-450scc: spectral and catalytic properties

The cytochrome P-450-dependent cholesterol side chain cleavage system of the brain has been studied using nonsynaptic mitochondria as the source of enzymatic activity. The system has been found to bind cholesterol and 11-deoxycorticosterone, producing type I difference spectra, whereas the binding of pregnenolone induced a reverse type I difference spectrum. Inhibitors of cytochrome P-450-linked monooxygenase activities produced type II spectra. The formation of labeled pregnenolone after incubation of brain mitochondria with [4-14C]cholesterol has been obtained, and this formation was inhibited by glutethimide, a specific inhibitor of cytochrome P-450scc. The functional significance of this enzymatic activity is discussed.     

Expression of rat renal gamma-glutamyltransferase cDNA in Escherichia coli

To obtain the expression of rat kidney gamma-glutamyltransferase (GGT) cDNA in E. coli, plasmids containing the cDNA sequences coding for various parts of GGT were constructed. Transformation of E. coli cells by these hybrid vectors results in a production of unglycosylated recombinant proteins, immunologically recognized by specific antirat kidney GGT antibodies. Plasmid, expressing the complete coding sequence of GGT cDNA, allows the production of enzymatically active proteins localized in the periplasmic space, while the same sequence without the N-terminal hydrophobic region results in a production of cytoplasmic proteins. These recombinant proteins present a very basic isoelectric point (pI greater than 9). These results suggest that the presence of the N-terminal region seems to be necessary to direct the expressed proteins enzymatically active in the periplasmic space.     

Inhibition of bilirubin UDPglucuronosyltransferase activity by triphenylacetic acid and related compounds

Bilirubin UDPglucuronosyltransferase of rat or human liver microsomes was inhibited, in vitro, by triphenylacetic acid and by structurally related arylcarboxylic acids. This inhibition appeared to be competitive towards bilirubin, and mixed-type towards UDPglucuronic acid. A decrease in the number of phenyl rings or the absence of the carboxyl group in the molecule gave structures which did not affect enzyme activity, showing that both the triphenyl moiety and the carboxyl group were necessary for the inhibition. On the other hand, successive additions of methylene groups in the aliphatic chain progressively increased inhibitory potency. Kappi,bilirubin for triphenylacetic acid was 96 microM compared with 5 microM for 7,7,7-triphenylheptanoic acid. The inhibition of bilirubin UDPglucuronosyltransferase was not due to displacement of bilirubin from albumin. On the basis of these results an attempt was made to delineate the molecular events leading to glucuronidation of bilirubin.     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

Localization of Drug-Metabolizing Enzyme Activities to Blood-Brain Interfaces and Circumventricular Organs

Abstract: The brain, with the exception of the choroid plexuses and Circumventricular organs, is partially protected from the invasion of blood-borne chemicals by the specific morphological properties of the cerebral micro-vessels, namely, the tight junctions of the blood-brain barrier. Recently, several enzymes that are primarily involved in hepatic drug metabolism have been shown to exist in the brain, albeit at relatively low specific activities. In the present study, the hypothesis that these enzymes are located primarily at blood-brain interfaces, where they form an "enzymatic barrier," is tested. By using microdissection techniques or a gradient-centrifugation isolation procedure, the activities of seven drug-metabolizing enzymes in isolated microvessels, choroid plexuses, meningeal membranes, and tissue from three Circumventricular organs (the neural lobe of the hypophysis, pineal gland, and median eminence) were assayed. With two exceptions, the activities of these enzymes were higher in the three Circumventricular organs and cerebral microvessel than in the cortex. Very high membrane-bound epoxide hydrolase and UDP-glucuronosyltransferase activities (approaching those in liver) and somewhat high 7-benzoxyre-sorufin- O -dealkylase and NADPH-cytochrome P-450 reductase activities were determined in the choroid plexuses. The pia-arachnoid membranes, but not the dura matter, displayed drug-metabolizing enzyme activities, notably that of epoxide hydrolase: The drug-metabolizing enzymes located at these nonparenchymal sites may function to protect brain tissue from harmful compounds.     

Peroxisome proliferators as inducers and substrates of UDP-glucuronosyltransferases

Summary— Peroxisome proliferators, despite their chemically unrelated structures, share the common property of being able to stimulate the glucuronidation of bilirubin in rodents and, probably, also in man. The aryloxycarboxylic acids (clofibric acid, fenofibrate, bezafibrate, ciprofibrate), tiadenol and probucol, all of which have hypolipidemic properties, as well as the fatty acid-like perfluorodecanoic acid all enhanced the expression of the UDP-glucuronosyltransferase (UGT) form involved in the conjugation of the pigment. This induction is manifested by an increase in the mRNA species encoding the protein with a subsequent increase in the neosynthesis of the corresponding protein in the endoplasmic reticulum. The induction process is concomitant with that of cytochrome P-450-IVA1 and cytosolic epoxide hydrolase, which, like bilirubin UGT, are mainly involved in the metabolism of endogenous substrates. With a series of carboxylic acids related to clofibric acid, it was possible to demonstrate that induction was mediated via specific interactions based on the physicochemical properties of the inducers. Until now, the molecular basis of induction of bilirubin UGT is not known. The peroxisome proliferators that possess a carboxyl group are good substrates of UGT, especially in man. The acylglucuronides formed are known for their instability and reactivity which could contribute to the toxicity encountered in some patients treated with the drugs. There is convincing evidence that UGT bilirubin does not catalyze the glucuronidation of these substances even if the two types of substrate form acylglucuronides.     

Differential action of thyroid hormones and chemically related compounds on the activity of UDP-glucuronosyltransferases and cytochrome P-450 isozymes in rat liver

The effect of thyroid hormones and chemically related compounds, on the activity of UDP-glucuronosyltransferases (EC 2.4.1.17) and cytochrome P-450-dependent monooxygenases in rat liver microsomes was investigated. The animals were thyroidectomized and treated with different doses of the drugs for 3 weeks. Opposite effects were observed depending on the isoenzyme of UDP-glucuronosyltransferase considered. While 3,3',5-triiodo-L-thyronine, 3,3',5-triiodothyroacetic acid, 3,3',5-triiodothyropropionic acid, isopropyldiiodothyronine and L- and D-thyroxine strongly increased 4-nitrophenol glucuronidation in a dose-dependent fashion, they decreased markedly bilirubin glucuronidation. However, the activity toward nopol, a monoterpenoid alcohol, was not significantly changed regardless of which compound or dose was used. Variation of UDP-glucuronosyltransferase observed with 4-nitrophenol and bilirubin was related to the thyromimetic effect of the drugs estimated from the increase in alpha-glycerophosphate dehydrogenase. Thyronine and 3,5-diiodo-L-tyrosine, which did not enhance this activity, also failed to affect glucuronidation. Variations in UDP-glucuronosyltransferase activity were more likely due to changes in protein expression rather than changes in enzyme latency, since lipid organization of the microsomal membrane, as estimated from the mean anisotropy of 1,6-diphenyl-1,3,5-hexatriene by fluorescence polarization was not significantly modified by the drug administration. Although some of the drugs could significantly decrease the triacylglycerol and cholesterol contents in plasma, all failed to affect lauric acid hydroxylation. The activities of catalase, palmitoyl-CoA dehydrogenase (CN- insensitive) and carnitine acetyltransferase in the fraction enriched in peroxisomes were also not significantly affected by treatment with the thyroid hormone LT3. In contrast, the activity of 7-ethoxycoumarine O-deethylase was increased by large doses of thyronine and by 3,3',5-triiodothyropropionic acid. The concentration of total cytochrome P-450 was decreased in a dose-dependent fashion by all the compounds used, except thyronine. Finally, significant correlations were observed between glucuronidation of bilirubin and 4-nitrophenol and the content in cytochrome P-450. This suggests a possible coordinate regulation of the two processes, which depends on the physicochemical characteristics of the thyroid hormones and related compounds.