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Protocol no. 42
LIVER SLICE HEPATOTOXICITY SCREENING SYSTEM

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.

CONTACT

Dr Uri Wormser
Department of Pharmacology
The Hebrew University Hadassah Medical School
PO Box 1172 91010 Jerusalem, Israel
Tel: Israel - 2-428632 Fax: Israel - 2-431094

RATIONALE

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.

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 minute 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.

CRITICAL ASSESSMENT

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 hour exposure of mouse liver slices to acetaminophen correlate with the extent of multifocal hepatocytic degeneration observed in the slices. 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 LD50 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.

CHEMICALS TESTED

Acetaminophen
Amitriptyline HCl
Aspirin
Chlorpromazine HCl
Cimetidine
Furosemide
Imipramine HCl
Mechlorethamine (nitrogen mustard)
Nifedipine
Pentobarbital sodium
Phenacetine
Quinidine sulphate
Theophylline
Valproic acid
Verapamil HCl
Carbon tetrachloride
Methyl alcohol
Ethyl alcohol
Isopropyl alcohol
Cadmium chloride

TEST STATUS

In house development.

REFERENCES

  1. Davis, D.C., Potter, W.Z., Jollow, D.J. & Mitchell, J.R. (1974) Species differences in hepatic glutathione depletion, covalent binding and hepatic necrosis after acetaminophen. Life Sci., 14, 2099-2109
  2. Dujovne, C.J., Levy, R., & Zimmerman, H.J. (1968) Hepatotoxicity of phenothiazines in vitro as measured by loss of aminotransferases to surrounding media. Proc. Soc. Exp. Biol. Med., 128, 561-563
  3. Goering, P.L., & Klassen C.D. (1984) Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxic. Appl. Pharmac., 74, 308-313
  4. Gerson, R.J., Casini, A., Gilfor, D., Serroni, A. & Farber, J.L. (1985) Oxygen-mediated cell injury in the killing of cultured hepatocytes by acetaminophen. Biochem. Biophys. Res. Commun., 126, 1129-1137
  5. Mitchell, J.R., Nelson, W.L., Potter, W.Z., Sasame, H.A. & Jollow, D.J. (1976) Metabolic activation of furosemide to a chemically reactive, hepatotoxic metabolite. J. Pharmacol. Exp. Ther., 199, 41-52
  6. Moore, M., Thor, H., Moore, G., Nelson, S., Moldeus, P. & Orrenius, S. (1985) The toxicity of acetaminophen and N-acetyl-p-benzoquinone imine in isolated hepatocytes is associated with thiol depletion and increased cystolic calcium. J. Biol. Chem., 260, 13035-13040 Schwetz, B.A. & Plaa, G.L. (1969) Catecholamine potentiation of carbon tetrachloride-induced hepatotoxicity in mice. Toxicol. Appl. Pharmacol., 14, 495-509
  7. Smith, C.V. & Mitchell, J.R., (1985) Acetaminophen hepatotoxicity in vivo is not accompanied by oxidant stress. Biochem. Biophys. Res. Commun., 133, 329-336
  8. Wormser, U., Ben Zakine, S., Stivelband, E., Eisen, O. & Nyska, A. (1990a) The liver slice system: A rapid in vitro acute toxicity test for primary screening of hepatotoxic agents. Toxicology In Vitro, 4, 783-789
  9. Wormser, U. & Ben Zakine, S. (1990) The liver slice system: An in vitro acute toxicity test for assessment of hepatotoxins and their antidotes. Toxicology In Vitro, 4, 449-451
  10. Wormser, U., Ben Zakine, S. & Nyska, A. (1990) Cadmium-induced metallothionein synthesis in the rat liver slice system. Toxicology In Vitro, 4, 791-794
  11. Wormser, U., Ben Zakine, S., Eisen, O. & Nyska, A. (1990b) The liver slice system: a rapid and simple acute toxicity test for assessment of environmental toxic substances. Proceedings of the Second International Conference on Environmental Analytical Chemistry: January 17-19, 1990, Honolulu, Hawaii, US. US Environmental Protection Agency, US National Institute of Standards and Technology, The Center for Environmental Research, Cornell University.
  12. Younes, M., Cornelius, S. & Siegers, C-P. (1986) Ferrous ion supported in vivo lipid peroxidation induced by paracetamol - its relation to hepatotoxicity. Res. Commun. Chem. Path. Pharmac., 51, 89-99
    13. ?? IP-42 © January 1992