Activity profile of glutathione-dependent enzymes and respiratory chain complexes in rats supplemented with antioxidants and treated with carcinogens.

Appropriate dietary interventions may reduce the potentially damaging effects of free radicals generated during metabolism and various physiological conditions. We have investigated the effects of dietary vitamins C, E, beta-carotene, or selenium (Se) on the activity of endogenous antioxidant enzymes and respiratory chain complexes in rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an antineoplastic drug. These agents are known to generate DNA-reactive species during their metabolism, which may enhance oxidative stress in cells. Female Fischer 344 rats aged 4 months were given antioxidant supplements singly or as a mixture 2 weeks prior to mutagen treatments; antioxidant supplementation continued for an additional 4 weeks. In rats treated with mutagens, the antioxidant intake lowered the activity of Se-dependent glutathione peroxidase (Se-GPx) in liver cytosolic and mitochondrial fractions, compared to activity in rats treated with mutagens alone. However, the vitamins, but not Se supplement, persistently increased Se-GPx activity in untreated control animals. Treatment of animals with mutagen raised K(m) value of Se-GPx and this correlated with an increase in V(max). However, Se intake, either singly or mixture, significantly reduced K(m) value in mutagen-treated and untreated rats in both fractions. Se intake increased glutathione S-transferases (GST) activity (P < 0.05) in both liver fractions of mutagen-treated and untreated animals. Similar response was seen in Se-independent GPx. Since GST-alpha possesses Se-independent GPx activity, the enhanced effect observed in GST activity may be due, in part, to increased activity in Se-independent GPx. Also, selenium or the antioxidant vitamin supplementation increased the activity of all four respiratory chain complexes in untreated rats. Although BLM treatment significantly increased the activity of electron transport complexes III and IV, selenium or the vitamin supplements modulated the responses. These results indicate that the intake of dietary vitamins or Se enhances antioxidant capacity in chemically exposed animals compared to animals receiving antioxidants alone. Furthermore, in addition to being an enhancer of the catalytic function of glutathione peroxidase, selenium may directly play a role as an antioxidant.

Influence of dietary antioxidants on the mutagenicity of 7,12-dimethylbenz[a]anthracene and bleomycin in female rats.

Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreventive compounds and for predicting their protective efficacy in humans. In this study, we investigated the ability of the dietary antioxidant Vitamins C, E, beta-carotene and the mineral selenium to inhibit the mutant frequency (MF) induced by treatment of rats with 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an anti-tumor agent that can damage DNA by free radical mechanisms. Both chemicals have been previously shown to be mutagenic in the rat lymphocyte Hprt assay. Adult female Fischer 344 rats were given the antioxidants singly or in a combination 2 weeks prior to mutagen treatment. Antioxidant intake continued for an additional 4 weeks post-mutagen treatment. At sacrifice, spleens were aseptically removed for the isolation of lymphocytes to conduct the mutagenesis assay at the Hprt locus. The DMBA and BLM treatment induced a marked increase in MF, 52.8 x 10(-6) and 19.2 x 10(-6), respectively, over the controls. The MFs seen in the individual antioxidants alone (single or mixture) were relatively similar to the controls, with the exception of Vitamins C and E, that had 1.7- and 1.5-fold increase, respectively. The degree of inhibitory response was dependent on the type of mutagen and the particular antioxidant. BLM/antioxidant combination had inhibitions ranging from 44 to 80%, while DMBA/antioxidant system ranged from 60 to 93%, with Vitamins C and E achieving the highest inhibition in both systems. The mixture displayed low inhibitory responses, 44.6% for BLM/mix and 47% DMBA/mix. On the whole, the results indicate that the dietary constituents tested are antimutagenic; however, because of the gradations seen with the responses, the protective efficacy of these antioxidants may depend on the type of mutagen/carcinogen they encounter. Pending molecular analysis of mitochondrial DNA mutations will also indicate whether there is a shift in the mutational spectra produced by the carcinogens in the presence of antioxidants.

Hypothermia enhances bcl-2 expression and protects against oxidative stress-induced cell death in Chinese hamster ovary cells.

Oxidative stress is one of the major causes of cellular injury. Various reactive oxygen (ROS) and nitrogen (RNS) species such as superoxide, hydroxyl radical, peroxynitrite, and nitric oxide are involved in the manifestations of different types of organ toxicity and the resultant syndromes, symptoms, or diseases. Hypothermic conditions have been reported to reduce the oxidative stress in various in vitro and in vivo studies. In the present study, we sought to determine the effect of lowered temperatures on oxidative stress-induced cell death in Chinese hamster ovary (CHO) cells. We also investigated the oxidative stress-induced alterations in the expression of anti-apoptotic protein, bcl-2, in CHO cells at lowered temperatures. CHO cells were incubated at four different temperatures of 30, 32, 35, and 37 degrees C (control temperature) from 1 to 4 d. In another set, the cells were incubated with 100 microM hydrogen peroxide (H(2)O(2)) for 30 min before harvesting at different time points. The cells were harvested at 1, 2, 3, and 4 d. Cell survival was significantly higher at 30 degrees C as compared to 37 degrees C over 4 d of incubation. In cells incubated with H(2)O(2), significantly higher cell viability was observed at lower temperatures as compared to the cells incubated at 37 degrees C. The activity of glutathione peroxidase (GSH-Px) also increased significantly at lower temperatures. Lowered temperature also provided a significant increase in the expression of anti-apoptotic protein, bcl-2 after 4 d of incubation. These data suggest that hypothermic conditions lowers the risk of oxidative stress-induced cellular damage and programmed cell death by increasing the activity of GSH-Px and by the induction in the expression of the anti-apoptotic protein, bcl-2.

Effects of bleomycin on liver antioxidant enzymes and the electron transport system from ad libitum-fed and dietary-restricted female and male Fischer 344 rats.

Dietary restriction (DR) is the only known intervention that delays aging and age-related diseases. Mechanisms proposed to explain this DR effect include a decline in free radical production and an increase in free radical detoxification. In the present study the effect of bleomycin (BLM) as a reactive oxygen species-generating antitumor drug has been evaluated on antioxidant enzymes and the electron transport system in different cellular fractions of liver in female and male Fischer 344 rats. Animals were fed ad libitum (AL) or 60% of the AL intake (DR) and were given a single intraperitoneal injection of 2.5, 5, or 10 mg BLM/kg body wt. After four weeks, BLM significantly increased glutathione peroxidase and lactate dehydrogenase activities in liver cytosol of female AL rats and increased activity even more in male rats. Similar changes were also noted for glutathione reductase and glucose 6-phosphate dehydrogenase activities in BLM-treated AL rats. In liver mitochondria, glutathione peroxidase was increased in female and male AL rats but was increased more in female rats. Drug treatment had no significant effect on these enzyme activities in cytosolic or mitochondrial fractions of DR animals. Profound effects of BLM were noted in activities of complexes I, III, and IV of the electron transport system in AL and DR female and male rats; however, complex II demonstrated no significant diet or treatment effect. Induced antioxidant enzyme activities in BLM-treated AL rats may be a response to excessive free radical generation due to BLM metabolism in AL animals that is mitigated by DR. Furthermore, dysfunction of the electron transport system might suggest its role in a secondary generation of free radicals during BLM metabolism contributing to its toxicity.

Effects of dietary restriction on insulin resistance in obese mice.

In many cases, development of insulin resistance has been linked to obesity and may contribute to mechanism of aging. The role of diet, irrespective of degree of obesity, in modulating insulin resistance and development of age degeneration disease remains uncertain. Lowered blood glucose levels are commonly associated with diet restriction (DR), which is an intervention shown to successfully retard aging and age associated disease. The effects of DR on blood glucose and insulin resistance were measured in yellow obese (A(vy)/A), lean black (a/a) mice and in another common inbred strain (B6C3F1) (at three different ages). The yellow obese mice become diabetic as a result of an insulin receptor defect which is not clearly understood. Insulin responses and radioinsulin binding were assayed in yellow obese and lean black mice fed either ad libitum (AL) or DR diets (YAL, BAL, YDR and YAL, respectively) at four different circadian intervals. The B6C3F1 controls were fed either AL (CAL) or DR (CDR) and measures were made at six circadian stages and three different ages. Within 23 days, DR produced a significant loss in body weight and a time-dependent 22-55% reduction in basal blood glucose levels in the yellow obese mice. Additionally, exogenous insulin produced circadian stage dependent (at the time of food intake) reductions in blood glucose in the YDR animals that were not present in YAL animals. (125)I-Insulin binding in liver was increased nearly 2-fold in YDR and BDR mice during the time of day that animals were active and eating. (125)I-Insulin binding was two-fold-higher in CDR mice at 4, 12 and >24 months of age. Binding decreased as a function of age in both the CAL and CDR animals. However, even in the >24 month group the CDR animals were found to have levels of binding that were as high as those found in younger CAL liver. The mechanism of action appears to be through resolution of insulin resistance by modulating an insulin receptor defect.

Attenuation of bleomycin-induced Hprt mutant frequency in female and male rats by calorie restriction.

Calorie restriction modulates spontaneous and chemically induced tumors and increases maximal life span in experimental animals; however, the mechanism by which calorie restriction exerts its ameliorating effects is not fully elucidated, although reduced levels of reactive oxygen species (ROS) by calorie restriction has generated much interest. In the present study, we have determined whether or not calorie restriction would affect the mutagenic response in rats treated with bleomycin (BLM) a radiomimetic drug that is associated with DNA damage by a free radical mechanism. Fourteen weeks after weaning, the rats were divided into two groups; ad libitum (AL)-fed and 40% calorie restriction. Both AL and calorie-restricted animals were injected with 2.5, 5.0 and 10.0 mg BLM/kg, or with phosphate-buffered saline (PBS), and they were killed 4 weeks post drug treatment. Lymphocytes from the spleens were seeded in 96-well microtiter plates to determine mutant frequency in the hypoxantine guanine phosphoribosyl transferase (Hprt) gene. The mutant frequency in the BLM-treated rats was higher in AL males (P=0.001), and AL females (P=0.0174) than in their calorie-restricted counterparts. The difference in mutagenic response relative to AL males and AL females appeared unrelated to a low percent cloning efficiency seen in the males, since the mean absolute number of Hprt mutant clones was higher in the AL males compared to the females. A reduction in animal weight by calorie restriction was significant in both sexes (P<0.001), but the dose effect appeared non-significant. The results indicate that calorie intake of 60% reduced the mutagenic response of BLM, a compound known to induce oxidative DNA damage, and suggest a possible decrease in ROS as a function of calorie restriction.

Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils.

BACKGROUND: As assessed by flow cytometry, the increase in hydrogen peroxide in individual neutrophils from old volunteers was significantly greater than in neutrophils from young volunteers. To explain the discrepancy in previous reports that showed reduced superoxide generation with age and our finding, we measured the kinetics of antioxidative enzymes. METHODS: Neutrophils were obtained from young (ages 21-34) and old (ages over 65) volunteers. The increase in hydrogen peroxide following stimulation with formyl peptide in individual neutrophils was assessed by flow cytometry by using dihydrorhodamine 123. The enzyme kinetics was determined from the best fit curve using Michaelis-Menten equations. RESULTS: Aging was associated with a significant reduction in the Vmax for glutathione peroxidase. The decreased activity was not due to selenium deficiency as the serum and neutrophil concentrations were identical with age. Following activation, a significant increase in the Km was noted in neutrophils from young but not from old volunteers. CONCLUSIONS: These results account for the increased intracellular accumulation of hydrogen peroxide as a function of age in stimulated neutrophils. These results provide evidence in humans of an age-related impairment in antioxidative defense mechanisms that support the free radical theory of aging.

Modulation of antioxidant enzymes in bleomycin-treated rats by vitamin C and beta-carotene.

Bleomycin (BLM), an antineoplastic drug, is known to induce DNA strand breaks and is also mutagenic in mammalian cells; however, its mechanism of action is not well understood. It has been proposed that BLM cytotoxicity is mediated through the generation of reactive oxygen species. We have determined the effects of BLM on endogenous hepatic antioxidant enzymes such as glutathione peroxidase (GPx), glutathione reductase, and glucose-6-phosphate dehydrogenase in rats exposed to BLM in conjunction with dietary vitamins, vitamin C and beta-carotene (BC). Male Fischer 344 rats of two different age groups were treated with BLM in the presence or absence of antioxidant vitamins. In control animals, an age-associated decrease in GPx activity was noted (p < 0.05). The decrease in GPx activity observed in BLM-treated old animals given vitamin C was significant (p < 0.05) compared with BLM-treated young animals fed vitamin C. BC moderately induced GPx and glutathione reductase activities in old BLM-treated animals; however, the increase in GPx was statistically significant (p < 0.05) only compared with old controls. A similar increase was noted in the activities of all the enzymes examined in young animals. Our results indicate that BLM exposure was accompanied by alterations in the activities of endogenous antioxidant enzymes, with a profound increase in activities occurring in old animals. In addition, the observed enzyme activities were modulated by antioxidant vitamin administration. The observation that both vitamins displayed differential effects on the enzyme activities also suggests that vitamin C and BC exert their effects by separate mechanisms.

A circadian study of liver antioxidant enzyme systems of female Fischer-344 rats subjected to dietary restriction for six weeks.

We examined the influences of dietary restriction (DR) on the circadian profile of liver catalase (CAT), glutathione peroxidase (GPx), and interacting systems required for removal of H2O2 (support systems), in 18-week old female Fischer 344 rats fed 60% of their ad libitum (AL) diet for six weeks. Food was presented to the DR animals during the early light-span. Regardless of diet, enzyme levels were generally consistent with circadian patterns. In CR animals, maximum activities often occurred at the time of food presentation. CAT and GPx activities generally were significantly higher in DR animals than in AL animals at the time of feeding. When assessing glucose-6-phosphate dehydrogenase (G6PDH) activity using saturating substrate (NADP(+)) concentrations, higher activities were seen at all times of day in the AL animals; however, when activity was measured in the presence of lower (i.e., physiologic) NADP(+) concentrations, the reverse was true. In contrast, glutathione reductase (GR) activity was not influenced by DR. Cytosolic levels of NADPH peaked and were higher in DR than in AL rodents prior to feeding. NADH levels were not influenced by diet, but did manifest a significant circadian pattern with a maximum occurring toward the middle of the dark span. These data suggest that even at a young age and following only a relatively brief duration of DR, there exists an enhanced enzymatic capability in rats subjected to DR to remove free radicals generated as a consequence of normal oxidative metabolism. Further, these data support emerging trends suggesting metabolic regulation of antioxidant defense systems in response to free radical generation.

Influences of age and dietary restriction on gastrocnemius electron transport system activities in mice.

Alterations in the mitochondrial electron transport system (ETS) may contribute to aging. Dietary restriction (DR) provides a model to investigate retarded aging. ETS activities were measured in gastrocnemius from 10- and 20-month-old B6C3F1 female mice fed either ad libitum (AL) or DR diets (40% < AL). Older (26 month old) AL mice were studied for complex IV. Activities of complexes I, III, and IV decreased 54-74% from 10 to 20 months of age in AL mice. At 10 months, activities of complexes I, III, and IV were 33-64% lower in DR compared to AL mice. The Km for ubiquinol-2 of complex III increased 29% by 20 months of age in AL mice while no change occurred in DR mice. The Vmax of complex IV declined by 90% from 10 to 26 months of age in AL mice and this change was opposed by DR. Complex IV contains high- and low-affinity binding sites. The Km for high-affinity sites was not influenced by age or diet through 20 months; however, the Km was approximately twofold higher at 26 months in AL mice. The percentage of total binding sites which were of high affinity fell from 68% at 10 months in AL mice to 46% at 20 months and was even lower (33%) at 26 months. This value was 80% for DR mice at 10 and 20 months. These alterations with aging in mitochondrial ETS capacities may contribute to decreases in skeletal muscle function.

MPP(+)-induced neurotoxicity in mouse is age-dependent: evidenced by the selective inhibition of complexes of electron transport.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been demonstrated to cause selective neurotoxicity by inhibiting complex I in mitochondria, through its toxic metabolite 1-methyl-4-phenylpyridine (MPP+) which is formed during the bioactivation of MPTP by monoamine oxidase B. In this report, we have evaluated the effect of MPP+ on the 4 mitochondrial respiratory chain complexes by incubating brain mitochondria of mice at 3 different age groups with MPP+ (200 microM) and monitoring enzyme activities of complexes I, II, III, and IV at 5, 10, 15, 30, 60, and 120 min. Complexes I, III, and IV showed significant inhibition within 15 min in all the age groups studied, followed by some recovery in enzyme activities upon further incubation for complexes I and IV. However, complex II was not affected by MPP+ at any age. Our data suggest that inhibition of complexes I, III, and IV by MPP+ efficiently restrict the transport of electrons down the respiratory chain which ultimately leads to decreased ATP production. This could further aggravate oxidative stress as ATP is required for the synthesis of glutathione (GSH), one of the important scavengers of free radicals. In this study, inhibition was more severe in mitochondrial preparations from older rather than younger mice. Additionally, young animals showed faster recovery following inhibition than old animals for complex I. Impaired respiratory chain function in older animals compared to younger ones supports the hypothesis of accumulation of age-related mitochondrial DNA mutations which partly encode for subunits of complexes I, III, and IV. From this study, it seems that inhibition of complexes I, III, and IV may be the underlying cause of neurotoxicity due to MPP+ which could be intensified by age-associated dysfunction of electron transport.

Experience with the International League Against Epilepsy classifications of epileptic seizures (1981) and epilepsies and epileptic syndrome (1989) in epileptic children in a developing country.

Four hundred eighty-three epileptic children attending the Pediatric Epilepsy Clinic at Bai Jerbai Wadia Hospital for Children, Bombay, India were classified according to the International League Against Epilepsy (ILAE) classification of epileptic seizures (1981) and epilepsies and epileptic syndromes (1989). The predominant seizures were partial (53.6), generalized (40.3%), and unclassifiable (6%). In epilepsies and epileptic syndromes, 55.3% were partial, 27% were generalized, 13.5% were undetermined, and 4.1% were special syndromes. Although our results were similar in many respects to those of other reported series, some differences were observed in the incidence of partial and generalized seizures, and partial and generalized epileptic syndromes and their subgroups, such as idiopathic, symptomatic, and cryptogenic partial syndromes, idiopathic generalized syndromes, and symptomatic specific syndromes. These differences are probably due to different age limits, methods of case ascertainment and inclusion criteria, different genetic and environmental factors, variable interpretation of clinical and EEG features, and lack of facilities for investigation in developing countries. Despite various limitations, we were able to classify most cases; the ILAE classification can be used in developing countries so that comparison can be made with other studies.