RESUMO
AIMS: To preliminarily explore, whether glucocorticoids have a therapeutic effect on diquat-induced acute kidney injury in rats. METHOD: 150 Wistar rats were randomly divided into six groups: exposure model group (DQ group), dexamethasone control group (GC group), blank control group (Ctrl group), dexamethasone 2.1 mg/kg dose group (DQ+L-GC group), dexamethasone 4.2 mg/kg dose group (DQ+M-GC group), and dexamethasone 8.4 mg/kg dose group (DQ+H-GC group), with 25 rats in each group. Each group was further divided into five subgroups, 24 h, 3 d, 7 d, 14 d, and 21 d after exposure, according to the feeding time and the course of treatment, with five animals in each subgroup. The rats in DQ, DQ+L-GC, DQ+M-GC, and DQ+H-GC groups were administered 115.5 mg/kg diquat by gavage, respectively. Moreover, 30 min after gavage, rats in DQ+L-GC group, DQ+M-GC group, DQ+H-GC group and GC group were intragastric administered dexamethasone 2.1 mg/kg, 4.2 mg/kg, 8.4 mg/kg and 8.4 mg/kg, respectively. After 7 days, the intraperitoneal injection of dexamethasone was changed to 6.3 mg/kg prednisone by intragastric administration. Subsequently, 7 days later, it was changed to 3.15 mg/kg prednisone by intragastric administration until the end of the experiment on 21 days. After the start of the experiment, changes in the conditions of the rats in each group were observed at a fixed time every day, changes in the body weight of the rats were monitored at the same time, and the death of the rats was recorded at 24 h, 3 d, 7 d, 14 d, and 21 d after exposure. The rats were sacrificed by an intraperitoneal injection of 100 mg/kg sodium pentobarbital overdose. Blood was collected by puncture of the inferior vena cava, used to determine Cr and BUN. The upper segment of the left kidney was collected for histopathological examination. Elisa was used to detect neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in the lower segment of left kidney. TLR4, Myd88, and NF-κB were detected in the right kidney. RESULTS: (1) After exposure, most rats in DQ group, DQ+L-GC group, DQ+M-GC group and DQ+H-GC group showed shortness of breath, oliguria, diarrhea, yellow hair and other symptoms. No symptoms and related signs were observed in Ctrl group and GC group. (2) The weight of rats in the Ctrl group and the GC group increased slowly during the test. the body weight of the rats in the DQ, DQ+L-GC, DQ+M-GC, and DQ+H-GC groups continued to decrease after self-infection. Body weight dropped to the lowest point at approximately 7 d, and gradually increased from 7 d to 21 d. (3) A small amount of capillary congestion in the medulla was observed after 7 days in the GC group. The DQ group showed tubular atrophy, edema of the epithelial cells, and over time, the tubules were seen dilated and became irregular in shape; large amount of capillary congestion was also observed in the renal cortex and medulla. The renal injury in the DQ+L-GC group was less than that in the DQ group. DQ+H-GC group had no obvious injury before 7 d, but more renal tubules were seen in the DQ+H-GC group from 7 d to 14 d. (4) Compared with the DQ group, there was no difference before 14 d, and at 14 d-21 d, DQ+L-GC group, DQ+M-GC group, DQ+H-GC group all had different degrees of decline. NGAL content: Compared with the DQ group, the content of NGAL and KIM-1 in kidney tissue of the DQ+L-GC, DQ+M-GC, and DQ+H-GC groups decreased compared with the DQ group at each time node. (5) Compared with the Ctrl group, the expression of TNF-α, TLR4, MyD88, NF-κB in the DQ, DQ+L-GC, DQ+M-GC, and DQ+H-GC groups at each time node increased in the renal tissue. The content of TNF-α, TLR4, MyD88, NF-κB in kidney tissue of the DQ+L-GC, DQ+M-GC, and DQ+H-GC groups at each time node was lower than that in the DQ group. CONCLUSION: (1) Diquat can cause kidney damage in rats, mainly manifested as renal tubular atrophy, epithelial cell edema, capillary congestion and dilation, and the renal function damage indicators have been improved to varying degrees. (2) Glucocorticoids have therapeutic effects on acute kidney injury in rats exposed to diquat. During the treatment, the efficacy of glucocorticoids did not increase with increasing doses after reaching a dose of 4.2 mg/kg. (3) TLR4 receptor-mediated TLR4/Myd88/NF-κB signaling pathway is involved in the inflammatory response of acute kidney injury in diquat poisoning rats. Glucocorticoids can inhibit the inflammatory response, thereby affecting the expression of TLR4/Myd88/NF-κB signaling pathway-related proteins.
Assuntos
Injúria Renal Aguda , NF-kappa B , Ratos , Animais , Ratos Wistar , NF-kappa B/metabolismo , Glucocorticoides/toxicidade , Diquat/farmacologia , Lipocalina-2 , Prednisona/farmacologia , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/patologia , Rim , Dexametasona/farmacologia , Peso Corporal , Atrofia/patologiaRESUMO
The aim of this study was to investigate the protective effects of glutathione (GSH) against oxidative stress and intestinal barrier disruption caused by diquat (an oxidative stress inducer) in weaned piglets. Twenty-four piglets were randomly assigned to four treatments with six pigs per treatment for an 18-d trial. Treatments were basal diet, basal diet + diquat challenge, 50 mg/kg GSH diets + diquat challenge and 100 mg/kg GSH diets + diquat challenge. On day 15, piglets in basal diet group and diquat-challenged groups were intraperitoneally injected with sterile saline and diquat at 10 mg/kg body weight, respectively. The results showed that GSH supplementation improved growth performance of diquat-injected piglets from days 15 to 18 (p < 0.05), especially at a dose of 100 mg/kg GSH. Meanwhile, diquat also caused oxidative stress and intestinal barrier damage in piglets. However, GSH supplementation enhanced the antioxidant capacity of serum and jejunum, as evidenced by the increase in GSH content and total superoxide dismutase activities and the decrease in 8-hydroxy-2'-deoxyguanosine concentrations (p < 0.05). GSH also up-regulated the mRNA expressions of intestinal tight junction protein (zonula occludens 1, ZO1; occludin, OCLN; claudin-1, CLDN1) and mitochondrial biogenesis and function (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, PGC1α; mitochondrial transcription factor A, TFAM; cytochrome c, CYCS), compared with diquat-challenged piglets in basal diet (p < 0.05). Thus, the study demonstrates that GSH protects piglets from oxidative stress caused by diquat and 100 mg/kg GSH has a better protective role.
Assuntos
Dieta , Diquat , Animais , Suínos , Diquat/farmacologia , Dieta/veterinária , Suplementos Nutricionais , Ração Animal/análise , Estresse Oxidativo , Glutationa/farmacologiaRESUMO
This study was designed to evaluate the antioxidant and anti-inflammatory effects of Eucommia ulmoides flavones (EUF) using diquat-challenged piglet models. A total of 96 weaned piglets were randomly allotted to 1 of 3 treatments with 8 replication pens per treatment and 4 piglets per pen. The treatments were basal diet, basal diet + diquat, and 100 mg/kg EUF diet + diquat. On day 7 after the initiation of treatment, the piglets were injected intraperitoneally with diquat at 8 mg/kg BW or the same amount of sterilized saline. The experiment was conducted for 21 days. EUF supplementation improved the growth performance of diquat-treated piglets from day 14 to 21. Diquat also induced oxidative stress and inflammatory responses and then impaired intestinal morphology. But EUF addition alleviated these negative effects induced by diquat that showed decreasing serum concentrations of proinflammatory cytokines but increasing antioxidant indexes and anti-inflammatory cytokines on day 14. Supplementation of EUF also increased villi height and villous height, crypt depth, but decreased the histopathological score and MPO activity compared with those of diquat-challenged pigs fed with the basal diet on day 14. Results indicated that EUF attenuated the inflammation and oxidative stress of piglets caused by diquat injection.
Assuntos
Antioxidantes/metabolismo , Diquat/farmacologia , Eucommiaceae/efeitos dos fármacos , Flavonas/metabolismo , Animais , Suplementos Nutricionais , Estresse Oxidativo , SuínosRESUMO
Reactive oxygen species (ROS) are closely related to the follicular granulosa cell apoptosis. Grape seed procyanidin B2 (GSPB2) has been reported to possess potent antioxidant activity. However, the GSPB2-mediated protective effects and the underlying molecular mechanisms in granulosa cell apoptosis process remain unknown. In this study, we showed for the first time that GSPB2 treatment decreased FoxO1 protein level, improved granulosa cell viability, upregulated LC3-II protein level, and reduced granulosa cell apoptosis rate. Under a condition of oxidative stress, GSPB2 reversed FoxO1 nuclear localization and increased its level in cytoplasm. In addition, FoxO1 knockdown inhibited the protective effects of GSPB2 induced. Our findings suggest that FoxO1 plays a pivotal role in regulating autophagy in granulosa cells, GSPB2 exerts a potent and beneficial role in reducing granulosa cell apoptosis and inducing autophagy process, and targeting FoxO1 could be significant in fighting against oxidative stress-reduced female reproductive system diseases.
Assuntos
Apoptose/efeitos dos fármacos , Biflavonoides/farmacologia , Catequina/farmacologia , Proteína Forkhead Box O1/metabolismo , Células da Granulosa/patologia , Extrato de Sementes de Uva/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proantocianidinas/farmacologia , Substâncias Protetoras/farmacologia , Animais , Antioxidantes/metabolismo , Autofagia/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Diquat/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Peróxido de Hidrogênio/farmacologia , Malondialdeído/metabolismo , Camundongos Endogâmicos ICR , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Diquat toxicity causes iron-mediated oxidative stress; however, it remains unclear how diquat affects iron metabolism. Here, we examined the effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats, with particular focus on gene expression. Hepatic nonheme iron content was unchanged until 20 h after diquat treatment. Hepatic free iron levels increased markedly in the early stages following treatment and remained elevated for at least 6 h, resulting in severe hepatotoxicity, until returning to control levels at 20 h. The level of hepatic ferritin, especially the H-subunit, increased 20 h after diquat treatment due to elevated hepatic ferritin-H mRNA expression. These results indicate that early elevated levels of free iron in the liver of diquat-treated rats cause hepatotoxicity, and that this free iron is subsequently sequestered by ferritin synthesized under conditions of oxidative stress, thus limiting the pro-oxidant challenge of iron. The plasma iron concentration decreased at 6 and 20 h after diquat treatment, whereas the level of plasma interleukin-6 increased markedly at 3 h and remained high until 20 h. In the liver of diquat-treated rats, expression of hepcidin mRNA was markedly upregulated at 3 and 6 h, whereas ferroportin mRNA expression was downregulated slightly at 20 h. Transferrin receptor 1 mRNA expression was significantly upregulated at 3, 6, and 20 h. These results indicate that inhibition of iron release from iron-storage tissues, through stimulation of the interleukin-6-hepcidin-ferroportin axis, and enhanced iron uptake into hepatocytes, mediated by transferrin receptor 1, cause hypoferremia.
Assuntos
Diquat/farmacologia , Herbicidas/farmacologia , Ferro/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Alanina Transaminase/metabolismo , Animais , Apoferritinas/genética , Apoferritinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Interleucina-6/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
Melanin-based traits involved in animal communication have been traditionally viewed as occurring under strict genetic control. However, it is generally accepted that both genetic and environmental factors influence melanin production. Medical studies suggest that, among environmental factors influencing melanization, oxidative stress could play a relevant role. On the other hand, genetic control would be exerted by the melanocortin system, and particularly by the alpha-melanocyte-stimulating hormone (alpha-MSH), which triggers the production of eumelanins (black pigments). To determine how the melanocortin system and an exogenous source of oxidative stress interact in the expression of melanin-based plumage, developing red-legged partridges (Alectoris rufa) were manipulated. Some partridges were injected with alpha-MSH, while other birds received a pro-oxidant molecule (diquat) in drinking water. Controls and birds receiving both treatments were also studied. Both alpha-MSH- and diquat-treated individuals presented larger eumelanin-based traits than controls, but alpha-MSH+diquat-treated birds showed the largest traits, suggesting that oxidative stress and melanocortins promote additive but independent effects. Diquat also induced a decline in the level of a key intracellular antioxidant (glutathione), which is associated with high expression of eumelanin-based signals in other bird species. Some scenarios for the evolution of melanin-based traits in relation to oxidative stress are proposed.
Assuntos
Plumas/metabolismo , Galliformes/metabolismo , Melaninas/metabolismo , Melanocortinas/metabolismo , Estresse Oxidativo , Animais , Antioxidantes/metabolismo , Diquat/farmacologia , Eritrócitos/metabolismo , Plumas/anatomia & histologia , Plumas/efeitos dos fármacos , Galliformes/anatomia & histologia , Galliformes/crescimento & desenvolvimento , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , alfa-MSH/farmacologiaRESUMO
Biochemical and gross pathological effects of diquat were studied with special attention to cysteine proteinase inhibitor level which was often increased in acute and chronic disorder. Diquat was fed continuously to rats at the dose of 1000 ppm in the diet. After 10 days, anorexia and severe diarrhea were observed but epistaxis and hypokinesia were not apparent. The rats were killed after feeding the diet for 13.5 days and plasma components such as acute phase reactant proteins and some vitamins which act as antioxidants were examined. The results showed that alpha-cysteine proteinase inhibitor (alpha-CPI) increased to 9-fold and vitamin C radical increased to 1.6-fold, whereas alpha 1 proteinase inhibitor (alpha 1-PI) decreased to 0.9-fold and vitamins C and E were the same as the control. Among three components of alpha-CPI, the T kininogen level in intoxicated rat plasma was about 20-fold, whereas the high molecular weight kininogen level was about 2-fold of the control. Diquat also enhanced the cysteine proteinase inhibitor (CPI) level to 20-fold in kidney and to 7- to 10-fold in the other organs. The large increment of T kininogen in these organs was also confirmed immunologically. The kidney showed a granular degeneration and its weight increased to 1.2-fold of control. The other organs showed neither gross pathological alteration nor weight change, compared with the control. The diquat distribution was highest in spleen and next highest in kidney among several organs. These results were compared with those caused by paraquat.
Assuntos
Inibidores de Cisteína Proteinase/sangue , Inibidores de Cisteína Proteinase/farmacocinética , Diquat/farmacologia , Animais , Inibidores de Cisteína Proteinase/biossíntese , Imunodifusão , Masculino , Ratos , Ratos WistarRESUMO
Positively charged hydrophobic pesticides of the dipyridyl family [diquat, paraquat, benzylviologen (BV++), etc.] were shown to provoke accumulation of 2-methylbutane-1,2,3,4-tetraol-2,4- cyclopyrophosphate in the cells Corynebacterium (Brevibacterium) ammoniagenes while neutral dipyridyls were not. Hydrophobicity was also an important factor in this phenomenon. Of the other pesticides tested, only linuron was effective. BV++ also induced biosynthesis of the compound in Rhodococcus rhodochrous, Rh.ruber, Rh.sp. (Nocardia corynebacteroides). These microorganisms as well as most of the previously identified oxidative stress activated producers of this new cyclopyrophosphate were able to synthesize free radical generating compounds. The microorganisms concerned belong mainly to the order Actinomycetales.
Assuntos
2,2'-Dipiridil/farmacologia , Bactérias/metabolismo , Eritritol/análogos & derivados , Praguicidas/farmacologia , 2,2'-Dipiridil/química , Bactérias/efeitos dos fármacos , Benzil Viologênio/farmacologia , Fenômenos Químicos , Físico-Química , Corynebacterium/efeitos dos fármacos , Corynebacterium/metabolismo , Diquat/farmacologia , Eletroquímica , Eritritol/metabolismo , Linurona/farmacologia , Nocardia/efeitos dos fármacos , Nocardia/metabolismo , Paraquat/farmacologia , Praguicidas/química , Rhodococcus/efeitos dos fármacos , Rhodococcus/metabolismo , Relação Estrutura-AtividadeRESUMO
The potential toxicity of enhanced intracellular reactive oxygen formation was investigated in isolated perfused livers of male Fischer rats. The presence of the redox-cycling agent diquat in the perfusate (200 microM) increased the basal efflux of glutathione disulfide (GSSG) into bile (2.65 +/- 0.26 nmol GSH-equivalents/min per g liver wt.) and perfusate (0.55 +/- 0.15 nmol/min per g) approximately 10-fold. Since no evidence was found for degradation of GSSG in the biliary tract of these animals, it could be estimated that diquat induced a constant O2- generation of approximately 1000 nmol/min per g liver wt for 1 h. Thus, reactive oxygen formation under these conditions was 1-2 orders of magnitude higher than under various pathophysiological conditions. Only minor liver injury (release of lactate dehydrogenase activity) was observed. To increase the susceptibility of the liver to the oxidant stress, animals were pretreated in vivo with 200 mg/kg body wt. phorone, which caused a 90% depletion of the hepatic glutathione content, 100 mg/kg ferrous sulfate, a combination of phorone and ferrous sulfate, or 40 mg/kg BCNU, which caused a 60% inhibition of hepatic GSSG reductase. Only the combined treatment of phorone + ferrous sulfate or BCNU caused a significant increase of the diquat-induced liver injury. Our results demonstrated an extremely high resistance of the liver against intracellular reactive oxygen formation (even with impaired detoxification systems) and can serve as reference for the evaluation of potential contributions of reactive oxygen to liver injury in various disease states.
Assuntos
Fígado/metabolismo , Superóxidos/metabolismo , Animais , Bile/metabolismo , Carmustina/farmacologia , Doença Hepática Induzida por Substâncias e Drogas , Diquat/farmacologia , Compostos Ferrosos/farmacologia , Glutationa/metabolismo , Glutationa Redutase/antagonistas & inibidores , Cetonas/farmacologia , Fígado/efeitos dos fármacos , Masculino , Oxirredução , Ratos , Ratos Endogâmicos F344RESUMO
Metallothionein (MT) is a low-molecular-weight protein with a high cysteine content that has been proposed to play a role in protecting against oxidative stress. For example, MT has been shown to be a scavenger of hydroxyl radicals in vitro, and cells with high levels of MT are resistant to radiation. However, it is not known if compounds that cause oxidative stress affect MT levels. Therefore, mice were injected subcutaneously with 11 chemicals (t-butyl hydroperoxide, paraquat, diquat, menadione, metronidazole, adriamycin, 3-methylindole, cisplatin, diamide, diethyl maleate, and phorone) that produce oxidative stress by four main mechanisms. MT was quantitated in the cytosol of major organs (liver, pancreas, spleen, kidney, intestine, heart, and lung) by the Cd/hemoglobin radioassay 24 hr after administration of the chemicals. All agents significantly increased MT levels in at least one organ. Liver was the most responsive to these agents in that all 11 chemicals increased MT concentrations in liver, with diethyl maleate, paraquat, and diamide producing 20- to 30-fold increases. Pancreas and kidney were the next most responsive organs to these chemicals. The organ least responsive to these agents was the heart, as only 3 compounds caused significant increases in MT concentrations in heart. Diethyl maleate and diquat were the most general inducers of MT in that they increased MT in six of the seven organs examined. No treatment resulted in a significant decrease in MT concentration in any organ. In conclusion, chemicals that produce oxidative stress by one of four distinct mechanisms are very effective at increasing MT concentrations in a variety of organs. This suggests that MT might be involved in protecting against oxidative stress.
Assuntos
Fígado/metabolismo , Metalotioneína/biossíntese , Animais , Cisplatino/farmacologia , Citosol/metabolismo , Diamida/farmacologia , Diquat/farmacologia , Doxorrubicina/farmacologia , Cetonas/farmacologia , Fígado/efeitos dos fármacos , Masculino , Maleatos/farmacologia , Metronidazol/farmacologia , Camundongos , Camundongos Endogâmicos , Especificidade de Órgãos , Paraquat/farmacologia , Peróxidos/farmacologia , Escatol/farmacologia , Vitamina K/farmacologia , terc-Butil HidroperóxidoRESUMO
Steady-state and laser flash photolysis techniques have been used to examine the photoreduction of yeast glutathione reductase by the one-electron reduction products of 5-deazariboflavin and the viologen analogue 1,1'-propylene-2,2'-bipyridyl. Steady-state photoreduction of the enzyme with the viologen generates the two-electron-reduced form, whereas photoreduction with deazaflavin generates the anion semiquinone. Flash photolysis indicates that the product of viologen radical reduction is also a semiquinone, suggesting that this species is rapidly further reduced by viologen in the steady-state experiment to form the EH2 enzyme. This reduction is apparently inhibited when deazaflavin is the photoreductant, perhaps due to complexation of the anion semiquinone with deazaflavin. Steady-state experiments demonstrate that complexation of the anion semiquinone with NADP+ also inhibits further reduction. Both one-electron reduction reactions of oxidized glutathione reductase proceed at close to diffusion-controlled rates (second-order rate constants = 10(8)-10(9) M-1 s-1), despite the relatively buried nature of the FAD cofactor. Addition of NADP+ and oxidized glutathione produced no effects on the kinetics of the initial entry of the electron into the enzyme. No kinetic evidence of intramolecular electron transfer involving the FAD and the protein disulfide was obtained during or subsequent to the initial one-electron reduction process. Thus, if this reaction occurs in the semiquinone, it must be quite rapid (k greater than 8000 s-1).
Assuntos
Diquat/análogos & derivados , Glutationa Redutase/metabolismo , Riboflavina/análogos & derivados , Viologênios/farmacologia , Leveduras/enzimologia , Fenômenos Químicos , Química , Diquat/farmacologia , Elétrons , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/metabolismo , Radicais Livres , Glutationa/farmacologia , Cinética , Lasers , NADP/farmacologia , Oxirredução , Fotoquímica , Riboflavina/farmacologia , Fatores de TempoRESUMO
Formation of glutathione disulfide (GSSG) was used as an index of reactive oxygen generation in the isolated perfused liver of male Fischer rats during normoxia and hypoxia. Low oxygen tension may affect GSSG formation, rereduction, and transport mechanisms. The effect of short-term hypoxia (15 min) on the biliary and sinusoidal transport of GSSG was tested with the glutathione S-conjugates of sulfobromophthalein and 1-chloro-2,4-dinitrobenzene. Hypoxia inhibited S-conjugate excretion through both pathways by 15-20%. tert-Butyl hydroperoxide (75 microM tBHP) or diquat (200 microM) in the perfusate increased hepatic GSSG release by 430 and 1,550%, respectively, and increased the tissue GSSG content by 47 and 124%, respectively, under normoxia. Hypoxia reduced the stimulated GSSG export by 38 (tBHP) and 83% (diquat) and also caused an additional increase of the tissue GSSG content by 112% during tBHP infusion but caused a reduction by 32% during diquat infusion. Inhibition of the biliary export of GSSG and S-conjugates is mainly compensated by the sinusoidal efflux. Therefore, it is concluded that hypoxia reduces GSSG formation predominantly through suppression of reactive oxygen formation with only marginal effects on the biliary and sinusoidal excretion mechanism. Thus hepatic GSSG formation is a sensitive indicator of oxidant stress during normoxia and hypoxia. Because single parameters may vary considerably, simultaneous monitoring of GSSG in bile, perfusate, and tissue is essential for qualitative and quantitative estimation of reactive oxygen formation.
Assuntos
Biomarcadores/análise , Glutationa/análogos & derivados , Hipóxia/metabolismo , Fígado/metabolismo , Animais , Diquat/farmacologia , Glutationa/análise , Glutationa/metabolismo , Dissulfeto de Glutationa , Técnicas In Vitro , Cinética , Fígado/efeitos dos fármacos , Masculino , Perfusão , Peróxidos/farmacologia , Ratos , Ratos Endogâmicos F344 , Valores de Referência , terc-Butil HidroperóxidoRESUMO
A new bioassay has been developed that allows rapid, sensitive detection of chemicals such as paraquat and adriamycin, which manifest their acute toxicity, mutagenicity or carcinogenicity by inducing a pro-oxidant state in vivo. Submitochondrial particles isolated from bovine myocardium are used to catalyze NADH-dependent enzymatic reduction of these chemicals to free radicals. The highly reactive species generated in this system reduce molecular dioxygen to the superoxide anion radical, which is detected spectrophotometrically using the adrenochrome reaction. The anticancer drug adriamycin, the herbicides paraquat and diquat, the analytical dye sulfonazo III, and the experimental carcinogen 4-nitroquinoline-N-oxide have been used to test the sensitivity of this new method. This assay can be used to screen fresh water samples for the presence of pollutants that can generate oxygen-centered free radicals in vivo, or to test newly synthesized chemicals for this activity, and may therefore be valuable for environmental monitoring and preliminary toxicity evaluation of industrial or pharmaceutical products.
Assuntos
Mitocôndrias Cardíacas/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Toxicologia/métodos , 4-Nitroquinolina-1-Óxido/farmacologia , Adrenocromo , Animais , Antimicina A/farmacologia , Compostos Azo/farmacologia , Bioensaio , Bovinos , Diquat/farmacologia , Doxorrubicina/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Paraquat/farmacologia , Rotenona/farmacologia , Superóxido Dismutase/metabolismoRESUMO
The aim of this study has been to define cytotoxic mechanisms that may cause clonal expansion in the liver of pre-carcinogenic cells. An in vitro model, which has been described previously, was used. Hepatocytes were isolated from carcinogen-treated rats and a high proportion of the cells were gamma-glutamyltranspeptidase (GGT)-positive. The cells were incubated in suspension and exposed to toxic agents in concentrations that induced a moderate increase in cellular leakage within 3 h. Samples were withdrawn and sampled cells were then allowed to attach to collagen-coated plates. Attached cells were stained and the ratio of GGT-positive/GGT-negative cells (GGT-ratio) was determined. The initial GGT-ratio was 10.4 +/- 4.7% and an increased ratio was taken as a sign of toxicity that resulted in a selection of GGT-positive cells. In a first series of experiments it was shown that hydroquinone and menadione increase the GGT-ratio, while diquat, sodium selenite, diethyl maleate or phorone do not. However, diethyl maleate in combination with diquat increased the GGT-ratio. Hydrogen peroxide (5 mM) increased the GGT-ratio as effectively as hydroquinone (0.3 mM). Lower concentrations of H2O2 (0.05 mM) increased the GGT-ratio in GSH-depleted cells. The changes induced by hydroquinone and H2O2 in low concentration were reversible. In another series of experiments, plates coated with antibodies against beta 1-integrin were used. An increase in the GGT-ratio was obtained with anti beta 1-integrin, but not with broad spectrum anti-rat hepatocyte or anti-rat beta 2-microglobulin antibodies as substrata. These data suggested an involvement of the beta 1-integrin in the selection. Taken together, these data indicate that GGT-positive hepatocytes are protected against GSH depletion and oxidative stress that may result in reversible receptor alterations.
Assuntos
Biomarcadores Tumorais/análise , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Fígado/enzimologia , Receptores de Superfície Celular/metabolismo , gama-Glutamiltransferase/análise , Animais , Transformação Celular Neoplásica , Células Cultivadas , Colágeno/metabolismo , Dietilnitrosamina/toxicidade , Diquat/farmacologia , Feminino , Hidroquinonas/farmacologia , Cetonas/farmacologia , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Maleatos/farmacologia , Fenobarbital/farmacologia , Ratos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Colágeno , Selênio/farmacologia , Selenito de Sódio , Vitamina K/farmacologiaRESUMO
The hypothesis that intracellular generation of reactive oxygen species in hepatocytes or reticuloendothelial cells may cause ischemia-reperfusion injury was tested in isolated perfused livers of male Fischer rats. GSSG was measured in perfusate, bile, and tissue as a sensitive index of oxidative stress. After a preperfusion phase of 30 min, the perfusion was stopped (global ischemia) for various times (30, 120 min) and the liver was reperfused for another 60 min. The bile flow (1.48 +/- 0.17 microliters/min X gram liver weight), the biliary efflux of total glutathione (6.54 +/- 0.94 nmol GSH eq/min X g), and GSSG (1.59 +/- 0.23 nmol GSH eq/min X g) recovered to 69-86% after short-term ischemia and to 36-72% after 2 h of ischemia when compared with values obtained from control livers perfused for the same period of time. During reperfusion, the sinusoidal efflux of total glutathione (16.4 +/- 2.1 nmol GSH eq/min X g) and GSSG (0.13 +/- 0.05 nmol GSH eq/min X g) did not change except for an initial 10-30-s increase during reperfusion washout. No increased GSSG secretion into bile was detectable at any time during reperfusion. The liver content of total glutathione (32.5 +/- 3.5 nmol GSH eq/mg protein) and GSSG (0.27 +/- 0.09 nmol GSH eq/mg protein) did not change significantly during any period of ischemia or reperfusion. We conclude, therefore, that at most only a minor amount of reactive oxygen species were generated during reperfusion. Thus, reactive oxygen species are unlikely to cause ischemia/reperfusion injury in rat liver by lipid peroxidation or tissue thiol oxidation.
Assuntos
Glutationa/metabolismo , Isquemia/metabolismo , Fígado/irrigação sanguínea , Fígado/metabolismo , Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bile/metabolismo , Diquat/farmacologia , L-Lactato Desidrogenase/metabolismo , Masculino , Oxirredução , Perfusão , Ratos , Fatores de TempoRESUMO
The glutathione peroxidase (GSH-Px)-like reduction of H2O2 by the selenoorganic compound 2-phenyl-1,2-benzoisoselenazol-3(H)-one (PZ51: Ebselen) was studied using glutathione (GSH) and the therapeutic agent N-acetylcysteine (NAC) to provide reducing equivalents. In a purely chemical system containing H2O2 and in an enzymatic system of glucose/glucose oxidase-generated H2O2 Ebselen alone did not reduce H2O2. Ebselen in combination with either GSH (1 mM) or NAC (1 mM) was capable of reducing H2O2 in both systems. In these non-cellular systems GSH was a more effective source of reducing equivalents than NAC. The GSH-Px-like activity of Ebselen was further investigated in a cellular system. The redox-cycling bipyridylium compound diquat generates active oxygen species, depletes intracellular glutathione, and is cytotoxic in isolated hepatocytes pretreated with the glutathione reductase inhibitor 1,3-bis(Z-chloro-ethyl)-1-nitrosourea (BCNU). Ebselen alone did not ameliorate diquat cytotoxicity, but in combination with either GSH (1 mM) or NAC (1 mM) it produced a significant delay in diquat-induced cytotoxicity. Further additions of either GSH (0.5 mM) or NAC (0.5 mM) at 30 min intervals provided significantly more protection against diquat-induced cytotoxicity and intracellular GSH depletion than the single 1 mM addition. Thus, the combination of Ebselen and NAC may provide an effective antidote in cases of overexposure to bipyridylium herbicides, such as diquat and paraquat.
Assuntos
Acetilcisteína/farmacologia , Azóis/farmacologia , Diquat/farmacologia , Glutationa/farmacologia , Fígado/efeitos dos fármacos , Compostos Organosselênicos , Compostos de Piridínio/farmacologia , Selênio/farmacologia , Animais , Azóis/metabolismo , Carmustina/farmacologia , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Isoindóis , Peróxidos Lipídicos/metabolismo , Fígado/metabolismo , Masculino , Oxirredução , Ratos , Ratos Endogâmicos , Selênio/metabolismoRESUMO
Although diquat produces massive oxidant stress in both Fischer and Sprague-Dawley rats, the Fischer rats sustain hepatic necrosis and the Sprague-Dawley rats do not. A previous example of probable hepatic necrosis produced by an oxidant stress-generating compound was demonstrated in animals in which glutathione peroxidase activity had been decreased by a dietary deficiency of selenium. In the present study the susceptible Fischer rats had hepatic peroxidase activities equal to the resistant Sprague-Dawley rats. Hepatotoxic doses of diquat did not diminish hepatic glutathione peroxidase or reductase activities or hepatic content of ascorbic acid, NADPH or protein sulfhydryls. Hepatic nonprotein sulfhydryls were decreased by 50% but recovered to control values by 6 h. Biliary excretion of oxidized glutathione in the Fischer rat after administration of diquat was 4 times that observed after administration in Sprague-Dawley rats. The diquat-induced peroxidation of hepatic lipids was indicated by small increases in the 11-, 12-, and 15-hydroxyeicosatetraenoic acids, as quantitated by a new gas chromatography-mass spectrometry assay. Thus, acute lethal injury caused by redox cycling compounds that generate reactive oxygen species does not exhibit a number of the biochemical alterations in vivo that occur with cell death produced by similar compounds in isolated hepatocyte systems.
Assuntos
Diquat/farmacologia , Fígado/patologia , Compostos de Piridínio/farmacologia , Estresse Fisiológico/patologia , Alanina Transaminase/sangue , Animais , Ácido Ascórbico/farmacologia , Aspartato Aminotransferases/sangue , Glutationa/análogos & derivados , Glutationa/metabolismo , Dissulfeto de Glutationa , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Hidroxiácidos/metabolismo , Masculino , NADP/metabolismo , Necrose , Ratos , Ratos Endogâmicos F344 , Ratos EndogâmicosRESUMO
Plasma GSH and GSSG concentrations were examined after the administration of compounds that deplete intracellular GSH either by adduct formation or by production of oxidative stress. A modified assay based on the GSSG reductase method was developed that minimizes the artifactual auto-oxidation of GSH to GSSG and mixed disulfides by rapid addition of bis(3-carboxy-4-nitrophenyl)disulfide or N-ethylmaleimide directly to whole blood or tissue samples. Control arterial plasma GSH and GSSG concentrations were found to be 16.5 +/- 0.7 and 0.3 +/- 0.1 microM, respectively. Depletion of GSH by fasting or by the administration of acetaminophen or diethyl maleate was associated with a proportional decrease in the arterial plasma GSH concentrations (r = 0.94) consistent with the hypothesis that the liver in vivo is a major source of plasma GSH. Diquat and t-butyl hydroperoxide, but not acetaminophen or diethyl maleate, elicited large increases in arterial plasma GSSG concentrations (17- and 115-fold, respectively) and several-fold increases in biliary GSSG levels without markedly increasing hepatic GSSG levels (2.7- and 1.2-fold, respectively). In contrast, treatment with paraquat produced substantial increases in arterial plasma GSSG levels (22-fold) without large increases in the bile (3-fold). Assessment of the arteriovenous difference for GSSG across the lungs after paraquat administration demonstrated that the lung may be a significant source of plasma GSSG. In conclusion, plasma GSH concentrations appear to reflect mainly intrahepatic GSH concentration, whereas plasma GSSG appears to arise from both hepatic and extrahepatic sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glutationa/análogos & derivados , Glutationa/sangue , Acetaminofen/farmacologia , Animais , Diquat/farmacologia , Glutationa/metabolismo , Dissulfeto de Glutationa , Inativação Metabólica , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Maleatos/farmacologia , Paraquat/farmacologia , Peróxidos/farmacologia , Ratos , Ratos Endogâmicos , terc-Butil HidroperóxidoAssuntos
Cloroplastos/metabolismo , Concentração de Íons de Hidrogênio , Fotofosforilação/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Benzoatos/farmacologia , Calorimetria , Cloroplastos/efeitos dos fármacos , Cianetos/farmacologia , Escuridão , Diquat/farmacologia , Ferricianetos/farmacologia , Luz , Fenil-Hidrazinas/farmacologia , Radioisótopos de Fósforo , Plantas/efeitos dos fármacos , Plantas/metabolismo , Termodinâmica , Fatores de Tempo , Desacopladores/farmacologiaRESUMO
1. Administration of diquat (NN'-ethylene-2,2'-bipyridilium) or paraquat (NN'-dimethyl-4,4'-bipyridilium) prevents the normal depletion of liver glycogen in starved rats. 2. There is an increase in blood glucose, which returns to normal values after approx. 7h. 3. After administration of diquat or paraquat, plasma corticosteroids increase to very high concentrations and remain high for at least 24h, but plasma ACTH (adrenocorticotrophin) is only increased for 4h. 4. Adrenal cyclic AMP is considerably increased after administration of diquat and remains significantly higher than control values for at least 24h. 5. It is suggested that diquat and paraquat increase the response of the adrenal cortex to ACTH.