Levels Of Oxidative DNA Lesions Research Articles

The association between inefficient repair of oxidative DNA lesions and common polymorphisms of the key base excision repair genes as well as their expression levels in patients with Rheumatoid Arthritis

IntroductionRA is a common autoimmune heterogeneous joint disease of still unknown etiology. One of the characteristic features of RA is oxidative stress, most probably induced and stimulated by inappropriate B and T-cell activity. We have hypothesized, that oxidative stress together with impaired DNA damage response to oxidative DNA lesions could be responsible for increased incidence in RA patients of some diseases with genetic instability background such as lymphoma.Material and methodsWe determined the level of oxidative DNA lesions and the kinetics of removal of DNA damage induced by TBH in PBMCs of 30 RA patients and 30 healthy individuals. The metrics from DNA repair study were correlated with genotypes of common polymorphisms of key BER genes as well as their expression levels. DNA repair were evaluated by comet assay, the genotypes of the polymorphism were determined by Taqman Assay and PrimeTime qPCR Assay was used to analyze the expression profile of genes related to BER.ResultsWe observed an association between RA occurrences and impaired DNA repair in PBMCs. After dividing the subjects by controls’ quartiles of DNA repair efficiency, we found an association between an increased risk of RA and inefficient DNA Repair. We also identified interaction between inefficient DNA Repair and polymorphism of the UNG gene (rs246079), and lower expression of key BER genes – MUTH, NEIL3 and UNG.ConclusionsOur result suggest that the genetic variations within BER genes as well as epigenetic factors may be linked with RA by the modulation of the cellular response to oxidative stress.

Oxidative DNA Damage - the possible use of biomarkers as additional prognostic factors in oncology

Oxidative stress represents a deregulation of the homeostasis between the reactive oxygen species and the mechanisms of detoxification and repair. By analyzing the level of oxidatively damaged DNA bases and nucleosides in urine we can assess the extent of DNA repair within the whole body. High levels of markers of oxidative DNA damage excreted in the urine indicate elevated levels of oxidative stress, but can also reflect a high level of efficiency of the processes that work to repair this damage (oxidative stress can be high and the repair processes eliminate its effects). In the present review we discuss the role of oxidative stress, oxidative DNA damage repair mechanisms, potential sources of 8-oxoGua and 8-oxodG (basic markers of oxidative stress) content in urine, effect of antioxidant supplementation on the levels of oxidative DNA lesions, potential application of oxidative DNA damage determination in clinical practice.

Deletion of the major peroxiredoxin Tsa1 alters telomere length homeostasis

Reactive oxygen species (ROS) are proposed to play a major role in telomere length alterations during aging. The mechanisms by which ROS disrupt telomeres remain unclear. In Saccharomyces cerevisiae, telomere DNA consists of TG(1-3) repeats, which are maintained primarily by telomerase. Telomere length maintenance can be modulated by the expression level of telomerase subunits and telomerase activity. Additionally, telomerase-mediated telomere repeat addition is negatively modulated by the levels of telomere-bound Rap1-Rif1-Rif2 protein complex. Using a yeast strain defective in the major peroxiredoxin Tsa1 that is involved in ROS neutralization, we have investigated the effect of defective ROS detoxification on telomere DNA, telomerase, telomere-binding proteins, and telomere length. Surprisingly, the tsa1 mutant does not show significant increase in steady-state levels of oxidative DNA lesions at telomeres. The tsa1 mutant displays abnormal telomere lengthening, and reduction in oxidative exposure alleviates this phenotype. The telomere lengthening in the tsa1 cells was abolished by disruption of Est2, subtelomeric DNA, Rap1 C-terminus, or Rif2, but not by Rif1 deletion. Although telomerase expression and activity are not altered, telomere-bound Est2 is increased, while telomere-bound Rap1 is reduced in the tsa1 mutant. We propose that defective ROS scavenging can interfere with pathways that are critical in controlling telomere length homeostasis.

The role of the 148 Asp/Glu polymorphism of the APE1 gene in the development and progression of primary open angle glaucoma development in the Polish population

Glaucoma is an ocular disorder that is characterized by progressive degeneration of the optic nerve and loss of visual field (VF). Recent data have suggested that the level of oxidative DNA damage in human trabecular meshwork is significantly increased in glaucomatous patients as compared to controls. It was also noted that progressive loss of visual field may by connected with elevated levels of oxidative DNA lesions. This hypothesis may suggest the role of an inefficient base excision repair pathway in primary open angle glaucoma (POAG) pathogenesis. The aim of the study was to evaluate the association of the 148 Asp/Glu APE1 gene polymorphism with the risk of POAG development. One hundred fifty patients with POAG and 190 controls were enrolled in our study. Gene polymorphisms were analyzed by PCR-CTPP. We did not observe a statistically significant difference between the frequencies of alleles and genotypes of the 148 Asp/Glu APE1 gene polymorphism in POAG patients and controls. However, the presented study indicated that 148 Asp/Glu of the APE1 gene was associated with decreased risk of progression of POAG with reference to the parameter VF. We suggest that the 148 Asp/Glu APE1 gene polymorphism may decrease the risk of POAG progression.

The effects of hemodialysis treatment on the level of DNA strand breaks and oxidative DNA lesions measured by the comet assay

Hemodialysis patients have a higher risk for oxidative stress-related complications, such as cardiovascular disease and cancer. The increased level of oxidative stress is due to several factors, e.g., the hemodialysis treatment itself and the uremic state. In the present study, the effects of dialysis treatment on the level of DNA breaks and oxidative DNA lesions in mononuclear cells were measured with the comet assay. Factors possibly affecting DNA damage (reported as % DNA in tail) such as the duration of dialysis, time since last dialysis session, years of dialysis treatment, nutritional status (measured as protein catabolic rate), age, and diabetes were also investigated. The levels of DNA breaks (13.6 ± 4.7 before dialysis) and oxidative DNA lesions (7.9 ± 4.8 before dialysis) were significantly higher in dialysis patients (n = 31) compared to the levels of DNA breaks (5.8 ± 1.1) and oxidative DNA lesions (3.4 ± 1.7) in 10 healthy controls (P < 0.001). A decrease of DNA breaks was observed after dialysis (P = 0.038), and the level of oxidative DNA lesions was higher when the time between two treatment sessions were 68 hours compared to 44 hours (P < 0.001). Older subjects had a higher level of DNA breaks (P = 0.003), a good nutritional status predicted a lower level of DNA breaks (P < 0.001), and the duration of the dialysis session was inversely correlated with oxidative DNA lesions (P = 0.014). Diabetes or years of dialysis treatment did not affect DNA damage. The observations in the present study suggest that accumulation of uremic toxins induce DNA damage. The hemodialysis treatment seems to change the DNA damage.

Genomic Instability in CML-CP originates From the Most Primitive Imatinib-Refractory Leukemia Stem Cells

AbstractAbstract 909Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in the appearance of clones carrying BCR-ABL1 kinase mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or those harboring additional chromosomal aberrations, eventually leading to disease relapse and/or malignant progression to blast phase (CML-BP) [Skorski, T., Leukemia and Lymphoma, 2011]. We found that Lin−CD34+CD38− human leukemia stem cells (huLSCs), including the quiescent sub-population, and Lin−CD34+CD38+ human leukemia progenitor cells (huLPCs) accumulate high levels of reactive oxygen species (ROS) resulting in numerous oxidative DNA lesions such as 8-oxoguanine (8-oxoG) and DNA double-strand breaks (DSBs) [Nieborowska-Skorska, Blood, 2012]. huLSCs and huLPCs treated with TKIs continue to exhibit ROS-induced oxidative DNA damage suggesting the persistence of genomic instability in TKI-treated patients. Furthermore, genomic instability in TKI-refractory huLSCs and TKI-sensitive huLPCs may have a varying impact on disease progression and determining novel treatment modalities. To determine if TKI-refractory huLSCs are a source of genomic instability we employed a tetracycline-inducible murine model of CML-CP: SCLtTA/p210BCR-ABL1. Mice exhibiting CML-CP -like disease demonstrated splenomegaly, leukocytosis, and expansion of mature Gr1+/CD11b+ cells. ROS were elevated in Lin−c-Kit+Sca-1+ cells (muLSCs), but not Lin−c-Kit+Sca-1− cells (muLPCs), which was associated with higher mRNA expression of BCR-ABL1 in muLSCs. In addition, ROS levels were directly proportional to BCR-ABL1 kinase expression in transduced CD34+ human hematopoietic cells, thus confirming the “dosage-dependent” effect of BCR-ABL1 on ROS. Among the Lin−c-Kit+Sca-1+ cells, enhanced ROS were detected in TKI-refractory quiescent muLSCs, in CD34−Flt3− long-term and CD34+Flt3− short-term muLSCs, and also in CD34+Flt3+ multipotent progenitors. High levels of ROS in muLSCs were accompanied by aberrant expression of genes regulating ROS metabolism (mitochondrial electron transport, oxidative phosphorylation, hydrogen peroxide synthesis, and detoxification). In addition, muLSCs, including the quiescent sub-population, displayed high levels of oxidative DNA lesions (8-oxoG, and DSBs). ROS-induced oxidative DNA damage in muLSCs was accompanied by genomic instability in CML-CP –like mice, which accumulated a broad range of genetic aberrations recapitulating the heterogeneity of sporadic mutations detected in TKI-naive CML-CP patients. These aberrations include TKI-resistant BCR-ABL1 kinase mutations, deletions in Ikzf1 and Trp53 and additions in Zfp423 and Idh1 genes, which have been associated with CML-CP relapse and progression to CML-BP. Imatinib caused only modest inhibition of ROS and oxidative DNA damage in TKI-refractory muLSCs. In concordance, CML-CP –like mice treated with imatinib continued to accumulate genomic aberrations. Since BCR-ABL1(K1172R) kinase-dead mutant expressed in CD34+ human hematopoietic cells did not enhance ROS, it suggests that BCR-ABL1 kinase-independent mechanisms contribute to genomic instability. In summary, we postulate that ROS-induced oxidative DNA damage resulting in genetic instability may originate in the most primitive TKI-refractory huLSCs in TKI-naive and TKI-treated patients. Disclosures:Lange:Novartis: Honoraria, Research Funding. Müller:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Borneol administration protects primary rat hepatocytes against exogenous oxidative DNA damage

Experimental evidences suggest that most essential oils possess a wide range of biological and pharmacological activities that may protect tissues against oxidative damage. In this study, we investigated DNA-protective effect of borneol, a component of many essential oils, against oxidative DNA damage induced in primary cultures of rat hepatocytes. Borneol was added to drinking water of Sprague-Dawley rats and DNA resistance against oxidative agents was compared in hepatocytes originated from control and borneol-treated rats. Oxidative stress induced by visible light-excited methylene blue (MB/VL) or 2,3-dimethoxy-1,4-naphthoquionone (DMNQ) resulted in increased levels of DNA lesions measured by the modified single cell gel electrophoresis. Borneol (17 or 34 mg/kg body weight) added to drinking water of rats for 7 days reduced the level of oxidative DNA lesions induced in their hepatocytes by MB/VL or DMNQ. To explain the increased resistance of DNA towards oxidative stress, we measured the base-excision repair (BER) capacity in liver cell extracts of control and borneol-supplemented rats on DNA substrate of HepG2 cells containing oxidative damage. Our results showed that administration of borneol in drinking water had no effect on incision activity of hepatocytes isolated from supplemented rats. The spectrophotometric assessment of enzymatic antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the flow cytometric assessment of total intracellular glutathione (iGSH) in primary hepatocytes of borneol-supplemented rats showed no changes in SOD and GPx activities but higher iGSH content particularly in hepatocytes of higher borneol dose (34 mg/kg) supplemented rats in comparison to control animals. Despite the fact that borneol had no effect either on BER of oxidative DNA damage or on the levels of antioxidant enzymes and manifested no reducing power and radicals scavenging activity, it increased significantly the level of non-enzymatic antioxidant iGSH which could reduce the oxidative DNA lesions induced by MB/VL or DMNQ.

Carboxymethyl chitin-glucan (CM-CG) protects human HepG2 and HeLa cells against oxidative DNA lesions and stimulates DNA repair of lesions induced by alkylating agents

A large number of functional foods, including those that contain β- d-glucans, have been shown to prevent human DNA against genotoxic effects and associated development of cancer and other chronic diseases. In this paper, carboxymethyl chitin-glucan (CM-CG) isolated from Aspergillus niger was investigated from two standpoints: (1) DNA-protective effects against oxidative DNA damage induced by H 2O 2 and alkylating DNA damage induced by MMS and MNNG, and (2) a potential effect on rejoining of MMS- and MNNG-induced single strand DNA breaks. The results obtained by the comet assay in human cells cultured in vitro showed that CM-CG reduced significantly the level of oxidative DNA lesions induced by H 2O 2 but did not change the level of alkylating DNA lesions induced by MMS or MNNG. On the other side, the efficiency of DNA-rejoining of single strand DNA breaks induced by MMS and MNNG was significantly higher in HepG2 cells pre-treated with CM-CG. The antioxidative activity of carboxymethyl chitin-glucan was confirmed by the DPPH assay.

DNA Damaging Effects of the Dyes Used in Sentinel Node Biopsy: Possible Implications for Clinical Practice

This study investigates whether methylene blue (MB), patent blue V (PBV), and indigo carmine (IDC) commonly used in sentinel node biopsy cause DNA damage to breast epithelial cells in vitro. MCF-7 and HB-2 cells were exposed for 5 minutes to the above dyes at the same concentrations used in clinical practice. Following exposure, the comet assay was performed to detect DNA damage. The enzyme, Fapy-DNA glycosylase (FpG) was incorporated to enable the detection of additional oxidative damage. Both PBV and MB stimulated DNA strand breaks in both MCF-7 and HB2 cell lines (P < 0.05). Levels were elevated over 3-fold (P < 0.05) in MCF-7 and HB2 cells treated with 2.5% PBV and 1% MB, compared with untreated control cells. In contrast, IDC did not stimulate DNA strand break damage at clinically relevant concentrations in either cell line. Addition of Fapy-DNA glycosylase enzyme also revealed significantly (P < 0.05) increased levels of oxidative DNA lesions (ODL) in MCF-7 cells treated with PBV (17.6% ODL) compared with control cells (5.9% ODL). This study shows, for the first time, that certain dyes (MB and PBV) commonly used in SLNB have genotoxic effects on breast cells at clinically relevant concentrations in vitro. In vivo studies are now warranted to assess and minimize DNA damage caused by these dyes during SLNB.

Down-Regulation of Oxidative DNA Lesions in Human Mononuclear Cells After Antioxidant Supplementation Correlates to Increase of Gamma-Tocopherol

The protective effect of vitamin E supplements has been questioned, possibly because they often contain only alpha-tocopherol, and recent studies indicate that gamma-tocopherol also has important properties. The aim of this study was to investigate whether the levels of DNA lesions in middle-aged, overweight males could be reduced by consumption of low doses of an antioxidant supplement for six weeks, designed to imitate a balanced diet. The participants (n=60) were randomly divided into: placebo, single-, and double-dose groups. Genotoxic and oxidative DNA lesions in mononuclear cells were measured with the Comet assay, before and after supplement administration. Furthermore, a cell study was performed to investigate if pre-incubation of a human lung cell line (A549) with alpha- and gamma-tocopherol (5 and 50 microM for 23 hours) could protect against induced oxidative DNA lesions as measured by the Comet assay. The level of oxidative DNA lesions in the double-dose group was significantly lower than in the control group. Oxidative DNA lesions correlated only to changes in serum gamma-tocopherol, and not alpha-tocopherol. In the cell study, only gamma-tocopherol protected cells against induced oxidative DNA lesions. We therefore hypothesize that gamma-tocopheol rather than alpha-tocopherol is involved in reducing oxidative DNA lesions.

Impaired DNA repair via the base-excision repair pathway after focal ischemic brain injury: a protein phosphorylation-dependent mechanism reversed by hypothermic neuroprotection

Cerebral ischemia and reperfusion induces rapid accumulation of oxidative DNA lesions in the brain, which, if not repaired promptly, may trigger cell death. The base-excision repair (BER) pathway is the main mechanism employed by neurons to repair various types of oxidative DNA damage. Recent studies have suggested that the cellular activity of BER is highly regulated (up- or down-regulated) after ischemic brain injury, and this regulation may contribute to the outcome of cell injury. The mechanism through which cellular BER is regulated in response to neuronal injury is currently poorly understood. In the present study, we have examined BER regulation in the rat model of focal ischemic brain injury induced by 2 hr of middle cerebral artery occlusion and 0-72 hr of reperfusion. As determined using cerebral nuclear extracts, focal ischemia resulted in a marked reduction in BER activities, including the overall BER activity, AP endonuclease activity and DNA polymerase-beta activity, indicating functional impairment of the BER pathway. BER reduction occurred as early as 0.5 hr after the onset of reperfusion. Thereafter, BER activity failed to recover, and there were persistent accumulations of apurinic/apyrimidinic abasic sites and DNA single-strand breaks in ischemic tissues. The reduction in BER during the early reperfusion phase (less than 6 hr) was not accompanied by any alterations in the levels of essential BER enzymes in brain extracts. However, increased serine- and threonine-specific phosphorylation was detected for both AP endonuclease and DNA polymerase-beta after ischemia, with the time course of serine phosphorylation closely correlated to that of changes in BER activity. Furthermore, dephosphorylation of nuclear extracts with alkaline phosphatase largely restored AP endonuclease and DNA polymerase-beta activities. Taking advantage of the neuroprotective effect of mild hypothermia (33 degrees C), which was induced in the brain during the first 2 hr of reperfusion, we found that the post-ischemic suppression of BER activity is a reversible event. Hypothermic treatment diminished the serine-specific phosphorylation of AP endonuclease and DNA polymerase-beta, promoted BER activities, and attenuated the levels of oxidative DNA lesions after ischemia. These results suggest that the functional impairment of the BER pathway after severe focal cerebral ischemia is due to the loss-of-function post-translational modifications of repair enzymes. Further investigations elucidating the precise mechanism underlying the post-translational regulation of BER enzymes may lead to novel therapeutic strategies for cerebral ischemia.

A comparative study on the antimutagenicity of atorvastatin and lovastatin against directly acting mutagens

Elevated levels of oxidative DNA lesions have been noted in many tumors and such damage is strongly implicated in the etiology of cancer. The cumulative risk of cancer increases with the fourth power of age and is associated with an accumulation of oxidative DNA damage. Many agents, synthetic or natural, that can inhibit mutation have been depicted as cancer chemopreventive agents. Antimutagenicity of the 3-hydroxy-3-methylgutaryl-CoA (HMG-CoA) reductase inhibitors atorvastatin and lovastatin was studied using the Ames Salmonella typhimurium assay. Directly acting mutagens, sodium azide (NaN(3)) and 4-nitro-o-phenylenediamine (NPDA), were used to induce mutation in Salmonella strains TA98 and TA100. The antimutagenicity of lovastatin and atorvastatin was found to be significant (p < 0.01) and dose-dependent. The percentage inhibition of a 3 mg lovastatin-treated plate was found to be 79.9% and 61.8% against NPDA- and NaN(3)-induced mutation to TA98 and TA100, respectively. Atorvastatin (0.5 mg/plate) inhibited NPDA-and NaN(3)-induced mutation to TA98 and TA100 by 78.6% and 45.5%, respectively. Atorvastatin showed antimutagenic activity at lower concentrations than lovatstatin. The results of the present study regarding the antimutagenic activity of atorvastatin and lovastatin suggested their therapeutic application as cancer chemopreventive agents.

Oxidative DNA modifications

Oxidative DNA modifications are frequent in mammalian DNA and have been suggested an important mechanism in carcinogenesis, diabetes and ageing. The foundations for this suggestion are: Evidence for the importance of oxidative DNA modifications in cancer development is: high levels of oxidative lesions in cancer tissue; highly conserved and specific DNA repair systems targeting oxidative lesions; high levels of oxidative DNA lesions in oxidative DNA repair knock-out animals; defective repair of oxidative lesions in cancer-prone progeria syndromes; reduced cancer incidence in populations with high dietary antioxidant intake; and increased oxidative stress to DNA in tobacco smokers. Conflicting evidence for a relation between oxidative stress to DNA and cancer is: disagreement about the true levels and occurrence of the oxidative lesions in vivo; failure to identify the localization of oxidative lesions in important genes, e.g. tumor suppressor and oncogenes; lack of evidence that the oxidative lesions induce mutations in vivo; no cancer development in animals knocked-out for specific DNA repair enzymes in spite of high tissue levels of oxidative lesions; and unchanged cancer rates after antioxidant interventions in large clinical controlled and randomized trials. The rate of DNA oxidation has been estimated from urinary excretion of repair products and it is evident that if these lesions were not repaired, a large part of DNA would be oxidized to a degree not compatible with living. The methodologies by which oxidative DNA modifications are measured cover a wide and different range, advantages and disadvantages will be presented. One particular problem is artificial oxidation, and methods to prevent such artifacts will be presented together with results from a large interlaboratory standardization program. The methodology by which the lesions can be measured is complicated and prone to artifacts during DNA isolation, digestion, derivatization and maybe even during the separation procedure proper prior to detection. A large effort from 20+ laboratories supported by a grant from the EU has reduced artifacts considerably and work towards interlaboratory standardization of the methodology is in progress. The presently agreed “normal” levels of the most frequent known lesion 8-oxodG is about 5 per million dG's in DNA. A comprehensive evaluation of the evidence, from chemistry to clinical and epidemiological trials, linking oxidative modifications to cancer will be given. Finally, an estimate of the quantitative role oxidative DNA modifications play among the multiplicity of other insults is given. While there is no question that all of these oxidative mechanisms do exist, quantitative data on their importance for the human situation do not exist. Prospective human studies that can provide such quantitative data on different mechanisms are underway.

Lignin-Stimulated Reduction of Oxidative DNA Lesions in Testicular Cells and Lymphocytes of Sprague-Dawley Rats In Vitro and Ex Vivo

: Lignin biopolymers constitute 30% of plant biomass and belong to the most abundant organic polymers on earth. We showed previously that this important component of dietary fiber exhibited a protective effect against the overall DNA damage induced by H2O2 or N-methyl-N′-nitro-N-nitrosoguanidine in hamster lung cells and human foreskin cells cultured in vitro. The objective of the present work was to examine DNA-protective effects of lignin in rat testicular cells and rat peripheral blood lymphocytes using in vitro and ex vivo experiments. H2O2 and visible light-excited methylene blue (MB) were used as DNA-damaging agents. Testicular cells were chosen because the germinal epithelium of testes is one of the most proliferately active tissues potentially susceptible to DNA-damaging effects. As a second target peripheral blood lymphocytes were chosen because dietary lignin or its metabolites circulate in the animal organism probably through the blood system. For the in vitro experiments, isolated cells were preincubated with lignin for 2 h before treatment with one of the oxidative agents. In ex vivo experiments, the cells were exposed to H2O2 or visible light-excited MB after isolation from rats fed either a common diet or a lignin-supplemented diet. The water-soluble, sulfur-free lignin used in experiments was obtained by fractionation of hardwood hydrolysate. The level of direct single-strand DNA breaks in H2O2-treated cells was measured by the classical comet assay, and the level of oxidative DNA lesions in visible light-treated cells was measured by a modified comet assay. We found that lignin reduced DNA lesions induced by H2O2 or visible light-excited MB both in vitro and ex vivo. The major conclusion of our study is that lignin polymer obtained by fractionation of hardwood hydrolysate manifested a specific type of antimutagenic effect.

Oxidative DNA Base Modifications and Polycyclic Aromatic Hydrocarbon DNA Adducts in Squamous Cell Carcinoma of Larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.

ROLE OF DNA REPAIR IN PARTICLE- AND FIBER-INDUCED LUNG INJURY

Lung injury after particle exposure is associated with the generation of reactive oxygen species (ROS), leading to increased levels of oxidative DNA damage. Furthermore, both stable and unstable DNA lesions may arise after metabolization of polycyclic aromatic hydrocarbons (PAH) frequently bound to particles. Oxidative DNA damage is predominantly removed by base excision repair (BER), which is usually comparatively fast and efficient. Nevertheless, even without exogenous exposure a measurable steady-state level of oxidative DNA lesions exists due to oxygen metabolism, and repair capacities may be exceeded upon particle exposure. Stable DNA adducts induced by PAHs are eliminated by nucleotide excision repair (NER), which is unequally distributed in the genome and incomplete in transcriptionally inactive DNA regions. Thus, even low adduct levels due to environmental exposure lead to persistent lesions in the human lung. Other factors that need to be considered are potential repair inhibitions by associated metal compounds and interindividual differences in repair capacities. Taken together, the extent of DNA repair is an important determinant in particle-induced genotoxicity and carcinogenicity.