Correlation Between the 8-hydroxy-2'-deoxyguanosine Level in the Peritoneal Solution of Patients Undergoing Peritoneal Dialysis and the Peritoneal Equilibration Test, Kt/V, Ferritin, and Albumin Levels.

INTRODUCTION: Peritoneal dialysis (PD) is an effective treatment  modality for advanced kidney failure, offering patients a significant  degree of independence. However, the long-term use of PD is  limited due to the degeneration of the peritoneal membrane,  resulting in reduced dialysis adequacy. Evaluating the peritoneal  membrane condition in patients with advanced kidney failure  who are undergoing PD is challenging with existing methods.  Therefore, this study aimed to investigate the correlation between  8-hydroxy-2'-deoxyguanosine (8OHDG) levels in the peritoneal  solution of patients undergoing PD and various factors, such  as peritoneal equilibration test (PET), dialysis adequacy (Kt/V),  underlying diseases, serum ferritin, and albumin levels. 8OHDG  is a sensitive marker of oxidative stress caused by DNA damage. METHODS: A total of 56 patients were included in this cross-sectional  study. Five milliliters of PD fluid were collected from the patients,  and 8-OHdG levels were measured using ELISA method. Then, they  were compared with PET, Kt/V, albumin, and ferritin markers in  the patients' files, and the results were analyzed by statistical tests. RESULTS: The study examined the correlation between 8OHDG  and other markers. It was found that this index had significant  associations with PET and underlying HTN (P < .05), whereas no  significant associations were identified with the other markers. CONCLUSION: The results of the present study demonstrate that  the level of 8OHDG, as one of the oxidative stress markers, could  be used to evaluate the function of the peritoneum in patients  undergoing PD. DOI: 10.52547/ijkd.7654.

Association of serum and hair antioxidant minerals with an oxidative stress marker in relation with characteristics of healthy adults: a cross-sectional study.

Excess oxidative stress generated in the body causes various types of cellular damage, including DNA damage. Certain trace minerals act as antioxidants by functioning as cofactors for antioxidant enzymes. This study was conducted to evaluate the serum and hair concentrations of major antioxidant trace minerals (zinc, manganese, selenium, and chromium) and to determine the association between the oxidative stress marker urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and serum or hair antioxidant trace mineral concentrations, according to the general characteristics of healthy adults. Study participants were selected after screening, and 108 participants aged 19-69 years were finally included. Serum and hair trace mineral concentrations were analyzed using inductively coupled plasma mass spectrometry, and urine 8-OHdG levels were quantified using an ELISA kit. Results showed that urinary 8-OHdG levels were significantly higher in exercisers than in those who did not exercise. Correlation analysis revealed that urinary 8-OHdG was negatively correlated with hair zinc in participants over 60 years of age and with poor health status, and positively correlated with hair chromium in participants with irregular dietary habits. In conclusion, these results suggest that urinary 8-OHdG is particularly correlated with hair zinc and chromium levels. Additional large-scale epidemiological studies are needed to generally confirm these findings.

Intracellular ascorbate is a safe-guard and/or reservoir for plasma vitamin C in prostate cancer patients undergoing radiotherapy.

Prostate cancer (PC) represents one of the most common cancer types worldwide and many patients suffering from this kind of cancer are treated with radiotherapy (RTH). Ionizing irradiation is closely associated with reactive oxygen species (ROS) production and oxidative stress. Over the years the role of vitamin C (VC) in cancer prevention has been highlighted as it may be mediated by its ability to neutralize pro-carcinogenic ROS. However, the debate concerning the presence of VC in blood and its beneficial effect on the survival of cancer patients is inconsistent and controversial. To our best knowledge until recently there have been no studies concerning such a role of intracellular VC (iVC). In the present study, blood and intracellular concentrations of vitamin C were analyzed along with the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), as an established marker of the stress condition, in leukocytes of PC patients during the course of radiotherapy. The level of intracellular vitamin C significantly decreased in PC patients in comparison with the healthy group, while there were no differences in blood VC. It was observed that a sub-group of the PC patients reacted to RTH decreasing VC in leukocytes (group A), while the other sub-group acted the other way round, significantly increasing its level (group B). Under stressful conditions (RTH) leukocytes react in two different ways. Both ways are in good agreement with two well recognized functions, proposed for iVC; it may serve as a save factor, to protect the cellular DNA, increasing its concentration inside the cell (group B), and as a reservoir decreasing the VC level inside leukocytes and releasing VC into the plasma to rescue its physiological level (group A). It was also demonstrated that there was a relationship between the level of 8-oxodG in leukocytes' DNA and the markers of RTH toxicity.

DNA oxidative damage in oral cancer: 8-hydroxy-2´-deoxyguanosine immunoexpression assessment

Background: The development and establishment of oral squamous cell carcinoma are confined to carcinogenesis, which involves oxidative stress via oxygen-free radical production as a hydroxyl radical (HO•), considered the most important cause of oxidative damage to basic biomolecules since it targets DNA strands. 8-Hydroxy-2´- deoxyguanosine (8-OHdG) is considered a free radical with a promutagenic capacity due to its ability to pair with adenosine instead of cytosine during replication. Material and Methods: We collected 30 paraffin-embedded tissue samples of OSCC from patients treated between 2013 and 2018. We recorded risk habits, disease stage, disease free survival and death with at least 3 years of followup. 8-Hydroxyguanosine was evaluated by immunohistochemistry and subsequently classified as weak-moderate or strong positive expression. Additionally, we noted whether it was expressed in the cytoplasm and/or nucleus. Results: Most of the cases expressed 8-OHdG with a strong intensity (80%). All neoplastic cells were preferentially stained in only the cytoplasm (70.0%), but nuclear positivity was found in 30%, independent of the intensity. Based on the location in the cytoplasm and/or nucleus, tumors >4 cm showed a high frequency (95.5%) of 8-OHdG expression in only the cytoplasm, with a significant difference (p value ≤ 0.001). Additionally, overall survival was affected when immunoexpression was present in the cytoplasm and nucleus because all deaths were in this group were statistically significant (p value = 0.001). Conclusions: All tumors showed DNA oxidative damage, and 8-OHdG was preferentially expressed in the cytoplasm. This finding was associated with tumor size and, when present in the nucleus, might also be related to death. (AU)

Chemical Insights into Oxidative and Nitrative Modifications of DNA.

This review focuses on DNA damage caused by a variety of oxidizing, alkylating, and nitrating species, and it may play an important role in the pathophysiology of inflammation, cancer, and degenerative diseases. Infection and chronic inflammation have been recognized as important factors in carcinogenesis. Under inflammatory conditions, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated from inflammatory and epithelial cells, and result in the formation of oxidative and nitrative DNA lesions, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-nitroguanine. Cellular DNA is continuously exposed to a very high level of genotoxic stress caused by physical, chemical, and biological agents, with an estimated 10,000 modifications occurring every hour in the genetic material of each of our cells. This review highlights recent developments in the chemical biology and toxicology of 2'-deoxyribose oxidation products in DNA.

DNA oxidative damage in oral cancer: 8-hydroxy-2´-deoxyguanosine immunoexpression assessment.

BACKGROUND: The development and establishment of oral squamous cell carcinoma are confined to carcinogenesis, which involves oxidative stress via oxygen-free radical production as a hydroxyl radical (HO•), considered the most important cause of oxidative damage to basic biomolecules since it targets DNA strands. 8-Hydroxy-2´-deoxyguanosine (8-OHdG) is considered a free radical with a promutagenic capacity due to its ability to pair with adenosine instead of cytosine during replication. MATERIAL AND METHODS: We collected 30 paraffin-embedded tissue samples of OSCC from patients treated between 2013 and 2018. We recorded risk habits, disease stage, disease free survival and death with at least 3 years of follow-up. 8-Hydroxyguanosine was evaluated by immunohistochemistry and subsequently classified as weak-moderate or strong positive expression. Additionally, we noted whether it was expressed in the cytoplasm and/or nucleus. RESULTS: Most of the cases expressed 8-OHdG with a strong intensity (80%). All neoplastic cells were preferentially stained in only the cytoplasm (70.0%), but nuclear positivity was found in 30%, independent of the intensity. Based on the location in the cytoplasm and/or nucleus, tumors >4 cm showed a high frequency (95.5%) of 8-OHdG expression in only the cytoplasm, with a significant difference (p value 0.001). Additionally, overall survival was affected when immunoexpression was present in the cytoplasm and nucleus because all deaths were in this group were statistically significant (p value = 0.001). CONCLUSIONS: All tumors showed DNA oxidative damage, and 8-OHdG was preferentially expressed in the cytoplasm. This finding was associated with tumor size and, when present in the nucleus, might also be related to death.

Single Cell Determination of 7,8-dihydro-8-oxo-2'-deoxyguanosine by Fluorescence Techniques: Antibody vs. Avidin Labeling.

An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG). Although several methods are available for the biochemical analysis of this molecule, its determination at the single cell level may provide significant advantages when investigating the influence of cell heterogeneity and cell type in the DNA damage response. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detection with the glycoprotein avidin has also been proposed because of a structural similarity between its natural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliability and sensitivity is not clear. In this study, we compared the immunofluorescence determination of 8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugated with the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different cell types by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species (ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, our results indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greater than that attained with avidin-AF488. These findings suggest that immunofluorescence techniques are best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage.

Increased dietary vitamin D3 and calcium partially alleviate heat stress symptoms and inflammation in lactating Holstein cows independent of dietary concentrations of vitamin E and selenium.

Twelve multiparous Holstein cows (42.2 ± 5.6 kg of milk/d; 83 ± 27 d in milk) were used in a split-plot design testing the effects of mineral and vitamin supplementation on the time course of animal performance, metabolism, and inflammation markers during heat stress. The main plot was the average concentrations of dietary vitamin E and Se (adequate: 11.1 IU/kg of vitamin E and 0.55 mg/kg of Se, and high: 223 IU/kg of vitamin E and 1.8 mg/kg of Se, respectively). Within each plot, cows were randomly assigned to (1) heat stress (HS) with adequate concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D3 and Ca (HS+D3/Ca; 3,764 IU/kg and 0.97%, respectively), or (3) pair-feeding (PF) in thermoneutrality with adequate concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73% Ca) in a Latin square design with 14-d periods and 7-d washouts. The highest rectal temperature was recorded at 1700 h for HS (39.4°C; mean of d 1 to 14), being 1.2 and 0.8°C greater than for PF and HS+D3/Ca, respectively. Respiratory rate and water intake were higher in HS (73 breaths/min and 115 L/d, respectively) relative to PF (28 breaths/min and 76 L/d). Heat stress decreased dry matter intake progressively, reaching a nadir on d 5 to 7 (33% reduction) and was not different between treatments. Milk yield decreased progressively in all treatments, but remained greater in PF relative to HS from d 3 to 14 (10%), whereas HS and HS+D3/Ca were not different. Milk fat, protein, and lactose concentrations and yields were lower in HS relative to PF from d 3 to 14, but not different between HS and HS+D3/Ca. Relative to PF, preprandial insulin concentrations were increased in HS, whereas plasma nonesterified fatty acids were decreased on d 7 and 14. Plasma lipopolysaccharide-binding protein concentrations increased in HS cows on d 7 and 14, respectively, relative to PF, whereas they were reduced in HS + D3/Ca on d 14. Plasma C-reactive protein, tumor necrosis factor-α, and fecal calprotectin were increased in HS relative to both PF and HS+D3/Ca on d 7 and 14. Rectal temperature was positively associated with plasma lipopolysaccharide-binding protein (r = 0.72), tumor necrosis factor-α (r = 0.74), C-reactive protein (r = 0.87), and with milk somatic cells (r = 0.75). Plasma 8-hydroxy-2-deoxyguanosine concentrations presented a 3-way interaction, where 8-hydroxy-2-deoxyguanosine was lower in HS than in PF on d 7 and 14, and lower in HS+D3/Ca relative to HS on d 14 in the adequate vitamin E and Se treatment, but no effects were observed in the high vitamin E and Se group. Plasma superoxide dismutase concentrations increased over time, and were higher in HS relative to PF on d 14, whereas HS+D3/Ca was similar to HS. Heat stress markedly reduced milk production and milk components while increasing markers of leaky gut and inflammation. In contrast, vitamin D3 and Ca supplementation reduced hyperthermia (d 7-14), markers of leaky gut, and inflammation independent of dietary concentrations of vitamin E and Se.

FapydG in the Shadow of OXOdG-A Theoretical Study of Clustered DNA Lesions.

Genetic information, irrespective of cell type (normal or cancerous), is exposed to a range of harmful factors, which can lead to more than 80 different types of DNA damage. Of these, oxoG and FapyG have been identified as the most abundant in normoxic and hypoxic conditions, respectively. This article considers d[AFapyGAOXOGA]*[TCTCT] (oligo-FapyG) with clustered DNA lesions (CDLs) containing both the above types of damage at the M06-2x/6-31++G** level of theory in the condensed phase. Furthermore, the electronic properties of oligo-FapyG were analysed in both equilibrated and non-equilibrated solvation-solute interaction modes. The vertical/adiabatic ionization potential (VIP, AIP) and electron affinity (VEA, AEA) of the investigated ds-oligo were found as follows in [eV]: 5.87/5.39 and -1.41/-2.09, respectively. The optimization of the four ds-DNA spatial geometries revealed that the transFapydG was energetically privileged. Additionally, CDLs were found to have little influence on the ds-oligo structure. Furthermore, for the FapyGC base-pair isolated from the discussed ds-oligo, the ionization potential and electron affinity values were higher than those assigned to OXOGC. Finally, a comparison of the influence of FapyGC and OXOGC on charge transfer revealed that, in contrast to the OXOGC base-pair, which, as expected, acted as a radical cation/anion sink in the oligo-FapyG structure, FapyGC did not significantly affect charge transfer (electron-hole and excess-electron). The results presented below indicate that 7,8-dihydro-8-oxo-2'-deoxyguanosine plays a significant role in charge transfer through ds-DNA containing CDL and indirectly has an influence on the DNA lesion recognition and repair process. In contrast, the electronic properties obtained for 2,6-diamino-4-hydroxy-5-foramido-2'deoxypyrimidine were found to be too weak to compete with OXOG to influence charge transfer through the discussed ds-DNA containing CDL. Because increases in multi-damage site formation are observed during radio- or chemotherapy, understanding their role in the above processes can be crucial for the efficiency and safety of medical cancer treatment.

Hydrogen exerts neuroprotective effects by inhibiting oxidative stress in experimental diabetic peripheral neuropathy rats.

Diabetic peripheral neuropathy (DPN) is a complex disorder caused by long-standing diabetes. Oxidative stress was considered the critical creed in this DPN pathophysiology. Hydrogen has antioxidative effects on diabetes mellitus and related complications. However, there is still no concern on the beneficial effects of hydrogen in DPN. This paper aimed to evaluate the effects of exogenous hydrogen to reduce the severity of DPN in streptozotocin-induced diabetic rats. Compared with hydrogen-rich saline treatment, hydrogen inhalation significantly reduced blood glucose levels in diabetic rats in the 4th and 8th weeks. With regard to nerve function, hydrogen administration significantly attenuated the decrease in the velocity of motor nerve conduction in diabetic animals. In addition, hydrogen significantly attenuated oxidative stress by reducing the level of malondialdehyde, reactive oxygen species, and 8-hydroxy-2-deoxyguanosine and meaningfully enhanced the antioxidant capability by partially restoring the activities of superoxide dismutase. Further studies showed that hydrogen significantly upregulated the expression of nuclear factor erythroid-2-related factor 2 and downstream proteins such as catalase and hemeoxygenase-1 in the nerves of diabetic animals. Our paper showed that hydrogen exerts significant protective effects in DPN by downregulating oxidative stress via the pathway of nuclear factor erythroid-2-related factor 2, which suggests its potential value in clinical applications.

Cross-sectional and Longitudinal Associations Between Propylene Oxide Exposure and Lung Function Among Chinese Community Residents: Roles of Oxidative DNA Damage, Lipid Peroxidation, and Protein Carbonylation.

BACKGROUND: Toxicologic studies have reported propylene oxide (PO) exposure may harm the respiratory system, but the association between PO exposure and lung function and potential mechanism remains unclear. RESEARCH QUESTION: What is the association between PO exposure and lung function and potential mediating mechanism? STUDY DESIGN AND METHODS: Urinary PO metabolite [N-Acetyl-S-(2-hydroxypropyl)-L-cysteine (2HPMA)] as PO internal exposure biomarker and lung function were measured for 3,692 community residents at baseline and repeated at 3-year follow up. Cross-sectional and longitudinal associations between urinary 2HPMA and lung function were assessed by linear mixed model. Urinary 8-hydroxy-deoxyguanosine, urinary 8-iso-prostaglandin-F2α, and plasma protein carbonyls as biomarkers of oxidative DNA damage, lipid peroxidation, and protein carbonylation, respectively, were measured for all participants to explore their potential roles in 2HPMA-associated lung function decline by mediation analysis. RESULTS: After adjustment for potential covariates, each threefold increase in urinary 2HPMA was cross sectionally associated with a 26.18 mL (95% CI, -50.55 to -1.81) and a 21.83 mL (95% CI, -42.71 to -0.95) decrease in FVC and FEV1, respectively, at baseline (all P < .05). After 3 years of follow up, 2HPMA was observed to be longitudinally associated with FEV1/FVC decline. No significant interaction effect of smoking or passive smoking was observed (Pinteraction > .05), and the associations between 2HPMA and lung function indexes were persistent among participants who were not smoking and those who were not passive smoking in both baseline and follow-up evaluations. We observed urinary 8-hydroxy-deoxyguanosine partially mediated the associations of 2HPMA with FVC (mediation proportion, 5.48%) and FEV1 (mediation proportion, 6.81%), and plasma protein carbonyl partially mediated the association between 2HPMA and FEV1 (mediation proportion, 3.44%). INTERPRETATION: PO exposure was associated with lung function decline among community residents, and oxidative DNA damage and protein carbonylation partially mediated PO exposure-associated lung function decline. Further attention on respiratory damage caused by PO exposure is warranted.

In vivo toxicity assessment of Remazol Gelb-GR (RG-GR) textile dye in zebrafish embryos/larvae (Danio rerio): Teratogenic effects, biochemical changes, immunohistochemical changes.

Dyes, which are very important for various industries, have very adverse effects on the aquatic environment and aquatic life. However, there are limited studies on the toxic properties of dyes on living things. This research elucidated the sublethal toxicity of acute exposure of the textile dye remazol gelb-GR (RG-GR) using zebrafish embryos and larvae for 96 h. The 96 h-LC50 for RG-GR in zebrafish embryos/larvae was determined to be 151.92 mg/L. Sublethal 96 hpf exposure was performed in RG-GR concentrations (0.5; 1.0; 10.0; 100.0 mg/L) to determine the development of toxicity in zebrafish embryos/larvae. RG-GR dye affected morphological development, and decreased heart rate, hatching, blood flow, and survival rates in zebrafish embryos/larvae. The immunopositivity of 8-hydroxy 2 deoxyguanosine (8-OHdG) in larvae exposed to RG-GR at high concentrations was found to be intense. Depending on the RG-GR dose increase, some biochemical parameters such as glutathione peroxidase (GSH) level, acetylcholinesterase (AChE) activity, catalase (CAT) activities, superoxide dismutase (SOD), and nuclear factor erythroid 2 (Nrf-2) levels were detected to be decreased in larvae, while malondialdehyde (MDA) content, nuclear factor kappa (NF-kB), tumor necrosis factor-α (TNF-α), DNA damage (8-OHdG level), interleukin-6 (IL-6) and apoptosis (Caspase-3) levels were found to be increased. The experimental results revealed that RG-GR dye has high acute toxicity on zebrafish embryo/larvae.

Ameliorative effects of the phytochemicals in dates (Phoenix dactylifera) against the toxicological changes induced by fipronil in male albino rats.

Scientific evidence has shown that fipronil induces oxidative stress and genotoxicity. Our study aimed to evaluate the potential oxidation in redox parameters and DNA, as well as determine the protective effect of date extract of increasing resistance to cellular damage. 30 Male albino rats were divided into six groups ( n = 5): 1) control group; 2) treatment group with date extract (1 g/kg B.W.); 3) treatment group with 1/20 LD50 of fipronil; 4) treatment group with 1/40 LD50 of fipronil; 5) treatment group with 1/20 LD50 of fipronil + 1 g/kg date extract; and 6) treatment group with 1/40 LD50 of fipronil + 1 g/kg dates extract. Date extract showed a high content of phenolic compounds and antioxidant properties. Fipronil increased 8-hydroxy-2-deoxyguanosine levels and lipid peroxidation by malondialdehyde but decreased the total antioxidant capacity in plasma. Moreover, glutathione, catalase, and superoxide dismutase levels in the liver and kidney decreased, along with histopathological abnormalities. Additionally, tail moment parameters of liver DNA and micronucleus frequencies in the bone marrow increased. This study showed that fipronil-induced various health hazards in vivo, whereas date extract alleviated the said toxicological effects. However, date extract failed to reduce genotoxicity.

OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION: Sperm telomeres, oxidative stress, and infertility.

In brief: Oxidative stress is recognized as an underlying driving factor of both telomere dysfunction and human subfertility/infertility. This review briefly reassesses telomere integrity as a fertility biomarker before proposing a novel, mechanistic rationale for the role of oxidative stress in the seemingly paradoxical lengthening of sperm telomeres with aging. Abstract: The maintenance of redox balance in the male reproductive tract is critical to sperm health and function. Physiological levels of reactive oxygen species (ROS) promote sperm capacitation, while excess ROS exposure, or depleted antioxidant defenses, yields a state of oxidative stress which disrupts their fertilizing capacity and DNA structural integrity. The guanine moiety is the most readily oxidized of the four DNA bases and gets converted to the mutagenic lesion 8-hydroxy-deoxyguanosine (8-OHdG). Numerous studies have also confirmed oxidative stress as a driving factor behind accelerated telomere shortening and dysfunction. Although a clear consensus has not been reached, clinical studies also appear to associate telomere integrity with fertility outcomes in the assisted reproductive technology setting. Intriguingly, while sperm cellular and molecular characteristics make them more susceptible to oxidative insult than any other cell type, they are also the only cell type in which telomere lengthening accompanies aging. This article focuses on the oxidative stress response pathways to propose a mechanism for the explanation of this apparent paradox.

Polycyclic aromatic hydrocarbon exposure and DNA oxidative damage of workers in workshops of a petrochemical group.

The petrochemical industry has promoted the development of economy, while polycyclic aromatic hydrocarbons (PAHs) produced by the industry become the threat for environment and humans. Data on human occupational exposure in petrochemical industry are limited. In the present study, urinary hydroxylated PAH metabolites (OH-PAHs) and a biomarker of DNA oxidative damage (8-hydroxy-2'-deoxyguanosine (8-OHdG)) were measured in 546 workers of a petrochemical group in Northeast China, to investigate PAH exposure and related potential health risk. The concentrations of ∑9OH-PAH in all workers were 0.25-175 µg/g Cre with a median value of 4.41 µg/g Cre. Metabolites of naphthalene were the predominant compounds. The levels of PAH metabolites were significantly different for workers with different jobs, which were the highest for recycling workers (13.7 µg/g Cre) and the lowest for agency managers (5.12 µg/g Cre). Besides, higher levels of OH-PAHs were usually found in males and older workers. There was a dose-response relationship between levels of 8-OHdG and ∑9OH-PAHs (p < 0.01). No difference was observed in concentrations of 8-OHdG for workers of different gender or ages, work history as well as noise. Furthermore, workers simultaneously exposed to other potential pollutants and higher levels of ∑9OH-PAH had significantly higher levels of 8-OHdG compared with those in the corresponding subgroups. Our results suggested that exposure to PAHs or co-exposure to PAHs and potential toxics in the petrochemical plant may cause DNA damage. We call for more researches on the associations among noise, chemical pollution and oxidative stress to workers in the real working environment.

A Silent Operon of Photorhabdus luminescens Encodes a Prodrug Mimic of GTP.

With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured Eleftheria terra, a betaproteobacterium; odilorhabdins, from Xenorhabdus, are broad-spectrum inhibitors of protein synthesis, and darobactins from Photorhabdus target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. Xenorhabdus and Photorhabdus are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed in vitro. To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3'-amino 3'-deoxyguanosine (ADG). Mutants resistant to ADG mapped to gsk and gmk, kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. IMPORTANCE Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on Photorhabdus, gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express in vitro. To gain access to silent operons, we first fractionated supernatant from Photorhabdus and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3'-amino 3'-deoxyguanosine (ADG), active against E. coli. ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2'-deoxyguanosine.

The aptamer portions of previously reported riboswitch classes that sense guanine, adenine, or 2'-deoxyguanosine are formed by a highly similar three-stem junction with distinct nucleotide sequences in the regions joining the stems. The nucleotides in these joining regions form the major features of the selective ligand-binding pocket for each aptamer. Previously, we reported the existence of additional, rare variants of the predominant guanine-sensing riboswitch class that carry nucleotide differences in the ligand-binding pocket, suggesting that these RNAs have further diversified their structures and functions. Herein, we report the discovery and analysis of three naturally occurring variants of guanine riboswitches that are narrowly distributed across Firmicutes. These RNAs were identified using comparative sequence analysis methods, which also revealed that some of the gene associations for these variants are atypical for guanine riboswitches or their previously known natural variants. Binding assays demonstrate that the newfound variant riboswitch representatives recognize xanthine, guanine, or 2'-deoxyguanosine, with the guanine class exhibiting greater discrimination against related purines than the more common guanine riboswitch class reported previously. These three additional variant classes, together with the four previously discovered riboswitch classes that employ the same three-stem junction architecture, reveal how a simple structural framework can be diversified to expand the range of purine-based ligands sensed by RNA.

LC-MS/MS for Assessing the Incorporation and Repair of N2-Alkyl-2'-deoxyguanosine in Genomic DNA.

Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N2-methyl-dG (N2-MedG) and N2-n-butyl-dG (N2-nBudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N2-MedG and N2-nBudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N2-MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N2-MedG and N2-nBuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N2-alkyl-dG lesions in mammalian cells.

The influence of cdG on 8-oxodG excision by OGG1 and FPG glycosylases.

Human genome is exposed to the variety of damaging factors, such as ionizing radiation. 5',8-cyclo-2'-deoxypurines (cdPus) are well described unfavorable outcomes of DNA damage, especially devastating as a part of clustered DNA lesions (CDL). Since cdPus are not repaired by base excision repair (BER) and poorly repaired by nucleotide excision repair (NER), it is important to unveil the mechanisms of cdPus action within the genome. In this study the influence of both 5'S and 5'R diastereomers of 5',8-cyclo-2'-deoxyguanosine (cdG) on the activity of OGG1 and FPG was examined. Synthetic oligonucleotides containing cdG and two molecules of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were designed as model of single-stranded CDL. The activity of both enzymes increased in the presence of cdG, compared to the control DNA strands, and the increase was greater in the case of 5'R diastereomer. These results are supported by previous studies concerning cdPus and confirm the impact of lesions proximity on the DNA repair efficiency. Due to the biological importance of cdPus, it is necessary to understand the mechanisms of lesions recognition by repair proteins in further studies.

Site-Specific Synthesis of Oligonucleotides Containing 6-Oxo-M1dG, the Genomic Metabolite of M1dG, and Liquid Chromatography-Tandem Mass Spectrometry Analysis of Its In Vitro Bypass by Human Polymerase ι.

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M1dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M1dG in genomic DNA is enzymatically oxidized to 6-oxo-M1dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M1dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M1dG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M1dG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-M1dG blocks further replication by the enzyme.