Forms Of Cytochrome P-450 Research Articles

Investigation of EROD, CYP1A immunopositive proteins and SOD in haemocytes of Chamelea gallina and their role in response to B[ a

CYP1A sub-family represents the main form of cytochrome P450 involved in benzo[ a]pyrene (B[ a]P) detoxification, but there are no clear evidences about its presence in invertebrates. 7-Ethoxy resorufin O-deethylase (EROD) activity is strictly related to CYP1A presence, at the same time P450-dependent oxidative metabolism leads to reactive oxygen species (ROS) production, thought to be an important mechanism of pollutant-mediated toxicity in aquatic organisms. Superoxide dismutases (SODs), EROD and CYP1A activities and/or expressions were detected in haemocytes of pooled clams ( Chamelea gallina) and cell-free haemolymph after 24 h, 7 and 12 days of exposure to 0.5 mg/L of B[ a]P. After 24 h, B[ a]P content was maximum in whole tissues. A 61 kDa band was recognized in haemocytes and cell-free haemolymph by polyclonal anti-fish CYP1A, while 53.5 and 63.8 kDa CYP1A immunopositive proteins were discriminate without differences of expression. Differently, EROD, MnSOD activity/expression and ECSOD expression decreased in haemocytes and haemolymph. C. gallina immune system presents an interesting response dose/time exposure of B[ a]P and the 7 days condition highlights the major effects of xenobiotic action. The identification of basal EROD levels supports the possible presence of the CYP1A, never identified in C. gallina and more specifically never isolated in immune cells, as confirmed by CYP1A-immunopositive proteins identification.

In vitro hepatic conversion of the anticancer agent nemorubicin to its active metabolite PNU-159682 in mice, rats and dogs: A comparison with human liver microsomes

We recently demonstrated that nemorubicin (MMDX), an investigational antitumor drug, is converted to an active metabolite, PNU-159682, by human liver cytochrome P450 (CYP) 3A4. The objectives of this study were: (1) to investigate MMDX metabolism by liver microsomes from laboratory animals (mice, rats, and dogs of both sexes) to ascertain whether PNU-159682 is also produced in these species, and to identify the CYP form(s) responsible for its formation; (2) to compare the animal metabolism of MMDX with that by human liver microsomes (HLMs), in order to determine which animal species is closest to human beings; (3) to explore whether differences in PNU-159682 formation are responsible for previously reported species- and sex-related differences in MMDX host toxicity. The animal metabolism of MMDX proved to be qualitatively similar to that observed with HLMs since, in all tested species, MMDX was mainly converted to PNU-159682 by a single CYP3A form. However, there were marked quantitative inter- and intra-species differences in kinetic parameters. The mouse and the male rat exhibited V max and intrinsic metabolic clearance (CL int) values closest to those of human beings, suggesting that these species are the most suitable animal models to investigate MMDX biotransformation. A close inverse correlation was found between MMDX CL int and previously reported values of MMDX LD 50 for animals of the species, sex and strain tested here, indicating that differences in the in vivo toxicity of MMDX are most probably due to sex- and species-related differences in the extent of PNU-159682 formation.

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter — nmt1 (pREP42) and constitutive promoter — adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed by HPLC analysis, using testosterone as substrate.

Clinical utility of drug measurement and pharmacokinetics – therapeutic drug monitoring in psychiatry

Based on the assumption that a relationship between blood levels and clinical effects (therapeutic effects, adverse events and toxicity) can be defined and considering that after equal doses plasma concentrations vary markedly between individual patients, therapeutic drug monitoring (TDM) can assist to personalize dose adjustment. Taken together, drug levels and a knowledge of the pharmacological profile of the administered drugs can enable the optimal dosage to be tailored according to the need of the individual patient. Therapeutic drug monitoring has been established for a limited number of drugs. In psychiatry, it has a 40-year-long history, which started with nortriptyline. Evidence has accumulated which shows that TDM is a valid tool for the optimization of psychopharmacotherapy. When used adequately, TDM is helpful for many patients and in many situations. Combined with pharmacogenetic tests, the metabolic status of a patient can be well characterized. Several new observations have been made during routine TDM that have stimulated clinical pharmacological research, such as investigations on inherited differences in drug metabolism that are closely linked to TDM in psychiatry. The contributions of individual forms of cytochrome P450 (CYP) to the metabolism of drugs was elicited by clinical observations on pharmacokinetic drug interactions. Therapeutic drug monitoring requires a close collaboration between the prescribing physician, the laboratory specialist, the clinical pharmacologist and the patient. This complexity may result in errors which can be detected by analysing the appropriate use of TDM in clinical practice. More education has to be provided to the prescribing clinicians on the pharmacology of the drugs and the algorithm of TDM. Moreover, clinical trials should include measurements of blood concentrations during drug development to generate valid data on the relationships between drug concentrations and clinical outcomes under well-controlled conditions. This would merely increase the amount of work and costs, as high-throughput methods are now available in many laboratories. Any progress in TDM has direct benefits for the treatment of many individual patients.

In vivo study of the effect of antiviral acyclic nucleotide phosphonate (R)-9-[2- (phosphonomethoxy) propyl]adenine (PMPA, tenofovir) and its prodrug tenofovir disoproxil fumarate on rat microsomal cytochrome P450

The total content of rat liver microsomal cytochrome P450 (CYP) significantly decreased after repeated i.p. administration of the antiviral agent tenofovir ((R)-9-[2-(phosphonomethoxy)propyl] adenine) and tenofovir disoproxil at a daily dose 25 mg/kg, although the content of liver microsomal protein did not change. The decrease of the CYP content was accompanied by concomitant increase of the amount of inactive CYP form, cytochrome P420. This effect was confirmed by a parallel study of the activities of selected CYP forms, CYP2E1 (p-nitrophenol hydroxylation) and CYP1A2 (7-ethoxyresorufin deethylation). The activity (expressed relatively to the protein content) of both CYP forms decreased significantly following the decrease of the total CYP. On the other hand, the CYP2E1 activity expressed relatively to the decreasing total CYP content remained unchanged. However, CYP1A2 activity also decreased when calculated relatively to the total native CYP content indicating lower stability of this form. Semiquantitative RT-PCR showed no significant changes in expression of major rat liver microsomal CYP forms after tenofovir treatment. In conclusion, repeated administration of tenofovir in higher doses led to significant decrease of the relative proportion of active liver microsomal CYPs accompanied by a conversion of these enzymes to the inactive form (CYP420) maintaining the sum of CYP proteins unchanged.

Enzyme systems of detoxication — overview of recent approaches

The most important enzymes of detoxication are cytochromes P450 of Phase I and UDP-glucuronosyltransferases of Phase II of drug metabolism. The conventional division of drug, or xenobiotic, metabolism to two phases has survived almost fifty years and although not perfect, still is rather informative and practical. The recent addition of the next, third phase to the first two (Phase I as yielding a molecule with free functional groups ready for conjugation with another molecule or its part in Phase II) stressed the importance of drug transport across the membranes even if it is not a pure metabolic process (i.e. a process changing the polarity and structure of a compound). The importance of testing the toxicity of newly invented drugs and drug candidates is also given by the simple fact that it is the toxicity and unwanted pharmacokinetics incl. interactions which is the reason for declination of one half of new or promising compounds. All other reasons as low efficacy, adverse effects, marketing and commercial reasons, and other reasons are responsible for the second half of the rejections (Van de Waterbeemd & Gifford, 2003). According to the recent view of the regulation authorities, the in vitro testing of drug metabolism of any new chemical entity should be realized together with an evaluation of its interaction with the system of drug transporters, mainly with the ABC membrane based protein pumps which often decide on the drug bioavailability. The majority of known cases of drug toxicity are however ascribed to reactions in which the catalytic processes involving cytochromes P450 are involved. The death toll caused by malignant arrhythmias due to increased level of terfenadine or astemizole is well documented and is a part of each modern textbook of pharmacology and toxicology. Also, the recent case of mibefradil which has been withdrawn from the market by the company two years after successful introduction is also well known. In all cases, increased levels of the drug were caused by interactions with other drugs, competing for the same form of cytochrome P450. Cytochromes P450 are also known for their two-sided behavior often compared with Dr. Jekyll and Mr. Hyde of the R. L. Stevenson′s novel: The same form is responsible for detoxication processess as well as by activation of a carcinogen or a toxicant, in principle, by the same type of chemical reaction, i.e. by activation of molecular oxygen. It is of course not the fault of the cytochromes P450, but our own – as the enzyme is simply performing the same role independently on our wish. The ways and approaches used for in vitro studies of processes in which cytochromes P450 take place can be divided according to the degree of organization of the system studied. From the most simple to the most complex, the first system should comprise only the molecules of the enzyme with the necessary components assuring the proper assembly and function.

Predictive value of animal models for human cytochrome P450 (CYP)-mediated metabolism: A comparative study in vitro

One major challenge in drug development is defining of the optimal animal species to serve as a model of metabolism in man. The study compared the hepatic drug metabolism characteristics of humans and six widely used experimental animal species. Classical in vitro model enzyme assays with known human cytochrome P450 (CYP) enzyme selectivity were employed and optimized to target human hepatic CYP forms. The profile of CYP activities best resembling the human was seen in mouse followed by monkey, minipig, and dog liver microsomes, with rats displaying the most divergent. The widest interindividual variability was found in CYP3A-mediated midazolam α-hydroxylase, and omeprazole sulphoxidase activities in human and monkey liver microsomes. These data demonstrate that if hepatic xenobiotic-metabolizing characteristics were to be the sole reason for the selection of animal species for toxicity studies, then the rat might not be the most appropriate model to mimic human CYP activity patterns.

Effect of Pyrethrins on cytochrome P450 forms in cultured rat and human hepatocytes

High doses of Pyrethrins produce liver and thyroid gland tumours in rats by modes of action involving the induction of hepatic xenobiotic metabolising enzymes. The aim of this study was to compare the effects of Pyrethrins with those of the rat liver and thyroid tumour promoter sodium Phenobarbital on some cytochrome P450 (CYP) forms in cultured rat and human hepatocytes. The treatment of female Sprague–Dawley rat and human (both male and female) hepatocytes for 72 h with 0–1000 μM Pyrethrins and 0–1000 μM Phenobarbital did not result in any marked cytotoxicity. In rat hepatocytes both Pyrethrins and Phenobarbital produced an induction of 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase activity (a CYP1A/2B form marker) and CYP2B1 and CYP2B1/2 mRNA levels. Pyrethrins and Phenobarbital also induced CYP3A-dependent testosterone 6β-hydroxylase activity in rat hepatocytes. In human hepatocytes Pyrethrins and Phenobarbital induced both testosterone 6β-hydroxylase activity and CYP3A4 mRNA levels and also increased CYP2B6 mRNA levels. The effects of Pyrethrins and Phenobarbital were concentration-dependent and exhibited a threshold. These results demonstrate that the effects of Pyrethrins on CYP forms in cultured rat and human hepatocytes are qualitatively similar to those of Phenobarbital. Pyrethrins induce CYP2B and CYP3A forms in cultured rat hepatocytes and can induce CYP3A and CYP2B forms in human hepatocytes. While CYP form induction by Pyrethrins, Phenobarbital and related compounds can be associated with liver and thyroid gland tumour formation in rodents, epidemiological data for Phenobarbital suggests that such effects do not occur in humans.

RT-PCR-based cytochrome P450 expression profile of oral tissue samples1

To analyse the expression of individual forms of cytochrome P450 (CYP) at the mRNA level in five homogenized oral buccal tissue samples from four individuals with or without oral malignancy. Individual forms of CYPs were studied by reverse transcriptase-polymerase chain reaction (RT-PCR), using specific primers for CYPs 2B6, 2C, 2D6, 2E1, 3A3/4 and 3A5, and oral CYP expressions were compared with CYP expression in liver tissue. Consistent expression of CYPs 2C, 2E1 and 3A5 was observed in oral buccal tissue at mRNA level. These particular CYPs have possible roles in the protection of the body against orally ingested xenobiotics as well as influence the bioavailability of therapeutic compounds.

Inhibition of human recombinant cytochromes P450 CYP1A1 and CYP1B1 by trans‐resveratrol methyl ethers

CYP1A1 and CYP1B1 are the inducible forms of cytochrome P450 expressed in extrahepatic tissues, which are responsible for the biotransformation of polycyclic aromatic hydrocarbons, heterocyclic amines and estradiol to the carcinogenic intermediates. The aim of our research was to determine and compare the inhibitory effect of naturally occurring analogues of trans-resveratrol on the catalytic activities of human recombinant CYP1A1 and CYP1B1. Pinostilbene (3,4'-dihydroxy-5-methoxystilbene), desoxyrhapontigenin (3,5-dihydroxy-4'-methoxystilbene), and pterostilbene (3,5-dimethoxy-4'-hydroxystilbene) appeared to be very potent inhibitors of CYP1A1 catalytic activity with Ki values of 0.13, 0.16 and 0.57 microM, respectively. Results from this study indicate that trans-resveratrol analogues in which the hydroxy groups are substituted by methoxy groups exhibit a remarkably stronger inhibitory effect towards CYP1A1 in comparison to the parent compound. On the contrary, the potency of pinostilbene, desoxyrhapontigenin and pterostilbene towards CYP1B1 with Ki values of 0.90, 2.06 and 0.91 microM, respectively, was comparable to that of resveratrol. It appears that between these analogues, inhibition of CYP1A1 and CYP1B1 catalytic activities does not vary much regardless of the number and position of methylether substitution. The results suggest that the trans-resveratrol analogues: pinostilbene, desoxyrhapontigenin and pterostilbene, which occur in some food plants, might be considered as promising chemopreventive agents.

The Time to Move Cytochrome P450 Induction into Mainstream Pharmacology Is Long Overdue

The understanding of the processes of induction of human drug-metabolizing enzymes has advanced considerably over the past decade. If we concentrate on CYP3A4, the most abundant form of cytochrome P450 and the one most involved in the clearance of the majority of pharmaceuticals, a clear

Gadolinium chloride suppresses styrene-induced cytochrome P450s expression in rat liver

To assess the effect of gadolinium (Gd) on the expression of several forms of cytochrome P450 (P450s) and antioxidant enzymes, we treated rats with gadolinium chloride (25 mg as Gd/kg body weight) 4 h after styrene (a multiple P450 inducer) treatment (600 mg/kg). Gd treatment significantly suppressed styrene-inducible cytochrome P4502B1 (CYP2B1), CYP2B2, CYP2E1, and CYP3A2 mRNA expressions to 48.6%, 69.8%, 61.1%, and 38.5%, accompanying with the reduction of proteins expression to 1.42%, 31.2%, 21.1% and 21.1%, respectively, compared with styrene alone treatment. Gd suppressed styrene-inducible CYP1A2 expression, but only at the protein level. On the other hand, styrene treatment caused a decrease in reduced form of glutathione (GSH), as well as increases in lipid peroxide and serum ALT and AST activities, suggesting the occurrence of hepatic damage probably due to styrene-induced oxidative stress in rat liver. Post-treatment of Gd attenuated this styrene-caused hepatic damage. Moreover, mRNA expressions of cellular antioxidant enzymes such as catalase, CuZn-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GPX) were hardly changed by styrene and/or Gd treatment. In summary, Gd suppressed styrene-inducible expression of not only CYP2B1 but also several forms of P450 at both the mRNA and protein levels, along with attenuation of styrene-caused liver damage. These findings suggested that Gd is a chemo-preventive agent against hepatic damage caused by xenobiotics requiring biotransformation.

Prioritization of Clinical Drug Interaction Studies Using In Vitro Cytochrome P450 Data:“Rank Order” Approach

For any new chemical entity (NCE), in vitro inhibition constants (K(i)) for the different human cytochrome P450 (CYP) forms can be ranked (lowest to highest). The CYP form with the lowest in vitro K(i) is evaluated first clinically, employing a suitable probe drug like midazolam (CYP3A4), theophylline (CYP1A2), (S)-warfarin (CYP2C9) and desipramine (CYP2D6), and the NCE is classified as a "none", "weak", "moderate", or "strong" inhibitor. In turn, the classification governs the next steps. A two stage strategy, in vitro ranking followed by classification, has the potential to enable decision making within an industrial and regulatory setting. With additional refinement and validation, the approach could be applied to mechanism-based inhibitors, inducers and substrates of CYPs also.

Rofecoxib Is a Potent, Metabolism-Dependent Inhibitor of CYP1A2: Implications for in Vitro Prediction of Drug Interactions

Rofecoxib was recently found to greatly increase plasma concentrations of the CYP1A2 substrate drug tizanidine in humans, but there are no published in vitro studies on the CYP1A2-inhibiting effects of rofecoxib. Our objective was to investigate whether rofecoxib is a direct-acting or metabolism-dependent inhibitor of CYP1A2 in vitro. The effect of rofecoxib on the O-deethylation of phenacetin (20 microM) was studied using human liver microsomes. The effect of preincubation time on the inhibitory potential of rofecoxib was also studied, and the inhibitor concentration that supports half the maximal rate of inactivation (KI) and the maximal rate of inactivation (kinact) were determined. Rofecoxib moderately inhibited phenacetin O-deethylation (IC50 23.0 microM), and a 30-min preincubation with microsomes and NADPH considerably increased its inhibitory effect (IC50 4.2 microM). Inactivation of CYP1A2 by rofecoxib required NADPH, and was characterized by a KI of 4.8 microM and a kinact of 0.07 min(-1). Glutathione, superoxide dismutase, mannitol, or dialysis could not reverse the inactivation of CYP1A2 caused by rofecoxib. Fluvoxamine decreased the rofecoxib-caused inactivation of CYP1A2 in a concentration-dependent manner. In conclusion, rofecoxib is a potent, metabolism-dependent inhibitor of CYP1A2, a cytochrome P450 form contributing to rofecoxib metabolism. The results provide a mechanistic explanation for the interactions of rofecoxib with CYP1A2 substrates and may partially explain its nonlinear pharmacokinetics.

Interactive effects of methyl tertiary-butyl ether (MTBE) and tertiary-amyl methyl ether (TAME), ethanol and some drugs: Triglyceridemia, liver toxicity and induction of CYP (2E1, 2B1) and phase II enzymes in female Wistar rats

The abilities of the gasoline additives methyl tert-butyl ether (MTBE) and tert-amyl methyl ether (TAME) to cause liver damage following oral administration, dosed alone or in combination with model hepatotoxins, were investigated in the rat. Inducibility of liver drug-metabolizing enzyme activities was also studied. Exposure to these ethers (10–20 mmol/kg) for 3 days resulted in hepatomegaly (13–30%) and induction of cytochrome P450 (CYP) activity towards N-nitrosodimethylamine (NDMAD), 7-pentoxyresorufin (PROD), and 7-ethoxyresorufin (EROD). Immunoinhibition assays with monoclonal antibodies showed that the ethers were equipotent as inducers of CYP2E1 activity (2-fold increase) but not of CYP2B1, which was elevated up to 260-fold in TAME-treated rats but only by 20-fold in MTBE rats. A slight or no modifying effect was observed on the NADPH:quinone oxidoreductase (NQO1), glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UGT) activities. Alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) were elevated in blood plasma after administration of the ethers. No dramatic enhancement of liver damage could be detected by plasma enzyme analysis (ALT, AST, alkaline phosphatase, γ-glutamyltransferase) following ether administration (13.5 mmol/kg) to rats pretreated with mildly hepatotoxic dosages of ethanol, pyrazole, phenobarbital, acetaminophen (paracetamol), or 13- cis-retinoic acid (13- cis-RA or isotretinoin). Plasma triglycerides increased in TAME-treated rats (1.7-fold) and in all 13- cis-RA-treated groups (2.1–2.8-fold). The findings that MTBE and TAME exhibited a clear but differential inducing effect on two ether-metabolizing CYP forms (2E1 and 2B1) with no marked effect on phase II activities may reflect the importance of these pathways in vivo. The observation that only TAME by itself induced hypertriglyceridemia while acetaminophen- and 13- cis-RA-induced hypertriglyceridemia were aggravated by both ethers, points to differences in their effects on lipid metabolism. TAME was clearly a more potent CNS depressant than MTBE. There was no marked potentiation of drug/chemical-induced acute liver damage either by MTBE or TAME.

Metabolism of a liver-selective nitric oxide-releasing agent, V-PYRRO/NO, by human microsomal cytochromes P450

Endogenously generated nitric oxide (NO) mediates a host of important physiological functions, playing roles in the vascular, immunological, and neurological systems. As a result, exogenous agents that release NO have become important therapeutic interventions and research tools. O(2)-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a prodrug designed with the hypothesis that it might release nitric oxide via epoxidation of the vinyl group by cytochrome P450, followed by enzymatic and/or spontaneous epoxide hydration to release the ultimate NO-donating moiety, 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) ion. In this study, we investigated this hypothetical activation mechanism quantitatively for V-PYRRO/NO using cDNA-expressed human cytochrome P450 (CYP)2E1. Incubation with CYP2E1 and an NADPH-regenerating system resulted in a time-dependent decomposition of V-PYRRO/NO, with a turnover rate of 2.0 nmol/min/pmol CYP2E1. Nitrate and nitrite were detected in high yield as metabolites of NO. The predicted organic metabolites pyrrolidine and glycolaldehyde were also detected in near-quantitative yields. The enzymatic decomposition of V-PYRRO/NO was also catalyzed, albeit at lower rates, by CYP2A6 and CYP2B6. We conclude that the initial step in the metabolism of V-PYRRO/NO to NO in the liver is catalyzed efficiently but not exclusively by the alcohol-inducible form of cytochrome P450 (CYP2E1). The results confirm the proposed activation mechanism involving enzymatic oxidation of the vinyl group in V-PYRRO/NO followed by epoxide hydration and hydrolytic decomposition of the resulting PYRRO/NO ion to generate nitric oxide.

Characterization of human liver cytochromes P450 by combining the biochemical and proteomic approaches

Highly purified human liver microsomes were processed by a combination of the biochemical and proteomic methods. Microsomes were purified from the morphologically normal liver tissue obtained from the resected and discarded masses of surrounding liver upon surgical treatment for hemangioma (control) or hepatic metastases arising from colon cancer (pathology). Proteins of each sample were separated by two-dimensional (2-DE) and one-dimensional electrophoresis (1-DE); selected gel regions were excised, in-gel digested and analyzed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Analysis of collected fingerprints has revealed a total of 13 microsomal membrane proteins involved in the biotransformation of xenobiotics. These were disulfide isomerase, flavine monooxygenase, NADPH-cytochrome P450 reductase and 10 cytochrome P450 forms, namely: CYPs 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. These same samples were characterized by the enzymatic assays using the marker substrates for CYPs 1A, 2B, 3A4, 2C and 2E1. Correlations between mass spectrometric data and enzymatic activities were investigated to demonstrate the manner in which the functional and structural aspects of proteomics meet each other in the field of cytochromes P450.

Phenomenon of Activation of Cytochrome P450 by Nonionic Detergents

Mechanism of substitution of nonionic detergent Emulgen 913 for phospholipid as an activator of N-demethylase activity of cytochrome P450 form 2B4 (LM(2)) has been studied. It is shown that such an activation takes place at the detergent concentrations below values critical for micelle formation. Under these conditions, Emulgen does not affect the hexameric state of the cytochrome. The stimulating effect proved to be similar in reconstituted monooxygenase systems containing (a) cytochrome P450 2B4 and NADPH-cytochrome P-450 reductase and (b) cytochrome 2B4 and organic hydroperoxides. These results indicate that the activation is due to an effect of the detergent upon P450 2B4 per se rather than upon P450/flavoprotein complex formation. The above conclusion is supported by the sedimentation data and measurement of the CD spectra of cytochrome P450 2B4 at 380-450 nm.

Enhanced Role of Grapefruit Juice on the Antischistosomal Activity of Artemether on the Liver of Schistosoma haematobium Infected Hamsters

Artemether (ART) is an efficacious anti-malarial drug that also displays antischistosomal properties. Laboratory studies have found that ART curtails the development of schistosoma worms and thus prevents morbidity. Grapefruit juice was found to interact with various drugs that have been metabolized by a form of Cytochrome P450, CYP3A4, thus increasing the plasma drug concentration. This work aimed to study the effect of grapefruit juice when administered before infection with Schistosoma haematobium and/or treatment with ART on its anti-schistosomal activity. Golden hamsters were infected each with 300 S. haematobium cercariae and divided into 6 groups (A – F), as follows: Infected control (A); infected received grapefruit juice before infection (B); or received grapefruit juice before infection and treated with ART (200 mg/k) at 5,6 and 7 weeks post infection (WPI) (C); or for 3 successive doses at 12 WPI (D); infected treated with ART alone (200 mg/k) at 5,6 and 7 WPI (E); infected received grapefruit juice (0.5 ml) half an hour before treatment with ART (200 mg/k) at 5,6 and 7 WPI (co-administration) (F). All groups were sacrificed 14 WPI. Some parasitological, biochemical and histopathological estimations were done. Results revealed that, the highest percent of worm reduction was observed in-group F (94.2%) compared to group C,E & D (87.3%, 77.6% & 65.9% respectively). The level of ALT, GGT, urea, thiol, albumin and alkaline phosphatase tend to normalize in accordance with the parasitological results. Neither hepatic granuloma nor prominent histopathological changes could be detected in group F. A minimal number of granulomas (1-3/animal) was observed in group E; meanwhile, the least diameter and collagen content of hepatic S. haematobium granulomas were observed in group C (125.6±5.5 & 2.4±0.572 respectively). The results of the present study demonstrated that, co-administration of grapefruit juice just before treatment with ART at 5, 6& 7 WPI enhanced its anti-schistosomal activity and improved the histopathological changes.

Cytochrome P450 expression—induction profile and chemically mediated alterations of the WIF‐B9 cell line

WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma cells (Fao) and human fibroblasts (WI38). It exhibits the structural and functional characteristics of differentiated hepatocytes, including active bile canaliculi. The aim of the present study was to characterize the WIF-B9 cell line as a model for analysing drug-induced hepatic effects. The drug metabolism potential of WIF-B9 cells was identified by studying the rat and human CYP (cytochrome P450) mRNA constitutive expression profile and induction potential after exposure to reference inducers. The morphological alterations provoked by chemical entities were also characterized. Competitive reverse transcriptase-PCR revealed that four rat (1A1, 2B1/2, 2E1 and 4A1) and four human (1A1, 2Cs, 2D6 and 2E1) CYP mRNA isoforms were constitutively expressed in WIF-B9 cells. The rat CYP forms were expressed at levels 2-4 orders of magnitude higher than the human forms. Exposure for 20-72 h to increasing concentrations of CYP reference inducers (beta-naphthoflavone, 3-methyl cholanthrene, dexamethasone, phenobarbital, clofibrate and pregnenolone 16alpha-carbonitrile) revealed that the rat CYP 1A1, 1A2, 3A1, 3A2 and 4A1 and human CYP 1A1 and 2Cs mRNAs were inducible. Rat CYP 1A1 and 1A2 were the most inducible isoforms since they were overexpressed up to 100-fold after 20-48 h of treatment with beta-naphthoflavone. Human CYP 1A1 and 2Cs mRNAs were induced 3-fold after 48 h of treatment with phenobarbital. Other mechanisms involved in hepatotoxicity were explored using microscopy and immunofluorescence. The WIF-B9 cell line exhibited fragmentation and dilatation of bile canaliculi upon exposure to erythromycin, and to isoniazid and cytochalasins, respectively. Monensin promoted cell depolarization and cytoplasmic granulation. Ethionine promoted cytoplasmic vacuolation and dilatation of the Golgi structures. These results indicate that the CYP expression and induction profiles and the morphological features of WIF-B9 cells allow prediction in vitro of the induction and hepatotoxicity profiles of chemical entities.