Chronic obstructive pulmonary disease (COPD) includes chronic bronchitis and emphysema. Environmental exposure, primarily cigarette smoking, can cause high oxidative stress and is the main factor of COPD development. Cigarette smoke also contributes to the imbalance of oxidant/antioxidant due to exogenous reactive oxygen species (ROS). Moreover, endogenously released ROS during the inflammatory process and mitochondrial dysfunction may contribute to this disease progression. ROS and reactive nitrogen species (RNS) can oxidize different biomolecules such as DNA, proteins, and lipids leading to epithelial cell injury and death. Various detoxifying enzymes and antioxidant defense systems can be involved in ROS removal. In this review, we summarize the main findings regarding the biological role of ROS, which may contribute to COPD development, and cytoprotective mechanisms against this disease progression.

Tyrosyl-DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdp1 in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5′ extensions showed a high frequency of misrepair in Δtdp1 cells. The joining errors in Δtdp1 cells were predominantly 2-4 nucleotide insertions. Ends with 3′ extensions or blunt ends did not show enhanced frequencies of errors, although Δtdp1 cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV, as well as polymerase IV. Our results show that yeast Tdp1 is a component of the NHEJ pathway. We suggest that Tdp1p 3′ nucleosidase activity regulates the processing of DNA ends by generating a 3′ phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3′ phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3′ phosphate is a key step in Tdp1-mediated error prevention during NHEJ.Tpp1 | nucleosidase | repair accuracy | break repair N onhomologous end joining (NHEJ) is a critical pathway for repairing DNA double-strand breaks. In higher eukaryotes, it functions as a primary repair pathway for repairing doublestrand breaks from exogenous DNA damage and is also required for gene rearrangements in the immune system (1). This pathway is conserved in lower eukaryotes and bacteria where it functions mainly as a secondary repair pathway for the repair of doublestrand breaks (2, 3). Because NHEJ does not rely on DNA homology for carrying out repair, it is an intrinsically error-prone pathway. The detailed steps of how accuracy is maintained during NHEJ processes remain poorly understood.NHEJ is carried out by a set of well-conserved proteins including the Ku70/Ku80 proteins that bind to DNA ends, serving as a scaffold for subsequent repair reactions, and DNA ligase IV (4). In mammalian cells, a DNA-dependent protein kinase DNAPKcs also plays critical roles (5). In addition to the core factors that are absolutely required for the NHEJ pathway, other factors such as nucleases and DNA polymerases participate in NHEJ (6, 7).Tdp1p was identified on the basis of its ability to remove peptides covalently bound to DNA (8-10). Yeast Tdp1 can remove phosphotyrosyl-linked peptides from both the 3′ and 5′ ends of DNA (8,11). Tdp1 also has the ability to remove damaged and undamaged nucleosides from the 3′ end of DNA through an activity that is mechanistically identical to the esterase activity that removes peptides (12). Because human Tdp1 is mutated in a DNA repair disorder [spinocerebellar ataxia with axonal neuropathy (13)], we assessed additional roles of this enzyme by using yeast repair mutants. In this paper, we demonstrate that yeast Tdp1 plays an important and unique role in NHEJ and affects the accuracy of the formation of repair junctions. ResultsYeast Tdp1 is Required for Accurate Joining of some NHEJ Subst...

Background: Cigarette smoke is the main risk factor of pulmonary emphysema development, which is characterized by alveolar wall destruction. Mitochondria are important for alveolar type II (ATII) cell metabolism due to ATP generation. Methods: We isolated ATII cells from control non-smoker and smoker organ donors, and after lung transplant of patients with emphysema to determine mitochondrial function, dynamics and mitochondrial (mt) DNA damage. Findings: We found high mitochondrial superoxide generation and mtDNA damage in ATII cells in emphysema. This correlated with decreased mtDNA amount. We also detected high TOP1-cc and low TDP1 levels in mitochondria in ATII cells in emphysema. This contributed to the decreased resolution of TOP1-cc leading to accumulation of mtDNA damage and mitochondrial dysfunction. Moreover, we used lung tissue obtained from areas with mild and severe emphysema from the same patients. We found a correlation between the impaired fusion and fission as indicated by low MFN1, OPA1, FIS1, and p-DRP1 levels and this disease severity. We detected lower TDP1 expression in severe compared to mild emphysema. Interpretation: We found high DNA damage and impairment of DNA damage repair in mitochondria in ATII cells isolated from emphysema patients, which contribute to abnormal mitochondrial dynamics. Our findings provide molecular mechanisms of mitochondrial dysfunction in this disease.

Cigarette smoke (CS) is a main risk factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by CS causes DNA and lung damage. Oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD. We studied the effect of oxidative stress generated by CS both in vivo and in vitro on murine primary alveolar type II (ATII) cells isolated from nuclear erythroid 2-related factor-2 (Nrf2)−/− mice. We determined human primary ATII cell injury by CS in vitro and analyzed ATII cells isolated from smoker and non-smoker lung donors ex vivo. We also studied whether trolox (water-soluble derivative of vitamin E) could protect murine and human ATII cells against CS-induced DNA damage and/or decrease injury. We analyzed oxidative stress by 4-hydroxynonenal expression, reactive oxygen species (ROS) generation by Amplex Red Hydrogen Peroxide Assay, Nrf2, heme oxygenase 1, p53 and P53-binding protein 1 (53BP1) expression by immonoblotting, Nrf2 nuclear translocation, Nrf2 and p53 DNA-binding activities, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cytokine production by ELISA. We found that ATII cells isolated from Nrf2−/− mice are more susceptible to CS-induced oxidative DNA damage mediated by p53/53BP1 both in vivo and in vitro compared with wild-type mice. Therefore, Nrf2 activation is a key factor to protect ATII cells against injury by CS. Moreover, trolox abolished human ATII cell injury and decreased DNA damage induced by CS in vitro. Furthermore, we found higher inflammation and p53 mRNA expression by RT-PCR in ATII cells isolated from smoker lung donors in comparison with non-smokers ex vivo. Our results indicate that the Nrf2 and p53 cross talk in ATII cells affect the susceptibility of these cells to injury by CS. Trolox can protect against oxidative stress, genotoxicity and inflammation induced by CS through ROS scavenging mechanism, and serve as a potential antioxidant prevention strategy against oxidative injury of ATII cells in CS-related lung diseases.

Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2-related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.