Suppression of 8-oxo-2'-deoxyguanosine formation and carcinogenesis induced by N-nitrosobis (2-oxopropyl)amine in hamsters by esculetin and esculin

Effects of esculetin (6,7-dihydroxycoumarin) and its glycoside, esculin, on 8-oxo-2'-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were administered esculetin by gastric intubation into the stomach 30 min before BOP administration or ingestion of a diet containing esculin for 7 days before BOP administration, and killed 1 or 4 h after BOP treatment, and the contents of thiobarbituric acid-reacting substrates (TBARS) and 8-oxodG in the pancreas were determined. Both compounds suppressed significantly the BOP-induced increases in 8-oxodG and TBARS contents in hamster pancreas. We further investigated the effect of esculin on pancreatic carcinogenesis by the rapid production model induced by augmentation pressure with a choline-deficient diet, ethionine, methionine and BOP. Esculin was given ad libitum as a 0.05% aqueous solution in either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly smaller than in the control group, while esculin given during the promotion phase showed no apparent effects. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a reliable oxidative stress marker in hypertension

The potential use of oxidative stress products as disease markers and progression is an important aspect of biomedical research. In the present study, the quantification of urine 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) concentration has been used to express the oxidation status of hypertensive subjects.

8-oxo-dG has been simultaneously isolated and assayed in nuclear (nDNA) and mitochondrial DNA (mtDNA). In addition, oxidative stress of mononuclear cells has been estimated by means of GSH and GSSG levels and GSSG/GSH ratio in hypertensive subjects before and after antihypertensive treatment. It is shown that oxidative stress decreases significantly in hypertensive patients after treatment the effect being accompanied by reduction of their blood pressure.

A significant correlation is observed comparing the yield of urine 8-oxo-dG and that isolated from mitochondria DNA. Moreover, urinary excretion of 8-oxo-dG also correlates with the GSSG/GSH ratio of cells. Conclusion: urine 8-oxo-dG assay is a good marker for monitoring oxidative stress changes in hypertensives.     

Recognition and detection of 8-oxo-rG in RNA using the DNA/OMeRNA chimera probes containing fluorescent adenosine-diazaphenoxazine analog

Recent studies indicate that oxidative damage to RNA results in dysfunction of translation and eventual pathogenesis. A representative oxidized base in RNA is 8-hydroxyguanosine (8-oxo-rG), however, unlike its DNA counterpart (8-oxo-dG), its role in pathogenesis has not attracted much attention until recently. The 2′-deoxyadenosine derivative with a diazaphenoxazine skeleton at the 6-amino group (Adap) was shown to be selective for 8-oxo-dG in DNA. In this study, the 2′-O-methoxy derivative of Adap (2′-OMeAdap) was designed as a selective molecule for 8-oxo-rG in RNA. 8-Oxo-rG in the homopurine RNA was selectively recognized by the ODN probe incorporating Adap. In contrast, although it was not possible by the Adap-containing ODN prove due to the instability of the corresponding duplex, 8-oxo-rG in homopyrimidine RNA was selectively detected by the 2′-OMeRNA probe incorporating 2′-OMeAdap.     

Isotope dilution high-performance liquid chromatography–electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in biological samples

A sensitive and specific assay aimed at measuring 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) has been developed by associating a reversed-phase liquid chromatographic separation with an electrospray tandem mass spectrometric detection. The HPLC–MS approach in the single ion monitoring (SIM) mode and the HPLC–MS/MS assay in the multiple reaction monitoring (MRM) mode have been compared, using isotopically labeled [M+4] 8-oxodGuo as the internal standard. The limit of detection of 8-oxodGuo was found to be around 5 pmol and 20 fmol for the HPLC–MS and HPLC–MS/MS methods, respectively. The HPLC–MS/MS assay is sensitive enough to allow the determination of the level of 8-oxodGuo in cellular liver DNA and in urine samples.     

Combination of azathioprine and UVA irradiation is a major source of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine

Thiopurine antimetabolites, such as azathioprine (Aza) and 6-thioguanine (6-TG), are widely used in the treatment of cancer, inflammatory conditions and organ transplantation patients. Recent work has shown that cells treated with 6-TG and UVA generate ROS, with implied oxidatively generated modification of DNA. In a study of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in renal transplant patients, we provided the first in vivo evidence linking Aza and oxidatively damaged DNA. Using the hOGG1 comet assay, we herein demonstrate high levels of 8-oxodG and alkali-labile sites (ALS) in cells treated with biologically relevant doses of 6-TG, or Aza, plus UVA. This damage was induced dose-dependently. Surprisingly, given the involvement of 6-TG incorporation into DNA in its therapeutic effect, significant amounts of 8-oxodG and ALS were induced in quiescent cells, although less than in proliferating cells. We speculate that some activity of hOGG1 towards unirradiated, 6-TG treated cells, implies possible recognition of 6-TG or derivatives thereof. This is the first report to conclusively demonstrate oxidatively damaged DNA in cells treated with thiopurines and UVA. These data indicate that Aza-derived oxidative stress will occur in the skin of patients on Aza, following even low level UVA exposure. This is a probable contributor to the increased risk of non-melanoma skin cancer in these patients. However, as oxidative stress is unlikely to be involved in the therapeutic effects of Aza, intercepting ROS production in the skin could be a viable route by which this side effect may be minimised.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

Assessment of oxidative DNA damage formation by organic complex mixtures from airborne particles PM(10)

The free radical generating activity of airborne particulate matter (PM₁₀) has been proposed as a primary mechanism in biological activity of ambient air pollution. In an effort to determine the impact of the complex mixtures of extractable organic matter (EOM) from airborne particles on oxidative damage to DNA, the level of 8-oxo-2′-deoxyguanosine (8-oxodG), the most prevalent and stable oxidative lesion, was measured in the human metabolically competent cell line Hep G2. Cultured cells were exposed to equivalent EOM concentrations (5–150 μg/ml) and oxidative DNA damage was analyzed using a modified single cell gel electrophoresis (SCGE), which involves the incubation of whole cell DNA with repair specific DNA endonuclease, which cleaves oxidized DNA at the sites of 8-oxodG. EOMs were extracted from PM₁₀ collected daily (24 h intervals) in three European cities: Prague (Czech Republic, two monitoring sites, Libuš and Smíchov), Košice (Slovak Republic) and Sofia (Bulgaria) during 3-month sampling periods in the winter and summer seasons. No substantial time- and dose-dependent increase of oxidative DNA lesions was detected in EOM-treated cells with the exception of the EOM collected at the monitoring site Košice, summer sampling. In this case, 2 h cell exposure to EOM resulted in a slight but significant increase of oxidative DNA damage at three from total of six concentrations. The mean 8-oxodG values at these concentrations ranged from 15.3 to 26.1 per 10⁶ nucleotides with a value 3.5 per 10⁶ nucleotides in untreated cells. B[a]P, the positive control, induced a variable but insignificant increase of oxidative DNA damage in Hep G2 cell (approximately 1.6-fold increase over control value).

Based on these data we believe that EOM samples extracted from airborne particle PM₁₀ play probably only a marginal role in oxidative stress generation and oxidative lesion formation to DNA. However, adsorbed organic compounds can undergo various interactions (additive or synergistic) with other PM components or physical factors (UV-A radiation) and in this way they might enhance/multiply the adverse health effects of air pollution.     

Dilazep analogues for the study of equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2)

As ENT inhibitors including dilazep have shown efficacy improving oHSV1 targeted oncolytic cancer therapy, a series of dilazep analogues was synthesized and biologically evaluated to examine both ENT1 and ENT2 inhibition. The central diamine core, alkyl chains, ester linkage and substituents on the phenyl ring were all varied. Compounds were screened against ENT1 and ENT2 using a radio-ligand cell-based assay. Dilazep and analogues with minor structural changes are potent and selective ENT1 inhibitors. No selective ENT2 inhibitors were found, although some analogues were more potent against ENT2 than the parent dilazep.     

Renal excretion of 8-oxo-7,8-dihydro-2(')-deoxyguanosine: degradation rates of RNA and metabolic rate in humans

Renally excreted 8-oxo-7,8-dihydro-2(')-deoxyguanosine (oxo(8)dG) is a potential marker of oxidative DNA damage by reactive oxygen species. Whole-body degradation rates of t- and rRNA are potential indicators of the resting metabolic rate (RMR). Excretion rates of oxo(8)dG and degradation rates of t- and rRNA were determined in healthy non-smoking adults and children. RMR (indirect calorimetry; 14 children, 16 adults), total energy expenditure (TEE; doubly labelled water technique; 4 children, 6 adults), and lean body mass (LBM; dual-energy X-ray absorptiometry; 14 children, 16 adults) were also measured. Degradation of t- and rRNA (micromol/d/kg LBM; 4 children, 6 adults) was highly correlated with RMR (kJ/d/kg LBM), r=0.867 (p<0.005) and 0.959 (p<0.001), respectively. Excretion of oxo(8)dG (pmol/d/kg LBM; 14 children, 16 adults) was not significantly correlated with RMR (p>0.05). Neither excretion of oxo(8)dG nor degradation of RNA was significantly correlated with TEE (kJ/d/ kg LBM) (p>0.05). In healthy subjects further factors, other than the metabolic rate, seem to influence the excretion rate of oxo(8)dG. The degradation rates of t- and rRNA seem to be appropriate indicators of the RMR.     

Recent developments in analytical methodology for 8-hydroxy-2'-deoxyguanosine and related compounds

When biomolecules such as proteins, lipids, and DNA are subjected to oxidative attack by free radicals or other reactive species, a number of measurable biomarkers may be produced. The study of oxidative DNA damage is valuable in research concerning cancer and aging. The current review includes methodology involving various separation science techniques for the analysis of DNA oxidation biomarkers, mainly 8-hydroxy-2'-deoxyguanosine. This review will present recent analytical developments with respect to sample preparation and instrumental considerations, noting key outcomes and biological relevance where appropriate.     

Comparison of results from different laboratories in measuring 8-oxo-2'-deoxyguanosine in synthetic oligonucleotides

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up in 1997 to resolve methodological problems and to reach agreement on the basal level of 8-oxo-2'-deoxyguanosine (8-oxodG) in biological samples. In the present ESCODD trial 6 samples of 8-oxodG-containing oligonucleotides with different ratios of 8-oxodG/2'-deoxyadenosine (dAdo) were sent to 25 laboratories throughout Europe. The methods used were HPLC with electrochemical detection (amperometric or coulometric), GC-MS or LC-MS-MS. The LC-MS-MS and the coulometric HPLC analyses gave 8-oxodG concentrations within 53 and 73% of expected values, respectively, whereas the amperometric HPLC and GC-MS consistently overestimated the 8-oxodG concentration by several fold. As the oligonucleotides contained no 2'-deoxyguanosine (dGuo), this was not due to artificial oxidation. On the contrary, in most cases the concentrations of dAdo and thymidine (dThd), used as estimates for non-oxidised DNA bases were underestimated, though a few laboratories overestimated the lowest concentration samples containing 8 and 20 μM, respectively. In one-third of the reported results, the ratio of 8-oxodG/10 5 dAdo was within 25% of the calculated value in the oligonucleotide samples and in half of the results the coefficient of variation in duplicate samples was less than 10%. The coefficients of variation were higher for the dAdo concentrations than for 8-oxodG. Our findings strongly indicate that careful quality control must be applied to the analytical procedures for 8-oxodG and very importantly also to the procedures for non-modified 2'-deoxyribonucleosides. We recommend the use of synthetic oligonucleotides for this purpose.     

Purine nucleoside phosphorylase (Np) in the mouse: Linkage relationship of Np-2 to esterase-10 (Es-10) and Np-1 on chromosome 14

An improved method for detecting four Np-1 (purine nucleoside phosphorylase) alleles in mouse erythrocytes by cellulose acetate electrophoresis is described. The previous linkage of Np-1 and Es-10 (esterase-10) was confirmed, with a map distance of 13.0±2.6 cM. Np-2 was detected by either specific activity assay or starch gel electrophoresis and shown to be linked to Es-10, 15.9 ± 3.1 cM, on chromosome 14. No recombinants between Np-1 and Np-2 were observed in 52 offspring, indicating either that these loci are either closely associated or that Np-2 represents simply a property of existing allelic products of the Np-1 locus.This research was supported by Medical Research Council of Canada grants to F.G.B. and F.F.S.     

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2) inhibitors: Structure-based virtual screening,optimization and biological evaluation

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders and pulmonary hypertension. Herein, we identified that clofarabine (4), an FDA-approved drug, displayed potential PDE2 inhibitory activity (IC₅₀?=?3.12?±?0.67?μM) by structure-based virtual screening and bioassay. Considering the potential therapeutic benefit of PDE2, a series of purine nucleoside derivatives based on the structure and binding mode of 4 were designed, synthesized and evaluated, which led to the discovery of the best compound 14e with a significant improvement of inhibitory potency (IC₅₀?=?0.32?±?0.04?μM). Further molecular docking and molecular dynamic (MD) simulations studies revealed that 5′-benzyl group of 14e could interact with the unique hydrophobic pocket of PDE2 by forming extra van der Waals interactions with hydrophobic residues such as Leu770, Thr768, Thr805 and Leu809, which might contribute to its enhancement of PDE2 inhibition. These potential compounds reported in this article and the valuable structure-activity relationships (SARs) might bring significant instruction for further development of potent PDE2 inhibitors.     

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.     

Interrelationship between oxidative stress,DNA damage and cancer risk in diabetes (Type 2) in Riyadh,KSA

Type 2 Diabetes Mellitus (T2DM) is the most widely known type of disorder of the endocrine system marked by hyperglycemia resulting either due to deficiency of insulin and or resistance. Persistent hyperglycemia induces oxidative stress and is suggested to play a prominent role in the pathophysiology underlying T2DM. Besides, oxidative stress can result in DNA damage leading to high cancer risk. Current study aimed to evaluate status of oxidative damage, damage to DNA and cancer biomarkers in regard to increased glucose in T2DM patients and to correlate the glycemic state with cancer. A total of 150 subjects consisting of control (50) and T2DM patients (1 0 0) were enrolled. Additionally, three tertiles were created among the two groups based on levels of HbA1c (Tertile I = 5.37 ± 0.34, n = 50; Tertile II = 6.74 ± 0.20, n = 50; Tertile III = 9.21 ± 1.47, n = 50). Oxidative stress parameters including malondialedehyde (MDA) and antioxidant enzymes were measured. Damage to DNA was analyzed by measuring the levels of DNA damage adduct-8 hydroxy deoxy Guanosine (8-OHdG). To detect cancer resulting from oxidative stress, cancer biomarkers CEA, AFP, CA125, CA-15, CA19-9, prolactin were measured in these subjects. All measurements were analysed by SPSS software. Levels of MDA and antioxidant enzymes altered significantly in T2DM group at p < 0.001 and p < 0.05 level of significance. Significant DNA damage accompanied with elevated levels of CEA, CA19-9 and decreased CA125, AFP and prolactin were noted in T2DM group. CA 19-9 and CEA levels increased at p < 0.05, whereas levels of prolactin decreased significantly (p < 0.001) in T2DM group compared to control. Additionally the mean values of DNA damage adduct 8-OHdG differ significantly at P < 0.01 between the two groups. However, no significant correlation in oxidative stress parameter, antioxidant enzymes, DNA damage and neither with the highest tertile of HbA1c (>7.5%) was noted. Based on the results obtained in the present study, we conclude that there is considerable change in oxidative stress and DNA damage in T2DM patients. Hence, assumption that the oxidative stress could cause cancer in T2DM as a result of hyperglycemic state was not speculated in this study.     

Spectroscopic and kinetic studies of interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, and its 9-(2-phosphonylmethoxyethyl) derivative

Spectroscopic and kinetic studies of interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, an excellent fluorescent/fluorogenic substrate for the synthetic pathway of the reaction, and its 9-(2-phosphonylmethoxyethyl) derivative, a bisubstrate analogue inhibitor, were carried out. The goal was to clarify the catalytic mechanism of the enzymatic reaction by identification of ionic/tautomeric forms of these ligands in the complex with PNP.     

Hydantoin derivative formation from oxidation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and incorporation of 14C-labeled 8-oxodG into the DNA of human breast cancer cells

One-electron oxidation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) yielded a guanidinohydantoin derivative (dGh) and a spiroiminodihydantoin derivative (dSp), both putatively mutagenic products that may be formed in vivo. The nucleoside dGh was the major product at room temperature, regardless of pH. The results are contrary to previously published model studies using 2',3',5'-triacetoxy-8-oxo-7,8-dihydroguanosine (Luo, W.; Miller, J. G.; Rachlin, E. M.; Burrows, C. J. Org. Lett. 2000, 2, 613; Luo, W.; Miller, J.G.; Rachlin, E.M.; Burrows, C.J. Chem. Res. Toxicol. 2001, 14, 927), who observed a spiroiminodihydantoin derivative as the major product at neutral pH. Clearly, the functional groups attached to the ribose moiety of 8-oxodG influence the oxidation chemistry of the nucleobase derivative. To explore this chemistry in vivo, (14)C-labeled 8-oxodG was synthesized and incubated with growing MCF-7 human breast cancer cells, resulting in the incorporation of the compound into cellular DNA as measured by a novel accelerator mass spectrometry assay.     

New derivative of 2-(2,4-dihydroxyphenyl)thieno-1,3-thiazin-4-one (BChTT) elicits antiproliferative effect via p38-mediated cell cycle arrest in cancer cells

2-(2,4-Dihydroxyphenyl)thieno-1,3-thiazin-4-ones are a group of new compounds with potential anticancer activity. This type of derivatives was poorly investigated in the area of synthesis and biological activities. In the present study the antiproliferative action of the most active derivative BChTT was described. The aim of biological evaluation was to investigate the ability of the compound to inhibit cancer cell proliferation and identify mechanism involved in its action on the molecular level. BChTT inhibited the proliferation of lung cancer A549, colon cancer HT-29 and glioma C6 cells in the concentration-dependent manner. It was not toxic to normal cells including skin fibroblasts, hepatocytes and oligodendrocytes in the antiproliferative concentrations. BChTT decreased the DNA synthesis in the treated cancer cells and induced cell cycle arrest in the G0/G1 phase. Moreover, the ability of the compound to activate p38 kinase and decrease cyclin D1 expression was estimated. Participation of p38 kinase in the antiproliferative action of the compound was confirmed by the analysis of BChTT activity in the cells with the p38 silenced gene. The obtained results may suggest the ability of the tested derivative to inhibit cancer cells proliferation by induction of p38-mediated cyclin D1 downregulation.     

Oxidative DNA damage in tissues of English sole (Parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

Discovery of novel 4-(2-fluorophenoxy)quinoline derivatives bearing 4-oxo-1,4-dihydrocinnoline-3-carboxamide moiety as c-Met kinase inhibitors

A series of novel 4-(2-fluorophenoxy)quinoline derivatives containing 4-oxo-1,4-dihydrocinnoline-3-carboxamide moiety were designed, synthesized and evaluated for their in vitro biological activities against c-Met kinase and six typical cancer cell lines (A549, H460, HT-29, MKN-45, U87MG and SMMC-7721). All the prepared compounds showed moderate to excellent antiproliferative activity, and the analysis of their structure–activity relationships indicated that 2-chloro or 2-trifluoromethyl substituted phenyl group on the 1-position of cinnoline ring was more favorable for antitumor activity. In this study, a promising compound 33, with a c-Met IC₅₀ value of 0.59 nM, was identified as a multitargeted receptor tyrosine kinase inhibitor.