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

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

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

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

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.     

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.     

Serum cholesterol positively associated with oxidative DNA damage: a propensity score-matched analysis

Oxidative DNA damage pathogenically links to some major diseases. This study aimed to comprehensively assess the association between serum total cholesterol (TC) and oxidative DNA damage based on propensity score matching (PSM) method. A total of 407 participants chronically exposed to arsenic via drinking water from China were enrolled. Oxidative DNA damage was determined with urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG). Serum TC was classified into favourable TC (FTC, TC <5.18?mmol/L) and unfavourable TC (NFTC, TC ≥5.18?mmol/L) categories. Multivariable generalised linear regression model was applied to examine the association. Of 407 participants, 125 pairs with FTC and NFTC subjects were matched using PSM. Urinary 8-OHdG/creatinine levels in NFTC were significantly higher than those in FTC category (p?=?.002). As compared to the counterparts, additional adjusted log-transformed 8-OHdG/creatinine increase was observed in NFTC for unmatched (β?=?0.12, p?=?.052) and matched (β?=?0.17, p?<?.001) participants, respectively. We also detected obviously increased log-transformed urinary 8-OHdG/creatinine with per interquartile range raise of serum TC either in unmatched (β?=?0.10, p?=?.007) or matched (β?=?0.16, p?=?.003) subjects. In conclusion, serum TC was independently associated with oxidative DNA damage. Our findings provided new insights on the health promotion of lipids relevant to the early warning of diseases due to oxidative DNA damage.     

Mechanisms of DNA damage induced by morin,an inhibitor of amyloid β-peptide aggregation

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer’s disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on ³²P-5′-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical ?OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H₂O₂ generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.     

Anticancer effect of selenium/chitosan/polyethylene glycol/allyl isothiocyanate nanocomposites against diethylnitrosamine-induced liver cancer in rats

BackgroundNano-based drug delivery systems have shown several advantages in cancer treatment like specific targeting of cancer cells, good pharmacokinetics, and lesser adverse effects. Liver cancer is a fifth most common cancer and third leading cause of cancer-related mortalities worldwide.ObjectiveThe present study focusses to formulate the selenium (S)/chitosan (C)/polyethylene glycol (Pg)/allyl isothiocyanate (AI) nanocomposites (SCPg-AI-NCs) and assess its therapeutic properties against the diethylnitrosamine (DEN)-induced liver cancer in rats via inhibition of oxidative stress and tumor markers.MethodologyThe SCPg-AI-NCs were synthesized by ionic gelation technique and characterized by various characterization techniques. The liver cancer was induced to the rats by injecting a DEN (200 mg/kg) on the 8th day of experiment. Then DEN-induced rats treated with 10 mg/kg of formulated SCPg-AI-NCs an hour before DEN administration for 16 weeks. The 8-hydroxy-2′ -deoxyguanosine (8-OHdG) content, albumin, globulin, and total protein were examined by standard methods. The level of glutathione (GSH), vitamin-C & -E, and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities were examined using assay kits. The liver marker enzymes i.e., alanine transaminase (ALT), aspartate tansaminase (AST), γ-glutamyl transaminase (GGT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) activities, alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA), Bax, and Bcl-2 levels, and caspase-3&9 activities was examined using assay kits and the liver histopathology was assessed microscopically by hematoxylin and eosin staining method. The effect of formulated SCPg-AI-NCs on the viability and apoptotic cell death on the HepG2 cells were examined using MTT and dual staining assays, respectively.ResultsThe results of different characterization studies demonstrated the formation of SCPg-AI-NCs with tetragonal shape, narrowed distribution, and size ranging from 390 to 450 nm. The formulated SCPg-AI-NCs treated liver cancer rats indicated the reduced levels of 8-OHdG, albumin, globulin, and total protein. The SCPg-AI-NCs treatment appreciably improved the GSH, vitamin-C & -E contents, and SOD, CAT, GPx, and GR activities in the serum of liver cancer rats. The SCPg-AI-NCs treatment remarkably reduced the liver marker enzyme activities in the DEN-induced rats. The SCPg-AI-NCs treatment decreased the AFP and CEA contents and enhanced the Bax and caspase 3&9 activities in the DEN-induced rats. The SCPg-AI-NCs effectively decreased the cell viability and induced apoptosis in the HepG2 cells.ConclusionThe present findings suggested that the formulated SCPg-AI-NCs remarkably inhibited the DEN-induced liver carcinogenesis in rats. These findings provide an evidence that SCPg-AI-NCs can be a promising anticancer nano-drug in the future to treat the liver carcinogenesis.     

The Effects of Different Buffers on the Oxidation of DNA by Thiols and Ferric Iron

Because buffers can act as metal ligands, they can effect several reactions necessary for DNA oxidation by ferric iron and thiols, such as iron reduction. Therefore, these reactions were studied in Hepes and phosphate buffers and unbuffered NaCl. Reduction of Fe³⁺ by dithiothreitol (DTT) and cysteine was observed in either Hepes or NaCl solutions, but not in phosphate buffer. Thiyl radicals were observed in Hepes, but there was much less thiyl radical production in the saline or phosphate solutions. Redox cycling between either DTT or cysteine and Fe³⁺ also resulted in dioxygen consumption in Hepes buffer. Reduction of Fe³⁺ and O₂ resulted in the formation of an oxidant capable of producing 8-hydroxy-2′-deoxyguanosine (8-OHdG) in calf-thymus DNA. The highest levels of 8-OHdG were detected when DTT or cysteine and Fe³⁺ were incubated in Hepes, while much less DNA oxidation was detected when the experiment was done in a saline solution, and almost no DNA oxidation occurred in the phosphate buffer. These results demonstrate that the use of different buffers can greatly affect the ability of thiols to promote iron-dependent oxidations. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 125–132, 1998     

Oxidative stress-related risk of the multiple sclerosis development

BackgroundMultiple sclerosis (MS) is characterized by inflammation, demyelination and axonal degeneration. Oxidative stress (OS) plays a significant role in the pathogenesis of the disease. The aim of the study was to examine the association between OS and smoking on the MS development.MethodsThe study included 175 patients with relapsing-remitting multiple sclerosis (RRMS) (76 males, 99 females) and 254 healthy subjects (81 males and 173 females). Oxidative stress biomarkers in serum, Total Antioxidant Status (TAS) and Total Oxidative Status (TOS) were determined spectrophotometrically. Oxidative Stress Index (OSI) was calculated as the ratio of TOS and TAS. Urinary 8-oxo7,8-dihydro-2''-deoxyguanosine were determined by HPLC-MS/MS and expressed as 8-oxodG/creatinine.ResultsIn females with RRMS were higher TOS, OSI and 8-oxodG/creatinine than in females in control group. The group of males with RRMS had lower level of TAS than the males in control group. Higher levels of 8-oxodG/creatinine was obtained in active, passive and former smokers with RRMS than in control group with the same exposition to tobacco smoke. Independent predictors of MS are passive smoking, increased OSI and increased levels of urinary 8-oxodG/creatinine.ConclusionsOur results demonstrate that the OS parameters should be included in the assessment of the risk for MS development. Due to the more sensitivity to oxidative stress, females may be at higher risk of MS development. This data indicates the importance of introducing the antioxidant therapy as a complementary treatment in patients with RRMS.     

Age-related increases of 8-hydroxy-2′-deoxyguanosine and DNA–protein crosslinks in mouse organs

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and DNA–protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by ³²P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during ³²P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8±0.7 8-OH-dG/10⁵ normal nucleotides), brain (1.6±0.5) and heart (2.3±0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7±0.4; brain, 3.6±1.1; heart, 6.8±2.2 8-OH-dG/10⁵ normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P<0.01), and 3.0-fold increase in heart (P<0.001). A similar trend was observed for DPXL levels, which were the 1.8±0.3%, 1.2±0.2%, and 2.2±0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9±0.4%, 2.0±0.4%, and 3.4±0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P<0.01), and 1.5 (P<0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r=0.619, P<0.001) and heart (r=0.800, P<0.0001), but not in liver (r=0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.