RESUMO
Varicocele is a prevalent pathology among infertile men. The mechanisms linking this condition to infertility, however, are poorly understood. Our previous work showed a relationship between sperm functional quality and the ability of spermatozoa to respond to capacitating conditions with increased membrane fluidity and protein tyrosine phosphorylation. Given the reported association between varicocele, oxidative stress, and sperm dysfunction, we hypothesized that spermatozoa from infertile patients with varicocele might have a combined defect at the level of membrane fluidity and protein tyrosine phosphorylation. Semen samples from infertile patients with and without grade II/III left varicocele were evaluated for motion parameters (computer-assisted semen analysis [CASA]), hyperactivation (CASA), incidence and intensity of protein tyrosine phosphorylation (phosphotyrosine immunofluorescence and western blotting), and membrane fluidity (Laurdan fluorometry), before and after a capacitating incubation (6 hr at 37 degrees C in Ham's F10/BSA, 5% CO(2)). Spermatozoa from varicocele samples presented a decreased response to the capacitating challenge, showing significantly lower motility, hyperactivation, incidence and intensity of tyrosine phosphorylation, and membrane fluidity. The findings reported in this article indicate that the sperm dysfunction associated to infertile varicocele coexists with decreased sperm plasma membrane fluidity and tyrosine phosphorylation. These deficiencies represent potential new pathophysiological mechanisms underlying varicocele-related infertility.
Assuntos
Infertilidade Masculina/etiologia , Fluidez de Membrana/fisiologia , Proteínas Tirosina Quinases/metabolismo , Espermatozoides/metabolismo , Varicocele/complicações , Adulto , Membrana Celular/fisiologia , Humanos , Masculino , Fosforilação , Motilidade dos EspermatozoidesRESUMO
Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2'-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2'-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid-water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.
Assuntos
Antioxidantes/farmacologia , Flavonoides/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Membranas/efeitos dos fármacos , Amidinas/antagonistas & inibidores , Amidinas/química , Compostos Azo/antagonistas & inibidores , Compostos Azo/química , Flavonoides/química , Bicamadas Lipídicas/química , Lipídeos/química , Lipossomos/química , Membranas/química , Micelas , Nitrilas/antagonistas & inibidores , Nitrilas/química , Oxidantes/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Relação Estrutura-Atividade , Substâncias Reativas com Ácido Tiobarbitúrico/químicaRESUMO
The aim of the present study was to further understand how changes in membrane organization can lead to higher rates of lipid oxidation. We previously demonstrated that Al(3+), Sc(3+), Ga(3+), Be(2+), Y(3+), and La(3+) promote lipid packing and lateral phase separation. Using the probe Laurdan, we evaluated in liposomes if the higher rigidity of the membrane caused by Al(3+) can alter membrane phase state and/or hydration, and the relation of this effect to Al(3+)-stimulated lipid oxidation. In liposomes of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylserine, Al(3+) (10-100 microM) induced phase coexistence and displacement of T(m). In contrast, in liposomes of brain phosphatidylcholine and brain phosphatidylserine, Al(3+) (10-200 microM) did not affect membrane phase state but increased Laurdan generalized polarization (GP = -0. 04 and 0.09 in the absence and presence of 200 microM Al(3+), respectively). Sc(3+), Ga(3+), Be(2+), Y(3+), and La(3+) also increased GP values, with an effect equivalent to a decrease in membrane temperature between 10 and 20 degrees C. GP values in the presence of the cations were significantly correlated (r(2) = 0.98, P < 0.001) with their capacity to stimulate Fe(2+)-initiated lipid oxidation. Metal-promoted membrane dehydration did not correlate with ability to enhance lipid oxidation, indicating that dehydration of the phospholipid polar headgroup is not a mechanism involved in cation-mediated enhancement of Fe(2+)-initiated lipid oxidation. Results indicate that changes in membrane phospholipid phase state favoring the displacement to gel state can facilitate the propagation of lipid oxidation.
Assuntos
Alumínio/farmacologia , Lipossomos/efeitos dos fármacos , Lipossomos/metabolismo , Metais/farmacologia , Fosfolipídeos/metabolismo , Água/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/metabolismo , Alumínio/metabolismo , Encéfalo , Cátions/metabolismo , Cátions/farmacologia , Cristalização , Corantes Fluorescentes/metabolismo , Cinética , Lauratos/metabolismo , Lipossomos/química , Metais/metabolismo , Oxirredução/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Espectrometria de Fluorescência , Temperatura , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
In the present study we characterized the capacity of zinc to protect lipids and proteins from Fe2+-initiated oxidative damage. The effects of zinc on lipid oxidation were investigated in liposomes composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS) at a molar relationship of 60:40 (PC:PS, 60:40). Lipid oxidation was evaluated as the oxidation of cis-parinaric acid or as the formation of 2-thiobarbituric acid-reactive substances (TBARS). Zinc protected liposomes from Fe2+ (2.5-50 microM)-supported lipid oxidation. However, zinc (50 microM) did not prevent the oxidative inactivation of glutamine synthetase and glucose 6-phosphate dehydrogenase when rat brain supernatants were oxidized in the presence of 5 microM Fe2+ and 0.5 mM H2O2. We also studied the interactions of zinc with epicatechin in the prevention of lipid oxidation in liposomes. The simultaneous addition of 0.5 microM epicatechin (EC) and 50 microM zinc increased the protection of liposomes from oxidation compared to that observed in the presence of zinc or EC separately. Zinc (50 microM) also protected liposomes from the stimulatory effect of aluminum on Fe2+-initiated lipid oxidation. Zinc could play an important role as an antioxidant in biological systems, replacing iron and other metals with pro-oxidant activity from binding sites and interacting with other components of the oxidant defense system.
Assuntos
Antioxidantes/farmacologia , Catequina/farmacologia , Compostos Ferrosos/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Zinco/farmacologia , Animais , Interações Medicamentosas , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Ratos , Ratos Wistar , Substâncias Reativas com Ácido TiobarbitúricoRESUMO
In the present study we characterized the capacity of zinc to protect lipids and proteins from Fe2+-initiated oxidative damage. The effects of zinc on lipid oxidation were investigated in liposomes composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS) at a molar relationship of 60:40 (PC:PS, 60:40). Lipid oxidation was evaluated as the oxidation of cis-parinaric acid or as the formation of 2-thiobarbituric acid-reactive substances (TBARS). Zinc protected liposomes from Fe2+ (2.5-50 microM)-supported lipid oxidation. However, zinc (50 microM) did not prevent the oxidative inactivation of glutamine synthetase and glucose 6-phosphate dehydrogenase when rat brain supernatants were oxidized in the presence of 5 microM Fe2+ and 0.5 mM H2O2. We also studied the interactions of zinc with epicatechin in the prevention of lipid oxidation in liposomes. The simultaneous addition of 0.5 microM epicatechin (EC) and 50 microM zinc increased the protection of liposomes from oxidation compared to that observed in the presence of zinc or EC separately. Zinc (50 microM) also protected liposomes from the stimulatory effect of aluminum on Fe2+-initiated lipid oxidation. Zinc could play an important role as an antioxidant in biological systems, replacing iron and other metals with pro-oxidant activity from binding sites and interacting with other components of the oxidant defense system.
Assuntos
Ratos , Animais , Antioxidantes/farmacologia , Catequina/farmacologia , Compostos Ferrosos/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Zinco/farmacologia , Interações Medicamentosas , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Lipídeos de Membrana/metabolismo , Lipossomos/metabolismo , Ratos Wistar , Substâncias Reativas com Ácido TiobarbitúricoRESUMO
In the first part of the present study we investigated the effects of pre-natal and early postnatal exposure of mice to high levels of dietary Al3+ on myelin lipid composition and lipid oxidation. We found: (1) a significantly higher (104%; P<0.01) content of brain myelin galactolipids in the high-Al3+ group than in controls, and, (2) a significant correlation (r2=0.70; P<0.01) between the concentration of myelin galactolipids and TBARS (2-thiobarbituric acid-reactive substances) content, a parameter of lipid oxidation. Based on these results, we evaluated in an in vitro model (liposomes) whether galactolipids could affect the capacity of Al3+ to stimulate Fe2+-initiated lipid oxidation, and whether this effect could be due to the promotion of changes in membrane physical properties (membrane phase separation and rigidification). The presence of galactolipids (10-40 mol%) in the liposomes caused a concentration-dependent increase in the stimulatory effect of Al3+ on Fe2+-induced TBARS production, and on the ability of Al3+ to induce phase separation and membrane rigidification. The capacity of Al3+ (10-100 microM) to induce lateral phase separation in liposomes composed of phosphatidylcholine/phosphatidylserine/galactolipid (36:24:40, molar ratio) was correlated significantly (r2=0.99; P<0. 001) with the stimulatory action of Al3+ on Fe2+-induced TBARS production. We propose that the high content of galactolipids found in myelin from Al3+-intoxicated mice could favour Al3+-induced changes in membrane physical properties, with the subsequent acceleration of lipid oxidation rates.
Assuntos
Alumínio/toxicidade , Glicolipídeos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Alumínio/intoxicação , Animais , Encéfalo/anatomia & histologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Bovinos , Dieta , Feminino , Polarização de Fluorescência , Galactolipídeos , Galactose/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Fluidez de Membrana/fisiologia , Lipídeos de Membrana/metabolismo , Camundongos , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The capacity of Al3+ to promote oxidative damage to brain membranes was investigated both in vitro and in vivo. In vitro, Al3+ and related metals (Sc3+, Ga3+, In3+, Be2+, Y3+, and La3+) stimulated Fe2+-initiated lipid and protein oxidation in brain myelin and synaptic membranes. Al3+, Sc3+, Y3+, and La3+ significantly promoted protein-associated carbonyl production in myelin, while in synaptic membranes, the stimulatory effect was observed in the presence of Ga3+, In3+, Y3+, Sc3+, and La3+. In myelin the magnitude of the stimulation of lipid oxidation followed the order Sc3+, Y3+, La3+ > Al3+, Ga3+, In3+ > Be2+. When compared to mitochondria and microsomal and synaptic membranes, myelin showed a marked susceptibility to Al3+-mediated lipid peroxidation. The differential susceptibility of myelin compared to synaptic membranes could not be explained by differences in membrane composition, since the relative content of negatively charged phospholipids (binding sites) was similar for both membranes, and myelin had a lower content of poly-unsaturated fatty acids (substrates of lipid oxidation) and a higher concentration of alpha-tocopherol compared to synaptic membranes. In a model of Al3+ intoxication imposed to mice during pregnancy and early development, a 72% higher content of lipid peroxidation products was found in brain myelin. The fluidity of myelin evaluated by the polarization fluorescence of 1,3-diphenylhexatriene was significantly higher in the Al3+-intoxicated mice than in controls. Since myelin has a high relative content of lipid:protein compared to other membranes, these results support our hypothesis that ions without redox capacity can stimulate in vitro and in vivo lipid oxidation by promoting phase separation and membrane rigidification, thus accelerating lipid oxidation.
Assuntos
Alumínio/farmacologia , Encéfalo/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Bainha de Mielina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Ácidos Graxos/análise , Compostos Ferrosos/farmacologia , Técnicas In Vitro , Fluidez de Membrana/efeitos dos fármacos , Metais/farmacologia , Metais Terras Raras/farmacologia , Camundongos , Bainha de Mielina/química , Bainha de Mielina/efeitos dos fármacos , Oxirredução , Fosfolipídeos/análise , Ratos , Ratos Wistar , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The capacity of Al3+-related cations (Sc3+, Ga3+, In3+, Be2+, Y3+, and La3+) to promote membrane rigidification and lateral phase separation was evaluated in liposomes containing zwitterionic (phosphatidylcholine, PC) and negatively charged (phosphatidylserine, PS) phospholipids. These effects were correlated with the capacity of the ions to stimulate Fe2+-supported lipid peroxidation. A13+, Sc3+, Ga3+, In3+, Be2+, Y3+, and La3+ (50-200 microM) increased the order parameter of the fluorescent probe 1,3-diphenylhexatriene incorporated in PC:PS membranes. In addition, the electron paramagnetic resonance spectra of spin-labeled fatty acids indicated a reduction in lipid motion induced by Sc3+, Y3+, and La3+. The effect was found to extend down to carbon 16 on the acyl chain. The ions (10-200 microM) were also able to induce lateral phase separation, as evaluated from the increase in fluorescence quenching of the probe 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadec anoyl-sn-glycero-3-phosphocholine. The ability of the ions to alter membrane lipid packing and induce lateral phase separation correlated in a positive manner (r2 = 0.91 and 0.90, respectively) with their capacity to stimulate the production of Fe2+-initiated 2-thiobarbituric-reactive species, a measure of lipid peroxidation. These results show that Al3+-related metal ions cause membrane rigidification and phase separation, which could affect membrane-related processes. The results support the hypothesis that ions without redox capacity can stimulate Fe2+-initiated lipid peroxidation by increasing lipid packing and by promoting the formation of rigid clusters. Both processes will bring phospholipid acyl chains closer together, thus favoring the propagation step of lipid peroxidation.
Assuntos
Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/química , Metais/toxicidade , Alumínio/toxicidade , Animais , Berílio/toxicidade , Cátions/química , Cátions/toxicidade , Espectroscopia de Ressonância de Spin Eletrônica , Gálio/toxicidade , Técnicas In Vitro , Índio/toxicidade , Lipossomos , Fluidez de Membrana/efeitos dos fármacos , Metais/química , Estrutura Molecular , Escândio/toxicidade , Termodinâmica , Ítrio/toxicidadeRESUMO
The capacity of metals chemically and physically related to Al (Sc, Ga, In, Y, and Be) to promote liposome aggregation, fusion, and permeabilization and to stimulate Fe2(+)-initiated lipid peroxidation was investigated in negatively charged liposomes. The effects of Sc, Ga, In, Be, and Y were compared with those of trivalent (Al, La) and divalent cations. At 50 microM concentration, Al, Sc, Ga, In, Y, and La released 5(6)-carboxyfluorescein from liposomes and the magnitude of the effect was Al, In > Ga, Sc > La, Y. At concentrations between 10 and 200 microM, Sc, Ga, In, Y, and Be caused liposome aggregation and fusion. Al, Sc, Ga, In, and Be had their maximal effect on liposome fusion and aggregation at 100 microM; Y and La had their maximal effect at 20 microM. Metal-induced fusion was dependent on the negative charge density of the liposomes. Sc, Ga, In, Be, and Y stimulated Fe2(+)-initiated lipid peroxidation in a time- and dose-dependent manner. The fusogenic capacity of these nonredox metals was positively correlated with their capacity to induce Fe2(+)-supported lipid peroxidation. Results suggest that metals without redox capacity can promote lipid peroxidation, in the presence of an initiator of the oxidative chain, by affecting membrane physical properties.