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Forty isolates of Ophiocordyceps sinensis collected from Himalayan alpine meadows of Uttarakhand, India, and cultivated on Jhangora (Echinochloa crusgalli) grains were screened to identify the isolate(s) of high cordycepin content. The cultured mycelia were extracted with 50% methanol-chloroform and analyzed by HPTLC using chloroform:methanol (6:1 v/v) as mobile phase and densitometry scanning at 263 nm. Cordycepin varied from 0.002% to 0.029% was detected in twenty-one isolates. Compared to natural O. sinensis (0.004%, 0.006%), cordycepin was determined to be enhanced in twelve cultured samples.

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) and 1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2'-deoxyguanosine and ca. 500 amol of adducts in 100 microg of hydrolyzed DNA in the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2'-deoxyguanosine and 1,N(2)-propano-2'-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilondGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilondGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/10(8) dGuo; brain, 2.96 +/- 1.43 1,N(2)-epsilondGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanodGuo/10(8) dGuo; and lung, 0.87 +/- 0.34 1,N(2)-epsilondGuo/10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental animals with pathological conditions and after air pollution exposure.

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen on the basis of epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its carcinogenicity in laboratory rats and mice. BD is metabolically activated to epoxide intermediates that can react with nucleophilic sites of cellular biomolecules. Among these, 1,2,3,4-diepoxybutane (DEB) is considered the ultimate carcinogenic species of BD due to its potent genotoxicity and mutagenicity attributed to the ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. DEB mutagenesis studies suggest that adducts formed at adenine bases may be critically important, as DEB induces large numbers of A --> T transversion mutations. We have recently identified two regioisomeric exocyclic DEB-dA adducts, 1,N(6)-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-gamma-HMHP-dA) and 1,N(6)-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-alpha-HMHP-dA) ( Seneviratne et al. ( ( 2010 ) Chem. Res. Toxicol. 23 , 118 - 133 ), which were detected in DEB-treated calf thymus DNA and in tissues of BD-exposed laboratory animals. In the present work, we describe a column switching HPLC-ESI(+)-MS/MS methodology for the quantitative analysis of 1,N(6)-HMHP-dA isomers in the DNA of laboratory mice exposed to BD by inhalation. On the basis of their exocyclic structure, which prevents normal Watson-Crick base pairing, these adducts could be responsible for mutations at the A:T base pairs observed following exposure to DEB.

A sensitive, selective and reliable liquid chromatography-mass spectrometry coupled with electrospray ionization interface method for simultaneous separation and determination of thymine, adenine, adenosine and cordycepin in Cordyceps sinensis has been established. The optimum separation for these analytes was achieved using a gradient elution system and a 2.0 x 150 mm Shimadzu VP-ODS column. 2-Chloroadenosine was used as internal standard for this assay. [M+H]+ ions at m/z 127, 136, 268, 252 and 302 were chosen and selective ion monitoring (SIM) mode was used for quantitative analysis of the four main nucleosides. The regression equations were linear in the range of 1.0-117.5 microg x mL(-1) for thymine, 1.8-127.0 microg x mL(-1) for adenine, 0.6-114.0 microg x mL(-1) for adenosine and 0.5-107.5 microg x mL(-1) for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were 1.0 and 0.2 microg x mL(-1) for thymine, 1.8 and 0.6 microg x mL(-1) for adenine, 0.6 and 0.1 microg x mL(-1) for adenosine and 0.5 and 0.1 microg x mL(-1) for cordycepin, respectively. The recoveries of the four nucleosides ranged from 98.47 to 99.32%. The developed method was successfully used to determine nucleosides in Cordyceps sinensis from different sources.

It is known that there are several codes residing simultaneously on the DNA double helix. The two best-characterized codes are the genetic code--the code for protein production, and the code for DNA packaging into nucleosomes. Since these codes have to coexist simultaneously on the same DNA region, both must be degenerate to allow this coexistence. A-tracts are homopolymeric stretches of several adjacent deoxyadenosines on one strand of the double helix, having unusual structural properties, which were shown to exclude nucleosomes and as such are instrumental in setting the translational positioning of DNA within nucleosomes. We observe, cross-kingdoms, a strong codon bias toward the avoidance of long A-tracts in exon regions, which enables the formation of high density of nucleosomes in these regions. Moreover, long A-tract avoidance is restricted exclusively to nucleosome-occupied exon regions. We show that this bias in codon usage is sufficient for enabling DNA organization within nucleosomes without constraints on the actual code for proteins. Thus, there is inter-dependency of the two major codes within DNA to allow their coexistence. Furthermore, we show that modulation of A-tract occurrences in exon versus non-exon regions may result in a unique alternation of the diameter of the '30-nm' fiber model.

Oxidatively induced DNA lesions (5'R)- and (5'S)-8,5'-cyclo-2'-deoxyadenosines (R-cdA and S-cdA) are detectable and accumulate in vivo due to disease states and defects in DNA repair. They block transcription and inhibit gene expression, and may play a role in disease processes. Accurate measurement of these lesions in DNA in vivo is necessary to understand their biological effects. We report on a methodology using liquid chromatography/isotope-dilution tandem mass spectrometry to measure R-cdA and S-cdA in DNA. This methodology permitted the detection of these compounds at a level of 0.1fmol on-column. Levels of R-cdA and S-cdA in mouse liver DNA amounted to 0.133+/-0.024 and 0.498+/-0.065 molecules/10(7) DNA 2'-deoxynucleosides, respectively. The successful measurement of R-cdA and S-cdA in DNA in vivo suggests that this methodology will be used for understanding of their repair and biological consequences, and that these compounds may be used as putative biomarkers for disease states.

Sensitive and reliable methods are required for the assessment of oxidative DNA damage, which can result from reactive oxygen species that are generated endogenously from cellular metabolism and inflammatory responses, or by exposure to exogenous agents. The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) method is described, that utilises online column-switching valve technology for the simultaneous determination of two DNA adduct biomarkers of oxidative stress, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA). To allow for the accurate quantitation of both adducts the corresponding [(15)N(5)]-labelled stable isotope internal standards were synthesised and added prior to enzymatic hydrolysis of the DNA samples to 2'-deoxynucleosides. The method required between 10 and 40 microg of hydrolysed DNA on-column for the analysis and the limit of detection for both 8-oxodG and 8-oxodA was 5 fmol. The analysis of calf thymus DNA treated in vitro with methylene blue (ranging from 5 to 200 microM) plus light showed a dose-dependent increase in the levels of both 8-oxodG and 8-oxodA. The level of 8-oxodG was on average 29.4-fold higher than that of 8-oxodA and an excellent linear correlation (r = 0.999) was observed between the two adducts. The influence of different DNA extraction procedures for 8-oxodG and 8-oxodA levels was assessed in DNA extracted from rat livers following dosing with carbon tetrachloride. The levels of 8-oxodG and 8-oxodA were on average 2.9 (p = 0.018) and 1.4 (p = 0.018) times higher, respectively, in DNA samples extracted using an anion-exchange column procedure than in samples extracted using a chaotropic procedure, implying artefactual generation of the two adducts. In conclusion, the online column-switching LC/MS/MS SRM method provides the advantages of increased sample throughput with reduced matrix effects and concomitant ionisation suppression, making the method ideally suited when used in conjunction with chaotropic DNA extraction for the determination of oxidative DNA damage., ((c) 2008 John Wiley & Sons, Ltd.)

The frequent deletion of the human chromosomal region 9p21, including the methylthioadenosine phosphorylase (MTAP) gene, is hypothesized to lead to the intra- and/or extracellular accumulation of 5'-deoxy-5'-methylthioadenosine (MTA) in cancer cells and the subsequent promotion of tumor progression. The lack of sensitive methodology for the direct measurement of MTA in tumor cells has hampered the testing of this hypothesis to date. A liquid chromatography electrospray ionization tandem mass spectrometry method (LC-MS/MS) was developed for the absolute quantitative determination of MTA in cell culture media and cell extracts using stable isotope labeled MTA as an internal standard. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 62.5 pM and 2 nM, respectively, and allowed the direct measurement of MTA in biological samples without prior enrichment. Average imprecision of MTA extraction from cells and cell media, as well as LC-MS/MS analysis were 9.7, 3.8 and 1.9%, respectively. The method enabled the demonstration of the accumulation of MTA in melanoma cell culture media reaching a steady-state level within 24h. Only a slight difference in extracellular MTA concentrations was observed between cells with and without MTAP expression. However, there was a fourfold increase in intracellular MTA concentration in melanoma cells lacking MTAP, thus confirming the hypothesized accumulation of MTA in human cancer cells harboring a chromosome 9p21 deletion.

Whether chronic lymphocytic leukaemia (CLL) is a latent or a proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukaemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements indicate that B lymphocytes do divide at significant rates in CLL. However, an important and still unanswered question is whether CLL cells proliferate faster or slower compared with their normal counterparts. This report addressed directly this point and compared B-cell kinetics in CLL subjects and healthy controls, using a pulse-chase approach based on incorporation of deuterium from 6,6-(2)H(2)-glucose into DNA. We confirmed that B cells proliferated at significant levels in CLL but found that the proliferation rates were reduced compared with healthy subjects (mean 0.47 vs. 1.31%/d respectively, P = 0.007), equivalent to an extended doubling time of circulating B cells (147 d vs. 53 d). In conclusion, CLL B cells proliferate at reduced levels compared with healthy controls. CLL is thus characterized by an aberrant B-cell kinetics with a decrease in cell turnover, an observation that may impact on elaboration of efficient therapeutic strategies.

Sample preparation is the first and very important step, which can greatly influence the repeatability and accuracy of the analysis. To date, several sample preparation methods with different solvents have been used for quantitative determination of nucleosides in Cordyceps, but their data are greatly various. In this study, five nucleosides, including adenosine, guanosine, inosine, uridine and cordycepin, in Cordyceps were determined using three extraction methods i.e. organic solvent pressurized liquid extraction, boiling water extraction and ambient temperature water extraction and high performance liquid chromatography (HPLC)-diode array detection (DAD). The similar results were obtained when organic solvent pressurized liquid extraction and boiling water extraction were applied. However, the amounts of nucleosides in natural C. sinensis and cultured C. militaris extracted with ambient temperature water were greatly increased except those of adenosine in natural C. sinensis and cordycepin in cultured C. militaris. In addition, the amount of investigated nucleosides in cultured C. sinensis had no obvious variation among the three extraction methods. The results suggest that sample preparation has significant effect on the quantification of nucleosides in Cordyceps.

Objective: To study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products., Methods: To determinate 13 samples of Cordyceps sinensis and its similar products by HPLC, and analyze the HPLC results with similar appraisal method and graphical methods of multivariate sample in two dimensional plane such as the methods of profile, radar chart and constellation graph., Results: The similar appraisal method might synthesize the similar degree in quantification, while the graphical methods such as profile graph, radar chart and constellation graph could show more details about the classification and the characteristic of varieties directly., Conclusions: We recommend the combined application of similar appraisal method and the graphical methods due to its advantages on the judgment and characteristic analysis of fingerprint.

Intake of linoleic acid (LA) increased etheno-DNA adducts induced by lipid peroxidation (LPO) in white blood cells (WBC) of female but not of male volunteers [J. Nair, C.E. Vaca, I. Velic, M. Mutanen, L.M. Valsta, H. Bartsch, High dietary omega-6 polyunsaturated fatty acids drastically increase the formation of etheno-DNA adducts in white blood cells of female subjects, Cancer Epidemiol. Biomarkers Prev. 6 (1997) 597-601]. Etheno-adducts were measured in rats gavaged with LA, oleic acid (OA) and saturated fatty acid rich coconut oil for 30 days. DNA from organs and total WBC was analyzed for 1, N(6)-ethenodeoxyadenosine (varepsilondA) and 3, N(4)-ethenodeoxycytidine (varepsilondC) by immunoaffinity/(32)P-postlabeling. Colon was the most affected target with LA-treatment, where etheno-adducts were significantly elevated in both sexes. In WBC both adducts were elevated only in LA-treated females. Unexpectedly, OA treatment enhanced etheno-adduct levels in prostate 3-9 fold. Our results in rodents confirm the gender-specific increase of etheno-adducts in WBC-DNA, likely due to LPO induced by redox-cycling of 4-hydroxyestradiol. Colon was a target for LPO-derived DNA-adducts in both LA-treated male and female rats, supporting their role in omega-6 PUFA induced colon carcinogenesis.

Aims: To examine the effects of ammonium feeding on the production of cordycepin (3'-deoxyadenosine, a nucleoside analogue) and exopolysaccharides (EPS) in mycelial culture of a new Cordyceps sinensis fungus Cs-HK1., Methods and Results: Cs-HK1 fungus was cultivated in a liquid medium containing glucose, yeast extract, peptone and a few major inorganic salts. NH(4)Cl was fed to the mycelial culture at various concentrations from 5 to 40 mmol l(-1) on day 3 (during exponential phase). NH(4)Cl, fed at 10 mmol l(-1), stimulated the cordycepin production most significantly, with nearly fourfold increase in the cordycepin content of mycelia (from 28.5 to 117 microg g(-1)), and also increased the EPS production by 40% (from 2.6 to 3.7 g l(-1)). The ammonium feeding had a slightly positive effect at 5-10 mmol l(-1), but a negative effect at higher concentrations on the mycelium growth. Ammonium feeding also caused a sharp drop of the medium pH, owing perhaps to the uptake of NH(3) and the release of H(+) by the fungal cells., Conclusions: Ammonium feeding to the mycelial culture of Cs-HK1 fungus enhanced the intracellular cordycepin accumulation and the EPS production. The enhanced cordycepin production may be attributed to the uptake of ammonia for nucleoside synthesis, and the enhanced EPS to the increased uptake of glucose for EPS biosynthesis., Significance and Impact of the Study: It is useful for the production of bioactive metabolites and for understanding ammonium metabolism and its relationship to the biosynthesis of nucleosides in a precious medicinal fungus.

Persistent oxidative stress and excess lipid peroxidation (LPO), induced by inflammatory processes, impaired metal storage, and/or dietary imbalance, cause accumulations and massive DNA damage. This massive DNA damage, along with deregulation of cell homeostasis, leads to malignant diseases. Reactive aldehydes produced by LPO, such as 4-hydroxy-2-nonenal, malondialdehyde, acrolein, and crotonaldehyde, react directly with DNA bases or generate bifunctional intermediates which form exocyclic DNA adducts. Modification of DNA bases by these electrophiles, yielding promutagenic exocyclic adducts, is thought to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. Ultrasensitive detection methods have facilitated studies of the concentrations of promutagenic DNA adducts in human tissues, white blood cells, and urine, where they are excreted as modified nucleosides and bases. Thus, immunoaffinity-(32)P-postlabeling, high-performance liquid chromatography-electrochemical detection, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, immunoslotblot assay, and immunohistochemistry have made it possible to detect background concentrations of adducts arising from endogenous LPO products in vivo and studies of their role in carcinogenesis. These background adduct levels in asymptomatic human tissues occur in the order of 1 adduct/10(8) and in organs affected by cancer-prone inflammatory diseases these can be 1 or 2 orders of magnitude higher. In this review, we critically discuss the accuracy of the available methods and their validation and summarize studies in which measurement of exocyclic adducts suggested new mechanisms of cancer causation, providing potential biomarkers for cancer risk assessment in humans with cancer-prone diseases.

Objective: To develop a LC-MS-MS method for determination of adenosine and cordycepin in Cordyceps sinensis and it's substitutes., Method: The sawple was extracted with. 90% methanol. Multi-reactions monitoring (MRM) technique was adopted., Result: The regression equations and coefficients were Y = 89.04X + 506.85 (r = 0.999 7) for adenosine, Y = 99.66X + 1 251.34 (r = 0.998 8) for cordycepin respectively. The linear range was 5.0-1 000.0 microg x L(-1) for adenosine and cordycepin. The limits of detection (LOD) were 0. 44 microg x L(-1) for adenosine and 0.31 microg x L(-1) for cordycepin, respectively. The average recoveries of adenosine and cordycepin were 98.1% and 97.9%, respectively., Conclusion: The method was highly sensitive, selective and fast, which can be used for the determination of adenosine and cordycepin in C. sinensis and it's substitutes. This method can also be applied for the quality control of the medicinal materials.

Objective: To determine whether urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), an in vivo parameter of oxidative stress, is correlated with the outcome of critically septic patients., Design and Setting: Clinical outcome study in an adult medical ICU., Patients: Eighty-five consecutive septic patients: 59 men and 26 women., Measurements and Results: Urinary 8-OHdG was analyzed using isotope-dilution liquid chromatography with tandem mass spectrometry (LC/MS/MS). ICU mortality in these 85 septic patients was 25.9% (n = 22) and hospital mortality 38.8% (n = 33). APACHE II scores of survivors on day 1, on day 3, and the difference between them differed significantly from those of nonsurvivors (day 1, 21.0 +/- 7.1 vs. 25.9 +/-8.0; day 3, 15.0 +/- 5.8 vs. 23.2 +/- 8.3; difference, 6.0 +/- 5.5 vs. 1.7 +/- 6.6). Urinary 8-OHdG was significantly lower in survivors than in nonsurvivors on day 1 (1.8 +/- 2.4 vs. 3.0 +/- 2.4). The area under receiver operating characteristic curve analysis for the association between day 1 urinary 8-OHdG and ICU mortality was 0.71. The comparison performed upon discharge from hospital revealed similar results., Conclusions: This is a preliminary study. The excretion of the urinary 8-OHdG, as measured using isotope-dilution LC/MS/MS, as the APACHE II score, were correlated with the outcome of critically septic patients in medical ICU.

This study aimed to further analyse the potential role of oxidative stress in children and adolescents with type 1 diabetes at clinical onset, during disease progression and when early microvascular complications ( + DC) appeared. Compared with age-matched controls, diabetic patients had greater oxidative damage to lipids, proteins and DNA demonstrated by analysis of plasma and erythrocyte malondialdehyde, carbonyl proteins and leukocyte 8-hydroxy-deoxyguanosine, all of which were significantly raised at onset, decreased during the first 1.5 years of evolution and rose progressively thereafter. Plasma lipid levels were significantly associated with lipid and protein oxidation products. Erythrocyte glutathione and glutathione-peroxidase activity were significantly decreased with the lowest values at onset and in + DC sub-groups. Insulin therapy in the first year improved metabolic and oxidant-antioxidant status and, consequently, hyperglycaemia-derived biomolecular oxidative damage. Diabetes-associated hyperlipidaemia is related to lipid and protein oxidation, thereby supporting the concept of glucotoxicity and lipotoxicity being inter-related. The overall increase in lipid, protein and DNA oxidative damage in diabetic patients with microangiopathy could be pathogenetically relevant in the early development of diabetes-related complications.

Objective: To investigate the High Performance Capillary Electrophoresis (HPCE) fingerprint of cultured Cordyceps militaris (L.) Link., Methods: Separation was performed on a 50 cm x 75 microm uncoated capillary with 0.5 mmol/L borate solution (pH 9. 18) as HPEC buffer. The run voltage was 20 Kv, temperature 25 degrees C and the DAD detection was set at 254nm., Results: Fingerprint consisted of 11 common peaks. The validation of methods was satisfied with the requirements for SFDA's technical regulations., Conclusion: The method was accurate and simple and suitable to the quality control of cultured Cordyceps militaris (L.) Link.

(5'S)-8,5'-Cyclo-2'-deoxyadenosine (S-cdA), which arises from the reaction of the hydroxyl radical (*OH) with 2'-deoxyadenosine in DNA, is a lesion comprising a base-sugar linkage that distorts the DNA backbone. This structure impedes transcription and blocks polymerase action. Further, a single S-cdA lesion in the TATA box reduces gene expression. Considering the ability of S-cdA to disrupt DNA structure, which is likely associated with increased cancer risk, we determined S-cdA concentrations in the DNA of stroma, epithelium, and myoepithelium from normal breast tissues using liquid chromatography/mass spectrometry (LC/MS). We also identified differences in the base and backbone structures using Fourier transform-infrared (FT-IR) spectroscopy. LC/MS revealed that the lowest concentration of S-cdA in the stroma (0.04 +/- 0.02 lesions/10(6) bases) occurred in women ages 17 to 30. The highest concentration (0.13 +/- 0.07 lesions/10(6) bases) was found in women 33 to 46. FT-IR spectroscopy showed significant base and backbone differences in the stromal DNA between the women under 30 and those over 50. These findings imply that distortions in the geometry of the helix increase with age, reaching significant proportions in older women. No differences were found in the S-cdA concentrations between the three cell types, suggesting that the *OH attack on the base structure may be essentially random. Initial insight is provided on changes in DNA structure that potentially affect gene expression and increase breast cancer risk.

O(6)-Methyl-2'-deoxyguanosine (O(6)-mdGuo), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), and 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O(6)-[(2)H(3)]mdGuo was synthesized and used as internal standard. The limits of quantification for O(6)-mdGuo, 8-oxodGuo, and epsilondAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24-125 pmol/ml, 0.98-125 pmol/ml, and 0.49-62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O(6)-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for epsilondAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O(6)-mdGuo was above the limit of detection (37 adducts per 10(9) normal nucleosides) but could not be quantified. 8-oxodGuo and epsilondAdo showed background levels of 500 and 130 adducts per 10(9) normal nucleosides, respectively. DNA analyzed 1h after treatment of rats with dimethylnitrosamine by oral gavage of 50 microg/kg b.wt. did not affect the levels of 8-oxodGuo and epsilondAdo but resulted in 200 O(6)-mdGuo adducts per 10(9) normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.

Upon inflammation, activated neutrophils secrete myeloperoxidase, an enzyme able to generate hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. An analytical method, involving HPLC coupled to electrospray tandem mass spectrometry, has been set-up to detect low levels of HOCl-induced nucleic acids lesions, including both ribo and 2'-deoxyribonucleoside derivatives of 8-chloroguanine, 8-chloroadenine and 5-chlorocytosine. Validation of the developed method was achieved using isolated cells treated with HOCl. The method was found to be sensitive enough to allow the measurement of background levels of 5-chloro-2'-deoxycytidine in the DNA of human white blood cells isolated from 7 mL of blood.

A rapid and sensitive method to determine 8-oxoguanine (8oxoG) and 8-hydroxydeoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in cerebral cortex microdialysate samples using capillary electrophoresis (CE) with electrochemical detection (CEEC) was developed. Samples were concentrated on-column using pH-mediated stacking for anions. On-column anodic detection was performed with a carbon fiber working electrode and laser-etched decoupler. The method is linear over the expected extracellular concentration range for 8oxoG and 8-OHdG during induced ischemia-reperfusion, with R.S.D. values

The mechanism of DNA damage by photoexcited alkaloids, berberine and palmatine, was examined using 32P-labeled DNA fragments obtained from human genes. Berberine and palmatine easily bind to DNA, leading to the formation of strong fluorescent complexes. The binding constants of berberine and palmatine to DNA, estimated from an analysis of their fluorescence enhancements, indicate the formation of stable complexes. Photoexcited berberine and palmatine caused DNA cleavage, specifically at almost all guanine residues, under the aerobic condition after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment, suggesting the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), an oxidized product of 2'-deoxyguanosine, and further oxidized products. The formation of 8-oxodGuo was confirmed by HPLC measurement. The quantum yield of 8-oxodGuo formation by berberine was almost the same as that induced by palmatine. Berberine and palmatine did not cause DNA photodamage under anaerobic conditions. Scavengers of singlet oxygen (1O2), such as sodium azide and methional, inhibited DNA damage. These findings suggest that photoexcited berberine and palmatine give rise to 8-oxodGuo through 1O2 generation. The photosensitized 1O2 generation from these alkaloids was examined using near-infrared luminescence measurements. Emission at ca. 1270 nm was observed during photoexcitation of the DNA-alkaloid complexes. This emission was quenched by sodium azide, a scavenger of 1O2. In the absence of DNA, berberine and palmatine could not show the emission. This spectroscopic study has shown that photoexcited alkaloids can generate 1O2 only when the DNA-alkaloid complexes are formed. In conclusion, berberine and palmatine easily bind to DNA and induce guanine specific photooxidation via 1O2 formation. The present study suggests that berberine and palmatine can act as functional photosensitizers enabling a switch in phototoxicity via 1O2 formation by the interaction with DNA.

Objective: To analysis the nutrient and effective ingredients of in Cordyceps militaris and make the best use of its medical value., Method: Adenosine, cordycepin, polysaccharides, cordyceps acid, protein and fat in different parts of C. militaris were extracted, they are quantified by HPLC and other colorimetric analysis., Result: The contents of polysaccharide was found to be 86.49 mg x g(-1) in C. militaris, 6.82 mg x g(-1) of adenosine in stroma, 13.28 mg x g(-1) of cordycepin and 44.07 mg x g(-1) of cordyceps acid in sclerolium., Conclusion: In different parts of C. militaris, the biosynthesis of effective ingredients is different. The total amount of effective ingredients is highest in C. militaris, the production of cordycepin and cordyceps acid is highest in sclerotium in comparison with other parts. Growth of C. militaris largely relies on its capability to utilize fat and protein from silkworm.

Objective: HPLC-ESI-MS to establish a method for simultaneous determination of adenosine and cordycepin in Cordyceps sinensis and C. militarris., Method: HPLC-ESI-MS method. An electrospray ionization (ESI) interface and selective ion monitoring (SIM) mode were used. The analytical column was a 2.0 mm x 150 mm Shimadzu VP - ODS column and the mobile phase was water (94%), methanol (5%) and formic acid (1%). 2-Chloroadenosine was used as internal standard for this assay., Result: The regression equations and coefficient were Y = 0.134 6X + 0.001 29 (r = 0.998 4) for adenosine, Y = 0.216 4X + 0.021 5 (r = 0.999 1) for cordycepin. The liner range was 0.5 approximately 124.5 microg x mL(-1) and 0.5 approximately 136.5 microg x mL(-1) for adenosine and cordycepin, respectively. The average recoveries of adenosine and cordycepin were 95.8% and 98.1%, respectively., Conclusion: This method is highly sensitive, fast and selective, which can be used for the determination of nucleosides in C. sinensis and its substitutes. This method can also be applied for the quality control of above herbs.

The determination of proton affinity (PA) of 2'-deoxyadenosine (dA) is used as a case study for the evaluation of possible drawbacks in the determination of the PA of the nucleic acid molecules by the kinetic method. The observed Delta PA among the different values obtain for dA by applying this procedure in its different extensions was 0.64 Kcal/mol, which is within the uncertainties of any theoretical or experimental approach. It was demonstrated that nucleosides can be generally used as reference compounds to measure the PA of an unknown nucleoside. The evaluation of Delta Delta S value for two competing reaction channels taken by proton-bound heterodimers formed by two nucleosides provides clear information on the reference base which has to be discarded from the set of reference compounds used for the estimation of an unknown PA. The PA of dA obtained with the most elaborated kinetic method (237.00 +/- 0.07 kcal/mol) is consistent with the value of 237.0 kcal/mol obtained by a simple treatment of the relative intensities of the product ions generated by two couples of the proton bound dimers formed by the nucleoside and two reference amines. The kinetic method can be, therefore, confidently used to assess the proton affinity of the multifunctional molecules such as nucleosides and nucleobases.

8,5'-cyclopurine-2'-deoxynucleosides in DNA are repaired by nucleotide-excision repair, and act as strong blocks to DNA polymerases, RNA polymerase II and transcription factor binding. Thus, it is important to accurately determine the level of these lesions in DNA. There is controversy in the literature regarding the ability of different enzymes to release these compounds from oligodeoxynucleotides or DNA. We used liquid chromatography/mass spectrometry (LC/MS) to investigate the ability of several enzymes to release (5'S)-8,5'-cyclo-2'-deoxyadenosine [(5'S)-cdA] from dinucleotides and oligodeoxynucleotides and from DNA. The data show that (5'S)-cdA is completely released from DNA by hydrolysis with nuclease P1, snake venom phosphodiesterase and alkaline phosphatase. The identity of the normal nucleoside 5' to the (5'S)-cdA had a significant effect on its release. Using LC/MS, we also showed that the levels of (5'S)-cdA were within an order of magnitude of those of 8-hydroxy-2'-deoxyguanosine, and three times higher than those of 8-hydroxy-2'-deoxyadenosine in pig liver DNA. Different DNA isolation methods affected the levels of the latter two lesions, but did not influence those of (5'S)-cdA. We conclude that (5'S)-cdA can be completely released from DNA by enzymic hydrolysis, and the level of (5'S)-cdA in tissue DNA is comparable to those of other oxidatively induced DNA lesions.

Benzo[a]pyrene (B[a]P) is an archetypal member of the family of polycyclic aromatic hydrocarbons (PAHs) and is a widely distributed environmental pollutant. B[a]P is known to induce cancer in animals, and B[a]P-containing complex mixtures are human carcinogens. B[a]P exerts its genotoxic and carcinogenic effects through metabolic activation forming reactive intermediates that damage DNA. DNA adduction by B[a]P is a complex phenomenon that involves the formation of both stable and unstable (depurinating) adducts. One pathway by which B[a]P can mediate genotoxicity is through the enzymatic formation of B[a]P-7,8-quinone (BPQ) from B[a]P-7,8-diol by members of the aldo-keto-reductase (AKR) family. Once formed, BPQ can act as a reactive Michael acceptor that can alkylate cellular nucleophiles including DNA and peptides. Earlier studies have reported on the formation of stable and depurinating adducts from the reaction of BPQ with DNA and nucleosides, respectively. However, the syntheses and characterization of the stable adducts from these interactions have not been addressed. In this study, the reactivity of BPQ toward 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) nucleosides under physiological pH conditions is examined. The identification and characterization of six novel BPQ-nucleoside adducts obtained from the reaction of BPQ and dG or dA in a mixture of phosphate buffer and dimethylformamide are reported. The structures of these adducts were determined by ultraviolet spectroscopy, electrospray mass spectrometry, and NMR experiments including (1)H, (13)C, two-dimensional COSY, one-dimensional NOE, ROESY, HMQC, HSQC, and HMBC. The reaction of BPQ with dG afforded four unique Michael addition products: two diastereomers of 8-N(1),9-N(2)-deoxyguanosyl-8,10-dihydroxy-9,10-dihydrobenzo[a]pyren-7(8H)-one (BPQ-dG(1,2)) and two diastereomers of 10-(N(2)-deoxyguanosyl)-9,10-dihydro-9-hydroxybenzo[a]pyrene-7,8-dione (BPQ-dG(3,4)). The BPQ-dG(1,2)( )()adducts suggest a 1,6-Michael addition reaction of dG, an oxidation of the hydroquinone to the quinone, a 1,4-Michael addition of water, and an internal cyclization. The BPQ-dG(3,4)( )()adducts suggest a 1,4-Michael addition reaction of dG, an oxidation of the hydroquinone to the quinone, and a 1,6-Michael addition of water. Under similar but extended reaction conditions, the reaction of BPQ with dA produced only one diastereomeric pair of adducts identified as 8-N(6),10-N(1)-deoxyadenosyl-8,9-dihydroxy-9,10-dihydrobenzo[a]pyren-7(8H)-one (BPQ-dA(1,2)). The BPQ-dA(1,2)( )()adducts suggest a 1,4-Michael addition reaction of dA, an oxidation of the hydroquinone to the quinone, a 1,6-Michael addition of water, and an internal cyclization. As considerable efforts have been placed in documenting the genotoxic effects of BPQ, this first report of the identification and characterization of these stable adducts of BPQ formed under physiological pH conditions is expected to contribute significantly to the area of BPQ-mediated genotoxicity and carcinogenesis.

Levels of 8-oxo2'dG (HPLC), p53 mRNA (PCR) and p53 protein (Western Blot) were estimated in four structures of rat brain, including grey matter (GM) of cerebral cortex, cerebral white matter (WM), cerebellum (C) and medulla oblongata (MO) of control (3.0-3.5-month-old) rats, 12- and 24-month-old rats. The level of oxidative DNA was statistically significantly higher in C of 24-month-old animals. Expression of p53 gene increased in C and also in the all other investigated brain parts, while the protein level of p53 was enhanced only in GM of 24-month-old rats. These data indicated that DNA oxidative damage and p53 gene expression increased significantly in aged brain. The higher expression of p53 gene in aged brain may suggest the activation of DNA repair processes.

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0 x 150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]+ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4-140.0 microg ml(-1) for adenine, 0.6-117.5 microg ml(-1) for hypoxanthine, 0.5-128.5 microg ml(-1) for adenosine and 0.5-131.5 microg ml(-1) for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 microg ml(-1) for adenine, 0.6 and 0.2 microg ml(-1) for hypoxanthine, 0.5 and 0.1 microg ml(-1) for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.

Aim: To rapidly separate and determine the nucleosides from natural and cultured Cordyceps kyushuensis Kob., and to compare the content of cordycepin and adenosine in different parts of Cordyceps kyushuensis Kob., which are the main nucleoside active components in medicinal fungus belonging to Cordyceps (Fr.) Link., Methods: The nucleosides were separated and determined by the high performance capillary zone electrophoresis (CZE). Beckman P/ACE system MDQ apparatus equipped with a PDA detector and a uncoated fused-silica capillary (41 cm x 45 microns ID, 30 cm effective length) were used. The experimental conditions were as follows: the running buffer was borax solution (adjust to pH 9.4 with sodium hydroxide), applied voltage was 20 kV, operated temperature was 20 degrees C and the detector wavelength was 258 nm. The content of cordycepin and adenosine in the fruiting body, stroma and host worm of natural and cultured C. kyushuensis were respectively investigated and quantitatively analyzed., Results: There are at least 8 kinds of nucleoside or nitrogen base in Cordyceps kyushuensis Kob. The content of cordycepin which is a bio-active substance with anti-tumor activity in C. kyushuensis is significantly higher than that in C. sinensis and C. militaris, and furthermore the cordycepin in the cultured C. kyushuensis is notably higher than the natural one. Adenosine was mainly found from the stroma of C. kyushuensis, While the cordycepin content is high in the stroma of both natural and cultured C. kyushuensis as well as in the host worm of the cultured one., Conclusion: There are some differences about the nucleoside components between the natural and cultured C. kyushuensis and between the different parts of them. With a high cordycepin content, C. kyushuensis should have a considerable medicinal potential.

3'-aminoacylamino-3'-deoxyadenosines, analogues of the antibiotic puromycin, have been synthesized from adenosine. They key 3'-azido derivative 10 was obtained through a 3'-oxidation/reduction/substitution procedure. A modified purification protocol on a larger scale was developed for the oxidation step using the Garegg reagent. The coupling reaction between an Fmoc-l-amino acid and the fully protected form of 3'-amino-3'-deoxyadenosine 11 furnished the aminoacylated compounds 12 in high yields. The puromycin analogues were obtained in 10 steps and up to 23% (14c) overall yield.

Lung epithelial cells are a primary target for reactive oxygen species (ROS). ROS can cause oxidative deoxyribonucleic acid modification, such as 8-hydroxy-deoxyguanosine (8-OHdG). A human homologue of the MutT protein (hMTH1) prevents this modification. Mitochondria are the most important cellular source of ROS and may be susceptible to oxidative damage. The purpose of this study is to investigate oxidative stress and mitochondrial damage in lung epithelial cells from idiopathic interstitial pneumonias (IIPs). The authors analysed 8-OHdG, hMTH1, and mitochondrial proteins on lung specimens from 13 patients with IlPs consisted of eight patients with usual interstitial pneumonia and five patients with nonspecific interstitial pneumonia using Western blot analysis and immunohistochemistry. Immunoreactivity for 8-OHdG and hMTH1 was significantly increased in the lung epithelial cells from patients with IIPs compared with controls. The expression of hMTH1 was localised in the nuclear and cytoplasmic, but not the mitochondrial, fraction of lung homogenates. Immunoreactivity for mitochondrial protein and cytochrome c oxidase complex subunit IV was increased in the lung epithelial cells from patients with IIPs compared with controls. The current study concludes that oxidative stress may participate in epithelial cell damage in idiopathic interstitial pneumonia, and that increased mitochondrial mass may associate with increased reactive oxygen species production in idiopathic interstitial pneumonia.

Cigarette smokers inhale a broad range of carcinogens derived from tobacco and its pyrolysis products, including free radicals, which induce oxidative stress and subsequent lipid peroxidation (LPO). Miscoding carcinogen-DNA adducts are formed by cigarette smoke constituents and are thought to initiate lung carcinogenesis. The presence of various types of DNA damage was therefore analyzed in tumor adjacent uninvolved lung tissues of 13 smoking and 11 non-smoking operated lung cancer patients. O(4)-ethylthymidine (O(4)etT), 1,N(6)-ethenodeoxyadenosine ( epsilon dA) and 3,N(4)-ethenodeoxycytidine ( epsilon dC) were determined by immuno-enriched (32)P-postlabeling. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were measured as diagonal radioactive zones after nuclease P1 enriched (32)P-postlabeling. Mean O(4)etT and PAH-DNA adduct levels were higher in lung DNA of smokers than of non-smokers (O(4)etT/10(8) thymidine: 3.8 versus 1.6, P < 0.01; PAH-DNA adducts/10(8) nucleotides: 11.2 versus 2.2, P < 0.01). Pulmonary etheno-DNA adduct levels did not differ between smokers and non-smokers, but large inter-individual variations were observed (80- and 250-fold differences for epsilon dA and epsilon dC, respectively). As all smokers (except one) refrained from smoking at least for 1 week before surgery, our results demonstrate the persistence of O(4)etT and PAH-DNA adducts in human lung. A positive correlation obtained between O(4)etT and PAH-DNA adducts (R = 0.65, P < 0.01) suggests that both adducts are formed from cigarette smoke as the main exposure source. We conclude that in addition to the DNA adducts derived from PAH and tobacco-specific nitrosamines, miscoding O(4)etT lesions are formed by cigarette smoke that contribute to the increased genomic instability and increased lung cancer risk in smokers.

Although curcumin is known to exhibit antitumor activity, carcinogenic properties have also been reported. To clarify the potentiality of carcinogenesis by curcumin, we have examined whether curcumin can induce DNA damage in the presence of cytochrome P450 (CYP) using [32P]-5(')-end-labeled DNA fragments obtained from genes relevant to human cancer. Curcumin treated with CYP 2D6, CYP1A1, or CYP1A2 induced DNA damage in the presence of Cu(II). CYP2D6-treated curcumin caused base damage, especially at 5(')-TG-3('), 5(')-GC-3('), and GG sequences. The DNA damage was inhibited by both catalase and bathocuproine, suggesting that reactive species derived from the reaction of H(2)O(2) with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2(')-deoxyguanosine was significantly increased by CYP2D6-treated curcumin in the presence of Cu(II). Time-of- flight mass spectrometry demonstrated that CYP2D6 catalyzed the conversion of curcumin to O-demethyl curcumin. Therefore, it is concluded that curcumin may exhibit carcinogenic potential through oxidative DNA damage by its metabolite.

A method involving high performance liquid chromatography (HPLC) separation associated with tandem mass spectrometry (MS/MS) detection in the multiple reaction monitoring mode was set-up for the measurement of 2-hydroxy-2'-deoxyadenosine (2-OHdAdo). This modified nucleoside, arising from the radical oxidation of 2'deoxyadenosine (dAdo), has been described in the literature as a potential biological marker of the Fenton reaction. Using the specific and sensitive HPLC-MS/MS assay, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 4,6-diamino-5-formamidopyrimidine and 2-hydroxy-2-deoxyadenosine (2-OHdAdo) were measured within 2'-deoxyadenosine and DNA solutions either exposed to gamma-rays or treated under Fenton reaction conditions. It was found that the yield of 2-OHdAdo was low compared to that of 8-oxodAdo under most of the oxidative conditions studied. In particular and in contrast to previous works, the formation of 2-OHdAdo was shown to be a minor process both upon gamma irradiation and under Fenton reaction conditions. However, a significant yield of formation of 2-OHdAdo was observed either upon incubation with high concentrations of Fe2+ ions in the absence of hydrogen peroxide or upon gamma-radiolysis of a nucleoside solution in the presence of the copper/ (o)-phenanthroline complex.

8,5'-Cyclopurine-2'-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies using thin layer chromatography of (32)P-postlabeled DNA digests have detected several bulky oxidative lesions of unknown structure, called I-compounds, in DNA from normal mammalian organs. We investigated whether any of these type II I-compounds contained 8,5'-cyclo-2'-deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of the sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the technique resulted in detection of two additional I-compounds, pTp-cAp and pGp-cAp. Each I-compound isolated from neonatal rat liver DNA matched authentic (32)P-labeled cA-containing chromatographic standards under nine different chromatographic conditions. Their levels increased significantly after normal birth. The (32)P-postlabeling technique used here is capable of detecting 1-5 lesions/diploid mammalian cell. Thus, it should now be possible to detect changes of cA levels resulting from low level ionizing radiation and other conditions associated with oxidative stress, and to assess cA levels in tissues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair.

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.

A method for the determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and urine by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometry is described. For the urine samples there is no sample preparation except for addition of buffer and internal standards followed by redissolvation of precipitate containing 8-oxo-2'-deoxyguanosine and a centrifugation step before the samples are injected onto the HPLC column. The detection limit for 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine is approximately 0.3 nM corresponding to 7.5 fmol injected. Long runs, that is, > 50 samples, can be analyzed with only minimal loss of sensitivity. The concentrations excreted into urine samples from humans are between 1 and 100 nM for 8-oxo-2'-deoxyguanosine and below 0.3 nM for 8-oxo-2'-deoxyadenosine. In calf thymus DNA levels down to about 1 oxidized guanosine and adenosine per 10(6) unmodified bases can be detected. High levels of 8-oxo-2'-deoxyguanosine were found, 30 per 10(6) 2'-deoxyguanosine, levels of 8-oxo-2'-deoxyadenosine are at or below the detection limit. These findings indicate that High Performance Liquid Chromatography-Tandem Mass Spectrometry is a highly sensitive and specific method for analysis of oxidative DNA modifications in tissue as well as for analysis of excretion of oxidized nucleotides into urine that ensures a minimum artifact formation.

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 (5'R)- and (5'S)-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.

Etheno-DNA adducts are promutagenic lesions present in normal animal and human tissues that are believed to be important in the etiology of cancer related to diet and lifestyle. A method has been developed for the quantification of trace levels of etheno-DNA adducts using on-line sample preparation coupled with liquid chromatography and electrospray tandem mass spectrometry. The use of automated solid-phase extraction and stable labeled internal standards permitted the robust determination of ethenodeoxyadenosine contained in crude DNA hydrolysates from untreated rodent and human tissues at levels on the order of one adduct in 10(8) normal nucleotides from 100 microg of DNA. Inherent analyte response and matrix interference made sensitivity for simultaneous determination of ethenodeoxycytidine approximately 5-fold lower. The method was applied to the analysis of liver DNA from untreated and urethane-treated B6C3F1 mice, untreated rat liver, human placenta, and several commercial DNA preparations. Some sources of potential artifactual formation of etheno-DNA adducts were investigated.

An assay with high selectivity and sensitivity has been developed which, for the first time, allows quantitative, simultaneous measurement in DNA of both 8-oxo-2'-deoxyguanosine (8-oxodG) and 8-oxo-2'-deoxyadenosine (8-oxodA)-important biomarkers of oxidative DNA damage in vivo. Using reversed-phase HPLC coupled to electrospray tandem mass spectrometry (HPLC-MS/MS) in multiple reaction monitoring (MRM) mode it was possible to detect background levels of these lesions in commercially available calf thymus DNA (85 +/- 3 and 7.1 +/- 0.2 per 10(6) DNA bases for 8-oxodG and 8-oxodA respectively; n = 3). Levels of 8-oxodG determined by HPLC coupled to an electrochemical detection system (HPLC-EC) were found to be similar (75 +/- 6 per 10(6) DNA bases; n = 3) to those obtained using tandem mass spectrometry., (Copyright 2000 Academic Press.)

Formation of the lipoxygenase-catalyzed metabolites of arachidonic acid, 8-hydroxyeicosatetraenoic acid (8-HETE) and 12-hydroxyeicosatetraenoic acid (12-HETE), and of the exocyclic DNA adducts 1,N(6)-ethenodeoxyadenosine (epsilondA) and 3, N(4)-ethenodeoxycytidine (epsilondC) was investigated in NMRI mouse skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). In reversible papillomas obtained after 20 weeks of TPA treatment, 15- and 68-fold higher contents of 8-HETE and 12-HETE, respectively, were observed, which were paralleled by 12- and 9-fold increased amounts of epsilondA and epsilondC, respectively. When compared to the level in vehicle-treated control skin, these elevations were statistically significant. In irreversible papillomas harvested 20 weeks after the last TPA treatment, the levels of HETEs and etheno-DNA adducts were found to be slightly reduced, as compared to those in reversible papillomas, but were still increased over control levels in age-matched mice. Comparison of mean group values by simple regression analysis showed a close positive correlation between HETE and etheno-DNA adduct levels. Consistent with the miscoding properties of epsilondA causing mainly A --> T transversions, its increased formation in papillomas could thus contribute to this type of mutation in codon 61 of cHa-ras, shown to be a hallmark of DMBA-initiated and TPA-promoted mouse skin carcinogenesis. Although direct evidence that etheno adducts are derived from lipoxygenase-catalyzed metabolites of arachidonic acid is missing, our results implicate DNA damage by oxidative stress and lipid peroxidation as a cause of genetic instability observed at late stages of tumor promotion in mouse skin carcinogenesis.

Analysis of the reaction between 2'-deoxyadenosine and 4-oxo-2-nonenal by liquid chromatography/mass spectrometry revealed the presence of three major products (adducts A(1), A(2), and B). Adducts A(1) and A(2) were isomeric; they interconverted at room temperature, and they each readily dehydrated to form adduct B. The mass spectral characteristics of adduct B obtained by collision-induced dissociation coupled with multiple tandem mass spectrometry were consistent with those expected for a substituted etheno adduct. The structure of adduct B was shown by NMR spectroscopy to be consistent with the substituted etheno-2'-deoxyadenosine adduct 1' '-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]heptane-2' '-one. Unequivocal proof of structure came from the reaction of adducts A(1) and A(2) (precursors of adduct B) with sodium borohydride. Adducts A(1) and A(2) each formed the same reduction product, which contained eight additional hydrogen atoms. The mass spectral characteristics of this reduction product established that the exocyclic amino group (N(6)) of 2'-deoxyadenosine was attached to C-1 of the 4-oxo-2-nonenal. The reaction of 4-oxo-2-nonenal with calf thymus DNA was also shown to result in the formation of substituted ethano adducts A(1) and A(2) and substituted etheno adduct B. Adduct B was formed in amounts almost 2 orders of magnitude greater than those of adducts A(1) and A(2). This was in keeping with the observed stability of the adducts. The study presented here has provided additional evidence which shows that 4-oxo-2-nonenal reacts efficiently with DNA to form substituted etheno adducts.

Etheno adducts in DNA bases are formed from exogenous agents such as vinyl chloride and urethane, but also via endogenous lipid peroxidation products like trans-4-hydroxy-2-nonenal. An immunohistochemical method was developed to localize the promutagenic 1,N(6)-ethenodeoxyadenosine DNA adduct in liver of rats exposed to vinyl chloride or an iron overload with or without carbon tetrachloride. Six monoclonal antibodies, previously produced through collaborative efforts, were screened for their optimal adduct recognition and low background formation. The antibody generated by clone EM-A-4 was found to be most suitable. Semi-quantitative image analysis of relative pixel intensity showed approximately 1.5 times higher adduct levels (P < 0.05) in the livers of rats treated with vinyl chloride or an iron overload when compared with untreated controls. Significantly elevated adduct levels persisted in vinyl chloride-treated rat liver 14 days after cessation of exposure, suggesting that this adduct is not rapidly eliminated from rat liver DNA. Using the new immunohistochemical method it is possible to visualize this promutagenic etheno-DNA adduct that may play a role in oxidative stress and lipid peroxidation-induced DNA damage in carcinogenesis.

The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA-AAI and dA-AAII) and guanine adducts (dG-AAI and dG-AAII) by the (32)P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC-AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI-DNA and AAII-DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the (32)P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by (32)P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA.

A capillary electrophoretic method for the separation and on-line identification of closely related analytes using low-temperature fluorescence spectroscopy is reported for the eight diastereomeric deoxyadenosine (dA) adducts derived from dibenzo[a,l]pyrene diol epoxide (DB[a,l]PDE). Electrophoretic separation of stereoisomers was accomplished by application of a mixed surfactant buffer [dioctyl sulfosuccinate (DOSS) and Brij-S], which was below the critical micelle concentration (CMC) due to the high concentration (approximately 25%) of organic solvent. Addition of multiple surfactant additives to the separation buffer provided electrophoretic resolution, which was unattainable under single surfactant conditions. It is shown that the CE-separated analyte zones could be identified on-line via low-temperature (4.2 K) fluorescence non-line narrowing and fluorescence line-narrowing (FLN) spectroscopy. In addition, it was determined that in CE buffer trans-syn-,cis-syn- and cis-anti-DB[a,l]PDE-14-N6dA diastereomeric adducts exist mostly with the -dA and DB[a,l]P moiety in an "open"-type conformation while the trans-anti-DB[a,l]PDE-14-N6dA adducts exist in two different conformations whose relative distribution depends on matrix composition. The above conformations have also been revealed by selective laser excitation. Thus, the low-temperature methodology not only provides fingerprint structure via vibrationally resolved 4.2 K fluorescence spectra for adduct identification, but also provides conformational information on the spatial relationship of the carcinogen and dA moiety. These results, taken together with those for DB[a,l]P-DNA adducts formed in standard glasses and mouse epidermis exposed to DB[a,l]P, support our earlier findings that DB[a,l]P-derived adducts exist in different conformations [Jankowiak et al., Chem. Res. Toxicol. 11 (1998) 674]. Therefore, the combination of the separation power of CE and spectral selectivity of low-temperature fluorescence spectroscopy at NLN and FLN conditions provides a powerful methodology which should prove useful for identification of closely related DNA adducts formed at low levels in biological systems.

1,N6-Etheno-2'-deoxyadenosine (epsilon dA) and 3,N4-etheno-2'-deoxycytidine (epsilon dC) are DNA adducts formed by a number of genotoxic chemicals, including vinyl chloride. They are also formed endogenously in tissue DNA, probably from a reactive metabolite of lipid peroxidation. Both the qualitative and quantitative detection of endogenous adducts is important in order to place adduct formation by chemicals such as vinyl chloride in the context of this natural background level. Methods with sufficient sensitivity are therefore being developed to measure the natural background of epsilon dA and epsilon dC adducts. We have developed a high-performance liquid chromatography (HPLC)-32P-postlabelling method to measure epsilon dA and epsilon dC at alkylation frequencies of 1 adduct in 10(7)-10(8) nucleotides in 10-microgram samples of DNA. In HPLC-32P-postlabelling analysis of liver DNA from control Wistar rats, epsilon dA and epsilon dC were determined at levels of 1 adduct in 8.1 x 10(7) and 1 adduct in 1.8 x 10(7) nucleotides, respectively. The levels of epsilon dA and epsilon dC measured in liver DNA of animals exposed orally to five daily doses of 50 mg/kg body weight vinyl chloride were found by this method to be 1 adduct in 2.9 x 10(7) and 1 adduct in 1.4 x 10(7) nucleotides, respectively. In contrast, in a direct labelling study, radiolabelled epsilon dA and epsilon dC were not detected in liver DNA of rats exposed for 6 h by nose-only inhalation to [1,2-14C]vinyl chloride at up to 45 ppm v/v. Immunochemical procedures are also being developed for recognizing etheno adducts. Thus, a monoclonal antibody raised to protein conjugates of epsilon dC showed high selectivity in the recognition of this DNA adduct. When the antibody was immobilized on a solid support and used in an immunoenrichment procedure to purify epsilon dC from a large excess of normal nucleotides, one epsilon dC adduct from about 10(8) normal nucleotides could be resolved. Coupling the immunoaffinity enrichment procedure with capillary zone electrophoresis permitted the detection of approximately one epsilon dC adduct in 3 x 10(6) nucleotides.

Increased oxidative stress and lipid peroxidation are implicated at various stages of carcinogenic processes. Recent studies have shown that reactive hydroxyalkenals derived from lipid peroxidation form the promutagenic exocyclic etheno DNA adducts 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytidine (epsilon dC). A highly selective and sensitive immunoaffinity 32P-postlabelling method has been developed to detect epsilon dA and epsilon dC, with a detection limit of about 5 adducts per 10(10) parent nucleotides, which permitted their measurement in small amounts of human DNA. Background levels of epsilon dA and epsilon dC were detected in normal human tissue DNA, apparently as a result of lipid peroxidation under normal physiological conditions. High levels of epsilon dA and epsilon dC were found in the liver DNA of cancer-prone patients with Wilson disease or primary haemochromatosis. High dietary intake of omega-6 polyunsaturated fatty acids, which are readily oxidized to form enals, increased the epsilon dA and epsilon dC levels in DNA from leukocytes of women. An immunoaffinity-high-performance liquid chromatography-fluorescence method has been developed to measure epsilon dA in human urine. Etheno DNA adducts can now be used as biomarkers to investigate the potential role of oxidative stress and lipid peroxidation in human cancers associated with certain lifestyles or chronic infections and to verify whether the levels of these adducts can be reduced by chemopreventive regimens.