© ECVAM DB-ALM: Protocol DB-ALM Protocol n° 42 : Liver Slice Hepatotoxicity Screening System Hepatotoxicity / Metabolism-mediated Toxicity Leakage of lactate dehydrogenase and alanine aminotransferase from rat and mouse liver slices exposed to the test compound is used as a measure of hepatotoxicity. Résumé The assessment of hepatotoxic effects plays an important role in toxicological studies of new drugs and environmental substances. The liver slice system, which has been widely used for the study of drug mechanisms and interactions, is proposed for routine testing of acute hepatotoxicity. Experimental Description Endpoint and Endpoint Measurement: ALANINE AMINOTRANSFERASE (ALT) LEAKAGE LACTATE DEHYDROGENASE (LDH) LEAKAGE Endpoint Value: EC 50 Experimental System(s): Freehand-cut rat liver slices Freehand-cut mouse liver slices Basic Procedure Liver lobes from rats or mice are sliced, incubated for 1 hour in Krebs Ringer Hepes (KRH) medium and then further divided into 100-120mg portions and incubated with 10 minutes oxygen aeration periods for 1 hour. The tissue fragments are exposed to the test compound for 2 hours. Medium is assayed for enzyme activity at regular intervals during the incubation. Finally, the tissue fragments are homogenized, centrifuged and the total enzyme activity is determined in the supernatant. Test Compounds and Results Summary Acetaminophen Phenacetine Amitriptyline HCl Quinidine sulphate Aspirin Theophylline Chlorpromazine HCl Valproic acid Cimetidine Verapamil HCl Furosemide Carbon tetrachloride Imipramine HCl Methyl alcohol Mechlorethamine (nitrogen mustard) Ethyl alcohol Nifedipine Isopropyl alcohol Pentobarbital sodium Cadmium chloride Discussion This assay is a relatively simple, inexpensive and rapid procedure. Various hepatotoxins can be examined at a wide range of concentrations using only a single animal, since one liver can provide a large number of tissue fragments. The leakage of cellular components is accepted as a standard measure of early hepatic injury. It has been shown (Wormser et al, 1990a) that the activities of LDH and ALT in the medium after a 2 hours exposure of mouse liver slices to acetaminophen correlate with the extent of multifocal hepatocytic degeneration observed in the slices. The level of LDH activity is the usual parameter used in the present assay. However, it must be noted that some toxins can inhibit LDH activity, mainly through the production of free radicals, but sometimes due to other mechanisms. If such inhibition is suspected, it can be confirmed by measuring the activity of a homogenate of unexposed slices and of a homogenate following https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocols page 1 / 7 © ECVAM DB-ALM: Protocol the addition of the test substance to thehomogenate. In such a case, the activity of aspartate aminotransferase, alanine aminotransferase of alkaline phosphatase can be used as markers of cell integrity. The liver slice system has advantages over cultures of dispersed hepatocytes which are time-consuming and require expensive equipment. In the liver slice system, the microscopic structure of the original tissue is preserved, thus allowing the retention of normal intercellular communication. In addition, the hepatocytes are not subjected to the stress of collagenase treatment which is used in the preparation of single cell suspensions. The liver fragments should therefore more closely resemble the in vivo situation. Importantly, the liver slice technique enables histopathological assessment of the exposed tissue. Further evidence that the liver slice system reflects the in vivo situation is provided by the finding that incubation of rat liver slices with CdCl2 resulted in a dose-dependent elevation of tissue metallothionein, which is the typical response of the liver to cadmium exposure in vivo (Wormser, Ben Zakine & Nyska, 1990). While the drug concentrations required to elicit a toxic effect in the rat and mouse liver slice systems (Wormser et al., 1990a) and also in the rabbit liver slice system (Dujovne et al., 1976) have been found to be higher than those observed in vivo in the sera of intoxicated animals (Davis et al.; Mitchell et al., 1976), the ratios of EC50 values of CCl4 and CdCl2 in the mouse liver slice system (Wormser et al., 1990a) are similar to those obtained in vivo in acute toxicity studies (Goering & Klassen, 1984; Schwetz & Plaa, 1969). Furthermore, EC50 values obtained with this system showed a correlation of 0.946 when plotted against LD 50 values for in vivo acute toxicity data obtained from the literature (Wormser, et al., 1990b). This indicates the potential usefulness of the liver slice system as a preliminary screening test. The system has also been used to demonstrate the protective effects of N-acetylcysteine against damage induced by acetaminophen (Wormser & Ben Zakine, 1990; Wormser et al., 1990b), and to investigate age-related changes in the susceptibility of the neonatal mouse liver to furosemide (Wormser et al, 1990b). The liver slice system, therefore, also has potential use in the investigation of the mechanisms of toxicity and adverse drug interactions. The development of mechanical precision-cut slicers (e.g. Krumdieck tissue slicer, Brendel-Vitron tissue slicer) allows for the production of thin liver slices of defined thickness (about 250 µM), wich are superior to the thicker, inhomogenous hand-cut slices with respect to diffusion of nutrients and test substances. A lot of further endpoint measurements are possible (Bach et al., 1996; de Kanter et al., 2002; Gebhardt et al., 2003; ZEBET, 2003; Farkas and Tannenbaum, 2005). Status In house development. Last update: January 1992 https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocols page 2 / 7 © ECVAM DB-ALM: Protocol PROCEDURE DETAILS, January 1992 Liver Slice Hepatotoxicity Screening System DB-ALM Protocol n° 42 Contact Details Dr. Uri Wormser Department of Pharmacology The Hebrew University-Hadassah Medical School PO Box: 1172 Jerusalem 91010 telephone: Israel -2-428632 Animals Male Wistar-Sabra rat or mouse Equipment Prep blades - Weck Wecprep, Edward Weck and Company, Inc., NC, USA Erlenmeyer flasks Rotary water bath shaker G-76 New Brunswick 20ml capped glass beakers Tissue homogenizer - Kinematica homogenizer Centrifuge, 10,000g Light microscope Materials Ether anaesthetic Hepes NaCl KCl CaCl2 KH2PO 4 MgSO4 Glucose b-hydroxybutyrate Propylene glycol LDH commercial kit - Raichem, San Diego, USA ALT commercial kit - Raichem, San Diego, USA Haematoxylin/eosin stain Paraffin Formaldehyde Standard phosphate buffered saline (50mM Pi buffer + 0.9% NaCl, pH 7.0) Make up the following Krebs Ringer Hepes (KRH) Medium 2.5mM Hepes pH 7.4 118mM NaCl 2.85mM KCl 2.5mM CaCl2 1.15mM KH2PO 4 1.18mM MgSO4 5.0mM beta-Hydroxybutyrate 4.0mM Glucose https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocols page 3 / 7 © ECVAM DB-ALM: Protocol N.B. The solvent for the medium is double distilled water. The medium does not need to be sterilised and can be stored for 3 days at 40C, and for many months at -200C. Test solutions Perform a preliminary range-finding experiment using dilutions of test compound in water, or appropriate non-toxic solvent. Compounds that are poorly soluble in water may be initially dissolved in propylene glycol. A maximum permitted final concentration of propylene glycol in the cultures has not, as yet, been determined. A concentration of 16.6% propylene glycol has been used without causing any tissue damage. Vehicles other than propylene glycol are rarely used because of their toxicity. For the experiment make up the appropriate dilutions of test compound in water, or propylene glycol where necessary. Formaldehyde buffer 4% formaldehyde in PBS (50mm Pi, pH 7.0) PROCEDURE Preparation of liver slices Anaesthetize the animal with ether and sacrifice by cervical dislocation. Dissect out the liver lobes and place on a glass surface. Using prep blades, slice the liver into small pieces of about 0.5 x 0.5 x 0.5mm. N.B. One adult mouse liver gives approximately 20 slice systems, and one lobe of rat liver is sufficient for each experiment. Incubate the slices for 1 hour in a capped Erlenmeyer flask containing approx. 30 ml of KRH medium at 370C in a rotary water-bath shaker. During this incubation, wash the slices by carefully removing the medium and replacing it with fresh medium every 10 minutes. N.B. All media used for incubation and rinsing should be prewarmed. If the liver tissue is allowed to cool, this will cause the ion pumps to stop functioning and ion leakage could occur. Consequently, the characteristics of the hepatocytes would change, perhaps leading to irreversible damage within the slices. Divide the slices into small portions of 100-120mg wet weight, with the aid of a small spatula. Incubate the fragments for 1 hour in 2ml KRH medium in 20-ml capped beakers (approximately 20-30 slices/beaker) at 370C in a rotary water-bath shaker. N.B. Aerate the cultures in the Erlenmeyer flask and in the beakers with oxygen every 10 minutes by removing the cap, aerating the Erlenmeyer/beaker with an oxygen stream for 5 seconds and immediately replacing the cap. Exposure to test compounds Add the appropriate dilution of test compound to the cultures. N.B. Set up duplicate cultures for each dilution tested and controls. Incubate for 2 hours at 370C. Histopathology (if necessary) Remove liver slices from culture at regular intervals. Fix in 4% formaldehyde buffer. Embed tissue in paraffin, section and stain, with haematoxylin/eosin. Observe histopathology under light microscope if necessary. https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocols page 4 / 7 © ECVAM DB-ALM: Protocol Enzyme leakage assays Follow instructions in the kits to determine enzyme activity. Remove aliquots of culture medium at regular intervals and assay for LDH and ALT. It is not necessary to assay both enzymes in each test run. LDH is an appropriate parameter, although ALT may be added in special circumstances. N.B. Zero-time activity is obtained by assaying medium after 5 seconds of exposure.