Use Of Liver Slices Research Articles

The use of liver slices from the Cape vulture (Gyps coprotheres) to better understand the role of liver toxicity of non-steroidal anti-inflammatory drugs (NSAIDs) in vultures

Diclofenac, a non-steroidal anti-inflammatory drug (NSAID) was responsible for the death of millions of vultures on the Asian subcontinent, following the consumption of diclofenac contaminated carcasses. The aim of this research was to establish if liver slices could serve as an alternate means of predicting the toxicity of NSAIDs in Gyps vultures. The Cape vulture liver slices was prepared and incubated with four NSAIDs for 6 h. A percent clearance of 1.0 ± 0.253, 0.58 ± 0.153, 0.961 ± 0.312 and 1.242 ± 0.406 (%/h*g) was attained for diclofenac, carprofen, ketoprofen and meloxicam respectively. Both meloxicam and diclofenac exerted toxic effects on the hepatic cells. Protein content indicated that the vulture tissue had lower enzyme levels than expected for an animal of its size. The poor distinction between the ex vivo hepatic percent clearance of meloxicam and diclofenac indicates that liver slices is not an ideal model to investigate NSAIDs toxicity in Cape vulture.

Gene expression and enzyme function of two cytochrome P450 3A isoenzymes in rat and cattle precision cut liver slices

1. Precision-cut liver slices are one of the in vitro models used in studies concerning xenobiotic metabolism. Sparse information on this field is actually available for cattle and other veterinary species.2. The aim of the current work was to study the effect of dexamethasone (DEX) on the gene expression and function of CYP3A23 (in rat), CYP3A28 (in cattle) and the transcriptional factors involved in their regulation.3. DEX (at 100 µM) up-regulated CYP3A23 mRNA (3.2-fold, p = 0.028) in rat liver slices after 12 h culture, whereas the gene expression profiles of transcriptional factors involved in CYP3A regulation were unaffected. A CYP3A-dependent enzyme activity (triacetyl-oleandomycin N-demethylase) increased 3.4-fold (p < 0.05) in rat liver slices cultured in the presence of DEX.4. The protocol used for rat liver slices was used as reference to study the expression of a CYP3A isoenzyme in cattle liver slices. Oppositely, DEX did neither affect the gene expression profile of CYP3A28 nor the CYP3A activity tested in cattle liver slices.5. The data reported here are a further contribution to demonstrate the usefulness of liver slices as an in vitro tool for studies on the expression and function of xenobiotic metabolizing enzymes in cattle and in other ruminant species.

Analysis of bile acid‐induced regulation of FXR target genes in human liver slices

Information about the role of nuclear receptors has rapidly increased over the last decade. However, details about their role in human are lacking. Owing to species differences, a powerful human in vitro system is needed. This study uses for the first time precision-cut human liver slices in the nuclear receptor field. The farnesoid X receptor (FXR) was chosen as a model. We were able to demonstrate that human liver slices efficiently take up bile acids and show a stable expression of a wide variety of genes relevant for bile acid metabolism, including bile acid transporters, cytochrome P450 enzymes and transcription factors. Treatment with chenodeoxycholate induced small heterodimer partner, bile salt export pump and p-glycoprotein, ABCB4 and repressed cholesterol 7alpha hydroxylase, hepatocyte nuclear factor (HNF)1, HNF4 and organic anion transporting peptide (OATP)1B1. OATP1B3, FXR, HNF3beta and cytochrome P450 enzyme remained relatively constant. In contrast to what has been observed in mice and rat studies, SHP induction did not result in repression of sodium-dependent bile acid cotransporter expression. Further, regulation of genes seemed to be dependent on concentration and time. Taken together, the study shows that the use of liver slices is a powerful technique that enables to study nuclear receptors in the human liver.

Evaluation of Human Liver Slices and Reporter Gene Assays as Systems for Predicting the Cytochrome P450 Induction Potential of Drugs in Vivo in Humans

The aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods. The CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). In human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUCtot to PXR EC50 values. Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.

Quantitative PCR assay for cytochromes P450 2B and 3A induction in rat precision-cut liver slices: Correlation study with induction in vivo

Introduction In drug development, new chemical entities that cause cytochrome P450 induction are considered to be undesirable since P450 induction is linked to tumor formation and may compromise the evaluation of drug safety when autoinduction results in poor drug exposure. Methods We evaluated the use of the precision-cut liver slice as a model for measuring induction of cytochrome P450 in rats. Quantitative real-time reverse-transcription polymerase chain reaction was used to analyze the induction of selected forms of cytochrome P450 at the mRNA level. Firstly, the system was validated against known inducers of CYP2B and 3A. Subsequently, 26 proprietary compounds were tested in rat liver slices and rats in vivo for CYP2B and 3A induction. Results Exposure of liver slices to the known CYP2B inducers phenobarbital, benzoyl-pyridine, cabarmazepine, metyrapone, RU486 and dexamethasone caused elevation of CYP2B1/2 expression 10- to 40-fold compared to the control values. The CYP3A inducers PCN, dexamethasone, nicardipine, nifedipine, clotrimazole and RU486 induced a 4- to 50-fold expression of CYP3A14. For 26 proprietary compounds, a correlation with an R 2 value of 0.74 was established between the induction of CYP2B expression in liver slices and that in rats in vivo. When liver slice results were used to predict the induction of CYP2B in rats in vivo, the success rate was 91%. The induction of CYP3A in rats in vivo was analyzed by Western blot, then quantified by densitometry. There was a good correlation between CYP3A induction in liver slices and induction in vivo as assessed by Western blot, with an 86% positive prediction rate. Discussion The use of liver slices in combination with TaqMan technology provides a good model for predicting CYP induction in the rat. This method is useful for identifying compounds with CYP2B and 3A induction liability in the early phase of drug discovery.

Precision-cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues.

Metabolism of xenobiotics is often seen as an exclusive function of the liver, but some current findings support the notion that the lungs, kidneys and intestine may contribute considerably. After the establishment of the use of liver slices as a useful in vitro model to study metabolism and toxicity of xenobiotics, the same concept is currently being used for slices from lung, kidney and intestine. It is the aim of this review to discuss the use of organ slices in biotransformation research. The basic idea behind the use of tissue slices in biomedical research is the assumption that the cells under study will function optimally in vitro if they are cultivated in an environment that is most alike to their natural in vivo embedding, which is the case in tissue slices. Advantages in the use of organ slices are the relatively easy preparation as well as the potential standardization of both the preparation and use. Moreover, a direct interspecies comparison can be made between liver, lungs, kidneys and intestines, for example with respect to their metabolic capacity and their sensitivity for toxicants. Of major importance is that organ slices can be made with a similar procedure from organs/tissues originating from different species, including man. This latter aspect is useful in drug development in general but also for a better insight in the metabolic fate of compounds in man. Importantly the use of slices may largely contribute to a reduction in the use of experimental animals.

Liver slice technology as an in vitro model for metabolic and toxicity studies.

AbstractLiver Slice Technology is a relatively new addition to the battery of in vitro assays of xenobiotic metabolism and toxicity evaluation. As with any developing technology, evaluation and refinement of the technique will be an essential component of its implementation to routine use. Three goals of this article will be to: (1) provide a brief survey of currently used methods for immediate and cultured liver slices, (2) review the use of liver slices in the assessment and quantification of pharmacological and toxicological responses, and (3) discuss the possible use of Liver Slice Technology for the investigation of drug interactions.KeywordsMetabolic ProfileMicrosomal FractionLiver SliceXenobiotic MetabolismToxicity IndexThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Optimal oxygen tension conditions for viability and functioning of precision-cut liver slices

Optimal oxygenation of culture media is important for the successful use of liver slices as an in vitro tool for studying liver function. For this reason the influence of 20, 40, 70 and 95% O2 concentration on the viability and metabolism of liver slices was investigated. The slices were incubated in the roller system at 37 degrees C under continuous gassing for 2, 24 and 48 hrs. Protein, DNA and potassium contents were maintained or even increased over time without influence by O2 concentrations. The albumin secretion of slices incubated at 40-95% O2 did not differ, but was much lower at 20% O2. A slight non-significant decrease in albumin secretion after 24 hrs of cultivation could be observed, whereas a much steeper decline was found in all groups after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation (ECOD) did not differ between the various O2 concentrations, but declined from 2 to 48 hrs of incubation. It can be concluded that O2 concentration of 20% is not sufficient to maintain all cell functions of incubated rat liver slices, wheras 40, 70 and 95% are useful O2 concentrations to retain all parameters investigated.

Validation of Precision-Cut Liver Slices in Dynamic Organ Culture as anin VitroModel for Studying CYP1A1 and CYP1A2 Induction

The utilization of precision-cut liver slices in dynamic organ culture as anin vitromodel was validated by comparing the induction of the biomarker responses followingin vitro(rat liver slice) andin vivoexposure of rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The biomarker responses investigated were cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) mRNA, protein, and activities. Precision-cut rat liver slices were incubated in dynamic organ culture for 24 hr with medium containing 0.001–10 nMTCDD or medium without TCDD (control). The resultant mean TCDD concentration in the slices ranged from 19 to 80,925 ppt (wet wt), respectively. A concentration-dependent induction of CYP1A1 mRNA, protein, and activities and a more modest induction of CYP1A2 mRNA was observed in liver slices at all medium concentrations of TCDD. The O-demethylation of 7-methoxyresorufin, a marker for CYP1A2 activity, was induced at TCDD medium levels of 0.01 nMand greater, whereas a detectable increase in CYP1A2 protein occurred only at the higher concentrations. Comparable liver concentrations of TCDD (8–64,698 ppt wet wt) were achieved at 24 hr following a singlein vivoexposure of rats to TCDD at doses ranging from 0.002 to 5 μg/kg po. Concentration–effect and dose–response relationships for induction of CYP1A1 and CYP1A2 were similar followingin vitroandin vivoexposure to TCDD, although the magnitude of induction was greater forin vivoexposure. The data support the use of liver slices in dynamic organ culture for assessing the relativein vivopotency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in thisin vitromodel to predict the biological and toxicological effects of a givenin vivoexposure to TCDD.

Cryopreservation of Rat and Monkey Liver Slices

A method for the long-term storage of liver slices by cryopreservation was developed. The viability of liver slices was determined by analysing the leakage of alanine aminotransferase, urea production, and the metabolism of testosterone. Rat liver slices were found to be optimally cryopreserved by exposure for 30 minutes to 12% dimethyl sulphoxide (v/v) at 2°C before freezing. Subsequent direct immersion in liquid nitrogen was more effective than a cooling rate of ±1°C/mmute, which reduced viability. Storage at a temperature of -80°C lowered viability compared to storage at -196°C. These conditions for optimal cryopreservation were used to cryopreserve rat, rhesus monkey and cynomolgus monkey liver slices. The viability of these liver slices was maintained at: 74%, 86% and 85%, respectively, when alanine aminotransferase content was measured; 80%, 109% and 82%, respectively, when urea production was measured; and 109%, 60% and 85%, respectively, when the metabolism of testosterone was measured. Viability was maintained for at least one month. The results show that, by using the method presented here, liver slices from these species can be stored while maintaining viabilities similar to initial values. This method will facilitate the optimal use of liver slices and reduce the number of experimental animals used.

Human Liver Slices Express the Same Lidocaine Biotransformation Rate as Isolated Human Hepatocytes

In order to investigate whether liver slices are a valuable tool for the assessment of drug metabolism in human liver, we compared the phase I metabolism of lidocaine in human liver slices and hepatocytes prepared from three human livers. Lidocaine is mainly metabolised to monoethylglycinexykdide (MEGX) via a cytochrome P450-mediated N-deethylation. The results indicate that the three livers showed considerable inter-individual differences in the rate of formation of MEGX, and that this difference was equally reflected in slices and isolated cells. The use of liver slices is still under development, and optimal incubation conditions still need to be assessed. However, these results suggest that, in slices of 200–300μm thickness, virtually all hepatocytes are involved in the biotransformation of lidocaine, and that the metabolic activity is preserved equally well as in isolated hepatocytes.

Predictive Value of Liver Slices for Metabolism and Toxicity in Vivo: Use of Acetaminophen as a Model Hepatotoxicant

To establish the usefulness of liver slices as predictive models for in vivo metabolism and toxicity, acetaminophen was used as a model hepatotoxicant for which the role of metabolism in toxicity is well documented. Acetaminophen was incubated with liver slices prepared from the rat and hamster since these species differ in susceptibility to acetaminophen-induced hepatotoxicity. Formation of acetaminophen metabolites (sulfate, glucuronide, and glutathione conjugate), slice glutathione levels, and slice histopathology were assessed. Acetaminophen (0.5, 1, and 2 mM) induced a dose-dependent depletion of glutathione in hamster slices (sensitive species) but not rat slices (insensitive species). Formation of the acetaminophen toxic metabolite, as measured by glutathione conjugate formation, was much lower in rat slices compared with hamster slices. Rat slices also showed greater activity of the nontoxic sulfation and glucuronidation pathways. These data predicted an 11-fold greater susceptibility to toxicity in the hamster based on the proportion of the dose metabolized to the reactive species. In vivo data, using hepatic glutathione depletion as an indicator of toxic metabolite formation, would predict a similar difference (13.3-fold) between the hamster and the rat. To ascertain if the characteristic centrilobular lesion induced by acetaminophen in vivo could be reproduced in vitro, liver slice histopathology was assessed 6 and 12 hr after a 2-hr treatment with acetaminophen (2 mM). Discrete damage to the centrilobular regions of the liver were noted in hamster but not rat slices. Overall, based on metabolite formation, glutathione depletion, and slice histopathology, the liver slices were excellent predictors of acetaminophen hepatotoxicity in vivo.

Liver slices in dynamic organ culture. I. An alternativein vitrotechnique 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.

Dynamic organ culture of precision liver slices for [formula omitted] toxicology

The lack of a reproducible method for the production of thin tissue slices has hindered the use of liver slices as an in vitro tool for hepatotoxicity studies. Fresh human, rat, and rabbit liver was processed using a mechanical slicer. With this instrument, precision (5% of thickness) liver slices in the submillimeter range could be produced at a rapid rate. Slices were prepared from fresh livers in chilled, oxygenated buffer to minimize trauma. Following incubation for up to 20 h in a dynamic organ culture system, histology of incubated slices suggested that 250 m precision-cut slices were optimum in regard to morphology relative to liver slices incubated under conventional organ culture conditions. Addition of bromobenzene to the culture showed time-dependent hepatotoxicity based on two classic parameters of cell degeneration. Histological evidence is presented which suggests the usefulness of this system for hepatotoxicity studies and the production of focal necrosis in vitro .

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Dimethylnitrosamine (DMN)-induced liver damage, as measured by the increase in plasma isocitrate dehydrogenase as well as by histologic assessment of necrosis, was marked after DMN ip administration (70 mg/kg) in males of all noninbred species tested (BALB/c mouse, Sprague-Dawley rat, Syrian golden hamster, general purpose guinea pig) but not in the noninbred White Leghorn chicken. At 1 and 3 hours after DMN injection, liver DMN levels were not lower in the chicken as compared to levels in the other species. Furthermore, in all species except the chicken, significant decreases were found at 3 hours as compared to 1 hour after DMN administration. DMN metabolism to CO2 and to formaldehyde, as well as covalent binding of DMN-reactive metabolites to either proteins or nucleic acid, was measured with the use of liver slices, microsomes, and/or 9,000 X g supernatants. Results indicated that chicken liver had a very low capacity for metabolism and activation (29-3,166 times lower than comparable data in mice or hamsters).

The role of the mitochondria in rat liver mixed function oxidation reactions

Recombination experiments indicate a complex role by the mitochondria in controlling drug biotransformation reactions. The use of liver slices and homogenates demonstrates that the effect of Krebs cycle intermediates on aminopyrine N-demethylation is not via an increase in NADH, but probably by a well-defined route from the mitochondria to the endoplasmic reticulum.

GLUTAMIC-PYRUVIC TRANSAMINASE AND GLUCONEOGENESIS IN LIVER SLICES: A COMPARISON OF THE EFFECTS OF CORTISOL INJECTION WITH TREATMENT IN VITRO

For many years rat liver slices have been used in studies on the effect of corticosteroids in vitro, and the assumption has often been made that the biochemical response of cells in slices is the same as that in the intact animal. This applies particularly to the demonstration of an increased synthesis of carbohydrate with steroids of the cortisol/corticosterone type (Chui, 1950; Haynes, 1962; Azuma & Eisenstein, 1964). However, the biochemical properties of isolated cells may differ in several respects from those in intact tissues. In this respect, the usefulness of liver slices in studying gluconeogenesis has been questioned (Krebs, Notton & Hems, 1966). The following results show that the increased glucose synthesis which occurs in response to the addition of cortisol to rat liver slices is not a feature of the liver from an animal in which glycogen synthesis has been stimulated by an injection of cortisol. Treatment in