RESUMO
A phospholipase A2 (PLA2, EC 3.1.1.4) from rat lung has been isolated and characterized. PLA2 was purified with ion-exchange and affinity chromatography and the purified enzyme was characterized with regard to pH optimum and calcium dependence. The isolated enzyme was also analyzed with two-dimensional gel electrophoresis and identified by Western immunoblots. The enzyme activity was found to be highest at pH 9.5-10.0, with a requirement for calcium, and the molecular mass was estimated to be 12 kDa by means of SDS-polyacrylamide gel electrophoresis. The two-dimensional gel electrophoresis analysis revealed two isoforms of PLA2 with isoelectric points of 7.8 and 9.5. On DEAE-Sepharose, PLA2 eluted as two peaks, one in the flow-through fraction and the other with increased salt concentration. Both peaks contained the same two PLA2 isoforms, as judged by two-dimensional gel electrophoresis. These results demonstrate the presence in rat lung of two isoforms of a calcium-dependent 12 kDa PLA2 with alkaline pH optimum. Using two-dimensional gel electrophoresis, this enzyme can be identified also in rat bronchoalveolar lavage fluid.
Assuntos
Pulmão/enzimologia , Fosfolipases A/isolamento & purificação , Fosfolipases/isolamento & purificação , Animais , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Cinética , Masculino , Peso Molecular , Fosfolipases A/metabolismo , Fosfolipases A2 , Ratos , Ratos EndogâmicosRESUMO
The postnatal development of group I phospholipase A2 (group I PLA2) in the glandular stomach and pancreas of neonatal rats was investigated. The amounts of group I PLA2 mRNA (and also the PLA2 enzymatic activity) in the glandular stomach mucosa increased with age in 3-60-day-old animals. This postnatal development of rat stomach group I PLA2 mRNA agreed with that of group I PLA2 mRNA of the rat pancreas, and thus seems to follow the general development of the gastrointestinal tract during the neonatal period. The latter was further supported by the finding that maturation of group I PLA2 in both the stomach and pancreas was induced precociously in rats treated with cortisone acetate. It is suggested that the stomach group I PLA2 is involved in mucosal eicosanoid production.
Assuntos
Cortisona/análogos & derivados , Mucosa Gástrica/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Pâncreas/enzimologia , Fosfolipases A/genética , Animais , Animais Recém-Nascidos , Northern Blotting , Cortisona/farmacologia , Mucosa Gástrica/crescimento & desenvolvimento , Masculino , Pâncreas/crescimento & desenvolvimento , Fosfolipases A2 , RNA Mensageiro/genética , Ratos , Ratos EndogâmicosRESUMO
The influence of green tea polyphenols (GTP) on the formation of DNA strand breaks (DNA-SB) and lipid peroxidation products (LPP) in cultured human lung cells (A 549) exposed to different oxidants was investigated. Cells were pretreated with GTP for 2 h and then exposed to cigarette smoke solution, H2O2 or FeCl3 for 30 min. After exposure, the cells were analyzed for DNA-SB, LPP, and viability. In addition, the effects of GTP added directly to the incubation mixtures during exposure were examined, using the same end points. It appeared that pretreatment with GTP inhibited both cigarette smoke- and H2O2-induced DNA breakage; i.e., following exposure to cigarette smoke or H2O2, the fraction of DNA passing through a microfilter increased significantly in cells not subjected to GTP, but this effect was prevented or inhibited in GTP-treated cells. Pretreatment with GTP also reduced the overall toxicity of H2O2 as determined by cell growth after exposure. Moreover, addition of GTP during exposure reduced both cigarette smoke- and H2O2-induced DNA breakage as well as formation of LPP after exposure to Fe3+. These results indicate that GTP inhibit the formation of DNA-SB in cells exposed to oxidants. It is possible that this ability to GTP to inhibit DNA-SB formation might contribute to the antitumorogenic properties of green tea.
Assuntos
Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Dano ao DNA , Flavonoides , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Fenóis/isolamento & purificação , Fenóis/farmacologia , Polímeros/isolamento & purificação , Polímeros/farmacologia , Chá/química , Linhagem Celular , Cloretos , Compostos Férricos/toxicidade , Radicais Livres/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias/prevenção & controle , Oxidantes/toxicidade , Plantas Tóxicas , Polifenóis , Espécies Reativas de Oxigênio/metabolismo , Fumaça/efeitos adversos , NicotianaRESUMO
An automated analytical method has been developed for determination of the oxidative DNA adduct, 8-hydroxydeoxyguanosine (8OHdG) in human urine, based on coupled-column high performance liquid chromatography with electrochemical detection. Urine is concentrated on Bondelut CH by means of an automated sample processor, and the enriched sample injected on to a polymeric reversed phase column coupled in line with an electrochemical detector and a C18 reversed phase column. By use of the electrochemical detector, a suitable retention time interval is set for collection of the fraction containing 8OHdG from the chromatography on the first column; this fraction is collected in a 2 mL loop and injected onto the C18 column. The system is operated by an automatic valve station controlled by an integrator. The method has a large sample capacity and measures 31.1, 15.7, and 7.43 nmol 8OHdG/L urine with variation coefficients of 8, 8 and 24% within series and 8, 11 and 23% between series. Normal healthy individuals were found to excrete 14.9 +/- 7.8 nmol 8OHdG/24 h, or 1.11 +/- 0.62 mumol 8OHdG per mol creatinine, in their urine, whereas increased levels of 8OHdG were found in 24 h collections from a variety of cancer patients, both in samples taken before onset of oncological therapy (1.84 +/- 1.12 mumol/mol creatinine, P < 0.01 versus healthy individuals) and after therapy onset (2.18 +/- 1.44 mumol/mol creatinine, P < 0.001 versus healthy individuals). Moreover, mean values of 8OHdG in random urinary samples from cancer patients were significantly higher than from healthy individuals (2.42 +/- 2.28 versus 1.19 +/- 0.48 mumol/mol creatinine, P < 0.001), both in samples taken before therapy onset (1.91 +/- 0.96, P < 0.001 versus healthy individuals) and after (2.57 +/- 2.46, P < 0.001 versus healthy individuals). High levels of urinary 8OHdG were found in patients subjected to whole body irradiation, and in patients receiving chemotherapy with various cytostatic agents. The potential use of the method for detecting increased urinary 8OHdG excretion and conditions associated with increased oxidative DNA damage in humans is discussed.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dano ao DNA , Desoxiguanosina/análogos & derivados , Neoplasias/urina , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Idoso , Antineoplásicos/uso terapêutico , Desoxiguanosina/urina , Eletroquímica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Distribuição Aleatória , Sensibilidade e Especificidade , Irradiação Corporal TotalRESUMO
Nitric oxide (NO) has a number of important functions in biological systems and may play a role in the toxicity of mineral fibers. We investigated whether NO might be present on the surface of mineral fibers and if crocidolite could adsorb NO from NO gas or cigarette smoke. NO was determined with a new gas chromatography-ultraviolet spectrophotometric technique after thermal desorption from the fiber surface and injection in a gas flow cell. NO was found in different amounts on chrysotile B, crocidolite, amosite, and silicon carbide whiskers. There was a strong correlation between the amount of NO and the specific surface area of these fibers (r = 0.98). NO could not be demonstrated on rockwool fibers [man-made vitreous fiber(s) (MMVF)21 and MMVF22] or silicon nitride whiskers. NO on crocidolite, amosite, and silicon carbide whiskers was readily desorbed from the fibers at increased temperature, while NO on chrysotile B seemed to be more firmly adsorbed to the fiber and required a longer period of time to be desorbed. The amount of NO bound to crocidolite increased from 34 micrograms/g fiber to 85 and 474 micrograms/g after exposing the fibers to cigarette smoke and NO gas, respectively. These findings indicate that a) NO adsorbs to fiber surfaces, b) some fibers adsorb more NO than others, c) some fibers adsorb NO more strongly than others, and d) the amounts of NO on fibers may be increased after exposure of the fiber to cigarette smoke or other sources of NO. The biological significance of NO on mineral fibers remains to be investigated.
Assuntos
Compostos Inorgânicos de Carbono/análise , Fibras Minerais/análise , Óxido Nítrico/análise , Compostos de Silício/análise , Adsorção , Asbesto Crocidolita/análise , Cromatografia Gasosa , Espectrofotometria UltravioletaRESUMO
We investigated whether exposure to indoor [gamma]-radiation and radon might be associated with enough free radical formation to increase urinary concentrations of 8-hydroxydeoxyguanosine (8-OHdG), a sensitive marker of DNA damage, due to a hydroxyl radical attack at the C8 of guanine. Indoor radon and [gamma]-radiation levels were measured in 32 dwellings for 6 months by solid-state nuclear track detectors and thermoluminescent dosimeters, respectively. Urine samples for 8-OHdG determinations were obtained from 63 healthy adult subjects living in the measured dwellings. An overall tendency toward increasing levels of 8-OHdG with increasing levels of radon and [gamma]-radiation was seen in the females, presumably due to their estimated longer occupancy in the dwellings measured. Different models were considered for females, with the steepest slopes obtained for [gamma]-radiation with a coefficient of 0.500 (log nmol/l of 8-OHdG for each unit increase of [gamma]-radiation on a log scale) (p<0.01), and increasing to 0.632 (p = 0.035), but with larger variance, when radon was included in the model. In conclusion, there seems to be an effect of indoor radioactivity on the urinary excretion of 8-OHdG for females, who are estimated to have a higher occupancy in the dwellings measured than for males, for whom occupational and other agents may also influence 8-OHdG excretion. ree radicals; [gamma]-radiation; radon.
Assuntos
Contaminação Radioativa do Ar/efeitos adversos , Dano ao DNA/efeitos da radiação , Desoxiguanosina/análogos & derivados , Exposição Ambiental/efeitos adversos , Radônio/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Idoso , Análise de Variância , Biomarcadores/urina , Desoxiguanosina/urina , Feminino , Radicais Livres/efeitos da radiação , Raios gama/efeitos adversos , Humanos , Estilo de Vida , Modelos Lineares , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos da radiação , Características de Residência/classificação , Distribuição por Sexo , Fumar/efeitos adversosRESUMO
OBJECTIVE: To study blood and bronchoalveolar lavage (BAL) fluid levels of platelet activating factor (PAF-acether) and phospholipase A2 (PLA2) in patients with septic shock or following severe trauma. DESIGN: Prospective controlled clinical study. SETTING: An intensive care unit (ICU) of a university hospital. PATIENTS AND PARTICIPANTS: The study comprised 12 patients, 8 with septic shock and 4 with trauma, consecutively admitted to the ICU. Healthy volunteers were used as controls. MEASUREMENTS AND RESULTS: Blood PAF-acether and plasma PLA2 levels were measured within 24 h after the patients arrival to the ICU. The Apache II score and outcome were registered. Median values for PAF-acether and PLA2 in the septic shock patients were 10.5 x 10(-10) M and 5300 units/ml, respectively, whereas corresponding values in the trauma patients were 1.3 x 10(-10) M and 770 units/ml. Normal healthy individuals had no detectable PAF-acether in the circulating blood (< 0.5 x 10(-10) M), and normal plasma PLA2 activity was < 300 units/ml. Moreover, both PLA2 and PAF-acether levels correlated well with the severity of the disease as assessed by the Apache II scoring system (p < 0.01 for PLA2 and p < 0.05 for PAF-acether). In addition, PAF-acether and PLA2 were determined in BAL fluid of patients with septic shock (n = 5) and trauma (n = 3); increased PAF-acether levels were found in four patients with septic shock and one patient with trauma. CONCLUSION: These results demonstrate a significant increase of both PLA2 and PAF-acether in the circulation of trauma patients, and a further increase in septic shock patients. It is possible that PAF-acether and PLA2 can be used as markers for the severity of the disease in septic shock and following severe trauma.
Assuntos
Traumatismo Múltiplo/sangue , Fosfolipases A/sangue , Fator de Ativação de Plaquetas/metabolismo , Choque Séptico/sangue , APACHE , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido da Lavagem Broncoalveolar/química , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Traumatismo Múltiplo/enzimologia , Fosfolipases A2 , Prognóstico , Estudos Prospectivos , Choque Séptico/enzimologiaRESUMO
This study demonstrates the formation of platelet-activating factor (PAF) in rats with acute experimental pancreatitis (AEP). The AEP was induced by infusing sodium taurodeoxycholate and trypsin into the bile-pancreatic duct. The PAF content was increased in blood samples and in pulmonary and pancreatic tissue as compared with control animals. Significant amounts of PAF were also found in peritoneal fluid. The PAF content did increase in blood samples and in pulmonary tissue after administration of endotoxin intravenously. The effects of intraperitoneal PAF administration were also studied and showed an increase of polymorphonuclear cells in blood samples. These findings suggest that acute pancreatitis might generate and release PAF. Whether PAF release is associated with the pathophysiology of complications in acute pancreatitis remains to be elucidated.
Assuntos
Pancreatite/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Doença Aguda , Animais , Masculino , Pancreatite/induzido quimicamente , Ratos , Ratos WistarRESUMO
The influence of quinacrine, a phospholipase A2 inhibitor, and enzymatic scavengers of active oxygen metabolites (superoxide dismutase and catalase) on ischemic small intestinal mucosal damage has been investigated. In the absence of an inhibitor, ischemia and reperfusion caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content, and increased myeloperoxidase and phospholipase A2 (PLA2) activities in the mucosa. All these effects of ischemia were efficiently inhibited by the PLA2 inhibitor quinacrine. On the other hand, superoxide dismutase together with catalase, even if it totally prevented the increased formation of malondialdehyde, was only able to reduce 50 percent of the increases of the other parameters. These findings indicate that, in addition to free radicals, other factors are involved in the pathogenesis of small intestinal mucosal injury after ischemia and reperfusion. We suggest that one such factor is the activation of PLA2 and the generation of various PLA2-dependent compounds such as arachidonic acid metabolites, lysophosphatidyl choline, and platelet-activating factor.
Assuntos
Íleo/irrigação sanguínea , Mucosa Intestinal/enzimologia , Isquemia/metabolismo , Oxigênio/metabolismo , Fosfolipases A/metabolismo , Fosfolipases/metabolismo , Acetilglucosaminidase/metabolismo , Animais , Catalase/farmacologia , Radicais Livres , Isquemia/enzimologia , Malondialdeído/metabolismo , Permeabilidade , Peroxidase/metabolismo , Fosfolipases A2 , Quinacrina/farmacologia , Ratos , Reperfusão , Superóxido Dismutase/farmacologiaRESUMO
This study demonstrates the ability of cigarette smoke condensate to generate hydrogen peroxide and to hydroxylate deoxyguanosine (dG) residues in isolated DNA to 8-hydroxydeoxyguanosine (8-OHdG). Both the formation of hydrogen peroxide and that of 8-OHdG in DNA was significantly decreased when catalase or tyrosinase was added to the smoke condensates, and this also occurred when pure hydroquinone or catechol, two major constitutes in cigarette smoke, was used instead of smoke condensate. Moreover, pure hydroquinone and catechol both caused dose-dependent formation of hydrogen peroxide and 8-OHdG, and there was good correlation between the amounts of hydrogen peroxide and 8-OHdG formed. These findings suggest that (i) hydroquinone and catechol may be responsible for the ability of cigarette smoke to cause 8-OHdG formation in DNA, (ii) this oxidative DNA-damage is due to the action of hydroxyl radicals formed during dissociation of hydrogen peroxide and (iii) the hydrogen peroxide in cigarette smoke is generated via autooxidation of hydroquinone and catechol.
Assuntos
Catecóis/toxicidade , Dano ao DNA , Desoxiguanosina/análogos & derivados , Hidroquinonas/toxicidade , Nicotiana , Plantas Tóxicas , Fumaça/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina , Animais , Catalase/metabolismo , Catecóis/metabolismo , Bovinos , DNA/efeitos dos fármacos , Desoxiguanosina/metabolismo , Peróxido de Hidrogênio/metabolismo , Hidroquinonas/metabolismo , Hidroxilação , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Fumaça/análiseRESUMO
Cigarette smoke can cause DNA single strand breaks in cultured human lung cells (T. Nakayama et al., Nature, 314 (1985) 462-464) but the mechanisms behind this DNA damage have not been clearly elucidated. In the present study we have investigated the possibility that one of the major constituents in cigarette smoke, hydroquinone, may be important for mediating smoke-induced DNA damage in the human epithelial lung cell line, A 549, and the mechanisms behind this damage. Cells were exposed to cigarette smoke, hydrogen peroxide, or hydroquinone, in the absence and presence of different inhibitors, and the resulting DNA damage was assessed either as DNA single strand break formation or formation of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. It was found that (i) exposure to cigarette smoke, hydrogen peroxide or hydroquinone causes a rapid decrease in the intracellular thiol level and a considerable DNA single strand break formation, (ii) the formation of DNA single strand breaks in cells exposed to cigarette smoke is inhibited by catalase, dimethylthiourea, and o-phenantroline, suggesting that hydroxyl radicals generated from iron-catalyzed hydrogen peroxide dissociation are involved in the DNA damage, (iii) hydroquinone causes considerable DNA strand break formation that is blocked by aurintricarboxylic acid, an inhibitor of endonuclease activation, and by BAPTA, an intracellular calcium chelator, (iv) addition of hydroquinone to a smoke condensate greatly enhances its ability to cause DNA single strand breaks, and (v) smoke, but not hydroquinone, causes formation of 8-hydroxydeoxyguanosine, a DNA damage product induced by the action of hydroxyl radicals on the DNA base, deoxyguanosine. These findings suggest that the ability of cigarette smoke to cause DNA single strand breaks in cultured lung cells is due to mechanisms involving hydroxyl radical attack on DNA and endonuclease activation. They also suggest that hydroquinone is an important contributor to the DNA damaging effect of cigarette smoke on human lung cells.
Assuntos
Dano ao DNA , Hidróxidos/metabolismo , Fumaça/efeitos adversos , Fumar , Linhagem Celular , DNA de Neoplasias/análise , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/metabolismo , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Radicais Livres , Humanos , Peróxido de Hidrogênio/farmacologia , Hidroquinonas/farmacologia , Radical Hidroxila , Cinética , Neoplasias Pulmonares , Timidina/metabolismoRESUMO
BACKGROUND: Previous investigations indicate that engine room personnel on ships are exposed to polycyclic aromatic hydrocarbons (PAH) from oil and oil products, with dermal uptake as the major route of exposure. Several PAH are known carcinogens and mutagens. AIMS: To investigate the urinary excretion of a marker for oxidative DNA damage, 8-hydroxydeoxy-guanosine (8OHdG), in engine room personnel, and to study the association between 8OHdG and 1-hydroxypyrene (1OHP), a biological marker for PAH exposure. METHODS: Urine samples were collected from engine room personnel (n = 36) on 10 Swedish and Norwegian ships and from unexposed controls (n = 34) with similar age and smoking habits. The exposure to oils, engine exhaust, and tobacco smoke 24 hours prior to sampling was estimated from questionnaires. The urinary samples were frozen for later analyses of 8OHdG and 1OHP by high performance liquid chromatography. RESULTS: Excretion in urine of 8OHdG (adjusted to density 1.022) was similar for controls (mean 18.0 nmol/l, n = 33), and for those who had been in the engine room without skin contact with oils (mean 18.7 nmol/l, n = 15). Engine room personnel who reported skin contact with oil had increased excretion of 8OHdG (mean 23.2 nmol/l, n = 19). The difference between this group and the unexposed controls was significant. The urinary levels of ln 1OHP and ln 8OHdG were significantly correlated, and the association was still highly significant when the effects of smoking and age were accounted for in a multiple regression analysis. CONCLUSION: Results indicate that exposure to PAH or possibly other compounds from skin contact with oils in engine rooms may cause oxidative DNA damage.
Assuntos
Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Exposição Ocupacional/efeitos adversos , Hidrocarbonetos Policíclicos Aromáticos/intoxicação , Navios , 8-Hidroxi-2'-Desoxiguanosina , Adolescente , Adulto , Biomarcadores/análise , Dano ao DNA , Monitoramento Ambiental/métodos , Humanos , Óleos Industriais/toxicidade , Masculino , Pessoa de Meia-Idade , Mutagênicos/análise , Estresse Oxidativo/efeitos dos fármacos , Pirenos/análise , Absorção Cutânea , Fumar/efeitos adversosRESUMO
Four cases of rapidly progressive glomerulonephritis with similar clinical courses are presented. Examination of kidney biopsies from these patients showed severe glomerulitis with capillary necrosis, fibrin thrombi and interstitial inflammation but no vasculitis. Electron microscopy showed wrinkled capillary basement membranes which were irregularly thickened, homogenous and had an irregular fibrillar structure. No localized deposits were observed. Immunohistological examination demonstrated linear and diffuse deposition of IgG and C3 along glomerular basement membranes. Nephrectomized kidneys from these patients were eluted and shown to contain antibodies against glomerular basement membrane. These antibodies were also present in sera of three of the patients.
Assuntos
Anticorpos/análise , Glomerulonefrite/imunologia , Rim/imunologia , Adolescente , Adulto , Infecções Bacterianas , Membrana Basal/imunologia , Biópsia por Agulha , Feminino , Imunofluorescência , Glomerulonefrite/etiologia , Glomerulonefrite/patologia , Humanos , Rim/patologia , Glomérulos Renais/imunologia , Glomérulos Renais/ultraestrutura , Masculino , Pessoa de Meia-IdadeRESUMO
The influence of quercetin, chlorpromazine, aristolochic acid, and indomethacin on group I phospholipase A2 (PLA2) from porcine pancreas and on group II PLA2 from Vipera russelli was compared. Quercetin and chlorpromazine were found to inhibit PLA2 activity in lower concentrations (< 100 microM), while aristolochic acid and indomethacin were inhibitory only in higher concentrations (> 100 microM). The order of potency against Vipera PLA2 was: quercetin > chlorpromazine > aristolochic acid > indomethacin, while the order of potency against pancreatic PLA2 was: chlorpromazine > aristolochic acid > indomethacin >> quercetin. Thus, quercetin was a potent inhibitor towards group II PLA2 (IC50 = 2 microM), but a very weak inhibitor against group I PLA2, with maximum 30% inhibition. Aristolochic acid and indomethacin were three to four times more potent towards group II PLA2 than towards group I PLA2, while chlorpromazine was equally potent towards the two PLA2 types. Quercetin and chlorpromazine were also tested against two PLA2 fractions purified from the plasma of septic shock patients; chlorpromazine was then equally potent towards the two PLA2 fractions, whereas quercetin was a potent inhibitor of only one of the two PLA2 fractions (IC50 = 4 microM). Together, these results indicate that (1) different PLA2 inhibitors have different potency depending on which type of PLA2 they are used against, (2) quercetin selectively inhibits group II PLA2 and may therefore be used to discriminate between different PLA2 forms in biological materials, and (3) both PLA2 of group I and group II are present in septic shock plasma.
Assuntos
Ácidos Aristolóquicos , Fosfolipases A/antagonistas & inibidores , Quercetina/farmacologia , Animais , Clorpromazina/farmacologia , Humanos , Indometacina/farmacologia , Pâncreas/enzimologia , Fenantrenos/farmacologia , Fosfolipases A/sangue , Fosfolipases A2 , Choque Séptico/enzimologia , Especificidade por Substrato , Suínos , Venenos de Víboras/enzimologiaRESUMO
The inhibitory effect of the plant flavonoid, rutin, on group I phospholipase A2 (PLA2-I) from porcine pancreas and Naja naja, and on group II phospholipase A2 (PLA2-II) from Vipera russelli and Crotalus atrox was investigated. Rutin efficiently inhibited PLA2-II from both Vipera russelli and Crotalus atrox but was only a weak inhibitor of PLA2-I from porcine pancreas and Naja naja. The lack of strong inhibition of pancreatic PLA2-I was not due to contaminating proteins in the enzyme preparation, since the same weak inhibition was obtained against pancreatic PLA2 purified to homogeneity as judged by two-dimensional gel electrophoresis. Rutin also efficiently inhibited human PLA2-II from synovial fluid but was only a weak inhibitor of human PLA2-I from pancreatic juice, suggesting that rutin is a selective PLA2-I from porcine pancreas. The results obtained indicate that the hydroxyl group in 5-position as well as the double bond and the double-bonded oxygen in the oxane ring are all important for the overall ability of flavonoids to inhibit PLA2 activity, and that the hydroxyl groups in 3'- and 4'- position are required for selective inhibition of PLA2-II.
Assuntos
Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Fosfolipases A/antagonistas & inibidores , Rutina/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Crotalus , Elapidae , Flavonoides/química , Humanos , Técnicas In Vitro , Mediadores da Inflamação/antagonistas & inibidores , Estrutura Molecular , Pâncreas/enzimologia , Suco Pancreático/enzimologia , Fosfolipases A/classificação , Fosfolipases A2 , Daboia , Rutina/química , Relação Estrutura-Atividade , Suínos , Líquido Sinovial/enzimologiaRESUMO
Phospholipase A2 plays an important part in the generation of inflammatory lipid mediators and so it is of major interest to understand functional distinctions between structurally similar forms of phospholipase A2. In the present study, the influence of zinc (Zn2+) on the activity of group I and group II phospholipase A2 was examined in vitro. It appeared that Zn2+ (0.04-1 x 10(-3)M) increased group I phospholipase A2 activity from porcine pancreas and rat lung whereas the activity of group II phospholipase A2 from Crotalus atrox and Vipera russelli was unaffected. The presence of Cd2+ of Hg2+ (0.8-5 x 10(-3)M) also increased group I pancreatic phospholipase A2 activity while no augmentation was found with Cr2+, Fe2+ or Mg2+. The selective stimulation of group I phospholipase A2 by Zn2+ corresponded to a binding of these phospholipases A2 to a zinc-affinity column, while group II phospholipase A2 was not bound. Furthermore, the PLA2 activity in bronchoalveolar lavage fluid from rat was stimulated by Zn2+. These results indicate that Zn2+ binds to and increases the activity of group I, but not group II phospholipase A2. This difference in Zn(2+)-binding may be used to discriminate between group I and group II phospholipase A2 and to separate the enzymes from each other in complex biological materials. The possibility that activation of group I phospholipase A2 in the lung is important in zinc-induced metal fume fever is implied.
Assuntos
Isoenzimas/metabolismo , Fosfolipases A/metabolismo , Venenos de Serpentes/enzimologia , Zinco/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Cádmio/farmacologia , Cátions Bivalentes/farmacologia , Cromatografia de Afinidade , Venenos de Crotalídeos/enzimologia , Ativação Enzimática/efeitos dos fármacos , Fosfolipases A2 do Grupo II , Isoenzimas/classificação , Pulmão/enzimologia , Mercúrio/farmacologia , Especificidade de Órgãos , Pâncreas/enzimologia , Fosfolipases A/classificação , Fosfolipases A2 , Ligação Proteica , Ratos/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Suínos/metabolismo , Venenos de Víboras/enzimologia , Zinco/metabolismoRESUMO
The influence of two surface-active food additives on the integrity and permeability of rat ileal mucosa has been studied. We determined the activity of N-acetyl-beta-glucosaminidase, a lysosomal enzyme, in the rat intestinal lumen after deposition of polyoxyethylene (20) sorbitan monostearate (polysorbate 60; Tween 60) or polyoxyethylene (20) sorbitan monooleate (polysorbate 80; Tween 80) in a section of ligated, cannulated gut. We also determined the activities of N-acetyl-beta-glucosaminidase, alkaline phosphatase, 5'-nucleotidase and phospholipase A2 in mixtures of isolated mucosal cells and polysorbate 60 or polysorbate 80. The activity of N-acetyl-beta-glucosaminidase was increased in the luminal contents of the cannulated gut 15 min after deposition of either polysorbate 60 or polysorbate 80 (10 mg/ml fluid instilled into gut). It was also increased in mixtures of mucosal cells and polysorbate 60 or polysorbate 80 (0.1-10 mg/ml). In contrast, the activities of alkaline phosphatase and 5'-nucleotidase were unaffected and that of phospholipase A2 was decreased by the presence of either polysorbate. These findings indicated that polysorbate 60 and polysorbate 80 released lysosomal enzymes from the intestinal mucosal cells and that these agents might damage the intestinal mucosa and increase its permeability. We therefore determined the intestinal permeability to sodium fluorescein in the absence and presence of polysorbate 60 or 80 and found that the permeability was slightly increased in the presence of either of the compounds at concentrations of 10 mg/ml fluid instilled into gut. It is possible therefore that surface-active food additives might impair the function of the mucosal barrier and increase the permeability of the gut to potentially toxic and pathogenic molecules.
Assuntos
Acetilglucosaminidase/metabolismo , Fosfatase Alcalina/metabolismo , Aditivos Alimentares , Hexosaminidases/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Nucleotidases/metabolismo , Polissorbatos/farmacologia , 5'-Nucleotidase , Animais , Interações Medicamentosas , Feminino , Fluoresceína , Fluoresceínas/farmacologia , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Permeabilidade , Fosfolipases A/metabolismo , Fosfolipases A2 , Ratos , Ratos EndogâmicosRESUMO
OBJECTIVES: Airborne exposure to polycyclic aromatic hydrocarbons (PAH) in the potroom of an aluminum reduction plant was studied in relation to genotoxic or mutagenic effects, and the possibility of host genotypes of different metabolizing enzymes modifying associations between PAH exposure and genotoxic or mutagenic response was assessed. SUBJECTS AND METHODS: Ninety-eight male potroom workers and 55 male unexposed blue-collar workers constituted the study population. Micronuclei in CD4+ and CD8+ lymphocytes, DNA (deoxyribonucleic acid) single-strand breaks, hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation frequency, and genotype for cytochrome P-4501A1, glutathione transferases M1, T1 and P1, and microsomal epoxide hydrolase were analyzed using peripheral mononuclear cells. Urine samples were collected for the analysis of 8-hydroxydeoxyguanosine. RESULTS: Micronuclei in peripheral CD4+ and CD8+ lymphocytes, DNA single-strand breaks, HPRT mutation frequency, and 8-hydroxydeoxyguanosine in urine did not differ between the potroom workers and the unexposed referents. With the exception of an observed exposure-response relationship for potroom workers with Tyr/Tyr genotype for microsomal epoxide hydrolase, between airborne PAH and CD8+ micronuclei, no correlations were found between any of the genotoxicity biomarkers and any of the exposure measures (airborne particulate PAH, airborne gas phase PAH, length of employment in the potroom, 1-hydroxypyrene in urine, or PAH-DNA adducts in peripheral lymphocytes), also when genotypes for biotransforamtion enzymes were considered. CONCLUSIONS: The results indicate that the employed biomarkers of mutagenic or genotoxic effects are not appropriate for surveillance studies of potroom workers exposed to current airborne levels of PAH. The significance of the correlation between airborne PAH and CD8+ micronuclei in Tyr/Tyr genotype subjects should be evaluated.
Assuntos
Indústria Química , Desoxiguanosina/análogos & derivados , Glutationa Transferase/genética , Hidrocarbonetos Aromáticos , Leucócitos Mononucleares , Micronúcleos com Defeito Cromossômico , Mutação , Exposição Ocupacional , Polimorfismo Genético , 8-Hidroxi-2'-Desoxiguanosina , Biomarcadores , Biotransformação , Desoxiguanosina/urina , Eletroforese em Gel de Ágar , Genótipo , Humanos , MasculinoRESUMO
Lysosomes and brush border membranes from diseased ileal mucosa of a patient with Crohn's disease were separated by rate zonal density gradient centrifugation. The organelles were located and characterized in the density gradient of assay of marker enzymes, and their enzyme content compared to that of similar organelles from normal tissue. The activity and latency of lysosomal enzymes (N-acetyl-beta-glucosaminidase and acid phosphatase) was significantly increased, whereas the activity of brush border alkaline phosphatase was rather decreased. The possible relevance of these findings to the pathophysiology of Crohn's disease is discussed.
Assuntos
Doença de Crohn/enzimologia , Íleo/enzimologia , Mucosa Intestinal/enzimologia , Acetilglucosaminidase/metabolismo , Fosfatase Ácida/metabolismo , Idoso , Fracionamento Celular , Centrifugação Zonal , Feminino , Humanos , Lisossomos/enzimologia , Masculino , Microvilosidades/enzimologia , Pessoa de Meia-IdadeRESUMO
Exposure to zinc fume may cause metal fume fever, an acute reaction characterized by an invasion of neutrophils into the airways. This investigation was conducted to examine the possibility that Zn2+ and ZnO might stimulate the formation of oxygen radicals by human neutrophils. Luminol-amplified chemiluminescence (CL) was monitored during 2 h from human neutrophils exposed to Zn2+ or ZnO. The response was compared to that of other metal ions and to that of endotoxin and phorbol myristate acetate (PMA). Zn2+ (6-50 microM) gradually caused a 2-6-fold increase of CL that reached an optimum after 70- 80 min. By contrast, Cd2+, Cr2+, Cr3+, Fe2+, Fe3+, Ni2+ or Co2+ in corresponding concentrations did not increase the CL. Similar to Zn2+, endotoxin (40-640 micrograms/ml) caused a 2-5-fold increase of CL with an optimum after 70 min, and endotoxin (40 micrograms/ml) together with Zn2+ (50 microM) synergistically increased the CL. ZnO (12-100 micrograms/ml) also augmented CL, with a 1.5-5-fold increase at 25-100 micrograms/ml ZnO but with a time response similar to that found after PMA stimulation, in which CL peaked after 20-40 min incubation. Both Zn(2+)- and ZnO-induced CL was inhibited by manoalide, a phospholipase A2 inhibitor, with IC50 of 0.25 microM and 0.66 microM respectively. These results indicate that Zn2+ and ZnO both stimulates oxygen radical formation in human neutrophils and that this might contribute to the pathogenesis of zinc fume fever.