The chemistry of DNA damage from nitric oxide and peroxynitrite

Nitric oxide is a key participant in many physiological pathways; however, its reactivity gives it the potential to cause considerable damage to cells and tissues in its vicinity. Nitric oxide can react with DNA via multiple pathways. Once produced, subsequent conversion of nitric oxide to nitrous anhydride and/or peroxynitrite can lead to the nitrosative deamination of DNA bases such as guanine and cytosine. Complex oxidation chemistry can also occur causing DNA base and sugar oxidative modifications. This review describes the different mechanisms by which nitric oxide can damage DNA. First, the physiological significance of nitric oxide is discussed. Details of nitric oxide and peroxynitrite chemistry are then given. The final two sections outline the mechanisms underlying DNA damage induced by nitric oxide and peroxynitrite.     

Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage.

Diverse flavonoid compounds are widely distributed in angiosperm families. Flavonoids absorb radiation in the ultraviolet (UV) region of the spectrum, and it has been proposed that these compounds function as UV filters. We demonstrate that the DNA in Zea mays plants that contain flavonoids (primarily anthocyanins) is protected from the induction of damage caused by UV radiation relative to the DNA in plants that are genetically deficient in these compounds. DNA damage was measured with a sensitive and simple assay using individual monoclonal antibodies, one specific for cyclobutane pyrimidine dimer damage and the other specific for pyrimidine(6,4)pyrimidone damage.     

DNA damage caused by ionizing radiation.

A survey is given of continuous-time Markov chain models for ionizing radiation damage to the genome of mammalian cells. In such models, immediate damage induced by the radiation is regarded as a batch-Poisson arrival process of DNA double-strand breaks (DSBs). Enzymatic modification of the immediate damage is modeled as a Markov process similar to those described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete-exchange model. The model postulates that, after being induced by radiation, DSBs subsequently either undergo enzymatically mediated restitution (repair) or participate pairwise in chromosome exchanges. Some of the exchanges make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. Methods for analyzing the Markov chains include using an approximate model for expected values, the discrete-time Markov chain embedded at transitions, partial differential equations for generating functions, normal perturbation theory, singular perturbation theory with scaling, numerical computations, and certain matrix methods that combine Perron-Frobenius theory with variational estimates. Applications to experimental results on expected values, variances, and statistical distributions of DNA lesions are briefly outlined. Continuous-time Markov chains are the most systematic of those radiation damage models that treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g., ignore diffusion limitations). They contain virtually all other relevant homogeneous models and semiempirical summaries as special cases, limiting cases, or approximations. However, the Markov models do not seem to be well suited for studying spatial dependence of DSB interactions, which is known to be important in some situations.     

Immunoanalysis of ultraviolet radiation induced DNA damage and repair within specific gene segments of plasmid DNA.

The region-specific heterogeneity of repairing DNA damage has been established in several biological systems. A flexible and sensitive approach, based upon DNA damage specific antibodies, is described to monitor the repair of specific lesions within discrete genomic segments. Membrane transblotted DNA restriction fragments are immunoanalyzed for the initial formation and repair of 254 nm radiation induced pyrimidine dimers. Sensitivity of dimer immunodetection increases proportional to fragment concentration and size. Antibody binding was detectable in a 0.5 kb fragment obtained from approx. 100 ng of restriction digested phage lambda DNA irradiated with 50 J m-2. Dimers within larger fragments (greater than 5 kb) could be detected at ultraviolet doses as low as 1 to 2 J m-2. To determine the occurrence of preferential repair in prokaryotic cells, damage was assessed in DNA sequences established in various Escherichia coli strains. In vivo repair of 8.9 kb vector and 6.4 and 3.2 kb gene inserts occurred with an approximate t1/2 of 45 min in UvrABC excision repair-proficient strains. Antibody binding sites were retained by DNA within repair-deficient strains. Compared to UvrABC, the repair of DNA fragments mediated by T4 endonuclease V was rapid and complete within 30 min of cellular irradiation. The efficient repair in DenV+ strain is attributable to a highly processive repair enzyme rather than to selective repair of actively replicating target genes. The results demonstrate the exceptional ability of antibodies specific for altered biomolecular lesions to map damage and repair in gene segments episomally established within cells.     

Efficient DNA interstrand crosslinking by 6-thioguanine and UVA radiation

Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.     

Antioxidant and pro-oxidant actions of flavonoids: effects on DNA damage induced by nitric oxide, peroxynitrite and nitroxyl anion

Antioxidant and pro-oxidant activities of flavonoids have been reported. We have studied the effects of 18 flavonoids and related phenolic compounds on DNA damage induced by nitric oxide (NO), peroxynitrite, and nitroxyl anion (NO⁻). Similarly to our previous findings with catecholamines and catechol-estrogens, DNA single-strand breakage was induced synergistically when pBR322 plasmid was incubated in the presence of an NO-releasing compound (diethylamine NONOate) and a flavonoid having an ortho-trihydroxyl group in either the B ring (e.g., epigallocatechin gallate) or the A ring (e.g., quercetagetin). Either NO or any of the above flavonoids alone did not induce strand breakage significantly. However, most of the tested flavonoids inhibited the peroxynitrite-mediated formation of 8-nitroguanine in calf-thymus DNA, measured by a new HPLC-electrochemical detection method, as well as the peroxynitrite-induced strand breakage. NO⁻ generated from Angeli’s salt caused DNA strand breakage, which was also inhibited by flavonoids but at only high concentrations. On the basis of these findings, we propose that NO⁻ and/or peroxynitrite could be responsible for DNA strand breakage induced by NO and a flavonoid having an ortho-trihydroxyl group. Our results indicate that flavonoids have antioxidant properties, but some act as pro-oxidants in the presence of NO.     

UVA1 radiation inhibits calcineurin through oxidative damage mediated by photosensitization

The protein phosphatase calcineurin has been gradually revealing itself as the central controller of our immune response, although it is involved in a wide array of signaling pathways related to cellular development and cell cycle progression. As such, calcineurin is an attractive, yet delicate, therapeutic target for the prevention of allograft rejection and treatment of several inflammatory skin conditions. However, calcineurin activity is not only sensitive to immunosuppressants such as cyclosporin A and tacrolimus, but also subject to modulation by reactive oxygen species. We have recently shown, both in vivo and in vitro, that UVA1 radiation suppresses calcineurin activity. In this paper, we present evidence that this activity loss is due to singlet oxygen and superoxide generated by photosensitization and show that a closely related phosphatase, PP2A, is not affected. Furthermore, a survey of this damage reveals oxidation of several Met and Cys residues as well as an overall conformational change. These findings provide a mechanistic basis for the hypothesis that UVA1 and calcineurin inhibitors both affect the same signal transduction pathway in skin.     

DNA damage and nitric oxide production in mice following infection with L. chagasi

Leishmania chagasi, which causes visceral leishmaniasis in South America, is an obligate intracellular protozoan. Production of nitric oxide by macrophages during the inflammatory response is one of the main microbicidal mechanisms against this parasite. The goal of this study was to evaluate whether L. chagasi infection causes DNA damage in peripheral blood and spleen cells of Balb/c mice and whether such damage may be related to NO production. Balb/c mice were either infected with L. chagasi or maintained as controls. The single-cell gel electrophoresis (comet) assay was used to measure DNA damage in peripheral blood and spleen cells, and the Griess reaction was used to measure NO production in the spleen. L. chagasi infection induced DNA damage in peripheral blood and spleen cells of infected mice. Macrophages from the control group, challenged with L. chagasi, showed significantly (p<0.05) greater NO production, compared to non-challenged cells. Treatment of spleen cells with N(G)-monomethyl-l-arginine (LNMMA) caused a significant reduction of NO production and DNA damage (p<0.05). Our results indicate that L. chagasi induces DNA damage in the peripheral blood and spleen cells and that NO not only causes killing of the parasite but also induces DNA damage in adjacent cells.     

The role of nitric oxide in lung innate immunity: Modulation by surfactant protein-A

Surfactant protein A (SP-A) and alveolar macrophages are essential components of lung innate immunity. Alveolar macrophages phagocytose and kill pathogens by the production of reactive oxygen and nitrogen species. In particular, peroxynitrite, the reaction product of superoxide and nitric oxide, appears to have potent antimicrobial effects. SP-A stimulates alveolar macrophages to phagocytose and kill pathogens and is important in host defense. However, SP-A has diverse effects on both innate and adaptive immunity, and may stimulate or inhibit immune function. SP-A appears to mediate toxic or protective effects depending on the immune status of the lung. In contrast to mouse or rat cells, it has been difficult to demonstrate nitric oxide production by human macrophages. We have recently demonstrated that human macrophages produce nitric oxide and use it to kill Klebsiella pneumoniae. SP-A either stimulates or inhibits this process, depending on the activation state of the macrophage. Given its diverse effects on immune function, SP-A may prove to be an effective therapy for both infectious and inflammatory diseases of the lung.     

The role of nitric oxide in lung innate immunity: modulation by surfactant protein-A

Surfactant protein A (SP-A) and alveolar macrophages are essential components of lung innate immunity. Alveolar macrophages phagocytose and kill pathogens by the production of reactive oxygen and nitrogen species. In particular, peroxynitrite, the reaction product of superoxide and nitric oxide, appears to have potent antimicrobial effects. SP-A stimulates alveolar macrophages to phagocytose and kill pathogens and is important in host defense. However, SP-A has diverse effects on both innate and adaptive immunity, and may stimulate or inhibit immune function. SP-A appears to mediate toxic or protective effects depending on the immune status of the lung. In contrast to mouse or rat cells, it has been difficult to demonstrate nitric oxide production by human macrophages. We have recently demonstrated that human macrophages produce nitric oxide and use it to kill Klebsiella pneumoniae. SP-A either stimulates or inhibits this process, depending on the activation state of the macrophage. Given its diverse effects on immune function, SP-A may prove to be an effective therapy for both infectious and inflammatory diseases of the lung.     

Protein, lipid, and DNA radicals to measure skin UVA damage and modulation by melanin

Afro-Caribbeans have a lower incidence of skin cancer than Caucasians, but the effectiveness of melanin as a photoprotective pigment is debated. We investigated the UVA and solar irradiation of ex vivo human skin and DMPO using electron spin resonance spectroscopy, to determine whether pigmented skin is protected by melanin against free radical damage. Initial ascorbate radicals in Caucasian skin were superseded by lipid and/or protein radical adducts with isotropic (a(H)=1.8 mT) and anisotropic spectra comparable to spectra in irradiated pig fat (a(H)=1.9 mT) and BSA. DNA carbon-centered radical adducts (a(H)=2.3 mT) and a broad singlet were detected in genomic DNA/melanin but were not distinguishable in irradiated Caucasian skin. Protein and lipid radicals (n=6 in Caucasian skin) were minimal in Afro-Caribbean skin (n=4) and intermediate skin pigmentations were variable (n=3). In irradiated Afro-Caribbean skin a shoulder to the melanin radical (also in UVA-irradiated pigmented melanoma cells and genomic DNA/melanin and intrinsic to pheomelanin) was detected. In this sample group, protein (but not lipid) radical adducts decreased directly with pigmentation. ESR/spin trapping methodology has potential for screening skin susceptibility to aging and cancer-related radical damage and for measuring protection afforded by melanin, sunscreens, and antiaging creams.     

DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: Evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide

Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41°C and 45°C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24h), or acute (1h) exposure to each treatment followed by incubation at 37°C over a period of 24h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF.     

Enzyme-independent nitric oxide formation during UVA challenge of human skin: characterization, molecular sources, and mechanisms

Many of the local UV-induced responses including erythema and edema formation, inflammation, premature aging, and immune suppression can be influenced by nitric oxide synthase (NOS)-produced NO which is known to play a pivotal role in cutaneous physiology. Besides NOS-mediated NO production, UV radiation might trigger an enzyme-independent NO formation in human skin by a mechanism comprising the decomposition of photo-reactive nitrogen oxides. Therefore, we have examined the chemical-storage forms of potential NO-generating agents, the mechanisms and kinetics of their decomposition, and their biological relevance. In normal human skin specimens we find nitrite and S-nitrosothiols (RSNO) at concentrations 25- or 360-fold higher than those found in plasma of healthy volunteers. UVA irradiation of human skin leads to high-output formation of bioactive NO due to photo-decomposition of RSNO and nitrite which represents the primary basis for NO formation during UVA exposure. Interestingly, reduced thiols strongly augment photo-decomposition of nitrite and are essential for maximal NO release. The enzyme-independent NO formation found in human skin opens a completely new field in cutaneous physiology and will extend our understanding of mechanisms contributing to skin aging, inflammation, and cancerogenesis.     

Bleomycin-iron complexes and DNA radiation damage.

Radiation in the form of high-energy electrons dose-dependently activates bleomycin-Fe3+ in oxygen-containing DNA solutions. This activation causes a DNA fragmentation and a release of oxidative degradation products from the DNA. During irradiation, bleomycin-chelated Fe3+ is reduced to Fe2+. The kinetics of DNA base propenal-formation (measured by reaction with thiobarbituric acid) and iron-reduction (measured by bathophenanthroline chelation) are similar, with a yield of 1 mol base propenal/6 mol Fe3+ reduced. The activation of bleomycin-Fe3+ by irradiation could be instrumental in the synergistic action of radiotherapy and bleomycin observed on simultaneous administration in vivo.     

Radiosensitization by nitric oxide at low radiation doses

Nitric oxide was shown to radiosensitize anoxic V79 and CHO hamster cells and MCF7 and UT-SCC-14 human cells, measuring clonogenic survival and/or DNA damage in vitro at low radiation doses (0.1-5 Gy). Radiosensitization was easily detected after 2 Gy in anoxic V79 cells exposed to 40 ppm ( approximately 70 nM) nitric oxide, indicating that nitric oxide is a significantly more efficient radiosensitizer than oxygen. The yield of double-strand breaks (as gamma-H2AX foci) in V79 and MCF7 cells was doubled by irradiation in 1% v/v nitric oxide/N(2), and there was a longer repair time in cells irradiated in nitric oxide than in air or anoxia; single-strand breaks ("comet" assay) also appeared to be enhanced. Potent radiosensitization by nitric oxide is consistent with near diffusion-controlled reaction of nitric oxide with purine and pyrimidine radicals observed by pulse radiolysis, with nitric oxide reacting two to three times faster than oxygen with the 5-hydroxy-uracil-6-yl radical. Stable NO/base adducts were formed with uracil radicals. Effects on the radiosensitivity of cells exposed to as low as 40 ppm v/v nitric oxide after doses of 1-2 Gy suggest that variations in radiosensitivity in individual patients after radiotherapy might include a component reflecting differing levels of nitric oxide in tumors.     

Sodium deoxycholate causes nitric oxide mediated DNA damage in oesophageal cells

Patients with chronic gastro-oesophageal reflux disease experience the reflux of acid and bile into the distal oesophagus. The secondary bile salt sodium deoxycholate (NDC) is implicated in the induction of mucosal injury during reflux episodes. This study hypothesized that NDC damages DNA in oesophageal cells by an oxidative mechanism. In the oesophageal cell line HET1-A, increased production of nitric oxide (NO) was measured in NDC-treated cells. Protection from DNA strand breaks induced by NDC (10 µm) was observed in cells coincubated with the nitric oxide scavenger C-PTIO (p<0.012) or pre-incubated with the NO synthase inhibitor L-NAME (p<0.009) or the NFκB inhibitor, TPCK (p<0.036). Collectively these data implicate the involvement of NFκB and nitric oxide synthase in the DNA damage induced by NDC in oesophageal cells. In conclusion, NDC-driven NO production may play an important role in inducing DNA damage during episodes of gastro-oesophageal reflux and thereby contribute to reflux-related carcinogenesis.