Dietary docosahexaenoic acid does not promote tissue lipid peroxide formation to the extent expected from the peroxidizability index of the lipids

Changes in susceptibility of tissues to lipid peroxidation were investigated in rats after ingestion of oxidation-prone docosahexaenoic acid (C22:6n-3). Lipid peroxide levels in the liver, kidney and testis increased concomitant with increases in the peroxidizability indices calculated from the fatty acid composition of tissue total lipids. However, even in these cases, the lipid peroxides were not increased to the levels expected from the peroxidizability indices of these tissues, and thus no tissue injury was recognized. When low level of vitamin E was given to rats, the lipid peroxide levels of liver, kidney and testis nearly coincided with the peroxidizability indices of these tissues, where the cell injuries were observed as well. The mechanisms of defense to suppress lipid peroxide levels below the peroxidizability indices in normal vitamin E administration were presumed to be due to enhanced antioxidative function in the tissues mediated primarily by vitamin E, ascorbic acid and glutathione, and also to increased incorporation of docosahexaenoic acid into neutral lipids and phosphatidylethanolamine in the tissues, probably leading to acquiring stability against oxidative attack. Owing to these suppressive mechanisms, physiological efficacy of n-3 fatty acids may be exerted effectively.     

Lipid peroxide levels in a murine adenocarcinoma exposed to hyperthermia: the role of glutathione depletion.

Increased lipid peroxide levels were obtained 1 h after a 60-min 43 degrees C hyperthermia treatment of a solid murine C3H mammary adenocarcinoma, grown subcutaneously in the hind paws of mice. Previous work from our group revealed that this heat treatment depletes the intracellular glutathione (GSH) content in this tumor. To investigate GSH depletion as one tentative mechanism behind the increased lipid peroxide levels obtained, we also measured the formation of lipid peroxidation products after extensive DL-buthionine-S,R-sulfoximine (BSO)-induced GSH depletion. The lipid peroxide effect provoked by BSO was less than that of the 60-min hyperthermia treatment. We therefore propose that the increased lipid peroxide levels induced by heat treatment do not correlate primarily with the observed decrease in GSH levels. Furthermore, in thermotolerance-induced tumors, lipid peroxide levels after a second heat treatment were observed to increase concomitantly with the cessation of thermotolerance. Lipid peroxide levels were also studied in liver, lung, and heart. Following BSO treatments, and up to 2-fold increase was observed in these organs in non-tumor-bearing mice. It was also observed that the intrinsic lipid peroxide levels in these organs from tumor-bearing mice were approximately 1.5- to 4-fold higher in comparison with non-tumor-bearing mice, thus indicating a systemic effect of the tumor implant.     

Inhibitory effect of gossypol on basal and luteinization factor-stimulated progesterone synthesis in porcine granulosa cells.

Gossypol, a polyphenolic aldehyde, inhibits steroidogenesis and the reproductive system in both sexes. The present study was undertaken to investigate whether gossypol may affect progesterone biosynthesis in cultured porcine granulosa cells isolated from small (1-2 mm) follicles (SGC). SGC were cultured with gossypol, NO donor S-nitroso-N-acetylpenicillamine (S-NAP) or the specific NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), in the presence or absence of follicular fluid isolated from large (5-8 mm) follicles (LFF) or conditioned media (CM) of granulosa cells isolated from large follicles (LGC). Gossypol enhanced the nitrite content in culture media of SGC and inhibited basal progesterone secretion by SGC. S-NAP (10(-3) M) inhibited progesterone secretion and enhanced the formation of cGMP by SGC. L-NAME had no effect on progesterone accumulation by SGC. The stimulatory effect of LFF or CM media on progesterone production by SGC in culture was also inhibited by S-NAP (10(-3)) and gossypol (10(-4) M). Moreover, gossypol inhibited forskolin-stimulated progesterone secretion, as well as substrate-enhanced conversion of 22-OH-cholesterol and pregnenolone to progesterone. These results suggest that the inhibitory effect of gossypol on progesterone secretion in culture of SGC may be mediated via NO generation.     

Gossypol inhibition of adenylate cyclase

Gossypol, a polyphenolic binaphthalene -dialdehyde reputed to exert contraceptive action in males, reversibly inhibits adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] in a concentration-dependent manner. In membranes prepared from a variety of organs, the half-maximal inhibitory concentration (IC50) ranges from 75 microM (rat Leydig tumor cells) to 250 microM (rat liver membranes). Kinetic studies using partially purified catalytic subunit isolated from bovine testis show that gossypol is competitive with ATP with an apparent Ki of 110 microM. These data suggest that gossypol inhibition of adenylate cyclase is due to direct interaction at the nucleotide-binding domain of the catalytic subunit of the enzyme.     

900 MHz pulse-modulated radiofrequency radiation induces oxidative stress on heart, lung, testis and liver tissues

Oxidative stress may affect many cellular and physiological processes including gene expression, cell growth, and cell death. In the recent study, we aimed to investigate whether 900 MHz pulse-modulated radiofrequency (RF) fields induce oxidative damage on lung, heart and liver tissues. We assessed oxidative damage by investigating lipid peroxidation (malondialdehyde, MDA), nitric oxide (NOx) and glutathione (GSH) levels which are the indicators of tissue toxicity. A total of 30 male Wistar albino rats were used in this study. Rats were divided randomly into three groups; control group (n = 10), sham group (device off, n = 10) and 900 MHz pulsed-modulated RF radiation group (n = 10). The RF rats were exposed to 900 MHz pulsed modulated RF radiation at a specific absorption rate (SAR) level of 1.20 W/kg 20 min/day for three weeks. MDA and NOx levels were increased significantly in liver, lung, testis and heart tissues of the exposed group compared to sham and control groups (p < 0.05). Conversely GSH levels were significantly lower in exposed rat tissues (p < 0.05). No significantly difference was observed between sham and control groups. Results of our study showed that pulse-modulated RF radiation causes oxidative injury in liver, lung, testis and heart tissues mediated by lipid peroxidation, increased level of NOx and suppression of antioxidant defense mechanism.     

Gossypol and testicular lipids of adult rats

Testicular lipids act as source of energy, structural components of spermatozoa and precursors of androgen biosynthesis. Treatment with antispermatogeneic agents cause accumulation of testicular lipids. Gossypol, an effective antispermatogenic agent causes marked accumulation of testicular neutral lipids. It did not affect testicular phospholipids. Gossypol treatment did not bring about marked changes in the key enzymes like HMG Co A reductase, glucose-6-phosphate dehydrogenase malic enzyme and cytosolic isocitrate dehydrogenase involved in sterol biosynthesis. Thus, gossypol brings about marked accumulation of glycerides and esterified cholesterol in the testis due to its effect on spermatogenic elements of adult rats.     

Gossypol mediated DNA degradation

Gossypol, a polyphenolic compound isolated from cotton plant was found to degrade pBR322 DNA in vitro in a reaction which required the presence of a metal ion, a reducing agent (2-mercaptoethanol) and oxygen as revealed after agarose gel electrophoresis. Fe3+ and Co2+ showed maximum degradation whereas addition of Ca2+ and Mg2+ prevented the gossypol mediated DNA damage. Gossypol caused degradation of rat liver DNA incubated in vitro even in the absence of added metal ions and 2-mercaptoethanol. Incubation of intact rat liver nuclei with gossypol revealed DNA degradation and nuclei isolated from rats treated with gossypol in vivo showed higher susceptibility to DNA fragmentation when incubated with gossypol in vitro than control nuclei. EcoR1 and AIuI digestion of DNA isolated from gossypol treated rats gave clear cut evidence for DNA degradation. These observations indicate that gossypol is genotoxic and considerable care has to be exercised in its use.     

长期低剂量γ线照射和照射加电击对老年前期大鼠性激素、肝功能和脂质过氧化物的影响

本文观察了长期低剂量γ射线照射和照射加电击对老年前期(18～21月龄)大鼠血浆性激素水平、肝微粒体混合功能氧化酶(MFO)活力以及组织脂质过氧化物的影响。长期照射加电击使雄性大鼠血浆睾酮水平明显降低(p<0.05),照射与照射加电击均使雄鼠肝微粒体MFO活力明显下降(p<0.05)。长期低剂量γ射线照射和照射加电击组的睾丸自由基浓度较对照组明显升高(p<0.05),长期照射使雄性大鼠肝匀浆与微粒体,睾丸匀浆与线粒体的脂质过氧化物较对照组明显增高,但照射加电击组的脂质过氧化物较照射组(以及对照组)明显下降。实验结果说明照射与照射加电击对老年前期大鼠的作用有所不同,这些环境因素具有加速老化的作用。     

Gossypol-induced free radical toxicity to isolated islet cells

1. The cytotoxicity of the polyphenolic potential male antifertility agent gossypol was investigated on isolated mouse islets cells. 2. Gossypol shared many properties with the diabetogenic agent alloxan. 3. Gossypol (0.1-1.0 mmol/l) induced a concentration-dependent increase of Trypan Blue uptake by the cells, indicating an increase of membrane permeability to the dye. 4. Trypan Blue uptake induced by 0.5 mmol/l gossypol was inhibited by concomitant incubation of the cells with enzymatic (200 mg/l superoxide dismutase, 200 mg/l catalase, 3 mmol/l cytochrome-c), or low-molecular weight (50 mmol/l D-mannitol) scavengers of oxygen radicals, and the metal chelator diethylenetriaminepentacetic acid (DTPA) (50 mumol/l). 5. The results support the hypothesis that gossypol is B-cytotoxic by generation of noxious free radicals and that when proposing gossypol as a male antifertility agent, studies to exclude gossypol as a diabetogenic agent should first be performed in vivo.     

Gossypol inhibition of acrosin and proacrosin, and oocyte penetration by human spermatozoa

Gossypol, a known antispermatogenic agent, was found to effectively inhibit the highly purified boar sperm proacrosin-acrosin proteinase enzyme system by irreversibly preventing the autoproteolytic conversion of proacrosin to acrosin and reversibly inhibiting acrosin activity. The agent appears to prevent the self-catalyzed by not the acrosin-catalyzed activation of proacrosin. In additional experiments, brief exposure of human semen to concentrations of gossypol, which did not visibly alter spermatozoal motility or forward progression, was found to irreversibly inhibit the conversion of proacrosin to acrosin although the activity of the nonzymogen acrosin was not decreased, and also to prevent the human spermatozoa from penetrating denuded hamster oocytes. Gossypol inhibition of proacrosin conversion to acrosin closely paralleled the decline in oocyte penetration. Racemic (+/-) gossypol was equally as effective as the enantiomer (+) gossypol. The results suggest that the inhibition of proacrosin conversion to acrosin is a mechanism by which gossypol exerts its antifertility effect at nonspermicidal concentrations and that low levels of gossypol should be tested for their contraceptive action when placed vaginally.     

Effect of cigarette smoke on lipid peroxidation and antioxidant enzymes in albino rat

Effect of cigarette smoke on lipid peroxidation (LPX) and antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) in various organs like brain, heart, lung, liver and kidney of the albino rats exposed to cigarette smoke for 30 min/day for a period of 30 days were assayed. It was observed that the lipid peroxide levels in liver, lung and kidney were enhanced in case of animals exposed to cigarette smoke, whereas brain and heart did not show any change as compared to control animals. The activity of the antioxidant enzymes was also elevated in liver, lung and kidney of the test animals whereas, brain and heart did not show any change in the activities of all of these antioxidant enzymes except glutathione-s-transferase which was increased in brain also. The level of reduced glutathione (GSH) was lowered in liver, lung and kidney of the tested animals when compared with the control animals but there was no significant change in brain and heart. The results of our study suggest that cigarette smoke induces lipid peroxidation in liver, lung and kidney, and the antioxidant enzymes levels were enhanced in order to protect these tissues against the deleterious effect of the oxygen derived free radicals. The depletion of reduced glutathione in these organs could be due to it's utilization by the tissues to mop off the free radicals.     

Mechanisms involved in reproductive damage caused by gossypol in rats and protective effects of vitamin E

Background

Gossypol is a chemical present in the seeds of cotton plants (Gossypium sp.) that reduces fertility in farm animals. Vitamin E is an antioxidant and may help to protect cells and tissues against the deleterious effects of free radicals. The aim of this study was to evaluate the mechanisms of reproductive toxicity of gossypol in rats and the protective effects of vitamin E. Forty Wistar rats were used, divided into four experimental groups (n = 10): DMSO/saline + corn oil; DMSO/saline + vitamin E; gossypol + corn oil; and gossypol + vitamin E.

Results

Fertility was significantly reduced in male rats treated with gossypol in that a significant decrease in epididymal sperm count was observed (P < 0.05) and the number of offspring was significantly reduced in females mated with them (P < 0.05). This dysfunction was prevented by vitamin E. Gossypol caused a significant increase in the activity of the enzymes glutathione peroxidase (P < 0.01) and glutathione reductase (P < 0.01), but vitamin E did not reduce the enzyme activities (P > 0.05). The levels of reduced glutathione and pyridine nucleotides in testis homogenate were significantly reduced by gossypol (P < 0.05 and P < 0.01, respectively) and this reduction was accompanied by increased levels of oxidized glutathione (P < 0.05). Vitamin E showed a preventive effect on the changes in the levels of these substances. Gossypol significantly increased the levels of malondialdehyde (P < 0.01), a lipid peroxidation indicator, whereas treatment with vitamin E inhibited the action of the gossypol. Vitamin E prevented a decrease in mitochondrial ATP induced by gossypol (P < 0.05).

Conclusions

This study suggests that the reproductive dysfunction caused by gossypol may be related to oxidative stress and mitochondrial bioenergetic damage and that treatment with vitamin E can prevent the infertility caused by the toxin.     

Influence of alloxan-induced diabetes and selenite treatment on blood glucose and glutathione levels in mice

Many clinical studies reported that diabetic patients had lower glutathione contents in erythrocytes or plasma. Recently, selenium, an essential trace element with well-known antioxidant characteristics, has been found to have insulin-mimetic properties. But seldom information is available about the influence of selenium on glutathione changes induced by diabetes mellitus in animals. Therefore, this study was designed to compare the impacts of selenite treatment on glutathione (GSH) levels of blood and tissues such as brain, kidney, liver, spleen and testis in mice. Four groups were used in this study: a control group, a diabetic group, a selenite-treated normal group and a selenite-treated diabetic group. Selenite was administered to the mice for 4 weeks with an oral dose of 2 mg kg(-1) day(-1) by gavage. The blood glucose level, and GSH level in blood and tissues were determined. The results show that the selenite-treated diabetic group had significantly lower blood glucose levels than the diabetic group. Moreover, alloxan-induced diabetes significantly decreased GSH levels in blood, kidney, liver and testis compared to the controls. Selenite treatment of the diabetic mice only improved the GSH levels in liver and brain. On the other hand, selenite administered to the normal mice reduced GSH levels in the liver compared to the controls. In conclusion, this study suggests that selenite treatment of diabetic mice with an effective dose would be beneficial for the antioxidant system of liver and brain although it exerts a toxic effect on the liver of normal mice.     

Biochemical effects of gossypol in isolated mitochondria: monovalent cations and ATPase activity

1. The effect of gossypol acetic acid upon the energy-producing system of isolated liver mitochondria was studied. Gossypol was found to exert an uncoupling effect. 2. Oxygen consumption and ATPase activity were stimulated only when a monovalent alkali metal cations was present. 3. The mitochondrial proton gradient, produced by the enzymatic hydrolysis of ATP, was lower in the gossypol-treated mitochondria than in the control experiments. 4. It is proposed that gossypol stimulates oxygen consumption and the ATPase activity in mitochondria by a protonophoretic effect and a cation translocation process.     

The antifertility agent,gossypol, changes several mitochondrial functions in the presence of Mg2+

1. The effect of gossypol in the presence of K⁺ or Mg²⁺, or both, was studied on ATPase activity and respiration of rat liver mitochondria.2. Respiration was uncoupled in the presence of gossypol, Mg²⁺, and K⁺, whereas in the presence of gossypol and Mg²⁺ a partial inhibition was observed.3. Gossypol stimulated ATPase activity in the presence of K⁺ or Mg²⁺, but maximal activity was observed when both cations were in the incubation medium.4. Stimulation of ATPase activity in the presence of Mg²⁺ was dose related.5. EDTA reverted the stimulation produced by gossypol on ATPase activity.6. Gossypol had no effect on the ATPase activity of submitochondrial particles, which suggests an indirect action of gossypol on the enzyme.7. Mitochondrial membrane potential showed a higher collapse in the presence of gossypol and 1mM MgCl₂.8. The observed effects of gossypol could be explained by the collapse of the mitochondrial membrane potential.     

Effect of stress,adrenalectomy and changes in glutathione metabolism on rat kidney metallothionein content: comparison with liver metallothionein

Eighteen hours of immobilization stress, accompanied by food and water deprivation, increased liver metallothionein (MT) but decreased kidney MT levels. Food and water deprivation alone had a significant effect only on liver MT levels. In contrast, stress and food and water deprivation increased both liver and kidney lipid peroxidation levels, indicating that the relationship between MT and lipid peroxidation levels (an index of free radical production) is unclear. Adrenalectomy increased both liver and kidney MT levels in basal conditions, whereas the administration of corticosterone in the drinking water completely reversed the effect of adrenalectomy, indicating an inhibitory role of glucocorticoids on MT regulation in both tissues. Changes in glutathione (GSH) metabolism produced significant effects on kidney MT levels. Thus, the administration of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased kidney GSH and increased kidney MT content, suggesting that increased cysteine pools because of decreased GSH synthesis might increase kidney MT levels through an undetermined mechanism as it appears to be the case in the liver. However, attempts to increase kidney MT levels by the administration of cysteine or GSH were unsuccesful, in contrast to what is known for the liver. The present results suggest that there are similarities but also substantial differences between liver and kidney MT regulation in these experimental conditions.     

Kinetics of gossypol inhibition of bovine lactate dehydrogenase X

Gossypol, a polyphenolic binaphthalene dialdehyde isolated from cotton meal is a potent inhibitor of lactate dehydrogenase-X purified from bovine testis. For the conversion of pyruvate to lactate the IC50 for gossypol is 200 microM for the reverse reaction the IC50 is 12 microM. Gossypol is a competitive inhibitor of NADH, Ki = 30 microM (Km = 17 microM), and NAD+, Ki = 6 microM (Km = 130 microM), and noncompetitive for pyruvate, Ki = 220 microM (Km = 224 microM), and lactate, Ki = 52 microM (Km = 5.6 mM).     

Effect of pre-exposure to beta rays of tritium on some biochemical parameters measured in organs of rats subsequently irradiated with fast neutrons

The experiment examined biological responses produced by combined sequential exposure to low-level tritium contamination, followed by challenging irradiation with fast neutrons. Modifications of endogenous antioxidant potential of different organs in rats were discussed in relation to tissue radiosensitivity. Rats pre-contaminated to 7 cGy and 35 cGy have been additionally irradiated to 1 Gy with fast neutrons. Lipid peroxide level was determined in liver, kidney, small intestine, spleen, bone marrow, and plasma. Reduced glutathione (GSH) level and glucose-6-phosphate dehydrogenase (G6PDH) activity were determined in erythrocytes. An in vitro thymidine uptake assay was performed in isolated bone marrow cells. The lipid peroxide level decreased significantly only in liver and kidney from rats pre-exposed to 35 cGy. For small intestine and spleen, tissues of comparatively higher radiosensitivity, no induced radioprotection was observed, as reflected in the homeostasis of the lipid peroxides. The same behavior was observed in bone marrow, the most radiosensitive tissue studied. However, the bone marrow thymidine-incorporation assay revealed a possible adaptive-type reaction in rats pre-exposed to 35 cGy. We conclude that for radiosensitive tissues pre-exposure to chronic low doses of low linear energy transfer (LET) irradiation has no protective effect on their antioxidant status, whereas a protective effect is observed in radioresistent tissues.     

Glutathione S-transferase distribution and concentration in human organs

The concentration of basic, near-neutral and acid GSH S-transferase was measured in 18 organs from each of 9 male human subjects using radial immunodiffusion. Basic transferases were detectable in all tissues studied. Highest concentrations were found in liver, testis, kidney, adrenal and jejunum while low levels were found in bladder, muscle and thyroid. The concentration in liver was 230 times higher than that in thyroid. Near-neutral GSH S-transferase were absent in all tissues in 5 of the 9 individuals studied. When present they were widely distributed, highest concentrations being found in liver, testis, muscle, adrenal and brain and lowest levels in thyroid, lung, duodenum, stomach, heart and kidney. Acid GSH S-transferases were present in every individual studied although they were undetectable in the liver of a single subject. Highest concentrations were present in colon, jejunum, ileum, bladder, spleen and lung while low concentrations were found in liver. Our study provides conclusive evidence of marked inter-individual and inter-organ variation of the three groups of human GSH S-transferase.     

Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.