Evaluation of an in vitro toxicogenetic mouse model for hepatotoxicity

Abstract

Numerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to determine whether viable cells may be isolated from different mouse inbred strains, evaluate the reproducibility of cell yield, viability and functionality over subsequent isolations, and assess the utility of the model for toxicity screening. Hepatocytes were isolated from 15 strains of mice (A/J, B6C3F1, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, BALB/cByJ, AKR/J, MRL/MpJ, NOD/LtJ, NZW/LacJ, PWD/PhJ and WSB/EiJ males) and cultured for up to 7 days in traditional 2-dimensional culture. Cells from B6C3F1, C57BL/6J, and NOD/LtJ strains were treated with acetaminophen, WY-14,643 or rifampin and concentration-response effects on viability and function were established. Our data suggest that high yield and viability can be achieved across a panel of strains. Cell function and expression of key liver-specific genes of hepatocytes isolated from different strains and cultured under standardized conditions are comparable. Strain-specific responses to toxicant exposure have been observed in cultured hepatocytes and these experiments open new opportunities for further developments of in vitro models of hepatotoxicity in a genetically diverse population.

Figure 1

Functional characterization of cultured hepatocytes isolated from 3 mouse strains. Hepatocytes were isolated from B6C3F1/J, C57BL/6J and NOD/LtJ (males, n=3) and cultured in 24-well plates in a conventional monolayer up to day 7. Media was harvested daily and activity of lactate dehydrogenase, serum concentrations of urea, pyruvate and lactate, and intracellular concentrations (amount in all cells in each well on days 1, 3 and 7) of ATP and glutathione were quantified. Data shown as mean±SD of independent biological replicates.

Figure 2

Analysis of reproducibility of gene expression in cultured hepatocytes isolated from 3 mouse strains. mRNA levels of Alb, Cps1, Hnf4α, Cyp1a2, Cyp3a11, Cyp4a10, Ugt1a1, Gsta1, and Slco1b2 was assessed using quantitative RT-PCR in liver tissue (black bar) and hepatocytes cultured for 1 (white bar) or 3 (grey bar) days. Data (mean±SD of biological replicates) was normalized to expression levels in whole liver samples.

Figure 3

Effects of acetaminophen on hepatocytes derived from 3 mouse strains. Hepatocytes were isolated from B6C3F1/J, C57BL/6J and NOD/LtJ (males, n=3) and cultured in 96-well plates in a conventional monolayer for 24 or 72 hr and exposed to acetaminophen (0.3, 1, 3, 10 and 30 mM) or vehicle (dimethyl sulfoxide, 0.5%) for additional 24 hr. Hepatocytes were harvested on day 2 (filled circles or white bars), or 4 (filled squares or black bars) of culture. (A) Assessment of cytotoxicity using intracellular ATP and glutathione (GSH) levels. Data (mean±SD of biological replicates) was normalized to the values in vehicle-treated cells. (B) mRNA levels of Cyp2e1 and Nrf2 were assessed using quantitative RT-PCR. Data (mean±SD of biological replicates) was normalized to expression level of Gusb.

Figure 4

Effects of WY-14,643 (0.1, 0.3, 1, 3 and 10 mM) on hepatocytes derived from 3 mouse strains. Cells were isolated, cultured, and data collected as detailed in the legend to Figure 3.

Figure 5

Effects of rifampin (1, 3, 10, 30 and 100 µM) on hepatocytes derived from 3 mouse strains. Cells were isolated, cultured, and data collected as detailed in the legend to Figure 3.