Bilirubin-Induced Oxidative Stress Leads to DNA Damage in the Cerebellum of Hyperbilirubinemic Neonatal Mice and Activates DNA Double-Strand Break Repair Pathways in Human Cells

Unconjugated bilirubin is considered a potent antioxidant when present at moderate levels. However, at high concentrations, it produces severe neurological damage and death associated with kernicterus due to oxidative stress and other mechanisms. While it is widely recognized that oxidative stress by different toxic insults results in severe damage to cellular macromolecules, especially to DNA, no data are available either on DNA damage in the brain triggered by hyperbilirubinemia during the neonatal period or on the activation of DNA repair mechanisms. Here, using a mouse model of neonatal hyperbilirubinemia, we demonstrated that DNA damage occurs in vivo in the cerebellum, the brain region most affected by bilirubin toxicity. We studied the mechanisms associated with potential toxic action of bilirubin on DNA in in vitro models, which showed significant increases in DNA damage when neuronal and nonneuronal cells were treated with 140 nM of free bilirubin (Bf), as determined by γH2AX Western blot and immunofluorescence analyses. Cotreatment of cells with N-acetyl-cysteine, a potent oxidative-stress inhibitor, prevented DNA damage by bilirubin, supporting the concept that DNA damage was caused by bilirubin-induced oxidative stress. Bilirubin treatment also activated the main DNA repair pathways through homologous recombination (HR) and nonhomologous end joining (NHEJ), which may be adaptive responses to repair bilirubin-induced DNA damage. Since DNA damage may be another important factor contributing to neuronal death and bilirubin encephalopathy, these results contribute to the understanding of the mechanisms associated with bilirubin toxicity and may be of relevance in neonates affected with severe hyperbilirubinemia.

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Mn Inhibits GSH Synthesis via Downregulation of Neuronal EAAC1 and Astrocytic xCT to Cause Oxidative Damage in the Striatum of Mice

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/4235695 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15 ◽

Cited By ~ 7

Author(s):

Xinxin Yang ◽

Haibo Yang ◽

Fengdi Wu ◽

Zhipeng Qi ◽

Jiashuo Li ◽

...

Keyword(s):

Oxidative Stress ◽

Oxidative Damage ◽

Dependent Manner ◽

Protein Levels ◽

Key Factor ◽

Protein Sulfhydryl ◽

Mrna And Protein Expression ◽

The Brain

Excessive manganese (Mn) can accumulate in the striatum of the brain following overexposure. Oxidative stress is a well-recognized mechanism in Mn-induced neurotoxicity. It has been proven that glutathione (GSH) depletion is a key factor in oxidative damage during Mn exposure. However, no study has focused on the dysfunction of GSH synthesis-induced oxidative stress in the brain during Mn exposure. The objective of the present study was to explore the mechanism of Mn disruption of GSH synthesis via EAAC1 and xCT in vitro and in vivo. Primary neurons and astrocytes were cultured and treated with different doses of Mn to observe the state of cells and levels of GSH and reactive oxygen species (ROS) and measure mRNA and protein expression of EAAC1 and xCT. Mice were randomly divided into seven groups, which received saline, 12.5, 25, and 50 mg/kg MnCl2, 500 mg/kg AAH (EAAC1 inhibitor) + 50 mg/kg MnCl2, 75 mg/kg SSZ (xCT inhibitor) + 50 mg/kg MnCl2, and 100 mg/kg NAC (GSH rescuer) + 50 mg/kg MnCl2 once daily for two weeks. Then, levels of EAAC1, xCT, ROS, GSH, malondialdehyde (MDA), protein sulfhydryl, carbonyl, 8-hydroxy-2-deoxyguanosine (8-OHdG), and morphological and ultrastructural features in the striatum of mice were measured. Mn reduced protein levels, mRNA expression, and immunofluorescence intensity of EAAC1 and xCT. Mn also decreased the level of GSH, sulfhydryl, and increased ROS, MDA, 8-OHdG, and carbonyl in a dose-dependent manner. Injury-related pathological and ultrastructure changes in the striatum of mice were significantly present. In conclusion, excessive exposure to Mn disrupts GSH synthesis through inhibition of EAAC1 and xCT to trigger oxidative damage in the striatum.

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Vascular Dysfunction Induced by Mercury Exposure

International Journal of Molecular Sciences ◽

10.3390/ijms20102435 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2435 ◽

Cited By ~ 4

Author(s):

Tetsuya Takahashi ◽

Takayoshi Shimohata

Keyword(s):

Oxidative Stress ◽

Vascular Dysfunction ◽

Brain Parenchyma ◽

Transport Systems ◽

Mehg Exposure ◽

In Vivo Models ◽

The Central Nervous System ◽

The Brain

Methylmercury (MeHg) causes severe damage to the central nervous system, and there is increasing evidence of the association between MeHg exposure and vascular dysfunction, hemorrhage, and edema in the brain, but not in other organs of patients with acute MeHg intoxication. These observations suggest that MeHg possibly causes blood–brain barrier (BBB) damage. MeHg penetrates the BBB into the brain parenchyma via active transport systems, mainly the l-type amino acid transporter 1, on endothelial cell membranes. Recently, exposure to mercury has significantly increased. Numerous reports suggest that long-term low-level MeHg exposure can impair endothelial function and increase the risks of cardiovascular disease. The most widely reported mechanism of MeHg toxicity is oxidative stress and related pathways, such as neuroinflammation. BBB dysfunction has been suggested by both in vitro and in vivo models of MeHg intoxication. Therapy targeted at both maintaining the BBB and suppressing oxidative stress may represent a promising therapeutic strategy for MeHg intoxication. This paper reviews studies on the relationship between MeHg exposure and vascular dysfunction, with a special emphasis on the BBB.

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DNA double-strand break repair: a theoretical framework and its application

Journal of The Royal Society Interface ◽

10.1098/rsif.2015.0679 ◽

2016 ◽

Vol 13 (114) ◽

pp. 20150679 ◽

Cited By ~ 9

Author(s):

Philip J. Murray ◽

Bart Cornelissen ◽

Katherine A. Vallis ◽

S. Jon Chapman

Keyword(s):

Dna Damage ◽

Auger Electron ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Histone H2ax ◽

Dna Double Strand Break ◽

A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

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Arginine-deprivation–induced oxidative damage sterilizes Mycobacterium tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808874115 ◽

2018 ◽

Vol 115 (39) ◽

pp. 9779-9784 ◽

Cited By ~ 22

Author(s):

Sangeeta Tiwari ◽

Andries J. van Tonder ◽

Catherine Vilchèze ◽

Vitor Mendes ◽

Sherine E. Thomas ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Mycobacterium Tuberculosis ◽

De Novo ◽

Target Space ◽

Biosynthesis Pathway ◽

Arginine Biosynthesis ◽

Arginine Deprivation

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

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DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa

Biomolecules ◽

10.3390/biom10121680 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1680

Author(s):

Tassanee Lerksuthirat ◽

Rakkreat Wikiniyadhanee ◽

Sermsiri Chitphuk ◽

Wasana Stitchantrakul ◽

Somponnat Sampattavanich ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Fluorescent Proteins ◽

Strand Break ◽

Control Group ◽

Focus Formation ◽

Dna Damage And Repair ◽

Biological Compounds ◽

Repair Pathways ◽

Garcinia Cowa

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

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Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2

Brain ◽

10.1093/brain/awz202 ◽

2019 ◽

Vol 142 (8) ◽

pp. 2352-2366 ◽

Cited By ~ 15

Author(s):

Guo-zhong Yi ◽

Guanglong Huang ◽

Manlan Guo ◽

Xi’an Zhang ◽

Hai Wang ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Methyltransferase ◽

Molecular Mechanisms ◽

Progression Free Survival ◽

Recurrent Glioblastoma ◽

Dna Lesions ◽

Dna Repair Proteins

Abstract The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

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The Rational Design and Biological Mechanisms of Nanoradiosensitizers

Nanomaterials ◽

10.3390/nano10030504 ◽

2020 ◽

Vol 10 (3) ◽

pp. 504 ◽

Cited By ~ 3

Author(s):

Hainan Sun ◽

Xiaoling Wang ◽

Shumei Zhai

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

Dna Damage ◽

Rational Design ◽

Design Strategies ◽

Biological Mechanisms ◽

Normal Tissues ◽

Current State

Radiotherapy (RT) has been widely used for cancer treatment. However, the intrinsic drawbacks of RT, such as radiotoxicity in normal tissues and tumor radioresistance, promoted the development of radiosensitizers. To date, various kinds of nanoparticles have been found to act as radiosensitizers in cancer radiotherapy. This review focuses on the current state of nanoradiosensitizers, especially the related biological mechanisms, and the key design strategies for generating nanoradiosensitizers. The regulation of oxidative stress, DNA damage, the cell cycle, autophagy and apoptosis by nanoradiosensitizers in vitro and in vivo is highlighted, which may guide the rational design of therapeutics for tumor radiosensitization.

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Drug-induced DNA damage and tumor chemosensitivity.

Journal of Clinical Oncology ◽

10.1200/jco.1990.8.12.2062 ◽

1990 ◽

Vol 8 (12) ◽

pp. 2062-2084 ◽

Cited By ~ 65

Author(s):

R J Epstein

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Tumor Cell ◽

Malignant Disease ◽

Dna Lesions ◽

Drug Induced ◽

Cell Subpopulations ◽

Therapeutic Cell

Cytotoxic drugs act principally by damaging tumor-cell DNA. Quantitative analysis of this interaction provides a basis for understanding the biology of therapeutic cell kill as well as a rational strategy for optimizing and predicting tumor response. Recent advances have made it possible to correlate assayed DNA lesions with cytotoxicity in tumor cell lines, in animal models, and in patients with malignant disease. In addition, many of the complex interrelationships between DNA damage, DNA repair, and alterations of gene expression in response to DNA damage have been defined. Techniques for modulating DNA damage and cytotoxicity using schedule-specific cytotoxic combinations, DNA repair inhibitors, cell-cycle manipulations, and adjunctive noncytotoxic drug therapy are being developed, and critical therapeutic targets have been identified within tumor-cell subpopulations and genomic DNA alike. Most importantly, methods for predicting clinical response to cytotoxic therapy using both in vitro markers of tumor-cell sensitivity and in vivo measurements of drug-induced DNA damage are now becoming a reality. These advances can be expected to provide a strong foundation for the development of innovative cytotoxic drug strategies over the next decade.

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DDB2, an Essential Mediator of Premature Senescence

Molecular and Cellular Biology ◽

10.1128/mcb.01480-09 ◽

2010 ◽

Vol 30 (11) ◽

pp. 2681-2692 ◽

Cited By ~ 26

Author(s):

Nilotpal Roy ◽

Tanya Stoyanova ◽

Carmen Dominguez-Brauer ◽

Hyun Jung Park ◽

Srilata Bagchi ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Excision Repair ◽

Culture Shock ◽

Premature Senescence ◽

Antioxidant Genes ◽

Repair Protein ◽

High Level

ABSTRACT Reactive oxygen species (ROS) is critical for premature senescence, a process significant in tumor suppression and cancer therapy. Here, we reveal a novel function of the nucleotide excision repair protein DDB2 in the accumulation of ROS in a manner that is essential for premature senescence. DDB2-deficient cells fail to undergo premature senescence induced by culture shock, exogenous oxidative stress, oncogenic stress, or DNA damage. These cells do not accumulate ROS following DNA damage. The lack of ROS accumulation in DDB2 deficiency results from high-level expression of the antioxidant genes in vitro and in vivo. DDB2 represses antioxidant genes by recruiting Cul4A and Suv39h and by increasing histone-H3K9 trimethylation. Moreover, expression of DDB2 also is induced by ROS. Together, our results show that, upon oxidative stress, DDB2 functions in a positive feedback loop by repressing the antioxidant genes to cause persistent accumulation of ROS and induce premature senescence.

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Dual Roles of Helicobacter pylori NapA in Inducing and Combating Oxidative Stress

Infection and Immunity ◽

10.1128/iai.00991-06 ◽

2006 ◽

Vol 74 (12) ◽

pp. 6839-6846 ◽

Cited By ~ 56

Author(s):

Ge Wang ◽

Yang Hong ◽

Adriana Olczak ◽

Susan E. Maier ◽

Robert J. Maier

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Oxidative Dna Damage ◽

Wild Type Mouse ◽

Wild Type ◽

Oxygen Intermediates ◽

Physiological Analysis ◽

H Pylori

ABSTRACT Neutrophil-activating protein (NapA) has been well documented to play roles in human neutrophil recruitment and in stimulating host cell production of reactive oxygen intermediates (ROI). A separate role for NapA in combating oxidative stress within H. pylori was implied by studies of various H. pylori mutant strains. Here, physiological analysis of a napA strain was the approach used to assess the iron-sequestering and stress resistance roles of NapA, its role in preventing oxidative DNA damage, and its importance to mouse colonization. The napA strain was more sensitive to oxidative stress reagents and to oxygen, and it contained fourfold more intracellular free iron and more damaged DNA than the parent strain. Pure, iron-loaded NapA bound to DNA, but native NapA did not, presumably linking iron levels sensed by NapA to DNA damage protection. Despite its in vitro phenotype of sensitivity to oxidative stress, the napA strain showed normal (like that of the wild type) mouse colonization efficiency in the conventional in vivo assay. By use of a modified mouse inoculation protocol whereby nonviable H. pylori is first inoculated into mice, followed by (live) bacterial strain administration, an in vivo role for NapA in colonization efficiency could be demonstrated. NapA is the critical component responsible for inducing host-mediated ROI production, thus inhibiting colonization by the napA strain. An animal colonization experiment with a mixed-strain infection protocol further demonstrated that the napA strain has significantly decreased ability to survive when competing with the wild type. H. pylori NapA has unique and separate roles in gastric pathogenesis.

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