Development of functional in vitro toxicity tests
In vitro toxicity tests which detect evidence of the formation of reactive metabolites have
previously relied upon cell death as a toxicity end point. Therefore these tests determine
cytotoxicity in terms of quantitative changes in specified cell functions.
In the studies involving the CaCO-2 cell model, there was no significant change in the
transport of [3H] l-proJine by the cell after co-incubation with either dapsone or
cyclophosphamide (50!!M) and rat liver microsomal metabolite generating system. The
pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase
activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation
Studies involving the L6 cell model showed that there was no significant effect in the cell
signalling pathway producing the second messenger cAMP, after incubation with dapsone
or cyclophosphamide (50!!M) and the rat microsomal metabolite generating system.
There was also no significant affect on the vasopressin stimulated production of the second
messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although
there were some morphological changes observed with the cells at the highest
concentration of dapsone hydroxylamine (1 OO!!M ) .
With the test involving the NG11 S-401l-C3 cell model, there was no significant changes
in DNA synthesis in terms of [3H] thymidine incorporation, after co-incubation with
either phenytoin or cyclophosphamide (50!!M) and the rat microsomal metabolite
In the one compartment erythrocyte studies, there were significant decreases in
glutathione with cyclophosphamide (SO!!M) (0.44 + 0.04 mM), sulphamethoxazole
(SOIlM) (0.43 + 0.08mM) and carbamazepine (SO!!M) (0.47 + 0.034 mM), when coincubated
with the rat microsomal system, compared to the control (0.52 + 0.07mM).
There was no significant depletion in glutathione when the erythrocytes were coincubated
with phenytoin and the rat microsomal system. In the two compartment
erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with
cyclophosphamide (50~lM) (0.953 + 011 OmM) when co-incubated the rat microsomal
system, compared to the control (1.124 + 0.032mM). Differences were considered
statistically significant for p