Metabolism Of 7-ethoxycoumarin Research Articles

Metabolism of 7-ethoxycoumarin, flavanone and steroids by cytochrome P450 2C9 variants.

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 (450nm) 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.

Drug Metabolite Heterogeneity in Cultured Single Cells Profiled by Pico-Trapping Direct Mass Spectrometry

We investigated the heterogeneity of tafluprost metabolism in primary human hepatocytes at a single-cell level by live single-cell mass spectrometry (MS). Picoliter volumes of cytoplasm were analyzed by nano-electrospray ionization MS in order to obtain single-cell metabolite profiles. The subcellular components of a single tafluprost-treated human hepatocyte were isolated and the single-cell metabolite profile was compared with those of traditional bulk hepatocyte analysis. In the bulk hepatocyte analysis, liquid chromatography-MS showed the averaged metabolism of tafluprost to tafluprost acid (TA) and β-oxidized metabolites. However, live single-cell MS showed that tafluprost metabolism varied among individual cells. In addition, there was significant variation in the quantities of TA and a major metabolite, dinor-TA, among cells, whereas there was no significant variation in 7-ethoxycoumarin metabolism. Thus, live single-cell MS successfully detected the heterogeneity of drug metabolism in individual living hepatocytes.

Gold Nanoparticles Uptake and Cytotoxicity Assessed on Rat Liver Precision-Cut Slices

A major obstacle in the field of nanotoxicology is the development of an in vitro model that accurately predicts an in vivo response. To address this concern, rat liver precision-cut slices were used to assess the impact of 5-nm gold nanoparticles (GNPs) coated with polyvinylpyrrolidone (PVP) on the mammalian liver, following exposure to different concentrations and for a duration of up to 24 h. The presence of GNPs inside endocytotic vesicles of hepatocytes was appreciable within 30 min of their addition. After 2 h, GNPs were clearly visualized inside endosome-like vesicles within the slice, not only in hepatocytes but also in endothelial and Kupffer cells located within the first two cellular layers. This uptake did not translate into modifications of either phase I or phase II of 7-ethoxycoumarin metabolism or alter activities of cytochrome P450 toward marker substrates. Furthermore, although the GNPs were rapidly internalized, no overt signs of cytotoxicity, assessed through lactate dehydrogenase release, reduction of methylthiazolyldiphenyl tetrazolium bromide, and glutathione levels, were observed. In conclusion, the use of rat liver slices successfully enhanced nanomaterial screening and determined that PVP-coated 5-nm GNPs were biocompatible with rat liver cells.

Gold nanoparticles uptake and cytotoxicity assessed on rat liver precision cut slices

Nanotechnology research and development have been rapidly growing worldwide for the past decade. Although nanomaterials (NM)s provide great potential of commercial benefits, some of them have been claimed to be toxic in in vivo and in vitro tests and our knowledge on their toxicological properties is far from being comprehensive. With the current technological capabilities toxicological and environmental risk assessment of NPs struggles to keep up with all newly synthesized NMs. For this reason several authors have underlined the need to develop reliable in vitro models for studying their toxic effects. In order to set up a novel tool for the risk assessment of nanoparticles toxicity we evaluated cell uptake and toxicity of 5 nm gold nanoparticles (GNP)s coated with polyvynylpirrolidone (PVP) on an in vitro model system represented by rat liver “precision cut” slices. Rat liver precision cut slices were incubated individually at 37°C in RPMI 1640 medium for up to 24 h in the presence of GNPs at different concentrations. The presence of GNPs inside endocytotic vesicles of hepatocytes was appreciable already after 30 min incubation. From 2h onwards, GNPs cell uptake was substantiated by their presence inside endosome-like vesicles not only in hepatocytes, but also in endothelial and Kuppfer cells. After 6h GNPs could be spotted within lysosomes located close to the biliary pole of the hepatocytes. These results were confirmed in vivo with few experiments carried out upon i.p injection of GNPs. This did not translate into modification of both phase I and phase II of 7-ethoxycoumarin metabolism. Similarly, CYPs activities of slices microsomal preparations towards marker substrates resulted unaffected. Although GNPs were promptly taken up by slices, no signs of cytotoxicity were observed. In fact, LDH release, MTT reduction and GSH levels in GNPs-treated slices were not different from those of control slices. In conclusion, rat liver precision cut slices can be employed for assessing the consequences of the biological impact of nanoparticles.

Evaluation of HepaRG Cells as an in Vitro Model for Human Drug Metabolism Studies

HepaRG cells, a newly developed human hepatoma cell line, differentiate into hepatocyte-like morphology by treatment with dimethyl sulfoxide (DMSO). The expression of cytochrome P450 (P450) enzymes, transporter proteins, and transcription factors was stable in differentiated HepaRG cells over a period of 6 weeks when cultured with DMSO. Compared with human hepatocytes, expression of P450 in HepaRG cells was in general lower with the exception for a considerably higher expression of CYP3A4 and CYP7A1. The expression of P450s generally decreased when DMSO was removed from the medium, whereas transporters and liver-specific factors were unaffected. The relative mRNA content of drug-metabolizing P450s displayed the highest resemblance between human hepatocytes and differentiated HepaRG cells 1 day after removal of DMSO from the medium. The metabolism of midazolam, naloxone, and clozapine in HepaRG cells was similar to human hepatocytes, indicating the function of CYP3A4, CYP1A2, and UDP-glucuronosyltransferase enzymes. However, the metabolism of 7-ethoxycoumarin and dextromethorphan was low, confirming low levels of CYP2E1 and CYP2D6 in HepaRG cells. The P450 probe substrates indicate a decrease in CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activities in HepaRG cells 1 day after removal of DMSO from the medium. The activities were then relatively stable in DMSO-free medium for up to 14 days. Based on the stable expression of liver-specific functions over a long period in culture, the relative mRNA content of drug-metabolizing P450s, and metabolic properties, HepaRG cells provide a valuable in vitro model for human drug metabolism studies.

Metabolic and structural viability of precision-cut rat lung slices in culture

The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72 h.Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with β-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600 µm.Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8 h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72 h incubation.Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24 h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24 h; longer incubations did not result in further leakage.It is concluded that precision-cut rat lung slices, of 600 µm thickness, can be maintained metabolically viable in culture for some 8 h.

Functional co-expression of xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 and UDP-glucuronosyltransferase 1A6, in yeast microsomes.

Xenobiotic Phase I and Phase II reactions in hepatocytes occur sequentially and cooperatively during the metabolism of various chemical compounds including drugs. In order to investigate the sequential metabolism of 7-ethoxycoumarin (7EC) as model substrate in vitro, xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 (P450 1A1) and UDP-glucuronosyltransferase 1A6 (UGT1A6) were co-expressed in Saccharomyces cerevisiae AH22. Rat P450 1A1 and yeast NADPH-P450 reductase were expressed on a multicopy plasmid (pGYR1) in the yeast. Rat UGT1A6 cDNA with a yeast alcohol dehydrogenase I promoter and terminator was integrated into yeast chromosomal DNA to achieve the stable expression. Co-expression of P450 1A1 and UGT1A6 in yeast microsomes was confirmed by immunoblot analysis. Protease treatment of the microsomes showed the correct topological orientation of UGT to the membranes. The metabolism of 7EC to 7-hydroxycoumarin (7HC) and its glucuronide in yeast microsomes was analyzed by reverse phase HPLC. In a co-expression system containing 7EC, NADPH and UDP-glucuronic acid, glucuronide formation was detected after a lag phase, following the accumulation of 7HC. In the case of P450 1A1 and UGT1A6, efficient coupling of hydroxylation and glucuronidation in 7EC metabolism was not observed in the co-expression system. This P450 and UGT co-expression system in yeast allows the sequential biotransformation of xenobiotics to be simulated in vitro.

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

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.

Drug-metabolizing activity of human and rat liver, lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo Vmax in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.

Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers.

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.

Effects of waterborne nitrite onphase I–II biotransformation in channel catfish( Ictalurus punctatus)

The effects of waterborne nitrite (3 mg/l NO 2) on channel catfish were studied to evaluate changes in hematological parameters and phase I–II biotransformation in liver slices. Nitrite-exposed fish had significantly higher methemoglobin, blood and liver nitrite, and significantly lower pO 2 than control fish. Total phase I-mediated metabolism of 7-ethoxycoumarin (EC) was not altered in nitrite-exposed fish compared with control fish (291±43 and 312±20 pmol/mg/h, respectively). However, phase II glucuronosyltransferase-mediated metabolism of 7-hydroxycoumarin (HC), both as a phase I metabolite of EC and as a parent substrate, was elevated in nitrite-exposed fish (204±17 and 1007±103 pmol/mg/h, respectively) as compared to control fish (149±14 and 735±87 pmol/mg/h) ( P<0.05). Sulfotransferase-mediated metabolism of HC (as a metabolite of EC and as a parent substrate) was not notably altered in nitrite-exposed fish (95±16 and 617±33 pmol/mg protein/h, respectively) as compared with control fish (118±24 and 575±55 pmol/mg/h, respectively). These studies indicate that in vivo nitrite exposure and associated changes in hematological parameters do not appear to affect hepatic phase I EC biotransformation in channel catfish. However, subtle but significant changes in phase II glucuronidation, but not sulfation activity, were observed. The mechanism of these alterations is unclear. However, the data suggest that environmentally realistic concentrations of nitrite may affect the dynamics of conjugative metabolism in exposed fish.

Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes.

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7x10(7) cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.

Structural and functional integrity of precision-cut liver slices in xenobiotic metabolism: a comparison of the dynamic organ and multiwell plate culture procedures

1. Objectives were two-fold: (1) to compare the viability of precision-cut liver slices in two culture systems, namely the dynamic organ and the multiwell plate; and (2) to evaluate whether increasing the number of slices per incubation results in a proportional increase in the extent of metabolism. 2. With both culturing systems, the major products of 7-ethoxycoumarin metabolism were the sulphate and glucuronide conjugates of 7-hydroxycoumarin with very low levels of the free compound. When the multiwell plate procedure was used, metabolism increased linearly for at least 10h, whereas it tended to plateau after 6h in the dynamic organ culture system. At preincubations 10h, significantly more metabolism of 7-ethoxycoumarin was seen in the slices cultured using the multiwell system compared with the dynamic organ system. 3. Morphological evaluation employing light and electron microscopy revealed that liver slices incubated using the multiwell system were structurally better preserved compared with those incubated using the dynamic organ system. 4. Using the multiwell system, increasing the number of slices per incubation from one to two resulted in only a modest increase in the metabolism of 7-ethoxycoumarin. The rate of metabolism of this substrate was much higher with one liver slice when expressed per mg homogenate protein. 5. It is concluded that (1) the multiwell plate culture system for culturing slices is superior to the dynamic organ system in studying the metabolism of xenobiotics following long-term incubations, (2) increasing the number of slices per incubation does not result in a corresponding increase in the rate of metabolism, and (3) in both culture systems optimal viability appears to be within 24h of incubation.

Influence of Slice Thickness and Culture Conditions on the Metabolism of 7-Ethoxycoumarin in Precision-cut Rat Liver Slices

This study investigated the effects of some experimental variables on the rate of xenobiotic metabolism in precision-cut rat liver slices. Liver slices of 123 ± 8μm (mean ± SEM of six slices), 165 ± 3μm, 238 ± 6μm and 515 ± 14μm thickness were prepared from male Sprague-Dawley rats, and incubated in RPMI 1640 medium in an atmosphere of 95% O2/5% CO2by using a dynamic organ culture system. Liver slices of all thicknesses metabolised 10μM 7-ethoxycoumarin to total (free and conjugated) 7-hydroxycoumarin in a time-dependent manner. The rate of 7-ethoxycoumarin metabolism was greatest in 165μm thick slices and slowest in 515μm thick slices, being 2.74 ± 0.19pmol/minute/mg slice protein and 0.69 ± 0.07pmol/minute/mg slice protein, respectively. No marked effects on the rate of 7-ethoxycoumarin metabolism in liver slices were observed either by changing the medium to Earle's balanced salt solution (EBSS) or by changing the gas phase to 95% air/5% CO2. Moreover, the perfusion of rat livers with EBSS at 2–4°C, prior to preparation of tissue cores, did not enhance 7-ethoxycoumarin metabolism in rat liver slices. In this study, the optimal slice thickness was 175μm, with higher rates of 7-ethoxycoumarin metabolism being observed than with 250μm thick slices, which are often used for studies of xenobiotic metabolism. Variable results were obtained with slices of around 100–120μm thickness, which may be attributable to the ratio between intact hepatocytes and cells damaged by the slicing procedure in these very thin slices.

Stoichiometry of 7-ethoxycoumarin metabolism by cytochrome P450 2B1 wild-type and five active-site mutants

Recombinant P450 2B1 wild-type and the active-site mutants I114V, F206L, V363A, V363L, and G478S were purified and studied. The efficiency of coupling of reducing equivalents to 7-hydroxycoumarin formation was decreased for all the mutants except I114V. Uncoupling to H 2O was increased for F206L, V363A, and G478S, decreased for V363L, and unchanged for I114V. Uncoupling to H 2O 2 was increased for V363L and decreased for I114V, F206L, and V363A. The findings from this study provide firm biochemical evidence that residues 206, 363, and 478 comprise part of the substrate binding site of P450 2B1.

Metabolism of [ring-U- 14C] agaritine by precision-cut rat, mouse and human liver and lung slices

Agaritine {(β- N-[γ- l(+)glutamyl]-4-hydroxymethylphenylhydrazine)} is present in the common cultivated mushroom Agaricus bisporus and several agaritine derivatives have been shown to produce tumours in experimental animals. In this investigation the metabolism of [ring-U- 14C]agaritine has been studied in precision-cut rat, mouse and human liver slices and in precision-cut rat and mouse lung slices. To confirm the functional viability of the tissue slice preparations, the metabolism of 7-ethoxycoumarin was also studied. Liver and lung slices from all species metabolized 50 μM 7-ethoxycoumarin to 7-hydroxycoumarin, which was conjugated with D-glucuronic acid and sulfate. Incubation of rat, mouse and human liver slices, and rat and mouse lung slices with 25 μM [ 14C]agaritine resulted in a time-dependent formation of metabolite(s), which bound covalently to tissue slice proteins. Agaritine metabolite covalent binding was greater in mouse liver than in rat and human liver slices and was greater in mouse lung than in rat lung slices. No correlation was observed between agaritine metabolite covalent binding and tissue slice γ-glutamyltransferase activity. Additional studies with mouse liver slices showed that [ 14C]agaritine was also metabolized to a number of unknown polar metabolites. These results demonstrate that agaritine can be metabolized by enzymes present in mammalian liver and lung.

Comparison of the metabolism of 7-ethoxycoumarin and coumarin in precision-cut rat liver and lung slices

The metabolism of 7-ethoxycoumarin and [3- 14C] coumarin was compared in precision-cut rat liver and lung slices. The lung slices were prepared using an agarose gel instilling technique enabling the production of tissue cylinders followed by lung slices employing a Krumdieck tissue slicer. Both 50μM 7-ethoxycoumarin and 50μM [3- 14C]coumarin were metabolized by rat liver and lung slices. 7-Ethoxycoumarin was converted to 7-hydroxycoumarin (7-HC) which was conjugated with both d-glucuronic acid and sulfate. 7-HC sulfate was the major metabolite formed by both liver and lung slices. [3- 14C]Coumarin was metabolized by rat liver and lung slices to both polar products and to metabolite(s) that bound covalently to tissue slice proteins. The polar products included unidentified metabolites and 3-hydroxylation pathway products, with only very small quantities of 7-HC being formed. These results demonstrate that precision-cut lung slices area useful model in vitro system for studying the pulmonary metabolism of xenobiotics. Moreover, the precision-cut tissue slice technique may be employed for comparisons of hepatic and extrahepatic xenobiotic metabolism.

Dynamic Organ Culture is Superior to Multiwell Plate Culture for Maintaining Precision-Cut Tissue Slices: Optimization of Tissue Slice Culture, Part 1

The increase in the use of precision-cut tissue slices for metabolism and toxicity studies has resulted in the development of various incubation systems and techniques for culturing the slices. This has led to inconsistencies in data obtained from different laboratories. For data to be comparable from one laboratory to another, reliable and consistent incubation systems must be used that will give the researcher the most optimal tissue slice viabilities. This study compares and contrasts the dynamic organ culture system (surface culture) and the multiwell plate culture system (submersion culture). Rat liver slices were produced using the Brendel/Vitron tissue slicer under oxygenated and ice-cold V-7 preservation solution. The slices (200 μm thick) were incubated in Waymouth's medium + 10% fetal calf serum (1.7 mL) containing either sodium bicarbonate or Hepes and gassed with either 95%/5% O2/CO2 or 95%/5% air/CO2. Slice viability was assessed at 6, 24, 48, and 72 h using ATP content, K+ retention, protein synthesis, alanine aminotransferase and lactate dehydrogenase leakage, and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reduction. 7-Ethoxycoumarin metabolism was also used to assess the xenobiotic metabolic activity of liver slices. The results indicated that the dynamic organ culture system maintained rat liver slices at a higher level of viability than the multiwell plate culture system and that Waymouth's medium gassed with 95% O2/5% CO2 was the best incubation condition for both systems. Thus, it is essential to optimize slice viability in order to obtain reliable data that will ultimately be used to predict what will be seen in humans.

Effect of peroxisome proliferators and inducers of xenobiotic metabolism on marker enzyme activities in cultured rat liver slices

Previous work has demonstrated that precision-cut liver slices represent a valuable in vitro system for studying the metabolism and toxicity of xenobiotics. We have investigated their use for enzyme induction studies. Liver slices were prepared from male Sprague-Dawley rats and by using a dynamic culture system could be maintained in RPMI 1640 medium for up to 72 hr. The addition of 0.02–0.5 m m concentrations of ciprofibrate, nafenopin and Wy-14,643 for 48 and 72 hr produced a concentration and time dependent induction of palmitoyl-CoA oxidation and carnitine acetyltransferase activities. Ultrastructural examination of liver slices revealed an increase in the number of peroxisomes. Liver slices were also cultured with 0.2–5 m m phenobarbitone, 5–50 μ m β-naphthoflavone and 0.5–20 μg Aroclor 1254/ml and effects on the metabolism of 7-ethoxycoumarin, 7-benzoxyresorufin and 7-ethoxyresorufin determined. All three compounds produced a concentration and time dependent induction of mixed-function oxidase enzyme activities. The results of this study demonstrate that rat liver slices can be cultured for up to 72 hr and appear to retain functional viability in that they respond to some known peroxisome proliferators and inducers of xenobiotic metabolism. Cultured liver slices may thus be a useful alternative in vitro system to hepatocytes for screening compounds for effects on various enzyme activities.

Metabolism of coumarin and 7-ethoxycoumarin by rat, mouse, guinea pig, cynomolgus monkey and human precision-cut liver slices.

1. The metabolism of 50 microM 7-ethoxycoumarin and 50 microM [3-14C]coumarin has been studied in precision-cut liver slices from the male Sprague-Dawley rat, female DBA/2 mouse, male Dunkin-Hartley guinea pig, male Cynomolgus monkey and man. 2. In liver slices from all five species 7-ethoxycoumarin was metabolized to 7-hydroxycoumarin (7-HC), which was extensively conjugated with D-glucuronic acid and sulphate. In rat and mouse, 7-HC was preferentially conjugated with sulphate, whereas rates of glucuronidation and sulphation were similar in the other three species. 3. [3-14C]coumarin was metabolized by liver slices from all five species to various polar products and to metabolite(s) that bound covalently to liver slice proteins. In Cynomolgus monkey and both human subjects studied, 7-HC was the major metabolite that was conjugated with D-glucuronic acid and sulphate, whereas in rat the major metabolites were products of the 3-hydroxylation pathway and unknown metabolites. Major metabolites in mouse liver slices were 7-HC, 3-hydroxylation pathway products and unknown metabolites, and in guinea pig liver slices, 7-HC and unknown metabolites. 4. The metabolism of 7-ethoxycoumarin to free and conjugated 7-HC and [3-14C]coumarin to total polar products was greater in liver slices from mouse and Cynomolgus monkey than the other three species. 5. With liver slices from all five species there appeared to be little difference in the extent of metabolism of 7-ethoxycoumarin and [3-14C]coumarin to various products in either a complex tissue culture medium (RPMI 1640 plus foetal calf serum) or a simple balanced salt solution (Earle's balanced salt solution). 6. These results demonstrate that precision-cut liver slices are a valuable in vitro model system for investigating species differences in xenobiotic metabolism. Generally, the observed species differences in coumarin metabolism in vitro agree well with available in vivo data.