Precision-cut Slices Research Articles

Influence of phenethyl isothiocyanate (PEITC) on carcinogen-metabolising enzymes in human liver: Studies with precision-cut slices

Influence of phenethyl isothiocyanate (PEITC) on carcinogen-metabolising enzymes in human liver: Studies with precision-cut slices

Up-regulation of the glutathione S-transferase system in human liver by polycyclic aromatic hydrocarbons; comparison with rat liver and lung

The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When activity was monitored using the alpha-class selective 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, B[b]F was the only PAH that caused an increase in activity, which was accompanied by a rise in the Ya immunoreacting band. In rat slices, in addition to B[b]F, B[a]P and D[a,h]A also enhanced activity, being paralleled with increased levels of the Ya immunoreacting band. In the rat, all PAHs elevated mRNA levels. In both human and rat liver slices, only B[b]F enhanced activity when 1-chloro-2,4-dinitrobenzene (CDNB) served as substrate. To investigate tissue differences, similar studies were undertaken in precision-cut rat lung slices, incubated with PAHs under identical conditions, using CDNB, as this was the only substrate for which activity could be detected; none of the PAHs studied stimulated activity. It is concluded that some PAHs have the potential to induce GST activity in human liver tissue and that species and tissue differences exist in the induction of this enzyme system in the rat. However, the extent of induction of GST activity is very modest compared with the effect these compounds have on CYP1 expression, the family responsible for their bioactivation, and it is unlikely to compensate for the enhanced production of reactive intermediates.

Induction of Metabolism and Transport in Human Intestine: Validation of Precision-Cut Slices as a Tool to Study Induction of Drug Metabolism in Human Intestine in Vitro

Induction of drug enzyme activity in the intestine can strongly determine plasma levels of drugs. It is therefore important to predict drug-drug interactions in human intestine in vitro. We evaluated the applicability of human intestinal precision-cut slices for induction studies in vitro. Morphological examination and intracellular ATP levels indicated tissue integrity up to 24 h of incubation, whereas in proximal jejunum slices, the metabolic rate toward most substrates remained at 40 to 50% of initial values. In colon slices, the cytochrome P450 conversions were below the detection limit, but conjugation rates remained relatively constant during incubation. The inducibility of drug-metabolizing enzymes and P-glycoprotein was evaluated using prototypical inducers for five induction pathways. beta-Naphthoflavone (aryl hydrocarbon receptor ligand) induced CYP1A1 (132-fold in colon and 362-fold in proximal jejunum) and UDP glucuronosyltransferase (UGT) 1A6 mRNA (9.8-fold in colon and 3.2-fold in proximal jejunum). In proximal jejunum, rifampicin (RIF) [pregnane X receptor (PXR) ligand] induced CYP3A4 (5.2-fold), CYP2B6 (2-fold), UGT1A6 (2.2-fold), and multidrug resistance-1 (MDR1)/ABCB1 mRNA (2.7-fold), whereas 6beta-hydroxytestosterone formation (CYP3A4) increased 2-fold. In colon, RIF induced UGT1A6 32-fold and MDR1 2.2-fold. Dexamethasone (glucocorticoid receptor and PXR ligand) induced CYP3A4 mRNA (3.5-fold) and activity (5-fold) in proximal jejunum. Phenobarbital (constitutive androstane receptor activator) induced CYP3A4 (4.1-fold, only in jejunum), CYP2B6 (4.9-fold in colon and 2.3-fold in proximal jejunum), and MDR1/ABCB1 mRNA and CYP3A4 activity (2-fold only proximal jejunum). Quercetin (nuclear factor-E2-related factor 2 activator) induced UGT1A6 mRNA (6.7-fold in colon and 2.2-fold in proximal jejunum). In conclusion, this study shows that human intestinal precision-cut slices are useful to study induction of drug-metabolizing enzymes and transporters in the human intestine.

Species differences in 3-methylsulphonyl-DDE bioactivation by adrenocortical tissue

The CYP11B1-activated adrenocortical toxicant 3-methylsulphonyl-DDE (3-MeSO2-DDE) is proposed as a lead compound for an improved chemotherapy for adrenocortical carcinoma. We compared the binding of 3-MeSO2-[14C]DDE in the adrenal cortex of four rodent species; hamster, guinea pig, mouse and rat, using a precision-cut adrenal slice culture system ex vivo. Localization and quantification of the bound radioactivity were carried out using light microscopy autoradiography and radioluminography. The results revealed major species differences since 3-MeSO2-[14C]DDE was extensively bound to the hamster adrenal tissue while the guinea pig adrenals were devoid of binding. A high binding in mouse adrenal cortex was confirmed while binding in rat adrenal cortex was very weak. The results support previous observations that metabolic activation of 3-MeSO2-DDE is highly species dependent. Since CYP11B1 could be expressed in tissues other than the adrenal cortex, final toxicological characterization should be carried out in a species that can bioactivate this compound.

In Vitro Methods to Study Intestinal Drug Metabolism

Although the liver has long been thought to play the major role in drug metabolism, also the metabolic capacity of the intestine is more and more recognized. In vivo studies eventually pointed out not only the significance of first-pass metabolism by the intestinal wall for the bioavailability of several compounds, but also the relevance of transporters in this process. Only a few methods are available to study drug metabolism in vivo or in situ and with most of these methods it remains difficult to discriminate between the contribution of liver and extrahepatic tissues. To study intestinal drug metabolism in vitro, apart from subcellular fractions, several intact cell systems are nowadays available. This review discusses the available intestinal in vitro methods to study drug metabolism. The advantages and limitations of intact cell systems (isolated intestinal perfusion, everted sac, Ussing chamber preparations, biopsies, precision-cut slices, primary cells), subcellular fractions (S9 fractions, microsomes) and intestinal cell lines (caco-2, LS180 cells amongst others) are discussed. Their applicability to different species and to study phase I and II metabolism/transport and drug-drug interactions are summarized. Furthermore, causes of variation within and between methods are discussed and metabolic rates obtained with different methods are compared. Whereas subcellular fractions and cell lines are efficient methods to study mechanistic aspects of drug metabolism at the enzyme level, the isolated intestinal perfusion, everted sac and Ussing chamber appear particularly useful for studying drug metabolism of rapidly metabolised drugs and interactions with transporters. Biopsies, precision-cut slices and primary cells seem all appropriate to study induction and metabolism of slowly metabolised drugs.

Up-regulation of CYP1A/B in rat lung and liver, and human liver precision-cut slices by a series of polycyclic aromatic hydrocarbons; association with the Ah locus and importance of molecular size

Exposure of precision-cut rat liver slices to six structurally diverse polycyclic aromatic hydrocarbons, namely benzo[ a]pyrene, benzo[ b]fluoranthene, dibenzo[ a, h]anthracene, dibenzo[ a, l]pyrene, fluoranthene and 1-methylphenanthrene, led to induction of ethoxyresorufin O-deethylase, CYP1A apoprotein and CYP1A1 mRNA levels, but to a markedly different extent. In liver slices, constitutive CYP1A1 mRNA levels were higher, as well as being markedly more inducible by PAHs, compared with CYP1B1, a similar profile to that observed in human liver slices following exposure to the PAHs. Increase in ethoxyresorufin O-deethylase and in CYP1A1 apoprotein levels was also observed when precision-cut rat lung slices were incubated with the same PAHs, the order of induction potency being similar to that observed in liver slices. Under the same conditions of exposure, CYP1B1 apoprotein levels were elevated in the lung. Up-regulation of CYP1A1 by the six PAHs correlated with their affinity for the Ah receptor, determined using the chemical-activated luciferase expression (CALUX) assay. It may be concluded that (a) precision-cut liver and lung slices may be used to assess the CYP1 induction potential of chemicals at the activity, apoprotein and mRNA levels; (b) rat is a promising surrogate animal for human in studies to evaluate CYP1 induction potential; (c) CYP1A1 is far more inducible than CYP1B1 in both rat liver and lung; (d) CYP1 up-regulation by PAHs is related to their affinity for the Ah receptor, and finally (e) computer analysis revealed that the ratio of molecular length/width is an important determinant of CYP1 induction potency among equiplanar PAHs.

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.

Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 °C/min) using 18% Me 2SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3–4 h of incubation at 37 °C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me 2SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50–80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.

Innovative Methods to Study Human Intestinal Drug Metabolism in Vitro: Precision-Cut Slices Compared with Ussing Chamber Preparations

Predictive in vitro methods to investigate drug metabolism in the human intestine using intact tissue are of high importance. Therefore, we studied the metabolic activity of human small intestinal and colon slices and compared it with the metabolic activity of the same human intestinal segments using the Ussing chamber technique. The metabolic activity was evaluated using substrates to both phase I and phase II reactions: testosterone, 7-hydroxycoumarin (7HC), and a mixture of cytochrome P450 (P450) substrates (midazolam, diclofenac, coumarin, and bufuralol). In slices of human proximal jejunum, the metabolic activity of several P450-mediated and conjugation reactions remained constant up to4hof incubation. In the colon slices, conjugation rates were virtually equal to those in small intestine, whereas P450-mediated conversions occurred much slower. In both organs, morphological evaluation and ATP content implied tissue integrity within this period. P450 conversions using the Ussing chamber technique showed that the metabolic rate (sum of metabolites measured in apical, basolateral, and tissue compartments) was constant up to 3 h. For 7HC conjugations, the metabolic rate remained constant up to 4 h. The distribution of the metabolites in the compartments differed between the substrates. Overall, metabolic rates were surprisingly similar in both techniques and appear similar to or even higher than in liver. In conclusion, this study shows that both human intestinal precision-cut slices and Ussing chamber preparations provide useful tools for in vitro biotransformation studies.

Advanced Generation Adenoviral Virotherapy Agents Embody Enhanced Potency Based upon CAR-Independent Tropism

In this current issue of Clinical Cancer Research , Reddy et al. ([1][1]) constructed an adenoviral virotherapy agent that uses the human E2F-1 gene promoter to regulate the adenoviral E1A gene. The E2F transcription factor plays an important role in cell proliferation and is repressed by the

Precision-cut organ slices to investigate target organ injury

Drug-induced organ injury is a multifaceted process, involving numerous cell types and mediators, and remains a significant safety issue in pharmaceutical development and clinical therapy. Organ slices, an in vitro model representing the multicellular, structural and functional features of in vivo tissue, is a promising model for elucidating mechanisms of drug-induced organ injury and for characterising species susceptibilities. Time- and concentration-dependent drug-induced effects on organ slice gene expression, function and morphology are providing insight into the molecular and biochemical pathways leading to organ dysfunction, an altered morphology and the induction of repair pathways. Human organ slice studies are valuable for bridging the extrapolation of animal-derived data and for identifying mechanisms relevant for humans. The liver is the major organ used in organ slice studies; however, the utility of extrahepatic-derived slices, as well as cocultures for investigating multiple organ involvement in tissue injury is increasing. Organ slice investigations can further our understanding of the cell types and cell interactions involved in drug-induced injury and the consequences of drug-induced off-target effects for identifying compound liabilities that will impact safety.

A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity

Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver. The Krumdieck thin-slice tissue culture system was used to obtain and culture slices of tumor xenografts of ovarian cancer cell lines, human primary ovarian tumors, and human liver. We determined the viability of slices in culture over a period of 36 to 48 hours by ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt)]) (MTS) assay. In vitro Hey cells, slices of Hey xenografts, and human ovarian tumor or human liver slices were infected with 500vp/cell of either replication competent wild-type adenovirus (Ad5/3wt), conditionally replicative adenovirus (Ad5/3cox-2), or the replication deficient adenovirus (Ad5/3luc1). At 12-, 24-, and 36-hour intervals, the replication of adenoviruses in these slices was determined by quantitative reverse transcription-PCR of adenoviral E4 copy number. Primary tumor slices were able to maintain viability for up to 48 hours after infection with nonreplicative virus (Ad5luc1). Infection of Hey xenografts with Ad5/3cox-2 showed replication consistent with that seen in Hey cells infected in an in vitro setting. Primary tumor slices showed replication of both Ad5/3wt and Ad5/3cox over a 36-hour time period. Human liver slices showed replication of Ad5/3wt but a relative reduction in replication of Ad5/3cox-2 indicative of conditional replication "liver off" phenotype, thus predicting lower toxicity. The thin-slice model system represents a stringent method of ex vivo evaluation of novel replicative adenoviral vectors and allows assessment of human liver replication relative to human tumor replication. This is the first study to incorporate this system for evaluation of therapeutic efficacy and replicative specificity of conditionally replicative adenoviruses. Also, the study is the first to provide a valid means for preclinical assay of potential conditionally replicative adenovirus-based hepatotoxicities, thus providing a powerful tool to determine therapeutic index for clinical translation of conditionally replicative adenoviruses.

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.

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.

New Hepatocyte In Vitro Systems for Drug Metabolism: Metabolic Capacity and Recommendations for Application in Basic Research and Drug Development, Standard Operation Procedures

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4 hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this system is that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used for the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.

Culturing precision-cut human prostate slices as an in vitro model of prostate pathobiology.

Due to the complex morphology of the prostate, it was hypothesized that precision-cut tissue slices from human prostate would provide a unique in vitro model. Precision-cut slices were generated from zones of human prostate and their viability was assessed under conditions of different media for up to 120 h. Slices were also exposed to several concentrations of CdCI2, which was used as a model toxicant. Maintenance of both stromal and epithelial cells was noted; however, there was a gradual loss of luminal epithelial cells when the medium was not supplemented with dihydrotestosterone (DHT). Minimal leakage of lactate dehydrogenase occurred throughout the incubation. Prostate-specific antigen (PSA) was detected in the medium at all time points, although the rates of secretion fell over time. There was a loss of PSA-positive cells when the medium was not supplemented with DHT, consistent with a loss of luminal cells, whereas PSA-positive cells were maintained in the DHT-supplemented media. A proliferation of basal cells was observed in the presence of media containing 10% fetal bovine serum. Exposure of slices to CdCl2 demonstrated a dose-response effect ranging from proliferation to complete cellular necrosis. Given the retention of stromal-epithelial interactions and the use of acquired human tissue, prostate slices represent a unique in vitro model for investigating human prostate pathobiology.

Biotransformation of benzydamine by microsomes and precision-cut slices prepared from cattle liver

1. Benzydamine (BZ), a non-steroidal anti-inflammatory drug used in human and veterinary medicine, is not licensed for use in food-producing species. Biotransformation of BZ in cattle has not been reported previously and is investigated here using liver microsomes and precision-cut liver slices. 2. BZ was metabolized by cattle liver microsomes to benzydamine N-oxide (BZ-NO) and monodesmethyl-BZ (Nor-BZ). Both reactions followed Michaelis-Menten kinetics (Km = 76.4 ± 16.0 and 58.9 ± 6.4 µM, Vmax = 6.5 ± 0.8 and 7.4 ± 0.5 nmol mg−1 min−1, respectively); sensitivity to heat and pH suggested that the N-oxidation is catalysed by the flavin-containing monooxygenases. 3. BZ-NO and Nor-BZ were the most abundant products derived from liver slice incubations, and nine other BZ metabolites were found and tentatively identified by LCMS. Desbenzylated and hydroxylated BZ-NO analogues and a hydroxylated product of BZ were detected, which have been reported in other species. Product ion mass spectra of other metabolites, which are described here for the first time, indicated the formation of a BZ N+-glucuronide and five hydroxylated and N+-glucuronidated derivatives of BZ, BZNO and Nor-BZ. 4. The results indicate that BZ is extensively metabolized in cattle. Clearly, differences in metabolism compared with, for example, rat and human, will need to be considered in the event of submission for marketing authorization for use in food animals.

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 the calcium channel blocker verapamil and sulphydryl reducing agent dithiothreitol on atractyloside toxicity in precision-cut rat renal cortical and liver slices

The effects of dithiothreitol (DTT), a sulfhydryl-containing agent and verapamil (VRP), a calcium channel blocker as possible cytoprotectants against the atractyloside-induced toxicity were characterized in rat kidney and liver slices in vitro using multiple markers of toxicity. Precision-cut slices (200 μm thick) were either incubated with atractyloside (2 m m) or initially preincubated with either DTT (5 m m) or VRP (100 μ m) for 30 min followed by exposure to atractyloside (2 m m) for 3 h at 37°C on a rocker platform rotated at approximately 3 rpm. All of the toxicity parameters were sensitive to exposure to atractyloside, but treatment with DTT or VRP alone did not provide any indication of damage to the tissues. Preincubation of slices containing either DTT or VRP for 30 min provided total protection against atractyloside-induced increase in LDH leakage in both kidney and liver slices. Increased induction of lipid peroxidation by atractyloside in liver slices was completely abolished by DTT and VRP. Both DTT and VRP provided partial protection against atractyloside-induced inhibition of gluconeogenesis in both kidney and liver slices. Atractyloside-induced ATP depletion in both kidney and liver slices was partially abolished by VRP but not DTT. The significant depletion of GSH in the kidney slices by atractyloside was completely reversed by DTT only, while VRP alone reversed the same process in liver slices. Decreased MTT reductive capacity and significant increase in ALT leakage caused by atractyloside in liver slices was partially reversed. Complete protection was achieved with both DTT and VRP against atractyloside-induced inhibition of PAH uptake in kidney slices. These findings suggest that both DTT and VRP exert cytoprotective effects in atractyloside-induced biochemical perturbation, effects that differ in liver and kidney. The effect of these agents on atractyloside has provided us with a further understanding of the molecular mechanism of its action.

Cytochrome P450-catalysed irreversible binding examined in precision-cut adrenal slice culture.

In order to examine cell-specific localisation of cytochrome P450 (CYP) catalysed binding and toxicity in human and wild animal tissues, we required a suitable tissue culture system. Precision-cut tissue slice culture was introduced in the early 1980s (1). It provides a test system where cell-specific effects can be studied in vitro during several days (2–4).