Primary hepatocytes isolated from human and porcine donors display similar patterns of cytochrome p450 expression following exposure to prototypical activators of AhR, CAR and PXR

Abstract

The hepatic cytochrome p450's (CYP) are of major importance for the metabolism of xenobiotics and knowledge about their regulation is crucial. This knowledge often originates from cell models; primary human hepatocytes (PHH) being the gold standard. However, due to limited availability of high-quality human donor organs, basic knowledge on alternative models are needed. Primary porcine hepatocytes (PPH) have been suggested as an alternative to PHH. Unfortunately, data comparing the response in gene-transcription to standard CYP inducers between PHH and PPH are missing. In the present study we, cultured PHH and PPH under the same conditions, treated them with standard inducers of the CYP1-3 and determined the response in gene and protein expression. The results demonstrated that in both species TCDD and omeprazole caused an increase in CYP1A/B expression. In PPH, CITCO increased the content of CYP1A/B. For the CYP2B/C/D's, phenobarbital and rifampicin caused increases in expression. For the CYP2D's, TCDD and omeprazole caused increased gene expression in PPH, which were not the case for PHH. Both phenobarbital, rifampicin and omeprazole increased CYP3A expression in PHH and PPH. Moreover, TCDD increased the gene expression of CYP3A in PPH; this was not the case for PHH. Multivariate data analysis found no difference in gene expression between PHH and PPH for phenobarbital, rifampicin and CITCO. However, differential clustering was observed for TCDD and omeprazole. In conclusion, despite model specificity, there are a high number of similar responses, and experiments investigating mRNA regulation made in PPH permits for a reliable translation into human setting.

Keywords: Detoxification; Gene-expression; Liver cells; Model; Pig; RT-PCR.

Graphical abstract

Fig. 1

mRNA and protein expression of the CYP1A and CYP1B family in primary hepatocytes from human and porcine donors following treatment with 10 nM TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxine), 10 µM rifampicin (RIF), 500 µM phenobarbital (PB), 1 µM CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime) and 100 µM omeprazole (OM) for 24 (mRNA) or 48 (protein) hours. Data are given as mean + SEM relative to control. N = 4 for both species. *** (p < 0.001), ** (p < 0.01) and * (p < 0.05) significantly different from control treated hepatocytes.

Fig. 2

mRNA and protein expression of the CYP2B family in primary hepatocytes from human and porcine donors following treatment with 10 nM TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxine), 10 µM rifampicin (RIF), 500 µM phenobarbital (PB), 1 µM CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime) and 100 µM omeprazole (OM) for 24 (mRNA) or 48 (protein) hours. Data are given as mean + SEM relative to control. N = 4 for both species. ** (p < 0.01) and * (p < 0.05) significantly different from control treated hepatocytes.

Fig. 3

mRNA expression of the CYP2C family in primary hepatocytes from human and porcine donors following treatment with 10 nM TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxine), 10 µM rifampicin (RIF), 500 µM phenobarbital (PB), 1 µM CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime) and 100 µM omeprazole (OM) for 24 h. Data are given as mean + SEM relative to control. N = 4 for both species. *** (p < 0.001), ** (p < 0.01) and * (p < 0.05) significantly different from control treated hepatocytes.

Fig. 4

mRNA expression of the CYP2D family in primary hepatocytes from human and porcine donors following treatment with 10 nM TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxine), 10 µM rifampicin (RIF), 500 µM phenobarbital (PB), 1 µM CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime) and 100 µM omeprazole (OM) for 24 h. Data are given as mean + SEM relative to control. N = 4 for both species. * (p < 0.05) significantly different from control treated hepatocytes.

Fig. 5

mRNA and protein expression of the CYP3A family in primary hepatocytes from human and porcine donors following treatment with 10 nM TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxine), 10 µM rifampicin (RIF), 500 µM phenobarbital (PB), 1 µM CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime) and 100 µM omeprazole (OM) for 24 (mRNA) or 48 (protein) hours. Data are given as mean + SEM relative to control. N = 4 for both species. *** (p < 0.001), ** (p < 0.01) and * (p < 0.05) significantly different from control treated hepatocytes.

Fig. 6

Principal component analysis. The PCA analysis was based on the gene expression human and porcine primary hepatocytes after following treatment with 10 nM TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxine), 10 µM rifampicin (RIF), 500 µM phenobarbital (PB), 1 µM CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime) and 100 µM omeprazole (OM) for 24 h. A) Score plot showing the clustering of human and porcine hepatocyte following treatment based on gene expression. The ellipse shows Hotellings T2. B) Loading plot showing the distribution of gene expression level.