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

1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72?h.

2. 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.

3. 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.

4. 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.

5. It is concluded that precision-cut rat lung slices, of 600?µm thickness, can be maintained metabolically viable in culture for some 8?h.

     

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 V(max) 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.     

Liver slices in dynamic organ culture. II. An in vitro cellular technique for the study of integrated drug metabolism using human tissue

1. Precision cut human liver slices in dynamic organ culture have been used to study the integrated metabolism of 7-ethoxycoumarin and the conjugation of 7-hydroxycoumarin. 2. The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was monitored for 6 h. For both substrates there was a time-dependent increase in metabolites present in the incubation medium. The low levels of free 7-hydroxycoumarin found in the medium when 7-ethoxycoumarin was the substrate suggests good coupling of phase I and phase II metabolism. 3. With suitable incubation conditions, i.e. change of medium containing new substrate every 2 h, the metabolism of both 7-ethoxycoumarin and 7-hydroxycoumarin by human liver slices was found to proceed at similar rates for up to 24 h. This was demonstrated using five separate human liver preparations. 4. Human liver slices also metabolized mono-chlorobenzene and o-, m- and p-dichlorobenzene to aqueous soluble metabolites. There was a time-dependent increase in the appearance of aqueous soluble metabolites present in the incubation medium. Metabolites were not retained by the liver slices. 5. A cold-storage transit buffer has been described and used to maintain the levels of drug metabolism in both rat and human tissue for periods of up to 6 h. 6. The use of human liver slices in dynamic organ culture as a suitable method for the direct assessment of integrated hepatic drug metabolism is proposed.     

Induction of phase I and II drug metabolism in rat small intestine and colon in vitro.

The aim of this study was to evaluate drug metabolism in rat small intestinal and colon precision-cut slices during 24 h of incubation and the applicability of these slices for enzyme induction studies. Various parameters were evaluated: intracellular levels of ATP (general viability marker), alkaline phosphatase activity (specific epithelial marker), villin expression (specific epithelial marker), and metabolic rates of 7-ethoxycoumarin (CYP1A), testosterone (CYP3A and CYP2B), and 7-hydroxycoumarin (glucuronide and sulfate conjugation) conversions. ATP and villin remained constant up to, respectively, 5 and 8 h in small intestine and up to 24 h in colon. The metabolic rate remained constant in small intestinal slices up to 8 h and decreased afterward to 24 to 92%, depending on the substrate studied. The inducibility of metabolism in small intestinal and colon slices was tested with several inducers at various concentrations and incubation times. The following inducers were used: 3-methylcholanthrene, beta-naphthoflavone, indirubin, and tert-butylhydroquinone (aryl hydrocarbon receptor ligands), dexamethasone (glucocorticoid receptor/pregnane X receptor ligand) and phenobarbital (constitutive androstane receptor ligand). After incubation with inducers, metabolic rates were evaluated with 7-ethoxycoumarin and testosterone (phase I) and 7-hydroxycoumarin (phase II) as substrate. All inducers elevated the metabolic rates consistent with the available published in vivo induction data. Induction of enzyme activity was already detectable after 5 h (small intestine) and after 8 h (colon) for 3-methylcholanthrene and beta-naphthoflavone and was clearly detectable for all tested inducers after 24 h (up to 20-fold compared with noninduced controls). In conclusion, small intestinal and colon precision-cut slices are useful for metabolism and enzyme induction studies.     

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 V_max 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.     

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 10 h, whereas it tended to plateau after 6 h in the dynamic organ culture system. At preincubations > 10 h, 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 24 h of incubation.     

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon

1: The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included. 2: The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon. 3: The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance. 4: It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.     

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver,lung, kidney,small intestine and colon

1.?The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included.

2.?The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon.

3.?The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance.

4.?It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.     

Liver slices in dynamic organ culture. I. An alternative in vitro technique for the study of rat hepatic drug metabolism

1. Precision-cut liver slices in dynamic organ culture, a novel in vitro technique, is described and applied to the study of hepatic drug metabolism in the rat. 2. These slices catalysed the oxidative O-deethylation of the substrate, 7-ethoxycoumarin, over 6 h incubation. In addition, the direct conjugation of 7-hydroxycoumarin with either sulphate or glucuronic acid was maintained over 6 h. 3. The formation of 7-hydroxycoumarin and the presence of the sulphate and glucuronide conjugates in slices exposed to 7-ethoxycoumarin demonstrated integrated phase I and phase II drug metabolizing activities in this system. 4. Minor modifications of the incubation system allowed for the metabolism of four volatile chlorinated benzenes: monochlorobenzene 1,2-, 1,3-, and 1,4-dichlorobenzenes to aqueous soluble metabolites. 5. The use of liver slices in dynamic organ culture as an alternative preparation for the study of xenobiotic metabolism is discussed.     

Stability of cytochromes P450 and phase II conjugation systems in precision-cut rat lung slices cultured up to 72 h

The objective of the present study was to evaluate the stability of cytochrome P450 enzymes and of the conjugation enzyme systems epoxide hydrolase, glucuronosyl transferase, sulphotransferase and glutathione S-transferase in precision-cut rat lung slices incubated in RPMI media for different time periods up to 72 h. Moreover, the effect of culturing of lung slices on total glutathione levels and glutathione reductase was also investigated. Monitoring of cytochrome P450 activity was achieved using established diagnostic probes, but when activity in the lung was low the maintenance of the various enzymes in culture was determined immunologically using Western blotting. The dealkylation of pentoxyresorufin declined markedly during the first 4h of incubation but in the case of ethoxyresorufin loss of activity was more gradual and less severe. Western blot analysis revealed that the rate of decrease in cytochrome P450 apoprotein levels was isoform-specific with CYP2E1 being the most stable and CYP3A the least stable. Generally, phase II activities, especially cytosolic sulphotransferase, were relatively more stable throughout the incubation period compared with cytochromes P450. Finally, glutathione reductase activity and total glutathione levels were maintained throughout the 72 h incubation. The present studies indicate that xenobiotic-metabolising enzymes in precision-cut rat lung slices decline in culture, but the rate of loss differs and depends on the nature of the enzyme.     

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

1. The metabolism of 50 μM 7-ethoxycoumarin and 50 μM [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 avaluable 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.     

Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.     

HISTOLOGICAL AND BIOCHEMICAL EVALUATION OF PRECISION-CUT LIVER SLICES

Human and animal precision-cut tissue slices are being widely used to obtain drug metabolism and toxicity profiles in vitro. These data are then used to predict what might be seen in human patients. The accuracy of the extrapolation of findings based on human or animal in vitro systems to the findings that occur in vivo is dependent on the quality of the in vitro system. This study investigates the optimal thickness of tissue slices and the optimal incubation system, as determined by histological evaluation (light and electron microscopy) and two biochemical parameters (adenosine triphosphate [ATP] content and potassium [K +] retention). The three incubation systems tested were the dynamic organ culture system and the roller incubation system for volatiles, which are surface culture systems, and the multiwell plate culture system, which is a submersion culture system. The cellular glycogen content of canine liver slices was determined using slices fixed in 10% buffered formalin and a periodic-acid Schiff stain. After determining the optimal slice thickness (≈150 μm) and incubation system (the dynamic organ culture system) using gross histological evaluation, the liver slices were incubated for 6, 12, 24, 48, and 72 h, and the slices were evaluated using electron microscopy. The micrographs showed that the hepatocytes displayed normal morphology for up to 72 h. These histological findings were compared to the biochemical findings. ATP content was found to be more sensitive in detecting cellular degeneration than K + retention but less sensitive than the histological evaluation. The use of tissue slices that are produced and incubated under optimal conditions can be a reliable in vitro method of investigating morphologic and metabolic alterations produced by a variety of agents, including drugs and toxicants.     

Metabolism of nicotine and induction of CYP1A forms in precision-cut rat liver and lung slices.

The aim of this study was to investigate xenobiotic metabolism and induction of cytochrome P450 (CYP) forms in precision-cut rat liver and lung slices, employing nicotine as a model compound. Freshly cut rat liver and lung slices metabolised nicotine to the major metabolite cotinine. Observed Km values for cotinine formation in liver and lung slices were 323 and 41.7 microM, respectively, with corresponding V(max) values of 47.2 and 3.21 pmol/min/mg protein, respectively. Rat liver and lung slices were cultured for 48 h with Aroclor 1254, benzo(a)pyrene, nicotine and cotinine. Both Aroclor 1254 and benzo(a)pyrene produced a marked induction of CYP1A-dependent 7-ethoxyresorufin O-deethylase activity in both liver and lung slices. However, while nicotine induced 7-ethoxyresorufin O-deethylase activity in lung slices, but not in liver slices, cotinine did not induce enzyme activity in either liver or lung slices. Overall, while higher rates of nicotine metabolism were observed in rat liver slices, nicotine-induced CYP1A form induction was observed in lung slices. These results demonstrate the usefulness of precision-cut tissue slices for studying tissue differences in xenobiotic metabolism and CYP form induction.     

Toxicity of 3-methylindole, 1-nitronaphthalene and paraquat in precision-cut rat lung slices

 The toxicity of 3-methylindole, 1-nitronaphthalene and paraquat has been studied in precision-cut rat lung slice cultures. Lung slices were prepared from male Sprague-Dawley rats using an agarose gel instilling technique with a Krumdieck tissue slicer and cultured for 24 h in a dynamic organ culture system. Treatment of rat lung slices with 3-methylindole, 1-nitronaphthalene or paraquat produced concentration dependent decreases in lung slice protein synthesis and potassium content. EC₅₀ values (concentration to produce a 50% inhibition) for protein synthesis were 0.024, 0.27 and 0.57 mM for paraquat, 1-nitronaphthalene and 3-methylindole, respectively. These results demonstrate that precision-cut lung slices are a useful in vitro model system for studying the pulmonary toxicity of xenobiotics. Lung slices offer the potential as a rapid in vitro screen for identifying pulmonary toxicants and to evaluate species differences in response. Received: 10 October 1994 / Accepted: 30 November 1994     

A new technique for preparing precision-cut slices from small intestine and colon for drug biotransformation studies

INTRODUCTION: A new technique was developed to prepare precision-cut slices from small intestine and colon with the object of studying the biotransformation of drugs in these organs. METHODS: Rat intestinal slices were prepared in two different ways. In the first method, slices were punched out of the small intestine. In the second method, precision-cut slices were made from agarose-filled and -embedded intestines, using the Krumdieck tissue slicer. This method was also applied to colon tissue. Viability of the slices was determined by analysis of intracellular ATP and RNA levels and morphology. Drug metabolizing activity was studied using lidocaine, testosterone, and 7-ethoxycoumarin (7-EC) as phase I substrates, and 7-hydroxycoumarin (7-HC) as a phase II substrate. RESULTS: Precision-cut slices made from agarose-filled and -embedded intestine better preserved ATP levels than tissue that was punched out of the intestinal wall. After 24 h of incubation, morphology in precision cut-slices showed was quite well preserved while punched out tissue was almost completely autolytic after incubation. In addition, total RNA amount and quality was much better maintained in precision-cut slices, when compared to punched out tissue. Both intestinal slices and punched-out tissue showed high, and comparable, phase I and phase II biotransformation activities. DISCUSSION: It is concluded that preparing precision-cut 0.25 mm slices out of agarose-filled and -embedded intestine provides an improvement, compared with punched-out tissue, and that both intestinal and colon slices are useful preparations for in vitro biotransformation studies.     

Characterization of rat small intestinal and colon precision-cut slices as an in vitro system for drug metabolism and induction studies.

The aim of this study was to characterize rat small intestinal and colon tissue slices as a tool to study intestinal metabolism and to investigate gradients of drug metabolism along the intestinal tract as well as drug-induced inhibition and induction of biotransformation. Tissue morphology and the intestinal mucus layer remained intact in small intestinal and colon slices during 3 h of incubation, while alkaline phosphatase was retained and the rate of metabolism of three model compounds (7-hydroxycoumarin, 7-ethoxycoumarin, and testosterone) appeared constant. Phase I and phase II metabolic gradients, decreasing from stomach toward colon were shown to be clearly different for the model compounds used. Furthermore, the observed slice activities were similar or even higher compared with the literature data concerning metabolism of in vitro intestinal systems. Preincubation with beta-naphthoflavone for 24 h induced the O-deethylation of 7-ethoxycoumarin from nearly undetectable to 140 pmol/min/mg protein in small intestine (fresh slices, 43 pmol/min/mg protein) and to 100 pmol/min/mg protein in colon slices (fresh slices, undetectable). Ketoconazole inhibited metabolism of testosterone by 40% and that of 7-ethoxycoumarin by 100%. In conclusion, we showed that the intestinal slice model is an excellent model to study drug metabolism in the intestine in vitro, since we found that the viability parameters remain constant and the measured enzyme activities are relevant, sensitive to inhibitors, and inducible. Therefore, it is a promising tool to study intestinal drug metabolism in human intestine in vitro in the future.     

Beta2-agonist abuse in food producing animals: use of in vitro liver preparations to assess biotransformation and potential target residues for surveillance.

1. The biotransformation of [3H]clenbuterol, [3H]salbutamol, [14C]salmeterol and 7-ethoxycoumarin by bovine liver was investigated by incubation with freshly prepared microsomes, suspension and monolayer cultures of isolated hepatocytes, precision-cut (250 microm) and chopped (600 microm) tissue slices. 2. Radio-HPLC analysis indicated that the saligenin beta2-agonists salmeterol and salbutamol were extensively metabolized by all intact cell preparations. A single major product (SmM1) was evident for salmeterol and two unresolved products for salbutamol (SbM1 and SbM2). Differential enzyme hydrolysis studies with Helix pomatia beta-glucuronidase/aryl sulphatase indicated that the main metabolites were glucuronide conjugates. Consistent with this, analysis of metabolites by liquid chromatography-mass spectrometry showed molecular ions ([M+H]+) at m/z 592 for Sm1 and 416 for both Sb1 and Sb2. 3. Comparable studies with clenbuterol revealed three minor metabolites. Prolonged incubations generated products representing, at maximum, 27% biotransformation. Two of the products have been identified as a glucuronide ([M+H]+, m/z 453) and hydroxyclenbuterol ([M+H]+, m/z 293). 4. These findings indicate that in vitro studies provide simple and cost-effective means of evaluating xenobiotic metabolism, and thus of identifying potential target residues to enable surveillance of use of unlicensed veterinary drugs, or prohibited substances in farm animals.     

Establishment of rat precision-cut fibrotic liver slice technique and its application in verapamil metabolism

1. Liver fibrosis is the compensatory state of cirrhosis. In the long asymptomatic period, it is imperative to select a proper dosing regimen for drugs that are applicable to hepatic fibrosis. Otherwise, progressive deterioration to uncompensated cirrhosis may occur. The present study explored the characteristics of drug metabolism in fibrotic liver. 2. A rat precision-cut fibrotic liver slice (PCFLS) technique was established and the metabolism of verapamil was studied employing this technique. A rat hepatic fibrosis model was successfully induced integrating complex factors that included a high-fat diet, alcohol and CCl4. The PCFLS were incubated under different conditions and lactate dehydrogenase leakage, glutathione S-transferase activity and 3[4,5-dimethythiazole-2-yl]-2,5-diphenyltetrazolium bromide reduction were used as indices to assess PCFLS viability. Activities of phase I and phase II metabolizing enzymes were monitored following treatment with cytochrome P450 (CYP) inducers. Normal and fibrotic liver slices were incubated individually with 10 micromol/L verapamil. The concentration of verapamil in the medium was determined by high-performance liquid chromatography and intrinsic clearance (Cl(int)) was calculated on the basis of the concentration-time curve. 3. The results showed that the PCFLS viability remained steady throughout the 6 h of culture when the thickness of slices was 300 microm and pH of the medium was 7.0; CYP inducers (phenobarbital and ethanol) enhanced CYP2E1, CYP3A1/2 and uridine diphosphate-glucuronate transferase (UDPGT) activities, respectively, in a time-dependent manner. The Cl(int) (microL/min per mg) values differed significantly between normal (9.7 +/- 1.8) and fibrotic (5.6 +/- 1.4) liver slices (P < 0.01). 4. These results suggested that the PCFLS could remain viable for 2-6 h under appropriate conditions. The stability and inducibility of drug-metabolizing enzymes of PCFLS were also demonstrated. Furthermore, the metabolic rate of verapamil in PCFLS was decreased. These findings add further support to the use of PCFLS as a tool to study drug metabolism and to guide clinical medication.     

Monooxygenation, conjugation and other functions in cryopreserved rat liver slices until 24 h after thawing

For the extensive use of precision-cut liver slices (particularly of human origin) for toxicological investigations successful cryopreservation is necessary. But so far, survival of thawed slices was limited to few hours. This was now overcome by modification of previous procedures. The concentration of DMSO as a cryoprotectant was enhanced to 30%, and washing steps after rapid thawing were omitted. The slices were frozen in liquid nitrogen, thawed at 38 degrees C and incubated immediately in Williams medium E. Protein and potassium contents were stable until 24 h. Glutathione content, amounting to nearly 50% of fresh slices, increased during incubation. High initial lactate dehydrogenase leakage dropped after medium change to less than half during 2-24 h. Testosterone hydroxylation and 7-ethoxycoumarin O-deethylation rates were similar to fresh slices, the latter reaction was inducible by beta-naphthoflavone within 24 h. Methylumbelliferone glucuronidation and p-nitrophenol glucuronidation and sulfation were well measurable and either maintained or decreased by about 50% until 24 h.Altogether, the results are encouraging for further experiments to standardise cryopreservation conditions and to investigate the suitability of this cryopreservation protocol with human liver slices.