Porcine drug metabolism and toxicity in vitro model utilizing transformed hepatocyte cell lines (HepCRE)

A.K. De, K.A. Darfour-Oduro, L. Rund, K.M. Schachtschneider, K. Singh, L.B. Schook
American Association for Cancer Research Annual Meeting, April 16-20, 2016, New Orleans, LA

To date, in vitro cytotoxicity assays are not highly predictive of in vivo toxicity. The adverse effects of new drugs are often not discovered until preclinical animal safety studies or even clinical trials; 40% of drugs drop out in preclinical animal studies and 89% of those that reach clinical trials fail. There is a critical need for more predictive and reliable in vitro testing methods. Due to its physiological similarities with humans, pigs have emerged as a suitable and reliable animal model for pharmacological and toxicological studies. To further the pigs’ suitability, we have developed and characterized a transformed porcine hepatocyte cell line to support drug toxicity and metabolism assessments. Primary porcine hepatocytes isolated by a modified procedure of Panda’s method (Panda et al., 2015) express phase I and II drug metabolism, phase-III transport, and nuclear receptors involved in regulation of drug metabolism transcripts. However, primary hepatocytes have a limited life span in culture and usually within 8 days post culture more than 50% of cells undergo apoptosis. Moreover, normal gene expression declines from day 5 in culture. To overcome these limitations, we have generated and characterized transformed hepatocyte cell lines (HepCre) derived from the transgenic Oncopig. Hepatocytes were isolated from Oncopigs (Schook et at., 2015) and transformed by treatment with Cre recombinase. When cultured in the presence of a differentiating agent (DMSO), the HepCre cell lines maintained normal hepatic functions, as well as the potential to express all the main drug metabolism and regulation genes comparable to that of cultured primary hepatocytes. The effect of model CYP (cytochrome P450) inducers (3- methylcholanthrene, rifampicin and phenobarbital) on the expression of CYP enzymes in primary hepatocytes and HepCre were studied. Treatment of 3-methylcholanthrene caused a significant upregulation of CYPA1 and CYPA2 transcripts, but the transcript of other CYPs remain unchanged. Both rifampicin and phenobarbital exposure resulted in the upregulation of several CYP transcripts, including CYP2A19, CYP2B22, and CYP3A. The gene transcription patterns in HepCre were similar to those of primary hepatocytes and in vitro human models. Toxicity responses of HepCre cells to hepatotoxic drugs (i.e. aflatoxin B1, chlorpromazine, amiodarone, and acetaminophen) was evaluated. After 72 hr of exposure, cell viability decreased in a concentration-dependent manner with IC50 of 5 µm, 50 µm, 20 µm, and 70 µm for aflatoxin B1, amiodarone, chlorpromazine, and acetaminophen, respectively. These findings indicate that this porcine transformed HepCre cell line represents a useful and predictive model for high throughput screening of new drugs as well as studies on metabolism and hepatotoxicity of chemicals.