Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly impacts gene expression and serves as a pivotal factor in stimulating neurodegenerative disorders, cancer, and aging. Therefore, precise quantification of 8-oxoG has clinical significance in the investigation of DNA damage detection methodologies. However, at present, the existing approaches for 8-oxoG detection pose challenges in terms of convenience, expediency, affordability, and heightened sensitivity. We employed the sandwich enzyme-linked immunosorbent assay (ELISA) technique, a highly efficient and swift colorimetric method, to detect variations in 8-oxo-dG content in MCF-7 cell samples stimulated with different concentrations of hydrogen peroxide (H2O2). We determined the concentration of H2O2 that induced oxidative damage in MCF-7 cells by detecting its IC50 value in MCF-7 cells. Subsequently, we treated MCF-7 cells with 0, 0.25, and 0.75 mM H2O2 for 12 h and extracted 8-oxo-dG from the cells. Finally, the samples were subjected to ELISA. Following a series of steps, including plate spreading, washing, incubation, color development, termination of the reaction, and data collection, we successfully detected changes in the 8-oxo-dG content in MCF-7 cells induced by H2O2. Through such endeavors, we aim to establish a method to evaluate the degree of DNA oxidative damage within cell samples and, in doing so, advance the development of more expedient and convenient approaches for DNA damage detection. This endeavor is poised to make a meaningful contribution to the exploration of associative analyses between DNA oxidative damage and various domains, including clinical research on diseases and the detection of toxic substances.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker of oxidative DNA damage induced by occupational exposure to nanomaterials: a systematic review.

In nuclear and mitochondrial DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of reactive oxygen species (ROSs) lesions, which commonly used as a biomarker for oxidative stress. Studies showed that the different nanomaterials can induce toxicity by ROSs in human body. So, this study is going to review the studies about oxidative DNA damage caused by occupational exposure to nanomaterials, using 8-OHdG biomarker.Systematic review was managed based on Cochrane systematic review guideline. Literature search was conducted in scientific databases with the main terms of "biomarkers," "biological markers," combined with "occupational exposure" and "nanomaterials." All papers in the field of occupational exposure to nanomaterials until 2020 December were included. To evaluate the quality and bias of studies, GRADE method (Grading of Recommendations, Assessment, Development, and Evaluation) was used.Two hundred twenty-six studies were primarily achieved. By considering the inclusion criteria, overall 8 articles were selected. The majority of the studies were classified as the moderate quality studies (six studies). Also, the study-level bias was critical. This review shows that there is a significant relationship between job title and amount of produced nanomaterials and the existence of 8-OHdG. Also, the levels of 8-OHdG can be measured in urine, blood, and inhalation samples by instrumental procedures.Oxidative damages are an important threat for workers exposed to nanomaterial. Blood and EBC 8-OHdG level can be introduced as a biomarker for metal nanomaterials, but urinary 8-OHdG needs to be taken with caution. So, it is recommended that evaluation not be solely based on one biomarker.

Detection of an alcohol-associated cancer marker by single-molecule quantum sequencing.

Alcoholic beverages are a well-known risk factor for cancer. N2-Ethyl-2'-deoxyguanosine (N2-Et-dG) is a promising biomarker for alcohol-associated cancers. However, the lack of a convenient detection method for N2-Et-dG hinders the development of practical DNA damage markers. Herein, we develop a detection method for N2-Et-dG using a single-molecule quantum sequencing (SMQS) method and machine learning analysis. Our method succeeded in discriminating between N2-Et-dG and dG with an accuracy of 99%, using 20 signals. Our developed method quantified the mixing ratio of N2-Et-dG from a mixed solution of N2-Et-dG and dG. It is shown that our method has the potential to facilitate the development of DNA damage markers, and thus the early detection and prevention of cancers.

The Role of DNA Repair Glycosylase OGG1 in Intrahepatic Cholangiocarcinoma.

BACKGROUND/AIM: The effects of oxidative stress on various carcinomas were reported in previous studies, but those in intrahepatic cholangiocarcinoma (ICC) have not been fully elucidated. The purpose of this study was, thus, to reveal the effects of oxidative DNA damage and repair enzymes on ICC. MATERIALS AND METHODS: The levels of 8-hydroxydeoxyguanosine (8-OHdG) and 8-OHdG DNA glycosylase (OGG1) were immunohistochemically evaluated in specimens resected from 63 patients with ICC. RESULTS: Low OGG1 expression was related to tumour depth T4 (p=0.04), venous invasion (p=0.0005), lymphatic vessel invasion (p=0.03), and perineural invasion (p=0.03). Compared to the high-OGG1-expression group, patients with low OGG1 expression had a significantly poorer prognosis (overall survival: p=0.04, recurrence-free survival: p=0.02). Unlike for OGG1, the expression levels of 8-OHdG showed no association with prognosis. CONCLUSION: Oxidative DNA damage and DNA repair enzymes may be closely related to ICC progression.

Predictive value of the ratio of 8­hydroxydeoxyguanosine levels between cancerous and normal tissues in patients with stage II/III colorectal cancer.

Reactive oxygen species (ROS) accumulation is known to induce carcinogenesis and accelerate cancer progression. 8­Hydroxydeoxyguanosine (8­OHdG) is a specific marker of ROS­mediated DNA damage. Therefore, we analysed 8­OHdG levels in cancerous and normal tissue DNA via enzyme­linked immunosorbent assay (ELISA) using 97 tissue specimens obtained from surgically­treated patients with stage II/III colorectal cancer (CRC). Additionally, 8­OHdG levels in these tissues were also assessed via quantitative immunohistochemistry (qIHC). To eliminate individual background variables, the ratio of 8­OHdG levels between cancerous and normal tissues was calculated using both techniques. A comparative analysis demonstrated that the 8­OHdG ratio in DNA was significantly correlated with both lymph node metastasis and lymphatic invasion. Multivariate analysis revealed that a high 8­OHdG ratio in DNA was independently correlated with poor prognosis. These results suggest that the 8­OHdG ratio in DNA reflects ROS­induced cancer progression. Conversely, a low 8­OHdG ratio as estimated via qIHC was an independent factor for poor prognosis. In Kaplan­Meier analysis, the combination of a high 8­OHdG ratio in DNA (ELISA) and a low 8­OHdG ratio in cytoplasm (qIHC) was associated with markedly worse patient prognosis than other combinations. Combined evaluation of the 8­OHdG ratio using ELISA and qIHC may be pivotal for predicting surgical outcomes for patients with stage II/III CRC.

An ultra-high-performance liquid chromatography tandem mass spectrometry method for oxidative stress biomarker analysis in wastewater.

Reported herein is the development of an analytical method for the detection of four oxidative stress biomarkers in wastewater using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) and solid phase extraction (SPE). The following four biomarkers of oxidative stress and lipid peroxidation have been investigated: hydroxynonenal-mercapturic acid (HNE-MA), 8-iso-prostglandin F2beta (8-iso-PGF2ß), 8-nitroguanine (8-NO2Gua) and 8-hydroxy-2-deoxyguanosine (8-OHdG). The method showed very good performance: accuracy (> 87%), precision (> 90%), method quantification limits (1.3-3.0 ng L-1) and biomarker stability in wastewater in the case of HNE-MA, 8-OHdG and 8-iso-PGF2ß. In contrast, 8-NO2Gua was found to be less stable in wastewater, which affected its method performance: accuracy (> 63%), precision (> 91%) and method quantification limits (85.3 ng L-1). Application of the developed method resulted in, for the first time, HNE-MA being successfully observed and quantified within wastewater over a study period of a week (displayed average daily loads per capita of 48.9 ± 4.1 mg/1000/people/day). 8-iso-PGF2ß was detected with good intensity but could not be quantified due to co-elution with other isomers. 8-OHdG was detected, albeit at < MQL. This study demonstrates the potential for expanding on the possible endogenous biomarkers of health used in urban water fingerprinting to aid in measuring health in near-real time on a community-wide scale.

Evaluating clinical and laboratory effects of ozone in non-surgical periodontal treatment: a randomized controlled trial.

OBJECTIVE: This study aims to evaluate the clinical and biochemical (oxidative stress and pro-inflammatory mediators) effects of the gaseous ozone use accompanied by scaling and root planning (SRP) in periodontal treatment. MATERIAL AND METHODS: The study population consisted of 40 patients with chronic periodontitis (CP) randomly sorted into two groups of 20. The experimental group received SRP plus 3 watts gaseous ozone in two separate applications five days apart, whereas the control group received SRP plus placebo. Clinical periodontal parameters were assayed and saliva samples were taken before the initial and one month after the second treatment. Periodontal examination assessed plaque index (PI), gingival index (GI), probing depth, and clinical attachment level (CAL). Total antioxidant status (TAS), total oxidant status (TOS), nitric oxide (NO), 8-hydroxy-2'-deoxyguanosine (8-OHdG), myeloperoxidase (MPO), glutathione (GSH), malondialdehyde (MDA), and transforming growth factor-beta (TGF-ß) levels were evaluated from saliva samples. RESULTS: Changes following treatment in PI, GI, probing depth, and CAL scores were similar for both groups (p>0.05). Of note, TGF-ß levels were observed to be higher in the treatment group than in controls (p<0.05). Changes in 8-OHdG, TAS, TOS, NO, MPO, GSH and MDA levels, however, were not significantly different between groups (p>0.05). CONCLUSION: The findings of this study indicate that SRP plus gaseous ozone versus SRP alone does not correlate to a significant improvement in periodontal recovery.

Evaluating clinical and laboratory effects of ozone in non-surgical periodontal treatment: a randomized controlled trial

Abstract Objective: This study aims to evaluate the clinical and biochemical (oxidative stress and pro-inflammatory mediators) effects of the gaseous ozone use accompanied by scaling and root planning (SRP) in periodontal treatment. Material and Methods: The study population consisted of 40 patients with chronic periodontitis (CP) randomly sorted into two groups of 20. The experimental group received SRP plus 3 watts gaseous ozone in two separate applications five days apart, whereas the control group received SRP plus placebo. Clinical periodontal parameters were assayed and saliva samples were taken before the initial and one month after the second treatment. Periodontal examination assessed plaque index (PI), gingival index (GI), probing depth, and clinical attachment level (CAL). Total antioxidant status (TAS), total oxidant status (TOS), nitric oxide (NO), 8-hydroxy-2'-deoxyguanosine (8-OHdG), myeloperoxidase (MPO), glutathione (GSH), malondialdehyde (MDA), and transforming growth factor-beta (TGF-β) levels were evaluated from saliva samples. Results: Changes following treatment in PI, GI, probing depth, and CAL scores were similar for both groups (p>0.05). Of note, TGF-β levels were observed to be higher in the treatment group than in controls (p<0.05). Changes in 8-OHdG, TAS, TOS, NO, MPO, GSH and MDA levels, however, were not significantly different between groups (p>0.05). Conclusion: The findings of this study indicate that SRP plus gaseous ozone versus SRP alone does not correlate to a significant improvement in periodontal recovery.

Improving the fluorometric determination of the cancer biomarker 8-hydroxy-2'-deoxyguanosine by using a 3D DNA nanomachine.

The authors describe a fluorometric method for improving the determination of the cancer biomarker 8-hydroxy-2'-deoxyguanosine (8-OHdG). A nicking endonuclease (NEase)-powered 3-D DNA nanomachine was constructed by assembling hundreds of carboxyfluorescein-labeled single strand oligonucleotides (acting as signal reporter) and tens of swing arms (acting as single-foot DNA walkers) on a gold nanoparticle (AuNP). The activity of this DNA nanomachine was controlled by introducing the protecting oligonucleotides. In the presence of aptamer against 8-OHdG, the protecting oligonucleotides are removed from the swing arms by toehold-mediated strand displacement reaction. In the next step, detached DNA walker hybridizes to the labelled DNA so that the DNA nanomachine becomes activated. Special sequences of signal reporter in the formed duplex can be recognized and cleaved by NEase. As a result, the DNA walker autonomously and progressively moves along the surface of the AuNP, thereby releasing hundreds of signal reporters and causing a rapid increase in green fluorescence. This 3-D nanomachine is highly efficient because one aptamer can release hundreds of signal reporters. These unique properties allowed for the construction of a DNA nanomachine-based method for sensitively detecting 8-OHdG in concentrations as low as 4 pM. This is three orders of magnitude lower compared to previously reported methods. Graphical abstract Schematic of a fluorometric method for determination of the cancer biomarker 8-hydroxy-2'-deoxyguanosine. A nicking endonuclease powered 3D-DNA nanomachine was used to improve the sensitivity. Limit of detection is three orders of magnitude lower than reported methods.

Strong impact of sulfotransferases on DNA adduct formation by 4-aminobiphenyl in bladder and liver in mice.

Bladder cancer risk is 3-4 times higher in men than women, but the reason is poorly understood. In mice, male bladder is also more susceptible than female bladder to 4-aminobiphenyl (ABP), a major human bladder carcinogen; however, female liver is more susceptible than male liver to ABP. We investigated the role of sulfotransferase (Sult) in gender-related bladder and liver susceptibility to ABP. Sulfation reactions of aromatic amine bladder carcinogens catalyzed by Sult may generate highly unstable and toxic metabolites. Therefore, liver Sult may decrease bladder exposure to carcinogens by promoting their toxic reactions in the liver. Notably, the expression of several liver Sults is suppressed by androgen in male mice. Here, we show that two Sults are critical for gender-related bladder susceptibility to ABP in mice. We measured tissue level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), a principal ABP-DNA adduct, as readout of tissue susceptibility to ABP. We identified Sutl1a1 and to a lesser extent Sult1d1 as Sults that promote dG-C8-ABP formation in hepatic cells. In mice, gender gap in bladder susceptibility to ABP was narrowed by knocking out Sult1a1 and was almost totally eliminated by knocking out both Sutl1a1 and Sult1d1. This was accompanied by dramatic decrease in ABP genotoxicity in the liver (>97%). These results show the strong impact of the Sults on bladder and liver susceptibility to a human carcinogen. Because liver expression of both Sult1a1 and Sutl1d1 is suppressed by androgen in male mice, our results suggest that androgen renders bladder more exposed to ABP in male mice by suppressing Sult-mediated ABP metabolism in liver, which increases bladder delivery of carcinogenic metabolites.

Protective Effects of Hydrogen Sulfide Against Cigarette Smoke Exposure-Induced Placental Oxidative Damage by Alleviating Redox Imbalance via Nrf2 Pathway in Rats.

BACKGROUND/AIMS: Cigarette smoke exposure (CSE) during pregnancy is a well-recognized health hazard that causes placental damage. Hydrogen sulfide (H2S) has been reported to protect multiple organs from injury. However, the protective effects of H2S have not been tested in the placenta. This study aimed to explore the potential of H2S in protecting placenta against oxidative injury induced by CSE during pregnancy and the possible underlying mechanisms. METHODS: Pregnant SD rats were randomly divided into 4 groups: NaCl, NaHS (a donor of H2S), CSE and CSE+NaHS. Placental oxidative damage was detected by 8-hydroxy-2-deoxyguanosine (8-OHdG) stain and malondialdehyde (MDA) assay. Placental redox status was assessed by measuring reactive oxygen species (ROS), total antioxidant capacity (T-AOC) and glutathione (GSH) levels, as well as copper/zinc SOD (SOD1), manganese SOD (SOD2), catalase (CAT) and glutathione peroxidase (GPx) activities and expressions. Meanwhile, nuclear factor erythroid 2-related factor 2 (Nrf2) was analyzed by immunohistochemistry, real-time PCR and Western blot. RESULTS: We found that NaHS markedly reduced the elevated levels of 8-OHdG and MDA induced by CSE. Further, NaHS treatment effectively mitigated CSE-induced placental redox imbalance by inhibiting ROS production, restoring T-AOC level, increasing GSH/GSSG ratio, and augmenting SOD1 SOD2, CAT and GPx activities and expressions. More notably, NaHS administration also reversed the aberrant decrease of Nrf2 due to CSE in rat placentas. CONCLUSION: Our data demonstrate that H2S can protect against CSE-induced placental oxidative damage probably by alleviating redox imbalance via Nrf2 pathway.

[Sperm DNA damage is not related to seminal leukocyte subsets in infertile males with asymptomatic genital tract infection].

OBJECTIVE: To study the correlation of leukocyte subsets with sperm DNA damage in the semen of infertile men with asymptomatic genital tract infection (AGTI). METHODS: This study included 111 infertile males with AGTI. After routine semen analysis, we determined the concentration of CD45+ leukocytes in the semen by immunocytochemistry, measured the concentrations of CD14+ cells of the mononuclear / macrophagic system and activated macrophages and HLA-DR+ cells in the semen by flow cytometry, and examined the sperm DNA fragmentation index (DFI) and the expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) by TUNEL assay. Then we analyzed the correlation of seminal leukocyte subsets with sperm DNA damage and routine semen parameters. RESULTS: The concentration of CD45+ leukocytes was correlated significantly with those of CD14+ and HLA-DR+ cells in the semen (P <0.01), but not that of leukocyte subsets with routine semen parameters, sperm DFI, or the percentage of 8-OHdG-positive cells. The percentage of 8-OHdG-positive sperm was correlated positively with the sperm DFI (r = 0.48, P<0.01) but negatively with sperm concentration (r = －0.44, P <0.01). After adjusted for age, abstinence time and cigarette smoking, the percentage of 8-OHdG-positive sperm was correlated independently with sperm concentration (ß = －0.25, P = 0.008) and DFI (ß = 0.23, P = 0.05). CONCLUSIONS: Sperm DNA damage is associated with poor semen quality but not with seminal leukocyte subsets in infertile males with asymptomatic genital tract infection.

Recent technical and biological development in the analysis of biomarker N-deoxyguanosine-C8-4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) which is primarily formed during tobacco combustion and overheated meat is a major carcinogen responsible for various cancers. Its adducted form, N-deoxyguanosine-C8-4-aminobiphenyl (dG-C8-4-ABP), has long been employed as a biomarker for assessment of the risk for cancer. In this review, the metabolism and carcinogenisity of 4-ABP will be discussed, followed by a discussion of the current common approaches of analyzing dG-C8-4-ABP. The major part of this review will be on the history and recent development of key methods for detection and quantitation of dG-C8-4-ABP in complex biological samples and their biological applications, from the traditional 2P-postlabelling and immunoassay methods to modern liquid chromatography-mass spectrometry (LC-MS) with the latter as the focus. Many vital biological discoveries based on dG-C8-4-ABP have been published by using the nanoLC-MS with column switching platform in our laboratory, which has also been adopted and further improved by many other researchers. We hope this review can provide a perspective of the challenges that had to be addressed in reaching our present goals and possibly bring new ideas for those who are still working on the frontline of DNA adducts area.

Glutathione peroxidase 4 overexpression inhibits ROS-induced cell death in diffuse large B-cell lymphoma.

Regulation of oxidative stress and redox systems has important roles in carcinogenesis and cancer progression, and for this reason has attracted much attention as a new area of cancer therapeutic targets. Glutathione peroxidase 4 (GPX4), an antioxidant enzyme, has biological important functions such as signaling cell death by suppressing peroxidation of membrane phospholipids. However, few studies exist on the expression and clinical relevance of GPX4 in malignant lymphomas such as diffuse large B-cell lymphoma. In this study, we assessed the expression of GPX4 immunohistochemically. GPX4 was expressed in 35.5% (33/93) cases of diffuse large B-cell lymphoma. The GPX4-positive group had poor overall survival (P = 0.0032) and progression-free survival (P = 0.0004) compared with those of the GPX4-negative group. In a combined analysis of GPX4 and 8-hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker, there was a negative correlation between GPX4 and 8-hydroxydeoxyguanosine (P = 0.0009). The GPX4-positive and 8-hydroxydeoxyguanosine-negative groups had a significantly worse prognosis than the other groups in both overall survival (P = 0.0170) and progression-free survival (P = 0.0005). These results suggest that the overexpression of GPX4 is an independent prognostic predictor in diffuse large B-cell lymphoma. Furthermore, in vitro analysis demonstrated that GPX4-overexpressing cells were resistant to reactive oxygen species-induced cell death (P = 0.0360). Conversely, GPX4-knockdown cells were sensitive to reactive oxygen species-induced cell death (P = 0.0111). From these data, we conclude that GPX4 regulates reactive oxygen species-induced cell death. Our results suggest a novel therapeutic strategy using the mechanism of ferroptosis, as well as a novel prognostic predictor of diffuse large B-cell lymphoma.

Ultrafine particulate matter impairs mitochondrial redox homeostasis and activates phosphatidylinositol 3-kinase mediated DNA damage responses in lymphocytes.

Particulate matter (PM), broadly defined as coarse (2.5-10 µm), fine (0.1-2.5 µm) and ultrafine particles (≤0.1 µm), is a major constituent of ambient air pollution. Recent studies have linked PM exposure (coarse and fine particles) with several human diseases including cancer. However, the molecular mechanisms underlying ultrafine PM exposure induced cellular and sub-cellular repercussions are ill-defined. Since mitochondria are one of the major targets of different environmental pollutants, we herein aimed to understand the molecular repercussion of ultrafine PM exposure on mitochondrial machinery in peripheral blood lymphocytes. Upon comparative analysis, a significantly higher DCF fluorescence was observed in ultrafine PM exposed cells that confirmed the strong pro-oxidant nature of these particles. In addition, the depleted activity of antioxidant enzymes, glutathione reductase and superoxide dismutase suggested the strong association of ultrafine PM with oxidative stress. These results further coincided with mitochondrial membrane depolarization, altered mitochondrial respiratory chain enzyme activity and decline in mtDNA copy number. Moreover, the higher accumulation of DNA damage response proteins (γH2AX, pATM, p-p53), suggested that exposure to ultrafine PM induces DNA damage and triggers phosphatidylinositol 3 kinase mediated response pathway. Further, the alterations in mitochondrial machinery and redox balance among ultrafine PM exposed cells were accompanied by a considerably elevated pro-inflammatory cytokine response. Interestingly, the lower apoptosis levels observed in ultrafine particle treated cells suggest the possibility that the marked alterations may lead to the impairment of mitochondrial-nuclear cross talk. Together, our results showed that ultrafine PM, because of their smaller size possesses significant ability to disturb mitochondrial redox homeostasis and activates phosphatidylinositol 3 kinase mediated DNA damage response pathway, an unknown molecular paradigm of ultrafine PM exposure. Our findings also indicate that maneuvering through the mitochondrial function might be a viable, indirect method to modulate lymphocyte homeostasis in air pollution associated immune disorders.

Paper-Based Sensing Device for Electrochemical Detection of Oxidative Stress Biomarker 8-Hydroxy-2'-deoxyguanosine (8-OHdG) in Point-of-Care.

This work presents a cost-effective, label-free in point-of-care (POC) biosensor for the sensitive detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG), the most abundant oxidative product of DNA, that may allow a premature assessment of cancer disease, thereby improving diagnosis, prognostics and survival rates. The device targets the direct detection of 8-OHdG by using for the first time a carbon-ink 3-electrode on a paper substrate coupled to Differential Pulse Voltammetry readings. This design was optimized by adding nanostructured carbon materials to the ink and the conducting polymer PEDOT, enhancing the electrocatalytic properties of the sensor towards 8-OHdG detection. Meanwhile, the ability of this oxidative stress biomarker to undertake an oxidation reaction enabled the development of the sensing electrochemical device without the need of chemical probes and long incubation periods. This paper-modified sensor presented high electrochemical performance on the oxidation of 8-OHdG with a wide linear range (50-1000 ng/ml) and a low detection limit (14.4 ng/ml). Thus, our results showed the development of a direct and facile sensor with good reproducibility, stability, sensitivity and more importantly, selectivity. The proposed carbon-based electrochemical sensor is a potential candidate to be miniaturized to small portable size, which make it applicable for in-situ 8-OHdG sensing in real biological samples.

Ultrasensitive High-Resolution Mass Spectrometric Analysis of a DNA Adduct of the Carcinogen Benzo[a]pyrene in Human Lung.

Benzo[a]pyrene (BaP), an archetypical polycyclic aromatic hydrocarbon, is classified as "carcinogenic to humans" and is ubiquitous in the environment, as evident by the measurable levels of BaP metabolites in virtually all human urine samples examined. BaP carcinogenicity is believed to occur mainly through its covalent modification of DNA, resulting in the formation of BPDE-N2-dG, an adduct formed between deoxyguanosine and a diol epoxide metabolite of BaP, with subsequent mutation of critical growth control genes. In spite of the liquid chromatography-mass spectrometry (LC-MS)-based detection of BPDE-N2-dG in BaP-treated rodents, and indirectly through high-performance liquid chromatography (HPLC)-fluorescence detection of BaP-7,8,9,10-tetraols released from human DNA upon acid hydrolysis, BPDE-N2-dG adducts have rarely if ever been observed directly in human samples using LC-MS techniques, even though sophisticated methodologies have been employed which should have had sufficient sensitivity. With this in mind, we developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodology employing high-resolution/accurate mass analysis for detecting ultratrace levels of these adducts. These efforts are directly translatable to the development of sensitive detection of other small molecules using trap-based LC-ESI-MS/MS detection. The developed methodology had a limit of detection (LOD) of 1 amol of BPDE-N2-dG on-column, corresponding to 1 BPDE-N2-dG adduct per 1011 nucleotides (1 adduct per 10 human lung cells) using 40 µg of human lung DNA. To our knowledge, this is the most sensitive DNA adduct quantitation method yet reported, exceeding the sensitivity of the 32P-postlabeling assay (∼1 adduct per 1010 nucleotides). Twenty-nine human lung DNA samples resulted in 20 positive measurements above the LOD, with smoker and nonsmoker DNA containing 3.1 and 1.3 BPDE-N2-dG adducts per 1011 nucleotides, respectively.

Scavenging of reactive oxygen species by astaxanthin inhibits epithelial-mesenchymal transition in high glucose-stimulated mesothelial cells.

BACKGROUND: High glucose concentrations influence the functional and structural development of the peritoneal membrane. We previously reported that the oral administration of astaxanthin (AST) suppressed peritoneal fibrosis (PF) as well as inhibited oxidative stress, inflammation, and epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) in a chlorhexidine-induced PF rat model. This suggests that oxidative stress induction of EMT is a key event during peritoneal damage. The present study evaluated the therapeutic effect of AST in suppressing EMT, in response to glucose-induced oxidative stress. METHODS: Temperature-sensitive mesothelial cells (TSMCs) were cultured in the presence or absence of AST and then treated with 140 mM glucose for 3 or 12 hours. Expression levels of TNF-α, TGF-ß, and VEGF were determined at the mRNA and protein levels, and nuclear factor kappa B (NF-κB) activity was evaluated. We measured NO2-/NO3- concentrations in cellular supernatants and determined 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in mitochondrial and nuclear DNA. The expressions of E-cadherin and alpha-smooth muscle actin (α-SMA) were evaluated by double immunofluorescence and protein levels. RESULTS: High glucose concentrations induced overproduction of reactive oxidative species (ROS), increasing 8-OHdG mitochondrial DNA and cytokine levels. The NF-κB pathway was activated in response to high glucose concentrations, whereas de novo α-SMA expression was observed with decreased E-cadherin expression. AST treatment attenuated ROS production, inflammatory cytokine production, NF-κB activation, and EMT. CONCLUSION: The findings of the present study indicate that AST may have an anti-EMT effect due to anti-oxidative and anti-inflammatory activities by scavenging glucose-induced ROS from mitochondria in PMCs. AST may be an efficacious treatment for PF.

Sensitive Detection of DNA Lesions by Bulge-Enhanced Highly Specific Coamplification at Lower Denaturation Temperature Polymerase Chain Reaction.

Mutagenic modifications of nucleotides or DNA lesions that result from environmental stress have proven to be associated with a variety of diseases, particularly cancer. The method for accurately detecting the lesions is therefore of great importance for biomedical research and toxicity study. We develop a sensitive and low-cost bulge-enhanced coamplification at lower denaturation temperature polymerase chain reaction (COLD-PCR) method for detecting DNA lesions (uracil and 8-oxoguanine) by combining an in vitro base excision repair (BER) pathway and COLD-PCR. The modified bases are converted to bulge via the BER pathway involving converting modified bases to an apurinic/apyrimidinic (AP) site, cleavage at the AP site, and break ligation. The presence of the bulge induces a large change of the hybridization thermodynamics of double-stranded DNA, eventually enhancing the specificity of COLD-PCR. Besides, we used the free energy of hybridization as a reference to optimize the critical denaturation temperature (Tc) of COLD-PCR obtaining more specific amplification than empirical Tc. Taking advantage of the proposed bulge-enhanced COLD-PCR, we are able to identify the presence of DNA lesion-containing strands at low abundance down to 0.01%. This method also exhibits high sensitivity for glycosylase with a detection limit of 10-4 U/mL [3 S/N (signal-to-noise ratio)] that is superior than some recently reported methods. With the design of the repair guide probe, the level of oxidative damage in genomic DNA caused by chemicals and photodynamic therapy (PDT) can be evaluated, heralding more applications in clinical diagnosis and epigenetic study.

Lifestyle Changes and Oxidative Stress in a High-incidence Area of Amyotrophic Lateral Sclerosis in the Southwestern Kii Peninsula, Japan.

Objective Lifestyle changes may play an important role in the incidence reduction and delay of onset age of amyotrophic lateral sclerosis (ALS) in the Koza/Kozagawa/Kushimoto (K) area. The aim of this study was to evaluate recent lifestyle changes in the K area and to investigate the relationships between lifestyle and oxidative stress among the residents. Methods We conducted a medical checkup for elderly residents in the K area and the control area and evaluated the urinary 8-OHdG levels, cognitive function test scores and metal contents in serum and scalp hair, coupled with a lifestyle questionnaire survey between 2010 and 2015. Results Recent lifestyle changes among the K residents, including a decrease in the Japanese pickle consumption, increase in fresh vegetable consumption and decrease in farm work, were evaluated in this study. Low consumption of Japanese pickles, high consumption of fresh vegetables, rare farm work and low levels of 8-OHdG/creatinine were all associated with high scores in the cognitive function tests. Frequent farm work and consumption of Japanese pickles was associated with high contents of transition metals, such as Mn, Al and V, in the scalp hair. Conclusion These lifestyle changes among residents in the K area may be associated with their oxidative stress.