Cytochrome P450 Hydroxylase Research Articles

Transpulmonary uptake and bioavailability of 2,3,7,8-TCDD from respirable soil particles

Female Sprague-Dawley rats were intratracheally instilled with TCDD-contaminated soil (74.4 ppm TCDD) or recontaminated gallium oxide (GaO; 72.2 ppm TCDD; positive control) and sacrificed 1, 7, or 28 days later. Enzyme activity in hepatic microsomes was time-dependent with up to 56% and 918% increases in cytochrome P-450 and aryl hydrocarbon hydroxylase (AHH), respectively. There was no significant difference in AHH induction between animals which received soil or GaO. Hepatic concentration of TCDD in soil-exposed animals was 115%, 101%, and 186% of positive controls at 1, 7, and 28 days, respectively, suggesting that (a) pulmonary bioavailability of TCDD from respirable soil particles is 100% and (b) soil or cocontaminants influenced hepatic retention of the compound.

Angiotensin-II acts via the type 1 receptor to inhibit 17 alpha- hydroxylase cytochrome P450 expression in ovine adrenocortical cells

In this study we have investigated the effect of angiotensin-II (A-II) on cortisol production and 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha)) expression in primary cultures of ovine adrenocortical cells and the A-II receptor subtypes that mediate these responses. While A-II alone had no stimulatory effect on cortisol secretion, it inhibited the cortisol response to ACTH (10(-8) M) in a dose-dependent manner (Ki, less than 0.1 nM; maximum inhibition, 60-80%). While prolonged treatment with ACTH (10(-8) M) increased the expression of P450(17 alpha), cotreatment with A-II (10(-8) M) also inhibited ACTH-stimulated expression, as determined by changes in mRNA, immunoreactive P450(17 alpha), and 17 alpha-hydroxylase activity. A study of the effects of the AT1 and AT2 receptor antagonists, DuP 753 and PD 123319, on binding of [125I]A-II to ovine adrenocortical cells showed that the A-II receptor population was predominantly of the AT1 subtype. The effects of A-II on inhibition of cortisol secretion in response to ACTH and the activation of phosphoinositidase-C in response to A-II alone were both fully antagonized by DuP 753, but not by PD 123319. Furthermore, the inhibitory effects of A-II on expression of P450(17 alpha), as measured at the levels of mRNA, immunoreactive protein, and enzyme activity, were reversed by DuP 753 (10(-5) M), but not PD 123319 (10(-5) M). We conclude that A-II has a potentially important role in the control of cortisol secretion and long term maintenance of P450(17 alpha) expression in the ovine adrenal cortex, and that the effects of A-II on both cortisol secretion and P450(17 alpha) expression are mediated through the AT1 receptor, which is coupled to phosphoinositidase-C.

Alterations in hepatic Phase I and Phase II biotransformation enzymes by garlic oil in rats

Studies were conducted to examine the effect of a single and repeated administrations of garlic oil (diallyl sulfide) on Phase I and Phase II biotransformation enzymes in rats. Adult, male Sprague-Dawley rats treated with a single dose of garlic oil (500 mg kg i.p.) showed a significant depression of hepatic cytochrome P-450, aminopyrine Ndemethylase and aniline hydroxylase while microsomal protein content, cytochrome b 5, NADPH-cytochrome c reducase, benzphetamine N-demethylase and cytosolic glutathione, S-transferase remained unaffected 24 h following the treatment. Although certain microsomal enzymes were depressed, there was no liver damage caused by garlic oil as judged by the putative serum enzyme test. On the other hand, daily administration of garlic oil (50 mg kg ) i.p. for 5 days) produced a significant increase in hepatic cytochrome P-450, aminopyrine N-demethylase and benzphetamine N-demethylase activities, but not in the rest of the aforementioned parameters of biotransformation reactions. These data indicate that the effect of garlic oil on the hepatic drug-metabolizing enzyme system is dose-dependent.

Rapid identification of deoxyribonucleic acid sequence differences in cytochrome P-450 21-hydroxylase (CYP21) genes with denaturing gradient gel blots

Denaturing gradient gel electrophoresis was used to identify single-base differences in the cytochrome P-450 21-hydroxylase (CYP21) genes of 132 unrelated control individuals and family members of three unrelated patients with 21-hydroxylase deficiency. The salt-wasting variety was caused by gene deletion and gene conversion/deletion mutations in affected members of two families studied. The simple virilizing form, present in the third family, was caused by an apparent point mutation not detectable by routine Southern blots. We have detected many restriction fragment melting polymorphisms in the CYP21 genes of the members of both salt-wasting families and normal individuals with denaturing gradient gel electrophoresis. We also identified a restriction fragment melting polymorphism specific for the simple virilizing patient in the third family. The data demonstrate that the CYP21 genes are highly polymorphic and that denaturing gradient gel electrophoresis is useful for genomic deoxyribonucleic acid analysis of patients with 21-hydroxylase deficiency.

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases

The gene cluster (ery) responsible for production of the macrolide antibiotic erythromycin by Saccharopolyspora erythraea is also known to contain ermE, the gene conferring resistance to the antibiotic. The nucleotide sequence has been determined of a 4.5 kb portion of the biosynthetic gene cluster, from a region lying between 3.7 kb and 8.2 kb 3' of ermE. This has revealed the presence of four complete open reading frames, including the previously known ery gene eryG, which catalyses the last step in the biosynthetic pathway. Comparison of the amino acid sequence of EryG with the sequence of other S-adenosylmethionine (SAM)-dependent methyltransferases has revealed that one of the sequence motifs previously suggested to be part of the SAM-binding site is present not only in EryG but also in many other recently sequenced SAM-dependent methyltransferases. Previous genetic studies have shown that this region also contains gene(s) involved in hydroxylation of the intermediate 6-deoxyerythronolide B. One of the three other open reading frames (eryF) in fact shows very high sequence similarity to known cytochrome P450 hydroxylases. An adjacent gene (ORF5) shows a strikingly high degree of similarity to prokaryotic and eukaryotic acyltransferases and thioesterases.

Diminution in phase I and phase II drug metabolizing enzymes of rat lung by asbestos: Anin vitro study

In the present paper studies are presented concerning the effect of three varieties of asbestos namely, chrysotile, crocidolite and amosite, and of titanium dioxide, an inert non-asbestos dust, on the enzymes of phase 1 and phase 2 drug metabolism in isolated rat lung microsomes and post-microsomal fraction. Since 3-methylcholanthrene (3-MC) is known to induce cytochromes P-450 in the IA family and their associated activity, benzo(a)pyrene hydroxylase, therefore, the effect of these mineral fibers on cytochrome P-450 and benzo(a)pyrene hydroxylase in lung microsomes isolated from 3-MC-treated rats was studied.

Lauric acid hydroxylase activity and cytochrome P450 IV family proteins in human liver microsomes

Lauric acid hydroxylase activity and cytochrome P450 IV family proteins in human liver microsomes

Localization of the gene encoding steroid hydroxylase cytochrome P-450 from Rhizopus nigricans inside a HindIII fragment of genomic DNA

The gene encoding steroid inducible cytochrome P450 of Rhizopus nigricans ATCC 6227b has been found inside a HindIII fragment of the genomic DNA by hybridization with a partial length cDNA probe. The latter was isolated by immunoscreening a cDNA library prepared in the λgtll expression system and identified on the basis of inducibility and sequence analysis. The nucleotide sequence of the cDNA probe revealed a coding sequence for the heme binding segment characteristic of the P450 gene family.

Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450.

Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2 alpha (or 16 alpha)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This finding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH.

COMPARISON OF THE EFFECTS OF PIPERINE ADMINISTERED INTRAGASTRICALLY AND INTRAPERITONEALLY ON THE LIVER AND LIVER MIXED-FUNCTION OXIDASES IN RATS

Piperine, a major pungent constituent of black and red peppers, was administered to rats intragastrically and intraperitoneally to study whether it alters the activities of hepatic mixed-function oxidases (MFO) and serum enzymes as specific markers of hepatotoxicity. An intragastric dose of 100 mg/kg of piperine to adult, male Sprague-Dawley rats caused an increase in hepatic microsomal cytochrome P-450 and cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h following treatment. On the other hand, a 10 mg/kg dose given i.p. exhibited no effect on the activities of the aforementioned parameters of the hepatic drug-metabolizing enzyme system. However, when the intragastric and intraperitoneal doses were increased to 800 mg/kg and 100 mg/kg, respectively, the black pepper alkaloid produced a significant decrease in the levels of cytochrome P-450, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h after treatment. None of the treatments significantly elevated the activities of serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and isocitrate dehydrogenase (ICD), suggesting that piperine is not a hepatotoxic agent.

Effect of anti-relapse antimalarial compound CDRI 80/53 and primaquine on hepatic mixed function oxidase system of rhesus monkey

A potential anti-relapse antimalarial compound CDRI 80/53 [N1-(3-acetyl-4-5-dihydro-2-furanyl)-N4-(6-methoxy-8-quinolinyl)1,4- pentanediamine] at 7.5 mg/kg body weight and primaquine at 6.0 mg/kg body weight did not cause any significant change in the status of hepatic microsomal mixed function oxidase system of rhesus monkeys, when given orally for 7 days. Further, the extension of the treatment at the same dossier up to 21 days resulted in impairment of the different indices of the MFO system. The compound CDRI 80/53 inhibited cytochrome P-450, aminopyrine-N-demethylase, aniline and benzo(a)pyrene hydroxylases, cytochrome b5 and haem levels by 17, 11, 58, 0, 36 and 35% whereas the inhibition caused by primaquine was 34, 40, 72, 54, 39 and 38% respectively, establishing that the cytochrome P-450 dependent mono-oxygenase system of monkey liver was comparatively less suppressed by compound CDRI 80/53. The cessation of the compound/drug treatment resulted in almost complete reversal of all the MFO activities to normal in a period of about 6 weeks.

Incorporation of steroidogenic pathways which produce cortisol and aldosterone from cholesterol into nonsteroidogenic cells

Cortisol production from cholesterol requires the activity of four steroid hydroxylases: cholesterol side chain cleavage cytochrome P-450 ( P-450 scc), 17α-hydroxylase cytochrome P-450 ( P-450 17 α ), 21-hydroxylase cytochrome P-450 ( P-450 c21) and 11β-hydroxylase cytochrome P-450 ( P-450 11 β). We have previously shown that transformed, nonsteroidogenic COS 1 cells derived from monkey kidney are a useful system for expression of various forms of cytochrome P-450. The present study shows that COS 1 cell cultures multiply transfected with six plasmids containing all four steroid hydroxylases, 3β-hydroxysteroid dehydrogenase /Δ 5→4-isomerase (3βHSD) and adrenodoxin produce cortisol and aldosterone when 22( R)-hydroxycholesterol is supplied to the system. When pregnenolone is used as substrate, various intermediate metabolites are detected at different time points further establishing the incorporation of complete functional steroidogenic pathways into the nonsteroidogenic cell cultures. Since the first and the last reactions in these pathways take place in the mitochondrion, the movement of various intermediate metabolites from mitochondrion to endoplasmic reticulum and back to mitchondrion occurs in and between COS 1 cells.

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b 5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.

Adrenocorticotropin Increases Expression of c-fos and β-Actin Genes in the Rat Adrenals*

It is widely accepted that expression of protooncogenes is coupled with cellular proliferation and differentiation. Since ACTH stimulates not only steroidogenesis but also cellular proliferation, we investigated whether ACTH affects the expression of c-fos, c-myc, and beta-actin genes. The effect of ACTH on adrenal glands was studied in hypophysectomized rats. Changes in the mRNA levels were studied by Northern and dot blot analyses. It was demonstrated that ACTH induces increases in mRNAs encoding c-fos and beta-actin in adrenal glands of hypophysectomized rats. When stimulated by ACTH (5 IU/100 g BW), the mRNA levels of both genes increase rapidly; the maximum levels are observed at 30 min for c-fos and 6 h for beta-actin. Both mRNAs declined to near-control levels by 6-24 h. The levels of mRNAs encoding cholesterol side-chain cleavage cytochrome P-450 and 21-hydroxylase cytochrome P-450 began to increase 3 and 12 h after ACTH administration, respectively. This increase continued for 24 h after ACTH treatment. Increases in total adrenal RNA and adrenal weight occurred slowly after ACTH treatment. On the other hand, the levels of c-myc mRNA were very low and were not increased by ACTH administration. These results suggest that increased expression of c-fos and beta-actin genes by ACTH may have important roles in mediating its action on adrenals.

The Interference of Aroclor 1254 with Progesterone Metabolism in Guinea Pig Adrenal and Testes Microsomes

The effects of Aroclor 1254 on cytochrome P-450-mediated steroidogenic activities were investigated in adrenal and testis microsomes of male guinea pigs. A significant decrease was recorded in the tissue content of adrenal microsomal cytochrome P-450 as well as a significant reduction in the overall conversion of progesterone to steroid products. The effects of exposure to Aroclor 1254 on activities of cytochrome P450 21-hydroxylase and cytochrome P450 17 alpha-hydroxylase/C17,20-lyase were selective. Cytochrome P-450 21-Hydroxylase activity was inhibited, as reflected by a decrease in production of 11-deoxycortisol and 11-deoxycorticosterone, whereas the cytochrome P-450 17 alpha-hydroxylase/C17,20-lyase activities, represented by the production of 17 alpha-hydroxyprogesterone and androstenedione were elevated. The same and even more pronounced pattern of altered progesterone metabolism elicited by Aroclor 1254 was observed in vitro, when Aroclor 1254 was introduced into incubation mixtures prepared with adrenal microsomes from untreated animals. Under such experimental conditions, a decrease in the overall metabolism of progesterone was observed as well as a decrease in cytochrome P-450 21-hydroxylase activity, while there was significant elevation in the 17 alpha-hydroxylase/C17,20-lyase activities. The effect of Aroclor 1254 on the testes differed largely from its effect on the adrenal cortex. In testis microsomes, pretreatment with Aroclor 1254 resulted in no changes in the cytochrome P-450 content, contrary to the decrease observed in adrenal microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional and subcellular distribution of cytochrome P-450-dependent drug metabolism in monkey brain: The olfactory bulb and the mitochondrial fraction have high levels of activity

In brain of female monkey ( M. fascicularis ) the content of cytochrome P-450 and benzo(a)pyrene hydroxylase activity in the mitochondrial fraction exceeded that of the microsomes by more than 4-fold. The mitochondrial drug metabolism activity exhibited substrate specificity and, unlike the microsomes, did not catalyze 7-ethoxyresorufin O-deethylase reaction. Moreover, the rate of benzo(a)pyrene hydroxylase activity by both the mitochondrial and the microsomal fractions displayed regional variation with the olfactory bulb displaying the highest hydroxylase activity. In contrast, the microsomal 7-ethoxyresorufin O-deethylase activity was uniformally distributed in all brain regions.

Cytochrome P-450 monooxygenase system and benzo(a)pyrene metabolism in echinoderms

A comparative study on mixed-function oxygenase (MFO) systems was carried out on four echinoderm species: the asteroidsAsterias rubens andMarthasterias glacialis, a holothurianHolothuria forskali and an echinoidEchinus esculentus. Cytochromes P-450 andb 5 and the MFO system-associated NADH-ferricyanide reductases NADH-cytochromec reductase, NADPH-cytochromec reductase and benzo(a)pyrene (BP) hydroxylase activities were present in microsomal fractions of pyloric caeca ofA rubens andM. glacialis and in the haemal plexus ofH. forskali. In contrast, cytochrome P-450 and BP hydroxylase activity were not detectable in the gonads ofE. esculentus. The tissue and subcellular distribution of the MFO system was studied inA. rubens. MFO system components were found in the stomachs and gonads, although detection of cytochrome P-450 in the latter tissue was difficult. Sex-related differences were not significant. The contents of the MFO system components and BP hydroxylase activities were highest in the microsomal (100 000 ×g) fractions, but MFO system components were also found in the mitochondrial (12 000 ×g) and cytosolic fractions. The BP hydroxylase activity in pyloric caeca microsomes ofA. rubens was NADPH-dependent and was inhibited by several agents known to be inhibitors of vertebrate cytochromes P-450. In the former respect, the characteristics of the MFO system were more like those of vertebrates and crustaceans than that of molluscs.

The cytochrome P450 IV family: constitutive and inducible haemoproteins

The cytochrome P450 IV family: constitutive and inducible haemoproteins

Identification of regions functioning in substrate interaction of rabbit liver cytochrome P-450 (laurate (omega-1)-hydroxylase).

The nucleotide sequence of cDNA for rabbit liver cytochrome P-450 (laurate (omega-1) hydroxylase) was replaced with that for rabbit liver cytochrome P-450 (testosterone 16 alpha-hydroxylase) in various regions coding for the amino acid sequence between residues 43 and 261. Six chimeric cDNAs thus constructed were cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. Chimeric P-450s synthesized in the transformed yeast cells were purified partially and their catalytic and spectral properties were examined and compared with those of the chimeric P-450 which is considered to possess the same catalytic properties as the wild-type P-450. In the oxidized state the chimeric P-450s exhibited a low-and high-spin mixed-type absorption spectrum of cytochrome P-450 and the spectrum was converted to a typical high-spin type on addition of laurate or caprate, indicating the binding of the fatty acids to the substrate site of the chimeric P-450s. However the affinities of the fatty acids for the chimeras devoid of the sequence of P-450 (laurate (omega-1)-hydroxylase) in either of the regions spanning residues 90-125 and 210-261 were 10 to 20 times lower than those for the chimeras containing the sequence of the wild-type P-450 in both regions. The latter chimeras have about the same affinities as the chimera which is essentially the wild-type P-450.(ABSTRACT TRUNCATED AT 250 WORDS)

Human liver mephenytoin 4'-hydroxylase cytochrome P-450 proteins and genes.

Human liver mephenytoin 4'-hydroxylase cytochrome P-450 proteins and genes.