Exploring the molecular and functional cellular response to hydrazine via transcriptomics and DNA repair mutant cell lines.

Hydrazine is a rodent carcinogen and is classified as a probable human carcinogen by IARC. Though hydrazine is positive in both in vitro and in vivo DNA strand break (comet) assays, hydrazine was reported to be negative in an in vitro mutation Muta Mouse lung epithelial cell (FE1) test, as well as in a regulatory-compliant, in vivo Big Blue mouse mutation test. In this article, mechanistic studies explored the cellular response to hydrazine. When tested in a regulatory-compliant mouse lymphoma assay, hydrazine yielded unusual, weakly positive results. This prompted an investigation into the transcriptional response to hydrazine in FE1 cells via RNA sequencing. Amongst the changes identified was a dose-dependent increase in G2/M DNA damage checkpoint activation associated genes. Flow cytometric experiments in FE1 cells revealed that hydrazine exposure led to S-phase cell cycle arrest. Clonogenic assays in a variety of cell lines harboring key DNA repair protein deficiencies indicated that hydrazine could sensitize cells lacking homology dependent repair proteins (Brca2 and Fancg). Lastly, hprt assays with hydrazine were conducted to determine whether a lack of DNA repair could lead to mutagenicity. However, no robust, dose-dependent induction of mutations was noted. The transcriptional and cell cycle response to hydrazine, coupled with functional investigations of DNA repair-deficient cell lines support the inconsistencies noted in the genetic toxicology regulatory battery. In summary, while hydrazine may be genotoxic, transcriptional and functional processes involved in cell cycle regulation and DNA repair appear to play a nuanced role in mediating the mutagenic potential.

Genome-wide analysis of human constitutive androstane receptor (CAR) transcriptome in wild-type and CAR-knockout HepaRG cells.

The constitutive androstane receptor (CAR) modulates the transcription of numerous genes involving drug metabolism, energy homeostasis, and cell proliferation. Most functions of CAR however were defined from animal studies. Given the known species difference of CAR and the significant cross-talk between CAR and the pregnane X receptor (PXR), it is extremely difficult to decipher the exact role of human CAR (hCAR) in gene regulation, relying predominantly on pharmacological manipulations. Here, utilizing a newly generated hCAR-knockout (KO) HepaRG cell line, we carried out RNA-seq analysis of the global transcriptomes in wild-type (WT) and hCAR-KO HepaRG cells treated with CITCO, a selective hCAR agonist, phenobarbital (PB), a dual activator of hCAR and hPXR, or vehicle control. Real-time PCR assays in separate experiments were used to validate RNA-seq findings. Our results indicate that genes encoding drug-metabolizing enzymes are among the main clusters altered by both CITCO and PB. Specifically, CITCO significantly changed the expression of 135 genes in an hCAR-dependent manner, while PB altered the expression of 227 genes in WT cells of which 94 were simultaneously modulated in both cell lines reflecting dual effects of PB on hCAR/PXR. Notably, we found that many genes promoting cell proliferation and tumorigenesis were up-regulated in hCAR-KO cells, suggesting that hCAR may play an important role in cell growth that differs from mouse CAR. Together, our results reveal both novel and known targets of hCAR and support the role of hCAR in maintaining the homeostasis of metabolism and cell proliferation in the liver.

Regulation of MRP2/ABCC2 and BSEP/ABCB11 expression in sandwich cultured human and rat hepatocytes exposed to inflammatory cytokines TNF-{alpha}, IL-6, and IL-1{beta}.

In the present study MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly down-regulated by TNF-α, IL-6, or IL-1ß. In contrast to mRNA decrease, which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1ß. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-α and IL-6, whereas the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-α or IL-1ß. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/Abcb11 protein in liver at 24 h post-treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 h post-LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep and for the first time demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.

Preclinical and clinical evidence for the collaborative transport and renal secretion of an oxazolidinone antibiotic by organic anion transporter 3 (OAT3/SLC22A8) and multidrug and toxin extrusion protein 1 (MATE1/SLC47A1).

N-({(5S)-3-[4-(1,1-dioxidothiomorpholin-4-yl)-3,5-difluorophenyl]-2-oxo-1,3-oxazolidin-5-yl}methyl)acetamide (PNU-288034), an oxazolidinone antibiotic, was terminated in phase I clinical development because of insufficient exposure. Analysis of the drug pharmacokinetic and elimination profiles suggested that PNU-288034 undergoes extensive renal secretion in humans. The compound was well absorbed and exhibited approximately linear pharmacokinetics in the oral dose range of 100 to 1000 mg in human. PNU-288034 was metabolically stable in liver microsomes across species, and unchanged drug was cleared in the urine by an apparent active renal secretion process in rat and monkey (two to four times glomerular filtration rate) but not dog. In vitro studies conducted to characterize the transporters involved demonstrated PNU-288034 uptake by human organic anion transporter 3 (OAT3; K(m) = 44 +/- 5 microM) and human multidrug and toxin extrusion protein 1 (hMATE1; K(m) = 340 +/- 55 microM). The compound was also transported by multidrug resistance P-glycoprotein and breast cancer resistance protein. In contrast, human organic cation transporter 2, human OAT1, and hMATE2-K did not transport PNU-288034. Coadministration of PNU-288034 and the OAT3 inhibitor probenecid significantly increased PNU-288034 plasma area under the curve (170%) and reduced both plasma and renal clearance in monkey. Coadministration of PNU-288034 and cimetidine, a MATE1 inhibitor, also reduced plasma clearance in rat to a rate comparable with probenecid coadministration. Collectively, our results demonstrated a strong in vitro-in vivo correlation for active renal secretion coordinated through the vectorial transport process of OAT3 and MATE1, which ultimately resulted in limiting the systemic exposure of PNU-288034.

Lack of pharmacokinetic and pharmacodynamic interactions between a smoking cessation therapy, varenicline, and warfarin: an in vivo and in vitro study.

This study investigated the effect of varenicline on the pharmacokinetics and pharmacodynamics of a single dose of warfarin in 24 adult smokers and compared these findings with data generated using human in vitro systems. Subjects were randomized to receive varenicline 1 mg twice a day or placebo for 13 days and then switched to the alternative treatment after a 1-week washout period. A single dose of warfarin 25 mg was given on day 8 of each treatment period, and serial blood samples were collected over 144 hours postdose. Pharmacokinetic parameters for both (R)- and (S)-warfarin and international normalized ratio (INR) values were determined. Varenicline was assessed as an inhibitor and inducer of human cytochrome P450 activities using liver microsomes and hepatocytes, respectively. Consistent with the in vitro data, no alteration in human pharmacokinetics of warfarin enantiomers was observed with varenicline treatment. The 90% confidence intervals for the ratios of area under the concentration-time curve from zero hours to infinity and peak plasma concentrations were completely contained within 80% to 125%. Warfarin pharmacodynamic parameters, maximum INR, and the area under the prothrombin (INR)-time curve, were also unaffected by steady-state varenicline. Concomitant administration of varenicline and warfarin was well tolerated. Consequently, warfarin can be safely administered with varenicline without the need for dose adjustment.

Breast cancer resistance protein (Bcrp/abcg2) is a major determinant of sulfasalazine absorption and elimination in the mouse.

Sulfasalazine is used in the treatment of ulcerative colitis, Crohn's disease, and rheumatoid arthritis. When administered orally, sulfasalazine is poorly absorbed with an estimated bioavailability of 3-12%. Recent studies using the T-cell line (CEM) have shown that sulfasalazine is a substrate for the ATP-binding cassette (ABC) efflux pump ABCG2. ABCG2 is known to efflux a number of xenobiotics and appears to be a key determinant of efficacy and toxicity of ABCG2 substrates. To date, there has not been any systematic study on the mechanisms involved in the transport of sulfasalazine in vivo. Accordingly, we investigated whether Bcrp (abcg2) is involved in the disposition of sulfasalazine. After oral administration of 20 mg/kg sulfasalazine, the area under the plasma concentration (AUC) time profile in Bcrp1 (abcg2)-/- knockout (KO) mice was approximately 111-fold higher than that in FVB wild-type (WT) mice. After intravenous administration of 5 mg/kg sulfasalazine, the AUC in Bcrp1 (abcg2)-/- KO mice was approximately 13-fold higher than that in WT mice. Moreover, treatment of WT mice with a single oral dose of gefitinib (Iressa; 50 mg/kg), a known inhibitor of Bcrp, given 2 h prior to administering a single oral dose of sulfasalazine (20 mg/kg), resulted in a 13-fold increase in the AUC of sulfasalazine compared to the AUC in vehicle-treated mice. Since gefitinib is also an inhibitor of P-glycoprotein (P-gp), the impact of P-gp on sulfasalazine absorption in vivo was also examined. The sulfasalazine AUC in mdr1a-/- KO versus WT mice did not differ significantly after either an oral (20 mg/kg) or an intravenous dose (5 mg/kg). We conclude that Bcrp (abcg2) is an important determinant for the oral bioavailability and the elimination of sulfasalazine in the mouse, and that sulfasalazine has the potential to be utilized as a specific in vivo probe of Bcrp (abcg2).