Piotr Czarny

Autophagy in DNA Damage Response

DNA damage response (DDR) involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1). mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADP)ribose polymerase 1 (PARP-1), Mre11–Rad50–Nbs1 (MRN) complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

Elevated Level of DNA Damage and Impaired Repair of Oxidative DNA Damage in Patients with Recurrent Depressive Disorder

Background Depressive disorder (DD), including recurrent DD (rDD), is a severe psychological disease, which affects a large percentage of the world population. Although pathogenesis of the disease is not known, a growing body of evidence shows that inflammation together with oxidative stress may contribute to development of DD. Since reactive oxygen species produced during stress may damage DNA, we wanted to evaluate the extent of DNA damage and efficiency of DNA repair in patients with depression. Material/Methods We measured and compared the extent of endogenous DNA damage – single- and double-strand breaks, alkali-labile sites, and oxidative damage of the pyrimidines and purines – in peripheral blood mononuclear cells isolated from rDD patients (n=40) and healthy controls (n=46) using comet assay. We also measured DNA damage evoked by hydrogen peroxide and monitored changes in DNA damage during repair incubation. Results We found an increased number DNA breaks, alkali-labile sites, and oxidative modification of DNA bases in the patients compared to the controls. Exposure to hydrogen peroxide evoked the same increased damage in both groups. Examination of the repair kinetics of both groups revealed that the lesions were more efficiently repaired in the controls than in the patients. Conclusions For the first time we showed that patients with depression, compared with non-depresses individuals, had more DNA breaks, alkali-labile sites, and oxidative DNA damage, and that those lesions may be accumulated by impairments of the DNA repair systems. More studies must be conducted to elucidate the role of DNA damage and repair in depression.

Mutagenesis of mitochondrial DNA in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is an age-related, slowly progressive disease, which may lead to loss of vision resulting from apoptosis of corneal endothelial (CE) cells, dysfunction of Descemet membrane (DM) and corneal edema. A growing body of evidence suggests that oxidative stress may play a major role in the pathogenesis of FECD and that mitochondria of CE cells are its main target. Mitochondrial DNA (mtDNA) is particularly prone to oxidative stress and changes in mtDNA were reported in FECD patients. In the present work we studied mtDNA damage and repair, mtDNA copy number, and the 4977bp common deletion in mtDNA in DM cells and peripheral blood lymphocytes (PBLs) isolated from FECD patients. PBLs from 35 FECD patients and 32 controls were challenged for 10min with hydrogen peroxide at 20μM and then left in a fresh medium for 3h, resulting in a decrease in mtDNA copy number in both groups. Damage to mtDNA was not fully repaired after 3h and the extent of remaining lesions was significantly higher in the patients than the controls. We observed a higher copy number and an increased extent of mtDNA damage as well as a higher ratio of the common 4977bp deletion in DM cells of FECD patients than the controls. Our results confirm that mutagenesis of mtDNA may be involved in FECD pathogenesis and disturbance in mtDNA sensitivity to damaging agent as well as changes in mtDNA damage repair along with alternations in mtDNA copy number may underline this involvement.

The levels of 7,8-dihydrodeoxyguanosine (8-oxoG) and 8-oxoguanine DNA glycosylase 1 (OGG1) - A potential diagnostic biomarkers of Alzheimer's disease.

Evidence indicates that oxidative stress contributes to neuronal cell death in Alzheimers disease (AD). Increased oxidative DNA damage l, as measured with 8-oxoguanine (8-oxoG), and reduced capacity of proteins responsible for removing of DNA damage, including 8-oxoguanine DNA glycosylase 1 (OGG1), were detected in brains of AD patients. In the present study we assessed peripheral blood biomarkers of oxidative DNA damage, i.e. 8- oxoG and OGG1, in AD diagnosis, by comparing their levels between the patients and the controls. Our study was performed on DNA and serum isolated from peripheral blood taken from 100 AD patients and 110 controls. For 8-oxoG ELISA was employed. The OGG1 level was determined using ELISA and Western blot technique. Levels of 8-oxoG were significantly higher in DNA of AD patients. Both ELISA and Western blot showed decreased levels of OGG1 in serum of AD patients. Our results show that oxidative DNA damage biomarkers detected in peripheral tissue could reflect the changes occurring in the brain of patients with AD. These results also suggest that peripheral blood samples may be useful to measure oxidative stress biomarkers in AD.

Association between single nucleotide polymorphisms of MUTYH, hOGG1 and NEIL1 genes, and depression

BACKGROUND An elevated levels oxidative modified DNA bases and a decreased efficiency of oxidative DNA damage repair were found in patients with depression disorders, including recurrent type (rDD). The glycosylases are involved in base excision repair (BER), which eliminates oxidative DNA damage. Therefore, we genotyped the single nucleotide polymorphisms (SNPs) of genes encoding three glycosylases: hOGG1, MUTYH and NEIL1. METHODS We selected three polymorphisms: c.977C > G - hOGG1 (rs1052133), c.972G > C - MUTYH (rs3219489) and c.*589G > C - NEIL1 (rs4462560). A total of 555 DNA samples (257 cases and 298 controls) were genotyped using TaqMan probes. RESULTS The C/C genotype and allele C of the c.*589G > C decreased the risk of rDD occurrence, while the G/G genotype and allele G of the same SNP increased the risk. This polymorphism had a stronger association with early-onset depression (patients with first episode <35 years of age) than with late-onset depression (first episode ≥ 35 years of age). We did not find any significant differences in distribution of alleles and genotypes of other SNPs; however, the G/G genotype of the c.972G > C increased the risk of late-onset rDD. We also found that combined genotype C/C-C/C of c.977C > G and c.*589G > C significantly reduced the risk of rDD. LIMITATIONS Limited sample size and ethnic homogeneity of the studied population. CONCLUSION This is the first study to show that SNPs of genes involved in DNA repair, particularly in BER pathway, may modulate the risk of rDD. These results further support the hypothesis on the involvement of DNA repair mechanisms in pathogenesis of depression.

Variants of Base Excision Repair Genes MUTYH , PARP1 and XRCC1 in Alzheimer's Disease Risk

Background: Many clinical studies have shown that oxidative stress pathways and the efficiency of the oxidative DNA damage base excision repair (BER) system are associated with the pathogenesis of Alzheimers disease (AD). Reduced BER efficiency may result from polymorphisms of BER-related genes. In the present study, we examine whether single nucleotide polymorphisms (SNPs) of BER genes are associated with increased risk of AD. Methods: SNP genotyping was carried out on DNA isolated from peripheral blood mononuclear cells obtained from 120 patients with AD and 110 healthy volunteers. Samples were genotyped for the presence of BER-related SNPs, i.e. XRCC1-rs1799782, rs25487; MUTYH-rs3219489, and PARP1-rs1136410. Results: We found a positive association between AD risk and the presence of G/A genotype variant of the XRCC1 rs25487 polymorphism [odds ratio (OR) = 3.762, 95% CI: 1.793-7.891]. The presence of the A/A genotype of this polymorphism reduced the risk of AD (OR = 0.485, 95% CI: 0.271-0.870). In cases of the PARP1 gene rs1136410 polymorphism, we observed that the T/C variant increases (OR = 4.159, 95% CI: 1.978-8.745) while the T/T variant reduces risk (OR = 0.240, 95% CI: 0.114-0.556) of AD. Conclusions: We conclude that BER gene polymorphisms may play an important role in the etiology of AD. Diagnosing the presence or absence of particular genetic variants may be an important marker of AD. Further research on a larger population is needed. There is also a need to examine polymorphisms of other BER in the context of AD risk.

Genotoxicity and cytotoxicity of ZnO and Al2O3 nanoparticles

Abstract Objectives: Metal oxide nanoparticles (ZnO-NPs and Al2O3-NPs) are used in many fields, including consumer products and biomedical applications. As a result, exposure to these NPs is highly frequent, however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms are available. For this reason, we studied cytotoxic and genotoxic effects of ZnO-NPs and Al2O3-NPs on human peripheral blood lymphocytes. Materials and methods: We obtained our goals by using MTT assay, Annexin V-FITC flow cytometry, and alkaline, neural and pH 12.1 versions of comet assay. Results: Exposure of lymphocytes to both NPs for 24 h slightly decreased viability of lymphocytes at ≥0.5 mM. For the first time, we revealed using the comet assays that both ZnO-NPs and Al2O3-NPs caused a concentration-dependent increase of DNA single-strand breaks, but not alkali-labile sites. Treatment with DNA glycosylases showed that the NPs induced oxidative DNA damage. DNA damage caused by both nanoparticles at 0.05 mM was removed within 120 min, however lymphocytes did not repair DNA damage induced by 0.5 mM NPs. Studied nanoparticles did not induce apoptosis in lymphocytes. Conclusion: Our results suggest that ZnO-NPs and Al2O3-NPs at concentration up to 0.5 mM did not exhibit cytotoxic effect but may exert genotoxic effect on lymphocytes, at least partially by the generation of oxidative DNA damage and strand breaks.

Single-Nucleotide Polymorphisms of Genes Involved in Repair of Oxidative DNA Damage and the Risk of Recurrent Depressive Disorder

Background Depressive disorder, including recurrent type (rDD), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce DNA damage. This thesis is supported by the presence of increased levels of DNA damage in depressed patients. Such DNA damage is repaired by the base excision repair (BER) pathway. BER efficiency may be influenced by polymorphisms in BER-related genes. Therefore, we genotyped nine single-nucleotide polymorphisms (SNPs) in six genes encoding BER proteins. Material/Methods Using TaqMan, we selected and genotyped the following SNPs: c.-441G>A (rs174538) of FEN1, c.2285T>C (rs1136410) of PARP1, c.580C>T (rs1799782) and c.1196A>G (rs25487) of XRCC1, c.*83A>C (rs4796030) and c.*50C>T (rs1052536) of LIG3, c.-7C>T (rs20579) of LIG1, and c.-468T>G (rs1760944) and c.444T>G (rs1130409) of APEX1 in 599 samples (288 rDD patients and 311 controls). Results We found a strong correlation between rDD and both SNPs of LIG3, their haplotypes, as well as a weaker association with the c.-468T>G of APEXI which diminished after Nyholt correction. Polymorphisms of LIG3 were also associated with early onset versus late onset depression, whereas the c.-468T>G polymorphism showed the opposite association. Conclusions The SNPs of genes involved in the repair of oxidative DNA damage may modulate rDD risk. Since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of DNA damage and repair mechanisms in the development of this disease.

Impact of Single Nucleotide Polymorphisms of Base Excision Repair Genes on DNA Damage and Efficiency of DNA Repair in Recurrent Depression Disorder

Elevated level of DNA damage was observed in patients with depression. Furthermore, single nucleotide polymorphisms (SNPs) of base excision repair (BER) genes may modulate the risk of this disease. Therefore, the aim of this study was to delineate the association between DNA damage, DNA repair, the presence of polymorphic variants of BER genes, and occurrence of depression. The study was conducted on peripheral blood mononuclear cells of 43 patients diagnosed with depression and 59 controls without mental disorders. Comet assay was used to assess endogenous (oxidative) DNA damage and efficiency of DNA damage repair (DRE). TaqMan probes were employed to genotype 12 SNPs of BER genes. Endogenous DNA damage was higher in the patients than in the controls, but none of the SNPs affected its levels. DRE was significantly higher in the controls and was modulated by BER SNPs, particularly by c.977C>G–hOGG1, c.972G>C–MUTYH, c.2285T>C–PARP1, c.580C>T–XRCC1, c.1196A>G–XRCC1, c.444T>G–APEX1, c.-468T>G–APEX1, or c.*50C>T–LIG3. Our study suggests that both oxidative stress and disorders in DNA damage repair mechanisms contribute to elevated levels of DNA lesions observed in depression. Lower DRE can be partly attributed to the presence of specific SNP variants.

The c.469+46_56del mutation in the homeobox MSX1 gene--a novel risk factor in breast cancer?

PURPOSE The aim of this study was to investigate the association of a 11 nucleotide deletion, the c.469+46_56del mutation, in the intron of the homeobox MSX1 gene and breast cancer occurrence and characteristics. METHODS The mutation was genotyped in peripheral blood lymphocytes of 200 breast cancer patients and 203 controls by single-strand conformational PCR and DNA sequencing. RESULTS The del/del variant of the c.469+46_56del mutation increased the risk of breast cancer occurrence (OR 2.20; 95% CI 1.41-3.44, p<0.05). We did not observe any association between genotypes of this mutation and lymph node status, Bloom-Richardson grading, estrogen and progesterone receptors and HER2 expression. CONCLUSIONS The del/del genotype of the c.469+46_56del mutation in the MSX1 gene may be associated with the increased risk of breast cancer in Polish population and may be considered as an early marker in this disease.