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Biomedical subjects

B N Ames

Publications and source records attributed to B N Ames.

At least 19 recordsLinked to original sources
PubMed1992

Oxidants are a major contributor to aging.

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 aging and to the degenerative diseases associated with aging such as cancer. The oxidative damage rate in mammalian species with a high metabolic rate, short life span, and high age-specific cancer rate such as 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 enormously increased risk for mutations in part due to the conversion of DNA adducts to mutations. Mitogenesis also increases the probability of gene amplification and loss of 5-methylcytosine. Dietary interventions that lower mitogenesis, such as calorie restriction, decrease the incidence of cancer.

Aging↗
PubMed1992

Rodent carcinogens: setting priorities.

The human diet contains an enormous background of natural chemicals, such as plant pesticides and the products of cooking, that have not been a focus of carcinogenicity testing. A broadened perspective that includes these natural chemicals is necessary. A comparison of possible hazards for 80 daily exposures to rodent carcinogens from a variety of sources is presented, using an index (HERP) that relates human exposure to carcinogenic potency in rodents. A similar ordering would be expected with the use of standard risk assessment methodology for the same human exposure values. Results indicate that, when viewed against the large background of naturally occurring carcinogens in typical portions of common foods, the residues of synthetic pesticides or environmental pollutants rank low. A similar result is obtained in a separate comparison of 32 average daily exposures to natural pesticides and synthetic pesticide residues in the diet. Although the findings do not indicate that these natural dietary carcinogens are important in human cancer, they cast doubt on the relative importance for human cancer of low-dose exposures to synthetic chemicals.

Animals↗
PubMed1992

Protein oxidation associated with aging is reduced by dietary restriction of protein or calories.

The accumulation of unrepaired oxidative damage products may be a major factor in cellular aging. Both oxidative lesions in DNA and oxidatively damaged proteins have been shown to accumulate during aging. The accumulation of oxidized proteins in Fischer 344 rats was compared for animals consuming protein-restricted and calorically restricted diets--both of which have been shown to extend lifespan. Rats were fed diets restricted in either protein (5% or 10% of the diet as compared with the normal 20% casein), or calories (25% or 40% less than normal), or total diet (40% less than normal). In addition, some of the rats fed a diet providing 5% or 20% protein were irradiated twice weekly (125 rads per exposure; 1 rad = 0.01 Gy). The level of oxidative damage to proteins (protein carbonyls) was determined in rats sacrificed at various times. The oxidative damage to proteins increased with aging and with radiation. Either protein or calorie restriction markedly inhibited the accumulation of oxidatively damaged proteins. Protein restriction reduced the accumulation of oxidatively damaged proteins during the oxidative stress of chronic irradiation.

Aging↗
PubMed1992

Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column.

An immunoaffinity column is described that facilitates the analysis of oxidative damage products of DNA and RNA in urine, blood plasma, and medium isolated from cultures of Escherichia coli. In intact animals, lesions (adducts) excised from DNA are transported from the cell through the circulation and excreted in urine. In bacteria, DNA adducts are excreted directly into the medium. In either case, the adducts can be assayed as a measure of oxidative damage to DNA. A monoclonal antibody that recognizes 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG;8-hydroxy-2'-deoxyguanosine), a bio-marker of oxidative damage to DNA, has been isolated, and its substrate binding properties have been characterized. The relative binding affinities of this monoclonal antibody for oxo8dG, unmodified nucleosides, or derivatives of Gua made it suitable for the preparation of immunoaffinity columns that greatly facilitate the isolation of oxo8dG, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroguanosine from various biological fluids. Quantitative analysis of these adducts in urine of rats fed a nucleic acid-free diet and in the medium from cultures of E. coli suggests that oxo8-7,8-dihydroguanine is the principal repair product from oxo8-dG in DNA of both eukaryotes and prokaryotes. The results support our previous estimate of about 10(5) oxidative lesions to DNA being formed and excised in an average rat cell per day.

Animals↗
PubMed1992

Endogenous oxidative damage of deoxycytidine in DNA.

Three major oxidation products of 2'-deoxycytidine (dC)--5-hydroxy-2'-deoxycytidine (oh5dC), 5-hydroxy-2'-deoxyuridine (oh5dU), and 5,6-dihydroxy-5,6-dihydro-2'-deoxyuridine (dUg)--were analyzed from enzymatically hydrolyzed DNA with reversed-phase high-performance liquid chromatography coupled to electrochemical detection. oh5dC and oh5dU can be detected with high sensitivity (50 fmol) and selectivity (0-0.2 V) from hydrolyzed DNA. dUg is not electrochemically active but can be measured by dehydrating it into oh5dU. The quantities of oh5dC, dUg, and oh5dU in untreated commercial-grade calf thymus DNA are 10, 10, and 0.75 fmol/micrograms of DNA, respectively. These levels increased substantially when calf thymus DNA was exposed to ionizing radiation, H2O2 alone, H2O2 and combinations of Fe3+ or Cu2+ and ascorbate, near-UV light (365 nm), near-UV light in the presence of menadione, and OsO4, indicating that oh5dC, oh5dU, and dUg are major oxidative DNA damage products. The steady-state levels of these products were determined from freshly extracted rat tissues and ranged from less than 0.5 fmol/micrograms of DNA for oh5dU to about 10 fmol/micrograms of DNA for oh5dC and dUg in liver and kidney and 22 fmol/micrograms of DNA for oh5dC in brain. The levels of oxo8dG were also determined and in general were somewhat lower than the levels of oh5dC. These findings reinforce the link between DNA damage induced by oxidative metabolism and spontaneous mutagenesis leading to cancer and aging.

Animals↗
PubMed1992

Oxidative damage to plasma constituents by ozone.

The reaction of ozone (O3) with human blood plasma was studied to help understand possible events that could occur in the respiratory tract. Uric acid (quantitatively the most important scavenger) and ascorbic acid were oxidized quickly, protein-SH groups were lost more slowly, and there was no loss of bilirubin or alpha-tocopherol. There was little formation of lipid hydroperoxides and no detectable formation of 4-hydroxynoneal, hexanal or nonanal, or changes in lipoprotein electrophoretic mobility. Uric acid in human upper airway secretions may play a significant role in removing inhaled O3. Oxidative damage to lipids must not be assumed to be the key mechanism of respiratory tract O3 toxicity.

Adult↗
PubMed1992

Multidegenerate DNA recognition by the OxyR transcriptional regulator.

The Escherichia coli OxyR protein, a regulator of hydrogen peroxide-inducible genes, is a potent stimulator of transcription in its oxidized form but not in its reduced form. OxyR protein purified in its oxidized form was found to bind four of its non-homologous, functional DNA-binding sites with over 10(6)-fold higher affinity than random DNA sequences. A similarly high DNA binding specificity was observed for the reduced (transcriptionally inactive) form of OxyR, consistent with a model in which the OxyR protein is bound to its recognition sequences even in the absence of an oxidative stress. Alignment of five functional OxyR-binding sites revealed a marked lack of perfectly conserved positions, yet an unusually high number of degenerate homologies (positions at which only two of the four possible base pairs are represented). Methylation interference assays on two OxyR-binding sites showed that OxyR contacts its recognition sequences predominantly at positions of degenerate homology. These results suggest that the OxyR protein specifically recognizes seemingly dissimilar sequences through the use of a multidegenerate recognition code. The chemical basis for a plausible degenerate recognition system is discussed.

Bacterial Proteins↗
PubMed1992

Extrapolation of carcinogenicity between species: qualitative and quantitative factors.

Prediction of human cancer risk from the results of rodent bioassays requires two types of extrapolation: a qualitative extrapolation from short-lived rodent species to long-lived humans, and a quantitative extrapolation from near-toxic doses in the bioassay to low-level human exposures. Experimental evidence on the accuracy of prediction between closely related species tested under similar experimental conditions (rats, mice, and hamsters) indicates that: (1) if a chemical is positive in one species, it will be positive in the second species about 75% of the time; however, since about 50% of test chemicals are positive in each species, by chance alone one would expect a predictive value between species of about 50%. (2) If a chemical induces tumors in a particular target organ in one species, it will induce tumors in the same organ in the second species about 50% of the time. Similar predictive values are obtained in an analysis of prediction from humans to rats or from humans to mice for known human carcinogens. Limitations of bioassay data for use in quantitative extrapolation are discussed, including constraints on both estimates of carcinogenic potency and of the dose-response in experiments with only two doses and a control. Quantitative extrapolation should be based on an understanding of mechanisms of carcinogenesis, particularly mitogenic effects that are present at high and not low doses.

Animals↗
PubMed1992

Animal cancer tests and cancer prevention.

The toxicological significance of exposures to synthetic chemicals is examined in the context of exposures to naturally occurring chemicals. We calculate that 99.99% (by weight) of the pesticides in the US diet are chemicals that plants produce to defend themselves (nature's pesticides). Only 52 of these natural pesticides have been tested in high-dose animal cancer tests, and 27 are rodent carcinogens; these 27 are shown to be present in many common foods. The toxicology of synthetic chemicals is compared to that of natural chemicals, which represent the vast bulk of the chemicals to which humans are exposed. It is argued that animals have a broad array of inducible general defenses to combat the changing array of toxic chemicals in plant food and that these defenses are effective against both natural and synthetic toxins. Synthetic toxins (eg, dioxin) are compared to natural chemicals (eg, indole carbinol [in broccoli] and ethanol). The finding that, in high-dose tests, a high proportion of both natural and synthetic chemicals are carcinogens, mutagens, teratogens, and clastogens (30%-50% for each group) calls into question current efforts to use these tests to protect public health by regulating low doses of synthetic chemicals. The administration of chemicals at the maximum tolerated dose in standard animal cancer tests is postulated to increase cell division (mitogenesis), which in turn increases rates of mutagenesis and, thus, carcinogenesis. The animal data are consistent with this mechanism, because a high proportion--about 50%--of all chemicals tested (whether natural or synthetic) are indeed rodent carcinogens.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Ascorbic acid protects against endogenous oxidative DNA damage in human sperm.

Damage to the DNA of germ cells can lead to mutation, which may result in birth defects, genetic diseases, and cancer. The very high endogenous rate of oxidative DNA damage and the importance of dietary ascorbic acid (AA) in preventing this damage has prompted an examination of these factors in human sperm DNA. The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (8-oxo-7,8-dihydro-2'-deoxyguanosine; oxo8dG), 1 of approximately 20 major products of oxidative damage to DNA, was measured in DNA isolated from human sperm provided by healthy subjects and compared to the seminal fluid AA levels. This relationship was studied in two groups. In a group of 24 free-living individuals 20-50 years old high levels of oxo8dG were correlated with low seminal plasma AA. The endogenous level of oxo8dG in this group was 13 fmol per microgram of DNA or approximately 25,000 adducts per sperm cell. The second group of individuals was maintained on a controlled diet that varied only in AA content. When dietary AA was decreased from 250 to 5 mg/day, the seminal fluid AA decreased by half and the level of oxo8dG in sperm DNA increased 91%. Repletion of dietary AA for 28 days (from 5 mg/day to 250 or 60 mg/day) caused a doubling in seminal fluid AA and reduced oxo8dG by 36%. These results indicate that dietary AA protects human sperm from endogenous oxidative DNA damage that could affect sperm quality and increase risk of genetic defects, particularly in populations with low AA such as smokers.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1991

Gas phase oxidants of cigarette smoke induce lipid peroxidation and changes in lipoprotein properties in human blood plasma. Protective effects of ascorbic acid.

Cigarette smoke (CS) is known to contain a large number of oxidants. In order to assess the oxidative effects of CS on biological fluids, we exposed human blood plasma to filtered (gas phase) and unfiltered (whole) CS, and determined the rate of utilization of endogenous antioxidants in relation to the appearance of lipid hydroperoxides. Lipid peroxidation was measured with a specific and sensitive assay that can detect lipid hydroperoxides at plasma levels as low as 10 nM. We found that exposure of plasma to the gas phase of CS, but not to whole CS, induces lipid peroxidation once endogenous ascorbic acid has been oxidized completely. In addition, CS exposure caused oxidation of plasma protein thiols and albumin-bound bilirubin, whereas uric acid and alpha-tocopherol were not consumed at significant rates. In plasma exposed to the gas phase of CS, low-density lipoprotein exhibited slightly increased electrophoretic mobility, but there was no apparent degradation of apolipoprotein B. Our results support the concept of an increased vitamin C utilization in smokers, and suggest that lipid peroxidation induced by oxidants present in the gas phase of CS leads to potentially atherogenic changes in lipoproteins.

Adult↗
PubMed1991

Natural carcinogens and dioxin.

Toxicology studies of synthetic chemicals at high doses must be viewed in the context of the world of natural chemicals, which make up the vast bulk of the chemicals to which humans are exposed. In tests at near toxic doses (e.g., the maximum tolerated dose), one-half of all chemicals, whether synthetic or natural, are carcinogens; one-third are teratogens, and about half are clastogens. Natural and synthetic chemicals are similar in their toxicology, and at the low doses of most human exposures where cell-killing does not occur, the hazards may be much lower than are commonly assumed and often will be zero. It is shown that the comparative hazards of dioxin at the levels that are causing concern appear insignificant.

Animals↗
PubMed1991

Assays for 8-hydroxy-2'-deoxyguanosine: a biomarker of in vivo oxidative DNA damage.

HPLC with electrochemical detection (HPLC-EC) is a highly sensitive and a selective method for detecting 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative DNA damage that is formed from hydroxyl radical attack of guanine residues in DNA. We propose that the noninvasive measurement of oh8dG in urine can be used to estimate in vivo oxidative damage. Application of this assay to urine samples obtained from rats of different ages and various species provide examples of the utility of this assay. The measurement of steady-state levels of oh8dG in DNA combined with the urinary excretion rates of oh8dG and oh8Gua, offer a powerful approach for estimating oxidative DNA damage and its repair. This method will be useful for studies designed to investigate the relationship of oxidative stress in DNA damage and the role of this damage in aging and cancer.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1991

Immunocompetence and oxidant defense during ascorbate depletion of healthy men.

To determine nonscorbutic effects of moderate vitamin C deficiency we measured immune function and oxidative damage in eight healthy men (25-43 y) who consumed 5-250 mg/d of ascorbic acid over 92 d on a metabolic unit. During ascorbic acid intakes of 5, 10, or 20 mg/d, subjects attained a state of moderate ascorbic acid deficiency as ascorbic acid concentrations in plasma, leucocytes, semen, and buccal cells dropped to less than 50% of baseline with no scorbutic symptoms observed. No changes in cell proliferation, erythrocyte antioxidant enzymes, and DNA strand breaks were observed; however, blood levels of glutathione and NAD(P) decreased during ascorbic acid deficiency, as did delayed hypersensitivity responsiveness. Concentrations of the oxidatively modified DNA base, 8-hydroxydeoxyguanosine in sperm DNA and fecapentaenes, ubiquitous fecal mutagens, were increased during ascorbic acid depletion. Moderate vitamin C deficiency, in the absence of scurvy, results in alteration of antioxidant chemistries and may permit increased oxidative damage.

Adult↗
PubMed1991

Immunoaffinity isolation of urinary 8-hydroxy-2'-deoxyguanosine and 8-hydroxyguanine and quantitation of 8-hydroxy-2'-deoxyguanosine in DNA by polyclonal antibodies.

An immunoaffinity column is described that facilitates the analysis of oxidative DNA damage. DNA adducts excised from DNA are excreted in urine and can be assayed as a measure of DNA damage in individuals. Polyclonal antibodies that recognize 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative damage to DNA, have been produced and their binding properties characterized. The antibodies, raised in rabbits following immunization with protein carrier-hapten conjugates prepared by covalently linking periodate-treated 8-hydroxyguanosine (oh8G) to bovine serum albumin (BSA) or casein, bind oh8dG with high affinity and selectivity, as measured by a competitive radioimmunoassay (RIA). Antibodies obtained from the rabbits immunized with the casein conjugate exhibited a binding affinity for oh8dG of 6.9 x 10(8) M-1. Studies on the relative binding affinities of these polyclonal antibodies for oh8dG, unmodified nucleosides, or derivatives of guanine indicate that the antibodies are suitable for the preparation of immunoaffinity columns that permit us to rapidly isolate oh8dG and 8-hydroxyguanine (oh8Gua) from urine. The high selectivity of the antibodies for oh8dG and oh8G reduces the amount of urinary contaminants previously observed in samples prepared by solid phase extraction, thus greatly facilitating the isolation of these damage products from urine. The relative binding affinity of these antibodies for oh8Gua and 2'-deoxyguanosine were approximately 7.6 x 10(3) and 7.4 x 10(4) fold lower respectively, than the binding affinity for oh8dG. The antibody can be used to quantitate oh8dG in enzymatic hydrolyzates of DNA with values comparable to those obtained by HPLC with electrochemical detection (HPLC-EC).

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1991

The Carcinogenic Potency Database: analyses of 4000 chronic animal cancer experiments published in the general literature and by the U.S. National Cancer Institute/National Toxicology Program.

The Carcinogenic Potency Database (CPDB) is an easily accessible, standardized resource of positive and negative long-term animal cancer tests. The CPDB has been published in four earlier papers that include results for approximately 4000 experiments on 1050 chemicals. This paper describes the CPDB: goals, inclusion criteria, fields of information, and published plot format. It also presents an overview of our published papers using the CPDB. The CPDB as published in plot format readily permits comparisons of carcinogenic potency and many other aspects of cancer tests, including for each experiment the species and strain of test animals, 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. A combined plot of all results from the four separate papers, which is ordered alphabetically by chemical, is available from L. S. Gold, in printed form or on computer tape or diskette. A computer readable (SAS) database is also available. The overview of papers includes descriptions of work on methods of estimating carcinogenic potency, reproducibility of results in near-replicate cancer tests, correlation in potency between species, ranking possible carcinogenic hazards, comparison of positivity and target organ in rats and mice, comparison of mutagens and nonmutagens, proportion of chemicals positive in animal tests, natural compared to synthetic chemicals, and mechanistic issues in interspecies extrapolation.

Animals↗