The influence of bilirubin on mRNA expression of cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and nuclear receptors in human hepatocytes was investigated. The treatment of the hepatocytes with 40 μg/mL bilirubin, which corresponds to hyperbilirubinemia, resulted in 1.7-fold increase of CYP2A6 mRNA compared to the vehicle control while CYP2A6 mRNA did not change after treatment with 1 μg/mL bilirubin, corresponding to physiologically normal level. No significant change of mRNA expression by 40 μg/mL bilirubin treatment was observed for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5, UGT1A1, UGT1A3, UGT1A6, UGT2B4, UGT2B7, UGT2B10 and UGT2B15, constitutive androstane receptor (CAR), pregnane X receptor (PXR), retinoid X receptor α (RXRα) and hepatocyte nuclear factor-4α (HNF-4α). The induction profile of bilirubin was different from that of rifampicin, a typical PXR activator. This study demonstrated that CYP2A6 can be induced by bilirubin in a concentration dependent manner.
Induction of drug metabolizing enzymes is of concern for clinical application of medicines, especially with narrow therapeutic windows, such as immunosuppressants and anti-coagulants. For drugs whose effect is achieved by the parent drug, the enzyme induction would increase the systemic clearance of the drug, resulting in lower drug exposure, and reduction in the pharmacological efficacy. For instance, rifampicin causes acute transplant rejection in patients treated with cyclosporine, presumably because of induction of the CYP3A4-mediated metabolism of cyclosporine [
In our previous study, significant correlation was observed between total bilirubin level and the systemic clearance (CL/F) of an aromatase inhibitor letrozole in healthy postmenopausal women in a population pharmacokinetic analysis, although it was not significant when other factors were incorporated to the final analysis model [
Cryopreserved human hepatocytes (60-year-old Caucasian male, Celsis IVT, Baltimore, MD, USA) were thawed at 37˚C, suspended in thawing medium without glucose (Biopredic International, Rennes, France), and centrifuged at 160 × g for 2 min. Hepatocytes were resuspended in William’s medium E supplemented with 10% FBS, 4 μg/mL bovine insulin, 100 IU/mL penicillin and 100 μg/mL streptomycin (Biopredic International) and cultured in a collagen-coated 24-well plate (BD Biosciences, Franklin Lakes, NJ, USA) at a density of 2.5 × 105 cells/500μL/well in a 37˚C incubator with 5% CO2 and 95% air. After 4 hours, the culture medium was replaced with serum-free William’s medium E supplemented with 4 μg/mL bovine insulin, 100 IU/mL penicillin, 100 μg/mL streptomycin and 50 μM hydrocortisone hemisuccinate (incubation medium), and cultured for 20 hours in a CO2 incubator. Then, the medium was replaced with the incubation medium containing 1 or 40 μg/mL bilirubin (Wako Pure Chemical Industries, Osaka, Japan) or 50 μM rifampicin (Wako Pure Chemical Industries) and cultured for 48 hours in a CO2 incubator before total RNA extraction for RT-PCR. During the exposure to bilirubin or rifampicin, the culture medium was replaced with freshly-prepared one containing bilirubin or rifampicin every 24 h.
At the end of the culture period, the medium was removed and total RNA was extracted from human hepatocytes using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s protocols. The concentration and purity of RNA were determined spectrometrically. Reverse transcription was performed using the TaKaRa RNA PCR Kit (AMV) Ver.3.0 (Takara Bio Co., Ltd, Shiga, Japan), according to the manufacturer’s instruction. Total RNA (400 ng) was mixed with reaction buffer, 5 mM MgCl2, dNTP mixture (1 mM each), RNase inhibitor (1 U/μL), AMV reverse transcriptase XL (0.25 U/μL), and random 9 mers (2.5 μM) in a final volume of 20 μL. The reaction mixture was incubated at 30˚C for 10 min followed by 42˚C for 30 min and then heated at 95˚C for 5 min to inactivate the enzyme. PCR was carried out using the TaKaRa PrimeSTAR Max DNA Polymerase (Takara Bio Co., Ltd), according to the manufacturer’s instruction. The reaction was performed in a total volume of 10 μL consisting of 2 × PrimeSTAR Max Premix, 0.3 μM forward primer, 0.3 μM reverse primer and the reverse transcription product as a template corresponding to 2 ng RNA. The amplification was performed by denaturation at 98˚C for 10 sec, annealing at an appropriate temperature for 5 sec, and extension at 72˚C for 5 sec for appropriate cycles. The primers used, the annealing temperatures and number of cycles of the PCR were listed in
Data were expressed as mean + standard deviation (SD). Statistical analyses and significance were performed using the one-way ANOVA followed by Dunnett’s multiple comparision test. In all comparisons, p < 0.05 was considered statistically significant.
In order to investigate the influence of bilirubin on metabolic activity of CYP2A6 and CYP3A4, mRNA of these enzymes was measured in the human hepatocytes after treatment by 1 μg/mL bilirubin, corresponding to physiologically normal level, or 40 μg/mL bilirubin, corresponding to hyperbilirubinemia, for 48 hours. As a positive control, hepatocytes were also treated by 50 μM rifampicin. As shown in
To further explore the influence of bilirubin on other hepatic metabolic enzymes, mRNA of CYPs expressed in human liver was measured (
Because bilirubin is conjugated with glucuronic acid by several UGT enzymes in humans, influence of bilirubin on mRNA levels of UGT was examined as self-regulation of its metabolism (
In order to explore induction mechanisms of metabolic enzymes by bilirubin, influence of bilirubin on CAR, PXR, RXRα and HNF-4α, which are reported to contribute to the CYP2A6 induction, was investigated (
In our previous study, the positive correlation between bilirubin level and CL/F of letrozole was indicated by population pharmacokinetic analysis [
Although no mRNA induction was reported in the study, it must be noted that enzyme activities and protein and mRNA expression levels including several CYPs such as CYP2A6, CYP3A4 and CYP2D6 are remarkably low in HepG2 compared to primary human hepatocytes and induction profile by typical inducers are quite different from human hepatocytes [19,20]. Therefore, the contradicting observation to our study will be due to the difference of cells used in the examinations.
Rifampicin is well known as an agonist of PXR and induces CYP3A4 by activating it [21-23]. CYP2A6 is also reported to be induced by rifampicin via PXR. Actually, CYP3A4 and CYP2A6 were induced by rifampicin treatment in the current study.
Induction of CYP2B6, CYP2C8, UGT1A1 and UGT1A3, which are reported to be regulated by PXR [21-27], by rifampicin was also observed. However, these enzymes were not induced by bilirubin in the present study. The induction profile of the enzymes by bilirubin observed in the study was different from the profile of rifampicin, suggesting that induction of CYP2A6 by bilirubin may not be caused via activation of PXR.
Bilirubin is known to cause translocation of CAR from hepatocyte cytoplasm to nucleus and induces UGT1A1
which is responsible for the glucuronidation of bilirubin [
lower bilirubin by reporter gene assay in HepG2 cells suggesting threshold around 30 to 50 μg/mL [
Bilirubin is also known to be a ligand of human aryl hydrocarbon receptor (AhR) [32,33]. Actually, mRNA of CYP1A2, which is known to be regulated by AhR [
As shown in
In order to elucidate the induction mechanism of CYP2A6 by bilirubin, further studies e.g. knock down or overexpression of the nuclear receptors [15,16], should be required. Because CYP2A6 is well known for the genetic polymorphism [35-38] and involved in the metabolism of drugs with narrow therapeutic window such as tegafur and cyclophosphamide [39,40], difference of the bilirubin influence among the genotypes is of interest.
In summary, we present here an unprecedented finding of the CYP2A6 induction by bilirubin in human hepatocytes. In addition, we revealed the influence of bilirubin on CYPs, UGTs and nuclear receptors. Although the induction mechanism of bilirubin for CYP2A6 cannot be fully clarified, it will not be through PXR activation, or the induction of CAR, PXR, RXRα and HNF-4α expression. These findings can be utilized as a tool to predict a drug metabolizing capability in patients with hyperbilirubinemia.