Claudia Luckert

Polycyclic aromatic hydrocarbons stimulate human CYP3A4 promoter activity via PXR

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) is mediated mainly by cytochrome P₄₅₀ monooxygenases (CYP) CYP1A1, 1A2 and 1B1. Several PAH are known to induce these CYP via aryl hydrocarbon receptor (AhR) signaling. Recently, it was shown that the PAH benzo[a]pyrene (BaP) can induce CYP3A4 as well. The induction was suggested to be mediated by the pregnane X receptor (PXR) rather than AhR. Metabolism by CYP3A4 is only known for dihydrodiol metabolites of PAH but not for their parent compounds. In the present study, a CYP3A4 reporter gene assay, requiring the overexpression of PXR, was used to investigate whether the PAH parent compounds BaP, benzo[c]phenanthrene (BcP) and dibenzo[a,l]pyrene (DBalP) as well as their corresponding phase I metabolites, the respective dihydrodiols and diol epoxides, can induce CYP3A4 promoter activity. BaP, BcP and their dihydrodiols were found to significantly activate the CYP3A4 promoter. Moreover, activation of PXR by all four compounds was detected by using a PXR transactivation assay, supporting that PXR mediates CYP3A4 induction by PAH. Taken together, these results show that both PAH parent compounds as well as their phase I metabolites induce CYP3A4 promoter via the transcription factor PXR.

Dose‐dependent induction of signaling pathways by the flavonoid quercetin in human primary hepatocytes: A transcriptomic study

SCOPE Quercetin is widespread in plant kingdom and consumed regularly with human diet (16 mg/day). Due to reported positive effects on health, quercetin supplements with recommended doses up to 2 g/day are offered. However, molecular effects of such high doses on human liver have not been assessed yet. Therefore, molecular effects on human hepatocytes were analyzed to help assessing the risk of quercetin supplementation. METHODS AND RESULTS Molecular effects of three different quercetin concentrations on gene expression in human hepatocytes were investigated by microarray analysis. Possible new signaling pathways were investigated using reporter gene assays. Quercetin concentrations representing the normal intake showed weak effects on mRNA expression in liver cells. In contrast, supplemental doses affect immune response and p53 signaling and might be associated with cancer. Additionally, quercetin showed inhibition of transcriptional activation and mRNA-expression of HNF4α and its target genes. Inhibitory effects were also found for FXR, LXRα, and PXR. CONCLUSION Normal intake of quercetin seems to play a minor regulatory role, while supplement doses may have great effects on gene expression in hepatocytes. However, since it is not clarified whether such high doses of quercetin exert positive or negative effects, a careful handling of quercetin supplements is advised.

Regulation of drug metabolism by the interplay of inflammatory signaling, steatosis, and xeno-sensing receptors in HepaRG cells

Nonalcoholic fatty liver disease (NAFLD), which is characterized by triglyceride deposition in hepatocytes resulting from imbalanced lipid homeostasis, is of increasing concern in Western countries, along with progression to nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Previous studies suggest a complex, mutual influence of hepatic fat accumulation, NASH-related inflammatory mediators, and drug-sensing receptors regulating xenobiotic metabolism. Here, we investigated the suitability of human HepaRG hepatocarcinoma cells as a model for NAFLD and NASH. Cells were incubated for up to 14 days with an oleate/palmitate mixture (125 µM each) and/or with 10 ng/ml of the inflammatory mediator interleukin-6 (IL-6). Effects of these conditions on the regulation of drug metabolism were studied using xenobiotic agonists of the aryl hydrocarbon receptor (AHR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), nuclear factor (erythroid-derived 2)-like 2, and peroxisome proliferator-activated receptor α (PPARα). Results underpin the suitability of HepaRG cells for NAFLD- and NASH-related research and constitute a broad-based analysis of the impact of hepatic fatty acid accumulation and inflammation on drug metabolism and its inducibility by xenobiotics. IL-6 exerted pronounced negative regulatory effects on basal as well as on PXR-, CAR-, and PPARα-, but not AHR-dependent induction of drug-metabolizing enzymes. This inhibition was related to diminished transactivation potential of the respective receptors rather than to reduced transcription of nuclear receptor-encoding mRNAs. The most striking effects of IL-6 and/or fatty acid treatment were observed in HepaRG cells after 14 days of treatment, making these cultures appear a suitable model for studying the relationship of fatty acid accumulation, inflammation, and xenobiotic-induced drug metabolism.

PXR: Structure-specific activation by hepatotoxic pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) comprise a large group of more than 660 secondary metabolites found in more than 6000 plant species worldwide. Acute PA intoxication induces severe liver damage. Chronic exposure to sub-lethal doses may cause cumulative damage or cancer. Nuclear receptor activation often constitutes a molecular event for xenobiotic-induced toxicity. However, so far nothing is known about potential interactions of PAs with nuclear receptors as a toxicological mode of action. Thus, in the present study PA-dependent activation of a comprehensive panel of nuclear receptors (PPARs, LXRα, RARα, RXRα, FXR, CAR, PXR, ERα/β) was investigated using GAL4/UAS-based transactivation reporter gene assays. To cover the most frequently occurring PA structure types (retronecine, heliotridine and otonecine type; as well as monoester, open-chain diester and cyclic diester) different PAs were analyzed for interaction with nuclear receptors. Most of the nuclear receptors investigated were not affected by the tested PAs. However, significant activation was found for PXR, which was exclusively activated by the open-chain diesters, echimidine and lasiocarpine. Induction of the model PXR target gene CYP3A4 by PAs was verified at the mRNA, protein and enzyme activity level. In conclusion, PXR activation and PXR-mediated induction of CYP3A4 expression by PAs seem to be structure-dependent. Data suggest that only open-chain diesters act as PXR agonists. This might imply that a PXR-mediated mode of action may contribute to the hepatotoxicity of PAs that is dependent on PA structure.

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.