RESUMO
Conjugated linoleic acid (CLA; 18:2) is a group of isomers (mainly 9-cis, 11-trans and 10-trans, 12-cis) of linoleic acid. CLA is the product of rumen fermentation and can be found in the milk and muscle of ruminants. Animals fed CLA have a lower body fat content. The objective of this study was to establish the possible mechanisms by which CLA affects adipogenesis. 3T3-L1 is a well-established cell line that is used extensively in studying adipocyte biology. These cells typically grow in a culture medium until they reach confluence, at which time they are induced to differentiate by hormonal treatment (d 0). Treatment of 3T3-L1 cells with 25 to 100 micromol/L CLA inhibited differentiation in a dose-dependent manner, while linoleic acid treatment did not differ from DMSO-treated controls. Continuous treatment from d -2, -1, 0 or 2 to d 8 and treatment from d -2 to d 0 and from d 0 to d 2 inhibited differentiation. Differentiation was monitored morphologically (oil Red-O staining), enzymatically (reduction of activity of glycerol-3-phosphate dehydrogenase), and by northern analysis of peroxisome proliferator-activated receptor gamma2, CCAAT/enhancer binding protein alpha and adipocyte specific protein 2 mRNA. CLA inhibited cell proliferation of nonconfluent cells but did not affect cell division of confluent cells, as indicated by 5-bromo-2'-deoxyuridine incorporation and mitochondria metabolism. Therefore, CLA inhibited differentiation before confluence and during induction. However, cellular proliferation was only inhibited prior to induction. These results imply that fat reduction caused by CLA treatment may be attributed to its inhibition of both proliferation and differentiation of preadipocytes in animals.
Assuntos
Adipócitos/citologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ácido Linoleico/farmacologia , Células-Tronco/citologia , Células 3T3 , Adipócitos/efeitos dos fármacos , Animais , Compostos Azo , Proteínas Estimuladoras de Ligação a CCAAT , Corantes , Meios de Cultura , Proteínas de Ligação a DNA/genética , Dimetil Sulfóxido/farmacologia , Glicerolfosfato Desidrogenase/metabolismo , Camundongos , Proteínas Nucleares/genética , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/genética , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
The differentiation of 3T3-L1 preadipocytes is induced by the coordinate activation of trans-acting factors in response to inducers. Depending on the time of treatment, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was effective in inhibiting 3T3-L1 preadipocyte differentiation and the expression of differentiation-dependent trans-acting factors. Based on glycerol-3-phosphate dehydrogenase activity, in the differentiation of 3T3-L1 cells was decreased by 70% in cells treated with TCDD before the induction of differentiation, 25% during induction, and not at all after induction. This time-dependent inhibition of cell differentiation by TCDD was correlated with the levels of aryl hydrocarbon receptor (AhR). TCDD treatment decreased the mRNA levels of C/EBP alpha and PPAR gamma 2 but did not affect the mRNA levels of RXR alpha and RAR alpha. Furthermore, TCDD did not change the mRNA or protein levels of C/EBP beta, which is thought to play a role in inducing C/EBP alpha and PPAR gamma 2 expression. These results suggest that TCDD inhibited 3T3-L1 preadipocyte differentiation through the AhR pathway, and the change of C/EBP beta mRNA and protein was not involved in reducing mRNA expression of C/EBP alpha and PPAR gamma 2.
Assuntos
Adipócitos/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Dibenzodioxinas Policloradas/farmacologia , Células-Tronco/efeitos dos fármacos , Teratogênicos/farmacologia , Células 3T3 , Adipócitos/química , Adipócitos/citologia , Animais , Northern Blotting , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Camundongos , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/análise , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Células-Tronco/química , Células-Tronco/citologia , Fatores de Tempo , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Understanding the differentiation pathway of adipocytes is an important first step for controlling human and animal fat deposition. Although many studies have been done on adipogenesis, most have utilized established cell lines rather than isolated primary cells. We have studied primary preadipocyte differentiation to determine whether the cell lines reflect the situation in vivo. In this study, mRNA of several transcription factors and adipocyte-related enzymes, isolated from cultured differentiating primary rat inguinal and epididymal cells, followed the same pattern of change during differentiation as seen in differentiating 3T3-L1 cells. As the cells differentiated, mRNA for C/EBPalpha, PPARgamma2, aP2 and lipoprotein lipase (LPL) increased, C/EBPbeta decreased and CHOP remained at a low level. Previously we have shown that in vivo treatment with TCDD (2,3,7,8- tetrachlorodibenzo-p-dioxin) inhibits in vitro adipogenesis and the increase of mRNAs for glycerol-3-phosphate dehydrogenase and LPL (Tox. Lett. 84:55, 1996). TCDD treatment in vivo also inhibited the increase of mRNA for the PPARgamma2, aP2 and C/EBPbeta during differentiation of the isolated preadipocytes. C/EBPbeta and CHOP mRNAs were unaffected. Due to the similarity of changes of the transcription factor mRNAs for primary and 3T3-LI cells during differentiation and after TCDD treatment, 3T3-L1 cells appear to provide a good model for more clearly defining the route of adipogenesis and TCDD inhibition.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Dibenzodioxinas Policloradas/toxicidade , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Fatores de Transcrição/genética , Células 3T3 , Adipócitos/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossínteseRESUMO
Inhibition of preadipocyte differentiation by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) or retinoic acid (RA) identified another transcription factor which appears to be important for preadipocyte differentiation. Within 15 min of treating 3T3-L1 cells with TCDD, the aryl hydrocarbon receptor (AhR) is present within the cell nucleus, and increased binding of COUP-TF to an oligomer of the PPAR gamma 2/RXR binding sequence (ARE7) occurs. Following 2 days of RA treatment, increased binding of COUP-TF to the ARE7 oligomer also occurs. In untreated preadipocytes, COUP-TF mRNA increased at confluence and then decreased after induction. TCDD treatment did not alter COUP-TF mRNA changes. Dephosphorylating the nuclear extracts from TCDD and RA-treated cells eliminated binding of COUP-TF to ARE7. This is the first indication that COUP-TF may play a role in preadipocyte differentiation and that COUP-TF binding to DNA is correlated with TCDD and RA-induced phosphorylation.
Assuntos
Adipócitos/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Adipócitos/citologia , Animais , Fator I de Transcrição COUP , Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Camundongos , Dibenzodioxinas Policloradas/farmacologia , Ligação Proteica , Receptores X de Retinoides , Fatores de Transcrição/química , Tretinoína/farmacologiaRESUMO
Fat and liver are the major sites for the deposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) given in vivo to rats. Although a great deal of information is available on the effects of TCDD in liver, very little is known of the effects in fat. The epididymal fat pads were removed and the stromal-vascular cells, released by collagenase digestion, were put into primary culture, 2, 4, 6 and 8 days after intubating 175 micrograms/kg TCDD into male Sprague-Dawley rats. Following 7 days in culture, the cells were examined morphologically, and assayed for an early (lipoprotein lipase, LPL) and late marker of fat cell differentiation (glycerol-3-phosphate dehydrogenase, GPDH). With rats sacrificed 6 or 8 days after TCCD intubation, the harvested cells from pair-fed rats contained significantly more fat and had a significantly higher level of GPDH enzyme activity, indicating more differentiation. The mRNA for LPL and GPDH genes was also higher for cells from pair-fed rats. In addition, for the rats that were sacrificed 4-8 days after TCDD intubation, despite similar food intake, the pair-fed control rats gained more total body weight than the treated rats. Although there was a body weight difference, there was no significant different between the weights of the epididymal fat pads. This is the first report to demonstrate that TCDD inhibits the differentiation of fat cells.
Assuntos
Adipócitos/citologia , Dibenzodioxinas Policloradas/toxicidade , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Animais , Northern Blotting , Peso Corporal/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Epididimo , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Masculino , Dibenzodioxinas Policloradas/administração & dosagem , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
Stromal-vascular (S-V) cells isolated from adipose tissue of newborn pigs (NBPC) and mature pigs (MPC) by collagenase digestion were used to evaluate differences in preadipocyte culture and development. Cells were seeded at a density of 3 x 10(4) cells/cm2 on six-well (35-mm) tissue culture plates in 3 mL of DMEM/HAM's F12 medium plus 10% fetal calf serum and cultured at 37 degrees C under a humidified atmosphere of 95% air:5% CO2 for 24 h. Cells were then washed thoroughly in DMEM/HAM's F12 medium without fetal calf serum and maintained in serum free (SF) medium or SF medium supplemented with 2.5% newborn pig serum (NBPS) or mature pig serum (MPS) for 12 d. After 1 d, more NBPC adhered to the culture plates, as indicated by DNA values. After 12 d, protein per culture well was not significantly different, but DNA concentration per well remained higher (P < .05) in cultures of NBPC than in the MPC cultured in the same medium, indicating fewer MPC. Protein:DNA ratios were higher (P < .05) in cultures of MPC regardless of the medium, reflecting larger cell size. More cells containing fat deposits were seen with NBPC in all conditions in comparison with MPC, and more fat was deposited in NBPC in SF than in SF plus NBPS or MPS. The NBPC had higher (P < .05) sn-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) per protein than MPC regardless of the medium. For both cell types, GPDH activity in either serum was less than activity of cells grown in SF.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adipócitos/citologia , Envelhecimento/fisiologia , Adipócitos/química , Adipócitos/fisiologia , Animais , Contagem de Células/veterinária , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , DNA/análise , Masculino , Células Estromais/química , Células Estromais/citologia , Células Estromais/fisiologiaRESUMO
Weanling male Sprague-Dawley rats were fed diets for four weeks which differed in their content of n-6 (corn oil; CO) and n-3 fatty acids (fish oil; FO), but were similar in their content of saturated and monounsaturated fatty acids and vitamin E. At the end of the four-week feeding period, each dietary group was subdivided into two groups. One group received a single placebo injection of alpha-tocopherol-stripped corn oil (TSCO); the other group received a single injection of the free radical generator; methyl ethyl ketone peroxide (MEKP), in TSCO. Twenty-four hours after injection, the effect of dietary oil and MEKP treatment on endogenous lipid peroxide (LPO) production (measured as methylene blue formed by the "Determiner LPO" assay), glutathione (GSH) and vitamin E content, and fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in heart and liver from unfasted animals were measured. FO-fed rats had significantly heavier hearts and livers, increased levels of n-3 fatty acids in membrane phospholipids, and higher liver LPO levels than CO-fed rats. MEKP treatment resulted in significantly lower body weights and liver GSH levels. The data indicate that dietary n-3 fatty acids increase lipid peroxidation in liver somewhat more than in heart. The study also demonstrates that the effect of induced oxidative stress due to a single dose of MEKP on lipid peroxide formation and antioxidant status in tissues from unfasted animals was independent of the dietary oils.
Assuntos
Antioxidantes/metabolismo , Butanonas/farmacologia , Óleo de Milho/farmacologia , Gorduras na Dieta , Óleos de Peixe/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Miocárdio/metabolismo , Peróxidos/farmacologia , Animais , Ácidos Graxos/análise , Ácidos Graxos Monoinsaturados/análise , Ácidos Graxos Monoinsaturados/farmacologia , Glutationa/metabolismo , Coração/anatomia & histologia , Coração/efeitos dos fármacos , Peróxidos Lipídicos/metabolismo , Fígado/anatomia & histologia , Fígado/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Vitamina E/metabolismo , Vitamina E/farmacologia , Aumento de Peso/efeitos dos fármacosRESUMO
Tissue disruption methods were developed and serum-free cell culture media formulated for the maintenance in vitro of cells from juvenile worms (day 18 after infection) of Schistosoma mansoni. Cultures maintained viability for up to 6 mo when plated on a feeder layer of irradiated rat liver cells and survived primarily as clusters of small (2.5-4 microns diameter) cells with a high nuclear-to-cytoplasmic ratio and relatively few organelles identified by electron microscopy. Cultures synthesized a protein profile similar to that of intact worms, and the cell clusters maintained a time- and concentration-dependent contractile response to serotonin. Cells synthesizing DNA were detected by precursor incorporation and flow cytometry in cultures initially and also after several weeks in vitro, although the percentage of cells synthesizing DNA decreased with time. Efforts to identify peptide growth factor-responsive tyrosine phosphorylation were negative, and the overall amount of S. mansoni phosphotyrosine-containing proteins identified by western blot with anti-phosphotyrosine monoclonal antibody was much less than that found in a peptide growth factor-responsive mouse cell line.
Assuntos
Schistosoma mansoni/crescimento & desenvolvimento , Animais , Western Blotting , Adesão Celular , Linhagem Celular , Meios de Cultura Livres de Soro , DNA/biossíntese , Fator de Crescimento Epidérmico/farmacologia , Citometria de Fluxo , Proteínas de Helminto/biossíntese , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Microscopia Eletrônica , Fosforilação/efeitos dos fármacos , Ratos , Schistosoma mansoni/citologia , Schistosoma mansoni/ultraestruturaRESUMO
Treatment of isolated mitochondria from rat hepatoma tumor cells (AS-30D) with the oxidant, t-butyl hydroperoxide (tBuOOH, 1 or 5 mumol/ml) resulted in the oxidation of glutathione (GSH to GSSG) and the formation of protein-glutathione mixed disulfides (ProSSG). The GSSG was retained inside of the hepatoma mitochondria. In the presence of ADP+succinate (5 or 10 mM), or ketoglutarate (10 mM) or malate (5 mM), the GSSG was reduced to GSH, but the amount of ProSSG stayed constant. With saline or ADP+glutamate (10 mM)/malate (0.1 mm) no reduction of GSSG to GSH occurred. The presence of antimycin (5 micrograms/ml) with ADP+succinate inhibited reduction. At a concentration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 0.5 mM) which inhibited a major portion of the glutathione reductase activity, the reduction of GSSG to replenish GSH was also inhibited. NADPH may play a critical role as well, for the addition of 2.4 mM NADPH to permeabilized hepatoma mitochondria fostered the reduction of GSSG after tBuOOH treatment. Therefore, hepatoma mitochondria possess a glutathione reductase-dependent system to reduce GSSG to GSH. The reaction only occurs with actively respiring mitochondria.
Assuntos
Glutationa/análogos & derivados , Neoplasias Hepáticas Experimentais/metabolismo , Mitocôndrias Hepáticas/metabolismo , Peróxidos/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Carmustina/farmacologia , Glutationa/metabolismo , Dissulfeto de Glutationa , Glutationa Redutase/metabolismo , Ácidos Cetoglutáricos/farmacologia , Malatos/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , NADP/farmacologia , Oxirredução , Ratos , Ratos Sprague-Dawley , Succinatos/farmacologia , Ácido Succínico , terc-Butil HidroperóxidoRESUMO
Cultured human lung carcinoma cells (A549) were incubated in a calcium-free medium containing calcium chelators (EGTA, 1-10 mM or BAPTA, 5 mM) for 1 hour at 37 degrees C. With limited toxicity, the presence of calcium chelators resulted in a decrease of cellular GSH and detachment of the cells from the tissue culture flask. The permeable EGTA tetraacetoxymethyl ester (0.5mM-5 mM) caused a decrease in the cellular GSH content without cell detachment. GSH was not oxidized to GSSG nor formed mixed disulfides with protein thiols. AT-125, a gamma-glutamyl transpeptidase inhibitor, prevented detachment, but not the efflux of cellular GSH. Pretreatment with two impermeable compounds (ruthenium red, 100 microM and neomycin, 0.5-10 mM) protected the cells from detachment and prevented the decrease in intracellular GSH. The presence of calcium in the medium during the EGTA and BAPTA treatments also protected the cells. Calcium associated with the cytoplasmic membrane phospholipids or proteins appears important to limit membrane permeability for GSH efflux and to maintain cell attachment.
Assuntos
Cálcio/fisiologia , Ácido Egtázico/farmacologia , Glutationa/metabolismo , Neomicina/farmacologia , Rutênio Vermelho/farmacologia , Linhagem Celular , Glutationa/análogos & derivados , Dissulfeto de Glutationa , Humanos , Isoxazóis/farmacologia , Cinética , Neoplasias Pulmonares , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.
Assuntos
Ácido Etacrínico/farmacologia , Etilmaleimida/farmacologia , Glutationa/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Peróxido de Hidrogênio/farmacologia , Peróxidos/farmacologia , Compostos de Sulfidrila/metabolismo , Linhagem Celular , Cisteamina/farmacologia , Cisteína/farmacologia , Cistina/farmacologia , Ditiotreitol/farmacologia , Glutationa/análogos & derivados , Glutationa/farmacologia , Dissulfeto de Glutationa , Humanos , Cinética , Neoplasias Pulmonares , Oxirredução , Ultrassom , terc-Butil HidroperóxidoRESUMO
Human lung carcinoma cells (A549) were oxidatively stressed with mildly-toxic or non-toxic amounts of hydrogen peroxide (H2O2, 0.1 mM to 120 mM) for 5 min. Hydrogen peroxide exposure resulted in a dose dependent inhibition of binding (pH 7) of the thiol reagent iodoacetic acid (IAA) to a 38 kDa cell protein. Incubation of cells in saline for 60 min following H2O2 removal restored the ability of IAA to bind to the protein. Treatment with 20 mM dithiothreitol or 2 M urea also restored IAA binding, but 10% Triton X102 or 1 mM Brij 58 had no effect. Increasing to pH 11 during the IAA binding also increased thiol availability. Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) has been identified as the protein undergoing thiol/disulfide redox status and enzymic activity changes.
Assuntos
Ditiotreitol/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Peróxido de Hidrogênio/farmacologia , Neoplasias Pulmonares/enzimologia , Catalase/farmacologia , Linhagem Celular , Humanos , Iodoacetatos/metabolismo , Ácido Iodoacético , Cinética , Oxirredução , Ligação Proteica , Compostos de Sulfidrila/metabolismoRESUMO
Freshly isolated rat hepatocytes, which metabolize methionine through the cystathionine pathway, and cultured L5178Y cells, which do not, were compared for their response to the inhibition of S-adenosylhomocysteine (SAH) hydrolase (EC 3.3.1.1). When cells were incubated in Fischer's medium lacking cystine but containing 0.67 mM methionine and 10% serum, the addition of periodate-oxidized adenosine (POA), an inhibitor of SAH hydrolase, increased the level of SAH approximately 4-fold in L5178Y cells (5 mM POA) and 30-fold in hepatocytes (1 mM POA). POA treatment also decreased the amount of intracellular glutathione (GSH) in hepatocytes by 6-fold, and in L5178Y cells by 3-fold. Incubation of hepatocytes with adenosine plus homocysteine, 2-chloroadenosine, or 2',3'-acyclic adenosine increased intracellular SAH and also lowered GSH levels. Neither GSH oxidation nor efflux of GSH or GSH conjugates appeared to account for the GSH loss. Intracellular GSH, covalently bound to proteins as mixed disulfides, increased when hepatocytes were incubated with POA, but the increase was insufficient to account for the total GSH loss. In hepatocytes with prelabeled [35S]GSH, POA caused the cellular GSH content to decrease while the specific activity of [35S]GSH remained constant, suggesting that inhibitor treatments that caused elevated SAH levels may have increased the degradation of GSH while GSH synthesis was inhibited.
Assuntos
Glutationa/metabolismo , Hidrolases/antagonistas & inibidores , Adenosina/metabolismo , Adenosil-Homocisteinase , Animais , Antígenos de Neoplasias/farmacologia , Células Cultivadas , Cistationina/metabolismo , Homocisteína/metabolismo , Leucemia L5178/metabolismo , Fígado/metabolismo , Masculino , Metionina/metabolismo , Oxirredução , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Human monomorphonuclear leukocytes (MMNs) stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) were found to be toxic towards human A549 lung carcinoma cells which have been desensitized against the direct growth-inhibitory effect of TPA. This toxicity was dependent on the TPA concentration and the ratio of MMNs to A549 cells. Using a TPA concentration of 10(-7) M and an effector:target cell ratio of 30:1, experiments were performed to give clues as to the mechanisms by which TPA-stimulated MMNs cause toxicity. Levels of the endogenous thiol glutathione were reduced by 37% in MMNs exposed to TPA for 24 h, but the glutathione levels in the A549 target cells were not markedly affected by TPA-stimulated MMNs. The supernatant of incubations of MMNs with TPA contained a species which was capable of oxidizing the thiol agent 5-thio-2-nitrobenzoic acid. Within 2 h, 9 nmol of this oxidant were produced by 10(7) MMNs. The oxidant exhibited a half-life of 20 h, and its formation was abolished by adding catalase (150 units/ml), azide (1 mM), or cyanide (1 mM) to the incubations of MMNs with TPA. The addition of superoxide dismutase (100 units/ml) enhanced oxidant formation. These results indicate that its generation was dependent on the myeloperoxidase:H2O2:halide system. Large amounts of an oxidizing species with properties identical to those described here have been characterized recently in polymorphonuclear leukocytes [S. J. Weiss, M. B. Lampert, and S. T. Test. Science (Wash. DC), 222: 625-627, 1983]. The toxicity exerted by TPA-stimulated MMNs was partially inhibited by superoxide dismutase and by retinoic acid (30 microM) but not at all by catalase, azide, or cyanide. Therefore, the 5-thio-2-nitrobenzoic acid oxidant does not appear to be involved in the process which led to cytotoxicity by TPA-stimulated MMNs in A549 cells.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Forbóis/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Linhagem Celular , Radicais Livres , Glutationa/metabolismo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Oxirredução , Compostos de Sulfidrila/metabolismo , Tretinoína/farmacologiaRESUMO
Intracellular glutathione (GSH) content of human lung carcinoma cells, A549, in log phase was 25 +/- 5 nmol/10(6) cells, which is considerably higher than that reported in other tumor cells. After partial depletion of GSH with diethyl maleate (DEM), addition of cystine to the medium allowed full resynthesis of GSH in 4 hr, cysteine in the same time period led to less resynthesis, and methionine provided minimal resynthesis. Using cystine as the sole sulfur source and with buthionine sulfoximine (BSO, 5 mM) included in the medium after cells were depleted with DEM, inhibition of both cystine uptake and resynthesis of GSH occurred. BSO inhibited [35S]cystine uptake (as early as 10 min) in a concentration-dependent process, ranging from a 28% decrease for 1 microM BSO to an 85% decrease for 100 microM BSO compared to the control cells after 240 min of incubation. In addition, GSH resynthesis from [35S]cystine for 240 min was inhibited in a parallel dose-dependent manner, in that 1 microM BSO caused a 27% decrease and 100 microM BSO provided a 75% decrease from control values. BSO did not inhibit the uptake of [35S]methionine, but inhibited the low amount of resynthesis of GSH when methionine was the sole sulfur source. BSO did not inhibit the uptake of arginine, phenylalanine, and leucine. DL-, L-, and methyl ester-BSO each inhibited [35S]cystine uptake and incorporation into GSH to a similar extent. The half-life of GSH was 3.5 +/- 0.4 hr in A549 cells that were grown in complete medium with GSH synthesis occurring.
Assuntos
Cistina/metabolismo , Glutationa/biossíntese , Neoplasias Pulmonares/metabolismo , Metionina Sulfoximina/análogos & derivados , Butionina Sulfoximina , Linhagem Celular , Cisteína/metabolismo , Humanos , Metionina Sulfoximina/farmacologia , Radioisótopos de EnxofreRESUMO
Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]-cysteine for GSH synthesis. Lymphocytes preferentially utilized cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathway for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including gamma-glutamyl transpeptidase, GSH S-transferase and gamma-cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.