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Protocol no. 13
HEPATOMA CELL CULTURES AS IN VITRO MODELS FOR HEPATOTOXICITY
This test is designed to detect irreversible toxic effects on both cell growth and survival, by the evaluation of colony-forming (CF) efficiency, in hepatoma cell lines derived from man, rat and mouse
CONTACT
Professor Margherita Ferro
Istituto di Patologia Generale
Via L.B. Alberti, 2
16132 Genova
Italy
Tel: + 39 10 353 833
Fax: + 39 10 353 836
Rationale
The liver is a major target organ for the cytotoxicity of many xenobiotics. It has been suggested that hepatoma cell lines may provide an appropriate in vitro model for the assessment of likely hepatotoxicity in vivo. It should be noted, however, that the usefulness of such systems largely depends upon the ability of the cells to maintain differentiated functions.
The procedure presented here suggests a simple means of assessing the cytotoxicity of compounds to hepatoma cell lines. The method involves exposing the cells to xenobiotics, after which colony formation is monitored and compared to that of non-exposed control cultures. The method can easily be adapted for many hepatoma cell lines.
In this particular procedure, six hepatoma cell lines have so far been used:
HepG2 - a human hepatoblastoma cell line which shows basal and inducible levels of monooxygenases.
MH1C1 - a rat hepatoma cell line which shows basal and inducible levels of monooxygenases.
7777 - a rat hepatoma cell line which shows basal and inducible levels of monooxygenases.
HTC - a rat hepatoma cell line which does not show monooxygenase activities.
JM2 - a rat hepatoma cell line which does not show monooxygenase activities.
Hepa 1c1c7 - a murine hepatoma cell line which shows basal and inducible levels of monooxygenases.
Basic Procedure
A small number of cells (250-500) are plated into complete medium in 6-well (35 mm diameter) culture plates. After 24 hours, the medium is removed and the cells are exposed to different concentrations of the test compounds in serum-free medium. After 1, 6 or 24 hours, the experimental medium is removed and the cells are grown in standard conditions for 8-15 days. The colonies are then stained and those at least 0.3 mm in diameter are scored with the naked eye and the percentage survival is calculated with reference to control cultures. A decrease in the number of colonies formed is an indication of test compound toxicity.
Critical Assessment
Colony formation may provide a much more sensitive measure of toxicity than certain other commonly employed methods. For example, it was found to be more sensitive than the LDH leakage assay (Ferro et al., 1988), due to the fact that it depends upon cell growth-related mechanisms rather than cell membrane damage. Colony formation was also found to be a more sensitive parameter of toxicity than cell viability assessed by total macromolecular content of the attached monolayer (Bassi et al., 1991, 1993). The reason for this increased sensitivity could be the fact that colony formation is assessed while the cells are in a state of proliferation, and thus more susceptible to toxic effects. Moreover, the measurement of total macromolecular content is carried out in a larger number of cells, which may mask some dose-dependent effects since the test compound is distributed over a large population of target cells. The sensitivity of the colony-formation assay, and the fact that dose and time-dependent effects are detectable, enables acute and chronic exposure periods to be investigated as well as permitting recovery studies.
It has been found that exposure of the cells to test compounds before seeding rather than 24h post attachment is not an effective way of assessing toxicity.
Colony formation may be employed as an endpoint in many different hepatoma cell lines. The method must be standardised, however, for each cell type. There are cell lines, for example H4IIEC3, whose cells spread out from the colony, thus impairing distinction of separate colonies. Ideally the cells should be plated out at a suitable level so that over a period of 7-15 days they form between 50 and 100 colonies. The level of plating and period of incubation for any particular cell line can be established this way.
The different hepatoma cell lines vary in their sensitivity to different chemicals. A comparison of HTC (rat hepatoma) and HepG2 (human hepatoma) cells (Bassi et al., 1993) showed differences that arise from the fact that HepG2 cells retain some cytochrome P450-dependent functions, while HTC cells are practically devoid of bioactivating enzymes. Thus, the IC50 values in mM for paracetamol, amitryptiline and nicotine (see Table 3) are approximately ten times greater for HTC cells than for HepG2 cells, showing that metabolism by the latter contributes to the toxicity. However, HepG2 cells are also more sensitive than HTC to iron sulphate, digoxin and potassium cyanide, substances which are assumed to have a direct toxic effect. This could be due to less specific mechanisms operating in the human cell line. Similarly, a difference in sensitivity to benzaldehyde was found between the two rat cell lines MH1C1 and HTC (Ferro et al., 1988), due to the fact that aldehyde dehydrogenase is present in much greater quantities in HTC cells.
Thus, although the effects of a chemical on colony formation in a hepatoma cell line will provide an indication of cytotoxicity, interpretation of the results depends upon many factors, including the metabolic competence of the cells with respect to the test compound. The degree of expression of the various monooxygenase isozymes by different hepatoma cell lines must therefore be taken into consideration. The relevance of results obtained in various hepatoma cell lines to human toxicity in vivo, and therefore the definition of the most appropriate cell lines to use for specific testing purposes, has yet to be established. The inclusion of this system into the MEIC (Multicentre evaluation of in vitro cytotoxicity) programme may begin to answer these questions.
The cells are exposed to the test compounds in serum-free medium so that the direct toxic effect can be estimated in the absence of any confounding effects arising from the interaction of the compounds with serum proteins.
Test Status
Used routinely to assess cytotoxicity.
Chemicals Tested
Acetaldehyde
Acetaminophen
Amitryptiline
Arsenic trioxide
Aspirin
Benzaldehyde
Benzoic acid
Butylated hydroxyanisole
Butylated hydroxytoluene
Cupric sulphate
Cycloheximide
Cyclophosphamide
Dextropropoxyphene HCl
Diazepam
2,4-Dichlorophenoxyacetic acid
Digoxin
2,4-Dinitrophenol
Ethyl alcohol
Ethylene glycol
Ferrous sulphate
4-Hydroxynonenal
Isopropyl alcohol Lindane
Lithium sulphate
Malathion
Mercuric chloride
Methyl alcohol
Nicotine
Paraquat
Phenobarbital
Phenol
Potassium dichromate
Potassium cyanide
Propionaldehyde
Propranolol HCl
Sodium chloride
Sodium fluoride
Thallium sulphate
Theophylline
Thioridazine HCl
1,1,1-Trichloroethane
Valeraldehyde
Warfarin
Xylene
References
Ferro M., Bassi A.M., and Nanni G. (1988)
Hepatoma cell cultures as in vitro models for the hepatotoxicity of xenobiotics.
ATLA, 16(1), 32-37.
Bassi A.M., Piana S., Penco S., Bosco O., Brenci S, and Ferro M. (1991)
Use of an established cell line in the evaluation of the cytotoxic effects of various chemicals.
Boll. Soc. It. Biol. Sper. 8: 809-816.
Ferro M., Bassi A.M., Penco S., Piana S., Usiglio D., and Nanni G. (1992)
Comparative assessment of the cytotoxic effects of different xenobiotics in three hepatoma cell lines.
Arzneim.-Forsch./Drug Res. 442: 1053-1057.
Bassi A.M., Bosco O., Brenci S., Adamo D., Penco S., Piana S., Ferro M., and Nanni G. (1993)
Evaluation of the cytotoxicity of the first 20 MEIC chemicals in two hepatoma cell lines with different xenobiotic metabolism capacities.
ATLA 21: 65-72.
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