Development of excipients free inhalable co-spray-dried tobramycin and diclofenac formulations for cystic fibrosis using two and three fluid nozzles.

This study aims to investigate the effect of physicochemical properties and aerosol performance of two (2FN) and three-fluid nozzles (3FN) on the inhalable co-formulation of tobramycin and diclofenac dry powders. Combination formulations of tobramycin and diclofenac at 2:1 and 4:1 w/w ratios were prepared at a laboratory scale using a spray dryer in conjunction with a 2FN or 3FN. Powder size, morphology, solid-state characteristics, and aerodynamic and dissolution properties were characterised. The nozzle types and the formulation composition influenced the yield, particle size, solid-state properties, aerosolization behaviour and dissolution of the co-spray dried formulations. In particular, using the 2FN the co-spray dried formulation of tobramycin and diclofenac at 2:1 w/w showed smaller particle size (D50, 3.01 ± 0.06 µm), high fine particle fractions (FPF) (61.1 ± 3.6% for tobramycin and 65.92 ± 3 for diclofenac) and faster dissolution with approx. 70% diclofenac released within 3 h and approx. 90% tobramycin was released within 45 min. However, the 3FN for the co-spray dried formulation of tobramycin and diclofenac at a 2:1 w/w ratio showed a larger particle size (D50, 3.42 ± 0.02 µm), lower FPF (40.6 ± 3.4% for tobramycin and 36.9 ± 0.84 for diclofenac) and comparative slower dissolution with approx. 60% diclofenac was released within 3 h and 80% tobramycin was released within 45 min. A similar trend was observed when the tobramycin to diclofenac ratio was increased to 4:1 w/w. Overall results suggest that spray drying with 2FN showed a superior and viable approach to producing excipients-free inhalable co-spray dried formulations of tobramycin and diclofenac. However, the formulation produced using the 3FN showed higher enrichment of hydrophobic diclofenac and an ability to control the tobramycin drug release in vitro.

The application of in vitro cellular assays for analysis of electronic cigarettes impact on the airway.

Electronic (e)-cigarettes have been marketed for more than a decade as an alternative to conventional cigarettes. Their popularity and use among adolescents have grown significantly during recent years. While e-cigarettes do not release carcinogenic aromatic hydrocarbons, they can generate reactive carbonyls and radicals during the heating process in vitro. Emphasis has been placed in recent studies to introduce more rigorous and physiologically relevant in vitro models to characterise the toxicological profile of e-cigarettes. However, significant challenges are present due to difficulties for the developed systems to fully represent the in vivo inhalation settings. Furthermore, research protocols that fail to simulate the characteristics of e-cigarettes can affect the findings of in vitro studies. This review will illustrate the status quo of e-cigarette assays in vitro, discussing the various cellular assays used for evaluating the safety profile of e-cigarettes. Future directions will also be provided to better assist the scientific community in interpreting the health risks of e-cigarettes.

In vitro and in vivo testing of a novel, hyperelastic thin film nitinol flow diversion stent.

A flexible, low profile, flow diversion stent could replace endovascular coiling for the treatment of intracranial aneurysms. Micropatterned-thin film nitinol (TFN) is a novel biomaterial with high potential for use in next-generation endovascular devices. Recent advancements in micropatterning have allowed for fabrication of a hyperelastic thin film nitinol (HE-TFN). In this study, the authors describe in vitro and in vivo testing of novel HE-TFN based flow diverting stents. Two types of HE-TFN with expanded pores having long axes of 300 and 500 µm were used to fabricate devices. In vitro examination of the early thrombotic response in whole blood showed a possible mechanism for the device's function, whereby HE-TFN serves as a scaffold for blood product deposition. In vivo testing in swine demonstrated rapid occlusion of model wide-neck aneurysms. Average time to occlusion for the 300-µm device was 10.4 ± 5.5 min. (N = 5) and 68 ± 30 min for the 500-µm device (N = 5). All aneurysms treated with bare metal control stents remained patent after 240 min (N = 3). SEM of acutely harvested devices supported in vitro results, demonstrating that HE-TFN serves as a scaffold for blood product deposition, potentially enhancing its flow-diverting effect. Histopathology of devices after 42 days in vivo demonstrated a healthy neointima and endothelialization of the aneurysm neck region. HE-TFN flow-diverting stents warrant further investigation as a novel treatment for intracranial aneurysms.

In vitro hemocompatibility of thin film nitinol in stenotic flow conditions.

Because of its low profile and biologically inert behavior, thin film nitinol (TFN) is ideally suited for use in construction of endovascular devices. We have developed a surface treatment for TFN designed to minimize platelet adhesion by creating a superhydrophilic surface. The hemocompatibility of expanded polytetrafluorethylene (ePTFE), untreated thin film nitinol (UTFN), and a surface treated superhydrophilic thin film nitinol (STFN) was compared using an in vitro circulation model with whole blood under flow conditions simulating a moderate arterial stenosis. Scanning electron microscopy analysis showed increased thrombus on ePTFE as compared to UTFN or STFN. Total blood product deposition was 6.3 ± 0.8 mg/cm(2) for ePTFE, 4.5 ± 2.3 mg/cm(2) for UTFN, and 2.9 ± 0.4 mg/cm(2) for STFN (n = 12, p < 0.01). ELISA assay for fibrin showed 326 ± 42 µg/cm(2) for ePTFE, 45.6 ± 7.4 µg/cm(2) for UTFN, and 194 ± 25 µg/cm(2) for STFN (n = 12, p < 0.01). Platelet deposition measured by fluorescent intensity was 79,000 20,000 AU/mm(2) for ePTFE, 810 ± 190 AU/mm(2) for UTFN, and 1600 ± 25 AU/mm(2) for STFN (n = 10, p < 0.01). Mass spectrometry demonstrated a larger number of proteins on ePTFE as compared to either thin film. UTFN and STFN appear to attract significantly less thrombus than ePTFE. Given TFN's low profile and our previously demonstrated ability to place TFN covered stents in vivo, it is an excellent candidate for use in next-generation endovascular stents grafts.

Permanent Genetic Resources added to Molecular Ecology Resources database 1 January 2009-30 April 2009.

This article documents the addition of 283 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Agalinis acuta; Ambrosia artemisiifolia; Berula erecta; Casuarius casuarius; Cercospora zeae-maydis; Chorthippus parallelus; Conyza canadensis; Cotesia sesamiae; Epinephelus acanthistius; Ficedula hypoleuca; Grindelia hirsutula; Guadua angustifolia; Leucadendron rubrum; Maritrema novaezealandensis; Meretrix meretrix; Nilaparvata lugens; Oxyeleotris marmoratus; Phoxinus neogaeus; Pristomyrmex punctatus; Pseudobagrus brevicorpus; Seiridium cardinale; Stenopsyche marmorata; Tetranychus evansi and Xerus inauris. These loci were cross-tested on the following species: Agalinis decemloba; Agalinis tenella; Agalinis obtusifolia; Agalinis setacea; Agalinis skinneriana; Cercospora zeina; Cercospora kikuchii; Cercospora sorghi; Mycosphaerella graminicola; Setosphaeria turcica; Magnaporthe oryzae; Cotesia flavipes; Cotesia marginiventris; Grindelia Xpaludosa; Grindelia chiloensis; Grindelia fastigiata; Grindelia lanceolata; Grindelia squarrosa; Leucadendron coniferum; Leucadendron salicifolium; Leucadendron tinctum; Leucadendron meridianum; Laodelphax striatellus; Sogatella furcifera; Phoxinus eos; Phoxinus rigidus; Phoxinus brevispinosus; Phoxinus bicolor; Tetranychus urticae; Tetranychus turkestani; Tetranychus ludeni; Tetranychus neocaledonicus; Tetranychus amicus; Amphitetranychus viennensis; Eotetranychus rubiphilus; Eotetranychus tiliarium; Oligonychus perseae; Panonychus citri; Bryobia rubrioculus; Schizonobia bundi; Petrobia harti; Xerus princeps; Spermophilus tridecemlineatus and Sciurus carolinensis.

Understanding electron transport systems of Streptomyces cytochrome P450.

Streptomyces spp. are known to produce various types of biologically active compounds including antibiotics, antiparasitic agents, herbicides and immunosuppressants. P450 (cytochrome P450) enzymes may have key roles in these biosynthetic and biotransformation reactions. Recent genomic analysis of Streptomyces coelicolor A3(2) indicates that S. coelicolor may have six ferredoxins (Fdxs), four putative Fdx reductases (FdRs) and 18 P450 genes. However, there are few clues to explain the mechanisms and functions of Streptomyces P450 systems. To solve these questions, we have expressed and purified five S. coelicolor P450s, four FdRs and six Fdxs in Escherichia coli. Of the purified P450s, CYP105D5 has fatty acid hydroxylation activity in a system reconstituted with putidaredoxin reductase and Fdx4 or with spinach FdR and spinach Fdx, although the reconstitutions with FdR2 or FdR3 and any of the Fdxs did not support CYP105D5-catalysed oleic acid hydroxylation. Elucidation of the detailed mechanisms of electron transport system for Streptomyces P450 may provide the perspective for usefulness of P450s as a biocatalyst.

A new selective and potent inhibitor of human cytochrome P450 1B1 and its application to antimutagenesis.

Human cytochrome P450 (P450) 1B1 is found mainly in extrahepatic tissues and is overexpressed in a variety of human tumors. Metabolic activation of 17beta-estradiol (E(2)) to 4-hydroxy E(2) by P450 1B1 has been postulated to be a factor in mammary carcinogenesis. The inhibition of recombinant human P450 1B1 by 2,4,3',5'-tetramethoxystilbene (TMS) was investigated using either bacterial membranes from a human P450/NADPH-P450 reductase bicistronic expression system or using purified enzymes. TMS showed potent and selective inhibition of the ethoxyresorufin O-deethylation (EROD) activity of P450 1B1 with an IC(50) value of 6 nM. TMS exhibited 50-fold selectivity for P450 1B1 over P450 1A1 (IC(50) = 300 nM) and 500-fold selectivity for P450 1B1 over P450 1A2 (IC(50) = 3 microM). The inhibitory effects of TMS on EROD activity of human liver microsomes were determined. TMS inhibited EROD activity of human liver microsomes at the same concentration as with recombinant human P450 1A2. TMS also strongly inhibited 4- and 2-hydroxylation of E(2) by P450 1B1-expressing membranes or purified P450 1B1. TMS was a competitive inhibitor of P450 1B1 with a K(i) of 3 nM. The inhibition by TMS was not mechanism-based, and the loss of activity was not blocked by the trapping agents glutathione, N-acetylcysteine, or dithiothreitol. Using purified histidine-tagged P450 1B1, the binding kinetic analysis was performed with TMS, yielding a K(d) of 3 microM. The activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline in an Escherichia coli lac-based mutagenicity tester system containing functional human P450 1B1 was strongly inhibited by TMS. Our results indicate that TMS is a very selective and potent competitive inhibitor of P450 1B1. TMS is selective for inhibiting P450 1B1 among other human P450s including 1A1, 1A2, and 3A4 and warrants consideration as a candidate for preventing mammary tumor formation by E(2) in humans.

Induction of cytochrome P450 1A1 gene expression by a vitamin K3 analog in mouse hepatoma Hepa-1c1c7 cells.

Nine vitamin K3 analogs were compared with respect to the induction of the cytochrome P450 1A1 (CYP1A1) expression in mouse hepatoma Hepa-1c1c7 cells. 6-(4-Diethylamino)phenyl-7-chloro-5,8-quinolinedione (EA4) caused a significant induction of the CYP1A1-mediated ethoxyresorufin O-deethylase activity in a time- and concentration-dependent manner. The induction was accompanied by an increase of the Cyp1a1 mRNA transcription. The transient expression of the mouse Cyp1a1-CAT gene into cells showed that EA4 induced CAT activity. However, the aryl hydrocarbon receptor and its nuclear partner, aryl hydrocarbon receptor nuclear translocator mRNA transcription, were unaffected by the EA4 treatment. When the cells were incubated with EA4 in the presence of 1 nM TCDD, the ethoxyresorufin O-deethylase activity that was induced by TCDD was significantly suppressed by EA4. Inhibition of protein synthesis by cycloheximide strongly enhanced the EA4-dependent Cyp1a1 mRNA expression. Up-regulation of protein kinase C by a 2 h preincubation with phorbol 12-myristate 13-acetate increased the EA4-dependent expression of the Cyp1a1 gene. In human cells, such as HepG2 (human hepatocarcinoma), MCF-7 (human breast adenocarcinoma cell line), and HL-60 (human promyelocytic cell line), the expression of CYP1A1 mRNA was also induced by EA4 treatment. Moreover, CYP1B1 mRNA was increased by EA4 in MCF-7 cells. These results indicate that EA4 modulates CYP1A1 and CYP1B1 expressions by transcriptional activation. Also, protein kinase C may be involved in the induction mechanism of CYP1A1 by EA4.

Ionizing radiation can overcome resistance to TRAIL in TRAIL-resistant cancer cells.

Although the majority of cancer cells are killed by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand treatment), certain types show resistance to it. Ionizing radiation also induces cell death in cancer cells and may share common intracellular pathways with TRAIL leading to apoptosis. In the present study, we examined whether ionizing radiation could overcome TRAIL resistance in the variant Jurkat clones. We first selected TRAIL-resistant or -sensitive Jurkat clones and examined cross-responsiveness of the clones between TRAIL and radiation. Treatment with gamma-radiation induced significant apoptosis in all the clones, indicating that there seemed to be no cross-resistance between TRAIL and radiation. Combined treatment of radiation with TRAIL synergistically enhanced killing of TRAIL-resistant cells, compared to TRAIL or radiation alone. Apoptosis induced by combined treatment of TRAIL and radiation in TRAIL-resistant cells was associated with cleavage of caspase-8 and the proapoptotic Bid protein, resulting in the activation of caspase-9 and caspase-3. No changes in the expressions of TRAIL receptors (DR4 and DR5) and Bcl-2 or Bax were found after treatment. The caspase inhibitor z-VAD-fmk completely counteracted the synergistic cell killing induced by combined treatment of TRAIL and gamma-radiation. These results demonstrated that ionizing radiation in combination with TRAIL could overcome resistance to TRAIL in TRAIL-resistant cells through TRAIL receptor-independent synergistic activation of the cascades of the caspase-8 pathway, suggesting a potential clinical application of combination treatment of TRAIL and ionizing radiation to TRAIL-resistant cancer cells.

Bax-dependent apoptosis induced by ceramide in HL-60 cells.

Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. In this study, we show that antisense bax inhibits cytochrome c release, poly(ADP-ribose)polymerase cleavage and cell death induced by ceramide in HL-60 cells. In addition, ceramide induces translocation of Bax to mitochondria. The addition of the broad spectrum caspase inhibitor zVAD-fmk prevented ceramide-induced apoptotic cell death but did not inhibit translocation of Bax and mitochondrial cytochrome c release. Furthermore, ceramide inhibits the expression of the antiapoptotic protein Bcl-xL with an increase in the ratio of Bax to Bcl-xL. These data provide direct evidence that Bax plays an important role in regulating ceramide-induced apoptosis.

Mechanism-based inhibition of human cytochrome P450 1A1 by rhapontigenin.

Recently we reported that resveratrol (trans-3,4',5-trihydroxystilbene) showed selective inhibition of recombinant human cytochrome P450 (P450) 1A1 in a concentration-dependent manner. The inhibition of recombinant human P450 1A1, 1A2, or 1B1 by various hydroxystilbene compounds having a similar structure to resveratrol was investigated using bacterial membranes from a human P450/NADPH-P450 reductase bicistronic expression system to find new candidates for cancer chemopreventive agents. Of seven compounds tested, rhapontigenin (3,3',5-trihydroxy-4'-methoxystilbene) exhibited a potent and selective inhibition of human P450 1A1 with an IC50 value of 0.4 microM. Rhapontigenin showed 400-fold selectivity for P450 1A1 over P450 1A2 and 23-fold selectivity for P450 1A1 over P450 1B1. Rhapontigenin did not show any significant inhibition of ethoxyresorufin O-deethylation (EROD) activity in human liver microsomes, the other human P450s such as P450 2E1, P450 3A4, P450 2D6, P450 2C8, and P450 2C9, or human NADPH-P450 reductase. We have further investigated the inhibition kinetics of P450 1A1 by rhapontigenin. Rhapontigenin inhibited EROD activity of expressed human P450 1A1 in a competitive manner. The loss of EROD activity was time- and concentration-dependent. The values for K(i) and k(inactivation) were 0.09 microM and 0.06 min(-1), respectively. The loss was not blocked by the trapping agents glutathione, N-acetylcysteine, or dithiothreitol. These results suggest that rhapontigenin is a potent mechanism-based inactivator of human P450 1A1 and may be considered as a good candidate for a cancer chemopreventive agent in humans.

Structure and stress-related expression of two cDNAs encoding proteinase inhibitor II of Nicotiana glutinosa L.

Two cDNAs, pNGPI-1 and pNGPI-2, encoding Nicotiana glutinosa proteinase inhibitor II (PI-II) have been cloned, sequenced and identified. The deduced amino acid sequences are 54-82% identical to those of other plant PI-II. The NGPI-1 protein is composed of eight repeated domains, while NGPI-2 contains six repeated regions, each with a putative reactive site. The expression of NGPI-1 is highly regulated in a developmental- and tissue-specific manner, with the transcript being detected in young leaves and floral organs of N. glutinosa plants. In mature leaves, the NGPI-1 gene is rapidly activated by distinct temporal induction patterns in response to pathogen-related (biotic) and wound-related (abiotic) stresses.

Involvement of p27(kip1) in ceramide-mediated apoptosis in HL-60 cells.

Ceramide acts as a mediator of apoptosis in various cell lines, but little is known regarding the molecular mechanism linked to the cell cycle. In the present study, we examined the expression of p27(kip1) and its relationship to apoptosis induced by ceramide. We demonstrated that treatment of HL-60 cells with C6-ceramide resulted in G1 phase elevation followed by apoptotic cleavage associated with increase in the level of cdk inhibitor p27(kip1). Ceramide inhibited the kinase activities of cdk2 and cdk4 within 24 h of treatment. Ceramide-induced inhibition of cdk2 and cdk4 kinase activities was accompanied by increase of p27(kip1) in the cdks complexes. In addition, we have shown that both the cell death and expression of p27(kip1) protein induced by ceramide were significantly decreased in HL-60 cells overexpressing bcl-2. Furthermore, ceramide induced a significant increase in Bax protein expression coincided with increase in p27(kip1) protein level. These findings indicate that p27(kip1) may play important roles in mediating ceramide-induced apoptosis and its expression can be regulated by Bax and Bcl-2.

Resveratrol is a selective human cytochrome P450 1A1 inhibitor.

Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin compound found in juice and wine produced from dark-skinned grape cultivars and reported to have anti-inflammatory and anticarcinogenic activities. To investigate the mechanism of anticarcinogenic activities of resveratrol, the effects on cytochrome P450 (P450) were determined in human liver microsomes and Escherichia coli membranes coexpressing human P450 1A1 or P450 1A2 with human NADPH-P450 reductase (bicistronic expression system). Resveratrol slightly inhibited ethoxyresorufin O-deethylation (EROD) activity in human liver microsomes with an IC(50) of 1.1 mM. Interestingly, resveratrol exhibited potent inhibition of human P450 1A1 in a dose-dependent manner with IC(50) of 23 microM for EROD and IC(50) of 11 microM for methoxyresorufin O-demethylation (MROD). However, the inhibition of human P450 1A2 by resveratrol was not so strong (IC(50) 1.2 mM for EROD and 580 microM for MROD). Resveratrol showed over 50-fold selectivity for P450 1A1 over P450 1A2. The activities of human NADPH-P450 reductase were not significantly changed by resveratrol. In a human P450 1A1/reductase bicistronic expression system, resveratrol inhibited human P450 1A1 activity in a mixed-type inhibition (competitive-noncompetitive) with a K(i) values of 9 and 89 microM. These results suggest that resveratrol is a selective human P450 1A1 inhibitor, and may be considered for use as a strong cancer chemopreventive agent in humans.

Effects of beta-ionone on the expression of cytochrome P450s and NADPH-cytochrome P450 reductase in Sprague Dawley rats.

We have recently reported that beta-ionone induces cytochrome P450 (P450) 2B1 in rats. Effects of beta-ionone on the expression of other P450 isozymes and NADPH-P450 reductase were further investigated in Sprague Dawley rats. Administration of beta-ionone subcutaneously 72 and 48 h before sacrificing the animals not only significantly induced the liver microsomal activities of P450-associated enzymes and NADPH-P450 reductase, but also clearly increased in the level of P450 1A1/2, P450 2C, and NADPH-P450 reductase proteins. The induction of P450 1A1/2 and 2C by beta-ionone was much greater in male than in female as measured by western immunoblotting. Reverse transcriptase-polymerase chain reactions showed that, in addition to P450 2B1 and 2B2 mRNAs, P450 1A2, 2C6 and NADPH-P450 reductase mRNAs were increased when beta-ionone was administered. Our previous and present results indicated that beta-ionone may induce several P450s and NADPH-P450 reductase by the accumulation of their corresponding mRNAs.

Expression of recombinant human cytochrome P450 1A2 in Escherichia coli bacterial mutagenicity tester strain.

Human cytochrome P450 1A2 is one of the major cytochrome P450s in human liver. It is known to be capable of activating a number of carcinogens such as arylamines and heterocyclic amines. In order to develop the new bacterial mutagenicity test system with human P450, a full length of human P450 1A2 cDNA inserted into pCW bacterial expression vector was introduced to Escherichia coli WP2 uvrA strain which is a well-known E. coli strain for bacterial reverse mutagenicity assay. Expressed human P450 1A2 showed typical P450 hemoprotein spectra. Maximum expression was achieved at 48 hrs after incubating at 30 degrees C in terrific broth containing ampicillin, IPTG and other supplements. High level expression of P 450 1A2 in E. coli WP2 uvrA membranes was determined in SDS-PAGE. The well-known mutagens 2-aminoanthracene and MelQ increased the revertant colonies of E. coli WP2 uvrA expressing human P450 1A2 without an exogenous rat hepatic post-mitochondrial supernatant (S9 fraction) in a dose-dependent manner. The results show that the functional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagenesis and carcinogenesis of new drugs and environmental chemicals.

Protection of rat liver microsomes against carbon tetrachloride-induced lipid peroxidation by red ginseng saponin through cytochrome P450 inhibition.

A possible role of cytochrome P450 (P450) inhibition by red ginseng saponins in carbon tetrachloride (CCl4)-induced lipid peroxidation was investigated in liver microsomes prepared from male Sprague Dawley rats. The total saponin of red ginseng standardized on ginsenosides-Rb1, -Rb2, -Rc, -Rd, -Re, and -Rg1 whose composition was studied in our previous report was used in the present study. The standardized saponin of red ginseng showed inhibitory effects on P450-associated monooxygenase activities in a dose-dependent manner, particularly p-nitrophenol hydroxylase activity which has been known to represent CCl4-activating P450 2E1 enzyme. Meanwhile, silymarin enhanced the activity of P450 2E1 enzyme in liver microsomes. When the lipid peroxidation was induced by incubating rat liver microsomes with CCl4 in the presence of NADPH, the standardized saponin significantly blocked the formation of thiobarbituric acid-reactive substances at the same concentrations showing P450 inhibition in liver microsomes. Silymarin revealed more potent protection against CCl4-induced lipid peroxidation. When the lipid peroxidation was induced by FeCl3, in which metabolic activation may not be required, only silymarin could protect the lipid peroxidation in liver microsomes. Taken together, our present results indicated that the inhibitory effects of red ginseng saponin on P450 enzymes may have a critical role in CCl4-induced lipid peroxidation in rat liver microsomes and that the mechanism of hepatoprotection by red ginseng saponin may be distinct from that of silymarin.

Characterization of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 oxidoreductase in Escherichia coli.

A cDNA of fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase was genetically engineered and expressed in Escherichia coli DH5alpha cells under the control of an inducible tac promoter (Y. J. Chun, T. Shimada, and F. P. Guengerich, (1996) Arch. Biochem. Biophys. 330, 48-58). E. coli membranes of transformed cells showed much higher P450 1Al-dependent monooxygenase and NADPH-P450 reductase activities than pCW control vector or P450 1A1 expression vector-transformed cells. Ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase were 22-fold and 11-fold higher than the control activity, respectively. alpha-Naphthoflavone and beta-naphthoflavone strongly inhibited P450 1A1 activity of the fusion protein, with alpha-naphthoflavone being more potent than beta-naphthoflavone. Divalent cations (e.g. Ca2+ and Mg2+) increased P450 1A1 activity as well as NADPH-P450 reductase activity. These results demonstrate that this fusion protein in E. coli membrane may be a useful model for elucidating details of protein-protein interactions between P450 and NADPH-P450 reductase in the endoplasmic reticulum of mammalian cells.

Cooperativity in oxidations catalyzed by cytochrome P450 3A4.

Cytochrome P450 (P450) 3A4 is the most abundant human P450 and oxidizes a diversity of substrates, including various drugs, steroids, carcinogens, and macrolide natural products. In some reactions, positive cooperativity has been reported in microsomal studies. Flavonoids, e.g., 7,8-benzoflavone (alpha-naphthoflavone, alpha NF), have been shown to stimulate some reactions but not others. In systems containing purified recombinant bacterial P450 3A4, positive cooperativity was seen in oxidations of several substrates, including testosterone, 17 beta-estradiol, amitriptyline, and most notably aflatoxin (AF) B1. With these and other reactions, alpha NF typically reduced cooperativity (i.e., the n value in a Hill plot) while either stimulating or inhibiting reactions. With the substrate AFB1, alpha NF both stimulated 8,9-epoxidation and inhibited 3 alpha-hydroxylation. The same patterns were seen with AFB1 in a fused P450 3A4-NADPH-P450 reductase protein. alpha NF did not alter patterns of activity plotted as a function of NADPH-P450 reductase concentration in systems containing the individual proteins. The patterns of AFB1 oxidation to the two products were modified considerably in systems in which NADPH-P450 reductase was replaced with a flavodoxin or ferredoxin system, iodosylbenzene, or cumene hydroperoxide. AFB2, which differs from AFB1 only in the presence of a saturated 8,9-bond, was not oxidized by P450 3A4 but could inhibit AFB1 oxidation. These and other results are considered in the context of several possible models. The results support a model in which an allosteric site is involved, although the proximity of this putative site to the catalytic site cannot be ascertained as of yet. In order to explain the differential effects of alpha NF and reduction systems on the two oxidations of AFB1, a model is presented in which binding of substrate in a particular conformation can facilitate oxygen activation to enhance catalysis.

Construction of a human cytochrome P450 1A1: rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification.

A plasmid (pCW) was modified to code for a fusion protein consisting of the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH-P450 reductase beginning at amino acid 57. This plasmid was used to express the fusion protein in Escherichia coli DH5alpha cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2',5'-ADP agarose chromatography. The purified fusion protein catalyzed benzo[a]pyrene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolamine 6-hydroxylation. Catalytic activity was not increased in the presence of added NADPH-P450 reductase, cytochrome b5, or phospholipid. The fusion protein could also transfer electrons to cytochromes c and b5 but not P450 lA2. The same oxidation products of benzo[a]pyrene were formed with the purified fusion protein and the fusion protein functioning in bacterial cells. The catalytic activity of the human P450 1A1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line with the reported differences in catalytic activities of the rat and human P450 1A1 enzymes. The purified fusion protein also oxidized (+)- and (-)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic amines to genotoxic products, in the absence of added NADPH-P450 reductase. The demonstration of catalytic activities of the human fusion protein within bacterial cells suggests the prospect of utilizing such cellular systems for production of human P450 metabolites.