Expression and inducibility of cytochrome P450s in human hepatocytes isolated from chimeric mice with humanised livers

Figures & data

Table 1. Preparation of Hu-Liver cells from chimeric mice with humanised livers.

Human hepatocytes (as transplantation donor cells)					Hu-Liver mice (as liver cell donors)				Ratio of cells (%)		Hu-Liver cells
Hepatocytes name	Race	Age	Gender	ID^a	ID	BW (g)	ALT (U/L)	RI %^b	Mouse^c	Human^d	Viability (%)	Cell yield (×10^7)	Human albumin secretion (ng/2.5 × 10^5 cells/24 h)
35YM	Asian	35	Male	A	A1	19.9	210	67	21.4	71.3	75.0	1.7	144
					A2	22.8	199	>95	11.5	85.8	79.4	1.4	187
					A3	23.7	317	87	15.5	81.3	88.1	0.7	181
					A4	21.1	440	46	36.5	63.5	77.7	2.1	NA
					A5	17.3	285	>95	4.9	92.3	96.0	4.9	NA
30YF	African American	30	Female	B	B1	24.4	405	69	36.5	63.2	86.2	0.9	157
2YM	Caucasian	2	Male	C	C1	18.5	265	>95	6.5	93.5	78.4	8.0	234
5YF	Caucasian	5	Female	D	D1	23.3	530	>95	2.8	97.1	76.0	0.9	NA
					D2	27.8	435	19	70.5	26.2	91.0	0.6	NA
					D3	25.1	610	37	26.0	72.6	87.4	0.7	NA
5YM	Caucasian	5	Male	E	E1	20.0	370	>95	2.4	96.4	90.1	16.4	NA
					E2	18.0	260	>95	4.5	93.0	85.2	9.5	NA
					E3	21.4	375	59	10.5	87.7	70.4	3.7	NA
					E4	27.1	95	19	29.7	67.9	87.7	0.4	NA
			R squared values (p value) of correlation with RI%						.6630 (.0004)	.6579 (.0004)	.0074 (.7697)	.2580 (.0637)	NA

^aTransplantation donor IDs A to C were purchased from Lonza Walkersville, Inc., D and E were purchased from Biopredic International and BioreclamationIVT, respectively.

^bRI %, replacement index (RI) calculated based on human serum albumin levels as described previously (Hasegawa et al., 2011).

^cRatio of mouse liver cells stained with monoclonal mouse anti-mouse H-2kd antibody.

^dRatio of human liver cells stained with monoclonal mouse anti-HLA antibody.

NA: not available.

Figure 1. Comparison of the morphology of human hepatocytes and Hu-Liver cells. Phase contrast images show human hepatocytes (lot A) and Hu-Liver cells prepared from a Hu-Liver mouse (A1) at 24 h after plating (scale bar =50 μm).

Figure 2. Change in CYP3A4 mRNA levels during week-long culture of Hu-liver cells and human hepatocytes. Data are expressed as the ratio of the CYP3A4 mRNA to GAPDH mRNA. Data represent the mean of duplicate determinations. Open circles, closed triangles, closed squares and closed diamonds show donor A human hepatocytes and Hu-liver cells A2, A3 and A1, respectively.

Figure 3. Correlation of human P450 mRNA expression levels in human hepatocytes with those in Hu-Liver cells. The expression levels of human CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4A11, CYP4F2, CYP4F3B, CYP4F11 and CYP4F12 mRNAs in human hepatocytes and Hu-Liver cells after culture incubation for 48 hours are shown on the y axis and x axis, respectively. P450 mRNA expression levels showed significant correlation (p < .001) between human hepatocytes and Hu-Liver cells. Data represent the mean ± standard deviation of triplicate determinations. The solid line passing through the origin represents unity and the dashed lines represent five-fold differences above and below unity.

Figure 4. Induction of human CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 mRNAs in human hepatocytes and Hu-Liver cells by typical human P450 inducers. Induction of human CYP1A2 (A), CYP2B6 (B), CYP2C8 (C), CYP2C9 (D), CYP2C19 (E) and CYP3A4 (F) mRNAs in human hepatocytes and Hu-Liver cells by typical human P450 inducers: omeprazole, CYP1A2; phenobarbital, CYP2B6, CYP2C8, CYP2C9 and CYP2C19; rifampicin, CYP3A4. Each human P450 mRNA level was normalised to the human GAPDH mRNA level. Data represent the mean values of duplicate determinations. Open and closed bars show data for control samples and inducer-treated samples, respectively.

Table 2. Fold induction of human CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 mRNAs in three lots of human hepatocytes and seven lots of Hu-Liver cells.

P450	Inducer	Hepatocytes
		Donor A	Hu-Liver cells				Donor B	Hu-Liver cells B1	Donor C	Hu-Liver cells C1	Hu-Liver cells D1
			A1	A2	A3	A4
CYP1A2	Omeprazole	10.2	12.2	34.5	10.8	2.7	19.0	6.8	5.1	2.2	3.8
	β-naphthoflavone	5.9	12.6	38.8	9.5	2.7	6.6	5.3	2.5	2.1	5.1
CYP2B6	Phenobarbital	2.3	1.7	1.8	1.4	1.6	5.6	2.5	1.3	1.8	2.3
	Rifampicin	1.2	1.7	1.5	1.9	2.5	2.9	2.0	1.2	1.4	2.0
CYP2C8	Phenobarbital	1.5	1.9	1.0	1.5	1.9	2.6	1.9	1.0	1.9	1.5
	Rifampicin	1.1	1.7	1.4	1.4	2.1	1.5	1.7	1.5	2.1	1.5
CYP2C9	Phenobarbital	1.7	2.3	2.3	1.9	1.3	1.7	1.6	1.2	1.1	2.4
	Rifampicin	1.1	1.7	1.6	1.4	1.4	1.0	3.2	1.2	1.6	1.7
CYP2C19	Phenobarbital	1.5	1.9	1.5	1.6	1.4	1.8	1.7	2.1	1.5	2.5
	Rifampicin	1.3	1.5	1.4	1.1	1.4	1.1	1.4	1.8	1.3	2.1
CYP3A4	Rifampicin	8.8	6.5	10.1	5.8	4.5	40.9	5.3	48.4	17.6	2.6
	Phenobarbital	7.2	4.1	13.0	7.4	2.6	39.4	4.3	26.3	9.1	2.8

Human hepatocytes and Hu-Liver cells were treated with omeprazole, β-naphthoflavone, phenobarbital or rifampicin for 48 h. The induction level (fold induction normalized to 0.1% DMSO) for each P450 mRNA was determined. Values are shown as the mean of duplicate determinations.

Figure 5. Expression levels of CYP3A4 mRNA in human hepatocytes and Hu-Liver cells treated with eight chemical inducers. Human hepatocytes and Hu-Liver cells were treated with omeprazole (50 μM), β-naphthoflavone (50 μM), CITCO (1 μM), phenobarbital (1000 μM), rifampicin (10 μM), pregnenolone 16α-carbonitrile (PCN) (10 μM), phenytoin (250 μM) and flumazenil (50 μM) for 48 h. Flumazenil was used as a negative control for CYP3A4 mRNA induction (Ramachandran et al., 2015). Data represent the mean of duplicate determinations. The dashed line represents data (one-fold) from the control sample (0.1% DMSO).

Figure 6. Dose–response curves of CYP3A4 mRNA expression in human hepatocytes and Hu-Liver cells when treated with rifampicin. Expression levels of CYP3A4 mRNA were determined in human hepatocytes of lot A (open circles) and Hu-Liver cell lots A1 (closed circles), A2 (closed triangles) and A4 (closed diamonds) treated with rifampicin concentrations between 0.28 and 50 μM. Solid and dashed lines represent curve-fitting of the data for the human hepatocytes and Hu-Liver cells, respectively. Data represent the mean of duplicate determinations.

Figure 7. Testosterone 6β-hydroxylation activities in human hepatocytes and Hu-Liver cells treated with rifampicin. Filled and open squares indicate basal (0.1% DMSO-treated) and 20 μM rifampicin-treated hepatocyte lots, respectively. The fold induction in testosterone 6β-hydroxylation activities in human hepatocytes lot A and Hu-Liver cells lots A1, A2 and A3 was 2.5, 2.7, 4.1 and 3.7, respectively. *p < .05; **p < .01; ***p < .001. Data represent the mean ± standard deviation of triplicate determinations.

Table 3. EC₅₀ and E_max values for the induction of CYP3A4 mRNA by rifampicin in human hepatocytes and Hu-Liver cells.

Hepatocytes	EC50	Emax	Correlation coefficient (r)
	μM	Fold
Human hepatocytes (donor A)	1.1 ± 0.4	6.0 ± 1.0	0.94
Hu-Liver cells A1	1.7 ± 0.4	10 ± 1	0.96
Hu-Liver cells A2	4.0 ± 1.1	9.6 ± 0.9	0.98
Hu-Liver cells A3	1.2 ± 0.3	6.7 ± 0.5	0.97

EC₅₀ and E_max values were determined with a three-parameter Hill sigmoidal equation, Y = E_max × xⁿ/(EC₅₀ⁿ + xⁿ), using the mean ± standard error (n = 12) for rifampicin concentrations between 0.28 and 50 μM).

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