Prediction of drug-induced liver injury using keratinocytes

Abstract

Drug-induced liver injury (DILI) is one of the most common adverse drug reactions. DILI is often accompanied by skin reactions, including rash and pruritus. However, it is still unknown whether DILI-associated genes such as S100 calcium-binding protein A and interleukin (IL)-1β are involved in drug-induced skin toxicity. In the present study, most of the tested hepatotoxic drugs such as pioglitazone and diclofenac induced DILI-associated genes in human and mouse keratinocytes. Keratinocytes of mice at higher risk for DILI exhibited an increased IL-1β basal expression. They also showed a higher inducibility of IL-1β when treated by pioglitazone. Mice at higher risk for DILI showed even higher sums of DILI-associated gene basal expression levels and induction rates in keratinocytes. Our data suggest that DILI-associated genes might be involved in the onset and progression of drug-induced skin toxicity. Furthermore, we might be able to identify individuals at higher risk of developing DILI less invasively by examining gene expression patterns in keratinocytes. Copyright © 2017 John Wiley & Sons, Ltd.

Keywords: IL-1β; drug-induced liver injury; hepatotoxicity; keratinocyte; prediction.

Figure 1

Concentration-dependent induction of S100A8, S100A9, NALP3, IL-1β and RAGE in hepatotoxic drug-treated HaCaT cells. HaCaT cells were treated with diclofenac, ketoconazole and flutamide (10 and 100 μM) for 48 h. mRNA expression levels of S100A8, S100A9, NALP3, IL-1β and RAGE were measured by quantitative reverse transcription–polymerase chain reaction. Gene expressions were normalized with the GAPDH expression. Data are the means ± SD of three independent determinations. *P < 0.05 and **P < 0.01 compared to the expression level in the vehicle-treated cells.

Figure 2

Concentration- and time-dependent induction of S100A8, S100A9, NALP3, IL-1β and RAGE in hepatotoxic drug-treated mouse keratinocytes. Primary mouse keratinocytes were pooled (n = 3) and cultured for 48 h (A). Isolated keratinocytes were treated with 10 and 100 μM diclofenac, ketoconazole and flutamide for 6 and 48 h (B). mRNA levels of NALP3 and IL-1β were measured by quantitative reverse transcription–polymerase chain reaction. Expression was normalized with the cyclophilin mRNA expression. Data are the means ± SD of three independent determinations. *P < 0.05 and **P < 0.01 compared to the expression level in the vehicle-treated cells.

Figure 3

Induction of DILI-associated gene mRNA expressions in hepatotoxic drug-treated human keratinocytes. HaCaT cells were treated with hepatotoxic drugs (100 μM) for 48 h. mRNA levels of S100A8, S100A9, NALP3, IL-1β and RAGE were measured by quantitative reverse transcription–polymerase chain reaction. Sum of the relative expression levels of S100A8, S100A9, NALP3, IL-1β and RAGE mRNA was calculated. mRNA expression levels were normalized to the level of GAPDH. Drugs shown with a red color exhibited more than 5 of the sum score. DILI, drug-induced liver injury.

Figure 4

IL-1β mRNA expression in pioglitazone-treated mouse keratinocytes. Mouse keratinocytes were isolated from 19 mice. Individual mouse keratinocytes were treated with 100 μM PIO for 6 h and the IL-1β mRNA expression levels were determined by quantitative reverse transcription–polymerase chain reaction. Expression was normalized with the cyclophilin mRNA expression. Data are the means ± SD. PIO, pioglitazone.

Figure 5

IL-1β mRNA expression in keratinocytes isolated from LPS-treated mice. LPS was administered to newborn mice (0.3 or 3 mg kg⁻¹, s.c.). Six hours after the treatment, skins were collected and the pooled keratinocytes (n = 3) were cultured. Keratinocytes were treated with 100 μM PIO for 6 h and IL-1β expression levels were measured by quantitative reverse transcription–polymerase chain reaction (A). Sums of IL-1β basal expression levels and induction rates were calculated (B). Expression was normalized with the cyclophilin mRNA expression. Data are the means ± SD of three independent determinations. *P < 0.05 and **P < 0.01 between the groups. LPS, lipopolysaccharide; PIO, pioglitazone.

Figure 6

Sum of IL-1β basal expression levels and induction rates in individual mouse keratinocytes. LPS was administered to newborn mice (0.3 or 3 mg kg⁻¹ in saline, s.c., n = 4). Six hours after the treatment, skins were collected and the individual keratinocytes were cultured. Keratinocytes were treated with 100 μM pioglitazone for 6 h and IL-1β expression levels were measured by quantitative reverse transcription–polymerase chain reaction. The sum of IL-1β basal expression levels and induction rates in keratinocytes were calculated. Expression was normalized with that of cyclophilin. Data are the means. LPS, lipopolysaccharide.