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 , RatasRESUMEN
UNLABELLED: The role of the hepatocyte plasma membrane structure in the development of oxidative stress during alcoholic liver diseases is not yet fully understood. Previously, we have established the pivotal role of membrane fluidity in ethanol-induced oxidative stress, but no study has so far tested the involvement of lipid rafts. In this study, methyl-beta-cyclodextrin or cholesterol oxidase, which were found to disrupt lipid rafts in hepatocytes, inhibited both reactive oxygen species production and lipid peroxidation, and this suggested a role for these microstructures in oxidative stress. By immunostaining of lipid raft components, a raft clustering was detected in ethanol-treated hepatocytes. In addition, we found that rafts were modified by formation of malondialdehyde adducts and disulfide bridges. Interestingly, pretreatment of cells by 4-methyl-pyrazole (to inhibit ethanol metabolism) and various antioxidants prevented the ethanol-induced raft aggregation. In addition, treatment of hepatocytes by a stabilizing agent (ursodeoxycholic acid) or a fluidizing compound [2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate] led to inhibition or enhancement of raft clustering, respectively, which pointed to a relationship between membrane fluidity and lipid rafts during ethanol-induced oxidative stress. We finally investigated the involvement of phospholipase C in raft-induced oxidative stress upon ethanol exposure. Phospholipase C was shown to be translocated into rafts and to participate in oxidative stress by controlling hepatocyte iron content. CONCLUSION: Membrane structure, depicted as membrane fluidity and lipid rafts, plays a key role in ethanol-induced oxidative stress of the liver, and its modulation may be of therapeutic relevance.
Asunto(s)
Etanol/efectos adversos , Hepatocitos/metabolismo , Microdominios de Membrana/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Colesterol Oxidasa/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Fluidez de la Membrana/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/enzimología , Fosfoinositido Fosfolipasa C/metabolismo , Ratas , Ratas Sprague-Dawley , beta-Ciclodextrinas/farmacologíaRESUMEN
We previously reported that 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) elicited apoptotic cell death as well as non-apoptotic programmed cell deaths (PCDs) with paraptotic and necroptotic characteristics, respectively. In the present study, we have further confirmed and extended these findings. Flow cytometric analyses of 1-NP-exposed/3NF-exposed Hepa1c1c7 cells revealed that caspase-3 was only activated in the subpopulation of cells corresponding to that with classic apoptotic morphology. Immunocytochemical analysis indicated that leucocyte elastase inhibitor-derived DNaseII (LEI/L-DNaseII), apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were more clearly translocated to the nucleus following 3-NF exposure than after 1-NP. These 3-NF-induced changes in AIF and EndoG translocation were reduced by necrostatin-1, an inhibitor of necroptotic cell death. Both compounds lead to accumulation of lipid droplets and induced DNA damage. Activation of checkpoint kinase (CHK) 1 and H2AX, but not ataxia telangiectasia mutated and CHK2, were observed. Furthermore, inhibition of p53 using pifithrin-alpha reduced the cell death induced by both compounds, suggesting a role of DNA damage/CHK1/p53 pathway in the death process. 1-NP-induced cell death was in addition characterized by increased oxidative damage and intracellular accumulation of Ca(2+). These findings further support the notion that 1-NP elicited apoptotic cell death and PCD with paraptotic characteristics, while 3-NF induced apoptosis and a PCD with necroptotic features.
Asunto(s)
Fluorenos/toxicidad , Pirenos/toxicidad , Apoptosis , Factor Inductor de la Apoptosis/biosíntesis , Proteínas de la Ataxia Telangiectasia Mutada , Benzotiazoles/farmacología , Proteínas de Ciclo Celular/biosíntesis , Muerte Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Daño del ADN , Proteínas de Unión al ADN/biosíntesis , Endodesoxirribonucleasas/biosíntesis , Histonas/biosíntesis , Humanos , Proteínas Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Tolueno/análogos & derivados , Tolueno/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
In this study, we show that the environmental pollutant, 3-nitrofluoranthene (3-NF) but not its amine form, 3-aminofluoranthene (3-AF), induces apoptosis as well as regulated necrosis with necroptotic features in Hepa1c1c7 cells. Upon exposure to 3-NF, both typical apoptotic and necrotic cells were observed. A large number of the cells exhibited a characteristic partial nuclear chromatin condensation. Cycloheximide completely attenuated 3-NF-induced cell death. Activation of caspase-8, -9, and -3 were observed. Moreover, Z-VAD-FMK decreased the apoptotic cells, whereas the number of propidium iodide (PI)-positive cells with partial chromatin condensation was reduced by Nec-1, an inhibitor of receptor interacting protein (RIP-1). Cyp1a1, but not nitric oxide synthase (NOS), appears to be involved in activation of 3-NF to reactive metabolites. Increase in the number as well as size of lysosomes, myelinosomes, and activation of autophagy were also observed. 3-NF induced phosphorylation of ERK1/2, JNK and p38 MAPKs. Interestingly, while inhibitors of ERK1/2 and JNK reduced apoptotic as well as necrotic cell death, the p38 inhibitor, SB202190 reduced only the necrotic cell death. Taken together, 3-NF elicits both apoptosis and a caspase-independent programmed cell death (PCD) with autophagic characteristics. Conversely, with 3-AF, no apparent cytotoxic effects besides a reduction in cell proliferation was observed.
Asunto(s)
Apoptosis/efectos de los fármacos , Fluorenos/toxicidad , Animales , Autofagia , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ratones , Microscopía Electrónica de Transmisión , Proteínas Quinasas Activadas por Mitógenos/metabolismo , NecrosisRESUMEN
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/metabolismoRESUMEN
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/metabolismoRESUMEN
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/metabolismoRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]yrene (B[a]P) constitute a widely distributed class of environmental pollutants, responsible for highly toxic effects. Elucidating the intracellular mechanisms of this cytotoxicity thus remains a major challenge. Besides the activation of the p53 apoptotic pathway, we have previously found in F258 hepatic cells that the B[a]P (50 nM)-induced apoptosis was also dependent upon the transmembrane transporter NHE1, whose activation might result from membrane alterations in our model. We here demonstrate that: (1) B[a]P induces a membrane fluidization surprisingly linked to NHE1 activation; (2) membrane stabilization by exogenous cholesterol protects cells from B[a]P-induced apoptosis, via an effect on late acidification and iron uptake.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzo(a)pireno/farmacología , Fluidez de la Membrana , Animales , Línea Celular , RatasRESUMEN
Phenobarbital (PB) alters expression of numerous hepatic genes, including genes of cytochrome P450 2B1 and 2B2 (CYP2B). However, the intracellular mechanisms remain to be fully elucidated. The present study investigated the involvement of mitogen-activated protein kinases (MAPKs) in rat hepatocytes in primary culture. We showed that PB induced an early, dose-dependent activation of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 MAPKs. Regarding the PB (1mM) induction of CYP2B mRNA expression, while chemically inhibiting JNK had no effect, specific inhibitors of the ERK (U0-126) and p38 (SB-203580) pathways up- and down-regulated this expression, respectively. However, although such a regulation was confirmed when testing the effect of a dominant negative mutant of the ERK pathway on the CYP2B2 enhancer-promoter activity, no such transcriptional role was found with the p38 pathway. Moreover, upon arrest of transcription, the stability of CYP2B mRNA remained unaffected by SB-203580. In conclusion, we show that the ERK pathway negatively regulates CYP2B2 enhancer-promoter activity and that, despite p38 activation upon PB exposure, the sensitivity of CYP2B mRNA expression to SB-203580 appears to be unrelated to this kinase.
Asunto(s)
Citocromo P-450 CYP2B1/genética , Hepatocitos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fenobarbital/farmacología , Animales , Northern Blotting , Butadienos/farmacología , Células Cultivadas , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Citocromo P-450 CYP2B1/biosíntesis , Diclororribofuranosil Benzoimidazol/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Imidazoles/farmacología , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Masculino , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Piridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The ubiquitous environmental pollutants polycyclic aromatic hydrocarbons are responsible for important carcinogenic and apoptotic effects, whose mechanisms are still poorly understood, owing to the multiplicity of possible cellular targets. Among these mechanisms, alterations of ionic homeostasis have been suggested. In this work, the effects of benzo(a)pyrene [B(a)P] on pHi were tested in the rat liver F258 epithelial cell line, using the fluoroprobe carboxy-SNARF-1. After a 48-h treatment, B(a)P (50 nM) induced an alkalinization, followed by an acidification after 72 h and the development of apoptosis. Determinations of pH(i) recovery following an acid load showed an increased acid efflux at 48 h. Cariporide inhibited both the early alkalinization and the increased acid efflux, thus suggesting the involvement of Na+/H+ exchanger 1 (NHE1). Besides, alpha-naphtoflavone (alpha-NF), an inhibitor of CYP1A1-mediated B(a)P metabolism, prevented all pH(i) changes, and NHE1 activation was blocked by the antioxidant thiourea, which inhibited CYP1A1 metabolism-dependent H2O2 production. Regarding B(a)P-induced apoptosis, this was prevented by alpha-NF and bongkrekic acid, an inhibitor of mitochondria-dependent apoptosis. Interestingly, apoptosis was significantly reduced by cariporide. Taken together, our results indicate that B(a)P, via H2O2 produced by CYP1A1-dependent metabolism, induces an early activation of NHE1, resulting in alkalinization; this appears to play a significant role in mitochondria-dependent B(a)P-induced apoptosis.
Asunto(s)
Benzo(a)pireno/toxicidad , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Benzo(a)pireno/farmacología , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Guanidinas/farmacología , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Hígado/citología , Hígado/efectos de los fármacos , Modelos Biológicos , Ratas , Sulfonas/farmacología , Tiourea/farmacologíaRESUMEN
We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.
Asunto(s)
Radicales Libres/metabolismo , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/metabolismo , Sobrecarga de Hierro/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Animales , Inducción Enzimática , Glutatión Transferasa/genética , Hepatocitos/metabolismo , Hierro/administración & dosificación , Sobrecarga de Hierro/enzimología , Riñón/enzimología , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/agonistas , ARN Mensajero/genéticaRESUMEN
How pH(i) changes, more specifically alkalinization, affect the apoptotic cascade has yet to be determined. The aim of the present work was to test the involvement of mitochondria in the apoptotic cascade triggered by benzo(a)pyrene [B(a)P] and to determine the role of pH(i) changes and p53 relative to mitochondria. Our results indicate that B(a)P-induced apoptosis might rely upon a p53-dependent and a pH-sensitive mitochondrial dysfunction.
Asunto(s)
Apoptosis/fisiología , Benzo(a)pireno/toxicidad , Células Epiteliales/citología , Concentración de Iones de Hidrógeno , Hígado/citología , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Tolueno/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Benzotiazoles , Ácido Bongcréquico/farmacología , Línea Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Guanidinas/farmacología , Hígado/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Ratas , Sulfonas/farmacología , Tiazoles/farmacología , Tolueno/farmacología , Proteína p53 Supresora de Tumor/efectos de los fármacosRESUMEN
Ximelagatran, the first oral agent in the new class of direct thrombin inhibitors, was withdrawn from the market due to a potential risk of severe liver injury. Increased rates of liver enzyme elevations had been observed during clinical trials of chronic use. Despite intensive preclinical investigations the cellular mechanisms behind the observed hepatic effects remain unknown. The aim of this study was to investigate whether ximelagatran has an effect on the plasma membrane fluidity and the membrane lipid composition which may be important for the cell integrity. After 1h exposure of primary human hepatocytes with 10 or 100 microM ximelagatran, a significant elevation of membrane fluidity was observed. This elevation was maintained at 24h, but diminished at 48 h exposure. As changed membrane lipid composition could influence membrane fluidities, changes in membrane lipid profiles were also studied. After 1h exposure, the phosphatidylcholine/phosphatidylethanolamine molar ratio decreased, whereas the total cholesterol/phospholipid molar ratio decreased after a 48 h exposure. The change in membrane fluidity and lipid composition in human hepatocytes exposed to ximelagatran might indicate changes in plasma membrane properties that in susceptible subjects, could result in loss of membrane integrity and leakage of cellular proteins.
Asunto(s)
Anticoagulantes/farmacología , Azetidinas/farmacología , Bencilaminas/farmacología , Hepatocitos/efectos de los fármacos , Fluidez de la Membrana/efectos de los fármacos , Lípidos de la Membrana/química , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Hepatocitos/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Tacrina/farmacologíaRESUMEN
Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed that the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent.
Asunto(s)
Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Pirenos/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Western Blotting , Caspasas/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colorantes , Medios de Cultivo , Cicloheximida/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/ultraestructura , Citometría de Flujo , Humanos , Inmunohistoquímica , Microscopía Electrónica , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fagosomas/efectos de los fármacos , Propidio , Inhibidores de la Síntesis de la Proteína/farmacología , Transducción de Señal/efectos de los fármacos , Vacuolas/efectos de los fármacos , Vacuolas/ultraestructuraRESUMEN
While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation and cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzo(a)pireno/toxicidad , Células Epiteliales/efectos de los fármacos , Hierro/metabolismo , Lactoferrina/metabolismo , Lisosomas/efectos de los fármacos , Naranja de Acridina/farmacología , Animales , Benzo(a)pireno/química , Carcinógenos/química , Carcinógenos/toxicidad , Caspasa 3/metabolismo , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Deferiprona , Deferoxamina/farmacología , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Hierro/antagonistas & inhibidores , Quelantes del Hierro/farmacología , Lactoferrina/genética , Hígado/citología , Lisosomas/metabolismo , Microscopía Electrónica , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Piridonas/farmacología , ARN Interferente Pequeño/genética , Ratas , TransfecciónRESUMEN
Polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), are ubiquitous genotoxic environmental pollutants. Their DNA-damaging effects lead to apoptosis induction, through similar pathways to those identified after exposure to other DNA-damaging stimuli with activation of p53-related genes and the involvement of the intrinsic apoptotic pathway. However, at a low concentration of B[a]P (50 nM), our previous results pointed to the involvement of intracellular pH (pHi) variations during B[a]P-induced apoptosis in a rat liver epithelial cell line (F258). In the present work, we identified the mitochondrial F0F1-ATPase activity reversal as possibly responsible for pHi decrease. This acidification not only promoted executive caspase activation, but also activated leucocyte elastase inhibitor/leucocyte-derived DNase II (LEI/L-DNase II) pathway. p53 appeared to regulate mitochondria homeostasis, by initiating F0F1-ATPase reversal and endonuclease G (Endo G) release. In conclusion, a low dose of B[a]P induced apoptosis by recruiting a large panel of executioners apparently depending on p53 phosphorylation and, for some of them, on acidification.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzo(a)pireno/toxicidad , Hígado/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Caspasas/metabolismo , Línea Celular , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Concentración de Iones de Hidrógeno , Hígado/citología , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Fosforilación , ATPasas de Translocación de Protón/metabolismo , Interferencia de ARN , Ratas , TransfecciónRESUMEN
We hypothesized that glutathione transferases could be induced and may participate to cellular defenses against the oxidative stress occurring during liver regeneration. Here, we evidenced that murine GSTA1 (mGSTA1), A4, Pi, and Mu are up-regulated during mouse liver regeneration, exhibiting a biphasic pattern of induction correlating early G(1) phase and G(1)/S transition of the cell cycle. Using confocal microscopy immunolocalization and subcellular fractionation, mGSTA4 was demonstrated in both mitochondria and cytosol and found preferentially increased in cytosol during liver regeneration. In addition, mGSTA4 was induced in vivo and in cultured hepatocytes by tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), and epidermal growth factor (EGF), factors that play crucial roles in hepatocyte survival and proliferation during liver regeneration. However, the mitogenic effect of EGF was not responsible for the induction of mGSTA4. In transient transfections, IL-6 and EGF, but not TNFalpha, transactivated the human GSTA4 (hGSTA4) promoter cloned upstream of the luciferase reporter gene suggesting that IL-6 and EGF up-regulated hGSTA4 at a transcriptional level, whereas TNFalpha could rather act at a post-transcriptional level. The inhibition of phosphoinositide 3-kinase, p38 MAPK, and MEK/ERK signaling pathways, using specific inhibitors, prevented EGF-dependent induction of mGSTA4 and transactivation of hGSTA4 promoter. Altogether, these data favor the conclusion that, in regenerating hepatocytes, several GST isoforms are induced and that cytokines TNFalpha and IL-6 and survival factor EGF positively regulate mGSTA4 via survival signaling pathways.