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1.
Ecotoxicol Environ Saf ; 276: 116261, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38574644

RESUMEN

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.


Asunto(s)
Citocromo P-450 CYP3A , Hepatocitos , Succinato Deshidrogenasa , Humanos , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/genética , Hepatocitos/efectos de los fármacos , Succinato Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/metabolismo , Fungicidas Industriales/toxicidad , ARN Mensajero/metabolismo , ARN Mensajero/genética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Línea Celular
2.
Cell Biol Toxicol ; 39(2): 371-390, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-35412187

RESUMEN

Non-alcoholic fatty liver disease (NAFLD) is a worldwide epidemic for which environmental contaminants are increasingly recognized as important etiological factors. Among them, the combination of benzo[a]pyrene (B[a]P), a potent environmental carcinogen, with ethanol, was shown to induce the transition of steatosis toward steatohepatitis. However, the underlying mechanisms involved remain to be deciphered. In this context, we used high-fat diet fed zebrafish model, in which we previously observed progression of steatosis to a steatohepatitis-like state following a 7-day-co-exposure to 43 mM ethanol and 25 nM B[a]P. Transcriptomic analysis highlighted the potent role of mitochondrial dysfunction, alterations in heme and iron homeostasis, involvement of aryl hydrocarbon receptor (AhR) signaling, and oxidative stress. Most of these mRNA dysregulations were validated by RT-qPCR. Moreover, similar changes were observed using a human in vitro hepatocyte model, HepaRG cells. The mitochondria structural and functional alterations were confirmed by transmission electronic microscopy and Seahorse technology, respectively. Involvement of AhR signaling was evidenced by using in vivo an AhR antagonist, CH223191, and in vitro in AhR-knock-out HepaRG cells. Furthermore, as co-exposure was found to increase the levels of both heme and hemin, we investigated if mitochondrial iron could induce oxidative stress. We found that mitochondrial labile iron content was raised in toxicant-exposed larvae. This increase was prevented by the iron chelator, deferoxamine, which also inhibited liver co-exposure toxicity. Overall, these results suggest that the increase in mitochondrial iron content induced by B[a]P/ethanol co-exposure causes mitochondrial dysfunction that contributes to the pathological progression of NAFLD.


Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/genética , Etanol/toxicidad , Pez Cebra , Benzo(a)pireno/toxicidad , Larva , Transcriptoma , Mitocondrias , Hemo
3.
Semin Cancer Biol ; 43: 49-65, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28088583

RESUMEN

According to the World Health Organization, around 20% of all cancers would be due to environmental factors. Among these factors, several chemicals are indeed well recognized carcinogens. The widespread contaminant benzo[a]pyrene (B[a]P), an often used model carcinogen of the polycyclic aromatic hydrocarbons' family, has been suggested to target most, if not all, cancer hallmarks described by Hanahan and Weinberg. It is classified as a group I carcinogen by the International Agency for Research on Cancer; however, the precise intracellular mechanisms underlying its carcinogenic properties remain yet to be thoroughly defined. Recently, the pH homeostasis, a well known regulator of carcinogenic processes, was suggested to be a key actor in both cell death and Warburg-like metabolic reprogramming induced upon B[a]P exposure. The present review will highlight those data with the aim of favoring research on the role of H+ dynamics in environmental carcinogenesis.


Asunto(s)
Carcinogénesis , Carcinógenos/toxicidad , Exposición a Riesgos Ambientales , Homeostasis , Concentración de Iones de Hidrógeno , Humanos
4.
Int J Mol Sci ; 19(11)2018 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-30453624

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental contaminants, known to affect T lymphocytes. However, the molecular targets and pathways involved in their immunotoxic effects in human T lymphocytes remain unknown. Here, we analyzed the gene expression profile of primary human T lymphocytes treated with the prototypical PAH, benzo[α]pyrene (B[α]P), using a microarray-based transcriptome analysis. After a 48 h exposure to B[α]P, we identified 158 genes differentially expressed in T lymphocytes, including not only genes well-known to be affected by PAHs such as the cytochromes P450 (CYP) 1A1 and 1B1, but also others not previously shown to be targeted by B[α]P such as genes encoding the gap junction beta (GJB)-2 and 6 proteins. Functional enrichment analysis revealed that these candidates were significantly associated with the aryl hydrocarbon (AhR) and interferon (IFN) signaling pathways; a marked alteration in T lymphocyte recruitment was also observed. Using functional tests in transwell migration experiments, B[α]P was then shown to significantly decrease the chemokine (C-X-C motif) ligand 12-induced chemotaxis and transendothelial migration of T lymphocytes. In total, this study opens the way to unsuspected responsive pathway of interest, i.e., T lymphocyte migration, thus providing a more thorough understanding of the molecular basis of the immunotoxicity of PAHs.


Asunto(s)
Benzo(a)pireno/toxicidad , Genoma Humano , Linfocitos T/metabolismo , Transcripción Genética/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferones/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Migración Transendotelial y Transepitelial/efectos de los fármacos
5.
Carcinogenesis ; 37(6): 635-645, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26992899

RESUMEN

Epidemiological studies have associated red meat intake with risk of colorectal cancer. Experimental studies explain this positive association by the oxidative properties of heme iron released in the colon. This latter is a potent catalyst for lipid peroxidation, resulting in the neoformation of deleterious aldehydes in the fecal water of heme-fed rats. The toxicity of fecal water of heme-fed rats was associated to such lipid peroxidation. This study demonstrated that fecal water of hemoglobin- and beef-fed rats preferentially induced apoptosis in mouse normal colon epithelial cells than in those carrying mutation on Apc (Adenomatous polyposis coli) gene, considered as preneoplastic. Highlighting the importance of lipid peroxidation and neoformation of secondary aldehydes like 4-hydroxy-2-nonenal (HNE), we optimized the depletion of carbonyl compounds in the fecal water which turned out to abolish the differential apoptosis in both cell lines. To explain the resistance of preneoplastic cells towards fecal water toxicity, we focused on Nrf2, known to be activated by aldehydes, including HNE. Fecal water activated Nrf2 in both cell lines, associated with the induction of Nrf2-target genes related to aldehydes detoxification. However, the antioxidant defense appeared to be higher in preneoplastic cells, favoring their survival, as evidenced by Nrf2 inactivation. Taken together, our results suggest that Nrf2-dependent antioxidant response was involved in the resistance of preneoplastic cells upon exposure to fecal water of hemoglobin- and beef-fed rats. This difference could explain the promoting effect of red meat and heme-enriched diet on colorectal cancer, by initiating positive selection of preneoplastic cells.


Asunto(s)
Antioxidantes/metabolismo , Neoplasias Colorrectales/etiología , Hemoglobinas/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Carne Roja/efectos adversos , Aldehídos , Animales , Apoptosis , Colon/metabolismo , Colon/patología , Heces , Inactivación Metabólica , Masculino , Ratones , Factor 2 Relacionado con NF-E2/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Ratas Endogámicas F344
6.
Chemosphere ; 358: 142122, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38663675

RESUMEN

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.


Asunto(s)
Succinato Deshidrogenasa , Humanos , Succinato Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Transporte Biológico/efectos de los fármacos , Fungicidas Industriales/toxicidad , Fungicidas Industriales/farmacología , Inhibidores Enzimáticos/farmacología , Estrona/análogos & derivados , Estrona/metabolismo , Células HEK293 , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/antagonistas & inhibidores
7.
J Physiol ; 591(9): 2287-306, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23420656

RESUMEN

Membrane acid extrusion by Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC) is essential for maintaining a low cytoplasmic [H(+)] (∼60 nm, equivalent to an intracellular pH (pHi) of 7.2). This protects myocardial function from the high chemical reactivity of H(+) ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pHi regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pHi imaging reveals that NBC stimulation induces spatially uniform pHi recovery from acidosis, whereas NHE1 stimulation induces pHi non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pHi microdomains is favoured by low cytoplasmic H(+) mobility and long H(+) diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H(+)]i regulation in ventricular myocytes, suggesting that, by guarding pHi, NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t-tubular excitation-contraction coupling.


Asunto(s)
Miocitos Cardíacos/fisiología , Sarcolema/fisiología , Simportadores de Sodio-Bicarbonato/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Calcio/fisiología , Capacidad Eléctrica , Femenino , Cobayas , Ventrículos Cardíacos , Concentración de Iones de Hidrógeno , Masculino , Ratas , Ratas Sprague-Dawley
8.
J Biol Chem ; 287(49): 41458-68, 2012 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-23055519

RESUMEN

F1 domain of F(1)F(o)-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here we show for the first time the presence of the F(1)-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using surface plasmon resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin (G-gly)) as a new ligand for the F(1)-ATPase. By molecular modeling, we identified the motif in the peptide sequence (E(E/D)XY), that directly interacts with the F(1)-ATPase and the amino acids in the F(1)-ATPase that bind this motif. Replacement of the Glu-9 residue by an alanine in the E(E/D)XY motif resulted in a strong decrease of G-gly binding to the F(1)-ATPase and the loss of its biological activity. In addition we demonstrated that F(1)-ATPase mediates the growth effects of the peptide. Indeed, blocking F(1)-ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F(1)-ATPase, which is induced by G-gly. These results suggest an important contribution of cell surface F(1)-ATPase in the pro-proliferative action of this gastrointestinal peptide.


Asunto(s)
Membrana Celular/enzimología , Colon/enzimología , Células Epiteliales/metabolismo , ATPasas de Translocación de Protón/química , Adenosina Difosfato/química , Secuencia de Aminoácidos , Animales , Células CACO-2 , Dominio Catalítico , Bovinos , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Células Endoteliales/citología , Humanos , Mitocondrias/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie
9.
Cell Physiol Biochem ; 29(5-6): 657-66, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22613966

RESUMEN

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are ubiquitous toxic environmental pollutants capable of inducing cell death. Intracellular pH plays a key role in the regulation of cell survival and death. Our previous works have demonstrated that intracellular alkalinization mediated by Na(+)/H(+) exchanger 1 (NHE-1) is a critical event involved in B[a]P-induced apoptosis. The aim of this study was to further elucidate the mechanisms of NHE-1 activation upon B[a]P exposure. METHODS: We tested the effects of plasma membrane cholesterol enrichment or depletion on B[a]P-induced NHE-1 activation related to apoptosis. We isolated cholesterol-rich plasma membrane microdomains to assess NHE-1 submembrane location and immunoprecipitated NHE-1 from the different sub-membrane fractions obtained to examine NHE-1 protein interactions during B[a]P-induced apoptosis. RESULTS: We found that NHE-1 is preferentially located in cholesterol-rich microdomains and that B[a]P activates NHE-1 via its relocation and binding of calmodulin outside these specialized plasma membrane microstructures; these events are necessary for the execution of the apoptosis-related intracellular alkalinization. CONCLUSION: Plasma membrane location of NHE-1 affects its protein interactions and apoptotic function.


Asunto(s)
Apoptosis/efectos de los fármacos , Benzo(a)pireno/farmacología , Colesterol/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Western Blotting , Calmodulina/metabolismo , Línea Celular , Transporte de Proteínas , Ratas
10.
Chem Res Toxicol ; 24(11): 1984-93, 2011 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-21967605

RESUMEN

Animal and epidemiological studies suggest that dietary heme iron would promote colorectal cancer. Oxidative properties of heme could lead to the formation of cytotoxic and genotoxic secondary lipid oxidation products, such as 4-hydroxy-2(E)-nonenal (HNE). This compound is more cytotoxic to mouse wild-type colon cells than to isogenic cells with a mutation on the adenomatous polyposis coli (APC) gene. The latter thus have a selective advantage, possibly leading to cancer promotion. This mutation is an early and frequent event in human colorectal cancer. To explain this difference, the HNE biotransformation capacities of the two cell types have been studied using radiolabeled and stable isotope-labeled HNE. Apc-mutated cells showed better biotransformation capacities than nonmutated cells did. Thiol compound conjugation capacities were higher for mutated cells, with an important advantage for the extracellular conjugation to cysteine. Both cells types were able to reduce HNE to 4-hydroxynonanal, a biotransformation pathway that has not been reported for other intestinal cells. Mutated cells showed higher capacities to oxidize 4-hydroxynonanal into 4-hydroxynonanoic acid. The mRNA expression of different enzymes involved in HNE metabolism such as aldehyde dehydrogenase 1A1, 2 and 3A1, glutathione transferase A4-4, or cystine transporter xCT was upregulated in mutated cells compared with wild-type cells. In conclusion, this study suggests that Apc-mutated cells are more efficient than wild-type cells in metabolizing HNE into thiol conjugates and 4-hydroxynonanoic acid due to the higher expression of key biotransformation enzymes. These differential biotransformation capacities would explain the differences of susceptibility between normal and Apc-mutated cells regarding secondary lipid oxidation products.


Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Células Epiteliales/metabolismo , Hemo/toxicidad , Hierro/toxicidad , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Aldehídos/metabolismo , Aldehídos/toxicidad , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Colon/efectos de los fármacos , Colon/patología , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , Daño del ADN , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Glutatión/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Hemo/efectos adversos , Hemo/metabolismo , Humanos , Hierro/efectos adversos , Hierro/metabolismo , Marcaje Isotópico , Espectrometría de Masas , Ratones , Mutación , Oxidación-Reducción , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Células Tumorales Cultivadas
11.
Cancers (Basel) ; 13(17)2021 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-34503147

RESUMEN

To investigate environmental impacts upon colorectal carcinogenesis (CRC) by diet, we assessed two western diet food contaminants: 4-hydroxynonenal (HNE), a major lipid peroxidation product neoformed during digestion, and a mixture of pesticides. We used human colonic cell lines ectopically eliciting varied genetic susceptibilities to CRC: the non-transformed human epithelial colonic cells (HCECs) and their five isogenic cell lines with the loss of APC (Adenomatous polyposis coli) and TP53 (Tumor protein 53) and/or ectopic expression of mutated KRAS (Kristen-ras). These cell lines have been exposed for either for a short time (2-24 h) or for a long period (3 weeks) to 1 µM HNE and/or 10 µM pesticides. After acute exposure, we did not observe any cytotoxicity or major DNA damage. However, long-term exposure to pesticides alone and in mixture with HNE induced clonogenic transformation in normal HCECs, as well as in cells representing later stages of carcinogenesis. It was associated with genotoxic and non-genomic mechanisms (cell growth, metabolic reprogramming, cell mobility and epithelial-mesenchymal transition) depending on genetic susceptibility. This study demonstrated a potential initiating and promoting effect of food contaminants on CRC after long-term exposure. It supports that these contaminants can accelerate carcinogenesis when mutations in oncogenes or tumor suppressor genes occur.

12.
Toxicol Appl Pharmacol ; 243(1): 68-76, 2010 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-19931295

RESUMEN

Benzo[alpha]pyrene (B[alpha]P) often serves as a model for mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Our previous work suggested a role of membrane fluidity in B[alpha]P-induced apoptotic process. In this study, we report that B[alpha]P modifies the composition of cholesterol-rich microdomains (lipid rafts) in rat liver F258 epithelial cells. The cellular distribution of the ganglioside-GM1 was markedly changed following B[alpha]P exposure. B[alpha]P also modified fatty acid composition and decreased the cholesterol content of cholesterol-rich microdomains. B[alpha]P-induced depletion of cholesterol in lipid rafts was linked to a reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Aryl hydrocarbon receptor (AhR) and B[alpha]P-related H(2)O(2) formation were involved in the reduced expression of HMG-CoA reductase and in the remodeling of membrane microdomains. The B[alpha]P-induced membrane remodeling resulted in an intracellular alkalinization observed during the early phase of apoptosis. In conclusion, B[alpha]P altered the composition of plasma membrane microstructures through AhR and H(2)O(2) dependent-regulation of lipid biosynthesis. In F258 cells, the B[alpha]P-induced membrane remodeling was identified as an early apoptotic event leading to an intracellular alkalinization.


Asunto(s)
Apoptosis/efectos de los fármacos , Benzo(a)pireno/toxicidad , Membrana Celular/efectos de los fármacos , Animales , Línea Celular , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Microdominios de Membrana/efectos de los fármacos , Ácido Mevalónico , Ratas
13.
Cancer Res ; 67(1): 218-26, 2007 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-17210702

RESUMEN

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that induces apoptosis in cancer cells but not in most normal cells. How tumor physiology, particularly acidic extracellular pH (pH(e)), would modify sensitivity of cancer cells to TRAIL-induced cell death is not known. We have previously shown that cancer cells, resistant to TRAIL-induced apoptosis at physiologic pH(e) (7.4), could be sensitized to TRAIL at acidic pH(e) (6.5). However, at this acidic pH(e), cell death was necrotic. We show here that, in spite of a necrosis-like cell death morphology, caspases are activated and are necessary for TRAIL-induced cell death at acidic pH(e) in HT29 human colon cancer cells. Furthermore, we observed that, whereas receptor-interacting protein (RIP) was cleaved following TRAIL treatment at physiologic pH(e) (7.4), it was not cleaved following TRAIL treatment at acidic pH(e) (6.5). Moreover, RIP degradation by geldanamycin or decrease expression of RIP by small RNA interference transfection inhibited TRAIL-induced necrosis at acidic pH(e), showing that RIP was necessary for this necrotic cell death pathway. We also show that RIP kinase activity was essential for this cell death pathway. Altogether, we show that, under acidic pH(e) conditions, TRAIL induces a necrosis-like cell death pathway that depends both on caspases and RIP kinase activity. Thus, our data suggest for the first time that RIP-dependent necrosis might be a major death pathway in TRAIL-based therapy in solid tumors with acidic pH(e).


Asunto(s)
Caspasas/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Benzoquinonas/farmacología , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Activación Enzimática , Células HT29 , Humanos , Concentración de Iones de Hidrógeno , Lactamas Macrocíclicas/farmacología , FN-kappa B/metabolismo , Necrosis , ARN Interferente Pequeño/genética , Transfección
14.
Cancer Res ; 67(4): 1696-705, 2007 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-17308111

RESUMEN

Regulation of the balance between survival, proliferation, and apoptosis on carcinogenic polycyclic aromatic hydrocarbon (PAH) exposure is still poorly understood and more particularly the role of physiologic variables, including intracellular pH (pH(i)). Although the involvement of the ubiquitous pH(i) regulator Na(+)/H(+) exchanger isoform 1 (NHE1) in tumorigenesis is well documented, less is known about its role and regulation during apoptosis. Our previous works have shown the primordial role of NHE1 in carcinogenic PAH-induced apoptosis. This alkalinizing transporter was activated by an early CYP1-dependent H(2)O(2) production, subsequently promoting mitochondrial dysfunction leading to apoptosis. The aim of this study was to further elucidate how NHE1 was activated by benzo(a)pyrene (BaP) and what the downstream events were in the context of apoptosis. Our results indicate that the mitogen-activated protein kinase kinase 4/c-Jun NH(2)-terminal kinase (MKK4/JNK) pathway was a link between BaP-induced H(2)O(2) production and NHE1 activation. This activation, in combination with BaP-induced phosphorylated p53, promoted mitochondrial superoxide anion production, supporting the existence of a common target for NHE1 and p53. Furthermore, we showed that the mitochondrial expression of glycolytic enzyme hexokinase II (HKII) was decreased following a combined action of NHE1 and p53 pathways, thereby enhancing the BaP-induced apoptosis. Taken together, our findings suggest that, on BaP exposure, MKK4/JNK targets NHE1 with consequences on HKII protein, which might thus be a key protein during carcinogenic PAH apoptosis.


Asunto(s)
Apoptosis/efectos de los fármacos , Benzo(a)pireno/farmacología , Hexoquinasa/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Apoptosis/fisiología , Línea Celular , Núcleo Celular/metabolismo , Activación Enzimática/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Fosforilación , Ratas , Especies Reactivas de Oxígeno/metabolismo , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Proteína p53 Supresora de Tumor/metabolismo
15.
Biochimie ; 163: 171-183, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31228544

RESUMEN

Despite the improvement of diagnostic methods and anticancer therapeutics, the human population is still facing an increasing incidence of several types of cancers. According to the World Health Organization, this growing trend would be partly linked to our environment, with around 20% of cancers stemming from exposure to environmental contaminants, notably chemicals like polycyclic aromatic hydrocarbons (PAHs). PAHs are widespread pollutants in our environment resulting from incomplete combustion or pyrolysis of organic material, and thus produced by both natural and anthropic sources; notably benzo[a]pyrene (B[a]P), i.e. the prototypical molecule of this family, that can be detected in cigarette smoke, diesel exhaust particles, occupational-related fumes, and grilled food. This molecule is a well-recognized carcinogen belonging to group 1 carcinogens. Indeed, it can target the different steps of the carcinogenic process and all cancer hallmarks. Interestingly, H+ dynamics have been described as key parameters for the occurrence of several, if not all, of these hallmarks. However, information regarding the role of such parameters during environmental carcinogenesis is still very scarce. The present review will thus mainly give an overview of the impact of B[a]P on H+ dynamics in liver cells, and will show how such alterations might impact different aspects related to the finely-tuned balance between cell death and survival processes, thereby likely favoring environmental carcinogenesis. In total, the main objective of this review is to encourage further research in this poorly explored field of environmental molecular toxicology.


Asunto(s)
Benzo(a)pireno/toxicidad , Carcinogénesis/inducido químicamente , Carcinógenos/toxicidad , Neoplasias/metabolismo , Protones , Animales , Benzo(a)pireno/metabolismo , Carcinógenos/metabolismo , Exposición a Riesgos Ambientales , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología
16.
PLoS One ; 14(11): e0224132, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31697708

RESUMEN

Succinate dehydrogenase (SDH) inhibitors (SDHIs) are used worldwide to limit the proliferation of molds on plants and plant products. However, as SDH, also known as respiratory chain (RC) complex II, is a universal component of mitochondria from living organisms, highly conserved through evolution, the specificity of these inhibitors toward fungi warrants investigation. We first establish that the human, honeybee, earthworm and fungal SDHs are all sensitive to the eight SDHIs tested, albeit with varying IC50 values, generally in the micromolar range. In addition to SDH, we observed that five of the SDHIs, mostly from the latest generation, inhibit the activity of RC complex III. Finally, we show that the provision of glucose ad libitum in the cell culture medium, while simultaneously providing sufficient ATP and reducing power for antioxidant enzymes through glycolysis, allows the growth of RC-deficient cells, fully masking the deleterious effect of SDHIs. As a result, when glutamine is the major carbon source, the presence of SDHIs leads to time-dependent cell death. This process is significantly accelerated in fibroblasts derived from patients with neurological or neurodegenerative diseases due to RC impairment (encephalopathy originating from a partial SDH defect) and/or hypersensitivity to oxidative insults (Friedreich ataxia, familial Alzheimer's disease).


Asunto(s)
Transporte de Electrón/efectos de los fármacos , Plaguicidas/farmacología , Succinato Deshidrogenasa/antagonistas & inhibidores , Animales , Antioxidantes/metabolismo , Abejas/metabolismo , Células Cultivadas , Farmacorresistencia Fúngica/efectos de los fármacos , Proteínas Fúngicas/farmacología , Hongos/metabolismo , Humanos , Membranas Mitocondriales/efectos de los fármacos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Oligoquetos/metabolismo , Succinato Deshidrogenasa/metabolismo
17.
Microbiome ; 7(1): 72, 2019 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-31060614

RESUMEN

BACKGROUND: The World Health Organization classified processed and red meat consumption as "carcinogenic" and "probably carcinogenic", respectively, to humans. Haem iron from meat plays a role in the promotion of colorectal cancer in rodent models, in association with enhanced luminal lipoperoxidation and subsequent formation of aldehydes. Here, we investigated the short-term effects of this haem-induced lipoperoxidation on mucosal and luminal gut homeostasis including microbiome in F344 male rats fed with a haem-enriched diet (1.5 µmol/g) 14-21 days. RESULTS: Changes in permeability, inflammation, and genotoxicity observed in the mucosal colonic barrier correlated with luminal haem and lipoperoxidation markers. Trapping of luminal haem-induced aldehydes normalised cellular genotoxicity, permeability, and ROS formation on a colon epithelial cell line. Addition of calcium carbonate (2%) to the haem-enriched diet allowed the luminal haem to be trapped in vivo and counteracted these haem-induced physiological traits. Similar covariations of faecal metabolites and bacterial taxa according to haem-induced lipoperoxidation were identified. CONCLUSIONS: This integrated approach provides an overview of haem-induced modulations of the main actors in the colonic barrier. All alterations were closely linked to haem-induced lipoperoxidation, which is associated with red meat-induced colorectal cancer risk.


Asunto(s)
Aldehídos/metabolismo , Colon/metabolismo , Hemo/administración & dosificación , Mucosa Intestinal/metabolismo , Hierro/metabolismo , Microbiota , Animales , Hemo/metabolismo , Homeostasis , Inflamación , Peróxidos Lipídicos/metabolismo , Masculino , Pruebas de Mutagenicidad , Ratas , Ratas Endogámicas F344
18.
Biochemistry ; 47(51): 13674-85, 2008 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-19035652

RESUMEN

NHE-1 is a ubiquitous, mitogen-activatable, mammalian Na+/H+ exchanger that maintains cytosolic pH and regulates cell volume. We have previously shown that the kinetics of NHE-1 positive cooperative activation by intracellular acidifications fit best with a Monod-Wyman-Changeux mechanism, in which a dimeric NHE-1 oscillates between a low- and a high-affinity conformation for intracellular protons. The ratio between these two forms, the allosteric equilibrium constant L0, is in favor of the low-affinity form, making the system inactive at physiological pH. Conversely the high-affinity form is stabilized by intracellular protons, resulting in the observed positive cooperativity. The aim of the present study was to investigate the kinetics and mechanism of NHE-1 regulation by osmotic shocks. We show that they modify the L0 parameter (865 +/- 95 and 3757 +/- 328 for 500 and 100 mOsM, respectively, vs 1549 +/- 57 in isotonic conditions).This results in an activation of NHE-1 by hypertonic shocks and, conversely, in an inhibition by hypotonic media. Quantitatively, this modulation of L0 follows an exponential distribution relative to osmolarity, that is, additive to the activation of NHE-1 by intracellular signaling pathways. These effects can be mimicked by the asymmetric insertion of amphiphilic molecules into the lipid bilayer. Finally, site-directed mutagenesis of NHE-1 shows that neither its association with membrane PIP2 nor its interaction with cortical actin are required for mechanosensation. In conclusion, NHE-1 allosteric equilibrium and, thus, its cooperative response to intracellular acidifications is extremely sensitive to modification of its membrane environment.


Asunto(s)
Regulación de la Expresión Génica , Presión Osmótica , Intercambiadores de Sodio-Hidrógeno/química , Animales , Proteínas de Transporte de Catión/metabolismo , Línea Celular , Cricetinae , Citosol/metabolismo , Fibroblastos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Microscopía Fluorescente , Modelos Biológicos , Isoformas de Proteínas , Transducción de Señal , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo
19.
J Cell Physiol ; 216(1): 207-20, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18264982

RESUMEN

The Na+/H+ exchanger 1, which plays an essential role in intracellular pH regulation in most tissues, is also known to be a key actor in both proliferative and apoptotic processes. Its activation by H+ is best described by the Monod-Wyman-Changeux model: the dimeric NHE-1 oscillates between a low and a high affinity conformation, the balance between the two forms being defined by the allosteric constant L(0). In this study, influence of cholesterol- and caveolin-rich microdomains on NHE-1 activity was examined by using cholesterol depleting agents, including methyl-beta-cyclodextrin (MBCD). These agents activated NHE-1 by modulating its L(0) parameter, which was reverted by cholesterol repletion. This activation was associated with NHE-1 relocation outside microdomains, and was distinct from NHE-1 mitogenic and hormonal stimulation; indeed MBCD and serum treatments were additive, and serum alone did not change NHE-1 localization. Besides, MBCD activated a serum-insensitive, constitutively active mutated NHE-1 ((625)KDKEEEIRK(635) into KNKQQQIRK). Finally, the membrane-dependent NHE-1 regulation occurred independently of Mitogen Activated Protein Kinases, especially Extracellular Regulated Kinase activation, although this kinase was activated by MBCD. In conclusion, localization of NHE-1 in membrane cholesterol- and caveolin-rich microdomains constitutes a novel physiological negative regulator of NHE-1 activity.


Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Caveolinas/metabolismo , Colesterol/metabolismo , Microdominios de Membrana , Intercambiadores de Sodio-Hidrógeno/metabolismo , Regulación Alostérica , Animales , Proteínas de Transporte de Catión/genética , Línea Celular , Colesterol Oxidasa/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Gangliósido G(M1)/metabolismo , Humanos , Hidrógeno/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Sodio/metabolismo , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , beta-Ciclodextrinas/metabolismo
20.
Environ Mol Mutagen ; 59(7): 595-602, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30091211

RESUMEN

Even if the comet assay has been widely used for decades, there is still a need for controlled studies and good mathematical models to assess the variability of the different versions of this assay and in particular to assess potential intra-experimental variability of the high-throughput comet assay. To address this point, we further validate a high-throughput comet assay that uses hydrophilic polyester film (Gelbond®). Experiments were performed using human peripheral blood mononuclear cells (PBMC) either untreated or treated with different concentration of MMS (methyl methanesulfonate). A positive control for the Fpg (Formamidopyrimidine DNA glycosylase)-modified comet assay (Ro 19-8022 with light) was also included. To quantify the sources of variability of the assay, including intradeposit variability, instead of summarizing DNA damage on 50 cells from a deposit by the mean or median of their percentage DNA tail, we analyzed all logit-transformed data with a linear mixed model. The main source of variation in our experimental data is between cells within the same deposit, suggesting genuine variability in the response of the cells rather than variation caused by technical treatment of cell samples. The second source of variation is the inter-experimental variation (day-to-day experiment); the coefficient of this variation for the control was 13.6%. The variation between deposits in the same experiment is negligible. Moreover, there is no systematic bias because of the position of samples on the Gelbond® film nor the position of the films in the electrophoresis tank. This high-throughput comet assay is thus reliable for various applications. Environ. Mol. Mutagen. 59:595-602, 2018. © 2018 Wiley Periodicals, Inc.


Asunto(s)
Ensayo Cometa/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Mutágenos/toxicidad , Poliésteres/química , Daño del ADN/efectos de los fármacos , ADN-Formamidopirimidina Glicosilasa/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Leucocitos Mononucleares/metabolismo , Modelos Lineales , Metilmetanosulfonato/toxicidad
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