1. Several ozonides,
peroxides and
aldehydes are formed during
ozone therapy, recently introduced in medicine.
tert-Butyl hydroperoxide (t-BHP), H2O2 and
diamide were investigated as model substrate in rat
hepatoma-derived Fa32 cells. 2. The cytotoxicity was measured by the
neutral red uptake inhibition assay after 1 h or 24 h treatment. The relative toxicities were quantified by the determination of the NI50. This is the concentration of test compound required to induce an inhibition of 50% in
neutral red uptake as compared to the control cells. All test chemicals were more toxic after 24 h than after 1 h. 3. The influence of the
glutathione (GSH) alteration on the cytotoxicity was measured by treating the cells with
2-oxo-4-thiazolidine carboxylic acid (OTC) or L-
buthionine sulfoximine (BSO). OTC increased the endogenous GSH content in the cells. BSO pretreatment strongly decreased the NI50 of the three chemicals. OTC pretreatment increased the NI50 of H2O2 but not of t-BHP and
diamide. This can be explained by the strong GSH-depletion after 1 h by t-BHP and
diamide, which contrasted with a weak GSH-depletion by H2O2 after the same time period. 4. The three test chemicals increased the endogenous GSH content after 24 h. t-BHP and H2O2, but not
diamide, increased the total GSH
transferase (GST) activity. Several alterations of the GST subunits were observed. Most striking was the increase of class alpha GST subunits, also for
diamide. 5. Since H2O2 and t-BHP are
ozone metabolites thought to be responsible for the
therapeutic effects of well-dosed
ozone, the results show that Fa32 cells can be used as a valuable alternative model system for studying the effects encountered in human
ozone therapy.