RESUMO
Oxidative stress has been implicated in many physiopathologies including neurodegenerative diseases, cancer, cardiovascular and respiratory diseases, and in mechanisms of action of environmental toxicants. tert-butylhydroperoxide (t-BHP) is an organic lipid hydroperoxide analogue, which is commonly used as a pro-oxidant for evaluating mechanisms involving oxidative stress in cells and tissues. This study investigates mechanisms of apoptosis induced by oxidative stress in hepatocytes, in particular, the involvement of caspases and subcellular compartments. Freshly isolated hepatocytes were exposed to 0.4 mM t-BHP during 1 h. A general caspase inhibitor, Boc-D-FMK, reduced t-BHP-induced apoptosis (chromatin condensation), confirming the involvement of caspases in apoptosis. A caspase-9 inhibitor, Z-LEHD-FMK, also reduced t-BHP-induced apoptosis, suggesting that caspase-9 plays a critical role in this process. Procaspase-9 underwent cleavage in mitochondria and translocation to the nucleus, where increased caspase-9 activity was detected. The caspase-9 substrates, caspase-3 and caspase-7, were not activated. Caspase-7 was translocated from the cytosol to the endoplasmic reticulum (ER), where it underwent processing; however, enzymatic activity of caspase-7 was inhibited by t-BHP. t-BHP caused cleavage of procaspase-12 at the ER and its subsequent translocation to the nucleus, where increased caspase-12 activity was found. t-BHP caused translocation of calpain from the cytosol to the ER. Calpain inhibition reduced chromatin condensation and caspase-12 activity in the nucleus, suggesting that calpain is involved in caspase-12 activation and apoptosis. This study demonstrates that caspase-9 and caspase-12 are activated in t-BHP-induced apoptosis in hepatocytes. We highlight the importance of subcellular compartments such as mitochondria, ER and nuclei in the apoptotic process.
Assuntos
Apoptose/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , terc-Butil Hidroperóxido/toxicidade , Animais , Transporte Biológico , Calpaína/efeitos dos fármacos , Calpaína/metabolismo , Caspase 12/efeitos dos fármacos , Caspase 12/metabolismo , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Caspase 9/efeitos dos fármacos , Caspase 9/metabolismo , Núcleo Celular/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Hepatócitos/metabolismo , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl(-)/HCO(3)(-) exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes.
Assuntos
Apoptose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Compostos de Trialquitina/toxicidade , Animais , Western Blotting , Sinalização do Cálcio/efeitos dos fármacos , Calpaína/metabolismo , Caspase 12/metabolismo , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Genes bcl-2 , Concentração de Íons de Hidrogênio , Masculino , Potenciais da Membrana/efeitos dos fármacos , Microscopia Confocal , Ratos , Ratos Sprague-Dawley , Vinculina/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Over the past decade, parathyroid hormone-related protein (PTHrP) has been identified as a key survival factor for cells subjected to apoptotic stimuli. Its anti-apoptotic activity has been attributed to nuclear accumulation of the intact protein, or a synthetic peptide corresponding to its nuclear targeting sequence (NTS), which promotes rapid exit of nutrient deprived cells from the cell cycle. Intracellular PTHrP also inhibited apoptosis by blocking tumor necrosis factor alpha (TNFalpha)-induced apoptosis by blocking signaling from the "death receptor" and preventing damage to the mitochondrial membrane. In both cases, the anti-apoptotic activity was significantly reduced in the presence of a nuclear deficient form of PTHrP with a (88)K/E K/E.K/I(91) mutation in the NTS. The current work was undertaken to determine the mechanism by which nuclear PTHrP blocked mitochondrial-mediated apoptosis. Using sub-cellular fractionation and functional assays we showed that pre-treatment of HEK293 cells with exogenous NTS peptide before inducing apoptosis with TNFalpha was as effective as expression of the full-length protein in inhibiting apoptosis. Inhibition of apoptosis was associated with increased expression of protein kinase casein kinase 2 (CK2) and in sustained CK2 accumulation and activity in the nuclear fraction. In primary chondrogenic cells harvested from the limb buds of PTHrP(+/-) and PTHrP(-/-) embryonic mice, there was a dose-dependent decrease in CK2 expression and activity that correlated with increased susceptibility to apoptosis. Taken together the results indicate that nuclear accumulation of PTHrP effectively inhibits mitochondrial-mediated apoptosis through regulation of the expression, activity, and sub-cellular trafficking of CK2.
Assuntos
Apoptose , Caseína Quinase II/metabolismo , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Caseína Quinase II/genética , Caspases/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Sobrevivência Celular , Condrócitos/metabolismo , Ativação Enzimática , Humanos , Botões de Extremidades/citologia , Botões de Extremidades/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteína Relacionada ao Hormônio Paratireóideo/deficiência , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Transporte Proteico , RNA Mensageiro/metabolismo , Frações Subcelulares , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Parathyroid hormone related protein (PTHrP) is expressed at low levels in many fetal and adult tissues where it plays a central role in regulating cell proliferation, cell death, and tissue homeostasis. In vivo and in vitro, PTHrP has been shown to promote the survival of a variety of cells by regulating expression of the anti-apoptotic protein Bcl2. Additional work has shown that intra-nuclear accumulation of PTHrP in CFK2 (PTH1R positive) and 27m21 (PTH1R negative) condrogenic cells promotes their survival by closing down ribosome biogenesis and promoting quiescence. The current studies were undertaken to examine the role of wild-type PTHrP and a mutant form that cannot translocate to the nucleus in protecting cells from TNFalpha-induced apoptosis. Both forms of the protein were equally effective in blocking the extrinsic pathway by inhibiting expression of the TNF receptor death domain, activating Bid, and promoting cleavage of caspase 8. These observations suggest a direct mechanism of PTHrP action on components of the extrinsic pathway, involving a region of the protein outside of the NTS. PTHrP and M1PTHrP also inhibited the intrinsic pathway by preventing the exchange of anti-apoptotic for pro-apoptotic proteins at the mitochondrial membrane, thus maintaining its integrity and preventing the release of caspase-activating factors into the cytosol. In general, this mitochondrial-related activity was somewhat delayed and was mediated more effectively by PTHrP than by M1PTHrP, suggesting an indirect mechanism of action that might require the presence of an intact NTS.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/efeitos dos fármacos , Caspase 8/metabolismo , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Mutação/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Receptores de Morte Celular/efeitos dos fármacos , Receptores de Morte Celular/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Hepatocytes are an important physiological model for evaluation of metabolic and biological effects of xenobiotics. They do not proliferate in culture and are extremely sensitive to damage during freezing and thawing, even after the addition of classical cryoprotectants. Thus improved cryopreservation techniques are needed to reduce cell injury and functional impairment. Here, we describe a new and efficient cryopreservation method, which permits long-term storage and recovery of large quantities of healthy cells that maintain high hepatospecific functions. In culture, the morphology of hepatocytes cryopreserved with wheat protein extracts (WPE) was similar to that of fresh cells. Furthermore, hepatospecific functions such as albumin secretion and biotransformation of ammonium to urea were well maintained during 4 days in culture. Inductions of CYP1A1 and CYP2B in hepatocytes cryopreserved with WPEs were similar to those in fresh hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for primary hepatocytes. The extract was also found to cryopreserve other human and animal cell types such as lung carcinoma, colorectal adenocarcinoma, Chinese hamster ovary transfected with TGF-b1 cDNA, cervical cancer taken from Henrietta Lacks, intestinal epithelium, and T cell leukemia. WPEs have potential as a universal cryopreservant agent of mammalian cells. It is an economic, efficient and non-toxic agent.
Assuntos
Linhagem Celular/fisiologia , Criopreservação/métodos , Crioprotetores/isolamento & purificação , Hepatócitos/citologia , Proteínas de Plantas/farmacologia , Animais , Células CHO , Linhagem Celular/citologia , Sobrevivência Celular , Cricetinae , Dimetil Sulfóxido/toxicidade , Indução Enzimática , Hepatócitos/fisiologia , Humanos , Masculino , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proteínas de Plantas/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Triticum/química , Células Tumorais CultivadasRESUMO
The kinetics of the early subcellular distribution of cadmium (Cd) was characterized in primary cultures of rat hepatocytes exposed to 10, 50 and 100 microM Cd in a serum-free WME medium for 10, 30 or 60 min. Our results demonstrate a time- and concentration-dependent increase in Cd content with the highest metal concentration measured in the cytosol, whereas the lowest was observed in the mitochondria. With the exception of early localization in the plasma membrane, Cd concentrations in fractions were characterized by the following decreasing order of magnitude: cytosol > low density molecules approximately nuclei > lysosomes approximately mitochondria. We also found evidence for: (i) a two-step process for Cd distribution in the nuclei and mitochondria; and (ii) a time-dependent 'slow' process of transfer from the plasma membrane to the cytosol. Saturation in Cd uptake was observed at 50 microM in most cell fractions at 10 and 30 min, except for the plasma membrane. The lack of apparent saturation for Cd accumulation at 60 min was not related to an increase in metallothionein synthesis. Altogether, our data provide insights into the dynamics of transfer between intracellular compartments, and allow a better identification of the organelles that are the most subjected to Cd toxicity for early exposure conditions.
Assuntos
Cádmio/metabolismo , Hepatócitos/metabolismo , Animais , Cádmio/farmacocinética , Cádmio/farmacologia , Cádmio/toxicidade , Células Cultivadas , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Cinética , Masculino , Metalotioneína/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Espectrofotometria Atômica , Fatores de TempoRESUMO
Tri-n-butyltin (TBT), a biocide, is known for its immunotoxicity and hepatotoxicity and is a well-characterised mitochondrial toxin. This report investigates the mechanisms involved in induction of apoptosis by TBT in primary cultures of rat hepatocytes. Release of cytochrome c from mitochondria into the cytosol was apparent after 15 min of exposure to 2.5 microM TBT. In addition, activity of initiator caspase-9 increased after 30 min, representing activation of the mitochondrial pathway in hepatocytes. The death receptor pathway was also activated by TBT, as indicated by recruitment of the adaptor protein FADD from the cytosol to the membrane as soon as 15 min after treatment. In addition, levels of the pro-apoptotic protein Bid decreased in the cytosol, while there was an increase in levels of the cleaved form tBid, in TBT-treated hepatocytes. Activity of initiator caspase-8 increased after 30 min. The principal effector caspase-3 was activated following 30 min of treatment with TBT. Activation was confirmed by immunodetection of a 17-kDa cleaved fragment. Apoptotic substrates such as Poly(ADP-ribose) polymerase and DNA fragmentation factor-45 are cleaved by caspase-3 to ensure the dismantlement of the cell. Cleavage of Poly(ADP-ribose) polymerase into a 85-kDa fragment appeared after 30 min of TBT treatment. DNA fragmentation factor-45 disappeared in TBT-exposed rat hepatocytes. This is the first detailed study reporting the involvement of initiator and effector caspases, cleavage of their intracellular substrates and activation of both death receptor and mitochondrial pathways in TBT-induced apoptosis in rat hepatocytes. The comprehension of molecular events of apoptosis is important for the evaluation of the risk to humans and animals.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Hepatócitos/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Compostos de Trialquitina/farmacologia , Animais , Proteína de Domínio de Morte Associada a Fas , Hepatócitos/citologia , Hepatócitos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The worldwide use of the organochlorine pesticide heptachlor has led to widespread contamination in the environment. Like many other organochlorine pesticides, heptachlor is considered to pose a threat to human health. It has been shown that heptachlor is a tumor-promoting agent, but the mechanisms involved still remain unclear. The negative response of heptachlor in in vitro genotoxicity test suggests that this pesticide displays its carcinogenicity through epigenetic pathways. With the growing evidence that proliferation accounts for the tumor-promoting effects of many agents, the purpose of this work was to investigate the mechanisms involved in the mitogenic activity of heptachlor in quiescent rat hepatocytes and to understand the properties of this compound as a tumor promoter in the liver. Heptachlor triggered significant proliferation in quiescent rat hepatocytes. Two mechanisms were delineated to support the mitogenic effect in the hepatocyte: activation of key kinases in signaling pathways and inhibition of apoptosis. Exposure to heptachlor led to activation of protein kinase C mitogenactivated protein kinases. Moreover, these results indicate that like many tumor promoters, heptachlor strongly inhibited TGFbeta-induced apoptosis and cytochrome c release into the cytosol. The levels of the anti-apoptotic protein Bcl-2 were also increased in the presence of heptachlor. In conclusion, these results indicate that heptachlor alters basic cell function by interfering with key cellular signaling pathways.
Assuntos
Apoptose/efeitos dos fármacos , DNA/biossíntese , Hepatócitos/efeitos dos fármacos , Heptacloro/toxicidade , Mitógenos/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Citosol/enzimologia , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Técnicas In Vitro , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Rat hepatocytes were seeded on three-dimensional highly porous polyvinylalcohol (PVA) and aminoethyl-modified polyvinylalcohol (AE-PVA) matrices. Hepatocytes were cultured under static and dynamic conditions. The three-dimensional matrices offered an improved extracellular microenvironment for long-term (5 days) maintenance of hepatocytes, compared to reference monolayer cultures on collagen. Cellular adhesion exceeded 80% with a viability superior to 70%. The preservation of albumin secretion after 5 days of culture was two times higher for static cultures on three-dimensional matrices (18% on PVA, 13% on AE-PVA) and three times higher for dynamic three-dimensional cultures (25% PVA and AE-PVA), compared to the static two-dimensional culture on collagen film (8%). The biotransformation of ammonia into urea was also maintained throughout the culture period. The addition of the aminoethyl function demonstrated no toxicity for the hepatocyte cultures. This function could be suitable eventually to further improve the hepatocyte culture system by linking more specific adhesion molecules on the polymer surface. This study demonstrated the efficiency of polyvinylalcohol as a three-dimensional matrix coupled to a perfusion culture system, which improves extracellular conditions for hepatocyte survival and promotes preservation of long-term hepatospecific functions.
Assuntos
Hepatócitos/citologia , Álcool de Polivinil/química , Animais , Adesão Celular , Técnicas de Cultura de Células , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Cadmium/Hg interactions have been studied in the TC7 clone of the enterocytic-like Caco-2 cells to test the hypothesis that these metals may compete for intestinal transport. Comparison of the kinetic parameter values for 203Hg(II) and 109Cd(II) uptake in a serum-free medium revealed that Hg is accumulated much more rapidly and to higher concentrations. The very rapid uptake/binding step and the initial uptake rate of 109Cd were both significantly inhibited by an excess of unlabeled Cd or Hg (apparent K(i) for Hg of 9.3 +/- 1.2 microM) without reciprocal effects. 109cadmium uptake was highly sensitive to temperature and a significant fraction of accumulation (12%) was EDTA extractable. 203Hg uptake remained insensitive to temperature or the EDTA washing procedure. However, the uptake of both tracers was half-decreased when an excess of the respective unlabeled metal was added in the stop solution, suggesting an exchange mechanism for adsorption. Cell pretreatment with N-ethylmaleimide (NEM) led to a 30% decrease or a 73% increase in the 3-min specific transport of 109Cd when NEM was still present in or removed from the uptake medium, respectively. NEM had no effect on 203Hg uptake. Overall our results suggest the involvement of a saturable specific mechanism for Cd, which is highly sensitive to inhibition by Hg and NEM under some conditions, and a nonspecific passive diffusion for Hg. The Hg- or NEM-induced inhibition of Cd uptake likely involves a thiol-mediated reaction, but our results suggest that NEM pretreatment may activate other cellular mechanisms leading to a stimulatory effect.
Assuntos
Cádmio/antagonistas & inibidores , Cádmio/metabolismo , Mercúrio/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Células CACO-2/efeitos dos fármacos , Células CACO-2/metabolismo , Cádmio/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , TemperaturaRESUMO
In many African countries, the economy largely depends on agriculture. Pesticides are therefore likely to represent an important source of xenoestrogens in contaminated rivers and lagoons. The largely uncontrolled use of diverse pesticides led us to hypothesize that these agents, and particularly organochlorine compounds, may pose a serious problem in the Republic of Benin. To verify our hypothesis, tilapia (Sarotherodon melanotheron) from five sites in the southern part of the main Ouémé River were analyzed. Ouémé River drains the southern region of the country. Vitellogenin (Vtg) was used as an indicator of contaminated sites. This approach has its limitations, because there are a wide variety of man-made chemicals present in the aquatic environment likely to induce Vtg in male fish. Therefore, in this study this approach allows us to define potential contaminated target sites. In order to determine whether the presence of Vtg could be attributable to pesticides, organochlorine pesticides in the flesh of tilapia were also analyzed. Significant amounts of Vtg in fish from contaminated sites were detected, and were correlated with organochlorine pesticide levels in tissue. These results indicate that organochlorine pesticides are present in the Ouémé River and that these compounds can act as endocrine modulators in this ecosystem. Eating fish from contaminated rivers, such as the Ouémé River, may contribute to the accumulation of high concentrations of these pesticides in the body, leading to exposure to their negative effects.
Assuntos
Exposição Ambiental , Hidrocarbonetos Clorados , Inseticidas/efeitos adversos , Tilápia/fisiologia , Vitelogeninas/biossíntese , Poluentes Químicos da Água/efeitos adversos , Animais , Benin , Sistema Endócrino/efeitos dos fármacos , Inseticidas/farmacocinética , Masculino , Distribuição Tecidual , Poluentes Químicos da Água/farmacocinéticaRESUMO
Many organochlorine pesticides are suspected of impairing natural hormonal function in organisms by mimicking endogenous estrogen. The aim of this study was to assess the estrogenic activity of the organochlorine pesticides o,p'-DDT, dieldrin, aldrin, heptachlor, mirex and DDT in rainbow trout hepatocyte cultures using vitellogenin (Vtg) as the biomarker. A wide range of pesticide concentrations (0.0001-100 microM) was evaluated. Among the pesticides tested, o,p'-DDT was the most potent inducer of Vtg. The lower potency of technical grade DDT relative to o,p'-DDT could be explained by the fact that this pesticide is a mixture of two different pesticides (18% o,p'-DDT and 77% p,p'-DDT). This suggests that o,p'-DDT is a stronger inducer of Vtg than p,p'-DDT. A simple hypothesis could be that pesticides mixed together competed for the same receptor to favor the formation of a complex with reduced activity towards EREs. If these compounds are classified according to the level of Vtg secreted, we observed the following decreasing order: 17beta-estradiol (E(2))>o,p'-DDT>dieldrin>aldrin>DDT. Non-toxic levels of these compounds competed with E(2) for binding to the estrogen receptor. Heptachlor and mirex did not induce Vtg. Since the latter compounds failed to stimulate Vtg production, the possibility that they could interfere with the estrogenic response by inhibiting E(2) action was tested. In the presence of heptachlor, Vtg production triggered by E(2) significantly decreased. The EC50 value for inhibition of ER binding by heptachlor was cytotoxic for hepatocytes in culture, and this could in part explain the lack of Vtg response observed with this compound at the concentrations tested. Our results indicate that organochlorine pesticides can act as positive or negative modulators of estrogenic function in rainbow trout.
Assuntos
Estradiol/farmacologia , Hidrocarbonetos Clorados , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Vitelogeninas/biossíntese , Animais , Ligação Competitiva , Células Cultivadas , Relação Dose-Resposta a Droga , Estradiol/metabolismo , Inseticidas/metabolismo , Fígado/metabolismo , Masculino , Oncorhynchus mykiss , Receptores de Estrogênio/metabolismoRESUMO
The involvement of reduction/oxidation (redox) state in cell sensitivity to apoptosis has been suggested by several studies in which induction of apoptosis was shown to require oxidative stress or GSH extrusion. On the other hand, biochemical studies of caspases revealed that their activation necessitates a reduced cysteine in their active site. This is ensured by maintaining intact intracellular glutathione status during apoptotic induction as reported by in vivo studies. Therefore, we investigated the relationship between intracellular glutathione levels and the sensitivity of mouse hepatocytes in culture to Fas-induced apoptosis as well as potential mechanisms responsible for this sensitivity. We found that total and reduced glutathione levels are decreased by one-half after cell isolation procedure and further decline by 25% during cell culture for 2 h in normal Williams' E medium. Cell culture in medium supplemented with cysteine and methionine maintains glutathione at a level similar to that measured just after cell isolation. Results show that the capacity of Fas to activate caspase-8 and to induce apoptosis requires important intracellular glutathione levels and high GSH/total glutathione ratio. In conclusion, the present study shows that intracellular glutathione plays an important role in maintaining the apoptotic machinery functional and is thus capable of transmitting the apoptotic signal.
Assuntos
Apoptose/fisiologia , Glutationa/deficiência , Hepatócitos/fisiologia , Receptor fas/fisiologia , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/metabolismo , Caspase 8 , Caspase 9 , Caspases/metabolismo , Morte Celular/fisiologia , Separação Celular , Células Cultivadas , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Cisteína/farmacologia , Combinação de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Glutationa/metabolismo , Hepatócitos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Metionina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Proteína bcl-XRESUMO
The purpose of the present work was to study the mechanisms involved in apoptosis induced by oxidative stress in rat hepatocytes. We focused on the apoptotic signaling molecules cytochrome c, Bcl-2 and Bax. Rat hepatocytes were exposed for 1 h to increasing concentrations of tert-butylhydroperoxide (t-BHP). Using lactate dehydrogenase (LDH) leakage as a biomarker for necrosis, and DNA fragmentation as a biomarker for apoptosis, we observed that a concentration of t-BHP of 0.4-0.5 mM provides a transition point below which apoptosis is favored and beyond which necrosis is favored. Malondialdehyde and 8-oxo-guanine formation indicates that t-BHP induces oxidative stress and damage. However, at 0.4 mM t-BHP, these oxidative molecular changes as well as LDH leakage no longer progress after the first hour of t-BHP exposure, suggesting the activation of some defense mechanisms. Western blot analysis of cytochrome c shows that its level increases in the cytosol while that of Bax decreases in this fraction as a result of t-BHP treatment. Moreover, there is a loss of Bcl-2 from mitochondria while, in contrast, Bax accumulates in this organelle following t-BHP treatment. However, cytochrome c appears to be relocalized to the endoplasmic reticulum as its presence in microsomes is greatly enhanced. We suggest that t-BHP triggers apoptosis through a step that involves cytochrome c release from mitochondria. This event is stimulated by Bcl-2 disappearance from mitochondria and Bax recruitment. Neutralization of excess cytosolic cytochrome c is achieved by its relocalization to the endoplasmic reticulum, hence triggering the down-regulation of apoptotic signals.