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Biomedical subjects

B N Ames

Publications and source records attributed to B N Ames.

At least 37 records · Page 2Linked to original sources
PubMed2000

(R)-alpha-lipoic acid reverses the age-associated increase in susceptibility of hepatocytes to tert-butylhydroperoxide both in vitro and in vivo.

Hepatocytes were isolated from young (3-5 months) and old (24-28 months) rats and incubated with various concentrations of tert-butylhydroperoxide (t-BuOOH). The t-BuOOH concentration that killed 50% of cells (LC50) in 2 hr declined nearly two-fold from 721 +/- 32 microM in cells from young rats to 391 +/- 31 microM in cells from old rats. This increased sensitivity of hepatocytes from old rats may be due, in part, to changes in glutathione (GSH) levels, because total cellular and mitochondrial GSH were 37.7% and 58.3% lower, respectively, compared to cells from young rats. Cells from old animals were incubated with either (R)- or (S)-lipoic acid (100 microM) for 30 min prior to the addition of 300 microM t-BuOOH. The physiologically relevant (R)-form, a coenzyme in mitochondria, as opposed to the (S)-form significantly protected hepatocytes against t-BuOOH toxicity. Dietary supplementation of (R)-lipoic acid [0.5% (wt/wt)] for 2 weeks also completely reversed the age-related decline in hepatocellular GSH levels and the increased vulnerability to t-BuOOH as well. An identical supplemental diet fed to young rats did not enhance the resistance to t-BuOOH, indicating that antioxidant protection was already optimal in young rats. Thus, this study shows that cells from old animals are more susceptible to oxidant insult and (R)-lipoic acid, after reduction to an antioxidant in the mitochondria, effectively reverses this age-related increase in oxidant vulnerability.

Age Factors↗
PubMed2000

Ascorbate is depleted by smoking and repleted by moderate supplementation: a study in male smokers and nonsmokers with matched dietary antioxidant intakes.

BACKGROUND: Lack of reliable dietary data has hampered the ability to effectively distinguish between effects of smoking and diet on plasma antioxidant status. As confirmed by analyses of comprehensive food-frequency questionnaires, the total dietary intakes of fruit and vegetables and of dietary antioxidants were not significantly different between the study groups in the present study, thereby enabling isolation of the effect of smoking. OBJECTIVE: Our objective was to investigate the effect of smoking on plasma antioxidant status by measuring ascorbic acid, alpha-tocopherol, gamma-tocopherol, beta-carotene, and lycopene, and subsequently, to test the effect of a 3-mo dietary supplementation with a moderate-dose vitamin cocktail. DESIGN: In a double-blind, placebo-controlled design, the effect of a vitamin cocktail containing 272 mg vitamin C, 31 mg all-rac-alpha-tocopheryl acetate, and 400 microg folic acid on plasma antioxidants was determined in a population of smokers (n = 37) and nonsmokers (n = 38). The population was selected for a low intake of fruit and vegetables and recruited from the San Francisco Bay area. RESULTS: Only ascorbic acid was significantly depleted by smoking per se (P < 0.01). After the 3-mo supplementation period, ascorbic acid was efficiently repleted in smokers (P < 0.001). Plasma alpha-tocopherol and the ratio of alpha- to gamma-tocopherol increased significantly in both supplemented groups (P < 0.05). CONCLUSIONS: Our data suggest that previous reports of lower concentrations of plasma vitamin E and carotenoids in smokers than in nonsmokers may primarily have been caused by differences in dietary habits between study groups. Plasma ascorbic acid was depleted by smoking and repleted by moderate supplementation.

Adult↗
PubMed2000

Supplementation of postmenopausal women with fish oil rich in eicosapentaenoic acid and docosahexaenoic acid is not associated with greater in vivo lipid peroxidation compared with oils rich in oleate and linoleate as assessed by plasma malondialdehyde and F(2)-isoprostanes.

BACKGROUND: Although the replacement of dietary saturated fat with unsaturated fat has been advocated to reduce the risk of cardiovascular disease, diets high in polyunsaturated fatty acids (PUFAs) could increase lipid peroxidation, potentially contributing to the pathology of atherosclerosis. OBJECTIVE: The objective of this study was to examine indexes of in vivo lipid peroxidation, including free F(2)-isoprostanes, malondialdehyde (MDA), and thiobarbituric acid reacting substances (TBARS), in the plasma of postmenopausal women taking dietary oil supplements rich in oleate, linoleate, and both eicosapentaenoic acid and docosahexaenoic acid. DESIGN: Fifteen postmenopausal women took 15 g sunflower oil/d, providing 12.3 g oleate/d; safflower oil, providing 10.5 g linoleate/d; and fish oil, providing 2.0 g EPA/d and 1.4 g DHA/d in a 3-treatment crossover trial. RESULTS: Plasma free F(2)-isoprostane concentrations were lower after fish-oil supplementation than after sunflower-oil supplementation (P: = 0.003). When plasma free F(2)-isoprostane concentrations were normalized to plasma arachidonic acid concentrations, significant differences among the supplements were eliminated. Plasma MDA concentrations were lower after fish-oil supplementation than after sunflower-oil supplementation (P: = 0.04), whereas plasma TBARS were higher after fish-oil supplementation than after sunflower oil (P: = 0.003) and safflower oil (P: = 0.001) supplementation. When plasma MDA concentrations were normalized to plasma PUFA concentrations, significant differences were eliminated, but TBARS remained higher after fish-oil supplementation than after sunflower oil (P: = 0.01) and safflower-oil (P: = 0.0003) supplementation. CONCLUSIONS: With fish-oil supplementation, there was no evidence of increased lipid peroxidation when assessed by plasma F(2)-isoprostanes and MDA, although plasma TBARS was higher than with sunflower-oil and safflower-oil supplementation.

Aged↗
PubMed2000

Both iron deficiency and daily iron supplements increase lipid peroxidation in rats.

Numerous studies have shown that iron-loaded diets increase markers of lipid peroxidation in rats, but few have addressed the effects of oral iron supplements on these markers. We investigated the effects of daily and intermittent iron supplements on iron and vitamin E status, and lipid peroxidation. Iron supplements were administered in doses equivalent to those often given to pregnant women in the developing world. In Study 1, iron-deficient (D) and iron-normal (N) rats were fed either 0 or 8000 microgram of supplemental iron daily for 21 d. In Study 2, D rats were fed either the same supplements daily or once every 3 d (8 supplements total). Lipid peroxidation was assessed by breath ethane and pentane and by malondialdehyde (MDA) (using GC-MS). In Study 1, daily supplemented N and D rats had liver nonheme iron concentrations that were 1.8- and 2.7-fold higher, respectively, than those in unsupplemented N rats. Breath ethane levels were also higher in supplemented rats (P < 0.05), but MDA (in plasma, liver, kidney) and liver vitamin E did not differ. Unexpectedly, severely D, anemic rats had significant elevations in the levels of breath ethane, liver MDA and kidney MDA. In Study 2, liver iron and breath ethane decreased progressively (P < 0.05) from 1 d to 3 d after the last iron dose in intermittently supplemented rats. We conclude that iron deficiency results in lipid peroxidation, but that its correction with daily iron supplements results in abnormal iron accumulation and increased lipid peroxidation in rats. These effects are mitigated by intermittent iron supplementation.

Animals↗
PubMed2000

Chronically and acutely exercised rats: biomarkers of oxidative stress and endogenous antioxidants.

The responses to oxidative stress induced by chronic exercise (8-wk treadmill running) or acute exercise (treadmill running to exhaustion) were investigated in the brain, liver, heart, kidney, and muscles of rats. Various biomarkers of oxidative stress were measured, namely, lipid peroxidation [malondialdehyde (MDA)], protein oxidation (protein carbonyl levels and glutamine synthetase activity), oxidative DNA damage (8-hydroxy-2'-deoxyguanosine), and endogenous antioxidants (ascorbic acid, alpha-tocopherol, glutathione, ubiquinone, ubiquinol, and cysteine). The predominant changes are in MDA, ascorbic acid, glutathione, cysteine, and cystine. The mitochondrial fraction of brain and liver showed oxidative changes as assayed by MDA similar to those of the tissue homogenate. Our results show that the responses of the brain to oxidative stress by acute or chronic exercise are quite different from those in the liver, heart, fast muscle, and slow muscle; oxidative stress by acute or chronic exercise elicits different responses depending on the organ tissue type and its endogenous antioxidant levels.

Animals↗
PubMed2000

Effect of ebselen on Ca2+ transport in mitochondria.

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.

Animals↗
PubMed1999

Endogenous oxidative damage of mtDNA.

Almost a decade ago, based on analytical measurements of the oxidative DNA adduct 8-oxo-deoxyguanosine (oxo8dG), it was reported that mitochondrial DNA suffers greater endogenous oxidative damage than nuclear DNA. The subsequent discovery that somatic deletions of mitochondrial DNA occur in humans, and that they do so to the greatest extent in metabolically active tissues, strengthened the hypothesis that mitochondrial DNA is particularly susceptible to endogenous oxidative attack. This hypothesis was (and is) appealing for a number of reasons. Nevertheless, solid direct support for the hypothesis is lacking. Since the initial measurements, attempts to repeat the observation of greater oxidation of mitochondrial DNA have resulted in a range of measurements that spans over four orders of magnitude. Moreover, this range includes values that are as low as published values for nuclear DNA. In the last 2 years or so, it has become apparent that the quantification of oxidative DNA adducts is prone to artifactual oxidation. We have reported that the analysis of small quantities of DNA may be particularly susceptible to such interference. Because yields of mitochondrial DNA are generally low, a systematic artifact associated with low quantities of DNA may have elevated the apparent level of adduct oxo8dG in mitochondrial DNA relative to nuclear DNA in some studies. Whatever the cause for the experimental variation, the huge disparity between published measurements of oxidative damage makes it impossible to conclude that mitochondrial DNA suffers greater oxidation than nuclear DNA. Despite the present confusion, however, there are reasons to hypothesize that this is indeed the case. We briefly describe methods being developed by a number of workers that are likely to surmount current obstacles and allow the hypothesis to be tested definitively.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Assay of malondialdehyde and other alkanals in biological fluids by gas chromatography-mass spectrometry.

The method described in this chapter allows the accurate measurement of MDA in diverse biological samples and can be extended to measurements of other alkanals. The use of GC/MS-NCI ensures specificity and sensitivity, and the ability to prepare samples without heating limits oxidation artifact. Although the widely used TBA assay for MDA does not require sophisticated equipment, its results may be of limited value as the assay is hindered by the possibility of cross reactivity and by heat-induced oxidation artifact. This GC-MS technique offers the additional advantages of efficient processing of large numbers of samples and the elimination of recovery errors by inclusion of an internal standard.

Aldehydes↗
PubMed1999

Micronutrient deficiencies. A major cause of DNA damage.

Deficiencies of the vitamins B12, B6, C, E, folate, or niacin, or of iron or zinc mimic radiation in damaging DNA by causing single- and double-strand breaks, oxidative lesions, or both. The percentage of the population of the United States that has a low intake (< 50% of the RDA) for each of these eight micronutrients ranges from 2% to 20+ percent. A level of folate deficiency causing chromosome breaks occurred in approximately 10% of the population of the United States, and in a much higher percentage of the poor. Folate deficiency causes extensive incorporation of uracil into human DNA (4 million/cell), leading to chromosomal breaks. This mechanism is the likely cause of the increased colon cancer risk associated with low folate intake. Some evidence, and mechanistic considerations, suggest that vitamin B12 and B6 deficiencies also cause high uracil and chromosome breaks. Micronutrient deficiency may explain, in good part, why the quarter of the population that eats the fewest fruits and vegetables (five portions a day is advised) has about double the cancer rate for most types of cancer when compared to the quarter with the highest intake. Eighty percent of American children and adolescents and 68% of adults do not eat five portions a day. Common micronutrient deficiencies are likely to damage DNA by the same mechanism as radiation and many chemicals, appear to be orders of magnitude more important, and should be compared for perspective. Remedying micronutrient deficiencies is likely to lead to a major improvement in health and an increase in longevity at low cost.

Adult↗
PubMed1998

Micronutrients prevent cancer and delay aging.

Approximately 40 micronutrients are required in the human diet. Deficiency of vitamins B12, folic acid, B6, niacin, C, or E, or iron, or zinc, appears to mimic radiation in damaging DNA by causing single- and double-strand breaks, oxidative lesions, or both. The percentage of the US population that has a low intake (< 50% of the RDA) for each of these eight micronutrients ranges from 2% to > or = 20%; half of the population may be deficient in at least one of these micronutrients. Folate deficiency occurs in approximately 10% of the US population, and in a much higher percentage of the poor. Folate deficiency causes extensive incorporation of uracil into human DNA (4 million/cell), leading to chromosomal breaks. This mechanism is the likely cause of the increased cancer risk, and perhaps the cognitive defects associated with low folate intake. Some evidence, and mechanistic considerations, suggest that vitamin B12 and B6 deficiencies also cause high uracil and chromosome breaks. Micronutrient deficiency may explain, in good part, why the quarter of the population that eats the fewest fruits and vegetables (five portions a day is advised) has approximately double the cancer rate for most types of cancer when compared to the quarter with the highest intake. Eighty percent of American children and adolescents and 68% of adults do not eat five portions a day. Common micronutrient deficiencies are likely to damage DNA by the same mechanism as radiation and many chemicals, appear to be orders of magnitude more important, and should be compared for perspective. Remedying micronutrient deficiencies is likely to lead to a major improvement in health and an increase in longevity at low cost. Aging appears to be due, in good part, to the oxidants produced by mitochondria as by-products of normal metabolism. In old rats mitochondrial membrane potential, cardiolipin levels, respiratory control ratio, and overall cellular O2 consumption are lower than in young rats, and the level of oxidants (per unit O2) is higher. The level of mutagenic aldehydes from lipid peroxidation is also increased. Ambulatory activity declines markedly in old rats. Feeding old rats the normal mitochondrial metabolites acetyl carnitine and lipoic acid for a few weeks, restores mitochondrial function, lowers oxidants to the level of a young rat, and increases ambulatory activity. Thus, these two metabolites can be considered necessary for health in old age and are therefore conditional micronutrients. This restoration suggests a plausible mechanism: with age-increased oxidative damage to proteins and lipid membranes causes a deformation of structure of key enzymes, with a consequent lessening of affinity (Km) for the enzyme substrate; an increased level of the substrate restores the velocity of the reaction, and thus restores function.

Aging↗
PubMed1998

Mitochondrial aging: open questions.

Interest in the role of mitochondria in aging has intensified in recent years. This focus on mitochondria originated in part from the free radical theory of aging, which argues that oxidative damage plays a key role in degenerative senescence. Among the numerous mechanisms known to generate oxidants, leakage of the superoxide anion and hydrogen peroxide from the mitochondrial electron transport chain are of particular interest, due to the correlation between species-specific metabolic rate ("rate of living") and life span. Phenomenological studies of mitochondrial function long ago noted a decline in mitochondrial function with age, and on-going research continues to add to this body of knowledge. The extranuclear somatic mutation theory of aging proposes that the accumulation of mutations in the mitochondrial genome may be responsible in part for the mitochondrial phenomenology of aging. Recent studies of mitochondrial DNA (mtDNA) deletions have shown that they increase with age in humans and other mammals. Currently, there exist numerous important and fundamental questions surrounding mitochondria and aging. Among these are (1) How important are mitochondrial oxidants in determining overall cellular oxidative stress? (2) What are the mechanisms of mitochondrial oxidant generation? (3) How are lesions and mutations in mtDNA formed? (4) How important are mtDNA lesions and mutations in causing mitochondrial dysfunction? (5) How are mitochondria regulated, and how does this regulation change during aging? (6) What are the dynamics of mitochondrial turnover? (7) What is the relationship between mitochondrial damage and lipofuscinogenesis? (8) What are the relationships among mitochondria, apopotosis, and aging? and (9) How can mitochondrial function (ATP generation and the establishment of a membrane potential) and dysfunction (oxidant generation) be modulated and degenerative senescence thereby treated?

Aging↗
PubMed1998

Mitochondrial decay in aging. Reversal through supplementation of acetyl-L-carnitine and N-tert-butyl-alpha-phenyl-nitrone.

We show that mitochondrial function in the majority of hepatocytes isolated from old rats (24 mo) is significantly impaired. Mitochondrial membrane potential, cardiolipin levels, respiratory control ratio, and overall cellular O2 consumption decline, and the level of oxidants increases. To examine whether dietary supplementation of micronutrients that may have become essential with age could reverse the decline in mitochondrial function, we supplemented the diet of old rats with 1% (w/v) acetyl-L-carnitine (ALCAR) in drinking water. ALCAR supplementation (1 month) resulted in significant increases in cellular respiration, mitochondrial membrane potential, and cardiolipin values. However, supplementation also increased the rate of oxidant production, indicating that the efficiency of mitochondrial electron transport had not improved. To counteract the potential increase in oxidative stress, animals were administered N-tert-butyl-alpha-phenyl-nitrone (30 mg/kg) (PBN) with or without ALCAR. Results showed that PBN significantly lowered oxidant production as measured by 2,7'-dichlorofluorescin diacetate (DCFH), even when ALCAR was coadministered to the animals. Thus, dietary supplementation with ALCAR, particularly in combination with PBN, improves mitochondrial function without a significant increase in oxidative stress.

Acetylcarnitine↗
PubMed1998

Acetyl-L-carnitine fed to old rats partially restores mitochondrial function and ambulatory activity.

Mitochondrial function and ambulatory activity were monitored after feeding old rats acetyl-L-carnitine (ALCAR). Young (3-5 mo) and old (22-28 mo) rats were given a 1.5% (wt/vol) solution of ALCAR in their drinking water for 1 mo, were sacrificed, and their liver parenchymal cells were isolated. ALCAR supplementation significantly reverses the age-associated decline of mitochondrial membrane potential, as assessed by rhodamine 123 staining. Cardiolipin, which declines significantly with age, is also restored. ALCAR increases cellular oxygen consumption, which declines with age, to the level of young rats. However, the oxidant production per oxygen consumed, as measured by 2',7'-dichlorofluorescin fluorescence levels, is approximately 30% higher than in untreated old rats. Cellular glutathione and ascorbate levels were nearly 30% and 50% lower, respectively, in cells from ALCAR-supplemented old rats than in untreated old rats, further indicating that ALCAR supplementation might increase oxidative stress. Ambulatory activity in young and old rats was quantified as a general measure of metabolic activity. Ambulatory activity, defined as mean total distance traveled, in old rats is almost 3-fold lower than in young animals. ALCAR supplementation increases ambulatory activity significantly in both young and old rats, with the increase being larger in old rats. Thus, ALCAR supplementation to old rats markedly reverses the age-associated decline in many indices of mitochondrial function and general metabolic activity, but may increase oxidative stress.

Acetylcarnitine↗
PubMed1998

Molecular analysis of H2O2-induced senescent-like growth arrest in normal human fibroblasts: p53 and Rb control G1 arrest but not cell replication.

Human diploid fibroblasts lose the capacity to proliferate and enter a state termed replicative senescence after a finite number of cell divisions in culture. When treated with sub-lethal concentrations of H2O2, pre-senescent human fibroblasts enter long-term growth arrest resembling replicative senescence. To understand the molecular basis for the H2O2-induced growth arrest, we determined the cell cycle distribution, levels of p53 tumour suppressor and p21 cyclin-dependent kinase inhibitor proteins, and the status of Rb phosphorylation in H2O2-treated cells. A 2-h pulse of H2O2 arrested the growth of IMR-90 fetal lung fibroblasts for at least 15 days. The arrested cells showed a G1 DNA content. The level of p53 protein increased 2- to 3-fold within 1.5 h after H2O2 exposure but returned to the control level by 48 h. The induction of p53 protein was dose dependent, beginning at 50-75 microM and reaching a maximum at 100-250 microM. The induction of p53 did not appear to correlate with the level of DNA damage as measured by the formation of 8-oxo-2'-deoxyguanosine in DNA. The level of p21 protein increased about 18 h after H2O2 exposure and remained elevated for at least 21 days. During this period, Rb remained underphosphorylated. The induction of p53 by H2O2 was abolished by the iron chelator deferoxamine and the protein synthesis inhibitor cycloheximide. The human papillomavirus protein E6, when introduced into the cells, abolished the induction of p53, reduced the induction of p21 to a minimal level and allowed Rb phosphorylation and entry of the cells into S-phase. The human papillomavirus protein E7 reduced the overall level of Rb and also abolished H2O2-induced G1 arrest. Inactivating G1 arrest by E6, E7 or both did not restore the replicative ability of H2O2-treated cells. Thus H2O2-treated cells show a transient elevation of p53, high level of p21, lack of Rb phosphorylation, G1 arrest and inability to replicate when G1 arrest is inactivated.

Cell Division↗
PubMed1998

Quantitation of age-related mitochondrial DNA deletions in rat tissues shows that their pattern of accumulation differs from that of humans.

Three age-related mtDNA deletions were identified, and the competitive polymerase chain reaction (PCR) was used to quantitate their levels in different Fisher 344 rat tissues. Deletions that removed 4834; 13273; or 13415nt of the mitochondrial genome were shown to be associated with 16 (mtDNA4834), nine (mtDNA13273), or five (mtDNA13415) nt direct repeats, respectively. The mtDNA4834 deletion was detected in an age-related manner in all tissues screened; the mtDNA13415 deletion was detected in old heart, and in both young and old brain; and the mtDNA13273 deletion was only detected in old brain tissues. The mtDNA4834 deletion was found to be at its highest level in the liver (1.88x10-2%), followed by the brain (0.22x10-2%) and kidney (0.40x10-2%) of old animals. Much lower levels were observed in old heart (0.07x10-2%) and lung (0. 04x10-2%). This distribution of mtDNA deletions in old rat tissues is in contrast to work done in humans where age-related deletions are present at the highest levels in post-mitotic tissues with much lower levels in more mitotic tissues. An inverse relationship was observed between the level of mtDNA deletions and the size of the deleted region, since the mtDNA13415 deletion was present at about a 100-fold lower level (0.53x10-5%) than the smaller mtDNA4834 deletion in old heart tissue.

Aging↗