Methodological considerations and factors affecting 8-hydroxy-2′-deoxyguanosine analysis

Oxidative stress is related to a number of diseases due to the formation of reactive oxygen species (ROS). There are also several substances found in the occupational environment or as life style related situations that generates ROS. A stable biomarker for oxidative stress on DNA is 8-hydroxy-2′-deoxyguanosine (8-OH-dG).

A potential problem in the work-up and analysis of 8-OH-dG is oxidation of dG with false high levels as a result of analysis. This paper summarizes and discusses some of the critical moments in terms of auto-oxidation. The removal of transition metals, low temperatures, absence of isotopes (or 2′-deoxyguanosine) and incubation times are all important factors. Removal of oxygen is complicated while the problem is reduced if a nitroxide (TEMPO) is added during work-up. Certain reducing agents and enzymes could be critical if added during work-up.

The application of the ³²P-HPLC method to analyze 8-OH-dG is discussed. The ³²P-HPLC method is suitable for 8-OH-dG analysis and avoids several factors that oxidizes dG by removal of dG before addition of isotopes. Factors of crucial importance (columns, eluents, gradients and detection of ³²P) for the analysis of 8-OH-dG are commented upon and certain recommendations are made to make it possible to apply the ³²P-HPLC methodology for this type of analysis.     

Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine and 5-(hydroxymethyl) uracil in Smokers

Cigarette smoke is known to generate free radicals by various mechanisms. In this study involving 30 non-smokers and 30 smokers, we show that urinary excretion of 5-(hydroxymethyl) uracil (HMUra) was not different in the two groups (6.54±2.07 vs. 6.70±1.68 nmol/mmol creatinine). In contrast, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) excretion increased by 16% (1.16±0.35 vs. 1.35±0.50 nmol/mmol creatinine, p=0.039). Results concerning 8-oxo-dGuo are in agreement with those of previous studies. We observed significant multiple correlations between HMUra and creatinine (r_p=0.44), BMI (r_p=-0.27) and nicotine derivatives (r_p=0.26). Multiple correlation analysis showed relations between 8-oxo-dGuo on the one hand, and: creatinine (r_p=0.36), nicotine derivatives (r_p=0.29), BMI (r_p=-0.24) on the other.     

Evaluation of a Multi-parameter Biomarker Set for Oxidative Damage in Man: Increased Urinary Excretion of Lipid,Protein and DNA Oxidation Products after One Hour of Exercise

The objective of the present study was to evaluate a comprehensive set of urinary biomarkers for oxidative damage to lipids, proteins and DNA, in man. Eighteen moderately trained males (mean age 24.6±0.7) exercised 60?min at 70% of maximal O₂ uptake on a cycle ergometer. Urine fractions for 12?h were collected 1 day before, and for 3 consecutive days after exercise.

As biomarkers of lipid peroxidation, 8 aldehydes (i.e. propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal and malondialdehyde—MDA)and acetone were analyzed in urines by gas chromatography with electron capture detection (GC-ECD). As a biomarker of protein oxidation, o,o′-dityrosine was analyzed in urine samples by a recently developed isotope dilution HPLC-atmospheric pressure chemical ionization (APCI)-tandem-mass spectrometry (HPLC-APCI-MS/MS) methodology. As a biomarker of oxidative DNA damage, urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG) was measured by an ELISA method.

On the day of exercise, significant increases were observed in urinary excretions of acetone (?p<0.025, n=18) and butanal (?p<0.01, n=18) in the 12?h daytime fractions compared to the daytime fraction before exercise. The urinary acetone excretion was also significantly (?p<0.05) increased on the 1st day after exercise. Octanal and nonanal were increased in the daytime urine fraction on the 2nd day after exercise. However, these increases were of borderline significance (?p=0.09 and p=0.07, respectively).

Significantly elevated urinary o,o′-dityrosine amounts were observed in the daytime fraction on the day of exercise (?p<0.025) and on the 1st day after exercise (?p=0.07) compared to the before exercise daytime fraction.

Excretion of urinary 8-OHdG was statistically significantly increased in the daytime fractions on the day of exercise (?p=0.07) and on the 1st day after exercise (?p<0.025) compared to before exercise daytime fraction.

Increases in urinary excretions of acetone, propanal, pentanal, MDA and 8-OHdG significantly correlated with training status (hours of exercise/week) of the volunteers, while o,o′-dityrosine did not.

To our knowledge, the present study is the first to evaluate a multi-parameter non-invasive biomarker set for damage to three main cellular targets of ROS. It shows that 1?h of exercise may already induce oxidative damage in moderately trained individuals and that the chosen urinary biomarkers are sensitive enough to monitor such damage.     

Nitrative and oxidative DNA damage caused by K-ras mutation in mice

Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-κB, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

Profiling base excision repair glycosylases with synthesized transition state analogs

Two base excision repair glycosylase (BER) transition state (TS) mimics, (3R,4R)-1-benzyl (hydroxymethyl) pyrrolidin-3-ol (1NBn) and (3R,4R)-(hydroxymethyl) pyrrolidin-3-ol (1N), were synthesized using an improved method. Several BER glycosylases that repair oxidized DNA bases, bacterial formamidopyrimdine glycosylase (Fpg), human OG glycosylase (hOGG1) and human Nei-like glycosylase 1 (hNEIL1) exhibit exceptionally high affinity (K_d∼pM) with DNA duplexes containing the 1NBn and 1N nucleotide. Notably, comparison of the K_d values of both TS mimics relative to an abasic analog (THF) in duplex contexts paired opposite C or A suggest that these DNA repair enzymes use distinctly different mechanisms for damaged base recognition and catalysis despite having overlapping substrate specificities.     

Enhancement of antibody production by the addition of Coenzyme-Q<Subscript>10</Subscript>

Recently, there has been a growing demand for therapeutic monoclonal antibodies (MAbs) on the global market. Because therapeutic MAbs are more expensive than low-molecular-weight drugs, there have been strong demands to lower their production costs. Therefore, efficient methods to minimize the cost of goods are currently active areas of research. We have screened several enhancers of specific MAb production rate (SPR) using a YB2/0 cell line and found that coenzyme-Q₁₀ (CoQ₁₀) is a promising enhancer candidate. CoQ₁₀ is well known as a strong antioxidant in the respiratory chain and is used for healthcare and other applications. Because CoQ₁₀ is negligibly water soluble, most studies are limited by low concentrations. We added CoQ₁₀ to a culture medium as dispersed nanoparticles at several concentrations (Q-Media) and conducted a fed-batch culture. Although the Q-Media had no effect on cumulative viable cell density, it enhanced SPR by 29%. In addition, the Q-Media had no effect on the binding or cytotoxic activity of MAbs. Q-Media also enhanced SPR with CHO and NS0 cell lines by 30%. These observations suggest that CoQ₁₀ serves as a powerful aid in the production of MAbs by enhancing SPR without changing the characteristics of cell growth, or adversely affecting the quality or biological activity of MAbs.     

Repair efficiency of (5′S)-8,5′-cyclo-2′-deoxyguanosine and (5′S)-8,5′-cyclo-2′-deoxyadenosine depends on the complementary base

5′-R and 5′-S diastereoisomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24–32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N²-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N²-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.     

Inhibition of tubulin polymerization with ribose-modified analogs of GDP and GTP reduced inhibition with microtubule-associated proteins and magnesium

Inhibitory effects of ribose-modified GDP and GTP analogs on tubulin polymerization were examined to explore nucleotide structural requirements at the exchangeable GTP binding site. With microtubule-associated proteins and Mg²⁺, GTP-supported polymerization was only modestly inhibited by GDP, and still weaker inhibitory activity was found with two analogs, dGDP and 9-β-D-arabinofuranosylguanine-5′-diphosphate (araGDP). Omission of Mg²⁺ significantly enhanced the inhibitory effects of GDP, dGDP and araGDP and resulted in weak inhibition of the reaction by several other GDP analogs. The relative inhibitory activity of the GDP analogs had no discernable relationship to the relative activity of cognate GTP analogs in supporting microtubule-associated protein-dependent polymerization. One GTP analog, 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), supports polymerization both with and without microtubule-associated proteins. The inhibitory activity of GDP and GDP analogs in ddGTP-supported polymerization was much greater in the absence of microtubule-associated proteins than in their presence; and both reactions were more readily inhibited than was microtubule-associated protein-dependent, GTP-supported polymerization. Microtubule-associated protein-independent, ddGTP-supported polymerization was also potently inhibited by GTP and a number of GTP analogs. GTP was in fact twice as inhibitory as GDP. The relative inhibitory activity of the GTP analogs was comparable to the relative inhibitory activity of the cognate GDP analogs and very different from their relative activity in supporting polymerization.     

Neopterin, a marker of immune response, and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress, correlate at high age as determined by automated simultaneous high-performance liquid chromatography analysis of human urine

Using an established high-performance liquid chromatography (HPLC) method based on anion exchange chromatography, fraction collection, and electrochemical detection, the oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OH-dG) can be analyzed rapidly and precisely in human urine samples. In addition, by ultraviolet (UV) detection, it was shown recently that it is possible to simultaneously analyze creatinine and 7-methylguanine (m⁷Gua), an RNA degradation product, in urine. By adding a fluorescence detector to the HPLC system, we now report that it is also possible to detect pteridins such as neopterin and biopterin. The fluorescence detection was evaluated in detail for neopterin, an immune response and tumor marker. The urinary content of neopterin, assessed by using the HPLC method, was verified with a commercial neopterin enzyme-linked immunosorbent assay (ELISA) kit as indicated by the high correlation between the two methods (r = 0.98). In urinary samples from 58 young healthy individuals (male and female nonsmokers, ages 19-39 years), it was found that there was no significant correlation (r = −0.04) between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). In contrast, in urinary samples from 60 old healthy individuals (male and female nonsmokers, ages 60-86 years), there was a significant correlation (r = 0.47) found between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). These findings strongly indicate that the higher level of immune response that was correlating with old age contributes significantly to the higher level of oxidative damage as assessed in the form of 8-OH-dG. Using this type of HPLC system, it is possible to evaluate oxidative DNA damage and immune response simultaneously using the respective urinary markers. These data may contribute to understanding of the pathophysiology of diseases such as infections and tumor progression where both oxidative stress and immune response occur simultaneously.