Essential role of constitutive androstane receptor in Ginkgo biloba extract induced liver hypertrophy and hepatocarcinogenesis.

Ginkgo biloba extract (GBE) is commonly used as a herbal supplement. The National Toxicology Program (NTP) study of GBE reported clear evidence of hepatocarcinogenicity in mice. To clarify the mode of action (MOA) for hepatocarcinogenesis by GBE, we investigated the involvement of the constitutive androstane receptor (CAR) in hepatocarcinogenesis induced by GBE using CAR-knockout (CARKO) and wild type (WT) mice. We used the same lot of GBE that was used for the NTP study. In 1-week GBE dietary treatment, hepatocellular DNA replication was increased in WT mice but not in CARKO mice. In 4- or 13-week treatment, greater hepatic Cyp2b10 induction and hepatocellular hypertrophy were observed in WT mice, whereas these effects of GBE were much smaller in CARKO mice. In a two-stage hepatocarcinogenesis model initiated by diethylnitrosamine, 27-week treatment with GBE resulted in an increase of eosinophilic altered foci and adenomas in WT mice. By contrast, foci and adenomas were clearly less evident in CARKO mice. These results indicate that GBE-induced hepatocarcinogenesis is mainly CAR-mediated. Since CAR-mediated MOA for hepatocarcinogenesis in rodents is considered to be qualitatively implausible for humans, our findings would be helpful to evaluate the carcinogenic characterization of GBE to humans.

Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function.

Identification of the protopanaxatriol synthase gene CYP6H for ginsenoside biosynthesis in Panax quinquefolius.

Panax quinquefolius is one of perennial herbs and well known for its outstanding pharmacological activity. Ginsenosides are thought to be the main active ingredients in P. quinquefolius and exist in many kinds of plant genus Panax (ginseng). Protopanaxatriol synthase, which is considered cytochrome P450 (CYP450) in ginsenoside biosynthesis pathway can convert protopanaxadiol into protopanaxatriol. However, the protopanaxatriol synthase gene in P. quinquefolius has not been identified. Here, we cloned and identified a protopanaxatriol synthase gene from P. quinquefolius (CYP6H, GenBank accession no. KC190491) at the first time, reverse transcription-PCR (RT-PCR) analysis showed no obvious transcription change of CYP6H in methyl jasmonate (MeJA)-induced hairy roots. Ectopic expression of CYP6H in Saccharomyces cerevisiae resulted in the production of protopanaxatriol with added exogenous protopanaxadiol and confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Moreover, high-performance liquid chromatography (HPLC) analysis shows that RNA interferences of CYP6H in transgenic hairy roots could increase the accumulation of protopanaxadiol-type ginsenosides and decrease the accumulation of protopanaxatriol-type ginsenosides, whereas the effect of overexpression CYP6H in transgenic hairy roots was contrary. Our study indicated that CYP6H is a gene encoding protopanaxadiol 6-hydroxylase which could convert protopanaxadiol into protopanaxatriol in P. quinquefolius ginsenoside biosynthesis, we also have confirmed the function of CYP6H on effect accumulation of ginsenosides.

Cytochrome P450 2C epoxygenases mediate photochemical stress-induced death of photoreceptors.

Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids.

Mechanism-based inactivation of cytochrome P450 2A6 and 2A13 by Rhinacanthus nasutus constituents.

  Human cytochrome P450 CYP2A6 and CYP2A13 catalyze nicotine metabolisms and mediate activation of tobacco-specific carcinogens including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). In this study, we found rhinacanthins A, B, and C isolated from Rhinacanthus nasutus potentially inhibited coumarin 7-hydroxylation mediated by reconstituted purified recombinant CYP2A6 and CYP2A13. Rhinacanthins A-C are mechanism-based inactivators of CYP2A6 and CYP2A13 as they cause concentration, time and NADPH-dependent inhibition. Among the three rhinacanthins, rhinacanthin-B possessed highest inhibitory potency against CYP2A13 with apparent KI and kinact of 0.16 µM and 0.1 min(-1), respectively, while values of 0.44 µM and 0.12 min(-1) were found against CYP2A6. Rhinacanthin-C had least inhibition potency, with apparent KI and kinact of 0.97 µM and 0.07 min(-1) for CYP2A6, respectively, and values of 1.68 µM and 0.05 min(-1) for CYP2A13. Rhinacanthin-A inhibited CYP2A6 and CYP2A13 with apparent KI values of 0.69 and 0.42 µM, respectively and apparent kinact of 0.18 and 0.06 min(-1), respectively. The inhibition of both enzymes by rhinacanthins A-C could not be prevented by addition of trapping agents or reversed by dialysis or potassium ferricyanide. These findings demonstrated that rhinacanthins A-C, which are 1,4-naphthoquinone derivatives, irreversibly inhibited CYP2A6 and CYP2A13 in a mechanism-based inhibition mode.

Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: selectivity, kinetic characterization, and molecular modeling.

Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least Ki value of 44±16 nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC50 values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogen-bonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different.

Synthesis and evaluation of a 18F-labeled 4-ipomeanol as an imaging agent for CYP4B1 gene prodrug activation therapy.

We report the development of a (18)F-labeled 4-ipomeanol (4-IM), which is metabolized by the CYP4B1 enzyme, to image tumors and monitor enzyme-activating anticancer prodrugs. The fluorine-substituted derivative, 1-(3-furyl)-4-hydroxy-5-fluoro-1-pentanone (F-4-IM, 1), was synthesized from 3-furaldehyde. [(18)F]F-4-IM ([(18)F]1) was prepared in 20%-35% radiochemical yield by a fluorine-18 displacement reaction, followed by reduction and deprotection of the ketal group, and was shown to be stable (>96% at 2 hours) in human serum at 37°C. The biodistribution of [(18)F]F-4-IM in normal rats was high in the lung, where CYP4B1 gene is preferentially expressed. We transduced C6-glioma cells with a retrovirus-expressing CYP4B1 (C6-CYP4B1). Evaluation of CYP4B1 expression was confirmed by reverse transcription polymerase chain reaction and MTT assay. Cell assays were carried out using C6 and C6-CYP4B, and the uptake of [(18)F]F-4-IM in these cells was compared with that in parental controls. The uptake ratio of [(18)F]F-4-IM was 2.8-fold higher in C6-CYP4B1 compared with that in parental cells at 1 hour, whereas [(3)H]4-IM was taken up at similar rates in both cell lines after 6 hours. These results suggest that [(18)F]F-4-IM could be a promising PET imaging agent with potential to be used for imaging of CYP4B1-transfected tumor cells, as well as for monitoring CYP4B1 enzyme/prodrug interactions.

Theoretical investigation of differences in nitroreduction of aristolochic acid I by cytochromes P450 1A1, 1A2 and 1B1.

OBJECTIVES: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Human cytochrome P450 (CYP) 1A1 and 1A2 enzymes were found to be responsible for the AAI reductive activation to form AAI-DNA adducts, while its structurally related analogue, CYP1B1 is almost without such activity. However, knowledge of the differences in mechanistic details of CYP1A1-, 1A2-, and 1B1- mediated reduction is still lacking. Therefore, this feature is the aim of the present study. METHODS: Molecular modeling capable of evaluating interactions of AAI with the active site of human CYP1A1, 1A2 and 1B1 under the reductive conditions was used. In silico docking, employing soft-soft (flexible) docking procedure was used to study the interactions of AAI with the active sites of these human enzymes. RESULTS: The predicted binding free energies and distances between an AAI ligand and a heme cofactor are similar for all CYPs evaluated. AAI also binds to the active sites of CYP1A1, 1A2 and 1B1 in similar orientations. The carboxylic group of AAI is in the binding position situated directly above heme iron. This ligand orientation is in CYP1A1/1A2 further stabilized by two hydrogen bonds; one between an oxygen atom of the AAI nitro-group and the hydroxyl group of Ser122/Thr124; and the second bond between an oxygen atom of dioxolane ring of AAI and the hydroxyl group of Thr497/Thr498. For the CYP1B1:AAI complex, however, any hydrogen bonding of the nitro-group of AAI is prevented as Ser122/Thr124 residues are in CYP1B1 protein replaced by hydrophobic residue Ala133. CONCLUSION: The experimental observations indicate that CYP1B1 is more than 10× less efficient in reductive activation of AAI than CYP1A2. The docking simulation however predicts the binding pose and binding energy of AAI in the CYP1B1 pocket to be analogous to that found in CYP1A1/2. We believe that the hydroxyl group of S122/T124 residue, with its polar hydrogen placed close to the nitro group of the substrate (AAI), is mechanistically important, for example it could provide a proton required for the stepwise reduction process. The absence of a suitable proton donor in the AAI-CYP1B1 binary complex could be the key difference, as the nitro group is in this complex surrounded only by the hydrophobic residues with potential hydrogen donors not closer than 5 Å.

Identification of novel CYP2A6 inhibitors by virtual screening.

The human CYP2A6 enzyme metabolises several xenobiotics including nicotine, the addictive component in tobacco. Reduced activity of CYP2A6, either for genetic reasons or by administering inhibitors of CYP2A6, reduces tobacco smoking. The aim was to design novel inhibitors of CYP2A6 using 3D-QSAR analysis combined with virtual screening. A 3D-QSAR model was utilised to identify the most important features of the inhibitors, and this knowledge was used to design inhibitors for CYP2A6. Chemical database screening yielded several potent inhibitor candidates such as alkylamine derivatives (compound no. 5, IC(50)=0.1 µM) and 1-benzothiophene-3-carbaldehyde that can be used as lead compounds in the development of drugs for smoking reduction therapy.

Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay.

The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.

Adverse effects by artificial grapefruit seed extract products in patients on warfarin therapy.

OBJECTIVE: Grapefruit seed extract (GSE) is promoted as a natural product with antibacterial and antiviral properties. The aim of this study was to investigate the composition of some commercially available GSE products and evaluate their effect in vitro on two cytochrome P450 enzymes, CYP2C9 and CYP3A4. METHODS: A couple on lifelong treatment with warfarin and continuous regular follow-ups took some drops of a GSE product for 3 days. The female patient experienced a minor subcutaneous haematoma 3 days later, and her international normalised ratio (INR) value was 7.9. This was reported to the Swedish Medical Products Agency (MPA) as a spontaneous post-marketing report concerning adverse drug reactions/interactions. The composition of the GSE products was determined by proton and carbon-13 nuclear magnetic resonance spectroscopy (NMR). The inhibitory effect of the GSE products on the cytochrome P450 enzymes was tested in an in vitro baculosome assay. RESULTS: The NMR analysis showed that all three investigated GSE products contained the synthetic preservative benzethonium chloride (BTC) in addition to glycerol and water. No authentic GSE extract was found in any of the three GSE products analysed. Furthermore, BTC was found to be a potent inhibitor of CYP3A4 and CYP2C9 activity in vitro. CONCLUSION: Our results suggest that BTC in the GSE products is responsible for the increase in the INR value in a patient on warfarin treatment.

Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4.

A fundamental aspect of cytochrome P450 function is the role of the strictly conserved axial cysteine ligand, replacement of which by histidine has invariably resulted in mammalian and bacterial preparations devoid of heme. Isolation of the His-436 variant of NH2-truncated P450 2B4 partly as the holoenzyme was achieved in the present study by mutagenesis of the I-helix Ala-298 residue to Glu and subsequent conversion of the axial Cys-436 to His. The expressed A298E/C436H double mutant, cloned with a hexahistidine tag, had a molecular mass equivalent to that of the primary structure of His-tagged truncated 2B4 and the sum of the two mutated residues, and contained a heme group which, when released on HPLC, showed a retention time and spectrum identical to those of iron protoporphyrin IX. The absolute spectra of A298E/C436H indicate a change in heme coordination structure from low- to high-spin, and, as expected for a His-ligated hemeprotein, the Soret maximum of the ferrous CO complex is at 422 nm. The double mutant has no oxygenase activity with representative substrates known to undergo transformation by the oxene [(FeO)3+] or peroxo activated oxygen species, but catalyzes significant H2O2 formation that is NADPH- and time-dependent, and directly proportional to the concentration of A298E/C436H in the presence of saturating reductase. Moreover, the catalytic efficiency of A298E/C436H in the H2O2-supported peroxidation of pyrogallol is more than two orders of magnitude greater than that of wild-type 2B4 or the A298E variant. The results unambiguously demonstrate that the proximal thiolate ligand is essential for substrate oxygenation by P450.

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Cytochrome b(5) (cyt b(5)) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane alpha-helix at its carboxyl terminus. These two domains are connected by an approximately 15-amino acid linker domain, Ser(90)-Asp(104), which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b(5) to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b(5) to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b(5) except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b(5) to interact with cyt P450 2B4. A model for the membrane-bound cyt b(5)-cyt P450 complex is presented.

Reversible and irreversible inhibition of CYP3A enzymes by tamoxifen and metabolites.

1. Preliminary studies have identified cytochrome P450 (CYP) 3A4 and CYP1B1 as the human CYPs inhibited by tamoxifen. To quantify the inhibitory potency of tamoxifen and its major metabolites, the metabolism of three substrates of CYP3A, midazolam, diltiazem and testosterone, and 7-ethoxyresorufin as a substrate of CYP1B1 were examined in catalytic assays carried out using human liver microsomes and cDNA-expression systems. 2. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen reversibly inhibited midazolam 1'-hydroxylation, diltiazem N-demethylation and testosterone 6beta-hydroxylation with K(i) ranging from 3 to 37 micro M in human liver microsomes. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen also reversibly inhibited the activity of cDNA-expressed CYP3A4, CYP3A5 and CYP1B1. 3. Tamoxifen and N-desmethyltamoxifen exhibited time-dependent inactivation of testosterone 6beta-hydroxylation by cDNA-expressed CYP3A4 (+ cytochrome b5) yielding k(inact) and K(i) of 0.04 min(-1) and 0.2 micro M for tamoxifen and 0.08 min(-1) and 2.6 micro M for N-desmethyltamoxifen. A metabolic intermediate complex (MIC) was also formed by tamoxifen and N-desmethyltamoxifen with CYP3A4 (+ cytochrome b5) and CYP3A4 but not with CYP3A5 or CYP3A7. Pre-incubation with 4-hydroxytamoxifen and 3-hydroxytamoxifen did not result in any CYP3A inactivation or detectable MIC formation. There was no detectable time-dependent inactivation or MIC formation with tamoxifen or metabolites with CYP1B1. 4. These data indicate that tamoxifen and its three major metabolites are effective inhibitors of CYP3A in vitro and that tamoxifen and N-desmethyltamoxifen are effective mechanism-based inhibitors. Thus, caution should be exercised when tamoxifen is coadministered with other CYP3A substrates.

Characterization of mixtures of recombinant human cytochrome p450s as a screening model for metabolic stability in drug discovery.

1. Recombinant human cytochrome p450 (rhCYP) has become an important screening model in drug metabolism studies due to the high cost of human and animal hepatic tissue. Until now, rhCYPs have been evaluated and used as separate forms, but a mixture of CYP forms comparable with the human liver could be of value in early drug discovery. 2. In the present study, rhCYP2C9, rhCYP2D6 and rhCYP3A4 co-expressed with reductase in Escerichia coli were mixed and evaluated with regards to kinetic properties (K(m) and V(max)). Furthermore, antioxidant was added to investigate whether a free radical scavenger would affect the kinetic parameters. Results were compared with data obtained in human liver microsomes (HLM). 3. Results showed a good correlation between mixed rh CYP data and HLM data for K(m) and V(max). K(m) varied < 3-fold between matrices for CYP2C9 and CYP3A4, whereas the K(m) for CYP2D6 varied up to 4.5-fold. V(max) differed up to 3-fold between matrices for the CYP forms investigated. However, the discrepancy in V(max) may depend on the anticipated level of each form in HLM. The addition of antioxidant increased V(max) for CYP2C9 and CYP2D6 by 75 and 50%, respectively, whereas V(max) for CYP3A4 was unchanged. 4. In conclusion, the rhCYP mixture shows promising results as a predictor of CYP kinetic parameters. Furthermore, addition of antioxidant can in certain cases increase catalytic activity.