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Protocol no. 23

RAT HEPATOCYTE FLOW CYTOMETRIC CYTOTOXICITY TEST

Flow cytometry is used to monitor drug-induced changes in DNA and protein contents of hepatocytes cultured at physiological oxygen concentrations.

CONTACT

Dr. Peter Maier Institut für Toxikologie der Eidgenössischen Technischen Hochschule und der Universität Zürich Labors für Zelluläre Toxikologie Schorenstrasse 16 CH-8603 Schwerzenbach bei Zurich Switzerland Tel: Switzerland 825 7428 or 825 7511 Fax: Switzerland 825 0476

RATIONALE

The importance of the liver in the metabolism of xenobiotics suggests that the use of hepatocytes in in vitro tests increases the probability of detecting and characterising the toxicity of test substances. The use of long-term hepatocyte cultures permits chronic effects to be monitored. Two-parameter flow cytometry allows the simultaneous observation of DNA and protein contents of hepatocyte subpopulations, making it possible to detect the dynamic responses of individual ploidy classes to the test substance. A further approximation to the in vivo situation is achieved by maintaining the cultures at oxygen tensions equivalent to those found in the periportal (13% O2) and pericentral (4% O2) regions of the liver lobules.

BASIC PROCEDURE

Primary hepatocytes are cultured for up to 7 days in the presence or absence of test substance at oxygen tensions similar to those found in liver lobules in vivo. The cells are then fixed in ethanol, subjected to ultrasonication and stained with stains specific for DNA and for protein. Two-parameter flow cytometry analysis is used to monitor the DNA and protein contents of hepatocyte subpopulations.

CRITICAL ASSESSMENT

One aim of in vitro toxicity tests is to provide a well-defined, stable system in which specified cell types can be exposed to test substances. The primary role of the liver in xenobiotic metabolism makes hepatocytes an obvious choice for such test systems. However, the liver in vivo exhibits functional, dynamic and metabolic heterogeneity (Jungermann & Sasse 1978, Gumucio & Miller 1981). Subpopulations of hepatocytes may be distinguished on the basis of their ploidy, which is affected in vivo by hepatocarcinogens and tumour promoters (Digernes 1983, Romagna & Zbinden 1981, Schwarze et al. 1984, Styles et al. 1985, van Ravenzwaay et al. 1987). Furthermore, ploidy, together with cellular protein content, is an important indicator of ageing and regeneration processes in vivo (James et al. 1979). For this reason, alterations in DNA and protein content of individual cells in vitro, as monitored by flow cytometry, may be viewed as indicators of the senescence of hepatocyte cultures and also as indicators of cellular responses to toxic substances (Holzer & Maier 1987a). The two-parameter DNA/protein flow cytometry analysis chosen for this test system has proved to be a highly reproducible method to monitor alterations in DNA and protein content of freshly isolated and altered hepatocytes (Holzer & Maier 1987a). It permits the observation of cellular responses occurring after prolonged exposure to non-toxic concentrations of chemicals, which are considered comparable to responses occurring in vivo. It must be noted that a considerable variation in the distribution of different ploidy classes has been observed between individual animals. For this reason it is important to carry out an individual ploidy analysis for each primary hepatocyte culture. A further significant factor in relation to hepatotoxic chemicals is the manifestation in vivo of a zone-specific response within the liver lobules (Tulp, et al. 1978, Massey & Butler 1979, Sweeney 1981.) Oxygen could be the most important parameter responsible for this phenomenon, since oxygen tension varies from 9-13% in the periportal zone to 4-5% in the pericentral zone. Incubator atmospheres of 13% and 4% O2 (v/v) are therefore employed to reproduce the most extreme periportal and pericentral oxygen conditions respectively (Nauck et al. 1981). Pericentral oxygen tension has been found to delay protein degradation and/or to stimulate protein synthesis and to increase the contribution of 2C hepatocytes. From these results it is concluded that low (pericentral) oxygen tension is more efficient at maintaining regeneration and delaying ageing processed in cultured hepatocytes. This suggestion that low pO2, as found with mesenchymal cell so also in hepatocytes, may support proliferative and tumour-promoting processes, has implications for the testing of potential carcinogens (Holzer & Maier 1987b). The achievement of physiological oxygen concentrations in primary hepatocyte cultures is, however, complicated by the fact that, under conventional culture conditions, the culture medium and the plastic walls of the culture vessels act as diffusion barriers to prevent oxygen reaching the hepatocytes. It is assumed that conventional hepatocyte monolayer cultures in a CO2/ambient air atmosphere reach a pO2 similar to that of the periportal region, but the actual pO2 is affected by plating density, by the rate of cell detachment in acute toxicity reactions, and by the addition of other cells in co-culture systems. It has been demonstrated that the actual pO2 of the culture medium can be maintained at a stable level corresponding to the chosen O2 concentration in the incubator atmosphere if conventional plastic dishes are replaced by gas-permeable hydrophilic teflon membrane dishes (Holzer & Maier 1987b). There is however the disadvantage when using Petriperm dishes that the cells are exposed to too high an oxygen tension on the bench while medium is being changed. Attempts are now in progress to rectify this situation. The presence of serum in a cell culture system introduces an element that cannot be fully characterized, due to the variation that occurs between different batches of serum. Furthermore, serum may affect the interactions that occur between the cells and the test chemical. However, fetal calf serum does have a restorative effect on freshly isolated hepatocytes. For this reason, culture medium containing 10% FCS is used during the first three hours of hepatocyte culture, and serum-free media are used in all other cases. FCS at a concentration lower than 10% has also been found to be effective in the initial culture medium, although the amount required tends to vary with the batch used. This point is currently under investigation, as is the possibility of replacing FCS altogether. So far, however, no procedure has been established which is superior to the system as described in this protocol. One factor that will affect the results of the flow analysis is any change in cellular protein content that may occur for technical reasons, e.g. during trypsinisation of cultured cells. The protein content has to be stable over the dissociation period used. It is therefore necessary to check for protein loss, using trypan blue, at various stages in the procedure. Trained operators are required to carry out the flow analysis. One operator can handle 16 samples in the course of one afternoon. Simultaneously to the flow analysis, one should also check biochemical parameters, such as cytochrome P-450 content, liver-specific enzyme functions. The total investigation, i.e. liver perfusion, flow analysis, quality control of cultures and of drug-induced changes in the hepatocytes after exposure to a chemical, cannot be handled by just one person. Two-parameter flow cytometry is also used in another test system developed in the same laboratory (Maier & Schawalder 1988) which is based on the detection of drug-induced chromosome aberrations in primary cultures of rat fibroblast-like cells isolated from subcutaneous granulation tissue. These cells may also be used as the internal standard for the flow cytometry described in this protocol. Furthermore, these fibroblasts might serve as a reference for extrahepatic cells from the same animal, as an alternative (mesenchymal, proliferating, limited xenobiotic metabolism) to hepatocytes (epithelial, nonproliferating, complete xenobiotic metabolism). A further refinement (Maier 1988) used co-cultures in which freshly isolated hepatocytes interacted with isolated, in vitro cultured, rat liver epithelial cells. The resultant heterotypic cell-cell interaction was shown to stabilise the aldrin epoxidase enzyme system (involved in xenobiotic metabolism) for more than two weeks and to allow the demonstration of preferential toxicity for certain chemicals and cell populations. The introduction of heterotypic cell-cell interactions was attempted with the aim of developing a culture system that would be closer to the in vivo situation. However, the fact that morphological criteria are used to isolate the helper cells means that the procedure cannot be strictly defined. Variation in behaviour between different populations of isolated helper cells does indeed occur. In addition, in co-cultures, cytoskeletons different from those occurring in vivo are synthesised. Co-cultured hepatocytes can no longer be dissociated by means of trypsin or collagenase without affecting the cellular protein content. Both the pure definition of the helper cells and the non-dissociable cells make the system unsuitable at present for use as a routine procedure.

TEST STATUS

In-house development.

CHEMICALS TESTED

Dimethylsulphoxide (not tested at controlled pO2) Phenobarbital (not tested at controlled pO2) A study is currently (1990) under way to test various agents at different oxygen tensions.

REFERENCES

  1. Ahrens, O., Albrecht, U. & Rajewsky, M.F. (1980) Microprocessor-based data acquisition system for flow cytometers. in Flow cytometry IV (eds.: Laerumt, O.D., Linamot, T. & Thorud, E.), Universitetsforlaget ; Bergen, pp. 112-115.
  2. Begue, J.M., Guguen-Guillouzo, C., Pasedeloup, N. & Guillouzo, A. (1984) Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type. Hepatology, 4, 839-842.
  3. Digernes, V. (1983) Chemical liver carcinogenesis: monitoring of the process by flow cytometric DNA measurements. Environmental health perspectives, 50, 159-200.
  4. Gumucio, J.J. & Miller, D.L. (1981) Functional implications of liver cell heterogeneity. Gastroenterology, 80, 393-403.
  5. Holzer, C. & Maier, P (1987a) DNA and protein contents of hepatocytes in primary cultures monitored by flow cytometry: effect of phenobarbital and dimethylsulphoxide. Toxicology in vitro, 1, 203-213.
  6. Holzer, C. and Maier, P. (1987b) Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: influence on cellular DNA and protein content monitored by flow cytometry. Journal of cellular physiology ,133, 297-304.
  7. James, J., Tas, J., Bosch, K.S., de Meere, A.J.P. & Schuyt, H.C. (1979) Growth pattern of rat hepatocytes during postnatal development. European journal of cell biology , 19, 222-226.
  8. Jungermann, K. and Sasse, D. (1978) Heterogeneity of liver parenchymal cells. TIPS 3: 198-202.
  9. Maier, P. (1984) The granuloma pouch assay. In Chemical mutagens : Principles and methods for their detection Vol. 9 (ed. F.J. de Serres) Plenum; New York, pp. 233-260.
  10. Maier, P. (1988) Development of in vitro toxicity tests with cultures of freshly isolated rat hepatocytes. Experientia 3117.
  11. Maier, P. & Schawalder, H. (1988) Alterations in the cellular DNA and protein content determined by flow cytometry as indicators for chemically induced structural and numerical chromosome aberrations. Mutagenesis, 3, 219-226.
  12. Massey, E.D. & Butler, W.H. (1979) Zonal changes in the rat liver after chronic administration of phenobarbitone: An ultrastructural, morphometric and biochemical correlation. Chemical and biological interactions, 24, 329-244.
  13. Nauck, M., Wölfle, D. & Katz, N. (1981) Modulation of the glucagon-dependent induction of phosphoenolpyruvate carboxykinase and aminotransferase by arterial and venous oxygen concentration in hepatocyte cultures. European journal of biochemistry, 119, 657-661.
  14. Romagna, F. & Zbinden, G. (1981) Distribution of nuclear size and DNA content in serial liver biopsies of rats treated with N-nitrosomorpholine, phenobarbital and butylated hydroxytoluene. Experimental cell biology, 49, 294-305.
  15. Schwarze, P.E., Pettersen, E.O., Shoaib, M.C. & Seglen, P.O. (1984) Emergence of a population of small, diploid hepatocytes during hepatocarcinogenesis. Carcinogenesis, 5, 1267-1275.
  16. Seglen, P.O. (1972) Preparation of rat liver cells. Effect of Ca2+ on enzymatic dispersion of isolated perfused liver. Expl. cell res., 74, 450-454.
  17. Seglen, P.O. (1973) Preparation of rat liver cells. Expl. cell res., 82, 391-398.
  18. Stöhr, M., Vogt-Schaden, M., Knoblauch, M., Vogel, R. & Futtermann, G. (1978) Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analysis. Stain technology, 53, 205-215.
  19. Styles, J.A., Elliott, B.M., Lefevre, P.A., Robinson, M., Pritchard, N., Hart, D. & Ashby, J. (1985) Irreversible depression in the ratio of tetraploid:diploid liver nuclei in rats treated with 3'-methyl-4 dimethylaminoazobenzene (3'M). Carcinogenesis, 6, 21-28.
  20. Sweeney, G.D. (1981) Functional heterogeneity among liver cells: Implications for the drug induced toxicity and metabolism. TIPS, June, pp. 141-144.
  21. Tulp, A., Welagen, J.J.M.N. & Westra, J.G. (1978) Binding of the chemical carcinogen N-hydroxy-acetyl-aminofluorene to ploidy classes of rat liver nuclei as separated by velocity sedimentation at unit gravity. Chemical and biological interactions, 23, 292-303.
  22. Van Ravenzwaay, B., Tennekes, H., Stöhr, M. & Kunz, W. (1987) The kinetics of nuclear polyploidization and tumour formation in livers of CF-1 mice exposed to dieldrin. Carcinogenesis, 8, 265-269.

IP-23 © December 1990