Establishment of a multi-acaricide resistant reference tick strain (IVRI-V) of Rhipicephalus microplus.

Tick-borne diseases is a global threat and tick resistance to commonly used acaricides is a growing problem, thus calling for improved resistance monitoring tools. To aid in monitoring of resistance in field tick populations, a resistant colony of Rhipicephalus microplus was characterized with the aim to establish a reference multi-acaricide resistant tick strain. Using a standardized adult immersion test, the Lethal Concentration(LC)50 values for deltamethrin, cypermethrin, fenvalerate and diazinon against the laboratory selected resistant tick (LSRT) strain were determined as 306.7 ppm, 2776.9 ppm, 30262.1 ppm and 9458.7 ppm. Relative to the susceptible IVRI-I tick strain, the LSRT strain showed 4.78- and 5.84-fold increases in activity of esterases, a 6-fold increase for monooxygenases and a 2.24 fold increase for glutathione S-transferase. In the acetylcholinesterase 2 gene, 22 single nucleotide polymorphisms (SNPs) were identified in the LSRT strain. Four of these SNPs lead to amino acid substitutions and were consistently found in resistant field populations in India. A C190A mutation in the domain II S4-5 linker region of sodium channel gene resulting in a L64I amino acid substitution was recorded in the LSRT strain. Monitorable indicators for the maintenance of the strain, designated as the reference IVRI-V tick strain and representing the first established multi-acaricide resistant tick strain in India, were identified.

Glycine Betaine Monooxygenase, an Unusual Rieske-Type Oxygenase System, Catalyzes the Oxidative N-Demethylation of Glycine Betaine in Chromohalobacter salexigens DSM 3043.

Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

An in silico functional annotation and screening of potential drug targets derived from Leishmania spp. hypothetical proteins identified by immunoproteomics.

Leishmaniasis is a parasitic disease caused by the protozoan of the Leishmania genus. While no human vaccine is available, drugs such as pentavalent antimonials, pentamidine and amphotericin B are used for treat the patients. However, the high toxicity of these pharmaceutics, the emergence of parasite resistance and/or their high cost have showed to the urgent need of identify new targets to be employed in the improvement of the treatment against leishmaniasis. In a recent immunoproteomics approach performed in the Leishmania infantum species, 104 antigenic proteins were recognized by antibodies in sera of visceral leishmaniasis (VL) dogs. Some of them were later showed to be effective diagnostic markers and/or vaccine candidates against the disease. Between these proteins, 24 considered as hypothetical were identified in the promastigote and amastigote-like extracts of the parasites. The present study aimed to use bioinformatics tools to select new drug targets between these hypothetical proteins. Their cellular localization was predicted to be seven membrane proteins, as well as eight cytoplasmic, three nuclear, one mitochondrial and five proteins remained unclassified. Their functions were predicted as being two transport proteins, as well as five with metabolic activity, three as cell signaling and fourteen proteins remained unclassified. Ten hypothetical proteins were well-annotated and compared to their homology regarding to human proteins. Two proteins, a calpain-like and clavaminate synthase-like proteins were selected by using Docking analysis as being possible drug targets. In this sense, the present study showed the employ of new strategies to select possible drug candidates, according their localization and biological function in Leishmania parasites, aiming to treat against VL.

Characterization of MymA protein as a flavin-containing monooxygenase and as a target of isoniazid.

Tuberculosis is a global health problem especially with the emergence of drug-resistant Mycobacterium tuberculosis strains, creating an urgent need to identify new drug targets. The mycobacterial cell wall is an attractive target for chemotherapeutic agents. Gene products of mymA operon are known to be required for the maintenance of cell wall and play an important role in persistence, thus making them important drug targets. This study was undertaken to biochemically characterize the MymA as a flavin-containing monooxygenase (FMO). Our results established its enzymatic activity in vitro and found that the mycobacterial FMO requires NADPH and FAD as cofactors, similar to other characterized bacterial FMOs. The enzyme follows Michaelis-Menten kinetics to catalyze substrates such as trimethylamine and thiourea. We also propose that MymA could be one of the targets of the antituberculosis drug, isoniazid (INH), which is a cell wall inhibitor. Molecular docking studies revealed that INH targeted NADPH-binding site of the MymA. Further, experimental validation revealed that INH inhibits MymA with the IC50 value of 4.9 µm. Thus, this study characterizes for the first time that MymA is a mycobacterial FMO, which may be a target of INH.

Targeting deoxyhypusine hydroxylase activity impairs cap-independent translation initiation driven by the 5'untranslated region of the HIV-1, HTLV-1, and MMTV mRNAs.

Replication of the human immunodeficiency virus type 1 (HIV-1) is dependent on eIF5A hypusination. Hypusine is formed post-translationally on the eIF5A precursor by two consecutive enzymatic steps; a reversible reaction involving the enzyme deoxyhypusine synthase (DHS) and an irreversible step involving the enzyme deoxyhypusine hydroxylase (DOHH). In this study we explored the effect of inhibiting DOHH activity and therefore eIF5A hypusination, on HIV-1 gene expression. Results show that the expression of proteins from an HIV-1 molecular clone is reduced when DOHH activity is inhibited by Deferiprone (DFP) or Ciclopirox (CPX). Next we evaluated the requirement of DOHH activity for internal ribosome entry site (IRES)-mediated translation initiation driven by the 5'untranslated region (5'UTR) of the full length HIV-1 mRNA. Results show that HIV-1 IRES activity relies on DOHH protein concentration and enzymatic activity. Similar results were obtained for IRES-dependent translation initiation mediated by 5'UTR of the human T-cell lymphotropic virus type 1 (HTLV-1) and the mouse mammary tumor virus (MMTV) mRNAs. Interestingly, activity of the poliovirus IRES, was less sensitive to the targeting of DOHH suggesting that not all viral IRESs are equally dependent on the cellular concentration or the activity of DOHH. In summary we present evidence indicating that the cellular concentration of DOHH and its enzymatic activity play a role in HIV-1, HTLV-1 and MMTV IRES-mediated translation initiation.

Modifying the sugar icing on the transplantation cake.

As a transplant surgeon, my interest in glycobiology began through my research into ABO-incompatible allotransplantation, and grew when my goal became overcoming the shortage of organs from deceased human donors by the transplantation of pig organs into patients with terminal organ failure (xenotransplantation/cross-species transplantation). The major target for human "natural" (preformed) anti-pig antibodies is galactose-α(1,3)-galactose (the "Gal" epitope), which is expressed on many pig cells, including the vascular endothelium. The binding of human IgM and IgG antibodies to Gal antigens initiates the process of hyperacute rejection, resulting in destruction of the pig graft within minutes or hours. This major barrier has been overcome by the production of pigs in which the gene for the enzyme α(1,3)-galactosyltransferase (GT) has been deleted by genetic engineering, resulting in GT knockout (GTKO) pigs. The two other known carbohydrate antigenic targets on pig cells for human anti-pig antibodies are (i) the product of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene, i.e., N-glycolylneuraminic acid, and (ii) the product of the ß1,4 N-acetylgalactosaminyltransferase gene, i.e., the Sd(a) antigen. Expression of these two has also been deleted in pigs. These genetic manipulations, together with others directed to overcoming primate complement and coagulation activation (the latter of which also relates to glycobiology) have contributed to the prolongation of pig graft survival in nonhuman primate recipients to many months rather than a few minutes. Clinical trials of the transplantation of pig cells are already underway and transplantation of pig organs may be expected within the relatively near future.

A cell-surface ß-hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) has a poor prognosis as a result of widespread intra- and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(asparaginyl)-ß-hydroxylase (ASPH) is a cell-surface enzyme that generates enhanced cell motility, migration, invasion, and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer-assisted drug design. Candidate compounds were tested for inhibition of ß-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion, and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biological effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor, MO-I-1100, was selected because it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion, and anchorage-independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. CONCLUSIONS: These studies suggest that the enzymatic activity of ASPH is important for hepatic oncogenesis. Reduced ß-hydroxylase activity generated by the SMI MO-I-1100 leads to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease.

Chromium-induced tropane alkaloid production and H6H gene expression in Atropa belladonna L. (Solanaceae) in vitro-propagated plantlets.

Hyoscyamine and scopolamine tropane alkaloids found in several solanaceous plants are anticholinergic drugs. Hyoscyamine 6ß-hydroxylase (H6H) catalyzes two consecutive oxidation reactions. The first reaction is the hydroxylation of hyoscyamine to 6ß-hydroxyhyoscyamine and the second is epoxidation of 6ß-hydroxyhyoscyamine yielding scopolamine that is the final metabolite in the tropane alkaloid biosynthetic pathway. The effects of trivalent chromium as KCr (SO4)(2) on the production of tropane alkaloids and the expression of hyoscyamine 6ß-hydroxylase gene (h6h) were studied in micro-propagated Atropa belladonna L. plantlets. The results showed that chromium treatment decreased the growth parameters (weights and lengths of the plantlets) and chlorophyll contents and increased proline contents. Moreover, semiquantitave RT-PCR analysis showed that the transcript level of H6H increased under chromium treatment. This treatment also increased hyoscyamine and scopolamine contents as shown by HPLC analysis. Changes of scopolamine contents correlate with the expression levels of h6h gene under different concentrations of chromium.

In vivo and in vitro mixed-function oxygenase activity and vitellogenin induction in fish and in fish and rat liver cells by stilbenes isolated from scotch pine (Pinus sylvestris).

Many types of pulp and paper mill effluents have the ability to induce mixed-function oxygenase (MFO) activity and vitellogenin (VTG) protein in exposed male fish. The search for the compounds responsible for MFO induction has led to several classes of compounds, among them retene and stilbenes. The objective of this study was to investigate the biological activities of candidate stilbene compounds. Three stilbenes, 3,5-dihydroxystilbene (pinosylvin; P1), 3-hydroxy-5-methoxystilbene (P2), and 3,5-dimethoxystilbene (P3), were extracted from Scotch pine (Pinus sylvestris) and purified to evaluate their ability to induce MFO activity in vitro using ethoxyresorufin-O-deethylase (EROD) activity in a rat hepatoma cell line (H4IIE). As these compounds may be chlorinated during pulp bleaching, chlorination of P2 was undertaken, producing di- and trichlorinated isomers (Cl-P2), which were also tested. Compounds were tested for EROD-inducing ability in vivo by exposing juvenile rainbow tout (Oncorhynchus mykiss) to waterborne concentrations (0.010 to 1.0 mg/L) for 4 days. Compounds were also tested for their ability to induce VTG in trout primary liver cells in vitro. The stilbenes were weak inducers of EROD and VTG. H4IIE EROD was induced by all four compounds, with the most potent induction by P3, followed by P1, the Cl-P2 mixture, and then P2. Induction for all four stilbenes was from 3.13 × 10⁻³ to 3.57 × 10⁻4 as potent as retene and about 1.11 × 10⁻5 to 1.20 × 10⁻6 as potent as TCDD. Juvenile rainbow trout did not show EROD induction after exposures to P1, P2, or the Cl-P2 mixture, whereas P3 caused activity fourfold above that of controls. P1, P3, and Cl-P2 all weakly induced VTG in rainbow trout hepatocytes. The most potent inducer of VTG was Cl-P2, followed by P3 and P1. The results show the ability of wood-derived stilbenes to cause weak MFO induction in fish and in rat liver cells and to weakly induce vitellogenin in fish liver cells.

Pharmacogenetics of warfarin: development of a dosing algorithm for brazilian patients.

A dosing algorithm including genetic (VKORC1 and CYP2C9 genotypes) and nongenetic factors (age, weight, therapeutic indication, and cotreatment with amiodarone or simvastatin) explained 51% of the variance in stable weekly warfarin doses in 390 patients attending an anticoagulant clinic in a Brazilian public hospital. The VKORC1 3673G>A genotype was the most important predictor of warfarin dose, with a partial R(2) value of 23.9%. Replacing the VKORC1 3673G>A genotype with VKORC1 diplotype did not increase the algorithm's predictive power. We suggest that three other single-nucleotide polymorphisms (SNPs) (5808T>G, 6853G>C, and 9041G>A) that are in strong linkage disequilibrium (LD) with 3673G>A would be equally good predictors of the warfarin dose requirement. The algorithm's predictive power was similar across the self-identified "race/color" subsets. "Race/color" was not associated with stable warfarin dose in the multiple regression model, although the required warfarin dose was significantly lower (P = 0.006) in white (29 +/- 13 mg/week, n = 196) than in black patients (35 +/- 15 mg/week, n = 76).

Comparative petroleum hydrocarbons levels and biochemical responses in mussels from hydrothermal vents (Bathymodiolus azoricus) and coastal environments (Mytilus galloprovincialis).

Aliphatic hydrocarbons and PAHs in the whole soft tissues of Bathymodiolus azoricus from three Mid-Atlantic Ridge hydrothermal vents (Menez-Gwen, Lucky Strike and Rainbow), and Mytilus galloprovincialis from three contaminated coastal sites in South Portugal were analysed, and its effects on the digestive gland microsomes mixed-function oxygenase system (MFO) were assessed. Aliphatic hydrocarbons levels were present in the same magnitude in both coastal and hydrothermal environments, while the UCM (unresolved complex mixture) for coastal mussels were higher than in vent mussels. In general, significantly higher PAHs concentrations were found in coastal mussels, compared to B. azoricus where low molecular weight PAHs (2-3 rings) represented the majority of PAHs contrarily to what was observed in M. galloprovincialis. The MFO components were present in both mussel species, and were detected in vent mussels for the first time. However this system seems to have different roles in species from these contrasting environments. In coastal mussels MFO responded to hydrocarbon contamination while response in hydrothermal organisms appeared to be related mainly to endogenous factors.

Pharmacogenetics in critical care: atrial fibrillation as an exemplar.

Pharmacogenetic testing is currently not routine in critical care settings but recent changes in the warfarin label are likely to lead to critical care nurses encountering physician or nurse practitioner orders for such testing. Although the science for pharmacogenetics is complex, the components of patient teaching are not beyond that which nurses already provide about other laboratory, disease, and treatment-based information. It is reasonable to expect that as the science of pharmacogenetics and pharmacogenomics expands and discoveries are translated in clinical settings, the additional information from pharmacogenetic test results will help prescribers select or adjust medication doses to reduce the risk for adverse drug reactions and improve the chances of achieving therapeutic targets in a timely fashion.

Effect of CYP2C19 polymorphism on nelfinavir to M8 biotransformation in HIV patients.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * Nelfinavir is an HIV protease inhibitor, substrate of the transporter P-glycoprotein and metabolized via CYP2C19, CYP3A4 and CYP3A5 enzymes. * Pharmacokinetic studies have shown wide interindividual variability of nelfinavir concentrations, some of this variability perhaps caused by variant drug metabolism or transporter genes. * For CYP3A4*1B and CYP3A5*3 polymorphism, results from three studies are in agreement, showing no difference in nelfinavir concentrations between patients with these different genotypes. * However, for MDR1 and CYP2C19 polymorphism, there have been contradictory studies, showing either no impact on nelfinavir concentration or modified concentrations which could influence virological response. WHAT THIS STUDY ADDS: * Patients with an *1/*2 or *2/*2 genotype for CYP2C19 had a nelfinavir to M8 biotransformation divided by 2 compared with *1/*1 patients. * No evidence of any influence of MDR1 polymorphism on nelfinavir absorption could be detected. AIMS: To evaluate the effect of CYP2C19 polymorphism on nelfinavir and M8 pharmacokinetic variability in human immunodeficiency virus-infected patients and to study the link between pharmacokinetic exposure and short-term efficacy and toxicity. METHODS: Nelfinavir (n = 120) and M8 (n = 119) concentrations were measured in 34 protease inhibitor-naive patients. Two weeks after initiating the treatment, blood samples were taken before, 1, 3 and 6 h after drug administration. Genotyping for CYP3A4, 3A5, 2C19 and MDR1 was performed. A population pharmacokinetic model was developed to describe nelfinavir-M8 concentration time-courses and to estimate interpatient variability. The influence of individual characteristics and genotypes were tested using a likelihood ratio test. Estimated mean (C(mean)), maximal (C(max)) and trough (C(trough)) nelfinavir and M8 concentrations were correlated to short-term virological efficacy and tolerance using Spearman nonparametric correlation tests. RESULTS: A one-compartment model with first-order absorption, elimination and metabolism to M8 best described nelfinavir data. M8 was modelled by an additional compartment. Mean pharmacokinetic estimates and the corresponding intersubject variabilities were: absorption rate 0.17 h(-1) (99%), absorption lag time 0.82 h, apparent nelfinavir total clearance 52 l h(-1) (49%), apparent nelfinavir volume of distribution 191 l, M8 elimination rate constant 1.76 h(-1) and nelfinavir to M8 0.39 h(-1) (59%) in *1/*1 patients and 0.20 h(-1) in *1/*2 or *2/*2 patients for CYP2C19*2. Nelfinavir C(mean) was positively correlated to glycaemia and triglyceride increases (P = 0.02 and P = 0.04, respectively). CONCLUSIONS: The rate of metabolism of nelfinavir to M8 was reduced by 50% in patients with *1/*2 or *2/*2 genotype for CYP2C19 compared with those with *1/*1 genotype.

Artemisinin and CYP2A6 activity in healthy subjects.

OBJECTIVE: To investigate whether the antimalarial drug artemisinin affects CYP2A6 activity in healthy subjects and to compare the utility of coumarin and nicotine as in vivo probe compounds for CYP2A6. METHODS: Twelve healthy male Vietnamese subjects were given coumarin or nicotine in randomized sequence before and after 5 days of a repeated oral administration of artemisinin during two different treatment periods 1 month apart. Sequential blood samples were drawn at baseline 7 days prior to artemisinin treatment and on the first and fifth day of artemisinin treatment during both treatment periods. Plasma concentrations of 7-hydroxycoumarin glucuronide (7-OHCG), nicotine, cotinine and artemisinin were analysed by high-performance liquid chromatography and those of coumarin and 7-hydroxycoumarin (7-OHC) were determined by liquid chromatography-tandem mass spectrometry. Urine, collected in two time intervals on the days of coumarin intake, was treated with beta-glucuronidase and analysed for 7-OHC levels. RESULTS: Artemisinin AUC(0-infinity) values decreased significantly to 23% [95% confidence interval (CI) 18%-28%] on the fifth day of artemisinin administration as compared with the first. The sum of renally excreted 7-OHC and 7-OHCG increased by 1.55-fold (adjusted 95% CI 1.08-2.23) in the 3- to 8-h interval compared to baseline 7 days before. The 7-OHCG/7-OHC plasma AUC(0-infinity) ratio increased by 1.72-fold (adjusted 95% CI 1.16-2.54) following 5 days of artemisinin intake. There was no significant change in the cotinine/nicotine AUC(0-11 hr) ratio between study days. CONCLUSION: Artemisinin significantly increased the sum of renally excreted 7-OHC and 7-OHCG in one of the two collection intervals, suggesting an induction of CYP2A6. A significant increase in the 7-OHCG to 7-OHC AUC(0-infinity) ratio indicates artemisinin to be an inducer of glucuronidation.

Effects of eleven isothiocyanates on P450 2A6- and 2A13-catalyzed coumarin 7-hydroxylation.

Many isothiocyanates (ITCs), both naturally occurring and synthetic, are potent and selective inhibitors of carcinogenesis in animal models and are now viewed as a class of promising chemopreventive agents. We have investigated the ability of 11 ITCs to inhibit and/or inactivate P450 2A6- and 2A13-mediated coumarin 7-hydroxylation. Two of these 11 ITCs, phenylpropyl isothiocyanate (PPITC) and phenylhexyl isothiocyanate (PHITC), were potent inhibitors of P450 2A13. The K I values for the inhibition of P450 2A13-mediated coumarin 7-hydroxylation by PPITC and PHITC were approximately 0.14 and 1.1 microM, respectively. P450 2A6 was also inhibited by these two ITCs; however, the K I values indicated they were approximately 10-20-fold less potent for P450 2A6 than for P450 2A13. Most of the ITCs tested, including PPITC and PHITC, showed some degree of inactivation of both P450s; however, only one compound, tert-butyl isothiocyanate (tBITC), showed significant inactivation of P450 2A13 at a concentration of 10 microM. None of the ITCs caused significant inactivation of P450 2A6 at this concentration. tBITC inactivated P450 2A13 with an apparent K I of 4.3 microM and a k inact of 0.94 min (-1). Inactivation of P450 2A6 by tBITC was observed only at high concentrations and long incubation times. The observed differences in inhibition and/or inactivation of P450 2A6 and 2A13 by a few of the isothiocyanates suggest that these compounds may be useful for structure-function studies.

VKORC1: molecular target of coumarins.

The genetic diagnosis of a single family with combined vitamin K-dependent clotting factor deficiency (VKCFD2, OMIM #607473) finally led to the identification and molecular characterization of vitamin K epoxide reductase (VKORC1). VKORC1 is the key enzyme of the vitamin K cycle and the molecular target of coumarins, which represent the most commonly prescribed drugs for therapy and prevention of thromboembolic conditions. However, coumarins are known to have a narrow therapeutic window and a considerable risk of bleeding complications caused by a broad variation of intra- and inter-individual drug requirement. Now, 3 years after its identification, VKORC1 has greatly improved our understanding of the vitamin K cycle and has led to the translation of basic research into clinical practise in at least three directions: (i) Mutations within VKORC1 have been shown to cause a coumarin-resistant phenotype and a single SNP (rs9923231) within the VKORC1 promoter region has been identified as the major pharmacodynamic determinant of coumarin dose. Together with the previously described CYP2C9 variants and other dose-influencing factors, such as age, gender and weight, individualized dosing algorithms have become available. (ii) Preliminary studies indicate that concomitant application of low-dose vitamin K (80-100 microg day(-1)) and warfarin significantly improves INR stability and time of INR within the therapeutic range. (iii) Co-expression studies of FIX and FX with VKORC1 have shown that VKOR activity is the rate-limiting step in the synthesis of biologically active vitamin K-dependent factors. Thus, co-expression of VKORC1 leads to a more efficient production of recombinant vitamin K-dependent coagulation factors such as FIX and FVII. This could improve production of recombinant factor concentrates in the future.

Protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay.

The aim of this study was to investigate the protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. None of the vitamin C concentrations tested (1-10 microM) in presence or absence of formamidopyrimidine-DNA glycosylase (Fpg enzyme) caused DNA damage per se. HepG2 cells simultaneously treated with vitamin C and N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP) reduced the genotoxic effects of the N-nitrosamines in a dose-dependent manner. At concentrations of 1-5 microM vitamin C, the protective effect was higher towards NPYR-induced oxidative DNA damage (78-79%) than against NDMA (39-55%), NDBA (12-14%) and NPIP (3-55%), in presence of Fpg enzyme. However, a concentration of 10 microM vitamin C led to a maximum reduction in NDBA (94%), NPYR (81%), NPIP (80%) and NDMA (61%)-induced oxidative DNA damage, in presence of Fpg enzyme. The greatest protective effect of vitamin C (10 microM) was higher towards NDBA-induced oxidative DNA damage. One feasible mechanism by which vitamin C exerted its protective effect is that may interact with the enzyme systems catalyzing the metabolic activation of the N-nitrosamines, blocking the production of genotoxic intermediates. Vitamin C (10 microM) strongly reduced the coumarin hydroxylase (82%) activity. However, the p-nitrophenol hydroxylase and the ethoxyresorufine O-deethylation activities were slightly and weakly reduced (32-19%), respectively.

Influence of various phenolic compounds on phenol hydroxylase activity of a Trichosporon cutaneum strain.

The phenol-degrading strain Trichosporon cutaneum R57 utilizes various aromatic and aliphatic compounds as a sole carbon and energy source. The intracellular activities of phenol hydroxylase [EC 1.14.13.7] of a Trichosporon cutaneum R57 strain grown on phenol (0.5 g/l) were measured. Different toxic phenol derivatives (cresols, nitrophenols and hydroxyphenols) were used as substrates in the reaction mixture for determination of the enzyme activity. The data obtained showed that the investigated enzyme was capable to hydroxylate all applied aromatic substrates. The measured activities of phenol hydroxylase varied significantly depending on the aromatic compounds used as substrates. The rate of phenol hydroxylase activity with phenol as a substrate (1.0 U/mg total cell protein) was accepted as 100%.

Influence of rabeprazole and lansoprazole on the pharmacokinetics of tacrolimus in relation to CYP2C19, CYP3A5 and MDR1 polymorphisms in renal transplant recipients.

The objective of this study was to evaluate whether genetic polymorphisms of CYP2C19, CYP3A5 and MDR1 significantly impact the interaction between tacrolimus and rabeprazole or lansoprazole. Seventy-three recipients were randomly assigned after renal transplantation to receive repeated doses of tacrolimus for 28 days with a regimen of either 20 mg of rabeprazole or 30 mg of lansoprazole. Blood concentrations of tacrolimus were measured by microparticle enzyme immunoassay. The mean daily dose and the dose-adjusted area under the plasma concentration-time curves from 0 to 12 h (AUC(0-12)) of tacrolimus coadministered with rabeprazole or lansoprazole were the lowest and highest, respectively, in CYP2C19 poor metabolizers (PMs) having the CYP3A5*3/*3 genotype (0.084 and 0.112 mg/kg/day and 1.269 and 1.033 ng.h/ml/mg/kg, respectively). On the other hand, the mean dose-adjusted AUC(0-12) of tacrolimus coadministered with rabeprazole or lansoprazole were the highest in CYP2C19 PMs having the MDR13435CC+CT genotype, but not significantly. The present study indicates that there are significant interactions between tacrolimus and rabeprazole or lansoprazole in CYP2C19 PM renal transplant recipients bearing the CYP3A5*3/*3 genotypes. For recipients having these genetic polymorphisms, lower dosages of tacrolimus are required to achieve the target therapeutic index.

Differential alteration of drug-metabolizing enzyme activities after cyclophosphamide/adriamycin administration in breast cancer patients.

Cyclophosphamide (CPA) and adriamycin (ADR) are widely used drugs for cancer chemotherapy. It has been reported that CPA and ADR singly or in combination could alter activities of a variety of drug-metabolizing enzymes in animals via multiple mechanisms. However, the effects of CPA/ADR on drug metabolism are largely unknown in human beings. Losartan metabolism has been suggested as a marker for determination of CYP2C9 activity. Caffeine is a commonly used probe to assess the metabolic activities of CYP1A2, CYP2A6, N-acetyltransferase 2 (NAT2) and xanthine oxidase (XO). The present study was designed to analyze the effects of CPA/ADR on these drug-metabolizing enzymes by using losartan and caffeine as probe drugs. A single oral dose of 25 mg losartan and a cup of instant coffee was given to 15 breast cancer patients on three occasions (before, and 2-4 h and 3 weeks after the adjuvant CPA/ADR chemotherapy [600 mg CPA/m2/day, 60 mg ADR/m2/day]). Losartan, caffeine and their metabolites were analyzed by using high-pressure liquid chromatography. When compared with baseline, CYP1A2 activity was increased by 20% and CYP2C9 activity was decreased by 315% 3 weeks after the administration of CPA/ADR chemotherapy (p = 0.05). The chemotherapy did not change the activities of CYP2A6, NAT2 or XO. CPA/ADR treatment caused a differential effect on drug-metabolizing enzyme activities, and this may contribute to predicting the efficacy and toxicity of chemotherapeutics, as well as understanding the drug-drug interactions.