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Myclobutanil is currently used on the flowering plants. Little is known about how Apis cerana cerana respond to myclobutanil exposure. Hence, the acute toxicity of myclobutanil and its sublethal effects on respiration, flight and detoxification enzymes [7-ethoxycoumarin O-deethylase (ECOD) and glutathione S-transferases (GSTs)] in A. cerana cerana were investigated. The results indicated that formulation grade myclobutanil showed moderate toxicity to A. cerana cerana either contact (LD 50 =4.697μg/bee) or oral (LD 50 =2.154μg/bee) exposure. Sublethal dose of myclobutanil significantly reduced the respiration rate of workers at 24h and 48h regardless of the exposure method. However, myclobutanil didn't significantly affect the take-off flight. After nurse bees exposure to the dose (LD 5 ) of formulation-grade myclobutanil, ECOD activity was significantly induced when compared with control, but GST activity didn't change. In the forager bees, no enzyme markers response was obtained in this test. From the present study we can infer that myclobutanil disturb respiration and P450-mediated detoxification of the individual bees of A. cerana cerana. Thus, myclobutanil may has risk for A. cerana cerana, it should be cautiously used., (Copyright © 2017. Published by Elsevier Inc.)

In quinoa seedlings, the pigment betalain accumulates in the hypocotyl. To isolate the genes involved in betalain biosynthesis in the hypocotyl, we performed ethyl methanesulfonate (EMS) mutagenesis on the CQ127 variety of quinoa seedlings. While putative amaranthin and celosianin II primarily accumulate in the hypocotyls, this process produced a green hypocotyl mutant (ghy). This MutMap+ method using the quinoa draft genome revealed that the causative gene of the mutant is CqCYP76AD1-1. Our results indicated that the expression of CqCYP76AD1-1 was light-dependent. In addition, the transient expression of CqCYP76AD1-1 in Nicotiana benthamiana leaves resulted in the accumulation of betanin but not isobetanin, and the presence of a polymorphism in CqCYP76A1-2 in the CQ127 variety was shown to have resulted in its loss of function. These findings suggested that CqCYP76AD1-1 is involved in betalain biosynthesis during the hypocotyl pigmentation process in quinoa. To our knowledge, CqCYP76AD1-1 is the first quinoa gene identified by EMS mutagenesis using a draft gene sequence., (Copyright © 2018. Published by Elsevier Inc.)

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b 5 enhanced the k cat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min -1 ) with little effect on K M (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6β-hydroxylation, progesterone 6β-/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids., (Copyright © 2017 John Wiley & Sons, Ltd.)

The control program of codling moth (Cydia pomonella L.) in the Río Negro and Neuquén Valley is intended to neonate larvae. However, adults may be subjected to sublethal pesticide concentrations generating stress which might enhance both mutation rates and activity of the detoxification system. This study assessed the exposure effects of chlorpyrifos on target enzyme and, both detoxifying and antioxidant systems of surviving adults from both a laboratory susceptible strain (LSS) and a field population (FP). The results showed that the FP was as susceptible to chlorpyrifos as the LSS and, both exhibited a similar chlorpyrifos-inhibitory concentration 50 (IC 50 ) of acetylcholinesterase (AChE). The FP displayed higher carboxylesterase (CarE) and 7-ethoxycoumarine O-deethylase (ECOD) activities than LSS. Both LSS and FP showed an increase on CarE activity after the exposure to low-chlorpyrifos concentrations, followed by enzyme inhibition at higher concentrations. There were no significant differences neither in the activities of glutathione S-transferases (GST), catalase (CAT) and superoxide dismutase (SOD) nor in the reduced glutathione (GSH) content between LSS and FP. Moreover, these enzymes were unaffected by chlorpyrifos. In conclusion, control adults from the FP exhibited higher CarE and ECOD activities than control adults from the LSS. AChE and CarE activities were the most affected by chlorpyrifos. Control strategies used for C. pomonella, such as rotations of insecticides with different modes of action, will probably delay the evolution of insecticide resistance in FPs from the study area., (© 2015 Institute of Zoology, Chinese Academy of Sciences.)

The development of a high-throughput tool is required for screening of environmental pollutants and assessing their impacts on aquatic animals. Freshwater planarians can be used in rapid and sensitive toxicity bioassays. Planarians are known for their remarkable regeneration ability but much less known for their metabolic and xenobiotic biotransformation abilities. In this study, the activities of different phase I and II enzymes were determined in vivo by directly measuring fluorescent enzyme substrate disappearance or fluorescent enzyme metabolite production in planarian culture media. For phase I enzyme activity, O-deethylation activities with alkoxyresorufin could not be detected in planarian culture media. By contrast, O-deethylation activities with alkoxycoumarin were detected in planarian culture media. Increases in 7-ethoxycoumarin O-deethylase (ECOD) activities was only observed in planarians exposed to 1μM, but not 10μM, β-naphthoflavone for 24h. ECOD activity was inhibited in planarians exposed to 10 and 100μM rifampicin or carbamazepine for 24h. For phase II enzyme activity, DT-diaphorase, arylsulfatases, uridine 5'-diphospho (UDP)-glucuronosyltransferase or catechol-O-methyltransferase activity was determined in culture media containing planarians. The results of this study indicate that freshwater planarians are a promising model organism to monitor exposure to environmental pollutants or assess their impacts through the in vivo measurement of phase I and II enzyme activities., (Copyright © 2016. Published by Elsevier Inc.)

The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The common marmoset (Callithrix jacchus), a New World primate species, is potentially a useful animal model for preclinical studies in drug development. However, cytochrome P450 (P450) enzymes have not been fully identified and characterized in marmosets. In this study, we identified P450 2A6 cDNA with the sequence highly identical (91-94%) to human P450 2A6, 2A7, and 2A13 cDNA and cynomolgus monkey P450 2A23, 2A24, and 2A26 cDNA. Among the tissue types examined, marmoset P450 2A6 mRNA was most abundantly expressed in livers where P450 2A6 protein was also detected by immunoblotting. Phylogenetic analysis showed that marmoset P450 2A6 was more closely clustered with human and cynomolgus monkey P450 2As than P450 2As of dog, rat, and mouse (the species also used in drug metabolism). Marmoset P450 2A6 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation, similar to human P450 2A6 and 2A13 and cynomolgus monkey P450 2A23, 2A24, and 2A26, but much less effectively coumarin 7-hydroxylation, showing some difference as well. Interestingly, marmoset P450 2A6 and cynomolgus monkey P450 2A23 catalyzed phenacetin O-deethylation, which is catalyzed by human P450 1A2 and 2A13, but not by P450 2A6. Marmoset P450 2A6 also exhibited catalytic activity toward testosterone by the multiple sites, but not rat P450 2A-specific testosterone 7α-hydroxylation activity. These results indicated that marmoset P450 2A6 had functional characteristics different from those of human and cynomolgus monkey P450 2As in terms of partially different substrate specificities and catalytic activities, indicating its importance of further studies for P450 2A-dependent drug metabolism in marmosets., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

The acute toxicity of fipronil and its sublethal effects on detoxification enzymes (carboxylesterases (CarEs), glutathione S-transferases (GSTs), and 7-ethoxycoumarin O-deethylase (ECOD)) in zebrafish (Danio rerio) were investigated. The results indicated that the 24-h LC50 of fipronil for zebrafish was 220.4 μg/L (95% CI: 173.7-272.4 μg/L). Sublethal concentrations of fipronil did not cause significant changes in CarEs activities. In the liver and muscle tissues, GST activities at the tested concentrations did not significantly differ from those in the control. In the brain and gill tissues, GST activities at a concentration of 4 μg/L were significantly lower than those at a concentration of 2 μg/L. The results suggest that CarEs and GSTs were not suitable biomarkers for fipronil effects in D. rerio. A significant induction in the ECOD activities in the brain, gill, liver, and muscle tissues was observed compared with the control. Moreover, the dose-dependent responses of the ECOD activity were observed after treatment with sublethal concentrations of fipronil in the range of 2-20 μg/L. The results suggested that ECOD could be a suitable biomarker of fipronil effects in D. rerio., (Copyright © 2014. Published by Elsevier Inc.)

The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these findings remains to be clarified., (© The Author(s) 2014.)

Motorcycle exhaust (ME) is a major source of air pollution and a potential health hazard in urban areas where motorcycles are a popular means of transportation. The main objectives of this study were to determine the ability of ME to cause cardiotoxicity in rats and investigate the possible mechanisms of toxicity. Male rats were exposed to 1:10 diluted ME by inhalation 2 h daily and Monday through Friday for 8 weeks. Exposure to ME increased heart weight and decreased cardiac antioxidant enzymes glutathione S-transferase (GST), superoxide dismutase and glutathione peroxidase activities in a concentration- and time-dependent manner. Analysis of echocardiographic parameters indicated that ME increased left ventricle posterior wall thickness, interventricular septum thickness and left ventricle mass. Histopathological examinations of the hearts revealed that ME exposure caused focal cardial degeneration and necrosis, mononuclear cell infiltration, and fibrosis. The results of reverse transcriptase-polymerase chain reaction studies showed that ME decreased GST-M1 and GST-P1 mRNA expression and increased the expression of proinflammatory cytokine interleukin-1β, hypertrophy marker atrial natriuretic peptide, fibrosis markers type I and III collagen, profibrotic cytokine connective tissue growth factor, and hypertrophy and fibrosis mediator transforming growth factor (TGF)-β1 in the heart. The data of Western blot analysis showed that cardiac TGF-β1 protein was induced by ME. These findings demonstrate that subchronic ME exposure caused hypertrophy and fibrosis, and modulated GST and TGF-β1 expression in rat heart possibly by mechanisms involving oxidative stress and inflammation.

Significant efforts are invested in field studies using fish, and it is important to optimize the number of organisms collected to evaluate the possible impacts of contamination. This paper provides ecotoxicologists with the approximate numbers of fish needed to identify statistically significant differences among samples using physiological indices and biochemical markers of fish health. The numbers of fish to collect are reported for ethoxyresorufin-o-deethylase (EROD); ethoxycoumarin-o-deethylase (ECOD), serum sorbitol dehydrogenase (sSDH), stress proteins, gonadosomatic index, liver somatic index, condition factor, and biliary metabolites of polycyclic aromatic hydrocarbons. The number of fish to collect was as few as four for ECOD activity (with a power of 80%), but as high as 106 for CF (with a power of 95%). Achieving statistical significance between sites does not help in the interpretation of the biological significance of a parameter, but well-planned field samplings will maximize the chances of correctly identifying areas of concern., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The chemical study of the extracts from leaves and stems of Ouratea ferruginea allowed the identification of a new isoflavone, 5-hydroxy-7,3'4'5'-tetramethoxyisoflavone, and twenty two known compounds, including friedelin, 3β-friedelinol, lupeone, a mixture of sitosterol, stigmasterol and campesterol, sitosteryl- and stigmasteryl-3-O-b-D-glucopyranosides, 5,4'-dihydroxy-7,5',3'-trimethoxyisoflavone, 5,4'-dihydroxy-7,3'-di-methoxyisoflavone (7,3'-di-O-methylorobol), 5,7,4'-trihydroxy-3',5'-dimethoxyisoflavone (piscigenin), 2R,3R-epicatechin, syringic acid, 2,6-dimethoxybenzoquinone, 2,6-dimethoxyhydroquinone, syringic and ferulic aldehyde, a mixture of vanillic acid, 1-hydroxy-2-methoxy-4-(1E-3-hydroxy-1-propenyl)-benzene and 3,5-dimethoxy-4-hydroxy-dihydrocinamaldehyde, besides amenthoflavone and 7-O-methylamenthoflavone (sequoiaflavone) which are considered as chemotaxonomic markers of Ouratea. The structures were identified by IR, (1)H- and (13)C-NMR and GC-MS, HPLC-MS, besides comparison with literature data. The inhibitory effects of 5,4'-dihydroxy-7,5',3'-trimethoxyisoflavone, 7,3'-di-O-methylorobol, piscigenin and 7-O-methylamenthoflavone on cytochrome P450-dependent 7-ethoxycoumarin O-deethylase (ECOD) and glutathione S-transferase (GST) were evaluated in vitro. The 5,4'-dihydroxy-7,5',3'-trimethoxy-isoflavone was the best inhibitor, inhibiting almost 75% of GST activity. Sequoiaflavone was the most potent inhibitor, inhibiting ECOD assay in 75%. These activities allow us to consider both these flavonoids as potential anticancer and chemopreventive agents.

Since three-dimensional (3D) cell culture models better reflect tissues in vivo in terms of cell shape and microenvironment compared to conventional monolayer cultures, 3D tissue culture substrates gain more importance for a wide range of biological applications like drug discovery, toxicological studies, cancer and stem cell research. In this study we developed a method for the fabrication of 3D cell culture substrates in a multiwell plate format by microstructuring the bottom of 96-well cell culture plates using an ultrasonic embossing process. The resulting microstructured area consists of cubic microcavities in which adherent multicellular aggregates can be formed. We performed the biological evaluation of the system with the liver-derived human cell-line HepG2 and compared the novel substrate with a commercially available 3D culture system comprising porous alginate sponges. Metabolic activity (alamarBlue® reduction) and induction of four biotransformation enzymes (EROD, ECOD, UGT, SULT) were determined by fluorimetry or HPLC. Our results revealed that HepG2 cells in microstructured plates showed a higher mitochondrial activity, as well as enzyme activity of ECOD and UGT after treatment with an inducer when compared to cells cultured in alginate sponges at otherwise comparable conditions. Since we have modified standard cell culture plates, the obtained system is adaptable to automated screening and might be useful for all kinds of cultures including adult, progenitor and stem cells which need a 3D culture configuration to restore or maintain the differentiated status.

The gene expression and induction of cytochrome P450 (CYP)-enzymes following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) peroral administration was studied in the livers of two wild vole species--the bank vole (Myodes glareolus) and the field vole (Microtus agrestis). The dioxin-sensitive C57BL/6 mouse was used as a reference. Doses of 0.05, 0.5, 5.0, and 50 µg/kg were applied to ascertain a dose-response relationship, and the dose of 50 µg/kg was applied to the study time course for up to 96 h. The cytochrome P450 1A1 (CYP1A1) mRNA expression showed an expected dose-dependent increase equally in both vole species. Bank voles expressed notably higher CYP2A mRNA levels as compared with field voles. Both species exhibited dose-dependent increases in putative CYP1A-, CYP2B-, and CYP2A-associated activities as measured by fluorometric assays for ethoxyresorufin-O-deethylase (EROD), penthoxyresorufin-O-depenthylase (PROD), and 7-ethoxycoumarin-O-deethylase (ECOD), respectively. Putative CYP2A-associated coumarin-7-hydroxylase (COH) activity showed a slight increase at the two highest doses of TCDD in field voles but not in bank voles, and their basal COH activity was only one-fourth or less of that in field voles. Overall, however, bank voles tended to exhibit higher CYP-associated enzyme activities measured at the two largest doses of TCDD than field voles. A western blot analysis of aryl hydrocarbon receptor (AhR) revealed that the two vole species had differential band patterns, suggesting dissimilar structures for their AhRs., (Copyright © 2011 SETAC.)

Palaemonetes australis is a euryhaline shrimp found in southwestern Australian estuaries. To determine if P. australis is a suitable bioindicator species for monitoring the health of estuarine biota, they were exposed to measured concentrations of the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) at 0.01, 0.1, or 1 ppm for 14 days under laboratory conditions. At the end of exposure the shrimp were sacrificed for biomarker [ethoxycoumarin O-deethylase (ECOD), 8-oxo-dG concentration, and sorbitol dehydrogenase (SDH) activity] analyses. Gender did not appear to influence biomarker responses of the shrimp in this study. ECOD activity was induced in the treatment groups in a linear fashion from three (0.01 ppm) times to 12 (1 ppm) times the negative controls. 8-oxo-dG concentration was reduced three times in treatment groups below the controls suggesting impaired DNA repair pathways. There was no increase in SDH, signifying hepatopancreatic cell damage had not occurred in any treatment group. The response of P. australis to B[a]P exposure indicates that this crustacean is suitable bioindicator species for both laboratory studies and field monitoring. A combination of ECOD and SDH activities and 8-oxo-dG concentration represent a suitable suite of biomarkers for environmental monitoring of the sublethal effects of organic pollution to crustaceans from an estuarine environment.

During the last years an increasing number of in vitro models have been developed for drug screening and toxicity testing. Primary cultures of hepatocytes are, by far, the model of choice for those high-throughput studies but their spontaneous dedifferentiation after some time in culture hinders long-term studies. Thus, novel cell culture systems allowing extended hepatocyte maintenance and more predictive long term in vitro studies are required. It has been shown that hepatocytes functionality can be improved and extended in time when cultured as 3D-cell aggregates in environmental controlled stirred bioreactors. In this work, aiming at further improving hepatocytes functionality in such 3D cellular structures, co-cultures with fibroblasts were performed. An inoculum concentration of 1.2×10(5) cell/mL and a 1:2 hepatocyte:mouse embryonic fibroblast ratio allowed to improve significantly the albumin secretion rate and both ECOD (phase I) and UGT (phase II) enzymatic activities in 3D co-cultures, as compared to the routinely used 2D hepatocyte monocultures. Significant improvements were also observed in relation to 3D monocultures of hepatocytes. Furthermore, hepatocytes were able to respond to the addition of beta-Naphtoflavone by increasing ECOD activity showing CYP1A inducibility. The dependence of CYP activity on oxygen concentration was also observed. In summary, the improved hepatocyte specific functions during long term incubation of 3D co-cultures of hepatocytes with fibroblasts indicate that this system is a promising in vitro model for long term toxicological studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effects of crude oil (Szeged-Algyo, Hungary) and oil fractions (F1: rich in aromatics; F2 fraction: free from aromatics) were investigated on liver CYP1A isoenzymes and antioxidant defence system following their i.p. injection into different freshwater fish species: carp (Cyprinus carpio L.), silver carp (Hyphothalmichtys molitrix V.), and European eel (Anquilla anquilla). A dose of 2 mL kg -1 crude oil enhanced EROD activity 8-fold in carp and only 5-fold in eel after 3 days. Oil fraction F1 caused only a 2-fold induction in EROD activity only in carp, while F2 fraction caused significant inhibition in all three investigated fish species. The antioxidant parameters [lipid peroxidation (LP), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH)] were measured following the treatment. A decrease of 50% in CAT activity was observed after oil treatment. The GSH level enhanced, resulting the protective effects against LP. The same dose of crude oil but a longer duration time resulted in lower CYP1A induction in carp and antioxidant parameters had returned close to control. In all treatments the EROD isoenzymes proved to be more sensitive and the effects of oil treatment showed species to be different. Carp proved to be more sensitive than eel or silver carp.

This study was designed to analyze the effect of environmental oxidative stress on human placental monooxygenases, glutathione S-transferase (GST) activity and polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human term placentas from radioactivity-contaminated and chemically-polluted areas of the Ukraine and Belarus, and to compare these biomarkers to the newborn's general health status. Placental PAH-DNA adduct formation, GST activity, 7-ethoxycoumarin O-deethylase (ECOD) activity, and thiobarbituric reactive substances (TBARS), an index of lipid peroxidation, were measured in groups of women exposed to different levels of radioactivity and PAH pollution. The in vitro metabolism data, obtained from 143 human placental samples at term, were compared to indices of maternal and newborn health. The highest ECOD activity was recorded in placentas obtained from chemically-polluted areas and a radioactivity-contaminated area; the ECOD activity was 7-fold and 2-fold higher compared to the region considered to be "clean". Newborns with the most compromised health status displayed the greatest down-regulation of GST activity (144-162mUmgprotein(-1) vs. 258-395mUmgprotein(-1)), enhanced ECOD activity and the highest level of PAH-DNA adduct formation. The highest level of TBARS was observed in women exposed to the highest levels of radiation. The efficiency of placental detoxification negatively correlated with maternal age and the health status of the newborn. Environmental oxidative stress was related to an increase in anemia, threatened abortions, toxemia, fetal hypoxia, spontaneous abortions and fetal hypotrophy. Our data suggest that chemically- or radioactivity-induced oxidative stress enhance cytochrome P450-mediated enzymatic activities potentially resulting in increased formation of reactive metabolites. The activity of GSH-transferase is not enhanced. This imbalance in detoxification capacity can be measured as increased production of PAH-DNA adducts, decreased lipid peroxidation and compromised fetal health.

Catalytic activities of CYP1A1, CYP1A2 and CYP1B1, and inducibility of the activities were studied in intact human lung samples (from 23 human subjects) ex vivo. The activities [as measured by ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and 3-cyano-7-ethoxycoumarin O-deethylase (CECOD)] were present in the lungs and were induced by benzo[a]pyrene (B[a]P) and diesel exhaust particle extract (DEE) but with extensive inter-subject variability. All three activities were substantially inhibited (>or=75%) by the CYP1 inhibitor alpha-naphthoflavone, whereas only MROD and CECOD were substantially inhibited by the CYP1A2-preferential inhibitor fluvoxamine. None of the three activities was substantially inhibited by the CYP1B1-preferential inhibitor tetramethoxystilbene, indicating lack of involvement of CYP1B1 in the activities in the intact lung. CYP1A1 and CYP1A2 proteins were present in the lungs (by western blot analysis), also with extensive inter-subject variability, and were induced by B[a]P or DEE more extensively than by the combination of B[a]P and DEE (B[a]P+DEE). CYP1B1 was also present in the lungs and its level varied extensively between subjects. In contrast with CYP1A and CYP1A2 levels, CYP1B1 level was not significantly altered by B[a]P or DEE treatment and was diminished more extensively by treatment with B[a]P+DEE. The findings point to the potential usefulness of the intact lung for assessing in situ xenobiotic biotransformation reactions as well as the CYP1 specificity of the reactions ex vivo. The findings also suggest MROD and CECOD as potential markers of CYP1A2 activity in the intact lung.

Stem cell models offer an opportunity both for therapeutic use and for the assessment of alternative in vitro models. Human lipoaspirate is a source of adult stem cells (pre-adipocytes), which are able to differentiate into various phenotypes, such as neurogenic lineage. Here, we analyse the suitability of these in vitro models in screening exogenous compounds, such as environmental pollutants, that may affect adipose cells and neurogenic development. To evaluate neurogenic differentiation, we analysed expression of cholinergic system and acetylcholinesterase immunoreactivity. Heterocyclic derivatives of polycyclic aromatic hydrocarbons (PAHs) are often significant components of environmental contaminants. As they contain inducers of cytochrome P450 1A1 (CYP1A1), we explored the activity of CYP1A1-related enzymes, i.e. 7-ethoxycoumarin- and 7-ethoxyresorufin-O-deethylase (ECOD and EROD) in both cell systems in basal conditions and after exposure to non-cytotoxic doses of beta-naphthoflavone (BNF), a well-known PAH-type inducer. Both cell models showed basal and inducible levels of ECOD. Analysis of CYP1A1 protein expression and EROD-related enzyme activity confirmed the inducibility of the CYP1A1 isoform by BNF. These results demonstrate that mesenchymal adult stem cells can constitute innovative models. We therefore propose the use of pre-adipocytes and their neurogenic derivates to evaluate the cytotoxic/biological effects of unintended exposure to contaminants.

We investigated the presence and inducibility of CYP1A in suckermouth catfish (Hypostomus affinis and Hypostomus auroguttatus, Loricariidae), tilapia (Oreochromis niloticus, Cichlidae) and mice (Mus musculus, Muridae). Alkoxyresorufin-O-dealkylases (EROD, MROD, PROD and BROD) were detected and proved to be inducible (beta-naphthoflavone, BNF or dimethylbenz[a]anthracene, DMBA, 50 mg/kg bw ip) in liver microsomes from tilapia and mice. In loricariids, alkoxyresorufin-O-dealkylases were either undetectable (MROD/EROD) or very low (PROD/BROD), and so they remained after treatment with BNF or DMBA. Ethoxycoumarin-O-deethylase (ECOD) was recorded in all species and proved not to be inducible by BNF or DMBA. In loricariids and tilapia, ECOD was not depressed by a concentration of alpha-naphthoflavone (CYP1A-inhibitor) that markedly depressed EROD in tilapia. A CYP1A-like protein was detected by a monoclonal antibody in rats, mice and tilapia, but not in loricariids. A polyclonal antibody, however, detected a CYP1A-like protein in liver microsomes of loricariids. Suckermouth catfish, rats, mice and tilapia express a protein reactive with a polyclonal antibody against trout CYP3A. Loricariids and tilapia exhibited marked genotoxic responses (enhanced incidence of micronucleated erythrocytes) following treatment DMBA (50 mg/kg bw ip), a promutagen activated by CYP1A/1B. Therefore, although not exhibiting EROD, a CYP1A-mediated activity, loricariids converted DMBA into its genotoxic metabolites. Our findings suggest that the CYP1A-like protein of locariid catfish recognizes DMBA, but not ethoxyresorufin, as a substrate.

It was found previously that membranes based on co-polymers of acrylonitrile (AN) and 2-acrylamido-2-methyl-propansulfonic acid (AMPS) greatly stimulated the functionality and survival of primary hepatocytes. In those studies, however, the pure AN-AMPS co-polymer had poor membrane-forming properties, resulting in quite dense rubber-like membranes. Hence, membranes with required permeability and optimal biocompatibility were obtained by blending the AN-AMPS co-polymer with poly(acrylonitrile) homopolymer (PAN). The amount of PAN (P) and AN-AMPS (A) in the blend was varied from pure PAN (P/A-100/0) over P/A-75/25 and P/A-50/50 to pure AN-AMPS co-polymer (P/A-0/100). A gradual decrease of molecular cut-off of membranes with increase of AMPS concentration was found, which allows tailoring membrane permeability as necessary. C3A hepatoblastoma cells were applied as a widely accepted cellular model for assessment of hepatocyte behaviour by attachment, viability, growth and metabolic activity. It was found that the blend P/A-50/50, which possessed an optimal permeability for biohybrid liver systems, supported also the attachment, growth and function of C3A cells in terms of fibronectin synthesis and P-450 isoenzyme activity. Hence, blend membranes based on a one to one mixture of PAN and AN-AMPS combine sufficient permeability with the desired cellular compatibility for application in bioreactors for liver replacement.

Pyrazole can induce CYP2E1 and 2A5, which produce reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates important antioxidant enzymes to remove ROS. In this study, we applied Nrf2 knockout mice to test the hypothesis that pyrazole will cause hepatotoxicity and elevate oxidative stress to a greater extent in Nrf2 knockout mice compared to wild type mice. Pyrazole induced severe oxidative liver damage in Nrf2 knockout mice but not in wild type mice. Activities and levels of CYP2E1 and 2A5 were elevated by pyrazole in the wild type mice but not in the Nrf2 knockout mice. However, expression or activity of Nrf2-regulated antioxidant enzymes, such as gamma-glutamylcysteine synthetase (GCS), heme oxygenase-1 (HO-1) and glutathione-S-transferase (GST), were upregulated in the pyrazole-treated wild type mice, but to a lesser extent or not at all in the pyrazole-treated Nrf2 knockout mice. Treatment with antioxidants such as vitamin C or S-adenosyl-l-methionine (SAM) or an inhibitor of iNOS prevented the pyrazole-induced oxidative liver damage, thus validating the role of oxidative/nitrosative stress in the pyrazole induced liver injury to the Nrf2 knockout mice. In summary, even though ROS-producing CYP2E1/2A5 were not elevated by pyrazole, impaired antioxidant capacity resulting from Nrf2 deficiency appear to be sufficient to promote pyrazole-induced oxidative liver injury.

The effects of Cu(2+)-sulfate and Pb(2+)-acetate on carp (Cyprinus carpio L.), silver carp (Hypopthalmichtys molitrix V.) and wels (Silurus glanis L.) were studied. The liver microsomal Cyt P450 content, the EROD, ECOD and APND monooxygenase activities were measured. In vivo treatment with 1 mg L(-1) Cu(2+) significantly elevated the activities of these enzymes and Cyt P450 content in silver carp livers. The high-dose Cu(2+) treatment (10 mg L(-1)) on silver carp caused two-fold higher induction in the P450 dependent monooxygenase isoensymes than in wels. Although the 2 mg kg(-1) treatment with Pb(2+) in carp elevated significantly the P450 content, the EROD isoenzyme activities were significantly decreased after 1 day, showing the destructive effect of metal ion on the enzyme system. In vitro, Cu(2+) and Pb(2+) decreased the Cyt P450 content in the carp liver microsomes and the absorption peak shifted to higher wavelength. Fourier Transform Infrared (FTIR) spectroscopy was used to detect the damaging effects of the heavy metals. According to the inhibitory potency to Cu(2+), the most sensitive isoenzyme was the EROD in wels, the least was the silver carp's isoenzyme. The investigated fish P450 isoenzymes showed, that the Cu(2+) was a stronger inhibitor than Pb(2+).

The yellowtail trumpeter (Amniataba caudavittata) is an estuarine-dependent omnivorous fish found in the Swan-Canning Estuary, Western Australia. Thirty five fish were injected with either the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P), the synthetic flavenoid beta-naphthoflavone (BNF), or used as controls. The fish were then sampled at 3 and 7 days postinjection. Induction of the enzyme ethoxyresorufin O-deethylase (EROD) activity was nonsignificant while ethoxycoumarin O-deethylase (ECOD) activity induction differed amongst treatments. A high interindividual variability in the EROD activity was observed. The measurement of sorbitol dehydrogenase in the serum (s-SDH) was elevated (BNF 2.2 times and B[a]P 3.2 times the control fish) demonstrating that liver cell damage had occurred. Increases in biliary metabolites of both B[a]P-type and pyrene-type (19 times and 3.4 times the controls respectively) indicated that detoxification of pyrene-type compounds had taken place. Fish of the Terapontidae family, such as the yellowtail trumpeter, were found to be suitable for biomonitoring the health of the Swan-Canning Estuary. A combination of ECOD activity, s-SDH, and the measurement of biliary metabolites represents a suitable suite of biomarkers for environmental monitoring of the sublethal effects of PAH pollution in these fish.

The aim of this study was to investigate the cellular pathological responses of hepatopancreatic digestive cells from the periwinkle Littorina littorea exposed to the polycyclic aromatic hydrocarbon (PAH) fluoranthene and to ascertain whether any injurious effects were reversible within the experimental time scale. A secondary objective was to establish the relationship of the various reactions to animal health status, using lysosomal stability as an index of well-being. Exposure of snails to a concentration of 335 microgl(-1) (1.7 microM) fluoranthene (seawater renewed and spiked daily with fluoranthene) for 5 days resulted in a reduction in lysosomal stability (neutral red retention) and endocytosis; and an increase in smooth endoplasmic reticulum (ER) and 7-ethoxycoumarin-o-deethylase (ECOD; measured as cyano-ECOD) activity measured in isolated live digestive cells. Exposed snails treated with clean seawater for a further 8 days resulted in a return to control levels of lysosomal stability, ECOD and ER; endocytosis showed only a partial recovery. Multi-variate and uni-variate analysis showed that there were strong correlations between the various cellular biomarker responses. These findings are interpretable within the current framework of molluscan biomarker responses to PAHs. Principal component analysis was used to derive the first principal component for endocytosis, ER and ECOD reactions and these were plotted against lysosomal stability as a measure of cellular well-being. The resulting significant regression represents the mapping of the individual biomarkers within health status space for a gradient of fluoranthene toxicity. From this analysis, we concluded that endocytosis is an indicator of healthy snails while proliferation of ER and to a lesser extent induced ECOD are indicative of dysfunction and reduced health. Finally, the results indicate that stress induced by chronic exposure to a PAH is reversible.

Miniaturized bioassays offer many advantages in exploring the toxic potential of chemicals, including small sample volumes and compatibility with high-throughput screening. One problem common to miniaturized systems, however, is the loss of test chemicals because of sorption. The idea of the current study was to use the sorption phenomenon in a positive way. It was found that contaminants sorbed to the growth surface in wells of tissue-culture plates or to the surface of selected sorbent bead materials are available to vertebrate cells growing in direct contact with the contaminant-coated surface. The use of beads provided more flexibility with regard to surface area, materials, and assay format. Biosilon, a bead cell-culture carrier made of polystyrene, was found to be most suitable. It supported cell adherence and allowed the detection of reproducible dose-response curves of an increase in cytochrome CYP1A enzyme activity by sorbed polycyclic aromatic hydrocarbons in the rainbow trout (Oncorhynchus mykiss) liver cell line, RTL-W1. The resulting bead assay provides a miniaturized, solvent-free exposure system. Potential future applications include the coupling to environmental sampling, in which the bead material is used as solid receiving phase before serving as a surface for vertebrate cells to attach and respond.

The Swan-Canning Estuary, in southwestern Australia, undergoes distinct seasonal changes, with freshwater discharge predominant in the winter (wet) season and low flow with high salinity predominant in the dry summer season. To investigate seasonal variability in biomarkers of exposure in fish, black bream (Acanthopagrus butcheri) were collected from seven sites in the Swan-Canning Estuary in winter 2000 and in summer 2001. No interseasonal or intersite differences in serum sorbitol dehydrogenase concentration were found, indicating the measured mixed-function oxygenase (MFO) enzymes were not influenced by liver damage. The ethoxyresorufin-O-deethlyase (EROD) activity of the postspawning females was higher in summer than in winter but was significantly lower than that in males in both seasons, suggesting estradiol suppression in females. Sexual differences in ethoxycoumarin-O-deethylase (ECOD) activity were not evident in either season. Both EROD and ECOD activities and polycyclic aromatic hydrocarbons (PAH) biliary metabolites had significantly different patterns of induction between seasons. The ratio of naphthalene-type to benzo(a)pyrene-type biliary metabolites was significantly higher in summer, indicating the sources of petroleum hydrocarbons were petrogenic compared to in winter, when the source was a mixture of pyrogenic and petrogenic PAHs. There was no upstream or downstream gradient of response in any biomarker in either season, demonstrating that there were multiple sources of contaminant input into the estuary. Although winter biomarker levels were triggered by the discharge runoff from major roads and drains, summer biomarker levels appear to have been related to recreational boating use on the estuary., ((c) 2005 Wiley Periodicals, Inc.)

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.

During the past decade the Swan-Canning estuary, Western Australia, has shown signs of stress which has been attributed to high nutrient inputs. There is little information on the effect of nonnutrient contaminants on biota inhabiting the estuary. A suite of biomarkers was measured on black bream (Acanthopagrus butcheri) to determine whether annual variations in fish biomarkers exist in the wet (winter) and dry (summer) seasons. Serum sorbitol dehydrogenase showed no significant differences between years, indicating that measured mixed-function oxygenase (MFO) enzyme activities were not affected by annual variations in hepatic tissue damage. Ethoxyresorufin-O-deethylase activity was lower in female black bream than in male fish while ethoxycoumarin-O-deethylase activity was not influenced by gender. Biomarker levels measured at various sites confirm that major roads and drains are significant contributors of MFO-inducing chemicals into the Swan-Canning estuary. No consistent upstream or downstream gradient in biomarker response was identified. The ratio of naphthalene-type to benzo(a)pyrene-type biliary metabolites was linked to runoff from urban areas into the estuary. There was high annual variability in all biomarkers in both seasons, suggesting that biannual monitoring is required to evaluate the effect of contaminants on the biota in the estuary.

The effects of thyme (Thymus vulgaris L.) leaves and its phenolic compounds, thymol and carvacrol, on the activities of xenobiotic-metabolizing enzymes, i.e., phase I enzymes such as 7-ethoxycoumarin O-deethylase (ECOD) and phase II enzymes such as glutathione S-transferase (GST) and quinone reductase (QR), were investigated. Mice were fed with a diet containing thyme (0.5% or 2.0%) or treated orally with thymol (50-200 mg/kg) or carvacrol (50-200 mg/kg) once a day for 7 successive days, and then the enzyme activities in the livers were analyzed. Dietary administration of 2% thyme caused slightly but significantly higher ECOD, GST, and QR activities by 1.1-1.4-fold. Thymol (200 mg/kg) treatment resulted in significantly higher ECOD, GST, and QR activities by 1.3-1.9-fold, and carvacrol (200 mg/kg) treatment caused significantly higher ECOD, GST, and QR activities by 1.3-1.7-fold. Thymol-treated animals had significantly higher protein levels of GST alpha and GST micro, and carvacrol-treated animals had significantly higher levels of GST micro. These results imply that thyme contains bifunctional inducers (i.e., substances capable of inducing both phase I and phase II enzymes) and that thymol and carvacrol may account for the effects of thyme.

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.

We studied the profile of permethrin resistance in populations of head lice infesting children 6-12 yr old in schools and their homes in and around Buenos Aires, Argentina. Five permethrin-resistant populations with different levels of resistance were collected: Hogar Loyola (HL), Republica de Turquia (RT), Hogar Mitre (HM), Guardia de Honor (GH), and Ricardo Guiraldes (RG). One susceptible population, Bandera Argentina (BA), also was collected. Their level of resistance was evaluated, and results showed resistance ratios of 13 for HL, 16 for RT, 22 for HM, 61 for GH, and 69 for RG. To elucidate the possible involvement of the cytochrome P450 monooxygenase system in conferring permethrin resistance, ethoxycoumarin-O-deethylase (ECOD) activity was measured in abdomens of individual third instars and adults by using a fluorometric assay. The ECOD activity was lower in the susceptible BA population (4.7 ng per louse) than in the resistant ones (13.7 ng per louse for RG, 12.3 ng per louse for GH, 8.6 ng per louse for RT, and 8.2 ng per louse for HL). ECOD activity was significantly correlated with the level of resistance in the field populations (r = 0.97, P = 0.0009), suggesting a role for cytochrome monooxygenase P450 system in permethrin resistance by head louse, Pediculus humanus capitis De Geer.

The expression, inducibility, and activities of several cytochrome P450 (CYP) enzymes were investigated in a human tongue carcinoma cell model, CAL 27, and compared with the human liver model HepG2 cells. The modulation effects of green tea on various CYP isoforms in both cell lines were also examined. RT-PCR analysis of CAL 27 cells demonstrated constitutive expression of mRNA for CYPs 1A1, 1A2, 2C, 2E1, 2D6, and 4F3. The results were negative for CYP2A6, 2B6/7, 3A3/4, and 3A7. Both cell lines displayed identical expression and induction profiles for all of the isoforms examined in this study except 3A7 and 2B6/7, which were produced constitutively in HepG2 but not Cal-27 cells. CYP1A1 and 1A2 were both induced by treatment with beta-napthoflavone as indicated by RT-PCR and Western blotting, while CYP2C mRNA was upregulated by all-trans retinoic acid and farnesol. RT-PCR and Western blot analysis showed that the expressions of CYP1A1 and 1A2 were induced by green tea extract (GTE), which also caused an increase in mRNA for CYP2E1, CYP2D6, and CYP2C isoforms. The four tea catechins, EGC, EC, EGCG and ECG, applied to either HepG2 or Cal-27 cells at the concentration found in GTE failed to induce CYP1A1 or CYP1A2, as determined by RT-PCR. Of the isoforms that were apparently induced by GTE, only 7-ethoxycoumarin deethylase (ECOD) activity could be detected in CAL 27 or HepG2 cells. Interestingly, mRNA and protein for CYP1A1 and CYP1A2 were detected in both cell lines, and although protein and mRNA levels of CYP1A1 and CYP1A2 were increased by GTE, the observed ECOD activity in both cell lines was decreased.

In this study, we have examined the presence and inducibility of phase I and II drug-metabolizing enzymes in the liver and nasal mucosa of Italian water frogs of control and pretreated with beta-naphthoflavone, phenobarbital and dichlobenil by using typical substrates for these enzymes along with polyclonal antibodies mainly raised against mammalian enzymes. The CYP content and various monooxygenase and phase II enzyme activities in the liver of this frog were found similar, when reported, to those of largely aquatic and semiaquatic frogs. The treatment with beta-naphthoflavone resulted in an induction in the liver of a CYP1A and the induction was manifested by (a) immunoblot analysis using anti-rat CYP1A1, (b) an increase of CYP1A-mediated methoxyresorufin-O-demethylase and ethoxyresorufin-O-deethylase activities. The treatments with both phenobarbital and dichlobenil did not produce in the liver any effect on the assayed enzymes. When the nasal mucosa of water frogs was analyzed, various monooxygenase and phase II enzymatic activities, generally comparable to those of liver, were determined. However, by using antibodies anti-three GST different classes, we found a different reactivity into the cytosol of the two tissues indicating a differential tissue susceptibility to toxic effects of xenobiotics. In the nasal mucosa, a protein immunorelated to CYP2A and monooxygenase activities (i.e. ethoxycoumarin-O-deethylase and coumarin-7-hydroxylase) linked in mammals to this isoform have also been found. The treatment of water frogs with the herbicide dichlobenil decreased both the above-mentioned activities and the immunoreactive CYP2A apoprotein. The pretreatment with metyrapone, a CYP inhibitor, protected the CYP2A apoprotein and its linked activities from toxic effect of dichlobenil indicating a key role of this enzyme in the bioactivation of this herbicide. The findings of the present work suggest that the hepatic CYP1A induction and the nasal CYP2A-like inhibition profiles might provide two potential biomarkers of the Italian water frogs exposure to environmental and aquatic pollutants.

1. The O-dealkylation of 7-alkoxycoumarins is widely used as an assay to characterize cytochrome P450 (CYP) activity. These substrates can also undergo oxidative attack at additional sites on the coumarin nucleus, which may influence their apparent selectivity for particular CYP forms. 2. Accordingly, the effect of blockade of these additional sites was investigated on the selectivity towards rat hepatic CYP forms, with emphasis on the CYP1A and 2B forms. 3. Blockade of the 3-/4- and 6-positions resulted in substrates for which the CYP1A1/2 selectivity of the unsubstituted 7-alkoxycoumarins was altered to a CYP2B selectivity; this was achieved with little overall change in the molecular dimensions of the substrate. Limited analysis of other inducible CYP forms indicated at most only small effects of structure modification on activity. 4. The findings suggest that the sensitivity of probe substrates for CYP forms may be limited by the occurrence of competing side reactions of the substrate, and that better probes may be derived by blocking the sites of these side reactions.

Liver and kidney glutathione S-transferase (GST) activities to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activities) were measured in 280 dogs from five different breeders, and significant individual differences in this activity were observed in both organs. Interestingly, 34 out of the 280 dogs (i.e. 12.1%) were those in which liver GST-D activities were less than 10 nmol/min per mg cytosolic protein, "low GST dogs", and the other dogs were classified as "middle" and "high" GST dogs for which the liver GST-D activities were 10-80 and >80 nmol/min per mg protein, respectively, and occurred at similar percentages (41.4% for the middle GST dog and 46.4% for the high GST dog). Furthermore, the existence of the low GST dogs was not limited to one particular breeder. There was a good correlation (r=0.910) between the liver and kidney GST-D activities, showing low activity in not only the liver but also the kidney in the low GST dogs. Although liver GST activity to 1-chloro-2,4-dinitrobenzene as a substrate (GST-C activity), catalyzed by various GST isozymes in dogs, was significantly correlated with liver GST-D activity, GST-C activity showed more than 450 nmol/min per mg protein even in the low GST dogs. There was no significant difference in cytochrome P450 content, 7-ethoxycoumarin O-deethylase activity or UDP-glucuronosyltransferase activity to p-nitrophenol as a substrate between low GST dogs and the other dogs. Finally, remarkably high plasma concentrations of DCNB were observed in the low GST dogs after single doses of DCNB at 5 or 100 mg/kg. The individual differences in GST-D activity are probably attributable to the content and/or activity of the theta class GST isozyme Yd(f)Yd(f) since it has been reported that glutathione conjugation of DCNB is specifically catalyzed by GSTYd(f)Yd(f) in dogs. In conclusion, we identified a number of low GST dogs in which the GST-D activities were not observed either in vivo or in vitro. The feasibility of using a single low dose of DCNB to phenotype dogs based on GST-D activity was confirmed. It was also suggested that low GST dogs have high susceptibility, including unexpected toxicity or abnormal exposure, to chemicals metabolized by GSTYd(f)Yd(f).

Tissue engineering with three-dimensional biomaterials represents a promising approach for developing hepatic tissue to replace the function of a failing liver. Herein, we address cell seeding and distribution within porous alginate scaffolds, which represent a new type of porous biomaterial for tissue engineering. The hydrophilic nature of the alginate scaffold as well as its pore structure and interconnectivity enabled the efficient seeding of hepatocytes into the scaffolds, that is, 70-90% of the initial cells depending on the seeding method. Utilization of centrifugal force during seeding enhanced cell distribution in the porous scaffolds, consequently enabling the seeding of concentrated cell suspensions (>1 x 10(7) cells/mL). Cell density in scaffolds affected hepatocyte viability as judged by MTT assay. At a cell density of 0.28 x 10(6) cells/cm3 scaffold, the number of viable hepatocytes decreased to 33% of its initial value within 7 days, whereas at the denser cultures, 5.7 x 10(6) cells/cm3 scaffold and higher, the cells maintained higher viability while forming a network of connecting spheroids. In the high-density cellular constructs, hepatocellular functions such as albumin and urea secretion, and detoxification (cytochrome P-450 and phase II conjugating enzyme activities), remained high during the 7-day culture. Collectively, the results of the present study highlight the importance of cell density on the hepatocellular functions of three-dimensional hepatocyte constructs as well as the advantages of alginate matrices as scaffoldings.

New approaches for in vitro testing of hepato-mediated toxicity are undertaken to offer alternatives to in vivo animal testing. The described bioassay for hepato-mediated toxicity testing is based on a small scale hepatocyte-bioreactor with pig hepatocytes connected to a silicon sensor based microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells and the microphysiometer alone. EAR represents the metabolic activity of tested cells (hepatocytes and ZR 751 cells) under the influence of perfused media, compared to controls, which were set to 100%. Cyclophosphamide (CYCL), whose cytostatic effect is dependent on CYP 450 biotransformation was used as a model substrate. CYCL showed decrease of EAR in hepatocytes, but not in ZR 751 cells. Bioreactor supernatant including CYCL was pumped into the microphysiometer and EARs of the target ZR 751 cell line were recorded. After 7 h of bioreactor supernatant perfusion the ZR 751 cell line showed an EAR decrease of 18.68% +/- 10.18, as compared to controls (bioreactor supernatant from the identical set-up without CYCL). Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of CYCL by the bioreactor. This new bioassay serves as an example of future applications for hepatocyte bioreactors in automated toxicity testing devices, e.g. in preclinical drug studies or evaluation of hepato-mediated toxicity, not depending on cell destruction or further assays.

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.

The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites.

The effects of the subchronic administration of Panax ginseng extracts were examined on the hepatic cytochrome P450-dependent monooxygenase system of guinea pigs pre-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Panax ginseng extracts were intraperitoneally administered to guinea pigs at 100 mg/kg/day for 14 days from 1 week after a single intraperitoneal injection of 1 microg of TCDD/kg of body weight. TCDD treatment increased the total cytochrome P450 content 2.86-fold, and this was remarkably inhibited by the administration of Panax ginseng extracts. Treatment with ginseng extract alone also decreased the contents of cytochrome P450 by 33%, but both TCDD and ginseng extracts had no effect on cytochrome b(5) content. The administration of TCDD resulted in a 1.73-fold increase in microsomal NADPH-cytochrome P450 reductase activity in the guinea pig liver, and this was significantly inhibited by ginseng extracts, but treatment with ginseng extracts alone had no effect on its activity, and no statistical changes in the activity of NADPH-cytochrome b(5) reductase were observed in guinea pig liver due to TCDD and/or ginseng extract administration. Compared to the control, ECOD activity remarkably (1.76-fold) increased after TCDD administration, but this increase was completely inhibited by treatment with ginseng extract. Treatment with ginseng extract alone resulted in a 50% reduction of ECOD activity. TCDD administration remarkably induced benzphetamine demethylation (BPDM) activity, while ginseng extract also slightly increased the enzyme's activity, but the induction attributed to ginseng extracts was not statistically significant. Even though administration of ginseng extracts slightly inhibited TCDD-induced BPDM activity, the inhibition was not statistically significant. These results indicate that ginseng extract exerts different effect on the induction of P450 isozymes. From these results, we suggest that Panax ginseng extracts may act as an inhibitor of CYP1A rather than that of CYP2B.

Interactions of 27 steroids, among them 17 derivatives such as ethers, sulfates and amidosulfonates derived from 17 beta- and 17 alpha-estradiol, from testosterone and alpha- and beta-dihydrotesosterone and from dehydroepiandrosterone with rat liver microsomal cytochromes P450 (P450) were investigated in vitro by assessing binding to P450 and effects on P450 mediated monooxygenase functions as measured by different model reactions: ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD) and ethylmorphine N-demethylation (EMND). With the exception of 17 alpha-estradiol-3-dimethylamidosulfonate, estrone, its -3-methylether and -3-amidosulfonate and testosterone, all other steroids displayed type I or reverse type I binding to P450. All steroids inhibited EROD activity in micromolar concentrations. An additional strong inhibition of ECOD and EMND activities was only demonstrated for the androgens and progestins. Estriol, estrone and mestranol displayed less inhibitory actions on the model reactions than estradiol. No major differences in comparison to the parent compounds were noted with the other derivatives. The only exceptions were 17 beta-(8,9-dehydro-14 alpha,15 alpha-methylene)estradiol, which displayed stronger effects than estradiol, and dehydroepiandrosterone-3-sulfate, which was less effective than dehydroepiandrosterone. Possible antioxidant properties of the steroids were examined by the stimulated lipid peroxidation (LPO), H2O2 production, and lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) using rat liver microsomes. Additionally, the influence on rat whole blood chemiluminescence (WB-CL) was assessed. All the estrogens, but not their methylethers and amidosulfonates inhibited LPO in micromolar concentrations. The effects on the other oxidase model reactions or on WB-CL were less distinct. Only ethinylestradiol and 17 beta-(8,9-dehydro-14 alpha,15 alpha-methylene)estradiol displayed a strong inhibitory action on all model reactions. With the exception of dehydroepiandrosterone-3-sulfate, which in general had only weak effects, the androgen and progestin derivatives, in contrast, strongly decreased H2O2 formation and LM- and LC-CL, but were mostly ineffective on LPO and WB-CL.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.

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-ethoxy-resorufin 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 (> or = 50 mg/kg), the induction ratios were less than 1.6-fold. The activities of other cytochrome P450-dependent monooxygenases, namely testosterone 7 alpha-hydroxylase, testosterone 2 alpha-hydroxylase, testosterone 6 beta-hydroxylase and lauric acid omega-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.

The influence of single chain lipids on the 7-ethoxycoumarin O-deethyase activity of the reconstituted binary protein complex of isolated cytochrome P450 and NADPH-cytochrome P450 reductase has been examined. The enzyme activity of this binary enzyme complex has been shown to be influenced by (i) altering the complexation process of both proteins, (ii) by altering the catalytic cycle time of the active binary protein complex and (iii) by altering the fraction of substrate molecules at the catalytic center of the enzyme. Competitive inhibition was measured for all single chain molecules. The following dissociation coefficients of substrate and lipids used for the catalytic center of the protein were obtained: 110 microM 7-ethoxycoumarin (substrate), 1.1 microM MOG (1-monooleoyl-rac-glycerol), 0.3 microM SPH (D-sphingosine), 1.5 microM OA (oleic acid), 3.0 microM LPC (L-alpha-lysophosphatidyl-choline), 15.5 microM MSG (1-monostearoyl-rac-glycerol), 9.5 microM AA (arachidonic acid), 9.0 microM PaCar (palmitoyl-L-carnitine), 3.5 microM MPG (2-monopalmitoyl-glycerol), 1.5 microM LPI (L-alpha-lysophosphatidyl-inositol), 50 microM LA (lauric acid), 60 microM MA (myristic acid), 85 microM PA (palmitic acid), >100 microM SA (stearic acid). Only competitive inhibition with the substrate molecule 7-ethoxycoumarin was observed for the single chain lipids LA, MA, PA, SPH, SA, and OA. Non-competitive effects were observed for MPG (-0.03 microM(-1)), PaCar (-0.02 microM(-1)), MSG (-0.023 microM(-1)), LPC (-0.03 microM(-1)), AA (-0.03 microM(-1)), and MOG (+0.04 microM(-1)). The negative sign indicates that the cycle time of the working binary complex is enlarged. The positive sign indicates that the formation of the binary complex is enhanced by MOG.

Recently, researchers have focused on the use of bioartificial liver devices to support patients with fulminant hepatic failure. Our team developed a cell-based flat membrane bioreactor (FMB). In this, porcine liver cells were maintained in 3D-coculture between two gel layers in a sandwich configuration for 3 weeks to study the influence of this bioreactor technique on the preservation of basic, not induced activities of phase I and phase II enzymes. First, the time and substrate dependencies of the following enzymes were measured: ethoxyresorufin-O-deethylase (EROD, CYP 1A1/1A2) and ethoxycoumarin-O-deethylase (ECOD, CYP 2B6) as phase I enzymes, and glutathione-S-transferase (GST), UDP-glucuronosyltransferase (UGT) and sulfotransferase (ST) as phase II enzymes. To find optimal test conditions Michaelis-Menten kinetics were calculated. Next, different potential inducers were tested to find out the most effective compounds. Based on these results, the basic, not induced levels of the different enzymes were determined in the flat membrane bioreactor. Furthermore, the response of these enzyme activities to the chosen inducers was investigated to examine whether the cells keep their ability for drug-drug interactions. Basic, not induced activities of both phase I enzymes and the phase II enzymes GST and UGT were maintained at nearly the initial levels during the complete period of study. In addition, it was possible to induce these enzymes twice or three times in a weekly interval. In contrast, the basic, not induced activity of ST increased during the first 10 days of culture. It stabilized then and was maintained steady. As in short-term investigations, no reaction of the ST-activity towards any inducer could be obtained. These results prove that porcine liver cells preserve their phase I and phase II activities and respond to inducing drugs over 3 weeks in culture. Therefore, the flat membrane bioreactor is not only suitable for investigating drug metabolism, drug-drug interactions, and enzyme induction but also for supporting liver functions., (Copyright 2001 John Wiley & Sons, Inc.)

1. The role of endogenous nitric oxide in rat hepatocyte functionality and survival in cell culture was examined. Towards this aim, cytochrome P450 activities (CYP1A1/2, 2B1, 2A1, 2C11, 2D1, 2E1 and 3A1), liver-specific metabolic functions and cell survival were comparatively evaluated in hepatocytes isolated from the male Sprague-Dawley rat and/or cultured in control conditions or in the presence of N-omega-nitro-L-arginine methyl ester (NAME), an inhibitor of nitric oxide synthesis. 2. Suppression of nitric oxide production by NAME paralleled a substantial preservation of hepatocyte phenotype in culture. The presence of NAME was particularly important during isolation and/or the 6-24h culture. By 24h, beneficial effects were evident in parameters particularly unstable in culture (glycogen content, P450), whereas no changes were produced in well-preserved functions (glucose, urea and albumin synthesis, glutathione, drug-conjugating enzymes). 3. Long-term treatment of hepatocytes with NAME also produced a reduction in caspase 3 activation and in the percentage of spontaneous apoptotic cells, and an increase in cell survival and transcriptional activity as shown by attached cellular protein content and the protein-DNA ratio respectively. 4. In conclusion, inhibition of early endogenous nitric oxide formation is an efficient procedure for obtaining hepatocyte cultures with stable expression of differentiated functions, high cell survival and few signs of cell senescence.

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.