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

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

Effects of Dichlorvos on cardiac cells: Toxicity and molecular mechanism of action

International audienceIn this study we aimed to understand the underlying mechanism of Dichlorvos-induced toxicity in cardiac cells. For this end, cells were treated by 170 μM of Dichlorvos (DDVP) (corresponding to the IC50) and molecular events were monitored by flow cytometry and western blotting. We have first demonstrated that cell exposure to DDVP for 24 h induced cell death by necroptosis. In fact, cell treatment with DDVP upregulated RIP1 expression and we have shown that chemical inhibition of RIP1 kinase activity by necrostatin-1 (Nec-1) greatly prevented from the induced cell death. Besides, we have demonstrated that, while there was no observed cell death following short exposure to DDVP (6 h), autophagy was enhanced, as proven by the increase in the level of both Beclin-1 and LC3-II and the accumulation of the CytoID® autophagy detection probe. Besides, when autophagy was inhibited by chloroquine (CQ) the percentage of necroptosis was significantly increased, suggesting that autophagy acts to protect cardiac cells against the toxicity induced by this pesticide. Concurrently, we have shown that the inhibition of the deacetylase sirtuin 1 (SIRT1) by EX527 or its knockdown by siRNA significantly increased DDVP-induced necroptosis, whereas when SIRT1 was activated by resveratrol (RSV) a significant decrease in DDVP-induced cell death was observed. In addition, we revealed that when the autophagy was inhibited by CQ, we can't reveal the protective effect of RSV anymore. Altogether, these results suggest that activation of SIRT1 protects cardiac cells from the toxicity of DDVP through an autophagy-dependent pathway

Tebuconazole induces ROS-dependent cardiac cell toxicity by activating DNA damage and mitochondrial apoptotic pathway

International audienceTebuconazole (TEB) is a common triazole fungicide that is widely used throughout the world in agriculture applications. We previously reported that TEB induces cardiac toxicity in rats. The aim of this study was to investigate the underlying mechanism of the toxicity induced by TEB in cardiac cells. TEB induced dose-dependent cell death in H9c2 cardiomyoblasts and in adult rat ventricular myocytes (ARVM). The comet assay and western blot analysis showed a concentration-dependent increase in DNA damage and in p53 and p21 protein levels 24 h after TEB treatment. Our findings also showed that TEB triggered the mitochondrial pathway of apoptosis as evidenced by a loss of mitochondrial transmembrane potential (ΔΨm), an increase in Bax/Bcl-2 ratio, an activation of caspase-9 and caspase-3, a cleavage of poly (ADP-ribose) polymerase (PARP) and an increase in the proportion of cells in the sub-G1 phase. In addition, TEB promoted ROS production in cardiac cells and consequently increased the amounts of MDA, the end product of lipid peroxidation. Treatment of cardiomyocytes with the ROS scavenger N-acetylcysteine reduced TEB-induced DNA damage and activation of the mitochondrial pathway of apoptosis. These results indicate that the genotoxic and cytotoxic effects of TEB are mediated through a ROS-dependent pathway in cardiac cells

Cytotoxic effects induced by patulin, sterigmatocystin and beauvericin on CHO-K1 cells

Mycotoxins are produced by different genera of fungi; mainly Aspergillus, Penicillium and Fusarium. The natural co-occurrence of beauvericin (BEA), patulin (PAT) and sterigmatocystin (STE) has been proved in feed and food commodities. This study investigates the cytotoxicity of individual and combined mycotoxins BEA, PAT and STE. The cytotoxicity on immortalized ovarian cells (CHO-K1) was evaluated using the MTT assay. After 24, 48 and 72 h, the IC50 values were 2.9 μM for PAT and ranged from 10.7 to 2.2 μM and from 25.0 to 12.5 μM for BEA and STE, respectively. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index (CI) value as a quantitative measure of the three mycotoxin interaction's degree. Binary and tertiary combinations showed a dose dependent effect. At low fraction affected, mycotoxin combinations were synergetic; whereas, at higher fraction affected, the combinations showed additive effect. Our results indicate that the co-occurrence of low concentrations of mycotoxin in food may increase their toxic effects