A Time-Course Study on the Effects of Interleukin-6 on Gene Expression of P450 Isoforms, Uptake Transporters, and Efflux Transporters in Human Hepatocytes

Pro-inflammatory cytokines such as interleukin-6 (IL-6) are known to alter expression of drug metabolism enzymes and transporter in rodent liver. However, their effects on human hepatocytes remain poorly characterized. The current study was to conduct a comprehensive analysis of the time course of the effects of IL-6 on the expression of human major P450 enzyme and transporters in human hepatocytes. Cryopreserved human hepatocytes were plated into a 24 well plate at a density of 350,000 cells/well. Following attachment plating medium was removed and the cells were overlaid with 0.25 mg/ml Matrigel™ in plating medium and incubated for an addition 20 hrs. The cells were then treated with 0, 0.1, 0.5 and 5 ng/mL of IL-6 in serum-free William’s E medium supplemented with ITS and dexamethasone for the time periods of 2, 6, 12, 24 and 48 hrs. Total RNA was isolated individually from each well and cDNA was synthesized from each sample. Real-timer PCR was performed using Taqman primers for P450 isoforms (CYPs 1A2, 2B6, 2C8, 2C9, 2D6, 3A4, 3A5), uptake transporters (SLC10A1, SLC22A1m SLC22A7, SLCO1B1, SLCO1B3, SLCO2B1) and efflux transporters (ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2) using the ABI 7500 Real-Time System. Each PCR cycle threshold (Ct) was normalized to the average Ct of the endogenous housekeeping control gene GAPDH. The comparative ? Ct method was used to calculate relative quantification of gene expression. Results showed that in the control (untreated) human hepatocytes, P450 isoforms and uptake transporters in the untreated cultures decreased rapidly with time and stabilized between 2 and 12 hrs and remained relative stable from 12 to 48 hrs at approximately 20% of that for the 2 hr time point. In contrast, efflux transporters remained relatively stable or increased during the time period of 2 to 48 hrs. IL-6 caused down regulations of most of the genes studied. The most significant changes were observed at either 12, 24 or 48 hours, depending on each specific gene. The results showed that a time-course study is a useful approach to evaluate the effects of cytokines such as IL-6 on ADME gene expression.