Rat P450 Research Articles

Oxidation of Methyl and Ethyl Nitrosamines by Cytochrome P450 2E1 and 2B1

Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine [N,N-dimethylnitrosamine (DMN)], a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥8. The KIE was not attenuated in noncompetitive intermolecular experiments with rat liver microsomes {(D)V = 12.5; (D)(V/K) = 10.9 [nomenclature of Northrop, D. B. (1982) Methods Enzymol. 87, 607-625]} but was with purified human P450 2E1 [(D)V = 3.3; (D)(V/K) = 3.7], indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine [N,N-diethylnitrosamine (DEN)], the intrinsic KIE was slightly lower and was not expressed [e.g., (D)(V/K) = 1.2] in noncompetitive intermolecular experiments. The same general pattern of KIEs was also seen in the (D)(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH(3)NH(2), CH(3)CH(2)NH(2), and NO(2)(-)). Experiments with deuterated N-nitroso-N-methyl-N-ethylamine demonstrated that the lower KIEs associated with ethyl versus methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 [Chowdhury, G., et al. (2010) J. Biol. Chem. 285, 8031-8044]. These same features (no lag phase for HCO(2)H formation and a lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has a lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkyl nitrosamine oxidation appears to be shared by a number of P450s.

Carbon Monoxide–Bound Red Blood Cells Protect Red Blood Cell Transfusion-Induced Hepatic Cytochrome P450 Impairment in Hemorrhagic-Shock Rats

Red blood cell (RBC) transfusions for massive hemorrhage induce systemic ischemic-reperfusion and influence the disposition and pharmacological activity of drugs as a result of a reduction in the level of expression and activity of cytochrome P450s (P450). It was reported that, when organ-preserving solutions are exposed to carbon monoxide (CO), the treatment was effective in suppressing the postreperfusion reduction in renal P450 levels in cases of kidney transplantation. Therefore, we hypothesized that transfusions with RBC that contain bound CO (CO-RBC) would protect the hepatic level of rat P450 during a massive hemorrhage, compared with plasma expanders and RBC resuscitation. To achieve this, we created 40% hemorrhagic-shock model rats, followed by resuscitation, with use of recombinant human serum albumin, RBCs, and CO-RBCs. At 1 hour after resuscitation, the expressions of hepatic P450 isoforms (1A2, 2C11, 2E1, and 3A2) were significantly decreased in the RBC resuscitation group, compared with the sham group. Such alterations in hepatic P450 significantly resulted in an increase in the plasma concentrations of substrate drugs (caffeine [1A2], tolbutamide [2C11], chlorzoxazone [2E1], and midazolam [3A2]) for each P450 isoform, and thus, the hypnotic action of midazolam could be significantly prolonged. Of interest, the reductions in hepatic P450 activity observed in the RBC group were significantly suppressed by CO-RBC resuscitation, and consequently, the pharmacokinetics of substrate drugs and the pharmacological action of midazolam remained at levels similar to those under sham conditions. These results indicate that CO-RBC resuscitation has considerable potential in terms of achieving safe and useful drug therapy during massive hemorrhages.

Simultaneous alterations of brain and plasma serotonin concentrations and liver cytochrome P450 in rats fed on a tryptophan-free diet

Our previous study suggested involvement of the brain serotonergic system in the regulation of liver cytochrome P450 (CYP). The aim of the present study was to demonstrate simultaneous responsiveness of liver CYP and the peripheral and brain serotonergic systems to a tryptophan deficient diet during three days and one or three weeks of ingestion. The concentrations of serotonin, noradrenaline, dopamine and their metabolites were measured in blood plasma, the hypothalamus and brain stem of male rats. The enzyme activity and protein levels in the liver were determined for isoforms CYP1A, CYP2A, CYP2B, CYP2C6, CYP2C11, CYP2D and CYP3A. A three-day tryptophan-free diet increased serotonin content in the hypothalamus (but not in the brain stem or plasma). After one week, the level of serotonin was not changed in the brain, but was markedly increased in the plasma. A three week tryptophan restriction significantly reduced the concentration of serotonin in the brain and plasma. Changes in CYP2C6 and CYP2C11 (an increase and a decrease, respectively) were maintained throughout the experiment, while those found in other CYP isoforms varied, which usually resulted in a gradual increase in the enzyme activity within three weeks. The observed alterations in liver CYPs suggest involvement of both central and peripheral serotonin in the regulation of liver CYP expression whose mechanism is discussed. In conclusion, a deficit in tryptophan in the diet may be responsible for very serious food-cytochrome P450 and food–drug metabolism interactions. Interactions of this type may also refer to drugs acting via serotonergic system.

Functional analysis of cytochrome P450 2B isoforms using ticlopidine as an active site probe

A recent study of in vitro metabolism of ticlopidine by reconstituted human P450 2B6 and rabbit P450 2B4 indicated that both enzymes generated multiple and similar metabolites but in different ratios. In this study, we determined the metabolism of ticlopidine by additional cytochrome P450 2B enzymes, including rat P450 2B1 and dog P450 2B11. Based on the X‐ray crystal structure of the P450 2B4‐ticlopidine complex, two active site mutants, F206A and F297A were also created. All enzymes were expressed in E. coli as N‐terminal modified, and C‐terminal His‐tagged constructs termed 2B4dH. The separation and characterization of the different metabolites of ticlopidine were achieved using HPLC‐MS/MS. P450 2B1 and 2B11 produced ticlopidine N‐oxide as the major product, and the production of this metabolite by the P450 2B subfamily exhibited a rank order of 2B1 > 2B11 > 2B4 > 2B6. The pattern of the products observed for P450 2B1 and 2B11 was similar to that of P450 2B4. In contrast, the 2B4dH F206A and F297A mutants formed predominantly ticlopidine S‐oxide dimer previously seen with P450 2B6. The highest rate of the dimer formation was observed in F206A compared with other 2B isoforms including 2B4dH F297A. These results suggest that ticlopidine is a valuable probe for differentiating P450 2B subfamily members and for assessing the effect of site‐directed mutations.(Supported by NIH grant ES003619)

2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) Is Enantioselectively Oxidized to Hydroxylated Metabolites by Rat Liver Microsomes

Developmental exposure to multiple ortho-substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB)-, dexamethasone (DEX)- and corn oil (VEH)-treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzyme. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer did not affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of PCBs and their metabolites in wildlife and humans are due to metabolism by P450 enzymes but also suggest that the enantioselective formation of neurotoxic PCB 136 metabolites, such as 4-OH-PCB 136, may play a role in the developmental neurotoxicity of PCBs.

Different Metabolites of Human Hepatotoxic Pyrazolopyrimidine Derivative 5‐n‐Butyl‐Pyrazolo[1,5‐a]Pyrimidine Produced by Human, Rat and Monkey Cytochrome P450 1A2 and Liver Microsomes

Different Metabolites of Human Hepatotoxic Pyrazolopyrimidine Derivative 5‐<i>n</i>‐Butyl‐Pyrazolo[1,5‐<i>a</i>]Pyrimidine Produced by Human, Rat and Monkey Cytochrome P450 1A2 and Liver Microsomes

Proteome studies on liver tissue in a phenobarbital-induced rat model

Many drugs may affect the activity of cytochrome P450 (CYP), which is a major source of adverse drug interactions (ADR). Phenobarbital (PB) is the typical inducer of cytochrome P450. The aim of our study was to determine the changes in the cytosolic proteins expression in rat liver at a protein level following induction of cytochrome P450. Firstly, we made a phenobarbital-induced cytochrome P450 rat model. The total cytosolic proteins were then extracted from rat liver tissue and separated by 2-D gel electrophoresis (2-DE). Differentially expressed spots were identified by MALDI-TOF/TOF tandem mass spectrometry followed by database searching. Eight differentially expressed proteins were identified and these proteins were found to be involved in protein degradation, oxidative stress, energy metabolism, biotransformation, and the synthesis of quinolinic acid (QUIN). These findings should provide useful information for research into the regulation of cytochrome P450 gene expression, drug metabolism and drug interaction.

Kinetics of Trihalogenated Acetic Acid Metabolism and Isoform Specificity in Liver Microsomes

This study determined the metabolism of 3 drinking water disinfection by-products (halogenated acetic acids [HAAs]), bromodichloroacetic acid (BDCAA), chlorodibromoacetic acid (CDBAA), and tribromoacetic acid (TBAA), using rat, mouse, human liver microsomes, and recombinant P450. Metabolism proceeded by reductive debromination forming a di-HAA; the highest under nitrogen >>2% oxygen > atmospheric headspaces. V (max) for the loss of tri-HAA was 4 to 5 times higher under nitrogen than atmospheric headspace. Intrinsic metabolic clearance was TBAA>CDBAA>>BDCAA. At the high substrate concentrations, tri-HAA consumption rate was 2 to 3 times higher than the formation of di-HAA. Liberation of Br(-) from TBAA corresponded to the expected amount produced after DBAA formation, indicating retention of Br(-) by additional metabolite/metabolites. Subsequent experiments with CDBAA detected negligible formation of chlorodibromomethane (CDBM) and failed to account for the missing tri-HAA. Carbon monoxide and especially diphenyleneiodonium ([DPI] P450 reductase inhibitor) blocked CDBAA metabolism. Other chemical inhibitors were only partially able to block CDBAA metabolism. Most effective were inhibitors of CYP 2E1 and CYP 3A4. Immunoinhibition studies using human liver microsomes and anti-human CYP 2E1 antibodies were successful in reducing CDBAA metabolism. However, CDBAA metabolism in wild-type (WT) and CYP 2E1 knockout (KO) mouse liver microsomes was similar, suggesting significant interspecies differences in CYP isoform in tri-HAA metabolism. Additional assessment of CYP isoform involvement was complicated by the finding that recombinantly expressed rat and human P450 reductase was able to metabolize CDBAA, which may be a contributing factor in interspecies differences in tri-HAA metabolism.

Effects of Chronic Renal Failure on Kidney Drug Transporters and Cytochrome P450 in Rats

Chronic renal failure (CRF) leads to decreased drug renal clearance due to a reduction in the glomerular filtration rate. However, little is known about how renal failure affects renal metabolism and elimination of drugs. Because both depend on the activity of uptake and efflux by renal transporters as well as enzymes in tubular cells, the purpose of this study was to investigate the effects of CRF on the expression and activity of select renal drug transporters and cytochrome P450. Two groups of rats were studied: control and CRF (induced by 5/6 nephrectomy). Compared with control rats, we observed reductions in the expression of both protein and mRNA of Cyp1a, sodium-dependent phosphate transport protein 1, organic anion transporter (Oat)1, 2, and 3, OatK1/K2, organic anion-transporting polypeptide (Oatp)1 and 4c1, P-glycoprotein, and urate transporter 1, whereas an induction in the protein and mRNA expression of Mrp2, 3, and 4 and Oatp2 and 3 was observed. Cyp3a expression remained unchanged. Similar results were obtained by incubating a human proximal tubule cell line (human kidney-2) with sera from CRF rats, suggesting the presence of uremic modulators. Finally, the renal elimination of [(3)H]digoxin and [(14)C]benzylpenicillin was decreased in CRF rats, compared with controls, as shown by a 4- and 9-fold accumulation, respectively, of these drugs in kidneys of rats in CRF. Our results demonstrate that CRF affects the expression and activity of several kidney drug transporters leading to the intrarenal accumulation of drugs and reduced renal clearance that could, at least partially, explain the tubular toxicity of many drugs.

Sex-related differences in rat liver microsomal enzymes and their induction by doxapram.

The effects of doxapram on the hepatic microsomal mono-oxygenase system of male and female rats were investigated. Male and female rats were administered doxapram (10-120 mg kg-1 day-1, i.p.) for 4 days. In female rats, administration of doxapram (20, 40, 60, 80, 100 and 120 mg kg-1) elevated the parameters in a dose-dependent manner while doxapram (100 and 120 mg kg-1) elevated the levels of cytochrome P450 and hexobarbitone hydroxylase in male rats. Doxapram (40 mg kg-1) caused induction of hepatic drug metabolism typified by an increase of hepatic microsomal cytochrome P450 content and activities of hexobarbitone hydroxylase, benzphetamine N-demethylase and ethylmorphine N-demethylase in female rats, but no change in male rats. These findings were supported by the results of SDS/polyacrylamide-gel electrophoresis. However, 7-ethoxycoumarin O-de-ethylase and arylhydrocarbon hydroxylase activities were significantly increased in male rats. NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities, and cytochrome b5 content were unaffected in rats of both sexes. The sex-dependent cytochrome P450 species may be selectively sensitive to the action of doxapram.

Induction of P450 3A1/2 and 2C6 by gemfibrozil in Sprague-Dawley rats

Fibrates are a group of peroxisome proliferator-activated receptor α agonists used in the treatment of dyslipidemia; however, they have been reported to cause species-related hepatocarcinogenesis and clinical myotoxicity. Gemfibrozil is one of the most commonly used fibrates, and it shows the highest risk for myotoxicity among the fibrates. The inhibitory drug-drug interaction mechanism associated with gemfibrozil has been explored recently, and the induction of human P450 3A4 and 2C8 has been reported. In this study, in vivo induction of rat P450 by gemfibrozil was studied in Sprague-Dawley rats. After the rats were dosed with gemfibrozil by oral gavage, microsomes were prepared. The metabolic activities of P450 3A1/2,2C6, and 2D2 were assayed using probe substrates, and the systemic concentration of gemfibrozil during its administration was determined. P450 3A1/2 and 2C6 activities were induced 32-77% in the rats by gemfibrozil when the exposure concentration was in the clinical range. These data indicate that the inducibility of homologous P450 isoforms by gemfibrozil is similar in Sprague-Dawley rats and in humans. Inductive drug-drug interactions and inhibitory actions are involved in the co-administration of gemfibrozil with other drugs, which suggests the relevance for a fibrate-toxicology investigation.

Effect of Regular Organic Solvents on Cytochrome P450-Mediated Metabolic Activities in Rat Liver Microsomes: Fig. 1.

The effects of regular organic solvents on the metabolic activities of various human cytochromes P450 (P450s) have been reported. However, very little is known about their influence on metabolic activities mediated by P450s in the rat liver microsomes (RLM). The purpose of this study was to investigate the effects of organic solvents such as methanol, acetonitrile, dimethyl sulfoxide (DMSO), acetone, and ethanol on CYP1A, CYP2C, CYP2D, CYP2E, and CYP3A-mediated metabolism using RLM. The results showed that the activities of most rat P450 enzymes appeared to be organic solvent-dependent, and the metabolism of the tested probes were remarkably reduced when the concentration of organic solvents was up to 5% v/v, whereas most organic solvents demonstrated no significant interference when the concentration was below 1%, with the exception of DMSO. In addition, organic solvents exhibited different inhibitory effects, for example, CYP2D and CYP2E showed a significant reduction of activities at lower concentrations of organic solvents. Hence, this phenomenon should be taken into consideration when designing in vitro metabolism studies of new chemical entities. Therefore, we recommend acetonitrile as the most suitable solvent for RLM incubations, and the content of organic solvent should be kept lower than 1% v/v.

The Effects of Azole-Based Heme Oxygenase Inhibitors on Rat Cytochromes P450 2E1 and 3A1/2 and Human Cytochromes P450 3A4 and 2D6

Heme oxygenases (HOs) catalyze the degradation of heme to biliverdin, carbon monoxide (CO), and free iron. The two major isoforms, HO-1 (inducible) and HO-2 (constitutive), are involved in a variety of physiological functions, including inflammation, apoptosis, neuromodulation, and vascular regulation. Major tools used in exploring these actions have been metalloporphyrin analogs of heme that inhibit the HOs. However, these tools are limited by their lack of selectivity; they affect other heme-dependent enzymes, such as cytochromes P450 (P450s), soluble guanylyl cyclase (sGC), and nitric-oxide synthase (NOS). Our laboratory has successfully synthesized a number of nonporphyrin azole-based HO inhibitors (QC-xx) that had little or no effect on sGC and NOS activity. However, their effects on various P450 isoforms have yet to be fully elucidated. To determine the effects of the QC-xx inhibitors on P450 enzyme activity, microsomal preparations of two rat P450 isoforms (2E1 and 3A1/3A2) and two human P450 supersome isoforms (3A4 and 2D6) were incubated with varying concentrations of HO inhibitor, and the activity was determined by spectrophotometric or fluorometric analysis. Results indicated that some QC compounds demonstrated little to no inhibition of the P450s, whereas others did inhibit these P450 isoforms. Four structural regions of QC-xx were analyzed, leading to the identification of structures that confer a decreased effect on both rat and human P450 isoforms studied while maintaining an inhibitory effect on the HOs.

Age-related changes in hepatic expression and activity of cytochrome P450 in male rats

Age-related changes in hepatic expression and activity of cytochrome P450 (CYP) were investigated in male rats aged 3 (weanling), 12 (young), 26 (adult), and 104 (old) weeks. Levels of microsomal protein, total CYP, and cytochrome b(5) increased fully after puberty. CYP1A1 was detected only in 3-week-old rats, and CYP1A2, CYP2B1, and CYP2E1 were maximally expressed at 3 weeks but decreased at 12 and 26 weeks. CYP2C11 and CYP3A2 increased markedly after puberty and decreased with aging. Ethoxyresorufin-O-deethylase, methoxyresorufin-O-demethylase, pentoxyresorufin-O-depenthylase, and p-nitrophenol hydroxylase activities were at their highest in 3-week-old rats, and midazolam hydroxylase activity was at a maximum in 12-week-old rats but decreased with aging. The present results show that increasing age caused significant alterations in hepatic expression/activity of CYP isoforms in an isoform-specific manner. These results suggest that age-related changes in hepatic CYP isoforms may be an important factor for deciding the efficacy and safety of xenobiotics.

Changes in the Gene Expression and Enzyme Activity of Hepatic Cytochrome P450 in Juvenile Sprague-Dawley Rats

The developmental changes in the hepatic cytochrome P450 (CYP) content, mRNA expression of 12 hepatic CYP subtypes, and the enzyme activities of 5 hepatic CYP subfamilies in rats were investigated using non-treated male and female Sprague-Dawley rats of ages postnatal day (PD) 4, 16, 30 and 8 and 12 weeks. The hepatic proliferation kinetics was also determined by using the phospho-histone H3 (p-histon)-labeled hepatocyte index. The developmental changes in the enzyme activities of hepatic expression of CYP1A and CYP3A in rats were similar to those in humans, although there is no fetal-neonatal dominant CYP3A subtype in rat livers unlike human CYP3A7. On the other hand, the developmental pattern of expression of the CYP2C subfamily differed between humans and rats. Enzyme activity and mRNA expression of each hepatic CYP subtype in rats on PD 30 was similar to that after 8 weeks of age, except in the case of sex-dependent CYP subtypes. The p-histon-labeled hepatocyte index was approximately 10-fold higher in PD 30 rats than in 8-week-old rats. Therefore, the livers of juvenile rats, which have high hepatocellular proliferation activity and a sufficient amount of metabolic enzymes such as CYP, may be more sensitive to the cytotoxic and carcinogenic effects of chemicals than the livers of adult rats. Thus, our results on developmental difference of hepatic CYPs in juvenile rats are useful to identify underlying age-dependent susceptibility of chemical-induced toxicity, and to understand developmental change of chemical disposition.

Effect of stress on drug metabolism and its modulation by antistress agents

Event Abstract Back to Event Effect of stress on drug metabolism and its modulation by antistress agents Panagiotis Kourounakis1*, Eleni Rekka1 and Karyophyllis Tsiakitzis1 1 Aristotle University of Thessaloniki, Department of Pharmaceutical Chemistry, Greece Introduction: The effect of hormones and stress on the resistance of the body to xenobiotics has been studied in the work of Hans Selye since early 70s. We, then, found that application of steroids, other hormones or stress to rats often increased resistance to drugs. In most, but not all cases, this was due to an increased hepatic drug metabolic activity. Furthermore, we have indicated that, by stress and through the hypothalamus-pituitary-adrenal axis, this resistance to drugs could be induced as a response to need. In this presentation, we study the effect of vitamin E (1) and a novel compound, trolox-CO-NHCH2CH2CH2CO-O-lorazepam (2) on stressed animals. Methods: Experimental animals were stressed by a combination of cold, food and water deprivation and immobilization. They were treated by 1 or 2 and total hepatic CYP content was determined srectrophotometrically, erythromycin -demethylation and 4-nitrophenol aromatic hydroxylation were assessd in stressed, treated and normal animals. Results: It was found that stressed rats demonstrated augmented drug metabolic activity as this was expressed by the increased indices mentioned above. Vitamin E induced total P450 in normal rats, but had little effect on stressed animals. Erythromycin N-demethylation and nitrophenol hydroxylation were induced in both normal and stressed rats. Compound 2 reduced the increased effect of stress on drug metabolism towards normal values. Conclusion: We have confirmed that stress augments the resistance of the organism to xenobiotics, by increasing the drug metabolic activity of the liver, via CYP induction. This is attributed to the action of the increased corticoids during the established stress, as we have demonstrated. Thus, this resistance of the body to drugs can be a response to need. The effects of the two antistress agents most probably are due to the inhibition of the hypothalamic-pituitary-adrenal axis acting on cytochrome P450. Keywords: drug metabolism, stress, Vitamin E, lorazepame, cytochrome P450 Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Invited speaker Topic: Xenobiotic metabolism Citation: Kourounakis P, Rekka E and Tsiakitzis K (2010). Effect of stress on drug metabolism and its modulation by antistress agents. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00149 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Panagiotis Kourounakis, Aristotle University of Thessaloniki, Department of Pharmaceutical Chemistry, Thessaloniki, Greece, panoskur@pharm.auth.gr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Panagiotis Kourounakis Eleni Rekka Karyophyllis Tsiakitzis Google Panagiotis Kourounakis Eleni Rekka Karyophyllis Tsiakitzis Google Scholar Panagiotis Kourounakis Eleni Rekka Karyophyllis Tsiakitzis PubMed Panagiotis Kourounakis Eleni Rekka Karyophyllis Tsiakitzis Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Bioactivation of Lamotrigine in Vivo in Rat and in Vitro in Human Liver Microsomes, Hepatocytes, and Epidermal Keratinocytes: Characterization of Thioether Conjugates by Liquid Chromatography/Mass Spectrometry and High Field Nuclear Magnetic Resonance Spectroscopy

Previous studies suggested that lamotrigene (LTG) underwent bioactivation to a reactive aryl epoxide intermediate in rats. Nevertheless, definitive structures of these thioether conjugates, which are often needed to substantiate the mechanism of bioactivation and identity of reactive intermediate(s), were not fully established. In the present study, GSH, cysteinylglycine, and N-acetyl cysteine conjugates of LTG were isolated from bile of rats orally dosed with LTG (100 mg/kg), and their structures were fully elucidated by LC/MS and NMR. The definitive structural characterization of these metabolites provided evidence for the existence of a reactive aryl epoxide that was trapped as a GSH adduct. In vitro studies using various hepatic cellular and subcellular fractions obtained from human and rat were performed to demonstrate that LTG underwent bioactivation to form a GSH conjugate that was identical to the one initially characterized from in vivo studies. Human P450 2A6 and rat P450 2C11 appeared to be the primary enzymes activating LTG in human and rat liver microsomes, respectively. Interindividual variation in the bioactivation of LTG was demonstrated with 20 individual human liver microsomes. Furthermore, it was shown that human epidermal keratinocytes were capable of forming the same GSH conjugate, suggesting that LTG could be bioactivated in skin cells. The results from these studies suggest that LTG has the potential to undergo hepatic and nonhepatic bioactivation, leading to a reactive aryl epoxide intermediate in human. The bioactivation of LTG in epidermal cells provides a possible explanation for the idiosyncratic cutaneous reactions associated with LTG therapy.

Toxicity of hexachloronaphthalene (HxCN) and induction of CYP 1A in rats

The aim of the study was to investigate the toxicity of hexachloronaphthalene (HxCN) and its effect on cytochrome P-450 in rats and to make a comparison between HxCN and tetrachloronaphthalene (TeCN), an inactive congener. Our study provided evidence that the anorectic effect, with concurrent significant increase in relative liver mass was the most spectacular symptom of the toxic effect of hexachloronaphthalene in the rats after its single (250 mg/kg) and repeated (1 and 10 mg/kg) administration. Regardless of the kind of the experiment (acute or subacute toxicity), dose-dependent increase in lipid peroxidation in the liver was also observed, which may indicate that HxCN most probably generates oxidative stress in this organ. It was also observed that HxCN is a very strong inducer of cytochrome P-450, especially of CYP 1A, which is the most sensitive biomarker of exposure to this congener. In this study, LOAEL is 1 mg HxCN/kg b.w.

Impact of rat P450 genetic polymorphism on diazepam metabolism

Rats are an important tool in pharmacology and toxicology. The authors focus on rat P450s in relation to diazepam metabolism. In particular, considerable attention is devoted to the CYP2D subfamily, which is a group of highly polymorphic enzymes. First, the metabolic profiles of diazepam of humans and other animals are compared. In this review, the authors describe a novel genetic polymorphism of diazepam observed in commonly used rat strains and compare it to human genetic polymorphisms. The genetic basis underlying diazepam polymorphism in rats is also discussed. The authors conclude that the metabolic capacities and major metabolic pathways of diazepam are quite different among rat strains and in the Wistar strain due to CYP2D3 genetic polymorphism, which is independent of the debrisoquine polymorphism catalyzed by CYP2D2. The situation, in which major metabolites differ depending on animal strain, will be highly problematic not only in pharmacokinetic studies of test compounds, but also in pharmacological or toxicological tests. This may provide researchers who use experimental animals insights into important aspects of the genetic background of experimental animals. Thus, great caution must be taken in the choice of rat strains for studies of drug metabolism.

Expert Panel report on a study of Splenda in male rats

A recent study in rats investigated the retail sweetener product, Granulated SPLENDA ® No Calorie Sweetener (Splenda) (Abou-Donia et al., 2008. Splenda alters gut microflora and increases intestinal P-glycoprotein and cytochrome P-450 in male rats. J. Toxicol. Environ. Health A, 71, 1415–1429), which is composed of (by dry weight) maltodextrin (∼99%) and sucralose (∼1%). The investigators reported that Splenda increased body weight, decreased beneficial intestinal bacteria, and increased the expression of certain cytochrome P450 (CYP450) enzymes and the transporter protein, P-glycoprotein (P-gp), the latter of which was considered evidence that Splenda or sucralose might interfere with the absorption of nutrients and drugs. The investigators indicated that the reported changes were attributable to the sucralose present in the product tested. An Expert Panel conducted a rigorous evaluation of this study. In arriving at its conclusions, the Expert Panel considered the design and conduct of the study, its outcomes and the outcomes reported in other data available publicly. The Expert Panel found that the study was deficient in several critical areas and that its results cannot be interpreted as evidence that either Splenda, or sucralose, produced adverse effects in male rats, including effects on gastrointestinal microflora, body weight, CYP450 and P-gp activity, and nutrient and drug absorption. The study conclusions are not consistent with published literature and not supported by the data presented.