RESUMO
Dental amalgam fillings are known to release significant amounts of mercury (Hg) in saliva which could represent a continuous source of oxidative damage to mouth tissues. The present investigation was aimed at verifying this hypothesis by determining a possible correlation between salivary Hg levels and salivary total antioxidant activity (TAA), which is used as an index of oxidative stress. Samples of saliva from 34 healthy donors were analyzed for Hg content, by vapor atomic absorption spectrometry, and for TAA, by determining the ferric reducing ability ('FRAP' method). A significant correlation between Hg and the number of amalgam restorations or total amalgam surface was evident in both the male and female subjects. A significant negative correlation between TAA and Hg levels or number of amalgam restorations or amalgam surface was evident in females, indicating that small increases in salivary Hg were sufficient to produce a decrease in salivary TAA. On the other hand, no significant correlation was found in the males. The present study provides, for the first time, evidence of a pro-oxidant role of the amalgam Hg chronically released in saliva.
Assuntos
Antioxidantes/farmacologia , Amálgama Dentário/química , Mercúrio/efeitos adversos , Mercúrio/química , Adolescente , Adulto , Antioxidantes/análise , Feminino , Humanos , Masculino , Estresse Oxidativo , Saliva/química , Fatores Sexuais , Espectrofotometria AtômicaRESUMO
Although the anti-apoptotic effect of Bcl-2 is well established, the role of Bcl-2 in tumour response to therapy and drug resistance is still unclear. The post-translational modifications of Bcl-2 are likely involved in the control of the apoptotic pathway. In the present study we have investigated the role of Bcl-2 in cellular response to oxidative stress (hydrogen peroxide) and cisplatin using a clone of human metastatic melanoma, which, in spite of Bcl-2 (over)expression, exhibited a moderate chemosensitivity. With both treatments melanoma cells died through an apoptotic process, associated with detachment of cells from the monolayer. In the floating apoptotic cells generated by either hydrogen peroxide or cisplatin, along with morphological and biochemical features of apoptosis, we detected a significant Bcl-2 cleavage, yielding the Bax-like fragment of 23 kDa. Preincubation of cells with the caspase-3/-7 inhibitor DEVD-CHO completely suppressed Bcl-2 cleavage, thus confirming that such a specific proteolysis requires activation of caspase-3/-7. The oxidant- and cisplatin-induced processing of Bcl-2 documented in the present study may represent a regulatory mechanism to circumvent the survival function of Bcl-2 upon apoptosis triggering and to enhance apoptotic response. Since the Bcl-2 cleavage should be regarded as a pro-apoptotic event, Bcl-2 expression is expected to increase susceptibility to apoptosis. Thus, such a pathway could be exploited to improve the efficacy of cytotoxic therapy of melanomas expressing Bcl-2.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Peróxido de Hidrogênio/farmacologia , Melanoma/patologia , Oxidantes/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Dano ao DNA , Humanos , Melanoma/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/farmacologia , Estresse Oxidativo , Fragmentos de Peptídeos/análise , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Hyperacute rejection (HAR) occurring after transplantation within phylogenetically distant species is a severe reaction triggered by preexisting xenoreactive antibodies and complement activation, leading to the destruction of the donor organ. Expression of human complement inhibitors in transgenic pig organs prolongs the survival of xenograft in experimental models. Moreover, the extent of protection from hyperacute rejection is dependent on the level and site of expression of the transgenic molecules and, probably, on the combination of different molecules. In this regard a small animal model to test the efficacy of expression vectors and different human molecules could be very advantageous. A murine model developed in our laboratory was characterized by measurement of several parameters characteristic of HAR in the livers of control and transgenic mice expressing transgenic human DAF (CD55) or MCP (CD46) at the end of 2 h of perfusion with human plasma and after I day. The parameters studied were heamatological values of hepatic functions (GOT and GPT), induction of pro-inflammatory molecules and histopathological evaluation. Cytokines (IL-1alpha, IL-1beta, IL-6) induction and exposure of P-selectin on the endothelial cell surface, was only observed in control animals after 2 h of perfusion, as an early event. GOT and GPT values increase dramatically after 2 h perfusion and 1 day after the treatment according to the histopathological observation of liver damage. On the contrary, the livers of hDAF or hMCP transgenic mice, under the same treatment were significantly protected although the extent of this protection is dependent on the level of expression of transgenic human molecules.
Assuntos
Antígenos CD/genética , Antígenos CD55/genética , Proteínas Inativadoras do Complemento/genética , Rejeição de Enxerto/prevenção & controle , Fígado/metabolismo , Glicoproteínas de Membrana/genética , Alanina Transaminase/sangue , Animais , Antígenos CD/biossíntese , Aspartato Aminotransferases/sangue , Antígenos CD55/biossíntese , Complemento C3c/metabolismo , Primers do DNA/química , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto , Humanos , Interleucinas/metabolismo , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Transgênicos , Modelos Animais , Selectina-P/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The metabolism of glutathione by membrane-bound &ggr;-glutamyl transpeptidase (GGT) has been recently recognized as a basal source of hydrogen peroxide in the extracellular space. Significant levels of GGT activity are expressed by malignant tumours, and in melanoma cell lines they were found to correlate with the malignant behaviour. As hydrogen peroxide and other oxidants can affect signal transduction pathways at several levels, the present study was aimed to verify: (i) the occurrence of GGT-dependent production of hydrogen peroxide in melanoma cells; (ii) the effects of GGT-dependent prooxidant reactions on known redox-sensitive cellular targets, i.e. protein thiols, the nuclear transcription factor NF-kappa B and p53. Two melanoma Me665/2 cell clones, exhibiting traces of (clone 2/21) or high (clone 2/60) GGT activity, were studied. The occurrence of GGT-dependent production of hydrogen peroxide was apparent in 2/60 cells, in which it was accompanied by lower levels of cell surface protein thiols. In 2/60 cells, GGT expression was also associated with higher levels of NF-kappa B activation, as compared to GGT-poor 2/21 cell clone. Indeed, stimulation or inhibition of GGT activity in 2/60 cells resulted in progressive activation or inactivation of NF-kappa B, respectively. An analysis of the p53 gene product indicated lack of protein expression in 2/60 cells, whereas a mutant protein was highly expressed in 2/21 cells. Taken together, these results indicate that the expression of GGT activity can provide melanoma cells with an additional source of hydrogen peroxide, and that such prooxidant reactions are capable to modify protein thiols at the cell surface level. In addition, GGT expression results in an up-regulation of the transcription factor NF-kappa B, which could explain the higher metastatic behaviour reported for GGT-rich melanoma cells as compared to their GGT-poor counterparts.
Assuntos
Peróxido de Hidrogênio/metabolismo , Melanoma , NF-kappa B/metabolismo , Compostos de Sulfidrila/metabolismo , gama-Glutamiltransferase/metabolismo , Western Blotting , Membrana Celular/enzimologia , Células Clonais , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Oxirredução , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , gama-Glutamiltransferase/análiseRESUMO
Dental amalgam fillings are known to release significant levels of mercury (Hg) in saliva which could represent a continuous source of oxidative damage to tissues. The present investigation was aimed at verifying this hypothesis by determining a possible correlation between salivary Hg levels and salivary total antioxidant activity (TAA), used as an index of oxidative stress. Samples of saliva from 34 healthy donors were analyzed for Hg content, through vapor atomic absorption spectrometry, and for TAA, by determining the ferric reducing ability ('FRAP' method). A significant correlation between Hg and the number of amalgam restorations or total amalgam surface was evident in both the male and female subjects. A significant negative correlation between TAA and Hg levels or number of amalgam restorations or amalgam surface was evident in females, indicating that small increases in salivary Hg were sufficient to produce a decrease in salivary TAA. On the other hand, no significant correlation was found in the males. The present study provides, for the first time, evidence of a pro-oxidant role of the amalgam Hg chronically released in saliva.
Assuntos
Antioxidantes/metabolismo , Amálgama Dentário/química , Restauração Dentária Permanente , Mercúrio/química , Saliva/efeitos dos fármacos , Adolescente , Adulto , Hidroxitolueno Butilado/metabolismo , Feminino , Compostos Férricos/metabolismo , Humanos , Masculino , Mercúrio/farmacologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Análise de Regressão , Reprodutibilidade dos Testes , Saliva/química , Saliva/metabolismo , Fatores Sexuais , Espectrofotometria Atômica , Propriedades de SuperfícieRESUMO
The measurement of single parameters of oxidative stress in biological fluids can often give results difficult to interpret as to the real involvement of oxidative processes in a given disease condition. In the present study we propose a novel integrated parameter, called "redox compensation index", obtained by combining the results of two established and convenient procedures, i.e. the Fox-2 assay for plasma lipid hydroperoxides and the ferric reducing/antioxidant power (FRAP) assay for total antioxidant potential of plasma. These procedures were employed for the evaluation of oxidative stress in a group of patients with type 2 diabetes mellitus, a condition in which oxidative processes are implicated in the development of complications. In type 2 diabetic patients, plasma lipid hydroperoxides were directly correlated with levels of glycated hemoglobin. On the other hand, a significant inverse correlation was observed between levels of glycated hemoglobin and redox compensation values. The data reported suggest that the redox compensation index could represent a convenient parameter for the direct appraisal of oxidative status in clinical subjects, and are in support of the proposed role of protein glycation in production of oxidative alterations during type 2 diabetes mellitus.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Idoso , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Peróxidos Lipídicos/sangue , Masculino , Pessoa de Meia-Idade , OxirreduçãoRESUMO
Iron is released in a free desferrioxamine-chelatable form when erythrocytes are challenged by an oxidative stress. The release of iron is believed to play an important role in inducing destructive damage (lipid peroxidation and hemolysis) or in producing membrane protein oxidation and generation of senescent cell antigens (SCA). In this report, we further tested the hypothesis that intracellular chelation of iron released under conditions of oxidative stress prevents erythrocyte damage or SCA formation. Fluor-benzoil-pyridoxal hydrazone (FBPH), an iron-chelating molecule of the family of aromatic hydrazones, was prepared by synthesis and used for the above purpose after the capacity of the product to enter cells had been ascertained. GSH-depleted mouse erythrocytes were incubated with the oxidant drug phenylhydrazine in order to produce iron release, lipid peroxidation, and hemolysis. FBPH at a concentration of 200 microM prevented lipid peroxidation and hemolysis in spite of equal values of iron release. FBPH was active even at a lower concentration (100 microM) when the erythrocytes were preincubated with it for 15 min. No preventive effect was seen when FBPH saturated with iron was used. Prolonged aerobic incubation (60 hr) of erythrocytes produced iron release and formation of SCA as determined by autologous immunoglobulin G (IgG) binding. The IgG binding was detected by using an anti-IgG antibody labeled with fluorescein and by examining the cells for fluorescence by confocal microscopy. FBPH prevented SCA formation in a dose-related manner. These results lend further support to the hypothesis that iron release is a key factor in erythrocyte ageing.
Assuntos
Eritrócitos/efeitos dos fármacos , Hidrazonas/farmacologia , Imunoglobulina G/metabolismo , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Piridoxal/análogos & derivados , Animais , Antígenos de Diferenciação/imunologia , Eritrócitos/imunologia , Eritrócitos/metabolismo , Imunoglobulina G/imunologia , Técnicas In Vitro , Camundongos , Estresse Oxidativo , Substâncias Protetoras/farmacologia , Piridoxal/farmacologiaRESUMO
Incubation of rat erythrocytes with the hydroxylated metabolites of aniline and dapsone (4-4'-diaminodiphenylsulfone), phenylhydroxylamine and dapsone hydroxylamine, respectively, induced marked release of iron and methemoglobin formation. On the contrary, no release of iron nor methemoglobin formation was seen when the erythrocytes were incubated with the parent compounds (aniline and dapsone). The acute intoxication of rats with aniline or dapsone induced a marked increase in the erythrocyte content of free iron and methemoglobin, indicating that the xenobiotics are effective only after biotransformation to toxic metabolites in vivo. Prolonged administration of aniline or dapsone to rats produced continuous release of iron from erythrocytes. Marked iron overload was seen in the spleen and in the liver Kupffer cells, as detected histochemically. The spleen weight in these subchronically treated animals was significantly increased. The free iron pool was markedly increased in the spleen and to a lower extent in the liver. The possible relationships between iron release in erythrocytes, oxidative damage seen in senescent cells, hemolysis, overwhelmed capacity of spleen and liver to keep iron in storage forms and subsequent increase in low molecular weight, catalitically active iron is discussed.
Assuntos
Compostos de Anilina/toxicidade , Dapsona/toxicidade , Eritrócitos/efeitos dos fármacos , Hemólise , Ferro/sangue , Hansenostáticos/toxicidade , Fígado/efeitos dos fármacos , Oxidantes/toxicidade , Baço/efeitos dos fármacos , Compostos de Anilina/metabolismo , Animais , Dapsona/análogos & derivados , Dapsona/metabolismo , Dapsona/farmacologia , Eritrócitos/metabolismo , Hidroxilaminas/farmacologia , Hansenostáticos/metabolismo , Fígado/metabolismo , Masculino , Metemoglobina/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Oxidantes/metabolismo , Ratos , Ratos Sprague-Dawley , Baço/metabolismoRESUMO
The expression of gamma-glutamyl transpeptidase (GGT), a plasma membrane ectoenzyme involved in the metabolism of extracellular reduced glutathione (GSH), is a marker of neoplastic progression in several experimental models, and occurs in a number of human malignant neoplasms and their metastases. Because it favors the supply of precursors for the synthesis of GSH, GGT expression has been interpreted as a member in cellular antioxidant defense systems. However, thiol metabolites generated at the cell surface during GGT activity can induce prooxidant reactions, leading to production of free radical oxidant species. The present study was designed to characterize the prooxidant reactions occurring during GGT ectoactivity, and their possible effects on the thiol redox status of proteins of the cell surface. Results indicate that: (i) in U937 cells, expressing significant amounts of membrane-bound GGT, GGT-mediated metabolism of GSH is coupled with the extracellular production of hydrogen peroxide; (ii) GGT activity also results in decreased levels of protein thiols at the cell surface; (iii) GGT-dependent decrease in protein thiols is due to sulfhydryl oxidation and protein S-thiolation reactions; and (iv) GGT irreversible inhibition by acivicin is sufficient to produce an increase of protein thiols at the cell surface. Membrane receptors and transcription factors have been shown to possess critical thiols involved in the transduction of proliferative signals. Furthermore, it was suggested that S-thiolation of cellular proteins may represent a mechanism for protection of vulnerable thiols against irreversible damage by prooxidant agents. Thus, the findings reported here provide additional explanations for the envisaged role played by membrane-bound GGT activity in the proliferative attitude of malignant cells and their resistance to prooxidant drugs and radiation therapy.
Assuntos
Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/metabolismo , Compostos de Sulfidrila/metabolismo , gama-Glutamiltransferase/farmacologia , Corantes Fluorescentes , Radicais Livres/metabolismo , Glutationa/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Isoxazóis/farmacologia , Proteínas de Membrana/análise , Microscopia Confocal , Oxidantes/metabolismo , Oxirredução , Escopoletina , Células U937RESUMO
Our previous studies have shown that iron is released in a free (desferrioxamine-chelatable) form when erythrocytes undergo oxidative stress (incubation with oxidizing agents or aerobic incubation in buffer for 24-60 h (a model of rapid in vitro ageing)). The release is accompanied by oxidative alterations of membrane proteins as well as by the appearance of senescent antigen, a signal for termination of old erythrocytes. In hemolytic anemias by hereditary hemoglobin alterations an accelerated removal of erythrocytes occurs. An increased susceptibility to oxidative damage has been reported in beta-thalassemic erythrocytes. Therefore we have investigated whether an increased iron level and an increased susceptibility to iron release could be observed in the erythrocytes from patients with beta-thalassemia. Erythrocytes from subjects with thalassemia intermedia showed an extremely higher content (0 time value) of free iron and methemoglobin as compared to controls. An increase, although non-statistically-significant, was seen in erythrocytes from subjects with thalassemia major. Upon aerobic incubation for 24 h the release of iron in beta-thalassemic erythrocytes was by far greater than in controls, with the exception of thalassemia minor. When the individual values for free iron content (0 time) seen in thalassemia major and intermedia were plotted against the corresponding values for HbF, a positive correlation (P < 0.001) was observed. Also, a positive correlation (P < 0.01) was seen between the values for free iron release (24 h incubation) and the values for HbF. These results suggest that the presence of HbF is a condition favourable to iron release. Since in beta-thalassemia the persistance of HbF is related to the lack or deficiency of beta chains and therefore to the excess of alpha chains, the observed correlation between free iron and HbF, is consistent with the hypothesis by others that excess of alpha chains represents a prooxidant factor.
Assuntos
Eritrócitos/metabolismo , Ferro/metabolismo , Talassemia beta/metabolismo , Adulto , Eritrócitos/efeitos dos fármacos , Hemoglobina Fetal/metabolismo , Glutationa/metabolismo , Humanos , Monometilidrazina/farmacologia , Fenil-Hidrazinas/farmacologia , Valores de ReferênciaRESUMO
BACKGROUND: gamma-Glutamyl transpeptidase (gamma-GT) is found in serum and in the plasma membranes of virtually all cell types. Its physiologic role is to initiate the hydrolysis of extracellular glutathione (GSH), a tripeptide in which cysteine lies between alpha-glycine and gamma-glutamate residues. Cysteine and other thiol compounds are known to promote LDL oxidation by reducing Fe(III) to redox active Fe(II); therefore, we sought to determine whether similar reactions can be sustained by GSH and influenced by gamma-GT. METHODS: Fe(III) reduction and LDL oxidation were studied by monitoring the formation bathophenanthroline-chelatable Fe(II) and the accumulation of thiobarbituric acid-reactive substances, respectively. Human atheromatous tissues were examined by histochemical techniques for the presence of oxidized LDL and their colocalization with cells expressing gamma-GT activity. RESULTS: A series of experiments showed that the gamma-glutamate residue of GSH affected interactions of the juxtaposed cysteine thiol with iron, precluding Fe(III) reduction and hence LDL oxidation. Both processes increased remarkably after addition of purified gamma-GT, which acts by removing the gamma-glutamate residue. GSH-dependent LDL oxidation was similarly promoted by gamma-GT associated with the plasma membrane of human monoblastoid cells, and this process required iron traces that can be found in advanced or late stage atheromas. Collectively, these findings suggested a possible role for gamma-GT in the cellular processes of LDL oxidation and atherogenesis. Histochemical analyses confirmed that this may be the case, showing that gamma-GT activity is expressed by macrophage-derived foam cells within human atheromas, and that these cells colocalize with oxidized LDL. CONCLUSIONS: Biochemical and histochemical correlates indicate that gamma-GT can promote LDL oxidation by hydrolyzing GSH into more potent iron reductants. These findings may provide mechanistic clues to the epidemiologic evidence for a possible correlation between persistent elevation of gamma-GT and the risk of fatal reinfarction in patients with ischemic heart disease.
Assuntos
Arteriosclerose/metabolismo , Ferro/metabolismo , Lipoproteínas LDL/metabolismo , gama-Glutamiltransferase/metabolismo , Adulto , Idoso , Catalase/farmacologia , Quelantes/metabolismo , Células Espumosas/enzimologia , Células Espumosas/patologia , Glutationa/farmacologia , Humanos , Técnicas Imunoenzimáticas , Ferro/química , Peroxidação de Lipídeos/fisiologia , Lipoproteínas LDL/química , Masculino , Pessoa de Meia-Idade , Fenantrolinas/metabolismo , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Células U937RESUMO
It has been reported in several cell lines that exposure to low levels of reactive oxygen species can exert a stimulatory effect on their proliferation. We have previously shown that mild oxidative conditions can also counteract apoptotic stimuli. A constitutive cellular production of low levels of superoxide and hydrogen peroxide originates from various sources; among these, gamma-glutamyl transpeptidase (GGT), the plasma membrane-bound activity in charge of metabolizing extracellular reduced glutathione, has recently been included. Since the inhibition of GGT is a sufficient stimulus for the induction of apoptosis in selected cell lines, we investigated whether this effect might result from the suppression of the mentioned GGT-dependent prooxidant reactions, on the theory that the latter may represent a basal antiapoptotic and proliferative signal for the cell. Experiments showed that: 1) GGT activity in U937 monoblastoid cells is associated with the production of low levels of hydrogen peroxide, and two independent GGT inhibitors cause a dose-dependent decrease of such GGT-dependent production of H2O2; 2) GGT inhibition with acivicin results in cell growth arrest, and induces cell death and DNA fragmentation with the ladder appearance of apoptosis; 3) treatment of cells with catalase--and even more with Trolox C--is able to decrease their proliferative rate; 4) GGT inhibition (with suppression of H2O2 production) results in a down-regulation of poly(ADP-ribose) polimerase (PARP) activity, which precedes the proteolytic cleavage of PARP molecule, such as that typically induced by caspases. The reported data suggest that the low H2O2 levels originating as a by-product during GGT activity are able to act as sort of a 'life signal' in U937 cells, insofar as they can maintain cell proliferation and protect against apoptosis, possibly through an up-regulation of PARP activity.
Assuntos
Apoptose , Divisão Celular , Peróxido de Hidrogênio/metabolismo , gama-Glutamiltransferase/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Catalase/metabolismo , Catalase/farmacologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glutationa/metabolismo , Humanos , Isoxazóis/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase/farmacologia , Células U937 , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
The formation, reactivity and toxicity of aldehydes originating from lipid peroxidation of cellular membranes are reviewed. Very reactive aldehydes, namely 4-hydroxyalkenals, were first shown to be formed in autoxidizing chemical systems. It was subsequently shown that 4-hydroxyalkenals are formed in biological conditions, i.e. during lipid peroxidation of liver microsomes incubated in the NADPH-Fe systems. Our studies carried out in collaboration with Hermann Esterbauer which led to the identification of 4-hydroxynonenal (4-HNE) are reported. 4-HNE was the most cytotoxic aldehyde and was then assumed as a model molecule of oxidative stress. Many other aldehydes (alkanals, alk-2-enals and dicarbonyl compounds) were then identified in peroxidizing liver microsomes or hepatocytes. The in vivo formation of aldehydes in liver of animals intoxicated with agents that promote lipid peroxidation was shown in further studies. In a first study, evidence was forwarded for aldehydes (very likely alkenals) bound to liver microsomal proteins of CCl4 or BrCCl3-intoxicated rats. In a second study, 4-HNE and a number of other aldehydes (alkanals and alkenals) were identified in the free (non-protein bound) form in liver extracts from bromobenzene or allyl alcohol-poisoned mice. The detection of free 4-HNE in the liver of CCl(4) or BrCC1(3)-poisoned animals was obtained with the use of an electrochemical detector, which greatly increased the sensitivity of the HPLC method. Furthermore, membrane phospholipids bearing carbonyl groups were demonstrated in both in vitro (incubation of microsomes with NADPH-Fe) and in vivo (CC1(4) or BrCCl(3) intoxication) conditions. Finally, the results concerned with the histochemical detection of lipid peroxidation are reported. The methods used were based on the detection of lipid peroxidation-derived carbonyls. Very good results were obtained with the use of fluorescent reagents for carbonyls, in particular with 3-hydroxy-2-naphtoic acid hydrazide (NAH) and analysis with confocal scanning fluorescence microscopy with image video analysis. The significance of formation of toxic aldehydes in biological membranes is discussed.
Assuntos
Aldeídos/análise , Aldeídos/metabolismo , Peroxidação de Lipídeos , Aldeídos/química , Aldeídos/história , Animais , Bioquímica/história , Histocitoquímica/métodos , História do Século XX , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Oxidantes/toxicidade , Patologia Clínica/tendênciasRESUMO
Incubation of glutathione (GSH) depleted mouse erythrocytes with the oxidants phenylhydrazine, acrolein, divicine and isouramil resulted in the release of free iron and in lipid peroxidation and hemolysis. The addition of the flavonoid quercetin, which chelates iron and penetrates erythrocytes, resulted in remarkable protection against lipid peroxidation and hemolysis. The protection seems to be due to intracellular chelation of iron, since a semi-stoichiometric ratio between released iron and the amount of quercetin necessary to prevent lipid peroxidation and hemolysis was found. Incubation of GSH depleted human erythrocytes with divicine and isouramil did not induce lipid peroxidation and hemolysis in spite of a substantial release of iron. However, divicine and isouramil produced alterations of membrane proteins, such as spectrin and band 3, as well as formation of senescent cell antigen. The addition of quercetin prevented these alterations.
Assuntos
Quelantes/farmacologia , Membrana Eritrocítica/fisiologia , Eritrócitos/fisiologia , Glutationa/sangue , Ferro/sangue , Quercetina/farmacologia , Acroleína/farmacologia , Animais , Barbitúricos/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Flavonoides/química , Flavonoides/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Quelantes de Ferro/farmacologia , Cinética , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/sangue , Metemoglobina/metabolismo , Camundongos , Pirimidinonas/farmacologia , Quercetina/químicaRESUMO
Asbestos exposure causes pulmonary fibrosis by mechanisms that remain uncertain. There is increasing evidence that iron from asbestos is responsible for many of its effects. In this paper, we investigated the effect of iron mobilized from crocidolite asbestos on collagen content in rat lung fibroblast cultures under serum-free conditions. Crocidolite (2, 4, 6 microg/cm2 well) increased collagen content in a dose-dependent manner (+42 +/- 8, +92 +/- 10, and +129 +/- 13% vs controls). This effect was specific for collagen, since it did not alter total protein content and was inhibited by the iron chelator deferoxamine (DFO). Preincubation of crocidolite with citrate (1 mM) for 48 hr resulted in iron mobilization (51 microM) and increased collagen production (>3-fold) in treated cells. These effects occurred without the intervention of serum factors. The absence of cell damage, proliferation or lipid peroxidation leads to the supposition that iron from crocidolite per se may act as a profibrogenic agent. Although the in vivo participation of other cells and factors cannot be excluded, we conclude that iron released from crocidolite plays a role in collagen increase occurring during asbestosis.
Assuntos
Asbesto Crocidolita/toxicidade , Colágeno/biossíntese , Ferro/metabolismo , Pulmão/metabolismo , Animais , Asbesto Crocidolita/química , Asbestose/etiologia , Asbestose/metabolismo , Células Cultivadas , DNA/análise , Fibroblastos , Pulmão/efeitos dos fármacos , Proteínas/análise , RatosRESUMO
Following the subchronic intoxication of rats with phenylhydrazine, resulting in marked anemia, reticulocytosis, methemoglobinemia and increased hemocatheresis, the hepatic content of total iron was increased, as was hepatic ferritin and its saturation by iron. A striking increase (approximately 7-fold) was also observed in free iron which appeared to be redox-active. The increase in liver free iron involved the hepatocellular component of the liver. Since DNA is one of the cellular targets of redox active iron, liver DNA from phenylhydrazine-treated rats was analyzed by electrophoresis and found to be markedly fragmented. Experiments with isolated hepatocytes in culture or in suspension challenged with phenylhydrazine or Fe-nitrilotriacetate strongly suggested that the DNA damage was due to reactive iron rather than to the hepatic metabolism of phenylhydrazine. The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a specific marker of oxidative DNA damage, were significantly higher in phenylhydrazine-treated rats as compared to untreated controls. The prolongation of phenylhydrazine treatment over a period of 6 weeks resulted in a persistent damage to DNA and in phenotypic changes such as an increase in hepatocyte gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2) activity. Possible relationships between iron overload, iron release, DNA damage and tumor initiation are discussed.
Assuntos
Dano ao DNA , Sobrecarga de Ferro/induzido quimicamente , Ferro/metabolismo , Fígado/metabolismo , Fenil-Hidrazinas/toxicidade , Animais , Fragmentação do DNA , Eritrócitos/efeitos dos fármacos , Histocitoquímica , Peroxidação de Lipídeos , Fígado/enzimologia , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley , Baço/metabolismo , gama-Glutamiltransferase/análiseRESUMO
Gamma-glutamyltranspeptidase (GGT), a plasma membrane-bound enzyme, provides the only activity capable to effect the hydrolysis of extracellular glutathione (GSH), thus favoring the cellular utilization of its constituent amino acids. Recent studies have shown however that in the presence of chelated iron prooxidant species can be originated during GGT-mediated metabolism of GSH, and that a process of lipid peroxidation can be started eventually in suitable lipid substrates. The present study was undertaken to verify if a GGT-dependent lipid peroxidation process can be induced in the lipids of biological membranes, including living cells, and if this effect can be sustained by the GGT highly expressed at the surface of HepG2 human hepatoma cells. In rat liver microsomes (chosen as model membrane lipid substrate) exposed to GSH and ADP-chelated iron, the addition of GGT caused a marked stimulation of lipid peroxidation, which was further enhanced by the addition of the GGT co-substrate glycyl-glycine. The same was observed in primary cultures of isolated rat hepatocytes, where the lipid peroxidation process did not induce acute toxic effects. GGT-stimulation of lipid peroxidation was dependent both on the concentration of GSH and of ADP-chelated iron. In GGT-rich HepG2 human hepatoma cells, the exposure to GSH, glycyl-glycine, and ADP-chelated iron resulted in a nontoxic lipid peroxidation process, which could be prevented by means of GGT inhibitors such as acivicin and the serine-boric acid complex. In addition, by co-incubation of HepG2 cells with rat liver microsomes, it was observed that the GGT owned by HepG2 cells can act extracellularly, as a stimulant on the GSH- and iron-dependent lipid peroxidation of microsomes. The data reported indicate that the lipid peroxidation of liver microsomes and of living cells can be stimulated by the GGT-mediated metabolism of GSH. Due to the well established interactions of lipid peroxidation products with cell proliferation, the phenomenon may bear particular significance in the carcinogenic process, where a relationship between the expression of GGT and tumor progression has been envisaged.
Assuntos
Peroxidação de Lipídeos/fisiologia , Fígado/metabolismo , gama-Glutamiltransferase/metabolismo , Animais , Radicais Livres/metabolismo , Glutationa/metabolismo , Humanos , Técnicas In Vitro , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Células Tumorais CultivadasRESUMO
The proliferative capacity of immune cells is known to be sensitive to conditions of oxidative stress and lipid peroxidation. We tested the hypothesis that activated neutrophils can induce peroxidation in extracellular lipid substrates, and evaluated the effects of 4-hydroxy-2,3-trans-nonenal (4-HNE)--the most reactive aldehydic product of lipid peroxidation--on mitogen-induced proliferation of human T lymphocytes. Neutrophils activated in the presence of extracellular lipid substrates (liposomes, cellular membranes) induced lipid peroxidation. By means of cytoimmunofluorescent labeling and confocal microscopy, the binding of 4-HNE to surface and cytoplasmic proteins of activated neutrophils was observed. Short (20 min) pre-treatment of cells with low concentrations of 4-HNE were able to dose-dependently decrease the proliferation of human peripheral blood lymphocytes challenged with PHA or anti-CD3 monoclonal antibody OKT3, as well as the proliferation of a tetanus specific human T-cell line challenged with tetanus toxoid. In these conditions, the binding of 4-HNE to surface and cytoplasmic proteins of lymphocytes was also observed. When the proliferative capacity of peripheral blood lymphocytes was monitored over several days after 4-HNE treatment and PHA challenge, a recovery and a rebound in cell proliferation was observed. Data reported indicate that the lipid peroxidation promoted by activated neutrophils can exert modulatory effects on the responsivity of human T cells, through the action of its most reactive product, 4-HNE.