Prediction of Acute Toxicity in HPCT-1E3 Hepatocytoma Cells with Liver-like Transport Activities

Abstract

A battery of in vitro methods has been developed for the prediction of acute oral toxicity, to reduce the number of animals used for this purpose. However, the results of these tests correlate more closely with lethal serum concentrations than with lethal doses. To address this issue, we have further evaluated the HPCT-1E3 model, which may be better able to emulate toxicokinetic factors that occur in vivo, due to the presence in these hepatocytoma cells of endogenous transmembrane carriers and a basal activity of xenobiotic metabolism. IC50 values produced by using the MTT test after a 48-hour incubation with 20 randomly-selected MEIC substances, correlated better with human oral LD50 values than with LC50 data, supporting this hypothesis. As with other models, the toxicity of receptor-specific rather than cytotoxic substances, for example digoxin, was underpredicted. When digoxin was removed from the correlation analysis, the coefficient of determination (r2) improved to 0.81, and none of remaining chemicals were wrongly predicted by more than one order of magnitude. IC50 values obtained with HepG2 cells under similar conditions (MEIC Test No. 3, 24 hours, MTT) correlated with human LD50 data with a r2 value of 0.55. A direct comparison of HPCT-1E3 and HepG2 cells further suggested that the differences between them may be due to transport processes. In conclusion, the HPCT-1E3 model may be valuable in improving the prediction of lethal doses, rather than lethal serum concentrations.

Apoptosis Induction by OTA and TNF-α in Cultured Primary Rat Hepatocytes and Prevention by Silibinin

“…A test was performed to detect reactive oxygen species and also malondialdehyde as a natural end product of membrane lipid peroxidation. Cell cultures of immortalized rat hepatocytoma cells (HPCT) [51,52] were exposed to OTA and ActD/TNF-α. For control, samples were also analyzed after incubation with H 2 O 2 .…”

“…Because HPCT-1E3 cells are a good model for studying cytotoxicity and express several hepatocyte specific properties in contrast to other immortal cell lines, their suitability as an in vitro model has to be proven in order to replace in vivo experiments [51]. The rat hepatocytoma cell line HPCT-1E3 was maintained in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% ( v / v ) fetal calf serum, 2 mM L-glutamine, 10 µg/mL insulin, 10 µg/mL inosine, 1.5 µmol/L dexamethasone, 100 IU/mL penicillin and 100 mg/mL streptomycin [53].…”

Validation of the Hepatocyte-like HPCT-1E3 Cell Line as an In Vitro Model for the Prediction of Acute In Vivo Toxicity

“…Therefore, in order to test whether HPCT-1E3 cells better reflect the in vivo situation, a preliminary study was performed in our laboratory, by using 20 randomly-selected MEIC substances. HPCT-1E3 IC50 values produced by using the MTT assay showed better correlation with human oral LD50 than with human LC50 values (7). Furthermore, IC50 values obtained from HPCT-1E3 cells correlated better with human LD50 data (r 2 = 0.81; P < 0.001) than did the IC50 values obtained from HepG2 cells (r 2 = 0.55; P = 0.0002).…”

“…Furthermore, IC50 values obtained from HPCT-1E3 cells correlated better with human LD50 data (r 2 = 0.81; P < 0.001) than did the IC50 values obtained from HepG2 cells (r 2 = 0.55; P = 0.0002). As HepG2 cells lack important liver-specific import transporters, including OATP carriers (8,9), it was suggested that the differences in the predictivity of human toxicity between the two cell lines may Validation of the Hepatocyte-like HPCT-1E3 Cell Line as an In Vitro Model for the Prediction of Acute In Vivo Toxicity Sandra Halwachs, Cathleen Lakoma and Walther Honscha be at least partly due to the carrier-mediated uptake of tested substances in HPCT-1E3 cells (7). However, our previous study revealed variability in the results from a similar test series over a prolonged test period (7), which might be due to a time-dependent alteration of the HPCT-1E3 cell phenotype in long-term culture.…”

“…Then, we expanded the list of tested MEIC chemicals to 37 substances, for cytotoxicity testing in HPCT-1E3 cells by using the MTT assay. These 37 chemicals for cytotoxicity evaluation were randomly selected from the original list of 75 chemicals selected for the MEIC programme (10), excluding the chemicals tested in preliminary cytotoxicity studies with the HPCT-1E3 cell line (7). Finally, the cytotoxicities of selected MEIC chemicals in HPCT-1E3 and HepG2 cells were compared, in order to test the influence of carrier-dependent import or export activities on the susceptibilities of the two cell lines.…”