Henry Rodriguez

Free radical-induced damage to DNA: mechanisms and measurement.

Free radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells, including DNA. The resulting damage to DNA, which is also called oxidative damage to DNA, is implicated in mutagenesis, carcinogenesis, and aging. Mechanisms of damage involve abstractions and addition reactions by free radicals leading to carbon-centered sugar radicals and OH- or H-adduct radicals of heterocyclic bases. Further reactions of these radicals yield numerous products. Various analytical techniques exist for the measurement of oxidative damage to DNA. Techniques that employ gas chromatography (GC) or liquid chromatography (LC) with mass spectrometry (MS) simultaneously measure numerous products, and provide positive identification and accurate quantification. The measurement of multiple products avoids misleading conclusions that might be drawn from the measurement of a single product, because product levels vary depending on reaction conditions and the redox status of cells. In the past, GC/MS was used for the measurement of modified sugar and bases, and DNA-protein cross-links. Recently, methodologies using LC/tandem MS (LC/MS/MS) and LC/MS techniques were introduced for the measurement of modified nucleosides. Artifacts might occur with the use of any of the measurement techniques. The use of proper experimental conditions might avoid artifactual formation of products in DNA. This article reviews mechanistic aspects of oxidative damage to DNA and recent developments in the measurement of this type of damage using chromatographic and mass spectrometric techniques.

Primary fibroblasts of Cockayne syndrome patients are defective in cellular repair of 8-hydroxyguanine and 8-hydroxyadenine resulting from oxidative stress

Cockayne syndrome (CS) is a genetic human disease with clinical symptoms that include neurodegeneration and premature aging. The disease is caused by the disruption of CSA, CSB, or some types of xeroderma pigmentosum genes. It is known that the CSB protein coded by the CS group B gene plays a role in the repair of 8‐hydroxyguanine (8‐OH‐Gua) in transcription‐coupled and non‐strand discriminating modes. Recently we reported a defect of CSB mutant cells in the repair of another oxidatively modified lesion 8‐hydroxyadenine (8‐OH‐Ade). We show here that primary fibroblasts from CS patients lack the ability to efficiently repair these particular types of oxidatively induced DNA damages. Primary fibroblasts of 11 CS patients and 6 control individuals were exposed to 2 Gy of ionizing radiation to induce oxidative DNA damage and allowed to repair the damage. DNA from cells was analyzed using liquid chromatography/isotope dilution mass spectrometry to measure the biologically important lesions 8‐OH‐Gua and 8‐OH‐Ade. After irradiation, no significant change in background levels of 8‐OH‐Gua and 8‐OH‐Ade was observed in control human cells, indicating their complete cellular repair. In contrast, cells from CS patients accumulated significant amounts of these lesions, providing evidence for a lack of DNA repair. This was supported by the observation that incision of 8‐OH‐Gua‐ or 8‐OH‐Ade‐containing oligodeoxynucleotides by whole cell extracts of fibroblasts from CS patients was deficient compared to control individuals. This study suggests that the cells from CS patients accumulate oxidatively induced specific DNA base lesions, especially after oxidative stress. A deficiency in cellular repair of oxidative DNA damage might contribute to developmental defects in CS patients.

Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.

Identification and quantification of 8,5′-cyclo-2′-deoxy-adenosine in DNA by liquid chromatography/ mass spectrometry

Recent studies suggested that 8,5-cyclo-2-deoxyadenosine may play a role in diseases with defective nucleotide-excision repair. This compound is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the sugar moiety of 2-deoxyadenosine. It is likely to be repaired by nucleotide-excision repair rather than by base-excision repair because of a covalent bond between the sugar and base moieties. We studied the measurement of 8,5-cyclo-2-deoxyadenosine in DNA by liquid chromatography/isotope-dilution mass spectrometry. A methodology was developed for the analysis of 8,5-cyclo-2-deoxyadenosine by liquid chromatography in DNA hydrolyzed to nucleosides by a combination of four enzymes, i.e., DNase I, phosphodiesterases I and II, and alkaline phosphatase. Detection by mass spectrometry was performed using atmospheric pressure ionization-electrospray process in the positive ionization mode. Results showed that liquid chromatography/isotope-dilution mass spectrometry is well suited for identification and quantification of 8,5-cyclo-2-deoxyadenosine in DNA. Both (5R)- and (5S)-diastereomers of 8,5-cyclo-2-deoxyadenosine were detected. The level of sensitivity of liquid chromatography/mass spectrometry with selected-ion monitoring amounted to 2 fmol of this compound on the column. The yield of 8,5-cyclo-2-deoxyadenosine was measured in DNA in aqueous solution exposed to ionizing radiation at doses from 2.5 to 80 Gray. Gas chromatography/mass spectrometry was also used to measure this compound in DNA. Both techniques yielded similar results. The yield of 8,5-cyclo-2-deoxyadenosine was comparable to the yields of some of the other major modified bases in DNA, which were measured using gas chromatography/mass spectrometry. The measurement of 8,5-cyclo-2-deoxyadenosine by liquid chromatography/mass spectrometry may contribute to the understanding of its biological properties and its role in diseases with defective nucleotide-excision repair.

Measurement of 8-Hydroxy-2'-Deoxyadenosine In DNA by Liquid Chromatography/Mass Spectrometry

8-Hydroxyadenine (8-OH-Ade) is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the C-8 position of adenine followed by oxidation. We describe the measurement of the nucleoside form of this compound, 8-hydroxy-2-deoxyadenosine (8-OH-dAdo) in DNA by liquid chromatography/mass spectrometry (LC/MS). The developed methodology enabled the separation by LC of 8-OH-dAdo from intact and modified nucleosides in enzymic hydrolysates of DNA. Measurements by MS were performed using atmospheric pressure ionization-electrospray process. Isotope-dilution MS was applied for quantification using a stable isotope-labeled analog of 8-OH-dAdo. The level of sensitivity of LC/MS with selected-ion monitoring (SIM) for 8-OH-dAdo amounted to approximately 10 femtomol of this compound on the LC column. This level of sensitivity is similar to that previously reported using LC-tandem MS (LC/MS/MS) with multiple-reaction monitoring mode (MRM) (7.5 femtomol). This compound was quantified in DNA at a level of approximately one molecule/10(6) DNA bases using amounts of DNA as low as 5 microg. The results suggested that this lesion may be quantified in DNA at even lower levels, when more DNA is used for analysis. In addition, gas chromatography/isotope-dilution mass spectrometry with SIM (GC/IDMS-SIM) was applied to measure 8-OH-Ade in DNA following its removal from DNA by acidic hydrolysis. The background levels of 8-OH-dAdo and 8-OH-Ade measured by LC/IDMS-SIM and GC/IDMS-SIM, respectively, were nearly identical. In addition, DNA samples, which were exposed to ionizing radiation at different radiation doses, were analyzed by these techniques. Nearly identical results were obtained, indicating that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results. The level of sensitivity of GC/MS-SIM for 8-OH-Ade was also measured and found to be significantly greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS-MRM for 8-OH-dAdo. The results show that the LC/MS technique is well suited for the measurement of 8-OH-dAdo in DNA.

Mapping Oxidative DNA Damage at Nucleotide Level

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the il vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50mM H2O2, or purified genomic DNA was treated with 5 mM H2O2, 100 microM Ascorbate, and 50 microM, 100 microM, or 100 microM of Cu(II), Fe(II), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H2O2-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCRs unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H2O2.

Chlorella Virus Pyrimidine Dimer Glycosylase Excises Ultraviolet Radiation– and Hydroxyl Radical–induced Products 4,6-Diamino-5-formamidopyrimidine and 2,6-Diamino-4-hydroxy-5-formamidopyrimidine from DNA¶

Abstract A DNA glycosylase specific for UV radiation–induced pyrimidine dimers has been identified from the Chlorella virus Paramecium Bursaria Chlorella virus-1. This enzyme (Chlorella virus pyrimidine dimer glycosylase [cv-pdg]) exhibits a 41% amino acid identity with endonuclease V from bacteriophage T4 (T4 pyrimidine dimer glycosylase [T4-pdg]), which is also specific for pyrimidine dimers. However, cv-pdg possesses a higher catalytic efficiency and broader substrate specificity than T4-pdg. The latter excises 4,6-diamino-5-formamidopyrimidine (FapyAde), a UV radiation– and hydroxyl radical–induced monomeric product of adenine in DNA. Using gas chromatography–isotope-dilution mass spectrometry and γ-irradiated DNA, we show in this work that cv-pdg also displays a catalytic activity for excision of FapyAde and, in addition, it excises 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Kinetic data show that FapyAde is a better substrate for cv-pdg than FapyGua. On the other hand, cv-pdg possesses a greater efficiency for the extension of FapyAde than T4-pdg. These two enzymes exhibit different substrate specificities despite substantial structural similarities.

Mapping Oxidative DNA Damage and Mechanisms of Repair

Abstract: We developed a method to map oxidative‐induced DNA damage at the nucleotide level using ligation‐mediated polymerase chain reaction (LMPCR) technology. In vivo and in vitro DNA base modification patterns inflicted by reactive oxygen species (ROS) in the human P53 and PGK1 gene were nearly identical in vitro and in vivo. In human male fibroblasts, these patterns are independent of the transition metal used (Cu (II), Fe(II), or Cr(VI)). Therefore, local probability of H2O2‐mediated DNA base damage is determined primarily by DNA sequence. Moreover, in cells undergoing severe oxidative stress, extranuclear sites contribute metals that enhance nuclear DNA damage. The role of the base excision repair pathway in human cells responsible for the repair of the majority of ROS base damage is also discussed.

Stoichiometric preference in copper-promoted oxidative DNA damage by ochratoxin A.

The ability of the fungal carcinogen, ochratoxin A (OTA, 1), to facilitate copper-promoted oxidative DNA damage has been assessed using supercoiled plasmid DNA (Form I)-agarose gel electrophoresis and gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS-SIM). OTA is shown to promote oxidative cleavage of Form I DNA with optimal cleavage efficiency occurring under excess Cu(II) conditions. As the concentration of OTA was increased and present in excess of Cu(II) the cleavage was less effective. Parallel findings were found for the ability of the OTA-Cu mixture to facilitate oxidative base damage. Yields (lesions per 10(6) DNA bases) of modified bases upon exposure of calf-thymus DNA (CT-DNA) to OTA-H(2)O(2)-Cu(II) were diminished when the OTA:Cu ratio was increased to 5:1. Electrochemical studies carried out in methanol implicate a ligand-centered 2e oxidation of OTA in the presence of excess Cu(II), while product analyses utilizing electrospray mass spectrometry support the intermediacy of the quinone, OTQ (3), in Cu-promoted oxidation of OTA. The implications of these findings with regard to the mutagenicity of OTA are discussed.

Oxidative DNA Damage Biomarkers Used in Tissue Engineered Skin

The process of tissue engineering often involves the mixing of cells with polymers that may cause inflammation to the tissue and thus elevate the level of endogenous free radical production. In order to assure that such composite materials are free of genetic changes that might occur from inflammation during the development phase of the product, our laboratory is responding to the need for test methods used to assess the safety and performance of tissue-engineered materials. Specifically, we are identifying cellular biomarkers that could be used during thein vitrodevelopment phase of tissue-engineered materials to ensure that cells have not undergone any inflammatory response during the development or shipment of the product. Using GC/MS technology, we have screened for a total of five genomic modified base DNA biomarkers in tissue-engineered skin and compared the levels to control cells, neonatal fibroblasts and neonatal keratinocytes. No significant level of damage was detected compared to control cells. LC/MS technology was used in the validation of one of the oxidatively modified DNA lesions. Nearly identical results were obtained when measuring the nucleoside with LC/MS. Biomarker programs such as this can provide the basis for an international reference standard of cellular biomarkers that can aid in the development and safety of tissue engineered medical products.