Repair Enzyme Research Articles

Mitochondrial ROS Triggers KIN Pathogenesis in FAN1-Deficient Kidneys.

Karyomegalic interstitial nephritis (KIN) is a genetic adult-onset chronic kidney disease (CKD) characterized by genomic instability and mitotic abnormalities in the tubular epithelial cells. KIN is caused by recessive mutations in the FAN1 DNA repair enzyme. However, the endogenous source of DNA damage in FAN1/KIN kidneys has not been identified. Here we show, using FAN1-deficient human renal tubular epithelial cells (hRTECs) and FAN1-null mice as a model of KIN, that FAN1 kidney pathophysiology is triggered by hypersensitivity to endogenous reactive oxygen species (ROS), which cause chronic oxidative and double-strand DNA damage in the kidney tubular epithelial cells, accompanied by an intrinsic failure to repair DNA damage. Furthermore, persistent oxidative stress in FAN1-deficient RTECs and FAN1 kidneys caused mitochondrial deficiencies in oxidative phosphorylation and fatty acid oxidation. The administration of subclinical, low-dose cisplatin increased oxidative stress and aggravated mitochondrial dysfunction in FAN1-deficient kidneys, thereby exacerbating KIN pathophysiology. In contrast, treatment of FAN1 mice with a mitochondria-targeted ROS scavenger, JP4-039, attenuated oxidative stress and accumulation of DNA damage, mitigated tubular injury, and preserved kidney function in cisplatin-treated FAN1-null mice, demonstrating that endogenous oxygen stress is an important source of DNA damage in FAN1-deficient kidneys and a driver of KIN pathogenesis. Our findings indicate that therapeutic modulation of kidney oxidative stress may be a promising avenue to mitigate FAN1/KIN kidney pathophysiology and disease progression in patients.

Prognostic value of O6-methylguanine-DNA methyltransferase hypermethylation and expression in head and neck cancer: A systematic review and meta-analysis

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that maintains the stability of genetic information. MGMT is a strong prognostic biomarker in patients with glioblastoma. However, the effect of its gene hypermethylation and expression on the survival rate of head and neck cancer (HNC) patients is still disputed. Therefore, we conducted a meta-analysis to evaluate the prognostic value of MGMT hypermethylation and expression in HNC patients. This meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines and was registered at the International Prospective Register of Systematic Reviews (CRD42021274728). Literature related to the survival rate of HNC patients and MGMT was systematically searched in PubMed, Embase, The Cochrane Library and Web of Science electronic databases (published from inception to February 1, 2023). The association was evaluated by the combined hazard ratio (HR) and related 95% confidence interval (CI). Two authors independently screened all records and extracted the data. The certainty of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation system. All of the statistical tests used in this meta-analysis were conducted with Stata 12.0 software. We included 5 studies with 564 HNC patients for the meta-analysis. All of the included patients were primary tumors and underwent surgical resection without prior radiotherapy or chemotherapy therapy. No significant heterogeneity was noted between MGMT and overall survival, MGMT and disease-free survival, and a fixed-effects model was used. HNC patients with MGMT hypermethylation and low expression had a poor prognosis, with pooled HR for overall survival (HR = 1.23, 95% CI: 1.10-1.38, P < .001) and disease-free survival (HR = 2.28, 95% CI: 1.45-3.58, P < .001). Subgroup analysis stratified by molecular abnormalities, such as hypermethylation or low expression, showed similar results. The insufficient number of trials included in our study encountered high risk of bias and may increase the deviation of the final meta-analysis results. HNC patients with MGMT hypermethylation and low expression were more likely to exhibit poorer survival. MGMT hypermethylation and low expression can predict survival in patients with HNC.

Abstract 4915: Sensitivity to NAMPT inhibition: In vitro and in vivo characterization in ovarian cancer

Abstract Previous in vitro work on determining the unique sensitivities of various cancers to nicotinamide phosphoribosyltransferase inhibitors (NAMPTis) has demonstrated promising effects of treating cancer cells with NAMPTis such as FK866 and KPT9274. These inhibitors act by disrupting cellular energy metabolism and reducing intracellular NAD+ levels. NAMPT inhibition thereby suppresses the proliferation of cancer cells which depend more heavily on the NAMPT enzyme to produce NAD+ than noncancerous cells. Using pan-cancer in vitro screens in a large panel of molecularly characterized cell lines (n = 496) we identified a subset of ovarian cancer, a cancer with a high unmet need due to lack of diverse targeted therapies, as being extremely sensitive to NAMPT inhibition. Subsequent in vivo work using CD-1 nude mice demonstrate that NAMPT inhibitors can successfully reduce the ovarian tumor burden and may be an effective treatment option of some ovarian cancers. Moreover, NAD+’s function as a substrate to the poly ADP-ribose polymerase (PARP) enzyme makes NAMPTis rational candidates for including in combination therapies with PARP inhibitors, several of which are approved for maintenance therapy for ovarian cancer. By using NAMPTis to disrupt the cellular metabolic process while simultaneously inhibiting PARP activity, an essential DNA damage repair enzyme, it is possible to exploit the dependence of ovarian cancer cells on functional PARP activity. This is particularly important in homologous recombination deficient subtypes. In addition to a synergistic growth inhibitory response in ovarian cancer cells, preclinical combination studies of NAMPTis with olaparib, an approved PARP inhibitor, exhibited higher levels of DNA damage accumulation than with single drug treatments. Our in vitro and in vivo characterizations of NAMPT inhibition suggest that NAMPTis as either single agents or in combination treatments with PARP inhibitors should be investigated further as potential treatment options for ovarian cancer patient populations. Citation Format: Jenny J. Hong, Martina S. McDermott, Neil A. O'Brien, Enrique Guandique, Tong Luo, Dennis J. Slamon. Sensitivity to NAMPT inhibition: In vitro and in vivo characterization in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4915.

The Unique Spectrum of MUTYH Germline Mutations in Colombian Patients with Extracolonic Carcinomas.

Protein MUTYH, encoded by the gene MUTYH, is an important mismatch repair enzyme in the base-excision repair pathway of DNA repair. When genetically altered, different neoplastic conditions can arise. One of the widely known syndromes associated with MUTYH mutations is MUTYH-associated polyposis, a form of familial colorectal cancer syndrome. MUTYH may also be a driver in other familial cancer syndromes, as well as breast cancer and spontaneous cancer cases. However, some controversies about the role of these alterations in oncogenesis remain, especially when affected in a heterozygous way. Most available data on MUTYH mutations are on Caucasian patients. We analyzed a small cohort of non-Caucasian, Colombian cancer patients with MUTYH germline heterozygous mutations, clinical features suggestive of familial cancer, and extensive genetic studies with no other mutations and without MUTYH-associated polyposis. With this case series, we intended to provide important data for the understanding of MUTYH as a possible driver of familial cancer, even when only heterozygous mutations are found.

MP14-06 E-LIQUID EXPOSURE INDUCES THE RELEASE OF PRO-ONCOGENIC EXTRACELLULAR VESICLES FROM BLADDER CANCER CELLS THAT PROMOTE THE MALIGNANT TRANSFORMATION OF RECIPIENT UROTHELIAL CELLS

You have accessJournal of UrologyCME1 Apr 2023MP14-06 E-LIQUID EXPOSURE INDUCES THE RELEASE OF PRO-ONCOGENIC EXTRACELLULAR VESICLES FROM BLADDER CANCER CELLS THAT PROMOTE THE MALIGNANT TRANSFORMATION OF RECIPIENT UROTHELIAL CELLS Ryan Molony, Chia-Hao Wu, and Yi-Fen Lee Ryan MolonyRyan Molony More articles by this author , Chia-Hao WuChia-Hao Wu More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cigarette smoking is the best-established risk factor for bladder cancer (BC). While E-cigarette usage has risen rapidly in recent years and preclinical and clinical evidence suggests that exposed tissues may be subject to genotoxic, inflammatory, and oxidative stress, the potential oncogenic risks associated with vaping are not fully understood. Our lab has previously demonstrated that BC cell-derived extracellular vesicles (BCEVs) can promote the neoplastic transformation of recipient urothelial cells. This study was designed to explore the impact of smoking or E-cigarette use on the pro-oncogenic properties of BCEVs. METHODS: Human grade IV BC TCCSUP cells were exposed to cigarette smoke extract (CSE), unflavored E-liquid (UEL), or menthol-flavored E-liquid (MEL), and BCEV release was measured via nanoparticle tracking assay. BCEV-induced genotoxic damage, oxidative stress, inflammation, and apoptotic responses in recipient SV-HUC urothelial cells were quantified by γH2Ax staining, ELISAs, reactive oxygen species (ROS) assays, and flow cytometry. Protein cargos in these BCEVs were quantified by label-free quantitative LC-MS. SV-HUC cells were treated with these BCEVs for 12 weeks, after which a soft agar colony forming assay was used to assess their neoplastic transformation. RESULTS: CSE, UEL, and MEL treatment significantly increased rates of BCEV release. CSE- and MEL-BCEVs enhanced NF-κB activation, DNA double-strand break (DSB) formation, and ROS production in recipient SV-HUC cells. Proteomic analyses indicated that these CSE- and MEL-BCEVs contain high levels of known BC-related pro-oncogenic proteins including PGAM1 and VCP, as well as the dimeric DSB repair enzymes XRCC5/6. Notably, long-term MEL-BCEV treatment induced significantly higher rates of neoplastic urothelial cell transformation. CONCLUSIONS: CSE or MEL exposure significantly alter the number and composition of EVs released from BC cells. These BCEVs, in turn, can induce genotoxic, inflammatory, and oxidative stress in recipient urothelial cells, with MEL-BCEVs ultimately promoting urothelial cell transformation. These data highlight the important role that BCEVs may play as mediators of BC onset or recurrence, with the use of certain E-cigarettes potentially compounding this risk. Source of Funding: Department of Defense W81XWH-19-1-0198 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e184 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryan Molony More articles by this author Chia-Hao Wu More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement PDF downloadLoading ...

Ultraviolet B-Rays Induced Gene Alterations and DNA Repair Enzymes in Skin Tissue.

Ultraviolet (UV) radiation leads to deoxyribonucleic acid (DNA) damage and changes in gene expression. Topical DNA repair enzymes in liposomes are capable of undoing this damage. To evaluate gene expression changes induced by ultraviolent B-rays (UVB) light and assess the effect of topical DNA repair enzymes extracted from Micrococcus luteus (M. luteus) and photolyase in modifying these changes. Non-invasive, adhesive patch collection kits were used to sample skin on the right and left post-auricular areas before and 24 hours after UVB exposure (n=48). Subjects applied topical DNA repair enzymes to the right post-auricular area daily for 2 weeks. Subjects returned 2 weeks later for repeat non-invasive skin sample collection. Eight of 18 tested genes demonstrated significant changes 24 hours following UVB exposure. DNA repair enzymes from M. luteus or photolyase had no significant effect on genetic expression compared with the control at 2 weeks post UV exposure. UVB exposure causes acute changes in gene expression, which may play roles in photo-aging damage and skin cancer growth and regulation. While non-invasive gene expression testing can detect UV damage, additional genomic studies investigating recovery from UV damage at different time periods are needed to establish the potential of DNA repair enzymes to minimize or reverse this damage. J Drugs Dermatol. 2023;22(5): doi:10.36849/JDD.7070.

An ultra-sensitive and specific UCBiosensor via CRISPR-Cas12a and UDG-mediated polymerase chain reaction

DNA damage is an important cause of several diseases. Uracil DNA glycosylase (UDG) is a key enzyme in DNA damage repair and used as an early diagnostic marker and a prognostic indicator for cancer. Therefore, there is an urgent need to develop a simple, low-background, ultra-sensitive UDG activity detection method for clinical applications. At present, biosensors based on CRISPR-Cas12a have become a powerful diagnostic tool owing to their high sensitivity. Here, we have established an ultra-sensitive biosensor for detecting UDG activity based on CRISPR-Cas12a coupled UDG-mediated polymerase chain reaction (U-PCR), which has the advantages of simplicity, low background, ultra-sensitivity, and specificity. We used this biosensor to multiply amplify the UDG signal, and the detection limit reached 3.5 × 10−6 U/mL, which is one of the most sensitive methods for UDG activity detection. Finally, we detected UDG activity in the lysates of cancer cells, indicating that this method could be used to detect UDG activity in real samples. The UDG inhibition test showed that the method we established is a useful tool for screening UDG inhibitors. We believe that the UDG activity detection biosensor established in this study has potential applications in biomedical research and clinical diagnosis.

Molecular markers predicting the progression and prognosis of human papillomavirus-induced cervical lesions to cervical cancer.

Persistent Human Papillomavirus (HPV) infection is linked to 99% of cervical cancer (CC) cases. HPV types 16 and 18 aloneresult in 75% of CC cases and thus are considered to be high-risk types (HR-HPV). CC is thethird most common cancer among women globally. Approximately, 7000 patients die from it yearly. It is worthy to note that not every patient with HPV precancerous lesions will progress to CC. The objectives of this review is to explore the utilization ofmolecular and viral biomarkers asa tool for early detection and prediction of HPV-induced cervical lesions that might progress to CC. The data basesPubMed, Google Scholar, EBSCO were searched using keywords CC screening, HPV, and recent molecular biomarkers. The search time frame was within the last 7years. Studies on HPV-induced cancers other than CC were excluded; a total of 200 eligible articles were retrieved. In this review we explored the current literature about HPV virology, virulence genes and early diagnostic/prognostic molecular biomarkers in CC. The oncogenic property of HPV is attributed to viral expression of various early proteins (E5, E6, E7). The interaction between viral oncoproteins and the cellular genetic apparatus alters the expression of many genes at different phases of the disease. There was an association between cervical lesions induced by HR-HPV andtheoverexpression of markers of oxidative DNA damage and other proteins. The markersp16INK4a, programmed cell death-1 (PD-1)/programmed cell death ligand 1, mismatch repair enzymes (MMR), miRNA-377, claudin family (CLDN)are dysregulated and are associated with high risk lesions. Furthermore, advanced older cervical lesions were associated with high methylation levels and higherrisk to progress to CC. Adding differentthe abovemarkers to the CC screening program scheme might offera triage for prioritizing patient management.

The High Lead Level Role in the DNA Repair RAD 18 and OGG1 Gene Polymorphism in Gasoline Station Workers

The DNA repair enzymes–heavy metal interaction is an interesting project that can help elucidate several diseases. The current study aims to assess the lead effect in the two DNA repair genes RAD-18 Arg302Gln (rs373572) and OGG1 Ser326Cys in Gasoline station workers. The output showed that work types were Gasoline supply worker with a high percentage (73.52%) than other groups (11.76% and 14.7%) for a maintenance worker and station employee, respectively, significant lead (p 0.010) increasing in station worker than the control group, and significant lead changes among work types groups (p 0.000), the station employee has a low level of lead than other groups, while the Gasoline supply worker has a higher level than other groups, the RAD 18 gene showed two polymorphisms Gln/Gln and Gln/Arg, and OGG1 showed two haplotypes, single and double haplotypes, non-significant association although of high frequent of Glu/Arg in the gasoline supply worker, and significant association of allele frequency, significant association with station worker that have two types of haplotypes (single and double haplotypes) while lack of tri-haplotypes which prepared in higher percentage in control group. The lead level according to RAD 18 genotyping show non-significant (p 0.454) elevation in Gln/Arg genotyping, and according to OGG 1 haplotype lead level was non-significant, changed (p 0.481) between single and double haplotypes. From these outputs, it can be concluded that the lead level is a significant elevation in gasoline station worker and it did not affect RA18 and OGG 1 genotyping, the RAD18 did not associate with workers while OGG strongly associated with them.

Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase β during base excision repair

Base Excision Repair (BER) is carried out by a series of proteins that function in a step-by-step process to identify, remove, and replace DNA damage. During BER, the DNA transitions through various intermediate states as it is processed by each DNA repair enzyme. Left unrepaired, these BER intermediates can transition into double-stranded DNA breaks and promote genome instability. Previous studies have proposed a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme at each step of the BER pathway to protect the BER intermediate. The transfer of BER intermediates between enzymes, known as BER coordination or substrate channeling, remains poorly understood. Here, we utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase β (Pol β). When preformed complexes of APE1 and the incised abasic site product (APE1 product and Pol β substrate) were subsequently bound by Pol β, the Pol β enzyme dissociated shortly after binding in most of the observations. In the events where Pol β binding was followed by APE1 dissociation during substrate channeling, Pol β remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that transfer of the BER intermediate from APE1 to Pol β during BER is dependent on the dissociation kinetics of APE1 and the duration of the ternary complex on the incised abasic site.

Natural Products and Their Derivatives as Inhibitors of the DNA Repair Enzyme Tyrosyl-DNA Phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an important repair enzyme that removes various covalent adducts from the 3' end of DNA. Particularly, covalent complexes of topoisomerase 1 (TOP1) with DNA stabilized by DNA damage or by various chemical agents are an examples of such adducts. Anticancer drugs such as the TOP1 poisons topotecan and irinotecan are responsible for the stabilization of these complexes. TDP1 neutralizes the effect of these anticancer drugs, eliminating the DNA adducts. Therefore, the inhibition of TDP1 can sensitize tumor cells to the action of TOP1 poisons. This review contains information about methods for determining the TDP1 activity, as well as describing the inhibitors of these enzyme derivatives of natural biologically active substances, such as aminoglycosides, nucleosides, polyphenolic compounds, and terpenoids. Data on the efficiency of combined inhibition of TOP1 and TDP1 in vitro and in vivo are presented.

A DNA/RNA hybrid fluorescent probe for high-throughput quantification of the activity of human apurinic/apyrimidinic endonuclease 1 in subcellular extracts

As a multifunctional DNA repair enzyme, human apurinic/apyrimidinic endonuclease 1 (APE1) plays a pivotal role in many cellular processes. In recent years, it has been recognized as a potential prognostic biomarker and a therapeutic target for many cancers and other diseases. In this work, we report a DNA/RNA hybrid-based fluorescent probe which allows rapid and high-throughput detection of APE1 in the nuclear, cytoplasmic, and mitochondrial extracts of different types of cultured cells without any purification steps. As a noncanonical substrate, the DNA/RNA hybrid probe possesses inherent specificity toward APE1 without the need of additional chemical modification. A substantial acceleration effect of the 3′-flanking mismatches enabled rapid digestion of the abasic sites in the DNA/RNA hybrid by APE1 at a comparable rate to those in natural double-stranded DNA. The hybrid probe showed adequately high sensitivity to the target enzyme (linear working range: 0.02–1.0 U/mL; detection limit: 0.01 U/mL) and superior resistance to interference from cellular proteins. The mechanism for the 3′-mismatch enhancement was also clarified via kinetic study and single-molecule fluorescence analysis. By using the probe, significantly varied subcellular distribution of APE1 in different cells were disclosed, showing the importance of quantitative measurement of APE1 activity in subcellular compartments for drug screening and evaluation of the treatment efficacy. The method was simple and robust, which offered an easily accessible molecular tool for high-throughput quantification of the subcellular APE1 activity. It may also facilitate the development of effective anticancer drugs.

8-Oxoguanine DNA Glycosylase 1 Upregulation as a Risk Factor for Obesity and Colorectal Cancer.

DNA damage has been extensively studied as a potentially helpful tool in assessing and preventing cancer, having been widely associated with the deregulation of DNA damage repair (DDR) genes and with an increased risk of cancer. Adipose tissue and tumoral cells engage in a reciprocal interaction to establish an inflammatory microenvironment that enhances cancer growth by modifying epigenetic and gene expression patterns. Here, we hypothesize that 8-oxoguanine DNA glycosylase 1 (OGG1)-a DNA repair enzyme-may represent an attractive target that connects colorectal cancer (CRC) and obesity. In order to understand the mechanisms underlying the development of CRC and obesity, the expression and methylation of DDR genes were analyzed in visceral adipose tissue from CRC and healthy participants. Gene expression analysis revealed an upregulation of OGG1 expression in CRC participants (p < 0.005) and a downregulation of OGG1 in normal-weight healthy patients (p < 0.05). Interestingly, the methylation analysis showed the hypermethylation of OGG1 in CRC patients (p < 0.05). Moreover, expression patterns of OGG1 were found to be regulated by vitamin D and inflammatory genes. In general, our results showed evidence that OGG1 can regulate CRC risk through obesity and may act as a biomarker for CRC.

Radiotherapy and olaparib in combination for carcinoma of the oesophagus: A phase I study

Background and purposeRadical concurrent chemoradiotherapy (CRT) for oesophageal cancer is associated with significant morbidity and is unsuitable for some patients. Olaparib, an inhibitor of the DNA repair enzyme poly-(ADP)-ribose polymerase (PARP) has radiosensitising properties and may be better tolerated than chemotherapy. Materials and methodsWe performed a phase 1 study of olaparib with radiotherapy (RT) in oesophageal cancer patients unsuitable for conventional CRT to determine its maximum tolerated dose (MTD) in this setting. ResultsEight patients were recruited. One of 5 patients receiving olaparib 50 mg twice daily and two of 3 receiving 100 mg twice daily experienced dose limiting toxicity (DLT). ConclusionsOlaparib 100 mg twice daily exceeded the MTD in combination with RT in these patients. 50 mg twice daily may be the MTD but this cannot be stated with certainty as the study closed before full recruitment.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity

Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants (kcat/KM) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing.

Poly ADP-Ribose Polymerase-1 inhibition by 3-aminobenzamide recuperates HEI-OC1 auditory hair cells from blast overpressure-induced cell death.

Introduction: Poly ADP-Ribose Polymerase-1 (PARP1), a DNA repair enzyme is implicated as a key molecule in the pathogenesis of several neurodegenerative disorders. Traumatic insults inducing oxidative stress results in its over-activation causing inflammation and cell death (Parthanatos). As PARP1 inhibition is known to reduce oxidative stress, we hypothesized that PARP1 inhibition by a known inhibitor 3-aminobenzamide (3AB) might recuperate the damage in an in vitro model of blast injury using HEI-OC1 cells (mouse auditory hair cells). Methods: Here, we evaluated the protective effect of 3AB on HEI-OC1 cells following single and repetitive blast overpressures (BOPs). Results: We found that inhibition of PARP1 b 3AB inhibits the PARP1 enzyme and its action of a post-translational modification i.e. formation of Poly ADP-Ribose Polymers which leads to massive ATP depletion. PARP inhibition (3AB treatment) reduced the oxidative stress (4HNE, a marker of lipid peroxidation, and 8OHdG, a marker of oxidative DNA damage) in cells exposed to single/repetitive BOPS through up-regulation of Nrf2, a transcriptional regulator of antioxidant defense and the GCLC, a rate limiting enzyme in the synthesis of glutathione. Discussion: Overall, we found that PARP inhibition by 3AB helps to maintain the viability of BOP-exposed auditory hair cells by recuperating the ATP pool from both mitochondrial and glycolytic sources.

An Overview of Comet Assay Application for Detecting DNA Damage in Aquatic Animals

This review discusses several research studies that employed comet assay to evaluate the environmental impact of genotoxins in aquatic environments. It focuses on in vivo and in situ studies of aquatic animals. New chemicals are being added each year to the existing burden of toxic substances in the environment. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat, as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Comet assay is a quick, sensitive, and low-cost technique for detecting DNA strand breakage. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. Comparing data from studies that employed different approaches, such as empirical scoring or comet tail lengths, comet assay is one of the challenging techniques to be utilized in environmental studies. The relative amount of DNA in the comet tail indicates DNA break intensity. The assay has been modified to detect various base alterations by including the digestion of nucleoids with a lesion-specific endonuclease. The determination of DNA damage in these indicator species using the comet test would thus offer information on the genotoxic potential of their habitat at an early stage. This would enable intervention techniques to prevent or mitigate adverse health impacts in sentinel animals and humans.

The Cellular Response to Complex DNA Damage Induced by Ionising Radiation.

Radiotherapy (ionising radiation; IR) is utilised in the treatment of ~50% of all human cancers, and where the therapeutic effect is largely achieved through DNA damage induction. In particular, complex DNA damage (CDD) containing two or more lesions within one to two helical turns of the DNA is a signature of IR and contributes significantly to the cell killing effects due to the difficult nature of its repair by the cellular DNA repair machinery. The levels and complexity of CDD increase with increasing ionisation density (linear energy transfer, LET) of the IR, such that photon (X-ray) radiotherapy is deemed low-LET whereas some particle ions (such as carbon ions) are high-LET radiotherapy. Despite this knowledge, there are challenges in the detection and quantitative measurement of IR-induced CDD in cells and tissues. Furthermore, there are biological uncertainties with the specific DNA repair proteins and pathways, including components of DNA single and double strand break mechanisms, that are engaged in CDD repair, which very much depends on the radiation type and associated LET. However, there are promising signs that advancements are being made in these areas and which will enhance our understanding of the cellular response to CDD induced by IR. There is also evidence that targeting CDD repair, particularly through inhibitors against selected DNA repair enzymes, can exacerbate the impact of higher LET, which could be explored further in a translational context.

Roles of glutathione peroxidase 4 on the mercury-triggered ferroptosis in renal cells: implications for the antagonism between selenium and mercury.

Understanding of how mercury species cause cellular impairments at the molecular level is critical for explaining the detrimental effects of mercury exposure on the human body. Previous studies have reported that inorganic and organic mercury compounds can induce apoptosis and necrosis in a variety of cell types, but more recent advances reveal that mercuric mercury (Hg2+) and methylmercury (CH3Hg+) may result in ferroptosis, a distinct form of programmed cell death. However, it is still unclear which protein targets are responsible for ferroptosis induced by Hg2+ and CH3Hg+. In this study, human embryonic kidney 293T cells were used to investigate how Hg2+ and CH3Hg+ trigger ferroptosis, given their nephrotoxicity. Our results demonstrate that glutathione peroxidase 4 (GPx4) plays a key role in lipid peroxidation and ferroptosis in renal cells induced by Hg2+ and CH3Hg+. The expression of GPx4, the only lipid repair enzyme in mammal cells, was downregulated in response to Hg2+ and CH3Hg+ stress. More importantly, the activity of GPx4 could be markedly inhibited by CH3Hg+, owing to the direct binding of the selenol group (-SeH) in GPx4 to CH3Hg+. Selenite supplementation was demonstrated to enhance the expression and activity of GPx4 in renal cells, and consequently relieve the cytotoxicity of CH3Hg+, suggesting that GPx4 is a crucial modulator implicated in the Hg-Se antagonism. These findings highlight the importance of GPx4 in mercury-induced ferroptosis, and provide an alternative explanation for how Hg2+ and CH3Hg+ induce cell death.

DNA Probes for Analysis the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells

The important role of DNA damage in the occurrence of various diseases, including cancer, stimulates studies of the mechanisms of genetic information stability, carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and an increased risk of diseases developing still remains relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and AP endonuclease APE1. The sensitivity of DNA probes was determined on purified enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed a significant variability in the level of enzyme activity in these cell lines. Obtained results can become the test system platform for analyzing the activity of the base excision DNA repair system in the human body.