Steady-state Level Of DNA Damage Research Articles

A pooled analysis of molecular epidemiological studies on modulation of DNA repair by host factors

Levels of DNA damage represent the dynamics between damage formation and removal. Therefore, to better interpret human biomonitoring studies with DNA damage endpoints, an individual’s ability to recognize and properly remove DNA damage should be characterized. Relatively few studies have included DNA repair as a biomarker and therefore, assembling and analyzing a pooled database of studies with data on base excision repair (BER) was one of the goals of hCOMET (EU-COST CA15132). A group of approximately 1911 individuals, was gathered from 8 laboratories which run population studies with the comet-based in vitro DNA repair assay. BER incision activity data were normalized and subsequently correlated with various host factors. BER was found to be significantly higher in women. Although it is generally accepted that age is inversely related to DNA repair, no overall effect of age was found, but sex differences were most pronounced in the oldest quartile (>61 years). No effect of smoking or occupational exposures was found. A body mass index (BMI) above 25 kg/m2 was related to higher levels of BER. However, when BMI exceeded 35 kg/m2, repair incision activity was significantly lower. Finally, higher BER incision activity was related to lower levels of DNA damage detected by the comet assay in combination with formamidopyrimidine DNA glycosylase (Fpg), which is in line with the fact that oxidatively damaged DNA is repaired by BER. These data indicate that BER plays a role in modulating the steady-state level of DNA damage that is detected in molecular epidemiological studies and should therefore be considered as a parallel endpoint in future studies.

Abstract 1756: Electronic cigarettes induce DNA damage in the oral epithelium of users

Abstract Background: The use of electronic cigarettes (e-cigs) has skyrocketed among adolescents and young adults. E-cigs are promoted as safer than combustible cigarettes but their long term effects on human health are still unknown. E-cig aerosols contain significantly lower number of chemicals than tobacco smoke, however they also contain known carcinogens, reactive oxygen species (ROS), and unique chemicals poorly studied. In vitro and animal studies revealed that e-cig aerosols increases DNA damage and reduces DNA repair. There have been no studies assessing e-cig-induced DNA damage in human subjects. This reflects, at least in part, technical limitations that make it quite challenging to measure the in vivo steady-state levels of DNA damage, particularly for relatively low levels of genotoxic exposure. Our lab has previously developed and validated a novel quantitative primer anchored DNA damage detection assay (q-PADDA) to quantify in vivo DNA damage. We have also reported that, in an in vitro setting, q-PADDA has high sensitivity to detect e-cig-induced DNA damage. Here, we take advantage of this assay to start assessing the potential long-term impact of e-cig use on human health. Aim: To determine whether e-cig users have higher levels of DNA damage on oral epithelial cells than non-users. Methods: Ethics Committee approval was obtained. Participants were recruited through internet ads, leaflets, flyers and word-of-mouth. After written informed consent, a total of 35 individuals were included in this study: 20 exclusive e-cigarette users and 15 individuals reporting no e-cig or tobacco use. Self-report of tobacco use was biochemically confirmed by salivary cotinine and by measuring exhaled carbon monoxide (CO). Oral mucosa samples were collected by cytobrush and used for DNA extraction and damage quantification. DNA damage was quantified by q-PADDA within p53, the most frequently mutated gene in human cancer. Plasma nicotine levels were analyzed by liquid chromatography-mass spectrometry. Data was analyzed by Student’s t test. Results: The study including participants using high and low voltage e-cig devices. E-cig users (33 ± 8) and non-users (33 ± 7) had similar age distribution. We observed that oral epithelial cells collected from e-cig users have significantly higher levels of DNA damage in both transcribed and non-transcribed DNA strands of the p53gene when compared to never users. Conclusion: Our work is the first study to measure DNA damage in e-cig users. Our study shows for the first time that e-cig users have significant levels of DNA damage in their oral mucosa epithelium. These data has major clinical implications and suggest that e-cig users might have an increased cancer risk. Further studies are urgently needed to expand these kind of studies and to characterize the molecular mechanisms underlying e-cig induced DNA damage which may leads to cancer. Citation Format: Vengatesh Ganapathy, Theodore Wagener, Lurdes Queimado. Electronic cigarettes induce DNA damage in the oral epithelium of users [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1756.

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents.

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations.

The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences

The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism.

Phenols of virgin olive oil protects nuclear DNA against oxidative damage in HeLa cells

Oxidative DNA damage is an inescapable consequence for cells constantly exposed to oxidative stress derived from normal metabolic processes and from environmental factors. Phenolic compounds, which have strong antioxidant activity, prevent DNA damage by protecting the cells against harmful effects of oxidative stress. In this study, the effect of virgin olive oil phenolic extract (OOPE) was investigated on H2O2-induced mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in HeLa cells. DNA damage was assessed in mitochondria and two nuclear regions by using quantitative PCR (QPCR) assay. The cells were pre-treated with non-cytotoxic doses of OOPE for 4h, and DNA damage was determined. OOPE alone does not change the steady-state level of DNA damage. The oxidative stress generated with 750μM H2O2 caused two times greater damages in mtDNA compared to nDNA, which included the nonexpressed β-globin region (1.507±0.110 lesions/10kb) and the expressed APEX1 gene (1.623±0.243 lesions/10kb) with respect to the control region. When cells were preincubated with OOPE for 4h, nDNA damage under stress condition was completely inhibited; however, mtDNA damage was not affected by this procedure. These results suggest that OOPE has a protective effect against nDNA damage in HeLa cells.

Abstract 3458: Benzo[a]pyrene (BP)-induced DNA damage is elevated in mice lacking Xeroderma pigmentosum complementation group A (Xpa) and heterozygous for the tumor suppressor gene p53

Abstract In developing countries, esophageal cancer is the third most common cancer in men. We have employed a mouse model of esophageal cancer using DNA repair deficient mice. Unlike their wild type counterparts, C57BL/6J mice lacking Xpa and heterozygous for p53 [Xpa(−/−) p53(+/−)] have previously been shown to develop forestomach and esophageal tumors when fed BP (Hoogervorst et al., Carcinogenesis, 2003). Two methodologies were employed to examine BP-induced steady-state levels of DNA damage in esophagi of male mice fed 0 or 100 ppm BP for 4 weeks. HPLC, in conjunction with tandem mass spectrometry (HPLC-MS/MS), was used to measure 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) and a chemiluminiscence immunoassay (CIA), using antiserum elicited against r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA was used to evaluate BP-DNA adducts. In the esophagi of Xpa(−/−) p53(+/−) mice, there were 38.7 ± 4.8 BPdG adducts/108 nucleotides (mean ± SE, n=5), as measured by HPLC-MS/MS, while in the wild type mice there were 28.0 ± 1.8 BPdG adducts/108 nucleotides (mean ± SE, n=5). Furthermore, complete loss of p53 resulted in the greatest accumulation of BPdG adducts, with 58.1 ± 8.6 BPdG adducts/108 nucleotides (n=5) in Xpa(−/−) p53(−/−) mice (p<0.05 vs. wild type mice). Esophageal BP-DNA adduct values, as measured by CIA, were 23.3 ± 1.7 adducts/108 nucleotides (n=3) in the Xpa(−/−) p53(+/−) mice, compared to 14.6 ± 3.0 adducts/108 nucleotides (n=3) in the wild type mice. Since similar adduct levels were determined using both HPLC-MS/MS and CIA, this indicates that BPdG is the major adduct resulting from BP in mouse esophagus. The higher levels of DNA damage in DNA repair-deficient p53-deficient mice, compared to their wild type counterparts, may contribute to the enhanced susceptibility of these mice to esophageal tumor induction by BP. Currently we are examining the role of chlorophyllin, a chemopreventive agent, in reducing BP-induced DNA damage levels and tumor formation in this model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3458.

Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke

The objective of the present study was to use the comet assay to evaluate the steady-state level of DNA damage in peripheral blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke. A total of 20 rats were distributed into four experimental groups ( n = 5 rats/group): non-diabetic (control) and diabetic exposed to filtered air; non-diabetic and diabetic exposed to cigarette smoke. A pancreatic beta (β)-cytotoxic agent, streptozotocin (40 mg/kg b.w.) was used to induce experimental diabetes in rats. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. At the end of the 2-month exposure period, each rat was anesthetized and humanely killed to obtain blood samples for genotoxicity analysis using the alkaline comet assay. Blood leukocytes sampled from diabetic rats presented higher DNA damage values (tail moment = 0.57 ± 0.05; tail length = 19.92 ± 0.41, p < 0.05) compared to control rats (tail moment = 0.34 ± 0.02; tail length = 17.42 ± 0.33). Non-diabetic (tail moment = 0.43 ± 0.04, p > 0.05) and diabetic rats (tail moment = 0.41 ± 0.03, p > 0.05) exposed to cigarette smoke presented non-significant increases in DNA damage levels compared to control group. In conclusion, our data show that the exposure of diabetic rats to cigarette smoke produced no additional genotoxicity in peripheral blood cells of female Wistar rats.

Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair

Chronic inflammation, induced by biological, chemical, and physical factors, was associated with increased risk of human cancer at various sites. Chronic inflammatory processes induce oxidative/nitrosative stress and lipid peroxidation (LPO), thereby generating excess reactive oxygen species (ROS), reactive nitrogen species (RNS), and DNA-reactive aldehydes. Miscoding etheno- and propano-modified DNA bases are generated inter alia by reaction of DNA with these major LPO products. Steady-state levels of LPO-derived (etheno-) DNA adducts in organs affected by persistent inflammatory processes were investigated as potential lead markers for assessing progression of inflammatory cancer-prone diseases. Using ultrasensitive and specific detection methods for the analysis of human tissues, cells, and urine, etheno-DNA adduct levels were found to be significantly elevated in the affected organs of subjects with chronic pancreatitis, ulcerative colitis, and Crohn's disease. Patients with alcohol-related liver diseases showed excess hepatic DNA damage progressively increasing from hepatitis, fatty liver, to liver cirrhosis. Ethenodeoxyadenosine excreted after DNA repair in urine of hepatitis B virus-related chronic hepatitis and liver cirrhosis patients was increased up to 90-fold. Putative mechanisms that may control DNA damage in inflamed tissues including impaired or imbalanced DNA repair pathways are reviewed. Persistent oxidative/nitrosative stress and excess LPO are induced by inflammatory processes in a self-perpetuating process and cause progressive accumulation of DNA damage in target organs. Together with deregulation of cell homeostasis, the resulting genetic changes act as driving force in chronic inflammation-associated human disease pathogenesis. Thus steady-state levels of DNA damage caused by ROS, RNS, and LPO end products provide promising molecular signatures for risk prediction and potential targets and biomarkers for preventive measures.

Poly(ADP-ribose) polymerase-1, DNA repair and mammalian longevity

Cellular DNA repair activities can be expected to control the rate of the ageing process by keeping the steady-state levels of DNA damage, which is continuously induced by endogenous and exogenous damaging agents, at low levels. Poly(ADP-ribosyl)ation is one of the immediate biochemical reactions of eukaryotic cells to DNA damage and is functionally associated with DNA base-excision repair and strand break repair. Here we review the current state of the art concerning the relationship between DNA strand break repair, poly(ADP-ribosyl)ation, maintenance of genomic stability and mammalian life span.

Postlabeling analysis of indigenous aromatic DNA adducts in mouse myocardium during aging

The possible presence of aromatic chemicals covalently linked to DNA (aromatic adducts) was investigated in heart cells during aging of the C57BL/6Nia mouse. Heart DNAs were isolated from untreated mice of different ages and analyzed by 32P-postlabeling assays. To determine low levels of adducts, assays were carried out in which aromatic adducts were first isolated by phase transfer to 1-butanol, then labeled with excess, carrier-free [γ- 32P]ATP. This analysis showed that the number and frequency of aromatic adducts varied between DNA samples. Several adducts were present in all mouse DNA preparations and were more abundant in 32P-maps of senescent heart DNA. The results suggest that genomes of myocytes have a higher steady-state level of DNA damage in old animals which could adversely affect cell function.