Roger Rahmani

Is CYP1A1 induction always related to AHR signaling pathway

Humans are daily subjected to ever increasing amounts of exogenous compounds. Some of them are capable of inducing cytochrome P450s, a process that allows the cell to adapt to changes in its chemical environment. One of the most widely CYP studied is CYP1A1 because it metabolises a large number of xenobiotics to cytotoxic and/or mutagenic derivatives. To date, results from the literature indicate that induction of CYP1A1 does not only involve the classical activation cascade of the Ah receptor, e.g. binding of the ligand to the AhR, heterodimerisation with Arnt protein, constitution of a complex with XRE responsive element and subsequent gene activation. Indeed, some xenobiotics do activate CYP1A1 gene expression in spite of their inability to compete with TCDD for binding to the AhR. Other signaling pathways must therefore also be considered. Firstly, the CYP1A1 inducer compounds could be very weak AhR ligands or may be metabolized into a form which is in turn capable of binding to the Ah receptor. A second hypothesis would be that these molecules could act through other signaling cascades. At this time, two of them seem to be implicated. One concerns the RARs signal transduction pathway, as already described for retinoic acid. The second may involve tyrosine kinase activation, but the precise relationship between this activation and CYPA1 induction remains yet to be established. For the moment there is still a black box which needs to be investigated.

Induction of cytochrome P450 1A1 gene expression, oxidative stress, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblastoid cells.

Carbaryl and thiabendazole, two widely used pesticides, have been shown to induce cytochrome P450 1A1 (CYP1A1) expression, but neither compound is capable of displacing [3H] 2,3,7,8-tetrachlorodibenzo-P-dioxin from its aryl hydrocarbon receptor binding site. In the present study, we investigated the transcriptional regulation of CYP1A1 as well as other genes in various human hepatoma HepG2 cell lines stably transfected with the chloramphenicol acetyl transferase (CAT) reporter gene and cloned under the control of each of 14 promoters or response elements from relevant stress genes. Carbaryl and thiabendazole were found to activate CYP1A1 at the level of transcription, as demonstrated by the dose-dependent increase in reporter CAT and CYP1A1 mRNAs. Moreover, this effect appeared to be mediated via the xenobiotic responsive element (XRE), because both pesticides specifically activated various fusion constructs containing XRE sequences (CYP1A, glutathione S-transferase, and XRE). Carbaryl and to a lesser extent thiabendazole also activated other stress genes such as c-fos and NF-kappaBRE, HSP70 and GRP78, and GADD153 at a transcriptional level. These data suggest that these molecules induce early alert genes, including those known to be sensitive to oxidative stress. This led us to examine the genotoxic effect of carbaryl and thiabendazole by an in vitro DNA repair solid-phase assay. Both compounds provoked a strong DNA-damaging activity in the human lymphoblastoid cell line that constitutively expresses human CYP1A1 cDNA, but not in the parental line, indicating that CYP1A1 is chiefly implicated in carbaryl and thiabendazole genotoxicity. This effect was confirmed on HepG2 cells. These observations support the notion that intracellular signals leading to CYP1A1 induction, oxidative stress, and genotoxicity are intimately related.

Microcystin-LR causes the collapse of actin filaments in primary human hepatocytes

Microcystin-LR (MCLR) is a potent inhibitor of protein phosphatases 1 and 2A and causes alterations in cytoskeletal filaments and morphological changes that underlie apoptosis in rat hepatocytes. It has also been reported that it caused several cases of human deaths and illness. As no study on the effect of microcystins on human hepatocytes was done, yet, the aim of the study is to evaluate the toxicity of MCLR on primary human hepatocytes. The hepatocytes were incubated in 12.5-50 nM MCLR for 3, 6 and 9 h, fixed and stained with fluorescent probes for actin filaments and nuclei. Spectral laser-scanning confocal microscopy revealed that in the MCLR-treated primary human hepatocytes the actin mesh collapsed into the center of the cell, similarly as it has been described for rat hepatocytes. Cells were blebbing, fragmenting, and separated from each other. The nuclei in the affected cells condensed. In conclusion, this study confirms that MCLR is toxic to primary human hepatocytes, and it may be responsible for the liver failure cases observed after acute cyanobacterial poisoning.

Is bisphenol S a safe substitute for bisphenol A in terms of metabolic function? An in vitro study

As bisphenol A (BPA) has been shown to induce adverse effects on human health, especially through the activation of endocrine pathways, it is about to be withdrawn from the European market and replaced by analogues such as bisphenol S (BPS). However, toxicological data on BPS is scarce, and so it is necessary to evaluate the possible effects of this compound on human health. We compared the effect of BPA and BPS on obesity and hepatic steatosis processes using low doses in the same range as those found in the environment. Two in vitro models were used, the adipose cell line 3T3-L1 and HepG2 cells, representative of hepatic functions. We analyzed different parameters such as lipid and glucose uptakes, lipolysis, leptin production and the modulation of genes involved in lipid metabolism and energy balance. BPA and BPS induced an increase in the lipid content in the 3T3-L1 cell line and more moderately in the hepatic cells. We also observed a decrease in lipolysis after bisphenol treatment of adipocytes, but only BPS was involved in the increase in glucose uptake and leptin production. These latter effects could be linked to the modulation of SREBP-1c, PPARγ, aP2 and ERRα and γ genes after exposure to BPA, whereas BPS seems to target the PGC1α and the ERRγ genes. The findings suggest that both BPA and BPS could be involved in obesity and steatosis processes, but through two different metabolic pathways.

Cytochrome 1A1 induction by primaquine in human hepatocytes and HepG2 cells: absence of binding to the aryl hydrocarbon receptor

Malaria remains the most prevalent infectious disease of tropical and subtropical areas of the world. It represents a crucial problem in public health care, affecting 750 million people annually, of whom at least two million die. Various antimalarials currently used were studied for their capability to induce expression of the cytochrome P450 1A1 (CYP1A1) gene, an enzyme that plays an important role in the activation of xenobiotics to genotoxic derivatives. Studies on human hepatocytes and HepG2 cell lines showed that primaquine was capable of dose dependently increasing both the ethoxyresorufin-O-deethylase activity and CYP1A1 mRNAs, suggesting a transcriptional activation of this gene. Moreover, alpha-naphthoflavone, a partial aryl hydrocarbon receptor (AhR) antagonist, and 8-methoxypsoralen, which interferes with the binding of activated AhR to the xenobiotic responsive element, were shown to suppress CYP1A1 induction when added to the cultures. However, neither primaquine nor its metabolites were able to displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin from AhR in competitive binding studies using 9S-enriched fractions of human cytosol. These data, together with the induction of CYP1A1 promoter-directed chloramphenicol acetyl transferase gene expression, suggest that CYP1A1 induction involves the participation of the AhR but not a direct primaquine-receptor interaction. This supports the notion that an alternative ligand-independent mechanism has to be considered. Given the pharmaco-toxicological significance of CYP1A1 induction, these findings may have important implications in the treatment of malaria with primaquine and new analogs.

Cytotoxic effects and induction of cytochromes P450 1A1/2 by insecticides, in hepatic or epidermal cells: binding capability to the Ah receptor

Insecticides deserve particular attention since the general population is potentially exposed to such chemicals through many routes. We therefore tested the comparative acute and chronic toxicity of chemicals belonging to the major insecticides families (DDT, malathion and tetrachlorvinphos, carbaryl, cypermethrin, diflubenzuron), in hepatocytes, HepG2 and HaCaT cell lines. Two kinds of end-points were used: cytotoxicity parameters and CYP1A1 induction. Except for cypermethrin and diflubenzuron, all these chemicals exerted a cytotoxic effect in hepatocytes and HaCaT, but not in HepG2 cells. However, the induction of the EROD activity appeared more sensitive since a response was detected at lower concentrations. Significant differences were observed between the cell types and the insecticides. Furthermore, these chemicals were unable to displace [3H]TCDD from its binding sites, suggesting that they would not be a ligand of the Ah receptor. The experimental approach used herein may be a good means for predicting the acute and chronic toxicity of pesticides.

Endosulfan decreases cell growth and apoptosis in human hacat keratinocytes : Partial ROS-dependent ERK1/2 mechanism

Endosulfan is an organochlorine insecticide described as a potential carcinogen in humans. This insecticide was recently reported to alter the mitogen‐activated protein (MAP) kinase signaling pathways and is suspected to affect cell growth and differentiation in human keratinocytes. This study was designed to assess the mitogenic, apoptogenic, and genotoxic effects of endosulfan on the HaCaT cell line. We first found that 25 µM endosulfan led to persistent extracellular signal‐regulated kinase (ERK)1/2 phosphorylation with an accumulation of the phosphorylated form in the nucleus, probably caused by MAP kinase phosphatase (MKP) inhibition. As previously described under sustained ERK1/2 activation, cell growth was decreased: delayed confluency and 35% decrease of BrdU incorporation was demonstrated in endosulfan‐treated keratinocytes. In addition, endosulfan has been shown to generate transient reactive oxygen species (ROS), and blocking this oxidative stress by N‐acetyl cysteine (NAC) strongly prevented both persistent nuclear ERK1/2 phosphorylation and cell growth decrease. Additional experiments demonstrated that unchanged endosulfan rather than its metabolites has mutagenic effects (Ames positive without S9) and increased DNA strand breaks (Comet assay) in HaCaT cells, via a ROS‐dependent mechanism. Therefore, to assess the putative pro‐apoptotic response of damaged cells, caspases 3/7 activity and poly(ADP‐ribose)‐polymerase (PARP) cleavage were measured. The results clearly indicated that endosulfan inhibited both spontaneous and staurosporine‐induced apoptosis. Taken together, these findings strongly support that endosulfan induces ROS generation leading to sustained ERK1/2 phosphorylation and decrease in cell growth. Moreover, endosulfan was found to inhibit apoptosis and this could contribute to mutant cell survival and therefore have possible carcinogenic effects. J. Cell. Physiol. 213: 177–186, 2007.

Lindane and cell death: at the crossroads between apoptosis, necrosis and autophagy.

Lindane, a persistent organochlorine pesticide, is recognized as a major public health concern because of its potential toxic effects on human health. Despite observations pointing to the toxicity of lindane, mechanisms underlying its deleterious effects in liver have yet to be understood. In this study, we investigated the effects of lindane on autophagic, apoptotic and necrotic cell death in primary cultured rat hepatocytes. We found that lindane deregulated the autophagic process as demonstrated by (1) the formation of enlarged acidic vesicles labeled with LC3, Rab7 and LAMP1 (specific markers of autophagic vacuole maturation), (2) the conversion of LC3-I (the cytosolic form) into LC3-II (membrane bound), (3) the deregulation of the Beclin 1 protein expression and (4) the enhanced formation of the Bcl-xL/Beclin 1 complex. Lindane induced vacuolization together with the inhibition of spontaneous and intrinsic apoptosis. This disruption of cell suicide was linked to Bcl-xL up-regulation, Bax down-expression, prevention of cytochrome c release, and inhibition of caspase-9 and -3 activities. Lindane-induced disruption of apoptosis and autophagy occurred in parallel with necrosis induction in rat hepatocytes. In consequence, we proposed that lindane toxicity in primary rat hepatocytes could be jointly attributed to the disruption of autophagic process, the inhibition of apoptotic cell death and the induction of necrosis. These events account, at least in part, for the involvement of both cytotoxic and carcinogenic signaling pathways in the action of lindane in the liver.

The nitroxide stable radical tempo prevents metal-induced inhibition of CYP1A1 expression and induction.

Heavy metals are known to provoke oxidative stress in fish liver cells. Because H2O2, OH*- and intracellular superoxide are involved in this oxidation, we investigated the effect of nitroxide radical, 2,2,6,6-tetramethylpiperidinyl-N-oxyl (abbreviated as TEMPO), a cell-permeable agent possessing antioxidant properties, on CYP1A expression in trout (Oncorhynchus mykiss) hepatocytes. 3-methylcholanthrene (3-MC) induced the CYP1A-related EROD activity. This induction was inhibited by concomitant exposure to Cd (II), Cu (II), Pb (II) or Zn (II). CYP1A mRNA levels were also reduced. Simultaneous treatment with 3-MC, a heavy metal and TEMPO suppressed both the inhibition of EROD activity and the decrease of CYP1A mRNA expression. These results suggest a working hypothesis that heavy metals produce multiple oxidative effects, including generation of hydroxyl radicals, which could down-regulate CYP1A1 expression. This metal-induced inhibition was prevented by TEMPO, which might protect trout hepatocytes by scavenging free radicals and thus preventing their inhibitory effects on CYP1A induction and expression.

Crosstalk between Beta-Catenin and Snail in the Induction of Epithelial to Mesenchymal Transition in Hepatocarcinoma: Role of the ERK1/2 Pathway

Epithelial to mesenchymal transition (EMT) is an integral process in the progression of many epithelial tumors. It involves a coordinated series of events, leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis. The EMT of hepatocellular carcinoma (HCC) cells is thought to be a key event in intrahepatic dissemination and distal metastasis. In this study, we used 12-O-tet-radecanoylphorbol-13-acetate (TPA) to dissect the signaling pathways involved in the EMT of HepG2 hepatocarcinoma cells. The spectacular change in phenotype induced by TPA, leading to a pronounced spindle-shaped fibroblast-like cell morphology, required ERK1/2 activation. This ERK1/2-dependent EMT process was characterized by a loss of E-cadherin function, modification of the cytoskeleton, the acquisition of mesenchymal markers and profound changes to extracellular matrix composition and mobility. Snail was essential for E-cadherin repression, but was not sufficient for full commitment of the TPA-triggered EMT. We found that TPA triggered the formation of a complex between Snail and β-catenin that activated the Wnt pathway. This study thus provides the first evidence for the existence of a complex network governed by the ERK1/2 signaling pathway, converging on the coregulation of Snail and the Wnt/β-catenin pathway and responsible for the onset and the progression of EMT in hepatocellular carcinoma cells.