ACTIVTox vs Primary Hepatocytes

CYP1A induction in ACTIVTox compared to primary hepatocytes

ACTIVTox, based on a patented human liver cell line (1), faithfully replicates results found using human primary hepatocytes. ACTIVTox circumvents the problems associated with primary hepatocytes as an experimental system.

Primary hepatocytes, from both rat and human, are widely used as a method to identify hepatotoxins and provide an in vitro system that is regarded as the closest approximation to the liver. As toxicity testing moves earlier in the discovery process and is viewed in the context of structure toxicity relationships, a wider selection of structural variants needs to be profiled. This reveals two flaws in the use of primary hepatocytes as an experimental system. While animal cells are readily available, they often do not reflect human metabolism. Human cells are unavailable in the quality and quantity necessary for high throughput toxicity testing.

Procedure

Methylcholanthrene, TCDD and β-napthoflavone were dissolved in DMSO at 10 mM concentrations then diluted into cell culture medium to the concentrations shown. Using confluent, stationary cultures of ACTIVTox cells in 96-well plates, the compounds were incubated overnight with the cells then CYP1A activity was measured by the formation of resorufin from ethoxyresorufin (2).

Results obtained with ACTIVTox correspond to those found with primary hepatocytes

Figure 1. Induction of CYP1A activity by TCDD,
Methylcholanthrene, and β-Napthoflavone

Figure 1 shows the response of HepG2/C3A to three classical inducers of CYP1A activity: TCDD, methylcholanthrene and β-napthoflavone. Each of the three compounds showed a peak of activity followed by a fall off at higher concentrations. Both TCDD and methylcholanthrene became toxic and activated apoptosis at concentrations above their peak induction (data not shown). In contrast, β-napthoflavone did not appear to cause toxicity even at 100µM concentrations. The optimum inducing concentration for each of these compounds corresponds to values known from other induction systems (3,4).

More detailed information about this study can be obtained from Stem Cell Innovations.

References

1. Kelly, JH (1994) Permanent human hepatocyte cell line and its use in a liver assist device (LAD). US Patent No. 5,290,684.
2. Kelly, JH, Sussman, NL (2000) A fluorescent cell-based assay for cytochrome P-450 isozyme 1A2 induction and inhibition. J. Biomol. Screen. 5, 249 – 254.
3. Silkworth, JB, et al. (2005) Comparison of TCDD and PCB CYP1A induction sensitivities in fresh hepatocytes from human donors, sprague-dawley rats, and rhesus monkeys and HepG2 cells. Toxicol. Sci. 87, 508 – 519.
4. Vernhet, L, et al. (2003) Blockage of multidrug resistance-associated proteins potentiates the inhibitory effects of arsenic trioxide on CYP1A1 induction by polycyclic aromatic hydrocarbons. J. Pharmacol. Exp. Ther. 304, 145 – 155.