NADPH-cytochrome P-450 reductase: Preferential inhibition by ellipticine and other type II compounds having little effect on NADPH-cytochrome c reductase

doi:10.1016/0006-2952(80)90249-X

Biochemical Pharmacology

Volume 29, Issue 1, 1 January 1980, Pages 89-95

https://doi.org/10.1016/0006-2952(80)90249-X Get rights and content

Abstract

Ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b]carbazole) binds with an affinity greater than most other compounds known to interact with P-450. Control and 3-methylcholanthrene-induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) and acetanilide 4-hydroxylase and control and phenobarbital-induced ethylmorphine N-demethylase activities are all markedly inhibited by ellipticine to about the same extent. Ellipticine and other Type II compounds (metyrapone, octylamine-1, pyridine and aniline) preferentially inhibit NADPH-cytochrome P-450 reductase activity, while affecting NADPH-cytochrome c reductase activity very little. Butanol-1, a compound having pure Reverse Type I character, does not block P-450 reductase activity like these Type II compounds. These data suggest that Type II compounds bind to P-450 ferric iron in the sixth coordinate position in such a way as to impede transfer of the first electron from the hydrophobic binding site of the reductase to the P-450-substrate complex, while leaving unencumbered any transfer of electrons from the hydrophilic binding site of the reductase to soluble electron acceptors such as cytochrome c. These data indicate that ellipticine may be very useful in attempting to understand the mechanism by which electrons are transferred from the reductase to the cytochrome(s) P-450.

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Potential biological functions of cytochrome P450 reductase-dependent enzymes in small intestine: Novel link to expression of major histocompatibility complex class II genes
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Citation Excerpt :
This finding, which defines a new physiological/pathological role of intestinal POR/P450 enzymes in modulating the expression of regulators of intestinal immunity, may have important clinical significance. POR is a direct target of inhibition by various drugs and other xenobiotic compounds (e.g. cyclophosphamide (57), ellipticine (58), and cadmium (59)). Furthermore, numerous genetic polymorphisms of the human POR gene that affect either POR expression or POR activity have been identified (60–63).
NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine.
Indications for the involvement of a CYP3A-like iso-enzyme in the metabolism of chlorobornane (Toxaphene®) congeners in seals from inhibition studies with liver microsomes
2001, Aquatic Toxicology
The different isoforms of the cytochrome P450 (CYP) system can metabolise a suite of classes of lipophilic, anthropogenic compounds. The bioaccumulative potential as well as the toxicity of xenobiotics may be significantly altered in the process. To compare the metabolic ability of different wildlife species, it is important to identify the different iso-enzymes of CYP, which are responsible for the metabolism of different classes of compounds. This can be achieved with in vitro incubation assays. In the present study, preparations of hepatic microsomes of a harbour seal (Phoca vitulina) and a grey seal (Halichoerus grypus) demonstrated that the chlorobornane (CHB) congeners CHB-32 and -62 were metabolised enzymatically to their hydroxylated derivatives. These derivatives were partially characterised by their NCI mass-spectra. Inhibition studies were carried out to identify the specific CYP isoform(s) responsible for the metabolism of CHB-32 and -62. Ketoconazole has been shown to inhibit CYP3A enzymes in human and rat studies. In this study, ketoconazole caused concentration-dependent inhibition of metabolism of CHB-32 and -62, reaching 80% at the 1.0 μM treatment level. Ellipticine (1.0 μM), which has been shown to inhibit CYP1A1/2, also inhibited CHB-32 and -62 metabolism in the microsomes of grey seal, but to a much lower degree of less than 10 and 24%, respectively. In the same experiment the metabolism of 4,4′-dichlorobiphenyl was already inhibited 70% by ellipticine treatment at the same concentration. This non-ortho substituted PCB congener can easily attain a planar molecular configuration, and therefore served as a model CYP1A substrate. Inhibition of chlorobornane metabolism was not observed after the addition of goat anti-rat CYP2B antibodies or Aldrin, which is a model CYP2B substrate in rat. Cautious interpretation is advised for results obtained with so-called selective competitive inhibitors. Regardless, these studies indicated for the first time the possible involvement a CYP3A isoform in the mediation of chlorobornane metabolism in seals. The immunochemical cross-reactivity of mouse, rabbit or sheep anti-rat antibodies in the hepatic microsomes of harbour seal confirmed the presence of CYP1A1/2, CYP1A1, CYP2B1/2, CYP3A and CYP4A isoenzymes. Enantioselective metabolism by the microsomes of harbour seal was observed for both CHB-32 and -62. Stereochemical preferences of biotransformation enzymes can have an influence on the environmental distribution of both enantiomers of optically active compounds.
Conjugation of 1-naphthol in primary cell cultures of rat ovarian cells
2000, Chemico-Biological Interactions
Citation Excerpt :
The potent inhibition of glucuronidation by ELP, both in cell cultures and isolated microsomes, is more difficult to explain. This compound is known to inhibit catalysis by the CYPs by inhibiting electron transfer from NADPH-CYP reductase to the cytochrome [28]. On the basis of its chemical structure and the relatively low concentrations of ELP required to inhibit glucuronidation, it seems unlikely that this compound is a competitive inhibitor of P-UGT.
The present study concerns conjugation of 1-naphthol in primary cultures of rat ovarian cells. Two phase II enzymes catalyzing conjugation, i.e. phenol sulfotransferase (P-SULT) and phenol UDP-glucuronosyltransferase (P-UGT), were measured using 1-naphthol as substrate. The rates of conjugation by the different cell types of the rat ovary were the same at low concentrations and short incubation times. However, after 20 h of incubation the rate of conjugation in cells isolated from ovaries enriched in corpora lutea (CL) exceeded the rate in cells isolated from ovaries enriched in preovulatory follicles. In addition, when the granulosa cells were removed from the preovulatory follicles, the rate of conjugation was 1.7-fold higher, i.e. in the theca/stroma cells. When the cells were incubated with 1-[¹⁴C]naphthol and conjugates were subsequently separated by thin-layer chromatography, naphthyl glucuronide was the only conjugate observed. Pentachlorophenol (PCP), a commonly used inhibitor of P-SULT, inhibited 1-naphthol conjugation 50% in cell cultures, as well as in microsomal preparations. α-Naphthoflavone (ANF) and ellipticine (ELP), both cytochrome P450 (CYP) inhibitors, affected the conjugation of 1-naphthol in different ways; ANF did not affect P-UGT activity in microsomal preparations, but inhibited 1-naphthol conjugation in cell cultures by as much as 90%. On the other hand, ELP inhibited the conjugation of 1-naphthol up to 99% in the cell cultures, but only 75% in microsomal fractions. Testosterone (TST) and estradiol inhibited this activity ≈50% in both of these experimental systems. Clomiphene citrate (CLF), a drug used to induce ovulation and demonstrating both estrogenic and antiestrogenic effects, did not influence the conjugation of 1-naphthol significantly in the cell cultures. The present findings demonstrate that P-UGT is by far the major enzyme conjugating 1-naphthol in the rat ovary and that commonly used inhibitors of P-SULT and CYPs also inhibit P-UGT activity, either directly or via other mechanisms.
Cytochrome P450 monooxygenase system in echinoderms
1998, Comparative Biochemistry and Physiology - C Pharmacology Toxicology and Endocrinology
The results of a limited number of studies on echinoderms provide evidence for the presence of a cytochrome P450 monooxygenase system in representatives of three classes of the phylum Echinodermata: the asteroids (sea stars), holothuroids (sea cucumbers) and echinoids (sea urchins). The monooxygenase system has been demonstrated to be involved in the metabolism of xenobiotic compounds, but is assumed to have its primary function in the metabolism of endogenous substrates, such as steroids. Available data on P450 cofactor requirement, P450-dependent metabolism of benzo[a]pyrene, studies with classical inhibitors of P450, specificity of P450 induction by planar compounds, and the changes in the benzo[a]pyrene metabolite profile in induced animals suggest similarities with the MO system present in vertebrates. However, the relatively high capacity of the monooxygenase system in sea stars to catalyse reactions with organic hydroperoxide as donor for activated oxygen, and the low induceability during exposure to xenobiotics indicate also important differences between the echinoderm cytochrome P450 monooxygenase system and that of vertebrates. Some evidence was found for the existence of different forms of cytochrome P450 in sea stars. Catalytic functions of the cytochrome P450 monooxygenase system of sea stars in the metabolism of steroids may be suppressed as a result of the induction of cytochrome P450 by xenobiotics.
Redox cycling of aromatic hydrocarbon quinones catalysed by digestive gland microsomes of the common mussel (Mytilus edulis L.)
1997, Aquatic Toxicology
The NAD(P)H-dependent redox cycling of six aromatic hydrocarbon quinones (1,4-benzoquinone, tetramethyl-1,4-benzoquinone (duroquinone), 1,2- and 1,4-naphthoquinones, 9,10-phenanthrenequinone, anthraquinone) by digestive gland microsomes of M. edulis was assessed in terms of oxygen consumption (Clark electrode) and reactive oxygen species (ROS) production (conversion of Superoxide anion radical, O₂⁻ and hydrogen peroxide, H₂O₂ to hydroxyl radical, ·OH—the latter detected by oxidation of 2-keto-4-methiolbutyric acid (KMBA) to ethylene); additionally, oxygen consumption was determined for the known ROS-generating 2-methyl-1,4-naphthoquinone (menadione) and benzo[a]pyrene diones (1,6-, 3,6- and 6,12-). Stimulated oxygen consumption was detectable for duroquinone, naphthoquinones, menadione and phenanthrenequinone only, whereas stimulated ROS production was evident for all ten quinones, indicating that the latter is a more sensitive measure of redox cycling. ROS production was greatest for 1,2-naphthoquinone and least for anthraquinone. Stimulated ROS production and/or oxygen consumption for the six aromatic hydrocarbon quinones was greater for the NADH- than the NADPH-dependent reactions. Michaelis-Menten kinetics with respect to quinone concentration were evident for both stimulated oxygen consumption and ROS production, and correlations between the two measurements provide strong evidence for the process of redox cycling (i.e. conversion of O₂ to O₂⁻) occurring in digestive gland microsomes. Differences were also seen between the profiles for the two measurements, particularly for 1,4-naphthoquinone, indicative of other processes of oxygen consumption and/or ROS production occurring. Comparison of different quinones indicates that enzyme specificity and quinone structure are factors in determining ROS production. Overall, the results indicate a wide pro-oxidant potential for quinones formed from aromatic hydrocarbons by biotransformation and photo-oxidation processes.
Effects of the inhibitor ellipticine on cytochrome P450-reductase and cytochrome P450 (1A) function in hepatic microsomes of flounder (Platichthys flesus)
1995, Marine Environmental Research
The effects of the mammalian inhibitor ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b] carbazole) were examined in a mechanistic study of the cytochrome P450 monooxygenase system of control and β-naphthoflavone (βNF)-induced hepatic microsomes of Platichthys flesus. Ellipticine was indicated to bind to the haem moiety of cytochrome P450s (gave type II binding spectra) and to inhibit the transfer of electrons from both the hydrophobic binding site of cytochrome P450 reductase (P450R) to P450 (inhibited P450R reductase activity) and the hydrophilic binding site of P450R to soluble electron acceptors (inhibited NAD(P)H-cytochrome c reductase activity). No effect was seen on cytochrome b₅ reductase activity. Ellipticine inhibition indicated the involvement of (i) P450R (possibly also P450s) in NADPH- but not NADH- dependent hydroxyl radical production, and (ii) electron transfer and P450/P450R interaction in NADPH-dependent cytochrome P450 1A-catalysed monooxygenation (7-ethoxyresorufin O-deethylase activity and benzo(a)pyrene (BaP) metabolism). Differential effects of ellipticine on cumene hydroperoxide (CHP)-dependent BaP metabolism (P450 peroxidase activity) with CHP concentration indicated the existence of at least two forms of P450 with different substrate affinities for CHP, and different mechanisms of formation for protein adducts and free metabolites. Overall, the studies indicate the primary site of action of ellipticine in P. flesus is binding between Fe³⁺-P450 and P450R.

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NADPH-cytochrome P-450 reductase: Preferential inhibition by ellipticine and other type II compounds having little effect on NADPH-cytochrome c reductase

Abstract

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Biochem. Pharmac.

Meth. Enzym.

Analyt. Biochem.

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Archs. Biochem. Biophys.

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Biochem. biophys. Res. Commun.