Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes

Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4′-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug–drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10?µmol?L^–1 diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25?µmol?L^–1 and Ki?=?4.473?µmol?L^–1 in human liver microsomes. Curcumin’s mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100?µmol L^–1. The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment.     

Functional screening of cytochrome P450 activity and uncoupling by capillary electrophoresis

Cytochrome P450s (CYPs) are functionally diverse monooxygenases responsible for oxidation of endogenous and xenobiotic compounds. The function of nonmammalian CYPs are largely unknown and tools for characterization limited. CYPs critical for xenobiotic metabolism are prone to catalytic cycle uncoupling resulting in reactive oxygen species (ROS) generation that is highly dependent on the specific CYP isoform and substrate interaction. This study describes the rapid assessment of the activity and coupling efficiency of CYPs using capillary electrophoresis with UV detection. The coupling efficiency of five zebrafish (Danio rerio) CYP1 isoforms with a series of fluorogenic substrate probes was determined by the rate of NADP(+) formation and compared with fluorescent product turnover rates. In most cases, NADP(+) formation significantly overestimated CYP1 catalytic activity for substrate O-dealkylation suggesting uncoupling. ROS production was confirmed by elevated hydrogen peroxide generation in poorly coupled reactions. Reactions with β-estradiol confirmed that CYP1A, 1C1, and 1C2 have greater catalytic activity and coupling efficiency; CYP1B1 and 1D1 had coupling efficiencies under 4%. This work highlights the wide disparity in uncoupling induced by unproductive substrate binding among different CYP isoforms.     

Effect of sodium alterations on hepatic cytochrome P450 3A2 and 2C11 and renal function in rats

Numerous dietary supplements are known to modulate cytochrome P450 (CYP)-mediated metabolism and subsequently alter drug toxicity or efficacy in animals and humans. In the present study we investigated the effect of varying amounts of sodium intake on renal function and the metabolic activity of the hepatic CYP3A2 and CYP2C11 isoforms. Rats were maintained on standard rodent chow or a low-salt rice diet. Within each of these groups rats received either a single intraperitoneal injection of furosemide to initiate salt depletion, or saline. Additional groups included salt supplementation of 500 mg/300 g body weight/day and 1.25 g/300 g body weight/day of sodium chloride solution. Rats receiving the low-salt diet, both with and without a concomitant furosemide administration, had a significant reduction in creatinine clearance without changes in serum creatinine. In addition, urine flow rate was markedly reduced in rats maintained on the low-salt diet. Western blot analysis indicated that neither sodium supplementation nor deprivation altered hepatic microsomal CYP3A2 levels; however, hepatic CYP2C11 levels significantly increased in rats receiving the largest sodium supplement. In vitro metabolic activity of CYP3A2 was unchanged as compared with controls. Activity of CYP2C11 was significantly reduced in both rat groups receiving additional sodium supplements. Acute manipulation of daily sodium intake does alter renal function and specific hepatic CYP isoforms and should be considered when using these rat models.     

Effects of capsicine on rat cytochrome P450 isoforms CYP1A2, CYP2C19, and CYP3A4

Due to the frequent consumption of capsaicin (CAP) and its current therapeutic application, the correct assessment of this compound is important from a public health standpoint. The purpose of this study was to find out whether CAP affects rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5?mL/kg, which contained phenacetin (15?mg/kg), omeprazole (15?mg/kg), and midazolam (10?mg/kg), was given orally to rats treated for 7?d with oral administration of CAP. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS. The results showed that treatment with multiple doses of CAP had no significant effect on rat CYP1A2. However, CAP had a significant inhibitory effect on CYP2C19 and an inductive effect on CYP3A4. Therefore, caution is needed when CAP is co-administered with some CYP substrates clinically because of potential drug–CAP interactions.     

Assessment of effects of IR and IPC on activities of cytochrome P450 isozymes in rats by a five-drug cocktail approach

Background and objective: To evaluate the effects of ischemia and reperfusion (IR) and ischemic preconditioning (IPC) on the metabolic activities of cytochrome P450 (CYP) isozymes in rats by a five-drug cocktail approach.

Methods: Cocktail approach was used to evaluate the influence of IR and IPC on the activities of CYP1A2, CYP2C9, CYP2E1, CYP2D6 and CYP3A4, which were reflected by the changes of pharmacokinetic parameters of five specific probe drugs: caffeine, chlorzoxazone, tolbutamide, metoprolol and midazolam, respectively. Rats were randomly divided into IR, IPC and sham groups, and then injected the mixture of five probe drugs. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by a HPLC method with UV detection. The pharmacokinetic parameters were calculated by the software of DAS 2.0.

Results: The parameters including t_1/2β, CLs, AUC, MRT and K₁₀ exhibited a similar tendency for both IR and IPC groups. Compared with sham group, CLs and K₁₀ of five probe drugs were significantly lower (p?t_1/2β of five or some probe drugs were significantly increased in IR and IPC groups (p?p?Conclusion: IR can variably decrease the activities of CYP isozymes in rats and this decrease can be attenuated by IPC.     

Metabolic interactions of magnolol with cytochrome P450 enzymes: uncompetitive inhibition of CYP1A and competitive inhibition of CYP2C

Magnolol (MAG; 5,5′-diallyl-2,2′-biphenyldiol) is a major bioactive component of Magnolia officinalis. We investigated the metabolic interactions of MAG with hepatic cytochrome P450 monooxygenase (CYP) through in vitro microsomal metabolism study using human (HLM) and rat liver microsomes (RLM). CYP2C and 3A subfamilies were significantly involved in the metabolism of MAG, while CYP1A subfamily was not in HLM and RLM. The relative contribution of phase I enzymes including CYP to the metabolism of MAG was comparable to that of uridine diphosphate glucuronosyltransferase (UGT) in RLM. Moreover, MAG potently inhibited the metabolic activity of CYP1A (IC₅₀ of 1.62?μM) and 2C (IC₅₀ of 5.56?μM), while weakly CYP3A (IC₅₀ of 35.0?μM) in HLM and RLM. By the construction of Dixon plot, the inhibition type of MAG on CYP activity in RLM was determined as follows: uncompetitive inhibitor for CYP1A (K_i of 1.09–12.0?μM); competitive inhibitor for CYP2C (K_i of 10.0–15.2?μM) and 3A (K_i of 93.7–183?μM). Based on the comparison of the current IC₅₀ and K_i values with a previously reported liver concentration (about 13?μM) of MAG after its seven times oral administration at a dose of 50?mg/kg in rats, it is suggested that MAG could show significant inhibition of CYP1A and 2C, but not CYP3A, in the in vivo rat system. These results could lead to further studies in clinically significant metabolism-mediated MAG–drug interactions.     

Activation of phosphorothionate pesticides based on a cytochrome P450 BM-3 (CYP102 A1) mutant for expanded neurotoxin detection in food using acetylcholinesterase biosensors

A novel enzymatic in vitro activation method for phosphorothionates has been developed to allow their detection with acetylcholinesterase (AChE) biosensors. Activation is necessary because this group of insecticides shows nearly no inhibitory effect toward AChE in their pure nonmetabolized form. In contrast, they exert a strong inhibitory effect on AChE after oxidation as it takes place by metabolic activation in higher organisms. Standard chemical methods to oxidize phosphorothionates showed inherent disadvantages that impede their direct use in food analysis. In contrast, a genetically engineered triple mutant of P450 BM-3 (CYP102 A1) could convert the two frequently used insecticides parathion and chlorpyrifos into their oxo variants as was confirmed by GC/MS measurements. The wild-type protein was unable to do so. In the case of chlorpyrifos, the enzymatic activation was as good as the chemical oxidation. In the case of parathion, the P450 activation was more efficient than the oxidation by NBS but neither activation method yielded an AChE inhibition that was as high as with paraoxon. The application of the method to infant food in combination with a disposable AChE biosensor enabled detection of chlorpyrifos and parathion at concentrations down to 20 microg/kg within an overall assay time of 95 min.     

Isolation and characterization of a Cr(VI)-reduction Ochrobactrum sp. strain CSCr-3 from chromium landfill

A strain CSCr-3 with high Cr(VI)-reducing ability under alkaline conditions was isolated from a chromium landfill and identified as Ochrobactrum sp. on the basis of 16S rRNA gene sequence analysis. The cells were rod shaped, Gram-negative and motile. The physiological characteristics and Cr(VI)-reduction of the strain were also studied. The results showed that the Ochrobactrum sp. strain CSCr-3 was tolerant to very high concentration of Cr(VI) (800 mg/L) and capable of reducing different forms of Cr(VI) (chromate and dichromate), under a wide range of temperatures (25-40 degrees C) and pH (7-11) with optimum at 35 degrees C and initial pH 10. Higher rates of Cr(VI)-reduction were observed with higher initial cell and Cr(VI) concentrations. Strain CSCr-3 could reduce Cr(VI) very efficiently over a wide range of Cr(VI) concentrations (100-800 mg/L). The addition of glucose caused a dramatic increase in Cr(VI)-reduction by Ochrobactrum sp. CSCr-3, while the presence of sulfate or nitrate had no influence. The presence of other metals, such as Cu, Co, Mn, etc., significantly stimulated Cr(VI)-reduction ability by the strain CSCr-3. The results obtained in this study have significance for the bioremediation of chromate pollution.     

Effect of particle size and lattice strain on Debye-Waller factors of Fe3C nanoparticles

Fe₃C nanoparticle powders have been prepared by a high energized ball mill. The resulting nanoparticle powders were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) measurements. The high-energy ball milling of Fe₃C after 10 h resulted in crystalline size of about 5 nm. The Debye temperature, mean-square amplitudes of vibration, Debye-Waller factor, lattice parameters, particle size, lattice strain and vacancy formation of energies of Fe₃C nanoparticles prepared by ball mill have been obtained from X-ray integrated intensities. The integrated intensities have been measured with a Philips CWU 3710 X-ray powder diffractometer fitted with a scintillation counter using filtered CuKα radiation at room temperature and have been corrected for thermal diffuse scattering. The X-ray Debye temperatures obtained in the present investigation has been used to estimate the vacancy formation energies for Fe₃C nanoparticles.     

Differential effect of arecoline on the endogenous dioxin-responsive cytochrome P450 1A1 and on a stably transfected dioxin-responsive element-driven reporter in human hepatoma cells

Dioxin-responsive element-mediated chemical activated luciferase expression (DRE-CALUX) is one of alternative bioassays for the determination of dioxin levels. We have previously established a DRE-CALUX cell line, Huh7-DRE-Luc, by using stable transfection of Huh-7 cells with a reporter plasmid (4xDRE-TATA-Luc) carrying a DRE-driven firefly luciferase gene. It was also shown that arecoline, a major areca nut alkaloid, inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cytochrome P450 1A1 (CYP1A1) activation in Huh-7 cells. The TCDD-activated aryl hydrocarbon receptor (AhR) induces the DRE-CALUX activation and CYP1A1 gene expression via binding to DRE in promoter regions of these dioxin-responsive genes. In the present study, the effect of arecoline on the TCDD-induced activation of DRE-CALUX and CYP1A1 enzyme in Huh7-DRE-Luc and Huh-7 cells, respectively, was examined. It was found that arecoline inhibited TCDD-induced CYP1A1 activation and however enhanced TCDD-induced DRE-CALUX activation. This finding indicates the differential effect of arecoline on the endogenous dioxin-responsive CYP1A1 and on a stably transfected DRE-driven reporter in human hepatoma cells. The present study suggests that induction of DRE-CALUX alone does not necessarily parallel with endogenous CYP1A1 gene expression, and that the reporter assay may detect interactions that are not functional in endogenous gene.     

Toxic effects of dissolved heavy metals on Desulfovibrio vulgaris and Desulfovibrio sp. strains

Biological treatment of metal-containing wastewaters with sulphate-reducing bacteria (SRB) is an attractive technique for the bioremediation of this kind of medium. In order to design a suitable engineering process to address this environmental problem, it is crucial to understand the inhibitory effect of dissolved heavy metals on these bacteria. Batch studies were carried out to evaluate the toxic effects of several heavy metal ions [Cr(III), Cu(II), Mn(II), Ni(II) and Zn(II)] on two cultures of SRB (Desulfovibrio vulgaris and Desulfovibrio sp.). The experimental data indicate that SRB show different responses to each metal. At the highest metal concentration tolerated for each metal, the precipitation levels for D. vulgaris were as follows: 24.7%-15 ppm Cr(III), 45%-4 ppm Cu(II), 60%-10 ppm Mn(II), 96%-8.5 ppm Ni(II) and 9%-20 ppm Zn(II). The corresponding values for Desulfovibrio sp. were: 25.5%-15 ppm Cr(III), 71%-4 ppm Cu(II), 66.2%-10 ppm Mn(II), 96.1%-8.5 ppm Ni(II) and 93%-20 ppm Zn(II). Results obtained in batch studies will be taken into account for the subsequent design of a sulphate-reducing bioreactor to reduce levels of heavy metals present in different types of contaminated media.     

The effects of strain rate and CCVC interfacial layer on mechanical behaviours of CALL

In this paper, the loading and loading-unloading tests of CALL and CALL (CCVC) under tensile impact have been carried out by a self-designed Rotating Circular Disk Tensile impact Apparatus. The quasi-static tension and short beam bending tests are performed on the Shimadzu-5000 testing apparatus. Experiment results show that both CALL and CALL (CCVC) have positive hybrid effect. Under quasi-static tension, the two composites have no obvious yielding until fracture, but have an obvious yielding point on the dynamic tensile stress-strain curves. The dynamic unstable fracture strain is about three times the static unstable fracture strain. The interlaminar shear strength (ISS) of CALL (CCVC) is 10 more than that of CALL. At the same time, the tensile strength and unstable fracture strain of CALL (CCVC) are also higher than that of CALL. In this paper, some conclusions are also drawn from the SEM observation of the fracture specimen surfaces.     

Decolorization of different dyes by a newly isolated white-rot fungi strain Ganoderma sp.En3 and cloning and functional analysis of its laccase gene

A laccase-producing white-rot fungi strain Ganoderma sp.En3 was newly isolated from the forest of Tzu-chin Mountain in China. Ganoderma sp.En3 had a strong ability of decolorizing four synthetic dyes, two simulated dye bath effluents and the real textile dye effluent. Induction in the activity of laccase during the decolorization process indicated that laccase played an important role in the efficient decolorization of different dyes by this fungus. Phytotoxicity study with respect to Triticum aestivum and Oryza sativa demonstrated that Ganoderma sp.En3 was able to detoxify four synthetic dyes, two simulated dye effluents and the real textile dye effluent. The laccase gene lac-En3-1 and its corresponding full-length cDNA were then cloned and characterized from Ganoderma sp.En3. The deduced protein sequence of LAC-En3-1 contained four copper-binding conserved domains of typical laccase protein. The functionality of lac-En3-1 gene encoding active laccase was verified by expressing this gene in the yeast Pichia pastoris successfully. The recombinant laccase produced by the yeast transformant could decolorize the synthetic dyes, simulated dye effluents and the real textile dye effluent. The ability of decolorizing different dyes was positively related to the laccase activity. In addition, the 5′-flanking sequence upstream of the start codon ATG in lac-En3-1 gene was obtained. Many putative cis-acting responsive elements were predicted in the promoter region of lac-En3-1.     

The effects of strain rate and grain size on nanocrystalline materials: A theoretical prediction

The article describes a dislocation-based model for the coupling effects of strain rate and grain size on the mechanical behavior of nanocrystalline materials. Two important experimental findings about nanocrystalline materials are explained by the model: 1. the difference of the dependencies of strain rate sensitivity (SRS) on grain size between FCC and BCC nanocrystalline materials and 2. the abnormal dependency of activation volume on thermally activated stress. Our analysis shows that the strain rate-dependent behavior of nanocrystalline materials is mainly determined by the ratio between the activation volumes in grain and grain boundary regions.     

The effects of strain annealing on grain boundaries and secure triple junctions in nickel 200

Changes in the microstructure of 99.5% pure nickel as a function of strain annealing have been investigated. The application of moderate amounts of compressive strain (6–7%), and subsequent thermal treatment at 750°C under vacuum is shown to increase the density of Σ3 grain boundaries, where the coincident-site lattice rationale is adopted to characterize the boundaries. A preliminary, post-strain surface oxidation treatment to 500°C is shown to drastically increase the proportion of twin boundaries, a majority of which are identified as being coherent in nature. In addition, a high level of connectivity for these twins is observed, along with greatly increased numbers of secure triple junctions. It is postulated that the mechanism by which grain boundary energy minimization is effected involves grain rotations when treatment is conducted under vacuum, but that annealing twin formation is the predominant process when an oxygen-rich environment exists. This revised version was published online in November 2006 with corrections to the Cover Date.     

The effects of carbon sources and micronutrients in whey and fermented whey on the kinetics of phenanthrene biodegradation in diesel contaminated soil

This paper demonstrates significant effects on phenanthrene degradation in diesel contaminated soil by the addition of organic amendments such as whey and fermented whey. Both amount of amendment added and mode of administration was shown to be decisive. There was a strong positive effect on the ¹⁴C-mineralization of phenanthrene by multiple (bi-weekly) additions of fermented whey 210 mg dw kg⁻¹ soil dw (FW multi) and also by single dose addition of 2100 mg dw sweet whey kg⁻¹ soil dw (SW high). The most prominent effects on phenanthrene degradation kinetics were a five to fifteen fold increase in the linear growth term (k₂) and a 23-27% increase in bioavailability factor S₀ for SW high and FW multi respectively. Also, total mineralization at the end of the experiment increased from 46% in the control to 66 and 71% respectively and the lag time was reduced from 21 to 15 days by multiple addition of fermented whey. The most significant stimulating effects on phenanthrene degradation kinetics could be attributed to lactate and vitamins. This study demonstrates a more complex dependence of carbon sources and growth factors for an aromatic compound such as phenanthrene in comparison to hexadecane.     

Biosynthesised AgCl NPs using Bacillus sp. 1/11 and evaluation of their cytotoxic activity and antibacterial and antibiofilm effects on multi‐drug resistant bacteria

Silver nanoparticles (AgNPs) have attracted the attention of researchers due to their properties. Biological synthesis of AgNPs is eco‐friendly and cost‐effective preferred to physical and chemical methods, which utilize environmentally harmful agents and large amounts of energy. Microorganisms have been explored as potential biofactories to synthesize AgNPs. Bacterial NP synthesis is affected by Ag salt concentration, pH, temperature and bacterial species. In this study, Bacillus spp., isolated from soil, were screened for AgNP synthesis at pH 12 with 5 mM Ag nitrate (AgNO₃) final concentration at room temperature. The isolate with fastest color change and the best ultraviolet‐visible spectrum in width and height were chosen as premier one. AgNO₃ and citrate salts were compared in terms of their influence on NP synthesis. Spherical Ag chloride (AgCl) NPs with a size range of 35–40 nm were synthesized in 1.5 mM Ag citrate solution. Fourier transform infrared analysis demonstrated that protein and carbohydrates were capping agents for NPs. In this study, antimicrobial and antitumor properties of the AgNP were investigated. The resulting AgCl NPs had bacteriostatic activity against four standard spp. And multi‐drug resistant strain of Pseudomonas aeruginosa. These NPs are also cytotoxic to cancer cell lines MCF‐7, U87MG and T293.Inspec keywords: silver compounds, nanoparticles, nanomedicine, nanofabrication, particle size, biomedical materials, microorganisms, ultraviolet spectra, visible spectra, Fourier transform infrared spectra, proteins, macromolecules, antibacterial activity, tumours, cancer, cellular biophysics, toxicologyOther keywords: citrate salts, spherical Ag chloride, particle size, Ag citrate solution, Fourier transform infrared analysis, protein, carbohydrates, capping agents, antitumour properties, bacteriostatic activity, Pseudomonas aeruginosa, multidrug resistant strain, cancer cell lines MCF‐7,U87MG, size 35 nm to 40 nm, temperature 293 K to 298 K, AgCl, ultraviolet‐visible spectrum, colour change, room temperature, Ag nitrate final concentration, soil, bacterial species, temperature effect, pH, Ag salt concentration, biofactories, microorganisms, environmentally harmful agents, chemical methods, physical methods, antibacterial properties, electrical properties, mechanical properties, silver nanoparticles, multidrug resistant bacteria, antibiofilm effects, antibacterial effects, cytotoxic activity, Bacillus sp. 1/11, biosynthesised AgCl NPs