Cumulative DNA Damage Research Articles

Microbial DNA metabolism, genome dynamics and human disease

Genetic material is highly dynamic. Genome stability is constantly challenged by endogenous and environmental agents that induce DNA lesions, genome rearrangements and other types of genotoxic stress. At the same time, localized and global mutation events and horizontal gene transfer (HGT) contribute to alterations in DNA. The balancing act between these forces has a major impact on the fitness of cells. While many of the changes generated can be deleterious, the variations they create are the bread and butter of evolutionary processes. During infection of animals, bacterial pathogens encounter DNA-damaging compounds generated by the host as antibacterial defenses. Nitric oxide and reactive oxygen species are particularly important genotoxic agents and generate many DNA lesions. DNA lesions may also accumulate in genomes over time as byproducts of normal metabolic processes. Most such lesions are mended by DNA repair. However, there is still the potential for cumulative unrepaired DNA damage in bacterial genomes, which can play a critical role in generating mutator activity. Our view of microbial DNA metabolism has to a large extent derived from analyses of the model organism Escherichia coli , which has served as a paradigm for DNA repair. However, genome projects have revealed a remarkable variation in gene content among different bacterial species, therefore reflecting differential needs and aims for genetic stability in disparate organisms. Mycobacterium tuberculosis , for instance, compensates for its …

Constitutive histone H2AX phosphorylation and ATM activation are strongly amplified during mitogenic stimulation of lymphocytes

We recently postulated that constitutive activation of Ataxia Telangiectasia, Mutated (CAA) and constitutive histone H2AX phosphorylation (CHP) seen in cells not treated with genotoxic agents are the events triggered by DNA damage caused by endogenous reactive oxygen species (ROS), the product of mitochondrial oxidative metabolism. The aim of this study was to seek further evidence in support of this postulate, namely to test whether the levels of CAA and CHP correlate with cells metabolic activity. Peripheral blood lymphocytes are non-cycling (G(0)) cells characterized by minimal rate of oxidative metabolism. A dramatic rise in transcriptional and translational activity, an increase in number of mitochondria, and induction of DNA replication, occur during their mitogenic stimulation. This classic model of cell activation was chosen to study a possible correlation between CAA and CHP versus metabolic activity and generation of ROS. The levels of CAA and CHP in lymphocytes were increased many-fold during their stimulation. This increase was paralleled by the rise in extent of endogenously generated ROS. The growth of stimulated lymphocytes in the presence glucose antimetabolite 2-deoxy-D-glucose led to markedly lowered translational activity, decreased ROS generation and correspondingly attenuated CHA and CAA. The present data are consistent with our postulate that CHP and CAA report DNA damage by endogenous oxidants whose level correlates with metabolic activity. Because cumulative DNA damage by ROS generated via oxidative metabolism is considered the key mechanism responsible for cell ageing and senescence the data imply that these processes are delayed in G(0) quiescent lymphocytes or stem cells as compared with proliferating cells.

Constitutive histone H2AX phosphorylation on Ser-139 in cells untreated by genotoxic agents is cell-cycle phase specific and attenuated by scavenging reactive oxygen species

DNA damage, particularly when it involves formation of double-strand breaks (DSBs), triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX has been named gammaH2AX, and induction of gammaH2AX in cells exposed to genotoxic agents is considered a sensitive and specific reporter of DNA damage. However, in untreated normal cells as well in the cells of various tumor lines cells, a fraction of histone H2AX molecules remain phosphorylated. In the present study, we observed that the extent of this constitutive H2AX phosphorylation varies depending on the cell type (line) and on cell cycle phase and, in most cell types, S and G(2)/M phase cells exhibit greater levels of H2AX phosphorylation than do cells in the G(1) phase. Furthermore, constitutive H2AX phosphorylation in human pulmonary carcinoma A549, lymphoblastoid TK6, and in normal bronchial epithelial cells was reduced following cell exposure to N-acetyl-L-cysteine, a scavenger of reactive oxygen intermediates; the reduction was most pronounced for G(2)M cells. Growth of A549 cells in the presence of buthionine sulfoximine, an inhibitor of glutathione synthetase, amplified the level of constitutive H2AX phosphorylation in A549 cells. The observed constitutive H2AX phosphorylation may be a reflection of the ongoing DNA damage mediated by reactive oxygen species (ROS) generated by metabolic activity during progression through the cell cycle, leading to formation of DSBs during the S phase. Because cumulative DNA damage in proliferating cells mediated by ROS is considered the key mechanism for cell ageing, the present approach to estimate the degree of attenuation of constitutive H2AX phosphorylation by antioxidants may provide a convenient tool to assess the DNA-protective and possible anti-ageing properties of other agents.

Autoclave sterilization produces acrylamide in rodent diets: implications for toxicity testing.

Acrylamide (AA) is a neurotoxic and carcinogenic contaminant that is formed during the cooking of starchy foods. Assessment of human risks from toxicants is routinely performed using laboratory rodents, and such testing requires careful control of unintended exposures, particularly through the diet. This study describes an analytical method based on liquid chromatography with electrospray tandem mass spectrometry that was used to measure endogenous AA in rodent diets and to survey a number of commercial products for contamination. Method sensitivity permitted accurate quantification of endogenous levels of AA in raw diets below 20 ppb. Autoclaving a standard rodent diet (NIH-31) increased the AA content 14-fold, from 17 to 240 ppb. A nutritionally equivalent diet that was sterilized by irradiation was found to contain approximately 10 ppb of AA (NIH-31IR). A toxicokinetic study of AA and its epoxide metabolite, glycidamide, was performed by switching mice from NIH-31IR to the autoclaved diet for a 30 min feeding period (average AA dose administered was 4.5 microg/kg of body weight). The concentrations of AA and glycidamide were measured in serum collected at various times. The elimination half-lives and the areas under the respective concentration-time curves were similar for AA and glycidamide. Mice maintained on autoclaved NIH-31 diet, but otherwise untreated, showed elevated steady state levels of a glycidamide-derived DNA adduct in liver relative to mice maintained on the irradiated diet. This study demonstrates that a heat sterilization procedure used in laboratory animal husbandry (i.e., autoclaving) can lead to the formation of significant levels of AA in basal diets used for toxicity testing. AA in rodent diets is bioavailable, is distributed to tissues, and is metabolically activated to a genotoxic metabolite, which produces quantifiable cumulative DNA damage. Although the contribution of endogenous AA to the incidence of tumors in multiple organs of rodents otherwise untreated in chronic carcinogenicity bioassays (i.e., control groups) is not known, the reduction of endogenous AA through the use of a suitable irradiated diet was deemed to be critical for ongoing studies of AA carcinogenicity and neurotoxicity.

Smoking Status and Occupational Exposure Affects Oxidative DNA Injury in Boilermakers Exposed to Metal Fume and Residual Oil Fly Ash

Abstract Epidemiologic studies demonstrate increased cancer incidence among workers exposed to polycyclic aromatic hydrocarbons (PAH) and metals, probably through cumulative oxidative DNA damage in response to carcinogens. Boilermakers are exposed to particulates of residual oil fly ash (ROFA) and metal fume that contain carcinogenic PAH and metals. We conducted a repeated-measures cohort study in boilermakers during the overhaul of an oil-fired boiler to determine a possible association between the level of 8-hydroxy-2′-deoxyguanosine (8-OH-dG; an oxidative injury biomarker) and biomarkers of PAH (1-hydroxypyrene; 1-OHP) and metal exposure. Preshift and postshift urine samples were analyzed for 8-OH-dG, cotinine, 1-OHP, and metals. Generalized estimating equations were used to model the multivariate relationship of 8-OH-dG to the explanatory variables of interest. Biomarker levels were determined for 181 urine samples from 20 male subjects (mean age 45 years, 50% smokers). Metal and 1-OHP levels increased cross-week and were affected by smoking status. Levels of 8-OH-dG were higher in nonsmokers at the start of the workweek yet declined after occupational exposure to similar levels as in smokers. Multivariate analysis indicated that metal × cotinine interaction terms for nickel, vanadium, chromium, and copper were significantly associated with the 8-OH-dG level, but there were differential effects depending on the metal. This study suggests that oxidative DNA damage in boilermakers is influenced by the interaction between occupational exposures and smoking status. In addition, boilermakers may have reduced ability to repair damaged DNA after ROFA and metal fume exposure. This finding has clinical relevance because these exposures may increase the cancer susceptibility of boilermakers.

Les antioxydants et le vieillissement dans la nutrition des animaux de compagnie

Cumulative DNA damage is recognised as an important factor in the age-related decline in tissue and organ function. This paper will provide evidence that nutrition may play a role in reducing some of the detrimental effects associated with such damage. Specifically, can we ameliorate DNA damage in cats and dogs through dietary antioxidants?

Histopathologic Features Seen with Radiation Recall or Enhancement Eruptions

Background: Although a radiation recall or enhancement eruption has been associated with a number of chemotherapeutic drugs, the histologic features have rarely been described. Objective: Our goal was to define the histologic features of radiation recall and enhancement eruptions in order to better understand their pathogenesis. Methods: We present ten patients on chemotherapeutic agents who developed erythematous maculopapular to psoriasiform eruptions often with associated follicular pustules. These eruptions occurred at the sites of prior or concurrent radiation therapy. Results: The most common class of drugs inducing these reactions were antibiotic chemotherapeutic agents alone or in combination with other chemotherapeutic drugs. In addition to routine histology, in four patients immunohistochemical staining for p53 was performed at the sites of the eruptions after resolution and at noninvolved sites matched for ultraviolet radiation (UVR) exposure. Histologic features in patients receiving concurrent radiation therapy included epidermal dysplasia, keratinocytes showing features of necrosis, increased mitotic figures, and a mixed inflammatory infiltrate. At sites of prior radiation therapy, the biopsy specimens showed a similar spectrum of epidermal changes and, in some cases, psoriasiform dermatitis with clearing within cells in the upper layers of the epidermis. Additional dermal changes included dermal fibrosis, vasodilatation, and atypical fibroblasts. Moderate to marked solar elastosis was seen in the majority of biopsy specimens. Immunohistochemical studies after resolution showed only a modest increase in p53 staining in epidermal keratinocytes in 3 of 4 sites of recall and enhancement eruptions after resolution of the reactions compared to skin that was matched for similar UVR exposure. Conclusion: Cumulative direct DNA damage and oxidative stress are probably important in radiation recall and enhancement eruptions, and these changes may be modulated by underlying nutritional deficits. Cumulative p53 mutations may play some role but are probably not a major factor in these eruptions. Mitochondrial dysfunction, which is known to occur with prior and concurrent radiation and chemotherapy, may be important in these eruptions. In addition to improvements in general nutrition, topical or oral antioxidant therapy may be a potential therapy to avoid radiation enhancement and recall reactions.

DNA adducts as markers of exposure to carcinogens and risk of cancer

DNA adducts as markers of exposure to carcinogens and risk of cancer

Investigation of the formation and accumulation of liver DNA adducts in mice chronically exposed to tamoxifen.

Tamoxifen was administered to three strains of female mice (B6C3F1, C57BL/6 and DBA/2) in short- and long-term studies to determine their ability to activate tamoxifen and cause hepatic DNA damage. 32P-Postlabelling of liver DNA from mice treated for 4 days showed a group of major adducts that increased in a dose-dependent manner and co-chromatographed with the major adducts detected in rat liver. On cessation of dosing, the majority of adducts were cleared within 3 days. Binding of [14C]tamoxifen to DNA nucleotides was demonstrated by the use of accelerator mass spectrometry. In long-term studies of 12 months to 2 years duration, dependent on strain, tamoxifen was administered continuously in the diet to give a daily dose of approximately 40 mg/kg. DNA adducts were detected after 3 months, although the number of adducts decreased with time and by 2 years were not detectable in the tamoxifen treated mice. None of the treated groups showed a significantly increased incidence of liver tumours, with or without phenobarbital promotion and there was no sustained liver cell proliferation. Tamoxifen was detected in the mouse livers, but at levels 50 times lower than those reported in a comparable rat study. These results suggest that, in contrast to the rat, tamoxifen is non-carcinogenic in mice because it does not cause sufficient cumulative DNA damage, or act as a promoter by causing cell proliferation.

The effects of some redox-active metals and reactive aldehydes on DNA-protein cross-links in vitro

It has been suggested that the measurement of DNA-protein cross-links (DPCs) may be of value in the assessment of an individual's exposure to specific environmental insults. For a biomarker to be reliable, its results should be consistent and specific. In the present study, the precision and specificity of the K +-SDS precipitation assay as a measurement for DPCs was assessed. Chinese hamster ovary (CHO) and human fibroblast cells were exposed to a number of diverse oxidative insults, whose concentrations ranged from physiological to superphysiological levels. Only super-physiological concentrations of the insults induced the formation of DPCs. Formaldehyde, chromate, vanadate, acetaldehyde, and copper were found to be the greatest inducers of DPC formation, followed by manganese and iron. DPC induction was consistently higher in the CHO cells than in human fibroblast cells. While the K +-SDS assay may be of value as an indicator of cumulative DNA damage, its value as a biomarker for specific environmental insults may be limited.

The Role of Mitochondria in Ischemic Heart Disease

Summary Mitochondria in the heart play two roles essential for cell survival: ATP synthesis and maintenance of Ca2+ homeostasis. These two processes are driven by the same energy source, the H+ electrochemical gradient (ΔμH). Under aerobic physiologic conditions, mitochondria do not contribute to the beat-to-beat regulation of cytosolic Ca2+, although a Ca2+ transient in mitochondrial matrix has been described. Micromolar increases in mitochondrial Ca2+ concentration stimulate the Krebs cycle and the NADH redox potential and, therefore, ATP synthesis. Trimetazidine has been shown to improve the calcium transient and, in so doing, the overall myocardial energy production. Under pathologic conditions, mitochondrial Ca2+ overload causes a series of vicious cycles that lead to irreversible cell damage. During ischemia, an alteration in intracellular Ca2+ homeostasis occurs and mitochondria are able to buffer cytosolic Ca2+, suggesting that they retain the Ca2+-transporting capacity. Accordingly, once isolated, even after prolonged ischemia the majority of the mitochondria are able to use oxygen for ATP phosphorylation. When isolated after reperfusion, mitochondria are structurally altered, contain large quantities of Ca2+, and produce an excess of oxygen free radicals. Their membrane pores are stimulated and the capacity for oxidative phosphorylation is irreversibly disrupted. The role of mitochondrial DNA damage in progressive human diseases such as coronary atherosclerosis is receiving growing interest. The sequence of ischemia and reperfusion, through increased production of oxygen free radicals, causes mitochondrial deletions in several areas of the mitochondrial genome. This cumulative mitochondrial DNA damage is associated with induction of nuclear oxidative phosphorylation gene mRNA. These observations support the hypothesis that mitochondria and mitochondrial DNA damage play important roles in ischemic heart disease.

The role of mitochondria in ischemic heart disease.

Mitochondria in the heart play two roles essential for cell survival: ATP synthesis and maintenance of Ca2+ homeostasis. These two processes are driven by the same energy source, the H+ electrochemical gradient (delta microH). Under aerobic physiologic conditions, mitochondria do not contribute to the beat-to-beat regulation of cytosolic Ca2+, although a Ca2+ transient in mitochondrial matrix has been described. Micromolar increases in mitochondrial Ca2+ concentration stimulate the Krebs cycle and the NADH redox potential and, therefore, ATP synthesis. Trimetazidine has been shown to improve the calcium transient and, in so doing, the overall myocardial energy production. Under pathologic conditions, mitochondrial Ca2+ overload causes a series of vicious cycles that lead to irreversible cell damage. During ischemia, an alteration in intracellular Ca2+ homeostasis occurs and mitochondria are able to buffer cytosolic Ca2+, suggesting that they retain the Ca(2+)-transporting capacity. Accordingly, once isolated, even after prolonged ischemia the majority of the mitochondria are able to use oxygen for ATP phosphorylation. When isolated after reperfusion, mitochondria are structurally altered, contain large quantities of Ca2+, and produce an excess of oxygen free radicals. Their membrane pores are stimulated and the capacity for oxidative phosphorylation is irreversibly disrupted. The role of mitochondrial DNA damage in progressive human diseases such as coronary atherosclerosis is receiving growing interest. The sequence of ischemia and reperfusion, through increased production of oxygen free radicals, causes mitochondrial deletions in several areas of the mitochondrial genome. This cumulative mitochondrial DNA damage is associated with induction of nuclear oxidative phosphorylation gene mRNA. These observations support the hypothesis that mitochondria and mitochondrial DNA damage play important roles in ischemic heart disease.

Linking genotoxic responses and reproductive success in ecotoxicology.

The potential of genotoxicity biomarkers as predictors of detrimental environmental effects, such as altered reproductive success of wild organisms, must be rigorously determined. Recent research to evaluate relationships between genotoxic responses and indicators of reproductive success in model animals is described from an ecotoxicological perspective. Genotoxicity can be correlated with reproductive effects such as gamete loss due to cell death; embryonic mortality; and heritable mutations in a range of model animals including polychaete worms, nematodes, sea urchins, amphibians, and fish. In preliminary studies, the polychaete worm, Neanthes arenaceodentata, and the nematode, Caenorhabditis elegans, have also shown the potential for cumulative DNA damage in gametes. If DNA repair capacity is limited in gametes, then selected life history traits such as long and synchronous periods of gametogenesis may confer vulnerability to genotoxic substances in chronic exposures. Recommendations for future research include strategic development of animal models that can be used to elucidate multiple mechanisms of effect (multiend point) at varying levels of biological organization (multilevel).

DNA strand breaks and DNA repair response in lymphocytes after chronic in vivo exposure to very low doses of ionizing radiation in mice

In contrast to the well-documented negative effects of high-dose oxidant exposure, accumulating evidence supports a positive, perhaps essential physiologic role for very low-level oxidant stress. For example, low-level oxidant exposure, within or below the physiologic range, has been reported to stimulate membrane signal transduction, proliferation, antioxidant defense and DNA repair. In the present study, we have examined whether whole-body exposure to low-dose radiation (LDR) results in an alteration in constitutive (steady state) levels of DNA-strand breaks and whether an adaptive increase in DNA-repair response is induced. C57B1/6J mice were exposed to 0.04 Gy (4 cGy) of γ-radiation as a model of low level oxidant stress. End points measured after chronic in vivo LDR included: (1) constitutive expression of DNA-strand breaks in quiescent spleen cells; (2) sensitivity to DNA damage after high-dose radiation exposure in vitro; (3) repair of constitutive and radiation-induced DNA strand breaks after mitogen stimulation: (4) activity of the DNA-repair associated enzyme, poly(ADP-ribose)transferase (ADPRT) and its substrate, NAD. The results indicated that the constitutive expression of DNA-strand breaks is significantly decreased chronic LDR; however, DNA-repair capacity after high-dose radiation exposure is not increased above that observed in sham-irradiated mice. Associated with the reduction in consitutive DNA-strand break accumulation was a decrease in resting levels of the DNA-repair-associated enzyme poly(ADP-ribose) transferase (ADPRT). These results are consistent with the interpretation that cumulative DNA damage and associated DNA-repair activity in unstimulated cells are both reduced after chronic LDR exposure.

Studies on the adrenal cortex of hypophysectomized rats: A model for abnormal cellular atrophy and death

Biochemical and ultrastructural studies indicate that the atrophy of adrenal cortex in hypoyhysectomized rats involves the following changes: (1) One to two days after hypophysectomy, there is loss of "template activity" resulting from cumulative DNA-damage and heterochromatinization. In vivo ACTH-administration led to recuperation of these cells, indicating damage during hypophysectomized state to be reversible. (2) If the duration of hypophysectomy is prolonged, some of the cells become irreversibly damaged and can no longer recuperate after in vivo ACTH administration. (3) The period of most rapid cell death is from the third to seventh day after hypophysectomy. The cause of cell death is probably due to membrane damage in the absence of protein synthesis, leading to lysis of the cells. Lysozomes and macrophages are apparently not involved.