8-OHdG Formation In DNA Research Articles

Progression of Hepatic Adenoma to Carcinoma in Ogg1 Mutant Mice Induced by Phenobarbital.

The carcinogenic potential of phenobarbital (PB) was assessed in a mouse line carrying a mutant Mmh allele of the Mmh/Ogg1 gene encoding the enzyme oxoguanine DNA glycosylase (Ogg1) responsible for the repair of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Mmh homozygous mutant (Ogg1−/−) and wild-type (Ogg1+/+) male and female, 10-week-old, mice were treated with 500 ppm PB in diet for 78 weeks. Hepatocellular carcinomas (HCCs) were found in PB-treated Ogg1−/− mice, while Ogg1+/+ animals developed only hepatocellular adenomas (HCAs) at the same rate. This was coordinated with PB-induced significant elevation of 8-OHdG formation in DNA and cell proliferation in adjacent liver of Ogg1−/− mice. Proteome analysis predicted activation of transcriptional factor Nrf2 in the livers and HCAs of PB-administered Ogg1+/+ mice; however, its activation was insufficient or absent in the livers and HCCs of Ogg1−/− mice, respectively. Significant elevation of phase I and II metabolizing enzymes was demonstrated in both Ogg1−/− and Ogg1+/+ animals. Treatment of Ogg1−/− mice with PB resulted in significant elevation of cell proliferation in the liver. These results indicate that PB induced progression from HCA to HCC in Ogg1−/− mice, due to persistent accumulation of DNA oxidative base modifications and suppression of Nrf2-mediated oxidative stress response, resulting in significant elevation of cell proliferation.

What causes human cancer? Approaches from the chemistry of DNA damage.

To prevent human cancers, environmental mutagens must be identified. A common mechanism of carcinogenesis is DNA damage, and thus it is quite possible that environmental mutagens can be trapped as adducts by DNA components. It is also important to identify new types of DNA damaging reactions and clarify their mechanisms. In this paper, I will provide typical examples of our efforts to identify DNA damage by environmental agents, from chemistry-based studies. 1) Oxidative DNA damage: 8-Hydroxydeoxyguanosine (8-OHdG, 8-oxodG) was discovered during a structural study of DNA modifications caused in vitro by heating glucose, which was used as a model of cooked foods. We found that various oxygen radical-forming agents induced the formation of 8-OHdG in DNA, in vitro and in vivo. Analyses of the urinary 8-OHdG levels are useful to assess the extent of oxidative DNA damage in a human population. 2) Lipid peroxide-derived DNA adducts: We searched for mutagens that react with deoxynucleosides, in model systems of lipid peroxidation. The reaction mixtures were analyzed by high performance liquid chromatography (HPLC), and we discovered various lipid peroxide-derived mutagens, including new mutagens. Some of these adducts were detected in human DNA. These mutagens may be involved in lipid peroxide-related cancers. 3) Methylation of cytosine by free radicals: Methylation of the cytosine C-5 position is an important mechanism of carcinogenesis, in addition to gene mutations. However, the actual mechanisms of de novo methylation in relation to environmental agents are not clear. We found that cytosine C-5 methylation occurred by a free radical mechanism. The possible role of this radical-induced DNA methylation in carcinogenesis will be discussed, in relation to the presently accepted concept of cancer epigenetics. In these studies, chemical analyses of the adducts formed in model reactions led to the discoveries of new mutagens and important types of DNA modifications, which seem to be involved in human carcinogenesis.

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The accumulation of damages in the DNA of the cells along the time, seems to play an important paper in the pathogenesis of cancer patients. DNA damage accumulates especially in mtDNA over time, and hOGG1 is an enzyme responsible for the repair of DNA oxidative damage either in nuclei and mitochondria. The Ser326Cys polymorphism was shown to diminish the hOGG1 activity in vitro, and it was suggested that the 326Cys allele might pose a higher risk of 8-OHdG formation in DNA. Recently, some authors have demonstrated that the capacity to repair oxidative DNA damage was significantly decreased in healthy human subjects bearing the hOGG1 Cys326Cys homozygous variant genotype, compared to wild type individuals. In the present, we intend to study the possible relation of this polymorphism with Bladder Cancer, BC. Bladder tissue from 22 cases of BC, and 90 controls, from peripheral blood lymphocytes; genomic DNA was extracted by means of the DNA direct II (Dynal) kit. Genotyping for the hOGG1 Ser326Cys polymorphism was performed by PCR-RFLP technique, briefly, a 234 bp product was amplified using the forward: 5′-CCC AAC CCC AGT GGA TTC TCA TTG C-3′ and the reverse: 5′-GGT GCC CCA TCT AGC CTT GCG GCC CTT-3′ primers. PCR conditions were 4 min at 94 °C and followed by 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 1 min and 30 s at 72 °C, and a final elongation step of 5 min at 72 °C. Overnight digestion at 37 °C with Fnu4HI resulted in 213- and 21-bp products for the wild type allele (Ser326), and in 164-, 49- and 21-bp products for the mutant allele (Cys326). Digestion products were visualized after electrophoresis on a 2% agarose gel containing ethidium bromide. The distribution of polymorphism in BC was: 64,3% SS, 33% SC 2,7% CC, in the control group: 66,4 SS, 30,4 SC 3,2 CC. The Chi-square test, p < 0.001.There are not correlation in polymorphism in this study inter BC and control group.

Bio-effects of Nano-TiO 2 on DNA and cellular ultrastructure with different polymorph and size

In this paper, we investigated the bio-effect of three kinds of nano-TiO 2 on the aspects of DNA strand breaking activity, generation of 8-hydroxy-2-deoxyguanosine (8-OHdG), cytotoxicity and cell morphology. Different extents of strand scission and the formation of 8-OHdG in DNA were obtained after treated with different types of nano-TiO 2 (10 – 20 nm anatase, 50 – 60 nm anatase and 50 – 60 nm rutile). Furthermore, MTT assay and TEM detection showed the toxic effects on CHO cells with a toxic order as 10 – 20 nm anatase > 50 – 60 nm anatase > 50 – 60 nm rutile, which was consistent with that of 8-OHdG generation and DNA strand breaking activities. It was interesting that three kind of nano TiO 2 all had much higher toxic effect on tumor cells than on normal cells, and nano-TiO 2 was observed in the cytoplasm of CHO cells. These results indicated that the toxic effect of nano-TiO 2 relates to the size and type of particles, in addition, their potential application for cancer treatment was discussed.

Induction of reactive oxygen species without 8-hydroxydeoxyguanosine formation in DNA of initiated mouse keratinocytes treated with 12-O-tetradecanoylphorbol-13-acetate.

Evidence for the involvement of oxidative stress in 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated tumor promotion has focused on non-initiated immune cells, tumor cell lines and non-initiated epidermis treated in vivo. This paper reports the effects of TPA on 8-hydroxydeoxyguanosine (8OHdG) formation and the generation of reactive oxygen species (ROS) in cloned initiated mouse epidermal keratinocytes in order to determine whether TPA can directly damage DNA through ROS production within the keratinocytes. Using high performance liquid chromatography with electrochemical detection (HPLC-EC), TPA did not induce 8OHdG formation in DNA of initiated keratinocytes treated under a variety of conditions. The reliability of the HPLC-EC system is demonstrated by (i) the linearity of the 8OHdG standard curve; (ii) the consistency of 8OHdG measurements in calf thymus and cellular DNA; and (iii) the dose-dependent increase in 8OHdG in DNA of initiated keratinocytes treated with UVC in the presence and absence of H2O2. Though not DNA-damaging, TPA induced a 65% increase in ROS (P < 0.05) as detected by luminol-dependent chemiluminescence. These results support a mechanism for the role of oxidative stress in tumor promotion that does not involve direct DNA damage to the keratinocyte target cell. The relationship between ROS, signal transduction and tumor promotion is discussed in light of the above results which is consistent with the role of TPA-induced ROS as second messengers in signal transduction.

Inhibition of UV light- and Fenton reaction-induced oxidative DNA damage by the soybean isoflavone genistein.

We have investigated the effect of the soybean isoflavone genistein on 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in calf thymus DNA exposed to either UV irradiation or the Fenton reaction system. Under the conditions used we observed that UV light and the Fenton reaction significantly increase 8-OHdG formation in DNA. Co-incubation with genistein inhibits the formation of 8-OHdG induced by either UV light irradiation or the Fenton reaction in a dose-dependent manner. The quenching effect of genistein on 8-OHdG formation induced by UV light is much more potent than that by the Fenton reaction, suggesting that the mechanisms of 8-OHdG formation may differ between the two systems. We further compared the antioxidant activities and quenching effect on 8-OHdG formation of genistein with biochanin A. Genistein potently scavenges both hydrogen peroxide in the medium and superoxide anion generated by xanthine/xanthine oxidase, whereas biochanin A has either a weak or no scavenging effect on these reactive oxygen species. However, both genistein and biochanin A display a similar quenching effect on UV light-induced 8-OHdG formation. These results suggest that the quenching effect of genistein and biochanin A on UV light-induced 8-OHdG formation is different from their ability to scavenge hydrogen peroxide and superoxide anion. The potent inhibition of UV light-induced oxidative DNA damage by genistein suggests its potential anticarcinogenic role in photocarcinogenesis.

Formation of 8-hydroxydeoxyguanosine in calf thymus DNA treated in vitro with phenylhydroquinone, the major metabolite of O-phenylphenol.

The generation of 8-hydroxydeoxyguanosine (8OHdG) in calf thymus DNA treated with O-phenylphenol (OPP) or its major metabolites, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), was studied. The content of 8OHdG residues was increased in DNA treated with PHQ, and the generation of 8OHdG was highly dependent on PHQ concentration. PBQ had little effect on the formation of 8OHdG, and OPP had no effect. The formation of 8OHdG by PHQ was reduced by oxygen radical scavengers such as catalase, sodium benzoate and sodium azide. The PHQ-induced 8OHdG formation was accelerated by the addition of CuCl or CuCl2 to the reaction mixture, but was decreased by the addition of chelating agents such as EDTA, bathocuproinedisulfonic acid disodium salt (bathocuproine disulfonate) and O-phenanthroline. These results demonstrate that hydroxyl radicals generated in the process of oxidation of PHQ contribute to the formation of 8OHdG in DNA, and copper ions facilitate the oxidative DNA damage. Copper ions greatly accelerated the PHQ-induced DNA cleavage in vitro, although they had no effect on cleavage without PHQ. On the other hand, DNA cleavage occurred by the addition of FeCl2 in the absence and presence of PHQ. FeCl2 stimulates 8OHdG formation only slightly with or without PHQ. Furthermore, the stimulatory effect of FeCl2 on 8OHdG formation was observed even in the presence of EDTA. The formation of 8OHdG in bladder DNA is likely to be one of a series of events leading to bladder tumors seen in rats fed OPP-containing diet.

Mediation of 8-hydroxy-guanine formation in DNA by thiazin dyes plus light

We have discovered that methylene blue plus light mediates the formation of 8-OHdG in DNA. Methylene blue is one of several thiazin dyes and we report here that the other thiazin dyes tested, in combination with white light, are effective in mediating 8-OHdG formation in DNA. The effectiveness of light plus the thiazin dyes in forming 8-OHdG in DNA were as follows: methylene blue > azure B > aure A > toluidine blue > thionin. Two other compounds tested; riboflavin and fuschin acid, in combination with light, caused formation of very little, if any, 8-OHdG in DNA. Thiazin dye mediated formation of 8-OHdG in DNA was not inhibited by the spin trap α-phenyl-t-butyl nitrone, which supports our previous observations that oxygen free radical scavengers did not inhibit methylene blue plus light mediated 8-OHdG formation in DNA. Ascobate addition to methylene blue plus DNA, in the absence of light, was ineffective in mediating 8-OHdG formation in DNA.

FRRC Highlight: This note Highlights a Seminal Paper Published in Free Rad. Res. Comms. 1, 163–172 (1986): The Development of a Sensitive Analysis for 8-Hydroxy-2′-Deoxyguanosine

We were fortunate to be able to first show that 8-hydroxy-2'-deoxyguanosine (8OHdG) could be sensitively quantitated (to 20 femto moles) by the use of HPLC with electrochemical detection.' This method provides a means to help assess the role of activated oxygen attack on DNA (and RNA) and as such provides data to understand oxidative stress' in biological systems. Tiger in Blake's poem by that title could refer to sagacity, defiance and doubt, but it is equally clear that serendipity, dependence upon the creative and rigorous observations of other researchers and determination fueled by curiosity and dissatisfaction must play an equally important role in exploring new areas and approaches scientifically. Perhaps all three aspects were operating to help make our first observations possible. It is important to acknowledge the following set of circumstances which significantly contributed: (a) Dr. S. Nishimura's generosity in providing us with an authentic standard of 8-OHdG, (b) Dr. P. Wong's collaborative expertise whose training in HPLC-electrochemical detection (HPLC-ED) derives from the pioneering efforts on this analytical technique in biological systems by Drs. L. Blank and R. Adams, (c) our first efforts to use HPLC-ED to detect reduced spin-adducts' and hydroxyl free radical reaction products of phenol and salicylate4 and (d) a persistent fascination with the fundamental chemistry of the toxic aspects of oxygen on biological systems and especially oxygen free radical reactions with nucleic acids.' Since the earlier observations, 8-OHdG has been demonstrated to be formed in the DNA of several biological systems undergoing oxidative stress. Nishimura's group has found, using a wide range of systems, a positive correlation between 8-OHdG formation in DNA and conditions enhancing cancer We have demonstrated that certain chromium salts and betel nut quid, agents responsible for cancer development, cause formation of 8-OHdG in Of significance is the fact that Kuchino et al. demonstrated that 8-OHdG in the DNA template caused miscoding not only of the complementary base but bases on either side of 8-OHdG. It has also been demonstrated that the 8-OHdG content of mitochondria1 DNA is higher than that of nuclear Along this same line, we have noted that the 8-OHdG content of plant chloroplast DNA is higher than nuclear DNA, and that ozone injured plants have a higher content of 8-OHdG in their chloroplast DNA.

Cigarette smoke-induced DNA-damage: Role of hydroquinone and catechol in the formation of the oxidative DNA-adduct, 8-hydroxydeoxyguanosine

This study demonstrates the ability of cigarette smoke condensate to generate hydrogen peroxide and to hydroxylate deoxyguanosine (dG) residues in isolated DNA to 8-hydroxydeoxyguanosine (8-OHdG). Both the formation of hydrogen peroxide and that of 8-OHdG in DNA was significantly decreased when catalase or tyrosinase was added to the smoke condensates, and this also occurred when pure hydroquinone or catechol, two major constitutes in cigarette smoke, was used instead of smoke condensate. Moreover, pure hydroquinone and catechol both caused dose-dependent formation of hydrogen peroxide and 8-OHdG, and there was good correlation between the amounts of hydrogen peroxide and 8-OHdG formed. These findings suggest that (i) hydroquinone and catechol may be responsible for the ability of cigarette smoke to cause 8-OHdG formation in DNA, (ii) this oxidative DNA-damage is due to the action of hydroxyl radicals formed during dissociation of hydrogen peroxide and (iii) the hydrogen peroxide in cigarette smoke is generated via autooxidation of hydroquinone and catechol.

Increased 8-Hydroxyguanine Content of Chloroplast DNA from Ozone-Treated Plants

The mechanism of ozone-mediated plant injury is not known but has been postulated to involve oxygen free radicals. Hydroxyl free radicals react with DNA causing formation of many products, one of which is 8-hydroxyguanine. By using high performance liquid chromatography with electrochemical detection, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) content of a DNA enzymatic digest can be sensitively quantitated. Beans (Phaseolus vulgaris L.) and peas (Pisum sativum L.) were treated with an ozone regime that caused acute injury. Chloroplast DNA was obtained from plants harvested either immediately after ozone treatment or 24 hours later. Ozone-exposed plants in general had nearly two-fold higher levels of 8-OHdG as compared to control plants. In vitro treatment of DNA in buffer solution with ozone did not cause formation of 8-OHdG in DNA, even though ozone did react directly with the macromolecule per se. Exposure of isolated, illuminated chloroplasts to ozone caused nearly a seven-fold increase in the amount of 8-OHdG in the chloroplast DNA as compared to none-ozone-exposed chloroplasts. These results suggest that ozone exposure to plants causes formation of enhanced levels of oxygen free radicals, thus mediating formation of 8-OHdG in chloroplast DNA. The reaction of ozone with DNA per se did not cause formation of 8-OHdG. Therefore, it is the interaction of ozone with plant cells and isolated chloroplasts which mediates oxygen free radical formation.

Methylene blue plus light mediates 8-hydroxyguanine formation in DNA

Exposure to methylene blue (MB) plus light mediates formation of large levels of 8-hydroxyguanine in DNA. The amount of 8-hydroxy-2′-deoxyguanosine (8-OHdG) present in DNA increased as the amount of MB concentration increased throughout the 2 to 200 μ m range studied and was dependent on light exposure. As the time of light exposure increased so did the 8-OHdG content to levels of about 750 8-OHdG/10 5 deoxyguanosine after 15 min of light exposure when MB was at 20 μ m. Even though previous research has demonstrated that hydroxyl free radicals formed from a variety of sources mediate 8-OHdG formation in DNA, inclusion of mannitol, superoxide dismutase, catalase, and desferal in the MB plus light experiments demonstrated that these scavengers of oxygen free radical intermediates or precursors caused either no change or an increase in the 8-OHdG content of DNA exposed to MB plus light. These results appear to rule out the direct role of oxygen free radical intermediates in the primary events involved in the MB plus light mediated formation of 8-OHdG in DNA. Oxygen was essential to cause MB plus light mediated 8-OHdG formation in DNA. It was noted that when the reaction was carried out where the deuterium oxide content had been increased to 100%, the amount of 8-OHdG formed in DNA increased about threefold over that observed when comparable reactions were carried out in pure H 2O. Use of the singlet oxygen scavenger 2,5-dimethylfuran has yielded variable results on the MB plus light mediated formation of 8-OHdG in DNA. The data taken collectively clearly indicate that MB plus light mediates 8-OHdG formation in DNA. The D 2O data and the requirement for oxygen suggest that singlet oxygen may be an intermediate.