7-ethoxycoumarin O-deethylase Research Articles

Influence of Cigarette Smoke on the L-Ascorbic Acid Metabolism and the Activities of Drug-Metabolizing Enzyme in Rats

This study clarified the influence of cigarette smoke on the L-ascorbic acid (AsA) metabolism and the activities of drug-metabolizing enzyme in rats. The test rats (group T) were exposed to weak sidestream smoke from cigarettes for 2 h, everyday for 57 days. AsA concentration in the tissues and excreted amount of AsA in urine of group T tended to be higher than those of control group (group C). The plasma AsA concentration and the activities of aniline hydroxylase and 7-ethoxycoumarin O-deethylase of group T were significantly higher than those of group C. There was no significant difference in the activity of UDP glucuronosyltransferase or in the liver cytochrome P-450 content between these two groups.

Preliminary studies of biochemical changes (ethoxycoumarin O-deethylase activities and vitellogenin induction) in two species of shrimp (Farfantepenaeus duorarum and Litopenaeus setiferus) from the Gulf of Mexico

Several investigations have demonstrated that the increase in chemicals in the environment may have caused effects on aquatic life and wildlife. The impact from oil production activities on the benthic community structure and shrimp (Farfantepenaeus duorarum and Litopenaeus setiferus) biochemical markers were studied in the Gulf of Mexico, which is heavily contaminated by polycyclic aromatic hydrocarbons and heavy metals. Shrimp were collected from a control area and from an area close to oil production platforms during October and November 2002. There was no spatial difference in exposure and response probably because shrimp migrate, as results did not show significant differences in cytochrome P4501A (as measured by ethoxycoumarin O-deethylase (ECOD) metabolism) between the two sites. In October, shrimp ECOD activities were higher and statistically different from those measured in the samples taken in November. As for ECOD activities, with the concentration of vitellogenin as another biomarker, there were no differences between the shrimp collected from the control area and the shrimp collected on the oil production platforms. In this case the concentrations were higher in shrimp collected in November vs. shrimp collected in October. However, there are significant correlations between contaminants and responses (biomarkers), indicating an effect of pollution. One of the most important considerations brought up by this kind of study is that, although the majority of groups studying the effects of endocrine disruption have focused almost exclusively on human health or vertebrate wildlife-related issues, it is necessary to increase research focused on understanding the function of hormones in invertebrate species exposed in the field to different kinds of pollutants.

COMPARISON OF HUMAN MONOCYTE-DERIVED NEO-HEPATOCYTE WITH HUMAN PRIMARY HEPATOCYTES AS A PERSPECTIVE FOR AUTOLOGOUS CELL THERAPY

P671* Aims: There is growing interest in new therapeutic options for the treatment of end-stage liver diseases. Apart from bioreactors, as mechanical devices supporting liver function, first cases of human primary hepatocyte transplantations are reported. We have generated neo-hepatocytes out of terminally differentiated peripheral blood monocytes after undergoing a step of dedifferentiation which show specific in vitro functions comparable to those of primary hepatocytes Methods: Monocytes were isolated from human peripheral blood and after a six day treatment with MCSF and IL-3 were then exposed to a hepatocyte differentiation media containing FGF4 for 10 to 20 days. Neo-hepatocytes were analyzed for Albumin. Urea formation was shown using an urea-GLDH-method kit. Phase I biotransformation activity of neo-hepatocytes was verified using 7-ethoxycoumarin O-de-ethylase. The main phase II reactant, uridine di-phosphate (UDP)-glucuronyltransferase, was measured using 4-MU as a substrate protein expression, albumin production, urea metabolism for detoxification, UDP-glucuronidation and Phase I metabolism. All functions were compared with those of primary human hepatocytes. Results: 14-20 days after treatment with FGF-43 (HCM: hepatocyte conditioning medium) cells displayed a typical hexagonal shape and formed confluent layers with cell-cell contacts closely resembling those in primary human hepatocytes. RT-PCR analysis revealed that these “neo-hepatocytes”, expressed hepatocyte markers, e. g. α-fetoprotein, carbamyl phosphate synthetase I and coagulation factor II. An in-depth functional analysis of the neo-hepatocytes revealed a sustained albumin synthesis and a detoxifying capacity (urea production) similar in quantity and duration to that in genuine human hepatocytes. Furthermore, we found activity of 7-ethoxycoumarin O-deethylase in neo-hepatocytes, an enzyme that is catalyzed by a broad range of P450 isoenzymes such as CYP1A2, CYP2A6, CYP2B6, CYP2C8-9, CYP2E1, and CYP3A3-5. This was demonstrated by metabolization of ethoxy coumarin into 7OH-coumarin after stimulation with 3-methylcholanthren. In contrast, phase II metabolization through UDP-glucuronidation of 4-methyl-umbelliferone (4-MU) appears to be identical in primary human hepatocytes and neo-hepatocytes. Conclusions: These data convincingly show that neo-hepatocytes possess unique hepatocyte-specific cellular functions, namely synthesis, detoxification, phase I and II metabolism, which are quantitatively and qualitatively comparable to those of primary human hepatocytes in vitro. These cells could be clinically applied in an autologous setting for treatment of end stage liver diseases or to improve liver function prior to organtransplantation without the need for immunosuppressants or the fear of rejection

Influence of recipient gender on intrasplenic fetal liver tissue transplants in rats: cytochrome P450-mediated monooxygenase functions

Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern with consecutive differences in P450-mediated monooxygenase activities, which have been shown to be due to a differential profile of growth hormone (GH) secretion. Parallel to previous investigations on P450 isoforms expression, the aim of the present study was to elucidate the influence of recipient gender on P450-mediated monooxygenase activities in intrasplenic liver tissue transplants in comparison to orthotopic liver. Fetal liver tissue suspensions of mixed gender were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting transplant-recipients and age-matched controls were treated with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the vehicles and sacrificed 24 or 48 h thereafter. P450-dependent monooxygenase activities were assessed by a series of model reactions for different P450 subtypes in liver and spleen 9000 g supernatants. In spleens of male and female control rats only very low monooxygenase activities were detectable, whereas with most model reactions distinct activities were observed in transplant-containing organs. Livers and transplant-containing spleens from male rats displayed higher basal ethoxycoumarin O-deethylase and testosterone 2alpha-, 2beta-, 6beta-, 14alpha-, 15alpha-, 15beta-, 16alpha-, 16beta- and 17-hydroxylase activities than those from females. On the other hand, like the respective livers, spleens from female transplant-recipients demonstrated more pronounced p-nitrophenol- and testosterone 6alpha- and 7alpha-hydroxylase activities than those from male hosts. With nearly all model reactions gender-specific differences in inducibility by BNF, PB or DEX could be demonstrated in livers as well as in transplant-containing spleens. These results further confirm that the P450 system of intrasplenic liver tissue transplants and the respective orthotopic livers is similarly influenced by recipient gender.

P11 Catalytic activity, genotype of detoxifying enzymes and DNA-adducts in human placenta as risk factors for newborns

Induction of isozymes of drug-metabolizing enzymes by butylated hydroxytoluene (BHT) was studied in the male ddY mouse and Chinese hamster. In mice given 0.05% BHT in the diet for 14 days cytochrome P-450 contents and the activities of uridine diphosphate-glucuronyl transferase (UDP-GT) and pentoxyresorufin O-dealkylase were markedly increased, while in those fed 0.15% BHT testoterone 6α-, 16α- and 16β-hydroxylases were greatly increased, while indicated induction of cytochrome P-450 isozymes of the CYP2B family. Western blot analysis also showed and increased level of the isozyme immunorelated to rat CYP2B2 by BHT feeding. The activities of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase (ECOD), erythromycin N-demethylase and glutathione S-transferase (GST) remained unchanged. In Chinese hamster given 0.05 and 0.15% BHT in the diet for 14 days activities if ECOD and GST were induced, but cytochrome P-450 contents and the activities of other enzymes were unaffected. Testosterone 15α-hydroxylase was induced in hamster fed 0.15% BHT. These findings suggested that BHT administration in the hamster induced CYP2A2-type isozyme, which was confirmed by Western blot analysis. BHT treatment enhanced activation of benzo[a] pyrene (B[a]P) as determined by the mutagenicity test, especially in Chinese hamster. The results suggest that BHT treatment induces specific isozymes of drug-metabolizing enzymes and might modify the expression of toxicities of other chemicals.

P.1.198 Follow-up of patients with major depression treated with intravenous mirtazapine

Across all levels of l-triiodothyronine (l.-T3) treatment, 2,3.7.8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased hepatic cytochrome P-450-associated activities of 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-dealkylase (EROD) and aryl hydrocarbon hydroxylase (AHH). The treatment of thyroidectomized rats with l-T3 at physiologic replacement levels in concert with TCDD produced an increase in ECOD, EROD and AHH activity above that seen with only TCDD. TCDD as well as l-T3 enhanced the activity of hepatic 1-naphthol glucuronyl transferase (NGT). In addition, the combined effect of l-T3 and TCDD resulted in similar levels of induction of NGT at both physiologic and supraphysiologic doses of l-T3. TCDD treatment resulted in elevated serum T3 levels at both physiologic and supraphysiologic levels of l-T3. One TCDD dose inhibited hepatic microsomal 3,3',5'-triiodothyronine (reverse T3) 5'-deiodinase activity by 61 % in thyroidectomized, T3-untreated rats. The inhibition of 5'-deiodinase activity was partially overcome by increasing the T3 dose.

Effect of age and photoperiodic conditions on metabolism and oxidative stress related markers at different circadian stages in rat liver and kidney

It has been shown that some cytochrome P450-dependent enzyme activities could present daily fluctuations, particularly CYP3A isoenzymes which are enhanced during the dark period. The aim of this study was to investigate whether age and photoperiodic conditions at different circadian stages could influence these fluctuations. Young mature (10 weeks) and old (22 months) Wistar rats were initially exposed to light-dark cycles 12:12 during 4 weeks, and secondly 18:6 for either one week or six weeks. Erythromycin N-demethylase (CYP3A-dependent), 7-ethoxycoumarin O-deethylase (CYP1A-dependent) and aniline 4-hydroxylase (CYP2E-dependent) activities were determined in liver and kidney microsomes at different hours after darkness onset (HADO). In addition, liver and kidney GSH, GSHPx, ATP, TBARS were determined. During the LD 12:12 cycle, while no significant modification was observed in CYP1A- and 2E-dependent enzyme activities as functions of HADO, erythromycin N-demethylase activity (CYP3A-dependent) showed a significant increase during the second third of the dark period in both young and old rats. After switching to a LD 18:6 cycle, this variation was still observed during second third of the dark period, to a lesser but still significant degree, with no difference between one week and six weeks exposure to the new photoperiod. It can be noted that the old rats showed a significantly lower level of erythromycin N-demethylase activity than the young rats, in parallel to a decrease in GSH, GSHPx and ATP, and an increase in TBARS. These results confirm the lower resistance of old animals to oxidative stress. The observed variations in metabolism parameters underline the need for study designs in pharmaco-toxicology taking into account the possible risks induced by circadian changes, especially in aged subjects.

Biological Evaluation of Pollution in the Sagami River Using the 7-Ethoxycoumarin O-Deethylase Activity Induced by River Sediment Extracts in HepG2 Cells

The arylhydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells was employed in a longitudinal study of pollution in the Sagami River flowing into Sagami Bay using river sediment extracts. The six sampling points for river sediment were Ogurabashi, Shimomizo, Atsugi, Kurami, Shinomiya, and Hiratsuka (in descending order of distance from the estuary). In samples obtained at the four midpoints along the river, ECOD activity was induced almost to the same extent. Ogurabashi, the point farthest from the estuary, seemed less polluted than downstream areas based on ECOD activity, while a marginal increase in the activity above the baseline was found at the highest concentration in the sample from Hiratsuka. The points where the river sediment caused a marked induction of ECOD activity in HepG2 cells were surrounded by inland industrial areas scattered along the river.

Biological Evaluation of the Pollution of the Tsurumi River with 7-Ethoxycoumarin O-Deethylase Activity Induced by River Sediment Extracts in HepG2 Cells.

The pollution level of the Tsurumi River flowing into Tokyo Bay was studied longitudinally using river sediment extracts in an assay system based on the arylhydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells. The sampling points of river sediment were as follows: Namamugi, Kami-Sueyoshi, Tsunashima, Shin-Yokohama A and B (downstream and upstream, respectively, from the former open-air industrial waste incineration site), Kozukue and Nakayama (Fig. 1). ECOD activity was induced to different extents in all samples tested. Namamugi, located in the middle of Keihin Industrial District, was considered to be the most polluted, followed by Shin-Yokohama A and Tsunashima. Results from these samples showed reverse U-shaped dose-response curves in terms of ECOD activity as well as 3-methylcholantrene (3-MC) although the expression of CYP1A1 mRNA in HepG2 cells remained roughly constant at higher concentrations as in the case of 3-MC, thus implying post-transcriptional suppression.

Induction of liver monooxygenases by annatto and bixin in female rats

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and beta-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (beta-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin) and bixin (95% pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.

Treatment of acute liver failure in pigs reduces hepatocyte function in a bioartificial liver support system.

Several different types of bioartificial liver (BAL) support systems have been developed to bridge patients suffering from acute liver failure (ALF) to transplantation or liver regeneration. In this study we assessed the effects of ALF plasma on hepatocyte function in the BAL system that has been developed in our center. Pigs (40-60 kg) were anaesthetised and a total hepatectomy was performed. Cells were isolated from the resected livers and were transferred to the bioreactor of the BAL system. Twenty hours after cell isolation, hepatocytes in the BAL were tested for cell viability and functional activity by using a recirculating test medium in which assessment of LDH leakage, ammonia clearance, urea synthesis, 7-ethoxycoumarin O-deethylase (ECOD) activity and pseudocholine esterase production was performed. Subsequently, two groups were studied. In one group (I, n=5), the cell-loaded bioreactor was used to treat the donor pig, rendered anhepatic, for 24 hours. In the second group (II, n=5) the bioreactor was cultured for 24 h and served as a control. After 24 hours treatment or culturing, the cell viability count and functional activity tests were repeated. The results show that hepatocytes in the BAL remained viable after 24 h treatment of anhepatic pigs, as shown by the LDH release and pseudocholine esterase production. However, metabolic functions such as ammonia clearance, ECOD and urea synthesis were reduced after 24 h exposure of hepatocytes to autologous ALF plasma, whereas these functions were unaltered after 24 h culturing of the cells in the bioreactor.

Mixed function oxidases in kidney and duodenum of camel, guinea pig and rat

The activities of the drug-metabolizing enzymes, aniline 4-hydroxylase, benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase have been measured in vitro in kidneys and duodenum of camels ( Camelus dromedarius), guinea pigs ( Cavia porcellus) and rats ( Rattus norvegicus). In these species, levels of hepatic microsomal parameters namely microsomal protein, cytochrome P 450, cytochrome b 5 and NADPH–cytochrome c reductase have also been determined. In general, camels seemed to have the lowest enzyme activity when compared to rats and guinea pigs. Rats showed the highest activity in NADPH–cytochrome c reductase, aniline 4-hydroxylase and ethoxycoumarin O-deethylase among these species. However, guinea pigs showed the highest enzyme activity in cytochrome P 450, cytochrome b 5 and benzphetamine N-demethylase.

Effects of trivalent chromium on hepatic CYP‐linked monooxygenases in laying hens

Chromium is an essential nutrient required for the metabolism of carbohydrates and lipids in humans and many animal species. We evaluated whether feeding laying hens with high levels of different chemical forms of trivalent chromium may affect hepatic metabolizing cytochrome P-450 (CYP)-linked enzymes. Modulation of CYP-dependent monooxygenases (which play a pivotal role in the endogenous metabolism) by Cr(III) was tested using either specific substrates as probes of different CYPs or testosterone as a multi-bioprobe. The CYP-supported oxidases were studied in liver microsomes from laying hens fed with diet supplemented with either 25 or 50 ppm chromium chloride as a yeast or as aminoniacinate. Although at 25 ppm no appreciable effects were observed, at 50 ppm a general inactivation of the recorded monooxygenases (ranging from 34% loss for ethoxycoumarin O-deethylase with chromium chloride to 85% loss for O-deethylation of ethoxyresorufin with either chromium yeast or aminoniacinate) were achieved in the supplemented groups vs controls. The only exception was the O-dealkylation of pentoxyresorufin, which was significantly boosted by both chromium yeast (up to 65% increase) and chromium aminoniacinate (up to 141%). Measurements of the regio- and stereoselective hydroxylation of testosterone used as a multi-bioprobe corroborated the inactivating properties of Cr(III) at the higher dose. Taken as a whole, these findings seem to indicate that high levels of Cr(III) supplementation can substantially impair CYP-catalysed drug metabolism in laying hens.

EFFECT OF ALACHLOR ON HEPATIC CYTOCHROME P450 ENZYMES IN RATS

Alachlor ((2-chloro-N-methoxymethyl)-N-(2,6-diethylphenyl)acetamide) is a widely used preemergence herbicide which has been classified by the USEPA as a probable human carcinogen. The herbicide has been suggested to be metabolized by hepatic cytochrome P450 system. We examined the effects of alachlor on cytochrome P450 enzymes in rat liver microsomes. Rats were treated intraperitoneally with alachlor daily for 5 days, at doses of 25, 50 and 100 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, 7-pentoxyresorufin O-depentylase, which is associated with CYP2B1, was dose-dependently increased by alachlor. The induction relative to control activity was 1.7–4.2-fold. The activities of CYP1A-dependent monooxygenases such as 7-ethoxyresorufin O-deethylase and acetanilide 4-hydroxylase were also significantly increased by alachlor at doses of 50 and 100 mg/kg (1.7–2.1-fold). Furthermore, immunoblotting showed that alachlor significantly increased CYP2B1/2 and CYP1A1/2 protein levels by 4.2–6.3- and 1.8-fold, respectively. Although 7-ethoxycoumarin O-deethylase, bufuralol 1′-hydroxylase and 4-nitrophenol 2-hydroxylase activities were significantly increased by alachlor at higher doses (≥ 50 mg/kg), the induction ratios were less than 1.6-fold. The activities of other cytochrome P450-dependent monooxygenases, namely testosterone 7α-hydroxylase, testosterone 2α-hydroxylase, testosterone 6β-hydroxylase and lauric acid ω-hydroxylase, were not affected by alachlor at any dose. In addition, there was no significant change in the protein levels of CYP2C11/6, CYP2D1, CYP2E1, CYP3A2/1 and CYP4A1/2/3. These results suggest that alachlor selectively induces cytochrome P450 isoforms of the CYP1A and CYP2B subfamilies in rat liver microsomes, and that the expression of these isoforms is closely related to the toxicity of alachlor.

Effects of atmospheric exposure to naphthalene on xenobiotic-metabolising enzymes in the snail Helix aspersa

Polycyclic aromatic hydrocarbons are xenobiotics whose elevated toxicity for living organisms requires to efficiently monitor air pollution, either by evaluating their levels in the environment, or by assessing their biological impacts on sentinel organisms. We investigated the effects of naphthalene exposure on some xenobiotic-metabolising enzyme activities in three organs of Helix aspersa. Particular activities depending on cytochrome P450 (ethoxyresorufin O-deethylase, EROD; ethoxycoumarin O-deethylase, ECOD; pentoxyresorufin O-dealkylase, PROD) and associated with glutathione (glutathione S-transferase, GST; glutathione peroxidase, GPX; glutathione reductase, GR) were assessed. In control animals, the P450-dependent specific activities were distributed according to the range kidney > digestive gland > mantle cavity forming tissues (MCFT). Neither ECOD nor PROD activities could be detected in MCFT. In the two other organs, the major phase I activities were due to ECOD, the level of PROD being very low or null. The glutathione-associated activities showed comparable levels in the three organs, except GPX activity that was higher in the digestive gland. Naphthalene (NAP) exposure did not affect any activity in MCFT, but it significantly decreased EROD and ECOD activities in the kidney as opposed to their increase in the digestive gland, whereas PROD activities were not influenced by the treatment. Glutathione-dependent activities were not significantly affected by NAP exposure, except for GPX which activity diminished in the digestive gland. This study demonstrates that complex detoxification pathways should exist in Helix aspersa as in mammals and that they could be used as potential biomarkers of NAP exposure.

Deformation behaviour of PP hollow fibres

Interspecies difference is an important issue in toxicology research. We compared the potential in vitro metabolism of human, porcine and rat hepatocytes over 2 weeks in culture in an organotypical culture model which reflects the in vivo situation. All three species show similar LDH-rates. Albumin measurements showed that rat cells are about twice as active as human and porcine hepatocytes. The ethoxyresorufin-O-deethylase (EROD) activity of the rat hepatocytes is with about 14 μU/106 cells distinctly higher than those of porcine and human cells (1.8 and 0.5 μU/106 cells respectively), furthermore, the activity of the rat EROD increases slightly during the prolonged time in culture, whereas those of porcine and human enzymes slightly decrease. Concerning ethoxycoumarin-O-deethylase (ECOD), the enzyme activities are found to be in three different ranges where rat cells show the highest activity with 66 μU/106 cells, porcine hepatocytes exhibit an activity of about 23 μU/106 cells, and human activity is lowest with 0.7 μU/106 cells. All three species show a similar decreasing trend of ECOD during the period of study. Regarding the biotransformation of testosterone, human and porcine liver cells form three major metabolites whereas rat cells form a mixture of all measured metabolites. Hence, in vitro metabolism using porcine hepatocytes would be much more scientific sense than one using rat hepatocytes since the metabolic pathways are much closer to human metabolism.

Induction by xenobiotics of phase I and phase II enzyme activities in the human keratinocyte cell line NCTC 2544

This study analyses the expression and induction of several drug-metabolising enzyme activities involved in either phase I or phase II biotransformations in NCTC 2544 human keratinocytes. The phase I activities 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depenthylase (PROD) were easily detectable in basal conditions. During incubations lasting up to 144 h in the presence of the classical cytochrome P450 inducers β-naphthoflavone (BNF), 3-methylcholanthrene (MC) and phenobarbital (PB), a considerable and significant increase in all the three activities was observed. PROD activity was induced up to 4.5-fold after 96 h in the presence of PB. The MC-induced ECOD and EROD activities were also dose-dependently inhibited by α-naphothflavone, which was given to the cells during the incubation with CYP 1A1 inducers. Also the PB-induced PROD activity was decreased by the simultaneous addition of the CYP 2B inhibitor metyrapone. Both cytochrome P450 inhibitors were used at non-cytotoxic concentrations. The phase II enzymes glutathione S-transferase, aldehyde dehydrogenase and quinone reductase were all highly expressed and inducible by MC. The exposure (24 h) of the cells to four hair dyes used in cosmetic formulations resulted in a marked increase in ECOD activity. All data give sustained evidence for the suitability of NCTC 2544 cell line to skin toxicology studies.

Changes in the expression of cytochrome P450 2E1 and the activity of carcinogen -metabolizing enzymes in Schistosoma haematobium-infected human bladder tissues

The bioactivation of N-nitrosamines and polycyclic aromatic hydrocarbons (PAHs) is mediated primarily by the mixed-function oxidase system, which includes dimethylnitrosamine N-demethylase I, arylhydrocarbon [benzo( a)pyerne] hydroxylase, cytochrome P450, cytochrome b 5, and ethoxycoumarin deethylase. Most of carcinogens and xenobiotics are conjugated and detoxified by phase II drug-metabolizing enzymes such as glutathione S-transferase. The present study showed the influence of Schistosoma haematobium on the activity of the above-mentioned enzymes in 13 schistosome-infected human bladder tissues compared with those of 15 schistosome-free samples. The contents of cytochrome P450 and cytochrome b 5 were increased in the bladder tissues by 48 and 69%, respectively. Moreover, the activities of dimethylnitrosamine N-demethylase I and arylhydrocarbon hydroxylase, ethoxycoumarin O-deethylase, ethoxyresourfin O-deethylase, and pentoxyresorufin O-pentoxyresorufin were increased by 75, 159, 49, 63 and 44%, respectively. The signal intensity for cytochrome P450 2E1 was greatly increased over the control. Also, the activity of glutathione S-transferase was increased by 89%. On the other hand, the activity of glutathione reductase and the level of reduced glutathione were decreased by 40 and 57%, respectively. Interestingly, the level of free radical, thiobarbituric acid reactive substance, was increased in the schistosome-infected human bladder tissues by 125%. The present study clearly demonstrated that S. haematobium changes the activity of carcinogen-metabolizing enzymes. We conclude that S. haematobium could enhance the carcinogenicity of polycyclic aromatic hydrocarbons (e.g. benzo(a)pyrene) and N-nitrosamines (e.g. dimethylnitrosamine) through induction of their corresponding bioactivating enzymes in human bladder tissues.

A Single Nucleotide Polymorphism of CYP2B6 Found in Japanese Enhances Catalytic Activity by Autoactivation

A single nucleotide polymorphism (SNP) resulting in a substitution from Gln to His was found in exon 4 of the CYP2B6 gene in Japanese. The frequency of the variant allele was found to be 19.9%. The mutant- and the wild-type enzymes were expressed in Escherichia coli, and the effects of the single amino acid substitution on the catalytic activity were examined by investigating the kinetic profiles of 7-ethoxycoumarin O-deethylase activity. The wild-type enzyme showed typical Michaelis–Menten kinetics, while the mutant-type enzyme represented the sigmoidal kinetics with a higher Vmax value compared to that of the wild-type enzyme. Eadie–Hofstee plots further revealed an existence of allosteric effects for the reaction catalyzed by the variant. This is the first evidence demonstrating that only one amino acid substitution, Gln172His, caused by natural SNP enhances the catalytic activity of CYP by obtaining the character of homotropic cooperativity.

Carbamazepine: a 'blind' assessment of CYP-associated metabolism and interactions in human liver-derived in vitro systems

1. The ability of various in vitro systems for CYP enzymes (computer modelling, human liver microsomes, precision-cut liver slices, hepatocytes in culture, recombinant enzymes) to predict various aspects of in vivo metabolism and kinetics of carbamazepine (CBZ) was investigated. 2. The study was part of the EUROCYP project that aimed to evaluate relevant human in vitro systems to study drug metabolism. 3. CBZ was given to the participating laboratories without disclosing its chemical nature. 4. The most important enzyme (CYP3A4) and metabolic route (10,11-epoxidation) were predicted by all the systems studied. 5. Minor enzymes and routes were predicted to a different extent by various systems. 6. Prediction of a clearance class, i.e. slow clearance, was correctly predicted by microsomes, slices, hepatocytes and recombinant enzymes (CYP3A4). 7. The 10,11-epoxidation of CBZ by the recombinant CYP3A4 was enhanced by the addition of exogenous cytochrome-b5, leading to a considerable over-prediction. 8. Induction potency of CBZ was predicted in cultured hepatocytes in which 7- ethoxycoumarin O-deethylase was used as an index activity. 9. It seems that for a principally CYP-metabolized substance such as CBZ, all liverderived systems provide useful information for prediction of metabolic routes, rates and interactions.