Caroline Aninat

EXPRESSION OF CYTOCHROMES P450, CONJUGATING ENZYMES AND NUCLEAR RECEPTORS IN HUMAN HEPATOMA HepaRG CELLS

Most human hepatocyte cell lines lack a substantial set of liver-specific functions, especially major cytochrome P450 (P450)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the HepaRG cells, derived from a human hepatocellular carcinoma, which exhibit a high differentiation pattern after 2 weeks at confluency to determine whether they could mimic human hepatocytes for drug metabolism and toxicity studies. We show that when passaged at low density, these cells reversed to an undifferentiated morphology, actively divided, and, after having reached confluency, formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. By contrast, when seeded at high density, hepatocyte-like clusters retained their typical differentiated morphology. Transcripts of various nuclear receptors (aryl hydrocarbon receptor, pregnane X receptor, constitutive androstane receptor, peroxisome proliferator-activated receptor α), P450s (CYP1A2, 2C9, 2D6, 2E1, 3A4), phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1), and other liver-specific functions were estimated by reverse transcriptase-quantitative polymerase chain reaction and were found to be expressed, for most of them, at comparable levels in both confluent differentiated and high-density differentiated HepaRG cells and in cultured primary human hepatocytes. For several transcripts, the levels were strongly increased in the presence of 2% dimethyl sulfoxide. Measurement of basal activities of several P450s and their response to prototypical inducers as well as analysis of metabolic profiles and cytotoxicity of several compounds confirmed the functional resemblance of HepaRG cells to primary cultured human hepatocytes. In conclusion, HepaRG cells constitute the first human hepatoma cell line expressing high levels of the major P450s involved in xenobiotic metabolism and represent a reliable surrogate to human hepatocytes for drug metabolism and toxicity studies.

Long-term functional stability of human HepaRG hepatocytes and use for chronic toxicity and genotoxicity studies

The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and to express various liver-specific functions, including the major cytochromes P450. This study was aimed to determine whether differentiated HepaRG cells retained their specific functional capacities for a long time period at confluence. We show that expression of transcripts encoding CYP1A2, 2B6, 3A4, and 2E1, several phase II and antioxidant enzymes, membrane transporters, including organic cation transporter 1 and bile salt export pump, the nuclear receptors constitutive androstane receptor and pregnane X receptor, and aldolase B remained relatively stable for at least the 4-week confluence period tested. Similarly, activities of CYP3A4 and CYP1A2 and their responsiveness to prototypical inducers were well preserved. Aflatoxin B1, a potent hepatotoxicant and carcinogen, induced a dose-dependent and cumulative cytotoxicity. Furthermore, at a concentration as low as 0.1 μM, this mycotoxin caused a decrease in both CYP3A4 activity and intracellular ATP associated with morphological alterations, after 14 days following every 2-day exposure. Moreover, using the comet assay, a dose-dependent DNA damage was observed after a 3-h treatment of differentiated HepaRG cells with 1 to 5 μM aflatoxin B1 in the absence of any cell damage, and this DNA damaging effect was strongly reduced in the presence of ketoconazole, a CYP3A4 inhibitor. These results bring the first demonstration of long-term stable expression of liver-specific markers in HepaRG hepatocyte cultures maintained at confluence and show that these cells represent a suitable in vitro liver cell model for analysis of acute and chronic toxicity as well as genotoxicity of chemicals in human liver.

Clinical review: The liver in sepsis

During sepsis, the liver plays a key role. It is implicated in the host response, participating in the clearance of the infectious agents/products. Sepsis also induces liver damage through hemodynamic alterations or through direct or indirect assault on the hepatocytes or through both. Accordingly, liver dysfunction induced by sepsis is recognized as one of the components that contribute to the severity of the disease. Nevertheless, the incidence of liver dysfunction remains imprecise, probably because current diagnostic tools are lacking, notably those that can detect the early liver insult. In this review, we discuss the epidemiology, diagnostic tools, and impact on outcome as well as the pathophysiological aspects, including the cellular events and clinical picture leading to liver dysfunction. Finally, therapeutic considerations with regard to the weakness of the pertinent specific approach are examined.

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 xenobiotics 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 CYP1A1/2s 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 xenobiotics 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. Biotechnol. Bioeng. 2011; 108:1704–1715.

Catecholamines induce an inflammatory response in human hepatocytes.

Objective:The liver is an early target organ in sepsis, severe sepsis, and septic shock, contributing to multiple organ failure, and both lipopolysaccharide and gut-derived catecholamines are implicated in the occurrence of hepatocellular dysfunction. Treatment of septic shock involves administration of vasoactive agents such as exogenous catecholamines or vasopressin in order to reestablish blood pressure. As a prelude to clinical application, we tested the hypothesis that catecholamines could modulate the lipopolysaccharide-induced inflammatory response and function in human liver. Design:An in vitro human cell culture study. Setting:Research laboratory of an academic institution. Subjects:Primary human hepatocytes and human hepatoma HepaRG cells. Interventions:Primary human hepatocytes and human hepatoma HepaRG cells were exposed to lipopolysaccharide to evaluate effects of epinephrine and several other compounds (norepinephrine, dobutamine, dopamine, dopexamine, phenylephrine, clonidine, salbutamol, and vasopressin). Markers of inflammation (interleukin-6, C-reactive protein) and drug metabolism (cytochrome P450 [CYP] 3A4, CYP2B6, CYP1A2, CYP2E1, constitutive androstane receptor, pregnane X receptor) were analyzed. Measurements and Main Results:Transcripts of C-reactive protein and CYP3A4 were strongly increased and depressed respectively after a 24-hr treatment with 10 ng/mL lipopolysaccharide. Co-treatment with either of the catecholamines failed to reverse lipopolysaccharide effects, whereas when added alone, epinephrine, and to a lesser extent norepinephrine, salbutamol, and dobutamine, mimicked lipopolysaccharide effects. Suppression of CYP3A4 implicated &bgr;-adrenergic receptors and was mediated through overproduction of interleukin-6. By contrast, vasopressin did not elicit an inflammatory response or modify CYP3A4 expression. Conclusions:Some catecholamines can induce an inflammatory response and exacerbate the hepatic dysfunction observed during sepsis, favoring the idea that catecholamines could alter the biotransformation of drugs metabolized by CYP3A4 and that alternative vasoactive agents, such as vasopressin, merit further investigation in septic shock patients.

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. Biotechnol. Bioeng. 2013; 110: 597–608.

The glutathione transferase kappa family

Glutathione transferase (GST) kappa, also named mitochondrial GST, is a very ancient protein family with orthologs in bacteria and eukaryotes. Both the structure and the subcellular localization of GSTK1-1, in mitochondria and peroxisomes, make this enzyme distinct from cytosolic GSTs. Rodent and human GSTK1 exhibit activity towards a number of model GST substrates and, in Caenorhabditis elegans, this enzyme may be involved in energy and lipid metabolism, two functions related to mitochondria and peroxisomes. Interestingly, GST kappa is also a key regulator of adiponectin biosynthesis and multimerization suggesting that it might function as a chaperone to facilitate correct folding and assembly of proteins. Since adiponectin expression has been correlated with insulin resistance, obesity and diabetes, GSTK1 expression level which is negatively correlated with obesity in mice and human adipose tissues may be an important factor in these metabolic disorders. Furthermore, a polymorphism in the hGSTK1 promoter has been associated with insulin secretion and fat deposition.

Predictive toxicology using systemic biology and liver microfluidic “on chip” approaches: Application to acetaminophen injury

We have analyzed transcriptomic, proteomic and metabolomic profiles of hepatoma cells cultivated inside a microfluidic biochip with or without acetaminophen (APAP). Without APAP, the results show an adaptive cellular response to the microfluidic environment, leading to the induction of anti-oxidative stress and cytoprotective pathways. In presence of APAP, calcium homeostasis perturbation, lipid peroxidation and cell death are observed. These effects can be attributed to APAP metabolism into its highly reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). That toxicity pathway was confirmed by the detection of GSH-APAP, the large production of 2-hydroxybutyrate and 3-hydroxybutyrate, and methionine, cystine, and histidine consumption in the treated biochips. Those metabolites have been reported as specific biomarkers of hepatotoxicity and glutathione depletion in the literature. In addition, the integration of the metabolomic, transcriptomic and proteomic collected profiles allowed a more complete reconstruction of the APAP injury pathways. To our knowledge, this work is the first example of a global integration of microfluidic biochip data in toxicity assessment. Our results demonstrate the potential of that new approach to predictive toxicology.

Regulation of Signal Transduction by Glutathione Transferases

Glutathione transferases (GST) are essentially known as enzymes that catalyse the conjugation of glutathione to various electrophilic compounds such as chemical carcinogens, environmental pollutants, and antitumor agents. However, this protein family is also involved in the metabolism of endogenous compounds which play critical roles in the regulation of signaling pathways. For example, the lipid peroxidation product 4-hydroxynonenal (4-HNE) and the prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) are metabolized by GSTs and these compounds are known to influence the activity of transcription factors and protein kinases involved in stress response, proliferation, differentiation, or apoptosis. Furthermore, several studies have demonstrated that GSTs are able to interact with different protein partners such as mitogen activated protein kinases (i.e., c-jun N-terminal kinase (JNK) and apoptosis signal-regulating kinase 1 (ASK1)) which are also involved in cell signaling. New functions of GSTs, including S-glutathionylation of proteins by GSTs and ability to be a nitric oxide (NO) carrier have also been described. Taken together, these observations strongly suggest that GST might play a crucial role during normal or cancer cells proliferation or apoptosis.

Anticancer Drug Metabolism: Chemotherapy Resistance and New Therapeutic Approaches

Over the last decades, several studies have demonstrated that cancer cells have a unique metabolism compared to normal cells (Herling et al., 2011). Metabolic changes occurring in cancer cells are considered to be fundamental for the transformation of normal cells into cancer cells and are also responsible for the resistance to different types of chemotherapeutic drugs (Cree, 2011). Therefore, resistance to chemotherapy represents a major problem in the treatment of several tumor types. Among the different metabolic and signalling pathways that are altered in cancer cells, variations in the expression and activity of several drugmetabolizing enzymes play a critical role in drug resistance (Rochat, 2009). Resistance can occur prior to drug treatment (primary or innate resistance) or may develop over time following exposure to the drug (acquired resistance). In some patients, prolonged exposure to a single chemotherapeutic agent may lead to the development of resistance to multiple other structurally unrelated compounds, known as cross resistance or multidrug resistance.