Toxicology of Industrial Compounds

212 USE OF LONG-TERM CULTURES OF HEPATOCYTES IN TOXICITY TESTING
may activate as well as deactivate a number of important molecules such as
thioether-containing pesticides (Hajjar and Hodgson, 1980), 3,3′-dichlorobenzidine
(Iba and Thomas, 1988), N-methyl-4-aminobenzene (Kadlubar
et al., 1976) and others. The expression of FMOs is also maintained better
and for a longer time in co-cultures of rat hepatocytes than it is in monocultures
of these cells (Coecke et al., 1993). In co-cultures a steady state
situation is obtained at a level of approximately 40 per cent of its initial
value in freshly isolated hepatocytes (Coecke et al., 1993).
Hormonal regulation of FMO is retained (Coecke et al., 1995a,b). It was
shown that 17β-oestradiol significantly decreased FMO activity in cocultures
of male rat hepatocytes which was not the case for testosterone
and 5α-dihydrotestosterone. These data are in accordance with in vivo
results (Coecke et al., 1995b). In addition, the thyroid hormone thyroxine
and its metabolite L-triiodothyronine were found to cause a significant
decrease in FMO activity suggesting a suppressive role in the regulation of
FMO in rat liver. In vivo data on this subject are not available in the
literature.
Phase 2
reactions in co-cultured hepatocytes
The activity, expression and regulation of the different glutathione Stransferase
(GST) isoenzymes in co-cultured rat hepatocytes, has been
extensively studied by our group (Vandenberghe et al., 1988a,b, 1989,
1990a,b, 1991). As far as the enzymatic activity is concerned, it was
observed that the composition of the culture medium was of much less
influence in co-cultures than was the case in mono-cultures (Vandenberghe
et al., 1988b). In cocultures GST activity was maintained for a longer
period and at a more stable level, comparable to the in vivo situation. This
observation was confirmed later by Niemann et al. (1991) and Utesch and
Oesch (1992), although the latter investigators reported a high variability
in their results depending on the batch of epithelial cells. GST activity in cocultured
rat hepatocytes was found to be increased or decreased by
phenobarbital and valproate, respectively (Rogiers et al., 1988a; Rogiers et
al., 1992).
These results are in good accordance with previously obtained in vivo
data (Rogiers et al., 1988a; Rogiers et al., 1992). They were also confirmed
at the protein level using the Western blot technique (Rogiers et al., 1995).
Furthermore, GST activity is increased significantly, in a dose dependent
way by ethanol. Both GST protein and mRNA amounts (in particular, GST
subunits 3 and 4) were increased by this compound (Coecke et al., 1995c).
Results obtained using different substrates suggested that the GST isoenzyme
profile changes as soon as hepatocytes are seeded in culture
(Vandenberghe et al., 1988b). By a combination of GSH agarose affinity