Metabolism of triflumuron in the human liver: Contribution of cytochrome P450 isoforms and esterases.

Abstract Triflumuron (TFM) is a benzoylurea insecticide commonly used in Tunisian agriculture and around the world to control crop pests and flies as a promising alternative to conventional insecticides for its arthropod specificity and low toxicity. From the evidence available in animal models, it can be expected that the metabolism of TFM is catalyzed by cytochrome P450 (CYP) and esterases. However, no data are available on human metabolism of TFM with regards to phase I metabolism and CYP isoform specificity. Hence, this manuscript describes experimental investigations to underpin in vitro phase I TFM metabolism in human samples for the first time. TFM biotransformation by recombinant human CYPs was characterized, then human liver microsomes (HLM) and chemical specific inhibitors have been used to identify the relative contribution of CYPs and esterases. Our results showed that all CYP isoforms were able to metabolize TFM with different affinity and efficiency. The relative contribution based both on the kinetic parameters and the CYP hepatic content was 3A4 > >2C9 > 2C8 > 2A6 > 1A2 > 2B6 > 2D6 > 2C19 > 2C18 > 1A1 at low TFM concentration, whilst at high TFM concentration it was 1A2 > >2C9 = 3A4 = 2A6 > 2C19 > 2B6 = 2C8 > 2D6 > 1A1 > 2C18. Experiments with HLMs confirmed the involvement of the most relevant CYPs in the presence of specific chemical inhibitors with a catalytic efficiency (Cliapp) lower by an order of magnitude compared with recombinant enzymes. Esterases were also relevant to the overall TFM kinetics and metabolism, with catalytic efficiency higher than that of CYPs. It is foreseen that such isoform-specific information in humans will further support in silico models for the refinement of the human risk assessment of single pesticides or mixtures

The implication of ROS production on triflumuron-induced oxidative stress and genotoxicity in human colon carcinoma (HCT-116) cells

The aim of this study is to evaluate the cytotoxic and the genotoxic effects of triflumuron (TFM) on human colon carcinoma cells (HCT-116). Indeed, TFM is used to protect vegetables, fruits, and domestic animals against a large spectrum of parasites causing animal and human disorders. However, studies revealing its toxicity and its mode of action in mammalian systems remain very limited. We monitored our work with the cytotoxicity assay starting with the cell viability test, the ROS generation, the malondialdehyde (MDA) production, the DNA fragmentation, and the measurement of some antioxidant enzymes activities such as catalase, superoxide dismutase, and the glutathione S-transferase. Also, we measured the mitochondrial transmembrane potential. We showed that TFM induced a dose-dependent cell death. This decrease in cell viability was accompanied by a significant reduction in the mitochondrial membrane potential. We also have shown that TFM induced oxidative stress as revealed by the generation of reactive oxygen species, the increase of the MDA levels, and the activation of the antioxidant enzymes. Moreover, our results indicated that TFM induced DNA damage in HCT-116 cells as monitored by the comet assay. We demonstrate, for the first time, the cytotoxic and the genotoxic potentials of TFM on human cultured cells

In vitro interaction of the pesticides flupyradifurone, bupirimate and its metabolite ethirimol with the ATP-binding cassette transporter G2 (ABCG2)

[EN] ABCG2 is an ATP-binding cassette efflux transporter that is expressed in absorptive and excretory organs such as liver, intestine, kidney, brain and testis where it plays a crucial physiological and toxicological role in protecting cells against xenobiotics, affecting pharmacokinetics of its substrates. In addition, the induction of ABCG2 expression in mammary gland during lactation is related to active secretion of many toxicants into milk. In this study, the in vitro interactions between ABCG2 and three pesticides flupyradifurone, bupirimate and its metabolite ethirimol were investigated to check whether these compounds are substrates and/or inhibitors of this transporter. Using in vitro transepithelial assays with cells transduced with murine, ovine and human ABCG2, we showed that ethirimol and flupyradifurone were transported efficiently by murine Abcg2 and ovine ABCG2 but not by human ABCG2. Bupirimate was not found to be an in vitro substrate of ABCG2 transporter. Accumulation assays using mitoxantrone in transduced MDCK-II cells suggest that none of the tested pesticides were efficient ABCG2 inhibitors, at least in our experimental conditions. Our studies disclose that ethirimol and flupyradifurone are in vitro substrates of murine and ovine ABCG2, opening the possibility of a potential relevance of ABCG2 in the toxicokinetics of these pesticides.S

Moringa oleifera Protects SH-SY5YCells from DEHP-Induced Endoplasmic Reticulum Stress and Apoptosis

Moringa oleifera (MO) is a medicinal plant that has been shown to possess antioxidant, anticarcinogenic and antibiotic activities. In a rat model, MO extract (MOe) has been shown to have a protective effect against brain damage and memory decline. As an extending study, here, we have examined the protective effect of MOe against oxidative stress and apoptosis caused in human neuroblastome (SH-SY5Y) cells by di-(2-ethylhexyl) phthalate (DEHP), a plasticizer known to induce neurotoxicity. Our data show that MOe prevents oxidative damage by lowering reactive oxygen species (ROS) formation, restoring mitochondrial respiratory chain complex activities, and, in addition, by modulating the expression of vitagenes, i.e., antioxidant proteins Nrf2 and HO-1. Moreover, MOe prevented neuronal damage by partly inhibiting endoplasmic reticulum (ER) stress response, as indicated by decreased expression of CCAAT-enhancer-binding protein homologous protein (CHOP) and Glucose-regulated protein 78 (GRP78) proteins. MOe also protected SH-SY5Y cells from DEHP-induced apoptosis, preserving mitochondrial membrane permeability and caspase-3 activation. Our findings provide insight into understanding of molecular mechanisms involved in neuroprotective effects by MOe against DEHP damag

Mitochondrial and endoplasmic reticulum stress pathways cooperate in zearalenone-induced apoptosis of human leukemic cells

<p>Abstract</p> <p>Background</p> <p>Zearalenone (ZEA) is a phytoestrogen from <it>Fusarium </it>species. The aims of the study was to identify mode of human leukemic cell death induced by ZEA and the mechanisms involved.</p> <p>Methods</p> <p>Cell cytotoxicity of ZEA on human leukemic HL-60, U937 and peripheral blood mononuclear cells (PBMCs) was performed by using 3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Reactive oxygen species production, cell cycle analysis and mitochondrial transmembrane potential reduction was determined by employing 2',7'-dichlorofluorescein diacetate, propidium iodide and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. Caspase-3 and -8 activities were detected by using fluorogenic Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) and Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin (IETD-AMC) substrates, respectively. Protein expression of cytochrome c, Bax, Bcl-2 and Bcl-xL was performed by Western blot. The expression of proteins was assessed by two-dimensional polyacrylamide gel-electrophoresis (PAGE) coupled with LC-MS2 analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) approach.</p> <p>Results</p> <p>ZEA was cytotoxic to U937 > HL-60 > PBMCs and caused subdiploid peaks and G1 arrest in both cell lines. Apoptosis of human leukemic HL-60 and U937 cell apoptosis induced by ZEA was via an activation of mitochondrial release of cytochrome c through mitochondrial transmembrane potential reduction, activation of caspase-3 and -8, production of reactive oxygen species and induction of endoplasmic reticulum stress. Bax was up regulated in a time-dependent manner and there was down regulation of Bcl-xL expression. Two-dimensional PAGE coupled with LC-MS2 analysis showed that ZEA treatment of HL-60 cells produced differences in the levels of 22 membrane proteins such as apoptosis inducing factor and the ER stress proteins including endoplasmic reticulum protein 29 (ERp29), 78 kDa glucose-regulated protein, heat shock protein 90 and calreticulin, whereas only <it>ERp29 </it>mRNA transcript increased.</p> <p>Conclusion</p> <p>ZEA induced human leukemic cell apoptosis via endoplasmic stress and mitochondrial pathway.</p

Cytotoxic and genotoxic effects of epoxiconazole on F98 glioma cells

International audienceEpoxiconazole (EPX) is a very effective fungicide of the triazole family. Given its wide spectrum of use, the increased application of this pesticide may represent a serious risk on human health. Previous studies have found that EPX is cytotoxic to cells, although the exact mechanism remains elusive. In particular, the effect on the nervous system is poorly elucidated. Here we evaluated the implication of oxidative stress in the neurotoxicity and studied its apoptotic mechanism of action. We demonstrated that the treatment by EPX reduces the viability of cells in a dose dependent manner with an IC50 of 50 μM. It also provokes the reduction of cell proliferation. EPX could trigger arrest in G1/S phase of cell cycle with low doses, however with IC50, it induced an accumulation of F98 cells in G2/M phase. Moreover, EPX induced cytoskeleton disruption as evidenced by immunocytochemical analysis. It provoked also DNA fragmentation in a concentration dependent manner. The EPX induced apoptosis, which was observed by morphological changes and by positive Annexin V FITC-PI staining concurrent with a depolarization of mitochondria. Furthermore, the cell mortality provoked by EPX was significantly reduced by pretreatment with Z-VAD-FMK, a caspase inhibitor. Moreover, N-acetylcysteine (NAC) strongly restores cell viability that has been inhibited by EPX. The results of these findings highlight the implication of ROS generation in the neurotoxicity induced by EPX, indicating that the production of ROS is the main cause of the induction of apoptosis probably via the mitochondrial pathway.</p

Ochratoxine A et néphropathie humaine en Tunisie : dix ans d'étude

L'ochratoxine A (OTA) est une mycotoxine connue essentiellement pour ses effets néphrotoxiques. Des études réalisées dans les régions balkaniques ont fortement soupçonné l'OTA d'être le principal agent causal de la Néphropathie Endémique des Balkans (NEB). Cependant, malgré les nombreuses investigations réalisées, l'implication de l'OTA dans cette néphropathie humaine est encore sujette à controverse. En Tunisie, une néphropathie interstitielle chronique (NIC) à étiologie indéterminée, similaire à la NEB, où l'OTA semble également être impliquée, a été bien caractérisée. Dans ce travail, nous nous proposons d'apporter des preuves supplémentaires impliquant davantage l'OTA dans cette néphropathie. Nous présentons le bilan d'une étude rétrospective de dix ans (1991-2001) engageant 954 néphropathes et 205 individus sains. Dans ce bilan, nous comparons les contaminations sériques en OTA dans trois groupes : un groupe d'individus sains ne présentant aucune pathologie (205 sujets), un groupe de néphropathes présentant une NIC inexpliquée (383 sujets) et un groupe de néphropathes à étiologie connue (571 sujets). La détection et le dosage de l'OTA sont réalisés après extraction à partir des échantillons sériques suivie d'une analyse par chromatographie liquide haute performance (HPLC) avec détection de fluorescence. Nous montrons que l'incidence la plus importante de la contamination sérique en OTA est trouvée dans le groupe présentant une NIC inexpliquée. En effet, dans ce groupe, le pourcentage d'individus OTA-positifs est de 97 % et il n'est que de 73 % et de 86 % respectivement chez les individus sains et chez ceux présentant d'autres néphropathies à étiologie connue. Les concentrations moyennes sont également plus élevées dans le groupe de NIC d'étiologie inconnue (50.77 ± 4.75 ng/ml) que dans les autres groupes de contrôle (2.35 ± 0.90 ng/ml) ou de néphropathes à etiologies connues (9.96 ± 3 ng/ml). Notre étude renforce davantage le rôle de VOTA dans cette néphropathie et la désigne comme agent causal très probable