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Background/Aims: Bile acids damage the liver, essentially by inducing hepatocyte apoptosis. Clinical studies have shown that several activators of the pregnane X receptor (PXR) may induce the remission of cholestasis. However, the molecular mechanisms involved in this beneficial effect remain unclear. We analysed the effect of an activator of PXR, clotrimazole (CLO), on the apoptosis induced by bile acids in primary cultures of rat hepatocytes. Methods: Rat hepatocytes were isolated by collagenase perfusion of the liver. Then, cells were pretreated with CLO for 24 h, after which they were exposed to deoxycholic and glycochenodeoxycholic acids (DCA, GCDCA). Apoptosis and necrosis were monitored morphologically and biochemically using cytotoxicity assays, phase-contrast microscopy, Annexin V/propidium iodide staining and evaluations of lactate dehydrogenase release. The activation of caspases and the proteolysis of their substrates were analysed by enzyme assays and Western blot. The signal transductions involved in the protective effect of the PXR activation were analysed by assessing the phosphorylation status of kinases belonging to the ERK, Akt and p38 pathways and by analysing pro- and anti-apoptotic proteins. Results: CLO protected rat hepatocytes against DCA- and GCDCA-induced apoptosis, preventing morphological aspects of this process (membrane blebbing, nuclear and chromatin condensation and DNA breakdown). This effect was attributable, at least partly, to caspases inhibition, Bcl-xL induction, the activation of ERK and Akt signalling and p38 inhibition. Conclusion: This study provides the description of the cytoprotective effect of PXR activation against bile acid-induced apoptosis and highlights molecular pathways that could be targeted in the treatment of cholestasis. [ABSTRACT FROM AUTHOR]

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. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

In vivo distribution of the neonicotinoid insecticide, imidacloprid, was followed during 72 h in six biological compartments of Apis mellifera L: head, thorax, abdomen, haemolymph, midgut and rectum. Honeybees were treated orally with 100 µg of 14C-imidacloprid per kg of bee, a dose close to the median lethal dose. Elimination half-life of total radioactivity in honeybee was 25 h. Haemolymph was the compartment with the lowest and rectum that with the highest level of total radioactivity during the whole study, with a maximum 24 h after treatment. Elimination half-life of imidacloprid in whole honeybee was 5 h. Imidacloprid was readily distributed and metabolised only by Phase I enzymes into five metabolites: 4/5-hydroxy-imidacloprid, 4,5-dihydroxy-imidacloprid, 6-chloronicotinic acid, and olefin and urea derivatives. The guanidine derivative was not detected. The urea derivative and 6-chloronicotinic acid were the main metabolites and appeared particularly in midgut and rectum. The olefin derivative and 4/5-hydroxy-imidacloprid preferentially occurred in head, thorax and abdomen, which are nicotinic acetylcholine receptor-rich tissues. Moreover, they presented a peak value around 4 h after imidacloprid ingestion. These results explain the prolongation of imidacloprid action in bees, and particularly the differences between rapid intoxication symptoms and late mortality. [ABSTRACT FROM AUTHOR]

Induction of both CYP1A1 and metallothioneins (MTs) in fish liver is increasingly being used in ecotoxicological studies. The interaction of heavy metals (Cd, Cu, Zn, Pb) with the CYP1A induction response and MT levels was studied in trout (Oncorhynchus mykiss) hepatocyte cultures. Cells were exposed to 3-methylcholanthrene (3-MC) or to increasing heavy metal concentrations or to a mixture of both (3-MC and one heavy metal). Metal cytotoxicity was assessed by the neutral red test. Ranking of toxicity was Cd(II) < Cu(II) < Zn(II) < Pb(II) (EC50: 45, 222, 873, and 945 µM, respectively). CYP1A1 expression was monitored by ethoxyresorufin-O-deethylase (EROD) activity as well as by Western and Northern blots. As expected, 3-MC induced EROD activity in a time- and dose-dependent manner (maximal induction 5 times that of the control at 0.5 µM and after a 72-h exposure period). These data were confirmed by Western blot (intense band of 55-60 KDa) and Northern blot analyses. Induction caused by 0.5 µM 3-MC was reduced to less than 50% of control by the concomitant exposure to Cd, Cu, Pb, or Zn (EC50: from 1 µM for Cd(II) to 18 µM for Pb(II)). The MTs were significantly induced in hepatocytes exposed to heavy metals for 24 h. In the presence of 3-MC (0.5 µM), MT levels were significantly lower than those found in cells treated with metals alone at 24 h only. Our results lead to the conclusion that heavy metals significantly affect CYP expression and that a CYP1A1 inducer (3-MC) can modulate the induction of MTs. These data have to be taken into consideration in biomarker monitoring. [ABSTRACT FROM AUTHOR]