RESUMEN
4-Hydroxynonenal (HNE) is produced from arachidonic acid or linoleic acid during oxidative stress. Although HNE is formed in tissues as a racemate, enantiospecific HNE effects have not been widely documented, nor considered. Therefore, a panel of cellular responses was compared after treatment with (R)-HNE, (S)-HNE, or racemic HNE. The phosphorylation status of Jun kinase (JNK) or Akt increased 28-fold or 2-3-fold, respectively, after treatment with 100 µM (S)-HNE and racemic HNE compared to (R)-HNE. In contrast, the increase in phosphorylation of MAPK was greatest for (R)-HNE. Caspase-3-dependent cleavage of the glutamate cysteine ligase (GCL) catalytic subunit and focal adhesion kinase (FAK) were greater in cells treated with (S)-HNE at 48 h. (S)-HNE also caused a greater number of subG1 nuclei, a hallmark of apoptosis, at 30 h after treatment. Together, the results demonstrate different dose- and time-dependent responses to (R)-HNE and (S)-HNE. The results further suggest that HNE enantiomers could differentially contribute to the progression of different diseases or contribute by different mechanisms.
Asunto(s)
Aldehídos/toxicidad , Supervivencia Celular/efectos de los fármacos , Hepatocitos/enzimología , Aldehídos/química , Animales , Caspasa 3/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Hepatocitos/citología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , EstereoisomerismoRESUMEN
The polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants whose residues are increasing in fish, wildlife and human tissues. However, relatively little is known regarding the mechanisms of cell injury caused by PBDE congeners in fish. In the present study, we employed flow cytometry-based analyses to understand the onset and mechanisms of cell injury in rainbow trout gill cells (RTgill-W1 cells) exposed to 2,2',4,4'-tetrabromodiphenyl ether (BDE 47). Substantial optimization and validation for flow cytometry protocols were required during assay development for the trout gill cell line. Exposure to micromolar concentrations of BDE 47 elicited a significant loss in RTgill-W1 cell viability that was accompanied by a decrease in NAD(P)H autofluorescence, a marker associated with disruption of cellular redox status. This loss in NAD(P)H content was accompanied by a decrease in nonyl acridine orange fluorescence, indicating mitochondrial membrane lipid peroxidation. Furthermore, low doses of BDE 47 altered cellular forward angle light scatter (FS, a measure of cell diameter or size) and side light scatter properties (SS, a measure of cellular internal complexity), consistent with the early stages of apoptosis. These changes were more pronounced at higher BDE 47 concentrations, which led to an increase in the percentage of cells undergoing frank apoptosis as evidenced by sub-G1 DNA content. Apoptosis was also observed at a relatively low dose (3.2muM) of BDE 47 if cells were exposed for an extended period of time (24h). Collectively, the results of these studies indicate that exposure of rainbow trout gill cells to BDE47 is associated with the induction of apoptosis likely originating from disruption of cellular redox status and mitochondrial oxidative injury. The current report extends observations in other species demonstrating that oxidative stress is an important mechanism of BDE 47 mediated cellular toxicity, and supports the use of oxidative stress-associated biomarkers in assessing the sublethal effects of PBDEs and their replacements in fish. The application of flow cytometry endpoints using fish cell lines should facilitate study of the mechanisms of chemical injury in aquatic species.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Oncorhynchus mykiss/fisiología , Bifenilos Polibrominados/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Branquias/citología , Branquias/efectos de los fármacos , Éteres Difenilos Halogenados , Mitocondrias/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacosRESUMEN
The polybrominated diphenyl ethers (PBDEs) are a group of flame retardants whose residues have markedly increased in the environment and in human tissues during the last decade. Of the various congeners, BDE 47 (2,2',4,4'-tetrabromodiphenyl ether) is typically the predominant congener observed in fish and wildlife samples, as well as in human tissues. Several studies indicate in utero transfer of PBDEs during pregnancy with residues accumulating in fetal tissues, and thus the potential for BDE 47-mediated injury in utero is of concern. In this study, we examined the mechanisms of BDE 47-mediated injury to primary human fetal liver hematopoietic stem cells (HSCs), which comprise a large proportion of fetal hepatic cells and play a key role in hematopoiesis during fetal development. Incubation of fetal liver HSCs with BDE 47 led to a loss of mitochondrial membrane potential and the onset of apoptosis. These effects were observed in the low micromolar range of BDE 47 exposures. At higher concentrations, BDE 47 elicited a loss of viability, which was accompanied by the generation of reactive oxygen species and peroxidation of HSC lipids. Preincubation of fetal liver HSCs with N-acetylcysteine, a glutathione (GSH) precursor, caused an increase in cellular GSH concentrations, restored mitochondrial redox status, and ameliorated the toxicity of BDE 47. BDE 47-mediated cytotoxicity or oxidative injury was not evident at the lower concentrations (< 1microM). Collectively, these data support a role for oxidative stress in the cytotoxicity of BDE 47 and indicate that oxidative stress-associated biomarkers may be useful in assessing the sublethal effects of BDE 47 toxicity in other models. However, the fact that BDE 47 undergoes a concentration-dependent accumulation in other primary cells in media that can underestimate cellular concentrations (W. R. Mundy et al., 2004, Toxicol. Sci. 82, 164-169) suggests that the HSC cell injury observed in our study may be of less relevance to human in utero PBDE exposures.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Hígado/citología , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/fisiología , Bifenilos Polibrominados/toxicidad , Acetilcisteína/farmacología , Anexina A5/metabolismo , Antígenos CD34/metabolismo , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Depuradores de Radicales Libres/farmacología , Éteres Difenilos Halogenados , Células Madre Hematopoyéticas/patología , Humanos , Peroxidación de Lípido/efectos de los fármacos , Hígado/patología , Especies Reactivas de OxígenoRESUMEN
Hydrogen peroxide (H2O2) can cause single strand DNA breaks (ssDNA) in cells when the mechanisms normally in place to reduce it are overwhelmed. Such mechanisms include catalase, glutathione peroxidases (GPx), and peroxiredoxins. The relative importance of these enzymes in H2O2 reduction varies with cell and tissue type. The role of the GPx cofactor glutathione (GSH) in oxidative defense can be further understood by modulating its synthesis. The first and rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which has a catalytic subunit (Gclc) and a modifier subunit (Gclm). Using mouse hepatoma cells we evaluated the effects of GCL over expression on H2O2-induced changes in GSH and ssDNA break formation with the single cell gel electrophoresis assay (SCG or comet assay), and the acridine orange DNA unwinding flow cytometry assay (AO unwinding assay). Cells over expressing GCL had higher GSH content than control cells, and both SCG and AO unwinding assays revealed that cells over expressing GCL were significantly more resistant to H2O2-induced ssDNA break formation. Furthermore, using the AO unwinding assay, the prevalence of H2O2-induced breaks in different phases of the cell cycle was not different, and the degree of protection afforded by GCL over expression was also not cell cycle phase dependent. Our results support the hypothesis that GCL over expression enhanced GSH biosynthesis and protected cells from H2O2-induced DNA breaks. These results also suggest that genetic polymorphisms that affect GCL expression may be important determinants of oxidative DNA damage and cancer.
Asunto(s)
Roturas del ADN de Cadena Simple , Citometría de Flujo , Glutamato-Cisteína Ligasa/metabolismo , Peróxido de Hidrógeno/toxicidad , Animales , Línea Celular , Ensayo Cometa , Glutamato-Cisteína Ligasa/aislamiento & purificación , Ratones , Estrés OxidativoRESUMEN
Acetaminophen overdose is a leading cause of drug-related acute liver failure in the United States. Glutathione, a tripeptide antioxidant protects cells against oxidative damage from reactive oxygen species and plays a crucial role in the detoxification of xenobiotics, including acetaminophen. Glutathione is synthesized in a two-step enzymatic reaction. Glutamate-cysteine ligase carries out the rate-limiting and first step in glutathione synthesis. We have generated C57Bl/6 mice that conditionally overexpress glutamate-cysteine ligase, and report here their resistance to acetaminophen-induced liver injury. Indices of liver injury included histopathology and serum alanine aminotransferase activity. Male transgenic mice induced to overexpress glutamate-cysteine ligase exhibited resistance to acetaminophen-induced liver injury when compared with acetaminophen-treated male mice carrying, but not expressing glutamate-cysteine ligase transgenes, or to female glutamate-cysteine ligase transgenic mice. We conclude that glutamate-cysteine ligase activity is an important factor in determining acetaminophen-induced liver injury in C57Bl/6 male mice. Because people are known to vary in their glutamate-cysteine ligase activity, this enzyme may also be an important determinant of sensitivity to acetaminophen-induced liver injury in humans.
Asunto(s)
Acetaminofén/toxicidad , Glutamato-Cisteína Ligasa/genética , Hígado/lesiones , Alanina Transaminasa/sangre , Animales , Antioxidantes/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mifepristona/farmacología , Modelos Genéticos , Estrés Oxidativo , TransgenesRESUMEN
Photodynamic therapy (PDT) relies on light-dependent, tissue-targeted, oxidative stress in tumors that have accumulated a photosensitizing drug. Glutathione S-transferases (GSTs) are often up-regulated in tumors and they modulate oxidative stress by several isoform-dependent mechanisms. GSTs, therefore, are potential confounding factors in PDT. Therefore, we examined this possibility in human kidney 293 cells transfected with a plasmid encoding either green fluorescent protein alone (pIRES-GFP) or both GFP and GSTP1-1 (pIRES-GFP-GSTP). Cells were cultured and treated with light alone, the sensitizer hypericin (HYP) alone, or light and HYP. Cells harboring pIRES-GFP-GSTP exhibited a modest 2-fold increase in GSTP1-1 expression over control cells. On the basis of flow cytometry and microscopy, the light-dependent toxicity of HYP was reduced in cells over-expressing GSTP1-1. Paradoxically, the decreased toxicity in the cells with GSTP1-1 over-expression occurred concomitantly with a modest approximately 2-fold increase in cellular uptake of the drug. Immunoprecipitation of HYP and Western analysis indicated that GSTP1-1 is a major intracellular-binding site for HYP. These results are the first to demonstrate GST expression as a confounding variable of photodynamic therapy. Further, a high-affinity GST inhibitor reversed the GSTP1-1-dependent resistance, suggesting the possible utility of pharmacological strategies to optimize PDT.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Glutatión Transferasa/metabolismo , Riñón/efectos de los fármacos , Riñón/enzimología , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Antracenos , Línea Celular , Glutatión Transferasa/genética , Humanos , Riñón/citología , Riñón/efectos de la radiación , Luz , Perileno/administración & dosificación , Resultado del TratamientoRESUMEN
Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450(eryF). UV/vis absorbance spectra of P450(eryF) demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of K(S) = 4 and 200 microM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450(eryF) with 9-AP and TST indicated binding on intermediate and fast chemical exchange time scales, respectively, which was consistent with the Hill-equation-derived K(S) values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands "contact" the same phenylalanines within the active site of P450(eryF). This finding is in agreement with X-ray crystal structures of bound P450(eryF) showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450(eryF) conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.
Asunto(s)
Bacterias/enzimología , Sistema Enzimático del Citocromo P-450/química , Sitios de Unión , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/metabolismo , Eritromicina/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fenantrenos/química , Fenantrenos/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Espectrofotometría Ultravioleta , Testosterona/química , Testosterona/metabolismo , TermodinámicaRESUMEN
Bacterial glutamine synthetases (GSs) are dodecameric aggregates comprised of two face-to-face hexameric rings, which form a cylindrical aqueous channel. Available crystal structures indicate that each subunit provides a 'central loop' that protrudes into this channel. Residues on either side of this loop contribute directly to substrate or metal ion cofactor binding. Although it has been suggested that this conspicuous structural feature may be functionally important, a systematic structure-function analysis of this loop has not been done. Here, we examine the behavior of a cysteine mutant, E165C, which yields inter-subunit disulfide bonds connecting the central loops. The inter-subunit disulfide bonds are readily detected by electrospray ionization mass spectrometry. Based on molecular models, the disulfide bonds would form only if the engineered cysteines on adjacent subunits moved approximately 5 A. Surprisingly, inter-subunit disulfide bonds between the central loops caused no detectable changes in the KMs for glutamate or ATP, nor the KD for either ATP or the transition state analog (L)-methionine sulfoximine (MSOX). Furthermore, covalent and quantitative adduction of the E165C mutant with iodo-acetamido-pyrene yielded nearly fully active enzyme bearing fluorescent pyrene excimers. The relative contribution of pyrene monomers to excimers in the steady state fluorescence is temperature dependent, suggesting thermal equilibrium between loop conformational states. However, the monomer-excimer ratio is independent of ligands such as MSOX, glutamate, or Mn2+. These results validate the suspected flexibility of the central loop, but raise significant doubt about its direct functional role in GS catalysis via conformational switching, including the proposed regulation of GS via ADP-ribosylation within this loop.
Asunto(s)
Escherichia coli/enzimología , Glutamato-Amoníaco Ligasa/química , Adenosina Difosfato/química , Adenosina Trifosfato/química , Catálisis , Disulfuros/química , Ácido Glutámico/química , Cinética , Magnesio/química , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Pirenos/química , Ribosa/química , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray , TemperaturaRESUMEN
Glutathione (GSH) is important in free radical scavenging, maintaining cellular redox status, and regulating cell survival in response to a wide variety of toxicants. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which is composed of catalytic (GCLC) and modifier (GCLM) subunits. To determine whether increased GSH biosynthetic capacity enhances cellular resistance to tumor necrosis factor-alpha- (TNF-alpha-) induced apoptotic cell death, we have established several mouse liver hepatoma (Hepa-1) cell lines overexpressing GCLC and/or GCLM. Cells overexpressing GCLC alone exhibit modest increases in GCL activity, while cells overexpressing both subunits have large increases in GCL activity. Importantly, cells overexpressing both GCL subunits exhibit increased resistance to TNF-induced apoptosis as judged by a loss of redox potential; mitochondrial membrane potential; translocation of cytochrome c to the cytoplasm; and activation of caspase-3, caspase-8, and caspase-9. Analysis of the effects of TNF on these parameters indicates that maintaining mitochondrial integrity mediates this protective effect in GCL-overexpressing cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Glutamato-Cisteína Ligasa/metabolismo , Mitocondrias/patología , Factor de Necrosis Tumoral alfa/toxicidad , Animales , Carcinoma Hepatocelular , Línea Celular Tumoral , Glutatión/metabolismo , Humanos , Neoplasias Hepáticas , Ratones , Mitocondrias/efectos de los fármacos , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates.