A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA

A major controversy in the area of DNA biochemistry concerns the actual in vivo levels of oxidative damage in DNA. We show here that 8-oxo-2-deoxyguanosine (oxo8dG) generation during DNA isolation is eliminated using the sodium iodide (NaI) isolation method and that the level of oxo8dG in nuclear DNA (nDNA) is almost one-hundredth of the level obtained using the classical phenol method. We found using NaI that the ratio of oxo8dG/10(5 )deoxyguanosine (dG) in nDNA isolated from mouse tissues ranged from 0.032 +/- 0.002 for liver to 0.015 +/- 0.003 for brain. We observed a significant increase (10-fold) in oxo8dG in nDNA isolated from liver tissue after 2 Gy of gamma-irradiation when NaI was used to isolate DNA. The turnover of oxo8dG in nDNA was rapid, e.g. disappearance of oxo8dG in the mouse liver in vivo after gamma-irradiation had a half-life of 11 min. The levels of oxo8dG in mitochondrial DNA isolated from liver, heart and brain were 6-, 16- and 23-fold higher than nDNA from these tissues. Thus, our results showed that the steady-state levels of oxo8dG in mouse tissues range from 180 to 360 lesions in the nuclear genome and from one to two lesions in 100 mitochondrial genomes.     

Endogenous DNA damage as related to cancer and aging

The endogenous background level of oxidant-induced DNA damage in vivo has been assayed by measuring 8-hydroxydeoxyguanosine (oh8dG), thymine glycol and thymidine glycol in urine and oh8dG in DNA. The level of oxidative DNA damage as measured by oh8dG in normal rat liver is shown to be extensive (1/130,000 bases in nuclear DNA and 1/8000 bases in mitochondrial DNA), especially in mtDNA. The methylation adduct 7-methylguanine (m7G) has also been found. m7G is one of about 5 adducts found on methylating DNA, and oh8dG is one of about 20 adducts found on oxidizing DNA, e.g., by radiation. We also discuss 3 hitherto unrecognized antioxidants in man.     

DNA damage, repair, and antioxidant systems in brain regions: a correlative study

8-Hydroxy-2'-deoxyguanosine (oxo(8)dG) has been used as a marker of free radical damage to DNA and has been shown to accumulate during aging. Oxidative stress affects some brain regions more than others as demonstrated by regional differences in steady state oxo(8)dG levels in mouse brain. In our study, we have shown that regions such as the midbrain, caudate putamen, and hippocampus show high levels of oxo(8)dG in total DNA, although regions such as the cerebellum, cortex, and pons and medulla have lower levels. These regional differences in basal levels of DNA damage inversely correlate with the regional capacity to remove oxo(8)dG from DNA. Additionally, the activities of antioxidant enzymes (Cu/Zn superoxide dismutase, mitochondrial superoxide dismutase, and glutathione peroxidase) and the levels of the endogenous antioxidant glutathione are not predictors of the degree of free radical induced damage to DNA in different brain regions. Although each brain region has significant differences in antioxidant defenses, the capacity to excise the oxidized base from DNA seems to be the major determinant of the steady state levels of oxo(8)dG in each brain region.     

Endogenous Oxidative DNA Damage,Aging, and Cancer

Progress in identifying the important endogenous processes damaging DNA and developing methods to assay this damage in individuals is presented. This approach may aid studies on modulation of cancer and aging.

The endogenous background level of oxidant-induced DNA damage in vivo has been assayed by measuring 8-hydroxydeoxyguanosine (oh⁸dG), thymine glycol and thymidine glycol in urine and oh⁸dG in DNA. oh⁸dG is one of about 20 adducts found on oxidizing DNA, e.g., by radiation. The level of oxidative DNA damage as measured by oh⁸dG in normal rat liver is shown to be extensive, especially in mtDNA (1/130,000 bases in nuclear DNA and 1/8,000 bases in mitochondrial DNA). We also discuss three hitherto unrecognized antioxidants in man.     

Influence of flavan-3-ols and procyanidins on UVC-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in isolated DNA

The flavan-3-ols (-)-epicatechin (epicatechin) and (+)-catechin (catechin) and their related oligomers (procyanidins) isolated from cocoa were assayed for their capacity to inhibit the UVC-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)dG) in calf thymus DNA. The above-mentioned compounds inhibited oxo(8)dG production in a concentration- and time-dependent manner. After 30 min of irradiation (30 kJ/m(2)), 0.1, 1.0, 10, and 100 microM epicatechin inhibited oxo(8)dG formation by 20, 36, 64, and 74%, respectively. For the same dose of UVC, 0.1, 1.0, 10, and 100 microM catechin inhibited oxo(8)dG formation by 1, 23, 50, and 70%, respectively. Epicatechin was more efficient than catechin with respect to inhibiting oxo(8)dG formation (IC(50) 1.7 +/- 0.7 vs 4.0 +/- 0.7 microM). Monomer, tetramer, and hexamer fractions were equally effective in inhibiting oxo(8)dG formation when assayed at 10 microM monomer equivalent concentration. At similar concentrations (1-50 microM), the inhibition of the UVC-mediated oxo(8)dG formation by flavan-3-ols and procyanidins was in the range of that of alpha-tocopherol, Trolox, ascorbate, and glutathione. These results support the concept that flavan-3-ols and their related procyanidins can protect DNA from oxidation at concentrations that can be physiologically relevant. Both epimerism and degree of oligomerization are important determinants of the antioxidant activity of flavan-3-ols and procyanidins.     

DNA damage,p53 gene expression and p53 protein level in the rat brain aging

The aging induces free radicals leading to DNA damage (8‐oxo‐2′‐deoxyguanosine, 8‐oxo2dG). DNA injury causes increased expression of p53 gene and p53 protein. Levels of 8‐oxo2dG (HPLC), p53 mRNA (PCR) and p53 protein (Western blot) were estimated in gray matter (GM), white matter (WM), cerebellum (C) and medulla oblongata (MO) of control, 12‐ and 24‐month‐old rats. The level of 8‐oxo2dG increased with age in C (P < 0.05 in 12‐month‐old and P < 0.01 in 24‐month‐old rats) and MO. In 12‐month‐old animals the level of 8‐oxo2dG in GM and WM was higher than in controls. In 12‐month‐old animals p53 gene expression decreased while amounts of p53 protein increased, depending on the oxidative DNA damage. In 24‐month‐old rats, expression of p53 increased in all structures (P ≤ 0.05) while p53 protein showed decreased levels in most of structures of central nervous system (WM, C, MO). Aging leads to increased 8‐oxo2dG and augmented p53 gene expression, accompanied by a lowered expression of p53 protein.     

Ogg1 null mice exhibit age-associated loss of the nigrostriatal pathway and increased sensitivity to MPTP

Cumulative damage to cellular macromolecules via oxidative stress is a hallmark of aging and neurodegenerative disease. Whether such damage is a cause or a subsequent effect of neurodegeneration is still unknown. This paper describes the development of an age-associated mild parkinsonian model in mice that lack the DNA repair enzyme 8-oxoguanine glycosylase 1 (Ogg1). Aged OGG1 knock-out (OGG1 KO) mice show a decreased spontaneous locomotor behavior and evidence a decrease in striatal dopamine levels, a loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN), and an increase in ubiquitin-positive inclusions in their remaining SN neurons. In addition, young OGG1 KO mice are more susceptible to the dopaminergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) than their wild-type (WT) counterparts. Age-associated increases in 7,8-dihydro-2'-deoxyguanine (oxo(8)dG) have been reported in brain regions and neuronal populations affected in Parkinson's disease (PD), toxin-induced parkinsonian models, and mice harboring genetic abnormalities associated with PD. Because of these increased oxo(8)dG levels, the OGG1 KO mouse strain could shed light on molecular events leading to neuronal loss as a consequence of cumulative oxidative damage to DNA during aging and after toxicological challenge.     

Resveratrol, melatonin, vitamin E, and PBN protect against renal oxidative DNA damage induced by the kidney carcinogen KBrO3.

Free radical scavengers can protect against the genotoxicity induced by chemical carcinogens by decreasing oxidative damage. The protective effect of the antioxidants melatonin, resveratrol, vitamin E, butylated hydroxytoluene and 2-mercaptoethylamine, and the spin-trapping compound alpha-phenyl-N-tert-butyl nitrone (PBN) against oxidative DNA damage was studied in the kidney of rats treated with the kidney-specific carcinogen potassium bromate (KBrO3). KBrO3 was given to rats previously treated with melatonin, resveratrol, PBN, vitamin E, butylated hydroxytoluene, or 2-mercaptoethylamine. Oxidative damage to kidney DNA was estimated 6 hours afterwards by measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG) referred to deoxyguanosine (dG) by means of high performance liquid chromatography with electrochemical-coulometric and ultraviolet detection. Levels of oxo8dG in the renal genomic DNA significantly increased by more than 100% after the KBrO3 treatment. This increase was completely abolished by the treatment with resveratrol and was partially prevented by melatonin, PBN and vitamin E. Resveratrol and PBN also prevented the increase in relative kidney weight induced by KBrO3. These results show that various different antioxidants and a free radical trap, working in either the water-soluble or the lipid-soluble compartments, can prevent the oxidative DNA damage induced in the kidney by the carcinogen KBrO3.     

Renal excretion of 8-oxo-7,8-dihydro-2(')-deoxyguanosine: degradation rates of RNA and metabolic rate in humans

Renally excreted 8-oxo-7,8-dihydro-2(')-deoxyguanosine (oxo(8)dG) is a potential marker of oxidative DNA damage by reactive oxygen species. Whole-body degradation rates of t- and rRNA are potential indicators of the resting metabolic rate (RMR). Excretion rates of oxo(8)dG and degradation rates of t- and rRNA were determined in healthy non-smoking adults and children. RMR (indirect calorimetry; 14 children, 16 adults), total energy expenditure (TEE; doubly labelled water technique; 4 children, 6 adults), and lean body mass (LBM; dual-energy X-ray absorptiometry; 14 children, 16 adults) were also measured. Degradation of t- and rRNA (micromol/d/kg LBM; 4 children, 6 adults) was highly correlated with RMR (kJ/d/kg LBM), r=0.867 (p<0.005) and 0.959 (p<0.001), respectively. Excretion of oxo(8)dG (pmol/d/kg LBM; 14 children, 16 adults) was not significantly correlated with RMR (p>0.05). Neither excretion of oxo(8)dG nor degradation of RNA was significantly correlated with TEE (kJ/d/ kg LBM) (p>0.05). In healthy subjects further factors, other than the metabolic rate, seem to influence the excretion rate of oxo(8)dG. The degradation rates of t- and rRNA seem to be appropriate indicators of the RMR.     

Isolation of a malondialdehyde-deoxyguanosine adduct from rat liver DNA.

In previous studies, an adduct of malondialdehyde (MDA) with guanine was identified in rat and human urine. Subsequent detection of an adduct with deoxyguanosine (dG) in urine prompted an investigation of its possible occurrence in DNA. Rat liver DNA was hydrolyzed using nuclease P1 and alkaline P-ase and subjected to deoxyribonucleoside analysis using reverse phase high-pressure liquid chromatography (HPLC) with fluorescence detection. A compound was isolated that could not be separated from a synthetic pyrimidinopurine adduct of MDA and dG (dG-MDA). Partial hydrolysis released guanine (Gua), Gua-MDA, and dG in amounts that, in aggregate, were the molar equivalent of the starting material calculated by fluorescence analysis as dG-MDA. Complete acid hydrolysis of the isolate yielded an equimolar amount of MDA. Analysis of liver DNA isolated from growing rats yielded a value for dG-MDA content of 9.0 +/- 1.6 pmol/100 micrograms DNA (mean +/- SEM, N = 5). This value is approximately 7 times those reported for the 8-hydroxy deoxyguanosine content of rat liver nuclear DNA. This study demonstrates that DNA is modified in vivo by reactions of its guanylate moiety with MDA, and indicates that, at least in the case of rat liver DNA, the prevalence of such modifications is greater than those caused by reactions with hydroxyl radicals.     

Levels of 8-hydroxy-2'-deoxyguanosine in DNA of white blood cells from workers highly exposed to asbestos in Germany

Asbestos fibers have genotoxic effects and are a potential carcinogenic hazard to occupationally exposed workers. The ability of inhaled asbestos fibers to induce the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the DNA of white blood cells (WBC) of workers highly exposed at the workplace has been studied. The 8-OHdG adduct level of asbestos-exposed workers was significantly increased (p<0.001) compared to that in the control group in all three years of the study. Asbestos-exposed individuals showed a mean value of 2.61+/-0.91 8-OHdG/10(5) dG (median 2.49, n=496) in 1994-1995, 2.96+/-1.10 8-OHdG/10(5) dG (median 2.76, n=437) in 1995-1996 and 2.55+/-0.56 8-OHdG/10(5) dG (median 2.53, n=447) in 1996-1997. For the control subjects, a mean of 1.52+/-0.39 (median 1.51, n=214) was determined. The results indicate that human DNA samples from exposed individuals contain between 1.7 times and twice the level of oxidative damage relative to that found in control samples in all 3 years of the study. The studies presented here show that asbestos exposure can result in oxidative DNA damage. Our data confirm that oxidative DNA damage occurs in the WBC of workers highly exposed to asbestos fibers, thus supporting the hypothesis that asbestos fibers damage cells through an oxidative mechanism. These in vivo findings underline the importance of oxidative damage in asbestos-induced carcinogenesis and highlight the need for exploring the molecular basis of asbestos-induced diseases, and for more effective diagnosis, prevention and therapy of mesothelioma, lung cancer and pulmonary fibrosis. In addition, preventive and therapeutic approaches using antioxidants may be relevant.     

Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.

DNA damage is considered of paramount importance in aging. Among causes of this damage, free radical attack, particularly from mitochondrial origin, is receiving special attention. If oxidative damage to DNA is involved in aging, long-lived animals (which age slowly) should show lower levels of markers of this kind of damage than short-lived ones. However, this possibility has not heretofore been investigated. In this study, steady-state levels of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) referred to deoxyguanosine (dG) were measured by high performance liquid chromatography (HPLC) in the mitochondrial (mtDNA) and nuclear (nDNA) DNA from the heart of eight and the brain of six mammalian species ranging in maximum life span (MLSP) from 3.5 to 46 years. Exactly the same digestion of DNA to deoxynucleosides and HPLC protocols was used for mtDNA and nDNA. Significantly higher (three- to ninefold) 8-oxodG/dG values were found in mtDNA than in nDNA in all the species studied in both tissues. 8-oxodG/dG in nDNA did not correlate with MLSP across species either in the heart (r=-0.68; P<0.06) or brain (r = 0.53; P<0.27). However, 8-oxodG/dG in mtDNA was inversely correlated with MLSP both in heart (r=-0.92; P<0.001) and brain (r=-0.88; P<0.016) tissues following the power function y = a(.)x(b), where y is 8-oxodG/dG and x is the MLSP. This agrees with the consistent observation that mitochondrial free radical generation is also lower in long-lived than in short-lived species. The results obtained agree with the notion that oxygen radicals of mitochondrial origin oxidatively damage mtDNA in a way related to the aging rate of each species.-Barja, G., Herrero, A. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.     

Renal excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine in Wistar rats with increased O(2) consumption due to cold stress

DNA damage by reactive oxygen species is of special interest in the development of cancer and in aging. The renally excreted amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)dG) is a potential noninvasive marker of oxidative DNA damage. The respiratory chain of mitochondria is one source for the formation of reactive oxygen species. In the present study we investigated in Wistar rats (n = 7; mean body weight at start, 307.4 +/- 11 g) the effect of an increased O(2) consumption, i.e., energy expenditure, due to cold stress on the renally excreted amount of oxo(8)dG. First, the rats were housed for 4 days at 23.5 degrees C (basic period, BP), and then for 6 days at 10 degrees C (cold stress period, CSP), and finally for 3 days at 23.5 degrees C (recovery period, RP). The O(2) consumption (L O(2)/day/kg weight) was significantly (P < 0.0001) on average 50% higher in CSP (69.0 +/- 3.9) than in BP (45.8 +/- 4.8), and similar in BP and RP (44.3 +/- 5.4). The average renal excretion of oxo(8)dG (pmol/day/kg weight) was significantly (P < 0.025) on average 13% higher in CSP (375.5 +/- 27.7) than in BP (333.2 +/- 47. 4) and similar in BP and RP (331.8 +/- 34.3). Maximum increase in oxo(8)dG excretion of on average 17% was on the third to fifth day of the CSP. This study reveals that an increase in O(2) consumption of 50% resulted in a much lower increase in the renal excretion of oxo(8)dG.     

Lipids and DNA oxidation in Staphylococcus aureus as a consequence of oxidative stress generated by ciprofloxacin

Ciprofloxacin induced an increment of reactive oxygen species in sensitive strains of Staphylococcus aureus leading to oxidative stress detected by chemiluminescence while resistant strains did not suffer such stress. Oxidation of lipids was performed by employing thiobarbituric acid reaction to detect the formation of the amplified intermediate between reactive species oxygen and cytoplasmic macromolecules, namely malondialdehyde (MDA). The sensitive strain presented higher peroxidation of lipids than the resistant strain. The oxidative consequence for DNA was investigated by means of bacteria incubation with ciprofloxacin and posterior extraction of DNA, which was studied by high performance liquid chromatography (HPLC). Sensitive S. aureus ATCC 29213 showed an increase of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) respect controls without antibiotic; there was evident increase of the ratio between 8-oxodG and deoxyguanosine (dG) as a consequence of oxidation of dG to 8-oxodG considered the major DNA marker of oxidative stress. The resistant strain showed low oxidation of DNA and the analysis of 8-oxodG/dG ratio indicated lesser formation of 8-oxodG than S. aureus ATCC 29213.     

Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain.

Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long-Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30-90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N-glycosylase (Fpg protein)-sensitive sites (FPGSS), as labeled in situ using digoxigenin-dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8-hydroxy-2'-deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS-positive cells in the cortex from the sham-operated control group was 3 +/- 3 (mean +/- SD per mm(2)). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS-positive cells were significantly increased by 200-fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8-hydroxy-guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 microg/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham-operated control group. We detected few FPGSS of oh8dG-positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked-end labeling (TUNEL)-positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL.     

Senescence-associated decline in the intranuclear accumulation of hOGG1-alpha and impaired 8-oxo-dG repair activity in senescing normal human oral keratinocytes in vivo

We determined the mitochondrial membrane status, presence of reactive oxygen species (ROS), and oxidative DNA adduct formation in normal human oral keratinocytes (NHOK) during senescence. The senescent cells showed accumulation of intracellular ROS and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), a major oxidative DNA adduct. Exposure of cells to H2O2 induced 8-oxo-dG accumulation in cellular DNA, which was rapidly removed in replicating NHOK. However, the 8-oxo-dG removal activity was almost completely abolished in the senescing culture. Both replicating and senescing NHOK expressed readily detectable 8-oxo-dG DNA glycosylase (hOGG1), the enzyme responsible for glycosidic cleavage of 8-oxo-dG. After exposure to H2O2, however, the intranuclear level of the hOGG1-alpha isoform was decreased in senescing but not in replicating NHOK. These results indicated that senescing NHOK accumulated oxidative DNA lesions in part due to increased level of endogenous ROS and impaired intranuclear translocation of hOGG1 enzyme upon exposure to oxidative stress.     

Accumulation of M1dG DNA adducts after chronic exposure to PCBs, but not from acute exposure to polychlorinated aromatic hydrocarbons

Oxidative DNA damage is one of the key events thought to be involved in mutation and cancer. The present study examined the accumulation of M1dG, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, DNA adducts after single dose or 1-year exposure to polyhalogenated aromatic hydrocarbons (PHAH) in order to evaluate the potential role of oxidative DNA damage in PHAH toxicity and carcinogenicity. The effect of PHAH exposure on the number of M1dG adducts was explored initially in female mice exposed to a single dose of either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or a PHAH mixture. This study demonstrated that a single exposure to PHAH had no significant effect on the number of M1dG adducts compared to the corn oil control group. The role of M1dG adducts in polychlorinated biphenyl (PCB)-induced toxicity and carcinogenicity was further investigated in rats exposed for a year to PCB 153, PCB 126, or a mixture of the two. PCB 153, at doses up to 3000 microg/kg/day, had no significant effect on the number of M1dG adducts in liver and brain tissues from the exposed rats compared to controls. However, 1000 ng/kg/day of PCB 126 resulted in M1dG adduct accumulation in the liver. More importantly, coadministration of equal proportions of PCB 153 and PCB 126 resulted in dose-dependent increases in M1dG adduct accumulation in the liver from 300 to 1000 ng/kg/day of PCB 126 with 300-1000 microg/kg/day of PCB 153. Interestingly, the coadministration of different amounts of PCB 153 with fixed amounts of PCB 126 demonstrated more M1dG adduct accumulation with higher doses of PCB 153. These results are consistent with the results from cancer bioassays that demonstrated a synergistic effect between PCB 126 and PCB 153 on toxicity and tumor development. In summary, the results from the present study support the hypothesis that oxidative DNA damage plays a key role in toxicity and carcinogenicity following long-term PCB exposure.     

A formation mechanism for 8-hydroxy-2'-deoxyguanosine mediated by peroxidized 2'-deoxythymidine

The oxidative formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA is closely associated with the induction of degenerative diseases, including cancer. However, the oxidant species participating in the formation of 8-OHdG has yet to be fully clarified. On the basis that peroxyl radicals are a strong candidate for this species, we employed 2,2'-azobis(2-amidinopropane) (AAPH) as a peroxyl radical generator. Exposure of calf thymus DNA to AAPH formed 8-OHdG, but the exposure of 2'-deoxyguanosine (dG) alone did not. From the exposure of various combinations of nucleotides, 8-OHdG was formed only in the presence of dG and thymidine (dT). A mix of dG with an oxidation product of dT, 5-(hydroperoxymethyl)-2'-deoxyuridine, produced 8-OHdG, but the amount formed was small. In contrast, 8-OHdG was produced abundantly by the addition of dG to peroxidized dT with AAPH. Thus, the formation of 8-OHdG was mediated by the peroxidized dT. Instead of artificial AAPH, endogenous peroxyl radicals are known to be lipid peroxides, which are probably the oxidant species for 8-OHdG formation mediated by thymidine in vivo.     

Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis

Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. However, the molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells.