Microstructuring of multiwell plates for three-dimensional cell culture applications by ultrasonic embossing.

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.

In vivo indirect measurement of cytochrome P450-associated activities in freshwater gastropod molluscs.

The cytochrome P450 (CYP) system is widely distributed across phyla and plays a key role in the metabolism of xenobiotic compounds. However, most studies on CYP system were developed on vertebrates and among invertebrates, gastropod molluscs are rarely used. In this context, ethoxycoumarin-O-deethylase (ECOD), ethoxyresorufin-O-deethylase (EROD), and pentoxyresorufin-O-dealkylase (PROD) activities, which are indirect measurements of CYP system, were characterized in two freshwater gastropod molluscs, Potamopyrgus antipodarum, and Valvata piscinalis, to ascertain their potential interest as biomarkers of exposure to chemicals. Activities were measured using an in vivo non lethal method based on the measurement of formed product (resorufin or hydroxycoumarin). This in vivo assay allowed to measure the three activities in P. antipodarum and two of them (ECOD and PROD) in V. piscinalis. The detection of activities and the optimization of experimental design were carried out first and allowed to measure the selected activities for one individual. The modulation of the detected activities was secondly assessed using a polycyclic aromatic hydrocarbon (Benzo(a)pyrene). Based on this non destructive measurement, effect of BaP exposure could be detected on ECOD and EROD activity in P. antipodarum, as well on PROD activity of V. piscinalis after 96 h of exposure. Such an in vivo assay must be further developed to be valuably used to screen the exposure of gastropod species to CYP inducer chemicals and its consequences in terms of fitness of the organisms and of the population.

Pathological reactions and recovery of hepatopancreatic digestive cells from the marine snail Littorina littorea following exposure to a polycyclic aromatic hydrocarbon.

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.

Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP(+) reductase from plant chloroplasts.

Fusion proteins of rat cytochrome P4501A1 with maize ferredoxin I (Fd) and pea ferredoxin NADP(+) reductase (FNR), the last electron transfer proteins of the photosynthetic channel in plant chloroplasts, were obtained by gene fusion in the yeast expression vector pAAH5N. The encoded fusion proteins P4501A1-Fd, P4501A1-FNR, P4501A1-Fd-FNR and P4501A1-FNR-Fd were produced in microsomes of the yeast Saccharomyces cerevisiae AH22. Enzymatic assays were carried out in vitro with the isolated microsomes. P4501A1-Fd-FNR and P4501A1-FNR-Fd were found to catalyze P450-monooxygenase activities towards 7-ethoxycoumarin and the herbicide chlortoluron. P4501A1-Fd-FNR was the most efficient enzyme as measured in vitro in ferricyanide and cytochrome c reductions, as well as P450-monooxygenase assays. Apparent K(m) and k(cat) of P4501A1-Fd-FNR were 70 microM and 7800 min(-1) for NADPH, 13.2 microM and 51.1 min(-1) for 7-ethoxycoumarin, and 21.3 microM and 23. 8 min(-1) for the herbicide chlortoluron, respectively. Fd in P4501A1-Fd-FNR fusion enzyme was found to be a limiting factor compared to P4501A1 fused to the yeast NADPH-cytochrome P450 reductase, an artificial enzyme described previously. The efficiency of electron transfer in the P4501A1 fusion proteins and a possible in vivo molecular coupling of Fd and FNR with microsomal cytochrome P4501A1 produced in plant chloroplasts are discussed.

Sex differences in hepatic cytochrome P-450 monooxygenase activities in rainbow trout during an annual reproductive cycle.

Hepatic microsomal cytochrome P-450 monooxygenase activities were investigated in rainbow trout during an annual reproductive cycle. The fish were kept in tanks supplied with fresh water at a constant temperature of 10 degrees C. The daily light and darkness cycle was adjusted to follow the natural photoperiod. Sampling was performed once every month for 1 year. Higher benzo(a)pyrene-hydroxylase (or aryl hydrocarbon hydroxylase; AHH), ethoxycoumarin-O-deethylase (ECOD) and ethylmorphine-N-demethylase (END) activities and cytochrome P-450 content were found during the late stage of sexual development in rainbow trout. When monooxygenase activities were expressed on a per cytochrome P-450 basis, sex-dependent differences were observed only for AHH and ECOD activities. It was thus found that sex-dependent variations of END were closely correlated with the total amount of cytochrome P-450. The results indicate that differences exist in hepatic cytochrome P-450 isoenzyme patterns between the sexes in rainbow trout. The similarity of the annual pattern of plasma levels of oestradiol and testosterone to that of sex-dependent differences in the cytochrome P-450 monooxygenases support the contention that sex steroids play a role in regulating the cytochrome P-450 system.

Drug metabolizing capacity in vitro and in vivo--II. Correlations between hepatic microsomal monooxygenase markers in phenobarbital-induced rats.

Pretreatment with various doses of phenobarbital (PB) has been used to create a pool of rats with a wide range of hepatic microsomal monooxygenase activity to systematically examine relationships between and within in vivo and in vitro markers. The in vivo clearance of tolbutamide (TOL), theophylline (TH), antipyrine (AP) and its metabolites were determined in the same rats used for hepatic microsome preparation and assessment of P450 content and activities (via 7-ethoxycoumarin O-deethylase (ECOD), 7 ethoxyresorufin O-deethylase, 7-methoxycoumarin O-demethylase (MCOD) and aldrin epoxidase determinations). A graded dose-response relationship was found between PB treatment and most but not all parameters. The need for careful selection of in vivo and as well as in vitro markers is apparent from these studies. The most responsive parameters--TOL and AP clearances, MCOD and ECOD activities--were also those producing the strongest in vivo-in vitro correlations. Despite the diffuse nature of the PB induced response in P450 complement, good predictive relationships were apparent between ECOD and TOL clearance (r2 = 0.88).

Drug metabolizing capacity in vitro and in vivo--I. Correlations between hepatic microsomal monooxygenase markers in beta-naphthoflavone-induced rats.

Relationships between and within in vivo and in vitro markers of drug oxidative metabolism have been investigated in rats displaying a wide range of hepatic microsomal monooxygenase activity due to prior treatment with various doses of the inducing agent beta-naphthoflavone (BNF). BNF induction produced large dose-related changes in the in vivo clearance (CL) of theophylline (TH), antipyrine (AP) and the individual AP metabolite formation clearances, 4-hydroxyantipyrine (4H) and norantipyrine, and the in vitro parameters, 7-ethoxycoumarin O-deethylase, 7-ethoxyresorufin and P450. No trends were observed with the formation clearance of 3-hydroxymethylantipyrine and 7-methoxycoumarin O-demethylase whilst a negative response was observed with aldrin epoxidase. The selectivity of the markers towards BNF induction was coincident with the degree of covariance observed between these parameters. Strong correlations were observed in particular between CL(TH) and CL(4H) and ECOD and EROD indicating the high predictive value of these parameters. These studies demonstrate that under the well controlled conditions which may be imposed in animal environments predictively useful relationships (r2 greater than 0.8) can be established between in vitro and in vivo markers of hepatic microsomal monooxygenase activity.

Sexual dimorphism in avian hepatic monooxygenases.

Adult white Leghorn chickens exhibited a sexual dimorphism in hepatic microsomal monooxygenases determined from the concentrations of total cytochromes P450 and b5, and the metabolism of drug (hexobarbital, coumarin and ethoxyresorufin) and steroid (androstenedione and testosterone) substrates that were 2- to 4-fold greater in roosters than in hens. Caponizing at 6 weeks of age reduced the activities of the monooxygenases to levels comparable to those found in intact hens. In spite of the fact that testosterone replacement maximally stimulated comb growth in the capons and elevated (i.e. masculinized) hepatic monooxygenase activities in the hens to male-like levels, androgen replacement was ineffective in increasing the subnormal enzyme levels in the capons. While the failure of testosterone administration to restore monooxygenase levels in the capons may be explained by the immaturity of the birds at orchiectomy, the present results demonstrate, that like some mammals, birds may display gender differences in hepatic monooxygenases that are regulated by the testes.

Cytochrome P450 in trypanosomatids.

Post-mitochondrial supernatant extracts prepared from bloodstream forms of Trypanosoma brucei brucei, T. cruzi epimastigotes, Leishmania donovani promastigotes and Crithidia fasciculata have been found to catalyse cytochrome P450-dependent reactions. Appreciable ethoxycoumarin deethylase and ethoxyresorufin deethylase activities were found in all of the above trypanosomatids, with T. cruzi epimastigotes having the highest activity (57.1 and 10.7 pmol/min/mg protein, respectively). In all four species these reactions were inhibited by the cytochrome P450 inhibitors carbon monoxide, proadifen and metyrapone. In contrast to rat liver microsomes, the trypanosomatid extracts showed no detectable pentoxyresorufin depentylase or pentamidine hydroxylase activity. Both C. fasciculata and T. b. brucei post-mitochondrial supernatants showed carbon monoxide difference spectra consistent with the presence of cytochrome P450 (9.6 and 6.3 pmol/mg protein, respectively). An additional hemoprotein which gave a carbon monoxide difference peak at 420 nm was also detected in C. fasciculata and T. b. brucei microsomes and C. fasciculata mitochondria. Subcellular fractionation of both early and late log C. fasciculata showed that the ethoxycoumarin deethylase activity was enriched in the microsomal fraction.

Effect of eugenol on drug-metabolizing enzymes of carbon tetrachloride-intoxicated rat liver.

The chemoprotection extended by eugenol against carbon tetrachloride (CCl4) intoxication was established by studies on drug-metabolizing phase I and phase II enzymes. An overall decrease in drug-metabolizing enzymes, namely NADPH-cytochrome c reductase, NADH-cytochrome reductase, coumarin hydroxylase, 7-ethoxy coumarin-O-deethylase, UDP-glucuronyltransferase and glutathione-S-transferase, was observed with CCl4 intoxication, with a subsequent decrease in cytochrome P450 and cytochrome b5 content. CCl4 caused a significant decrease in microsomal phospholipids and the marker enzymes glucose-6-phosphatase and 5'-nucleotidase, and an increase in thiobarbituric acid reactive substances (TBARS). Simultaneous administration of eugenol with CCl4 inhibited the accumulation of TBARS and the decrease in the microsomal phospholipids and marker enzymes. Further, the chemical onslaught imposed by CCl4 on the drug-metabolizing system was removed successfully by eugenol. Eugenol appears to act as an in vivo antioxidant and as a better inducer of phase II enzymes than phase I enzymes. It is therefore suggested that eugenol could be an interesting basic structure for drug design.

Gamma-glutamyltransferase induction by dexamethasone in cytochrome P-450-depleted rat liver.

The effects of the cytochrome P-450 depletion by cobaltic protoporphyrin IX on the postnatal glucocorticoid-inducibility of the membrane-bound enzyme gamma-glutamyltransferase have been assessed in the rat liver. Dexamethasone-induced gamma-glutamyltransferase activity in 14-, 28- and 77-day-old rats was high, weak and absent, respectively, and inversely correlated with the physiological cytochrome P-450 activity. In the liver acinus, the enzyme was reexpressed by the zone 1 and zone 2 hepatocytes in suckling rats, substantially only by the zone 1-hepatocytes in just weaned rats. Following cytochrome P-450 depletion, gamma-glutamyltransferase induction by dexamethasone was more rapid, more intense and more extended in the liver acinus, occurring also in the zone 3 hepatocytes in suckling rats, in the zone 2 and a few zone 3 hepatocytes in just weaned rats. Further, the enzyme induction occurred also in adult rats in the zone 1 and in some zone 2 cells. This shows that cytochrome P-450 modulates the extent of hepatic gamma-glutamyltransferase induction by dexamethasone in postnatal rat-hepatocytes. The phenomenon may be consequent on hormone biotransformation changes caused by the cytochrome P-450 depletion.

Nerval influences on liver cytochrome P450.

In male young adult Wistar rats the influences of nucleus raphe electrocoagulation, spinal cord dissection (cordotomy between C7 and Th1), vagotomy and denervation of liver hilus by phenol on liver cytochrome P450-system (cytochrome P450 concentration, ethylmorphine N-demethylation and ethoxycoumarin O-deethylation activities, hexobarbitone sleeping time) were investigated. In general the influences were small or negligible when compared with sham operated controls, only after vagotomy the depressing effect of sham operation was abolished. In all cases sham operation had a depressing effect until up to five weeks after operation.

Influence of skin and muscle lesions on the hepatic cytochrome P450 function in 10 and 60 day-old male Wistar rats.

In 10 and 60 day-old male Wistar rats skin lesions of different extents and muscle lesions lead to decreases in cytochrome P450 concentrations and monooxygenase activities (ethylmorphine N-demethylation and ethoxycoumarin O-deethylation) at least up to 7 days after operation. The extent of the depression was related to the extent of the lesion and independent of the nature of the tissue involved.

Comparison of in vitro and in vivo biotransformation in patients with liver disease of differing severity.

The activity of 7-ethoxycoumarin O-deethylase (ECOD) has been measured in liver biopsy samples from 23 patients (smokers and non-smokers) with different degrees of structural liver damage. The results, which reflect in vitro cytochrome P450-dependent biotransformation, were correlated with various measures of the P450-dependent in vivo elimination of caffeine and metamizol. The relatively non-specific, low affinity component of ECOD activity was significantly correlated with the kinetics of metamizol (mean residence time, apparent clearance, half-life, area under the concentration-time curve, and metabolite excretion in the urine). Thus, metamizol elimination, which is mainly due to P450 IIB, and the low affinity component of ECOD both reflect, at least in part, the activity of the same form of P450. In contrast, caffeine biotransformation, which is via P450 IA, was not correlated with ECOD activity. There was no relation between the kinetics of metamizol and caffeine, perhaps because of the inducing effect that smoking has on caffeine elimination. In patients with liver disease, smoking appears to alter the elimination of caffeine more than the degree of liver disease.

[7-ethoxycoumarin o-deethylase and fibrosis in chronic liver diseases]. / Die 7-Ethoxycoumarin O-Deethylase und der fibrosierende Prozess bei chronischen Lebererkrankungen.

In the liver biopsy of 100 patients with chronic liver diseases, the activity of 7-ethoxycoumarin O-deethylase (ECOD) was determined as a parameter of hepatic monooxygenase system and was compared with some markers of fibrosis e.g. collagen peptidase and hydroxyproline. ECOD was significantly different in healthy liver, fatty liver, chronic active hepatitis (CAH) and cirrhosis. The importance of the fibrotic process was shown by the significant correlations between ECOD and the signs of fibrosis in the liver biopsy. A connection between ECOD and the markers of fibrosis was not found. Further research is necessary to clarify this difference.

Automated high-performance liquid chromatographic assay for the determination of 7-ethoxycoumarin and umbelliferone.

An improved high-performance liquid chromatographic assay is presented for the determination of 7-ethoxycoumarin O-deethylase activity. Following a 30-min microsomal incubation, 7-ethoxycoumarin, 4-methylumbelliferone (internal standard), and the metabolite umbelliferone were extracted with chloroform. Separation was achieved with an isocratic mobile phase using a microBondapak phenyl (300 mm x 3.9 mm I.D.) analytical column. The effluent was monitored by fluorescence detection with an excitation wavelength of 360 nm and an emission wavelength of 470 nm. The intra- and inter-assay coefficients of variation were 10 and 6%, respectively. A detection limit of 0.07 micrograms/ml was achieved, making this method suitable for characterizing P-450 activity of human livers.

Lack of effect of streptogramins on hepatic drug metabolism enzymes in the rat.

Male Sprague-Dawley rats were treated with streptogramin derivatives (RP 7293, RP 54476, RP 57669, and RP 59500) or with the macrolide troleandomycin. Liver cytosol and microsomes were prepared, and the in vitro transformation of several model substrates studied. Furthermore, total and complexed microsomal cytochrome P-450 levels were compared. Hepatic cytochrome P-450 metabolite complexes were detected 4 days after troleandomycin treatment (500 mg/kg/day po), whereas such effects were not observed with po RP 7293 (500 mg/kg/day, 4 days) or with iv RP 54476 (12 mg/kg/day, 7 days), RP 57669 (6 mg/kg/day, 7 days), or RP 59500 (6 and 18 mg/kg/day, 7 days). The administration of troleandomycin resulted in statistically significant increases in liver weight (+20%), microsomal protein (+70%), total cytochrome P-450 (+187%), and cytosolic glutathione S-transferase activity (+32%). The activities of aniline hydroxylase, aminopyrine N-demethylase, and the high and low phases of 7-ethoxyresorufin O-deethylase were markedly decreased by 36% to 56%. In contrast, none of these hepatic parameters was changed significantly after administration of each streptogramin. These results suggest that streptogramins have not, in contrast to many commonly used macrolide antibiotics, had potent or specific effects on hepatic drug metabolizing enzymes in rats.

Biochemical responses and environmental contaminants in breams (Abramis brama L.) caught in the river Elbe.

Adult breams (Abrama brama L.) were caught in October 1992 at seven stations in the river Elbe and at one nonpolluted reference site, the Belauer See. The locations of the sampling stations extended from the city of Steti (Tschechien Republic) to the city of Hamburg. Indices of biochemical effects in microsomal and cytosolic fractions of livers were studied by measuring cytochrome P450-dependent monooxygenases and glutathione-S-transferase (GST) activities. In addition, levels of mercury and 35 polychlorinated biphenyl (PCB) congeners were analyzed in livers of breams. Fish caught in the River Elbe exhibited a significant increase of cytochrome P450-mediated monooxygenase activities and the detoxifications enzyme GST compared to the reference site. At two stations of the river Elbe (Steti and Dresden) elevated activities of ethoxyresorufin-O-deethylase (EROD) were analyzed. These effects were discussed as effects from the pulp mill industries at station Steti and high concentrations of PCBs in the livers of breams at station Dresden. A significant reduction of GST activities was observed at station Dresden compared to those at Steti. These findings were probably a synergistic effect of high mercury concentrations at Dresden. The results presented in this study suggest that breams can be successfully employed for monitoring biological effects in the river Elbe.

A microfluorometric method for measuring ethoxycoumarin-O-deethylase activity on individual Drosophila melanogaster abdomens: interest for screening resistance in insect populations.

We developed a method for measuring ethoxycoumarin deethylase (ECOD) activity using a single Drosophila abdomen. The activities obtained were well correlated with the classic method from microsomes (r = 0.902). This new method, performed in microtitration plates, was at least six times more sensitive compared to the conventional cuvet fluorometric one. Moreover, it was possible among a large number of insects to differentiate those with low or high ECOD activities. This improved procedure has been checked upon crosses between resistant strain (with high ECOD activity) and susceptible strain (with low ECOD activity). The results demonstrate the possible separation of resistant phenotypes and emphasize the importance of this approach in assessing the spreading of insecticide resistance in natural populations of insects.

The use of porcine hepatocytes for biotransformation studies of veterinary drugs.

1. Cultures of porcine hepatocytes with high viability were isolated from a liver sample by a simple procedure. In ageing monolayer cultures the cytochrome P-450 content and 7-ethoxycoumarin-O-deethylase activity decreased gradually while glutathione levels increased. 2. The nitrofuran, furazolidone, was rapidly metabolized, partly resulting in the formation of 3-(4-cyano-2-oxobutylidene amino)-2-oxazolidone. 3. Acetylating and deacetylating activities towards sulphadimidine and its N4-acetyl metabolite were present in porcine hepatocytes. Relative and absolute levels of these activities varied in different batches of hepatocytes. 4. No differences were seen in a number of enzyme activities measured in cytosolic and microsomal fractions isolated from different lobes of one liver. Differences between livers from different animals were marked.