Measuring oxidative damage to DNA; HPLC and the comet assay compared

Depending on the analytical method employed estimates of background levels of base oxidation in human DNA vary over orders of magnitude. It is now realised that oxidation of guanine in vitro can result in serious overestimation of the nucleoside by HPLC (with electrochemical detection). We have modified procedures of isolation, hydrolysis and storage of DNA with the aim of eliminating this artefact. Vacuum- or freeze-drying, and dialysis, tend to encourage oxidation. We compare results obtained with HPLC and with the comet assay, which employs lesion-specific enzymes to introduce breaks in DNA at sites of oxidative damage. Although estimates of background levels of DNA oxidation using the comet assay are several-fold lower than the estimates by HPLC, both approaches have been used successfully to detect differences between human subjects or population groups that seem to relate to human disease and nutritional factors.     

Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells?

The most commonly measured marker of oxidative DNA damage is 8-oxo-7,8-dihydroguanine (8-oxoGua) or its deoxyribonucleoside (8-oxodGuo). Published estimates of the concentration of 8-oxoGua/8-oxodGuo in DNA of normal human cells vary over a range of three orders of magnitude. Analysis by chromatographic methods (GC-MS, HPLC with electrochemical detection (ECD) or HPLC-MS/MS) is beset by the problem of adventitious oxidation of guanine during sample preparation. An alternative approach, based on the use of the DNA repair enzyme formamidopyrimidine DNA N-glycosylase (FPG) to make breaks in the DNA at sites of the oxidised base, gives much lower values. ESCODD, the European Standards Committee on Oxidative DNA Damage, has been testing the ability of different laboratories using a variety of methods to measure 8-oxoGua in standard samples of 8-oxodGuo, calf thymus DNA, pig liver, oligonucleotides, and HeLa cells, and in lymphocytes isolated from blood of volunteers. HPLC-ECD is capable of measuring 8-oxodGuo induced experimentally in calf thymus DNA or HeLa cells with high accuracy. However, there is no sign of consensus over the background level of this damage, suggesting that, even though standard extraction procedures were used, variable oxidation of Gua is still occurring. GC-MS failed to detect a dose response of induced 8-oxoGua and cannot be regarded as a reliable method for measuring low levels of damage. HPLC-MS/MS as yet has not proved capable of measuring low levels of oxidative DNA damage. FPG-based methods seem to be less prone to the artefact of additional oxidation. Although they can be used quantitatively, they require careful calibration and standardisation if they are to be used in human biomonitoring. The background level of DNA oxidation in normal human cells is likely to be around 0.3-4.2 8-oxoGua per 10(6) Gua. An effort should be made to develop alternative, validated methods for estimating oxidative DNA damage.     

Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues

DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the “gold standard” of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.     

Repair kinetics of trans-4-hydroxynonenal-induced cyclic 1,N2-propanodeoxyguanine DNA adducts by human cell nuclear extracts

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of omega-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N(2)-gamma-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [(32)P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were approximately 1200/10(6) dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of alpha[(32)P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [(32)P]-postlabeling/HPLC method, showing that 50-60% of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 degrees C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3.     

MSAP markers and global cytosine methylation in plants: a literature survey and comparative analysis for a wild‐growing species

Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation‐sensitive amplification polymorphism (MS‐AFLP or MSAP) have been often used to assess methyl‐cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome‐wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome‐wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl‐cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context‐specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation‐based epigenetic processes in nonmodel plants.     

A comparison of trihydroxyindole and HPLC/electrochemical methods for catecholamine measurement in adrenal chromaffin cells

Three commonly used methods for the determination of epinephrine and norepinephrine levels in adrenal medullary tissue were compared. Two variations of the trihydroxyindole procedure, which utilized oxidation at room temperature or 0°C, underestimated levels of total catecholamines in certain standard solutions and were unable to determine correctly their norepinephrine:epinephrine ratios. However, both the variability and the underestimation of the trihydroxyindole procedure carried out at room temperature were more pronounced than that of the trihydroxyindole assay at 0°C. In addition, we tested an isocratic HPLC method utilizing electrochemical detection which separates epinephrine from norepinephrine. The ability of this method to measure correctly total and individual catecholamine levels was superior to either trihydroxyindole procedure, as was its variability. When the three assay methods were used to measure total and individual catecholamine levels in cultured adrenal bovine chromaffin cells, both the trihydroxyindole (0°C) method and the HPLC method yielded values in agreement with those in the literature. However, the HPLC method produced data with lower error estimates. The trihydroxyindole (room temperature) assay was unable to reliably measure levels of epinephrine and norepinephrine in chromaffin cells.These comparisons of catecholamine assays demonstrated that there are circumstances under which the use of each is appropriate. In experiments where the epinephrine:norephinephrine ratio may be changing, the more accurate and precise HPLC assay may be essential, since the trihydroxyindole assays underestimate total catecholamines to varying degrees depending on this ratio. However, the HPLC method suffers from a requirement for technical sophistication for routine use. Therefore, in some laboratories and for repetitive measurement of many samples, the trihydroxyindole assay has a distinct advantage due to its easy utilization and ubiquitous materials. However, the superior results obtained with the trihydroxyindole (0°C) assay over the trihydroxyindole (room temperature) assay emphasizes the need to evaluate the trihydroxyindole procedure for the required purpose, especially if differential oxidation is used for estimation of individual catecholamine levels.     

Measurement of DNA oxidation in human cells by chromatographic and enzymic methods

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve problems in the measurement of DNA oxidation that have resulted in varying estimates of the extent of this damage in humans. HeLa cells, sent to members for analysis, were either untreated, or treated with light in the presence of a photosensitizer to induce different amounts of 8-oxo-7,8-dihydroguanine (8-oxoGua) in DNA. Laboratories employing HPLC with electrochemical detection were able to measure the induced damage with similar efficiency; dose response gradients for seven of the eight sets of results were almost identical. GC-MS and HPLC-MS/MS, employed in three laboratories, did not convincingly detect the dose response. An alternative approach to measuring base oxidation employs the enzyme formamidopyrimidine DNA N-glycosylase (FPG) to convert 8-oxoGua to strand breaks, which are then measured by alkaline unwinding, alkaline elution, or the comet assay. Ten laboratories used this approach; five were able to detect the dose response in cells treated with photosensitizer plus light (at lower doses than for chromatographic methods, because the enzymic methods are more sensitive and less prone to spurious oxidation). Median values for 8-oxoGua (or FPG-sensitive sites) in untreated cells were 4.01 per 10(6) guanines for chromatographic methods, and 0.53 per 10(6) guanines for techniques based on FPG.     

Analysis of oxidative DNA damage and HPRT mutations in humans after hyperbaric oxygen treatment

DNA damage induced by reactive oxygen species (ROS) seems to play an important role in the induction of mutations and cancer. We have recently shown that hyperbaric oxygen (HBO) treatment of volunteers (i.e., exposure to 100% oxygen at a pressure of 2.5 ATA) induces DNA damage detected in leukocytes with the comet assay. Using formamidopyrimidine-DNA glycosylase (FPG protein) we provided indirect evidence for the induction of oxidative DNA base damage. We now comparatively evaluated FPG-sensitive sites with the comet assay and 7,8-dihydro-8-oxo-deoxyguanosine (8-OHdG) with HPLC analysis after a single HBO. As 8-OHguanine (8-OHgua) is one of the major DNA modifications induced by ROS and a pre-mutagenic lesion, we looked for HBO-induced mutations at the HPRT locus with the T cell cloning test. We also determined the genotypes for glutathione transferases (GST) and tested a possible influence of the GSTM1 and GSTT1 genotypes on the sensitivity of subjects against HBO-induced genotoxicity. Our results indicate that despite a clear induction of FPG-sensitive sites no increased levels of 8-OHdG and no induction of HPRT mutations was detected in lymphocytes after HBO. Furthermore, the DNA effects in the comet assay and the mutant frequencies in the HPRT test seem to be unrelated to the GST genotypes of the test subjects.     

Antioxidants; not the only reason to eat fruit and vegetables

There is a considerable body of evidence, from epidemiology, that fruits and vegetables help to prevent cancer. This is commonly attributed to the antioxidants that they contain, which are supposed to decrease cancer risk by protecting DNA against oxidative damage. It is certainly true that individual antioxidants, such as vitamin C, vitamin E (α-tocopherol) and carotenoids can decrease oxidative damage to DNA, in in vitro model systems, in cultured cells, and in humans given supplements. Real foods, including fried onions, carrot juice, soya milk and kiwifruit also decrease DNA oxidation in human lymphocytes tested ex vivo. The significance of these effects has to be examined in the light of revised estimates of the level of background damage in normal cells; this is hundreds of times less than has been suggested in the past. Other effects of phytochemicals found in these foods are potentially important, such as the enhancement or inhibition of phase I and phase II metabolising enzymes, and modulation of DNA repair. This revised version was published online in June 2006 with corrections to the Cover Date.     

Measuring oxidative damage to DNA and its repair with the comet assay

Background

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases.

Scope of review

With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells.

Major conclusions

There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay.

General significance

In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

What is the significance of increases in background levels of carcinogen-derived protein and DNA adducts? Some considerations for incremental risk assessment

Improvements in analytical methodology have led to the detection and quantification of 'background' levels of a number of DNA and protein adducts. Many of these adducts are derived from 'low molecular weight' reactive species which may be generated during normal physiological processes, metabolic pathways or inflammatory processes. The adducts have been detected using gas chromatography-mass spectrometry, HPLC in combination with various detection systems, 32P-postlabelling and immunoassay methods. The reliability and accuracy of many widely used methods for adduct measurements are discussed with reference to several examples where human data is available, namely 4-aminobiphenyl, malondialdehyde, methylating agents, ethylene oxide and hydroxyl radical damage. The accurate and specific quantitation of 'background' levels of damage is essential if reliable estimates of increases in risk associated with incremental increases in exposure to exogenous agents are to be calculated. In experimental studies using low dose exposures to carcinogens, such as N-nitrosodimethylamine, adduct levels in liver correlate closely with tumour incidence. In all likelihood, such relationships need to be established for each exposure and, in order to be relevant to human risk assessment, need to take into account factors such as DNA repair and mutagenic efficiency. Finally, in order to estimate the increase in cancer attributable to a given level of external exposure, it is clearly important to establish background levels of corresponding DNA damage so that the scale of the incremental increase can be calculated.     

Nephrotoxicity of Penicillium aurantiogriseum,a possible factor in the aetiology of Balkan Endemic Nephropathy

Water-soluble components of a nephrotoxic isolate of Penicillium aurantiogriseum have been fractionated by sequential ion-exchange, size-exclusion gel filtration, reverse-phase silica chromatography and HPLC. Nephrotoxicity in the rat was confined to a size-exclusion fraction approximating to 1500 daltons, which also inhibited DNA synthesis in cultured kidney cells. The more sensitive in vitro assay allowed toxicity to be followed to a sub-fraction from gradient-elution HPLC which in further HPLC resolved into a small group of glycopeptides. Recent Yugoslavian P. aurantiogriseum isolates, from a village in which the idiopathic human disease Balkan Nephropathy is hyperendemic, elicited a similar nephropathology and were acutely cytotoxic, reinforcing a need to regard this novel Penicillium nephrotoxin as a potential factor in human nephropathy.     

Measurement of oxidatively generated base damage to nucleic acids in cells: facts and artifacts

The search for DNA biomarkers of oxidative stress has been hampered for several decades by the lack of relevant information on base oxidation products and the challenging issue of measuring low amounts of lesions, typically a few modifications within the range 10⁶?C10⁸ normal bases. In addition and this was ignored for a long time, there is a risk of artifactual oxidation of overwhelming nucleobases during DNA extraction and subsequent workup that has led to overestimation of some base damage up to 2?C3 orders of magnitude. The main aim of the survey is to critically review the available methods that have been developed for measuring oxidatively generated base damage in nuclear and mitochondrial DNA. Among the chromatographic methods, high-performance liquid chromatography associated with tandem mass spectrometry (HPLC?CMS/MS) is the most accurate and versatile approach whereas HPLC?Celectrochemical detection (ECD) is restricted to electrochemically active modifications. These methods allow measuring several single oxidized pyrimidine and purine bases, tandem base lesions and interstrand DNA cross-links in nuclear DNA. As complementary analytical tools, enzymatic methods that associate DNA repair enzymes with either the alkaline comet assay or the alkaline elution technique are suitable for assessing low variations in the level of different classes of oxidatively generated DNA lesions. Most of the immunoassays suffer from a lack of specificity due to the occurrence of cross-reactivity with overwhelming normal bases. One major exception concerns the immunodetection of 5-hydroxymethylcytosine, produced in a relatively high yield as an epigenetic DNA modification. HPLC?CMS/MS is now recognized as the gold standard for measuring oxidized bases and nucleosides in human fluids such as urine, saliva, and plasma.     

A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater RNA than DNA oxidation in rat liver after doxorubicin administration

We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0 degrees C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro.     

An in vitro nucleoside analog screening method for cancer gene therapy

Suicide genes that sensitize cells to drugs that are normally nontoxic at therapeutic levels represent an important approach in human gene therapy research. We have developed an in vitro screening assay to assess the modulation of nucleoside analogs after transfection of a vector expressing the herpes simplex virus thymidine kinase gene (HSV-TK). The thymidine kinase gene enhances nucleoside phosphorylation to nucleotides that kill cells by blocking DNA elongation. Cells lines used are 3T3-NIH fibroblasts (parental cells) and 3T3-TKc3 (HSV-TK gene-transfected 3T3-NIH). Two types of analysis are performed: a cytotoxicity assay, the neutral red uptake assay to assess the IC₅₀ on the two cell lines, and an HPLC analysis coupled to a radiochemical flow detector to evaluate metabolic profiles after incubation of cells with tritiated analogs. Results show that cells expressing the HSV-TK gene are more sensitive than the parent cells to the effect of acyclovir or ganciclovir, the reference purine analog drugs, and also to the effect of pyrimidine analogs, bromodeoxyuridine, bromovinyldeoxyuridine, and ethyldeoxyuridine. Promising nucleoside analogs for gene therapy that can be achieved by HSV-TK could be evaluated using this model.Abbreviations ACV acyclovir - ACV-MP acyclovir monophosphate - ACV-DP acyclovir diphosphate - ACV-TP acyclovir triphosphate - BDU bromodeoxyuridine - BVDU bromovinyldeoxyuridine - EDU ethyldeoxyuridine - FDU fluorodeoxyuridine - GCV ganciclovir - HSV-TK herpes simplex virus thymidine kinase gene - IDU iododeoxyuridine - NA nucleoside analog     

Assay of ribonucleotide reductase activity in intact permeabilized hamster cells: an evaluation

An intact cell assay system, based on Tween-80 permeabilization can be used to investigate ribonucleotide reductase activity in a variety of mammalian cell lines. An important consideration in the use of intact cells is the presence of other nucleotide metabolizing activities. The influence of these activities on estimates of pyrimidine (CDP) and purine (ADP) reductase in permeabilized hamster cells has been examined. Studies on the incorporation of label from CDP and ADP into RNA indicated that a very small proportion of the reductase substrates was eventually incorporated into RNA during routine enzyme assays, and would have no detectable effect on activity estimates. The possibility that the products of the reaction (dCDP and dADP) were eventually phosphorylated and incorporated into DNA was also examined, and it was found that proper permeabilization of the cells eliminated or greatly reduced loss of deoxyribonucleotides to DNA. An analysis by HPLC of nucleotides present during CDP and ADP reductase reactions showed that various kinases and phosphatases were active in permeabilized cells, as all levels of phosphorylation of nucleotide substrates and allosteric effectors were detected. The base composition of the nucleotides added to the assay systems were not altered. Although movement of phosphates occurred during the assay, the concentrations of substrates quickly reached equilibrium (within 1 min) with their respective nucleosides and nucleotides, resulting in a relatively constant although reduced concentration of CDP or ADP substrates during the 20-min assay. Similarly the levels of allosteric effectors, ATP for pyrimidine and dGTP for purine reductase activities, declined within the first minute of the assays and quickly reached an equilibrium with their respective adenine or guanine containing nucleotides during most of the reaction time. Although useful approximations of intracellular reductase activity can be obtained without correcting for modified nucleotide concentrations, precise determinations can be calculated when these alterations are taken into consideration. For example, estimates of intracellular K_m values for CDP closely resembled those reported with highly purified mammalian enzyme preparations in other studies. Clearly, the intact cell assay system provides worthwhile information about mammalian ribonucleotide reductase in its physiologically relevant environment.     

Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses.

Estimates of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA vary at least one order of magnitude using different quantitative methods or even the same method. Our hypothesis is that an incomplete DNA hydrolysis to nucleosides by the conventional nuclease P1 (NP1) and alkaline phosphatase (AP) digestion system plays an important role in contributing to the variability of measurements using HPLC coupled with UV and electrochemical (EC) detection. We show here that factors, such as the amount of DNA, choice of enzymes, their activities, and incubation time, can affect DNA digestion and, thus, cause variability in 8-oxo-dG levels. The addition of DNase I and phosphodiesterases I and II to the NP1 + AP system improves the DNA digestion by completely releasing normal nucleosides and 8-oxo-dG, thereby reducing the interday variations of 8-oxo-dG levels. Diethylenetriamine pentaacetic acid (DTPA), an iron chelator, prevented background increases of 8-oxo-dG during DNA digestion, as well as during the waiting period in the autosampler when a batch of DNA samples is analyzed by HPLC. After optimization of the DNA digestion conditions, the interday variability of 8-oxo-dG measurements using commercially available salmon testes DNA (ST DNA) were 26% over a period of 2 years. Under these optimal conditions, our laboratory variability may contribute as little as 13% to the overall variability as shown by assessment of oxidative DNA damage in a population of smokers. Based on our results, we believe that the modified DNA digestion conditions will provide much more accurate 8-oxo-dG determinations and, thus, more reliable estimates of cancer risk.     

Oxidative DNA damage background estimated by a system model of base excision repair

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level, based on measuring 8-oxoguanine lesions as a biomarker, have led to estimates that vary over three to four orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our findings show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.     

Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA

The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 × 105 pmol dG, while antibody IF11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6-and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/105 dG using 100μg DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well (r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers (p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.