The Use of Tissue Slices for Pharmacotoxicological Studies

The Report and Recommendations of ECVAM Workshop 201,2

Reprinted with minor amendments from ATLA 24, 893-923.

Appendix 1

The Use of Tissue Slices Prepared by Conventional Cutting Methods

The literature on the use of slices prepared from non-nervous tissue by conventional cutting methods is extensive (over 4000 publications from between 1980 and 1994 can be retrieved by conducting an appropriate EMBASE®, MedLine® or similar literature search). Over 10,000 papers on brain slices have been published in the same period.

Data derived from conventional tissue slices (free-hand, guided, Stadie-Riggs or dermatome) are available for many species and for all of the major organs. Studies undertaken include assessment of the effects of sex, age, circadian rhythm, diet, and hibernation on biological processes. Relatively few studies have involved the use of human tissues, and limited comparisons between effects in human and animal slices have been conducted.

Detailed methodology for preparing and using the tissue slices is not often available, and the information on the long-term viability of many of the preparations is inadequate. There have been few attempts to optimise the existing protocols with respect to trying to enhance or prolong tissue slice viability. Despite these limitations, conventional tissue slices have been used for a broad range of biomedical applications.

 

Viability and Endpoints

Typical endpoints include: MTT reduction; leakage of marker enzymes; K+ levels; lipid peroxidation; glutathione (GSH) levels; oxygen consumption; ATP utilisation; and measurement of transport activities (such as transport of the organic anion, para-aminohippurate, and of organic cations, such as tetraethylamine). It is also possible to assess biochemical changes occurring within slices; for example, a Clark-type oxygen electrode has been used to show that nitric oxide release from vascular endothelial cells plays an important role in the regulation of mitochondrial respiration (1).

 

Applications

Anatomically and biochemically distinct parts of the same organ can be studied; for example, the renal cortex and, to a lesser extent, the papilla; the mucosa and smooth muscle of the duodenum, jejunum, ileum, and caecum of the gastrointestinal tract; the atrial, ventrical, and septal tissues of the heart; and the different regions of the respiratory tract, including the nasal septum, tracheal smooth muscle, and the lung.

Biochemical Processes

Slices from different organs have been used to assess most aspects of intermediary metabolism which are common to all cells, and also biochemical processes which are specific to certain tissues.

Physiology and Pharmacology

Many aspects of physiology and pharmacology have been assessed in slices. For example, data are available on: the classification of hormone responsiveness, receptor sub-types, and receptor agonists and antagonists; the effects of therapeutic agents on receptor function; second messengers and the production, modulation and effects of nitric oxide; and other aspects of cell signalling, including the inositol phosphate pathway and changes in protein kinase C activity. More recently, the role of biotechnology products (for example, insulin-like growth factors, interleukin-1β, endothelin-1, and tumour growth factor) has been assessed in terms of hormonal responsiveness in normal and diseased tissues.

There are also reports where slices have been used for investigations which are more usually conducted in vivo or with isolated cells. For example, slices from the cardiac ventricles of 1-14 day old rats can be used for single-channel patch-clamp recordings, in order to assess the electrophysiological properties of heart cells in situ (2). This can be achieved in slices without the need for complicated cell isolation procedures, which may alter channel properties due to the need for proteolytic enzymes. Steady-state membrane potentials and intracellular Na+, K+, H+, and Cl- concentrations have been studied by using a double-barrelled ion-selective microelectrode (3).

Receptor distribution studies are generally undertaken by using frozen or fixed tissue sections to avoid active uptake, metabolism, and unwanted regulation of the radiolabelled ligands which are intended to bind to receptors. Fresh tissue slices are especially suitable for the study of receptor regulation (4, 5) and receptor mapping studies have been undertaken in fresh tissue slices at 4°C, or by using metabolic inhibitors.

Toxicology Studies

The toxicities of a wide range of compounds have been investigated with tissue slices prepared by conventional cutting methods; heavy metals, antineoplastic agents, analgesics, antibiotics, organic solvents, and organic pollutants have been studied.

Biotransformation

Tissues from most organs have been used to study and compare many aspects of xenobiotic biotransformation, in terms of measuring the amount of chemical uptake, the formation of metabolites, and total covalent binding.

  1. Benzidine metabolism: There are important species differences in benzidine metabolism (6-10). In rat liver slices, N,N'-diacetylbenzidine formation is favoured, whereas human liver produces more N-acetylbenzidine. These differences relate to the acetylator status, which helps to explain the species and organ specificities of benzidine effects. The role of acetylation in benzidine metabolism and DNA adduct formation has been compared in dog, rat, and human liver slices. The metabolites detected include N-acetylbenzidine and N,N'-diacetylbenzidine. Rat liver slices contained only N'-(3'-monophosphodeoxyguanosine-8-yl)-N-acetylbenzidine adducts; no acetylated adducts were detected in dog liver slices (the dog is a non-acetylator of benzidine).

    N-Glucuronidation appears to represent a major pathway of benzidine metabolism in humans, with the extent of N-acetylation affecting the proportion of benzidine and N-acetylbenzidine which is subsequently glucuronidated. Human liver converts low concentrations of N-acetylbenzidine to the N-glucuronide, whereas this only occurs at high concentrations in rat liver. Dog liver slices detoxify benzidine to its N-glucuronide, which is stable in plasma and is weakly bound compared to the parent compound. It will be excreted in urine where, due to its acid lability, it will release benzidine; this will accumulate in acidic urine, be taken up by bladder epithelium, and be activated in the bladder (6-10).

  2. Autoradiography: tape-section and light microscope autoradiography have been used to map the covalent localisation of radiolabelled toxicants and carcinogens. For example, the patterns of covalent labelling of rodent respiratory and upper alimentary tract epithelium with activated N-nitrosamines and halogenated hydrocarbons have been reported (11, 12). The involvement of various forms of cytochrome P450 can be examined by using enzyme-selective inducers and inhibitors (13). The effects of factors which protect the tissues against highly reactive intermediates (for example, GSH) can be assessed by using animals pretreated with GSH-modulating agents (14).

Selective Toxicity

Data are available for renal and liver slices which show that a number of compounds which affect specific cell types in vivo also do so in vitro, as assessed by biochemical parameters, and morphological and ultrastructural changes. Lung slices warrant special mention, since they maintain their viability for prolonged periods (> 7 days), and at the same time display a high degree of functional and structural specificity (15-19).

Tumour Biology

The use of tumour tissue slices obtained from animals and humans has facilitated the assessment of metabolism, inter-species comparisons, and investigations of various aspects of tumourigenesis. Specific investigations which have been reported include: (a) the measurement of steady-state membrane potentials and intracellular ion concentrations (3); (b) studies on oestrogen receptors and oestrogen metabolism; (c) the role of growth factors in tumourigenesis; and (d) aspects of photodynamic therapy (20).

Sequential changes in gap junctional intercellular communication (GJIC) during multi-stage rat liver carcinogenesis, as determined by microinjection dye transfer and immunostaining of connexin-32 (the major liver gap junctional protein), have provided further evidence of the benefits of using tissue slices for assessing changes in intercellular communication as one of the stages in the carcinogenic process. Such studies have shown that, in hepatocellular carcinomas, GJIC is significantly reduced, and this is accompanied by a large decrease in connexin-32 expression (21-23).

References

  1. Shen, W., Hintze, T.H. & Wolin, M.S. (1995). Nitric oxide. An important signaling mechanism between vascular endothelium and parenchymal cells in the regulation of oxygen consumption. Circulation 92, 3505-3512.
  2. Burnashev, N.A., Edwards, F.A. & Verkhratskii, A.N. (1991). Primenenie tonkikh srezov miokarda dlia registratsii tokov cherez odinochnye ionnye kanaly [The use of thin slices of myocardium for recording the currents across single ion channels]. Fiziologicheskii Zhurnal 37, 119-122.
  3. Comolli, R., Rossetti, C. & Cremaschi, D. (1990). Measurement with microelectrodes of intracellular Na+, K+, H+, and Cl- activities and of membrane potential in normal rat liver slices and during 4-dimethylaminoazo-benzene-induced rat hepatocarcinogenesis. Cancer Biochemistry and Biophysiology 11, 13-22.
  4. Wilkinson, M., Jacobson, M. & Watson-Wright, W. (1986). Tissue slices in radioligand binding assays: studies in brain, pineal gland, and muscle. Life Sciences 39, 2037-2048.
  5. Shaw, C.A. & Wilkinson, M. (1994). Receptor characterization and regulation in intact tissue preparations. Pharmacological implications. Biochemical Pharmacology 47, 1109-1119.
  6. Babu, S.R., Wongsurawat, V.J., Zenser, T.V. & Davis, B.B. (1993). Benzidine glucuronidation in dog liver. Carcinogenesis 14, 893-897.
  7. Babu, S.R., Lakshmi, V.M., Hsu, F.F., Kane, R.E., Zenser, T.V. & Davis, B.B. (1993). N-Acetylbenzidine-N'-glucuronidation by human, dog, and rat liver. Carcinogenesis 14, 2605-2611.
  8. Babu, S.R., Lakshmi, V.M., Owens, I.S., Zenser, T.V. & Davis, B.B. (1994). Human liver glucuronidation of benzidine. Carcinogenesis 15, 2003-2007.
  9. Lakshmi, V.M., Zenser, T.V., Goldman, H.D., Spencer, G.G., Gupta, R.C., Hsu, F.F. & Davis, B.B. (1995). The role of acetylation in benzidine metabolism and DNA adduct formation in dog and rat liver. Chemical Research in Toxicology 8, 711-720.
  10. Lakshmi, V.M., Bell, D.A., Watson, M.A., Zenser, T.V. & Davis, B.B. (1995). N-Acetylbenzidine and N,N'-diacetylbenzidine formation by rat and human liver slices exposed to benzidine. Carcinogenesis 16, 1565-1571.
  11. Brittebo, E.B. & Brandt, I. (1994). Metabolic activation of the food mutagen 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole (Trp-P-1) in endothelial cells of cytochrome P-450-induced mice. Cancer Research 54, 2887-2894.
  12. Brittebo, E.B. & Brandt, I. (1996). Cell- and tissue-specific metabolic activation of chemicals: in vitro/in vivo correlations. In Vitro Toxicology, in press.
  13. Brittebo, E.B. (1994). Metabolic activation of the food mutagen Trp-P-1 in endothelial cells of heart and kidney in cytochrome P450-induced mice. Carcinogenesis 15, 667-672.
  14. Brittebo, E.B. (1987). Metabolic activation of phenacetin in rat nasal mucosa: dose-dependent binding to the glands of Bowman. Cancer Research 47, 1449-1456.
  15. Freeman, B.A. & O'Neil, J.J. (1984). Tissue slices in the study of lung metabolism and toxicology. Environmental Health Perspectives 56, 51-60.
  16. Placke, M.E. & Fisher, G.L. (1987). Adult peripheral lung organ culture - a model for respiratory tract toxicology. Toxicology and Applied Pharmacology 90, 284-298.
  17. Placke, M.E. & Fisher, G.L. (1987). Asbestos in peripheral lung culture. A species comparison of pulmonary tissue response. Drug and Chemical Toxicology 10, 133-156.
  18. Fisher, G.L. & Placke, M.E. (1988). Cell and organ culture models of respiratory toxicity. In Toxicology of the Lung (ed. D.E. Gardner, J.D. Crapo & E.J. Massaro), pp. 285-314. New York: Plenum Press.
  19. Sawyer, T.W., Wilde, P.E., Rice, P. & Weiss, M.T. (1995). Toxicity of sulphur mustard in adult rat lung organ culture. Toxicology 100, 39-49.
  20. Fukuda, H., Casas, A., Chueke, F., Paredes, S. & Batelle, A.M. (1993). Photodynamic action of endogenously synthesized porphyrins from aminolevulinic acid, using a new model for assaying the effectiveness of tumoral cell killing. International Journal of Biochemistry 25, 1395-1398.
  21. Krutovskikh, V.A., Mesnil, M., Mazzoleni, G. & Yamasaki, H. (1995). Inhibition of rat liver gap junction intercellular communication by tumor-promoting agents in vivo. Association with aberrant localization of connexin proteins. Laboratory Investigation 72, 571-577.
  22. Krutovskikh, V.A., Oyamada, M. & Yamasaki, H. (1991). Sequential changes of gap-junctional intercellular communications during multistage rat liver carcinogenesis, direct measurement of communication in vivo. Carcinogenesis 12, 1701-1706.
  23. Krutovskikh, V.A. & Yamasaki, H. (1995). Ex-vivo dye transfer assay as an approach to study gap junctional intercellular communication disorders in hepatocarcinogenesis. Progress in Cell Research 4, 93-97.

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