Thalidomide increases human hepatic cytochrome P450 3A enzymes by direct activation of the pregnane X receptor.

Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated by H. Yamazaki et al. ( ( 2013 ) Chem. Res. Toxicol. 26 , 486 - 489 ) using the model substrate midazolam in various in vitro and in vivo models. Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pretreatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting the activation of upstream transcription factors involved in detoxication, e.g., the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of coregulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 coregulator-derived nuclear receptor-interaction motifs and coregulator and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5-hydroxythalidomide displayed significant modulation of coregulator interaction with PXR and CAR ligand-binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation.

Investigation of ifosfamide nephrotoxicity induced in a liver-kidney co-culture biochip.

In this article, we present a liver-kidney co-culture model in a micro fluidic biochip. The liver was modeled using HepG2/C3a and HepaRG cell lines and the kidney using MDCK cell lines. To demonstrate the synergic interaction between both organs, we investigated the effect of ifosfamide, an anticancerous drug. Ifosfamide is a prodrug which is metabolized by the liver to isophosforamide mustard, an active metabolite. This metabolism process also leads to the formation of chloroacetaldehyde, a nephrotoxic metabolite and acrolein a urotoxic one. In the biochips of MDCK cultures, we did not detect any nephrotoxic effects after 72 h of 50 µM ifosfamide exposure. However, in the liver-kidney biochips, the same 72 h exposure leads to a nephrotoxicity illustrated by a reduction of the number of MDCK cells (up to 30% in the HepaRG-MDCK) when compared to untreated co-cultures or treated MDCK monocultures. The reduction of the MDCK cell number was not related to a modification of the cell cycle repartition in ifosfamide treated cases when compared to controls. The ifosfamide biotransformation into 3-dechloroethylifosfamide, an equimolar byproduct of the chloroacetaldehyde production, was detected by mass spectrometry at a rate of apparition of 0.3 ± 0.1 and 1.1 ± 0.3 pg/h/biochips in HepaRG monocultures and HepaRG-MDCK co-cultures respectively. Any metabolite was detected in HepG2/C3a cultures. Furthermore, the ifosfamide treatment in HepaRG-MDCK co-culture system triggered an increase in the intracellular calcium release in MDCK cells on contrary to the treatment on MDCK monocultures. As 3-dechloroethylifosfamide is not toxic, we have tested the effect of equimolar choloroacetaldehyde concentration onto the MDCK cells. At this concentration, we found a quite similar calcium perturbation and MDCK nephrotoxicity via a reduction of 30% of final cell numbers such as in the ifosfamide HepaRG-MDCK co-culture experiments. Our results suggest that ifosfamide nephrotoxicity in a liver-kidney micro fluidic co-culture model using HepaRG-MDCK cells is induced by the metabolism of ifosfamide into chloroacetaldehyde whereas this pathway is not functional in HepG2/C3a-MDCK model. This study demonstrates the interest in the development of systemic organ-organ interactions using micro fluidic biochips. It also illustrated their potential in future predictive toxicity model using in vitro models as alternative methods.

Improvement of HepG2/C3a cell functions in a microfluidic biochip.

Current developments in tissue engineering and microtechnology fields allow the use of microfluidic biochip as microtools for in vitro investigations. In the present study, we describe the behavior of HepG2/C3a cells cultivated in a poly(dimethylsiloxane) (PDMS) microfluidic biochip coupled to a perfusion system. Cell culture in the microfluidic biochip for 96 h including 72 h of perfusion provoked a 24 h delay in cell growth compared to plate cultures. Inside the microfluidic biochip, few apoptosis, and necrosis were detected along the culture and 3D cell organization was observed. Regarding the hepatic metabolism, glucose and glutamine consumptions as well as albumin synthesis were maintained. A transcriptomic analysis performed at 96 h of culture using Affymetrix GeneChip demonstrated that 1,025 genes with a fold change above 1.8 were statistically differentially expressed in the microfluidic biochip cultures compared to plate cultures. Among those genes, phase I enzymes involved in the xenobiotic's metabolism such as the cytochromes P450 (CYP) 1A1/2, 2B6, 3A4, 3A5, and 3A7 were up-regulated. The CYP1A1/2 up-regulation was associated with the appearance of CYP1A1/2's activity evidenced by using EROD biotransformation assay. Several phase II enzymes such as sulfotransferases (SULT1A1 and SULT1A2), UDP-glucuronyltransferase (UGT1A1, UGT2B7) and phase III transporters (such as MDR1, MRP2) were also up-regulated. In conclusion, microfluidic biochip could and provide an important insight to exploring the xenobiotic's metabolism. Altogether, these results suggest that this kind of biochip could be considered as a new pertinent tool for predicting cell toxicity and clearance of xenobiotics in vitro.