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Biomedical subjects

B N Ames

Publications and source records attributed to B N Ames.

At least 91 records · Page 5Linked to original sources
PubMed1994

Oxidative damage and mitochondrial decay in aging.

We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of aging. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Several mitochondrial functions decline with age. The contributing factors include the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Acetyl-L-carnitine, a high-energy mitochondrial substrate, appears to reverse many age-associated deficits in cellular function, in part by increasing cellular ATP production. Such evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.

Aging↗
PubMed1994

Heterocyclic amines formed by cooking food: comparison of bioassay results with other chemicals in the Carcinogenic Potency Database.

Results in the Carcinogenic Potency Database (CPDB) on 11 mutagenic heterocyclic amines (HA) tested for carcinogenicity in rats, mice and cynomolgus monkeys are compared to results for other chemicals. An analysis of strength of evidence of carcinogenicity for HA vs. other mutagenic carcinogens and vs. all rodent carcinogens, indicates strong carcinogenicity of HA in terms of positivity rates and multiplicity of target sites. The liver is the most frequent target site in each species. Despite several target sites in each species, concordance in target sites between rats and mice is restricted to the liver for each HA except one. In cynomolgus monkeys, liver tumors have been induced rapidly by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Human exposures to HA in cooked animal foods are small, in the low ppb range. A comparison of possible carcinogenic hazards from a variety of exposures to rodent carcinogens in the American diet is presented, using an index (Human Exposure/Rodent Potency, HERP) that relates human exposure to carcinogenic potency in rodents. Results indicate that there is a large background of exposures to naturally-occurring rodent carcinogens in typical portions of common foods, and that possible hazards from HA rank below those of most natural pesticides and products of cooking or food preparation; synthetic pesticide residues also rank low.

Animals↗
PubMed1994

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

Malondialdehyde (MDA) is assayed in femtomole quantities in biological samples by gas chromatography-mass spectrometry (GC-MS). The MDA trapped in protein as a Schiff base is released by H2SO4, the protein precipitated using Na2WO4, and the MDA derivatized with pentafluorophenylhydrazine to form the stable adduct, N-pentafluorophenylpyrazole. Negative chemical ionization (NCI) capability allows the sensitive detection of this MDA adduct in biological samples at a level of 5 nM on-column. A stable-isotope-labeled MDA, [2H2]MDA, was used as an internal standard for quantitation. MDA recovery from plasma was 76%. This assay provides two forms of confirmation of the analyte, retention time and mass ion, thus minimizing error due to interfering compounds. The commonly used thiobarbituric acid assay for MDA overestimates the MDA levels by over 10-fold, possibly resulting from cross-reactivity with other aldehydes and artifactual oxidation due to 100 degrees C temperature conditions. In our assay, all steps were performed at room temperature thereby suppressing artifactual oxidation of the sample. We have successfully applied this assay to biological samples including plasma, tissue homogenates, and sperm.

Animals↗
PubMed1994

Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells.

Human diploid fibroblast cells lose replicative potential after a certain number of population doublings. We use this experimental system to investigate the role of oxidative damage in cellular aging. Treating cells with H2O2 at < 300 microM did not affect the viability of the majority of cells when judged by morphology, trypan blue exclusion, and protein synthesis. However, the treatment caused a dose-dependent inhibition of DNA synthesis. After a 2-hr treatment with 200 microM H2O2, the cells failed to respond to a stimulus of serum, platelet-derived growth factor, basic fibroblast growth factor, or epidermal growth factor by synthesizing DNA, and the loss of response could not be recovered by 4 days. Subcultivation showed that, as in senescent cells, division of the treated cells was inhibited. The life-time cumulative growth curve showed that the loss of replication due to H2O2 treatment was cumulative and irreversible. The H2O2 treatment decreased the number of the population doublings in the rest of the life span by 35.3 +/- 10.3%. Enzymatic assays indicated that, like the cells in their senescent state, the treated cells were less able to activate ornithine decarboxylase and thymidine kinase. Furthermore, subcultivation after the H2O2 treatment showed that the cells developed the morphology of senescent cells. In conclusion, sublethal treatment of H2O2 "stunned" F65 cells and caused the cells to enter a state resembling senescence.

Amino Acids↗
PubMed1994

Oxidative susceptibility of low density lipoprotein subfractions is related to their ubiquinol-10 and alpha-tocopherol content.

The conjugated polyene fatty acid parinaric acid (PnA) undergoes a stoichiometric loss in fluorescence upon oxidation and can be used to directly monitor peroxidative stress within lipid environments. We evaluated the course of potentially atherogenic oxidative changes in low density lipoproteins (LDL) by monitoring the oxidation of PnA following its incorporation into buoyant (p = 1.026-1.032 g/ml) and dense (p = 1.040-1.054 g/ml) LDL subfractions. Copper-induced oxidation of LDL-associated PnA exhibited an initial lag phase followed by an increased rate of loss until depletion. Increased PnA oxidation occurred immediately after the antioxidants ubiquinol-10 and alpha-tocopherol were consumed but before there were marked elevations in conjugated dienes. Despite differences in sensitivity to early oxidation events, PnA oxidation and conjugated diene lag times were correlated (r = 0.582; P = 0.03), and both indicated a greater susceptibility of dense than buoyant LDL in accordance with previous reports. The greater susceptibility of PnA in dense LDL was attributed to reduced levels of ubiquinol-10 and alpha-tocopherol, which were approximately 50% lower than in buoyant LDL (mol of antioxidant/mol of LDL) and together accounted for 80% of the variation in PnA oxidation lag times. These results suggest that PnA is a useful probe of LDL oxidative susceptibility and may be superior to conjugated dienes for monitoring the initial stages of LDL lipid peroxidation. Differences in oxidative susceptibility among LDL density subfractions are detected by the PnA assay and are due in large part to differences in their antioxidant content.

Adult↗
PubMed1994

Analysis of uracil in DNA by gas chromatography-mass spectrometry.

A sensitive method using gas chromatography and negative chemical ionization mass spectrometry for the detection of uracil in DNA is described. Uracil DNA glycosylase is used to specifically cleave uracil from DNA. Once removed, uracil is derivatized with 3,5-bis(trifluoromethyl)benzyl bromide and the sample components are separated with gas chromatography. Negative chemical ionization mass spectrometry is used to quantitatively detect as little as 1 pg of uracil per 100 micrograms DNA. This assay is 10 times more sensitive than previously described quantitative methods for the detection of uracil in DNA. We apply this method as part of a project to determine why folic acid deficiency causes DNA breaks. We demonstrate that inhibition of folic acid metabolism induces an accumulation of uracil in DNA.

Animals↗
PubMed1994

Assays of oxidative DNA damage biomarkers 8-oxo-2'-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection.

High-performance liquid chromatography with electrochemical detection is a highly sensitive and selective method for detecting oxo8dG and oxo8Gua, biomarkers of oxidative DNA damage. When employed together with the DNA isolation and monoclonal antibody-based immunoaffinity purification methods described, oxo8dG and oxo8Gua in DNA and urine can be readily detected and quantitated, offering a powerful approach for assessing oxidative DNA damage in vivo. Application of the technique to the detection of oxo8dG from DNA permits quantitation of the steady-state levels of this oxidatively modified deoxynucleoside and overcomes the detection problems associated with the extremely low levels present in DNA. In addition, the selectivity gained by this detection method eliminates the problem of separating the signal for oxo8dG from those of normal deoxynucleosides. The quantitation of oxo8dG and oxo8Gua in biological fluids is noninvasive and complements the measurement of oxo8dG in DNA by estimating the rate of oxidative DNA damage occurring within the body or in a population of cells. This analytical approach may allow one to estimate oxidative DNA damage in an animal or individual exposed to prooxidant conditions associated with lifestyle, genetic predisposition, degenerative diseases, or environmental toxins. Furthermore, these assays may allow one to assess the potentially beneficial effects of intervention strategies that protect DNA from such damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Measurement of antioxidants in human blood plasma.

The concentration of antioxidants in human blood plasma is important in investigating and understanding the relationship between diet, oxidant stress, and human disease. The HPLC-EC technique combines selectivity with high sensitivity for measuring both water- and lipid-soluble antioxidants. The excellent sensitivity of the methods described here allows one to measure a panel of antioxidants in a small volume of plasma.

Antioxidants↗
PubMed1994

Intestinal cell proliferation is influenced by intakes of protein and energy, aflatoxin, and whole-body radiation.

Intestinal epithelial cell proliferation in young male F344 rats was measured in response to dietary protein content (5%, 10%, and 20% casein diets), energy restriction (energy intake was 60% of ad libitum energy intakes of animals consuming the 20% casein diet), total diet restriction (dietary intake was 60% of the ad libitum intake of 20% casein diet group), aflatoxin administration, and whole body irradiation. Cellular proliferation was measured in sections of jejunum, ileum, proximal colon, and distal colon with the [3H]thymidine technique. Restricting energy or total diet intakes by 40% from ad libitum levels reduced proliferation in epithelial cells throughout the intestine. In comparison to the 5% casein diet, the 20% casein diet resulted in modestly lower cellular proliferation in all intestinal segments. Radiation induced a decrease in cellular proliferation in the jejunum and ileum; this decrease was prevented by a 20% casein diet. Pretreatment with aflatoxin B1 decreased intestinal cell proliferation throughout the intestine, and this decrease was not influenced by the protein content of the diet.

Aflatoxins↗
PubMed1993

The oxidation of blood plasma and low density lipoprotein components by chemically generated singlet oxygen.

Human blood plasma and freshly isolated LDL were exposed to singlet oxygen (1O2) by thermal decomposition of synthetic endoperoxides. Exposure of blood plasma to 20 mM water-soluble 1O2 generator resulted in the depletion of ascorbate (100%), urate (75%), ubiquinol-10 (65%), protein thiols (50%), and bilirubin (25%), whereas under these conditions the levels of alpha-tocopherol, beta-carotene, and lycopene remained unchanged. The following rates of depletion were obtained by kinetic analysis (moles depleted per 100 mol of 1O2 consumed): protein thiols (5), urate (5), ascorbate (4), bilirubin (1), and ubiquinol-10 (0.008). In contrast, the rates of depletion using the lipid-soluble 1O2 generator were faster for bilirubin (13-fold), protein thiols (9-fold), ubiquinol-10 (8-fold), and ascorbate (5-fold), and slower for urate (2-fold). The formation of lipid hydroperoxides, including mostly cholesteryl linoleate hydroperoxide, was observed in 1O2-treated plasma (0.007-0.009 mol/100 mol 1O2) and LDL solutions (0.086 mol/100 mol 1O2). Based on competition kinetics, we estimate that 98% of 1O2 generated in the aqueous phase of plasma is quenched by components in this phase, mostly by plasma protein (63%; 6% by protein thiols), urate (9%; 5% by chemical quenching), and bilirubin (5%; 1% by chemical quenching). Ascorbate and ubiquinol-10 do not contribute to 1O2 quenching in plasma, and their oxidation is probably mediated secondary species. The remaining 1O2 generated in plasma (2%) diffuses into lipoprotein leading to the formation of lipid hydroperoxides with an efficiency of about 100-fold greater than that compared to aqueous generated 1O2. The principal 1O2 quenchers in LDL include apoB (42%), lycopene and beta-carotene (40%), and alpha-tocopherol (17%). The importance of carotenoids in the quenching of 1O2 in lipoprotein suggest that the beneficial effects of these compounds in health may in part be due to the elimination of this species in biology and medicine.

Adult↗
PubMed1993

Oxidants, antioxidants, and the degenerative diseases of aging.

Metabolism, like other aspects of life, involves tradeoffs. Oxidant by-products of normal metabolism cause extensive damage to DNA, protein, and lipid. We argue that this damage (the same as that produced by radiation) is a major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular disease, immune-system decline, brain dysfunction, and cataracts. Antioxidant defenses against this damage include ascorbate, tocopherol, and carotenoids. Dietary fruits and vegetables are the principal source of ascorbate and carotenoids and are one source of tocopherol. Low dietary intake of fruits and vegetables doubles the risk of most types of cancer as compared to high intake and also markedly increases the risk of heart disease and cataracts. Since only 9% of Americans eat the recommended five servings of fruits and vegetables per day, the opportunity for improving health by improving diet is great.

Aging↗
PubMed1993

Oxidants and mitogenesis as causes of mutation and cancer: the influence of diet.

A very high level oxidative damage to DNA occurs during normal metabolism. In each rat cell, the steady-state level of this damage is estimated to be about 10(6) oxidative adducts, and about 10(5) new adducts are formed daily. This endogenous DNA damage appears to be a major contributor to cancer and aging. The oxidative damage rate in mammalian species with a high metabolic rate, short life span, and high age-specific cancer rate such as in rats is much higher than the rate in humans, long-lived mammals with a lower metabolic rate, and a lower age-specific cancer rate. It is argued that deficiency of micronutrients, that protect against oxidative DNA damage, is a major contributor to human cancer. Epidemiological studies, a large body of experimental evidence, and theoretical work on the mechanisms of carcinogenesis point to mitogenesis as a major contributor to cancer. Dividing cells, compared to nondividing cells, are at an increased risk for mutations due to: 1.) conversion of DNA adducts to mutations; 2.) chance of mitotic recombination, gene conversion, and nondisjunction; and, 3.) increased exposure of DNA to mutagens. Mitogenesis also increases the probability of gene amplification and loss of 5-methylcytosine. Dietary interventions that lower mitogenesis, such as calorie restriction, decrease cancer incidence.

Aging↗
PubMed1993

Antioxidant status and indexes of oxidative stress during consecutive days of exercise.

We tested whether consecutive days of prolonged submaximal exercise would result in oxidant stress sufficient to alter blood antioxidant profiles, progressively change and exhaust blood and plasma antioxidants, and damage RNA. Eleven moderately trained males (24.3 +/- 1.1 yr) exercised 90 min at 65% peak O2 uptake on a cycle ergometer for 3 consecutive days. During day 1 exercise, blood reduced glutathione (GSH) declined 55 +/- 10% and oxidized glutathione (GSSG) increased 28 +/- 7% within 15 min. Total blood glutathione did not significantly change during exercise. GSH levels returned to baseline after 15 min of recovery. On day 3, preexercise GSH and GSSG levels were not significantly different from day 1 preexercise values; essentially similar results were obtained during exercise and recovery. During day 1 exercise, plasma total ascorbate (ascorbate + dehydroascorbate) increased from 53.8 +/- 9.3 to 59.0 +/- 11.3 microM, and percent reduced ascorbate increased from 77.6 +/- 9.3 to 87.3 +/- 9.7%. During day 3 exercise, plasma ascorbate changes were similar to those on day 1. Plasma vitamin E did not change due to exercise on either day 1 or 3. RNA adducts, urinary 8-hydroxyguanosine, did not change significantly due to exercise. Observed increases in GSH oxidation indicate the presence of oxidant stress during prolonged submaximal exercise. Similar redox changes on consecutive days of exercise, with recovery to preexercise values within 15 min, indicate no evidence of persistent or cumulative exercise effects on blood glutathione redox status.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

The fifth plot of the Carcinogenic Potency Database: results of animal bioassays published in the general literature through 1988 and by the National Toxicology Program through 1989.

This paper is the fifth plot of the Carcinogenic Potency Database (CPDB) that first appeared in this journal in 1984 (1-5). We report here results of carcinogenesis bioassays published in the general literature between January 1987 and December 1988, and in technical reports of the National Toxicology Program between July 1987 and December 1989. This supplement includes results of 412 long-term, chronic experiments of 147 test compounds and reports the same information about each experiment in the same plot format as the earlier papers: the species and strain of test animal, the route and duration of compound administration, dose level and other aspects of experimental protocol, histopathology and tumor incidence, TD50 (carcinogenic potency) and its statistical significance, dose response, author's opinion about carcinogenicity, and literature citation. We refer the reader to the 1984 publications (1,5,6) for a guide to the plot of the database, a complete description of the numerical index of carcinogenic potency, and a discussion of the sources of data, the rationale for the inclusion of particular experiments and particular target sites, and the conventions adopted in summarizing the literature. The five plots of the database are to be used together, as results of individual experiments that were published earlier are not repeated. In all, the five plots include results of 4487 experiments on 1136 chemicals. Several analyses based on the CPDB that were published earlier are described briefly, and updated results based on all five plots are given for the following earlier analyses: the most potent TD50 value by species, reproducibility of bioassay results, positivity rates, and prediction between species.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

DNA lesions, inducible DNA repair, and cell division: three key factors in mutagenesis and carcinogenesis.

DNA lesions that escape repair have a certain probability of giving rise to mutations when the cell divides. Endogenous DNA damage is high: 10(6) oxidative lesions are present per rat cell. An exogenous mutagen produces an increment in lesions over the background rate of endogenous lesions. The effectiveness of a particular lesion depends on whether it is excised by a DNA repair system and the probability that it gives rise to a mutation when the cell divides. When the cell divides, an unrepaired DNA lesion has a certain probability of giving rise to a mutation. Thus, an important factor in the mutagenic effect of an exogenous agent whether it is genotoxic or non-genotoxic, is the increment it causes over the background cell division rate (mitogenesis) in cells that appear to matter most in cancer, the stem cells, which are not on their way to being discarded. Increasing their cell division rate increases mutation and therefore cancer. There is little cancer from nondividing cells. Endogenous cell division rates can be influenced by hormone levels, decreased by calorie restriction, or increased by high doses of chemicals. If both the rate of DNA lesions and cell division are increased, then there will be a multiplicative effect on mutagenesis (and carcinogenesis), for example, by high doses of a mutagen that also increases mitogenesis through cell killing. The defense system against reactive electrophilic mutagens, such as the glutathione transferases, are also almost all inducible and buffer cells against increments in active forms of chemicals that can cause DNA lesions. A variety of DNA repair defense systems, almost all inducible, buffer the cell against any increment in DNA lesions. Therefore, the effect of a particular chemical insult depends on the level of each defense, which in turn depends on the past history of exposure. Exogenous agents can influence the induction and effectiveness of these defenses. Defenses can be partially disabled by lack of particular micronutrients in the diet (e.g., antioxidants).

Animals↗