Mutagenicity of a bi-stranded clustered DNA lesion containing (5'S) or (5'R) 5',8-cyclo-2'-deoxyAdenosine in Escherichia coli model.

Ionizing radiation induces DNA damage, including characteristic clusters and tandem lesions e.g., 5',8-cyclo-2'-deoxyPurines (cdPus). Clustered DNA Lesions (CDL) defined as 2 or more lesions within 1-2 helical turns resulting from a single radiation track contribute to the harmful effects of radiation. Moreover, the presence of CDL and cdPus in human DNA may decrease the efficiency of the DNA repair mechanisms, which in consequence may lead to, e.g., carcinogenesis. This preliminary study showed the mutagenic potential of CDL containing dU on one strand and 5',8-cyclo-2'-deoxyAdenosine (cdA) on a complementary strand separated by up to 4 bp. Mutagenicity was determined using Escherichia coli reporter assay and 40-mer model ds-oligonucleotides with CDL. Mutation frequencies were determined to be significantly higher for CDL than for single isolated lesions (cdA or dU placed only in one strand). The results demonstrated that the dU lesion located on the opposite DNA strand separated by 0 or 1 bp from cdA led to severe mutagenicity. The most frequent mutations observed comprised point deletions and transitions. Oligonucleotides with CDL containing ScdA/RcdA demonstrated even up to 100% mutation rate. Interestingly, increasing the distance between lesions within CDL to 4 bp led to full recovery of the correct sequence of ds-oligonucleotides, indicating an efficient repair process. The results obtained with the bacterial model are in agreement with previous in vitro studies on eukaryotic models. The high mutagenicity and/or inhibited repair process of clusters with lesions located in close proximity provides additional verification of the previously presented trends describing how the distance between cdPu and dU affects DNA repair processes.

The Influence of 5',8-Cyclo-2'-deoxypurines on the Mitochondrial Repair of Clustered DNA Damage in Xrs5 Cells: The Preliminary Study.

The 5',8-cyclo-2'-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered-the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5'-end side of cdPu than on its 3'-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.

Effects of 5',8'-Cyclo-2'-Deoxypurines on the Base Excision Repair of Clustered DNA Lesions in Nuclear Extracts of the XPC Cell Line.

Clustered DNA lesions (CDL) containing 5',8-cyclo-2'-deoxypurines (cdPus) are an example of extensive abnormalities occurring in the DNA helix and may impede cellular repair processes. The changes in the efficiency of nuclear base excision repair (BER) were investigated using (a) two cell lines, one of the normal skin fibroblasts as a reference (BJ) and the second from Xeroderma pigmentosum patients' skin (XPC), and (b) synthetic oligonucleotides with single- and double-stranded CDL (containing 5',8-cyclo-2'-deoxyadenosine (cdA) and the abasic (AP) site at various distances between lesions). The nuclear BER has been observed and the effect of both cdA isomers (5'R and 5'S) presence in the DNA was tested. CdPus affected the repair of the second lesion within the CDL. The BER system more efficiently processed damage in the vicinity of the ScdA isomer and changes located in the 3'-end direction for dsCDL and in the 5'-end direction for ssCDL. The presented study is the very first investigation of the repair processes of the CDL containing cdPu considering cells derived from a Xeroderma pigmentosum patient.

When UDG and hAPE1 Meet Cyclopurines. How (5'R) and (5'S) 5',8-Cyclo-2'-deoxyadenosine and 5',8-Cyclo-2'-deoxyguanosine Affect UDG and hAPE1 Activity?

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5',8-cyclo-2'-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5'S and 5'R diastereomers of 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2'-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome.

The Influence of 5'R and 5'S cdA and cdG on the Activity of BsmAI and SspI Restriction Enzymes.

Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5',8-cyclo-2'-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction-modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme's recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5'S)-5',8-cyclo-2'-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5'R)-5',8-cyclo-2'-deoxyadenosine (RcdA). Interestingly, in the case of 5',8-cyclo-2'-deoxyguanosine (cdG), both 5'S and 5'R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.

How (5'S) and (5'R) 5',8-Cyclo-2'-Deoxypurines Affect Base Excision Repair of Clustered DNA Damage in Nuclear Extracts of xrs5 Cells? A Biochemical Study.

The clustered DNA lesions (CDLs) are a characteristic feature of ionizing radiation's impact on the human genetic material. CDLs impair the efficiency of cellular repair machinery, especially base excision repair (BER). When CDLs contain a lesion repaired by BER (e.g., apurinic/apyrimidinic (AP) sites) and a bulkier 5',8-cyclo-2'-deoxypurine (cdPu), which is not a substrate for BER, the repair efficiency of the first one may be affected. The cdPus' influence on the efficiency of nuclear BER in xrs5 cells have been investigated using synthetic oligonucleotides with bi-stranded CDL (containing (5'S) 5',8-cyclo-2'-deoxyadenosine (ScdA), (5'R) 5',8-cyclo-2'-deoxyadenosine (RcdA), (5'S) 5',8-cyclo-2'-deoxyguanosine (ScdG) or (5'R) 5',8-cyclo-2'-deoxyguanosine (RcdG) in one strand and an AP site in the other strand at different interlesion distances). Here, for the first time, the impact of ScdG and RcdG was experimentally tested in the context of nuclear BER. This study shows that the presence of RcdA inhibits BER more than ScdA; however, ScdG decreases repair level more than RcdG. Moreover, AP sites located ≤10 base pairs to the cdPu on its 5'-end side were repaired less efficiently than AP sites located ≤10 base pairs on the 3'-end side of cdPu. The strand with an AP site placed opposite cdPu or one base in the 5'-end direction was not reconstituted for cdA nor cdG. CdPus affect the repair of the other lesion within the CDL. It may translate to a prolonged lifetime of unrepaired lesions leading to mutations and impaired cellular processes. Therefore, future research should focus on exploring this subject in more detail.

Nutrition Can Help DNA Repair in the Case of Aging.

Micronutrients such as vitamins and trace elements are crucial for maintaining the health of all organisms. Micronutrients are involved in every cellular/biochemical process. They play roles in proper heart and brain functioning, influence immunological responses, and antioxidant defense systems. Therefore, prolonged deficiency in one or more micronutrients leads to cardiovascular or neurodegenerative disorders. Keeping micronutrients at adequate levels is especially important for seniors. They are prone to deficiencies due to age-associated functional decline and often to a diet poor in nutrients. Moreover, lack of micronutrients has an indirect impact on the genome. Their low levels reduce the activity of antioxidant enzymes, and therefore inhibit the efficiency of defense against free radicals which can lead to the formation of DNA lesions. The more DNA damage in the genetic material, the faster aging at the cellular level and a higher risk of pathological processes (e.g., carcinogenesis). Supplementation of crucial antioxidative micronutrients such as selenium, zinc, vitamin C, and vitamin E seems to have the potential to positively influence the condition of an aging organism, including minimizing inflammation, enhancing antioxidative defense, and limiting the formation of DNA lesions. In consequence, it may lead to lowering the risk and incidence of age-related diseases such as cardiovascular diseases, neurodegenerative diseases, and malnutrition. In this article, we attempt to present the synergistic action of selected antioxidant micronutrients (vitamin C, vitamin E, selenium, and zinc) for inhibiting oxidative stress and DNA damage, which may impede the process of healthy aging.

8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a Cause of Autoimmune Thyroid Diseases (AITD) During Pregnancy?

The thyroid is not necessary to sustain life. However, thyroid hormones (TH) strongly affect the human body. Functioning of the thyroid gland affects the reproductive capabilities of women and men, as well as fertilization and maintaining a pregnancy. For the synthesis of TH, hydrogen peroxide (H2O2) is necessary. From the chemical point of view, TH is a reactive oxygen species (ROS) and serves as an oxidative stress (OS) promoter. H2O2 concentration in the thyroid gland is much higher than in other tissues. Therefore, the thyroid is highly exposed to OS. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) are DNA lesions resulting from ROS action onto guanine moiety. Due to their abundance, they are recognized as biomarkers of OS. As thyroid function is correlated with the level of OS, 8-oxodG and 8-OHdG has been taken under consideration. Studies correlate the oxidative DNA damage with various thyroid diseases (TD) such as Hashimoto's thyroiditis (HT), Graves' disease (GD), and thyroid cancer. Human sexual function and fertility are also affected by OS and TD. Hypothyroidism and hyperthyroidism diagnosed in pregnant women have a negative effect on pregnancy as it may increase the risk of miscarriage or fetus mortality. In the case of TD in the mother, fetal health is also at risk - neurodevelopment and cognitive function of the child may be impaired in its future life. This review presents thyroid function in the context of TD during pregnancy. The authors introduce OS and describe oxidative DNA lesions as a crucial marker of thyroid pathologies.

The role of AMPK in metabolism and its influence on DNA damage repair.

One of the most complex health disproportions in the human body is the metabolic syndrome (MetS). It can result in serious health consequences such as type 2 diabetes mellitus, atherosclerosis or insulin resistance. The center of energy regulation in human is AMP-activated protein kinase (AMPK), which modulates cells' metabolic pathways and protects them against negative effects of metabolic stress, e.g. reactive oxygen species. Moreover, recent studies show the relationship between the AMPK activity and the regulation of DNA damage repair such as base excision repair (BER) system, which is presented in relation to the influence of MetS on human genome. Hence, AMPK is studied not only in the field of counteracting MetS but also prevention of genetic alterations and cancer development. Through understanding AMPK pathways and its role in cells with damaged DNA it might be possible to improve cell's repair processes and develop new therapies. This review presents AMPK role in eukaryotic cells and focuses on the relationship between AMPK activity and the regulation of BER system through its main component-8-oxoguanine glycosylase (OGG1).

The Similarities between Human Mitochondria and Bacteria in the Context of Structure, Genome, and Base Excision Repair System.

Mitochondria emerged from bacterial ancestors during endosymbiosis and are crucial for cellular processes such as energy production and homeostasis, stress responses, cell survival, and more. They are the site of aerobic respiration and adenosine triphosphate (ATP) production in eukaryotes. However, oxidative phosphorylation (OXPHOS) is also the source of reactive oxygen species (ROS), which are both important and dangerous for the cell. Human mitochondria contain mitochondrial DNA (mtDNA), and its integrity may be endangered by the action of ROS. Fortunately, human mitochondria have repair mechanisms that allow protecting mtDNA and repairing lesions that may contribute to the occurrence of mutations. Mutagenesis of the mitochondrial genome may manifest in the form of pathological states such as mitochondrial, neurodegenerative, and/or cardiovascular diseases, premature aging, and cancer. The review describes the mitochondrial structure, genome, and the main mitochondrial repair mechanism (base excision repair (BER)) of oxidative lesions in the context of common features between human mitochondria and bacteria. The authors present a holistic view of the similarities of mitochondria and bacteria to show that bacteria may be an interesting experimental model for studying mitochondrial diseases, especially those where the mechanism of DNA repair is impaired.

Two Faces of Vitamin C-Antioxidative and Pro-Oxidative Agent.

Vitamin C has been known for decades. It is common in everyday use as an element of the diet, supplementation, and a preservative. For years, research has been conducted to precisely determine the mechanism of action of ascorbate in the cell. Available results indicate its multi-directional cellular effects. Vitamin C, which belongs to antioxidants scavenging free radicals, also has a 'second face'-as a pro-oxidative factor. However, whether is the latter nature a defect harmful to the cell, or whether a virtue that is a source of benefit? In this review, we discuss the effects of vitamin C treatment in cancer prevention and the role of ascorbate in maintaining redox balance in the central nervous system (CNS). Finally, we discuss the effect of vitamin C supplementation on biomarkers of oxidative DNA damage and review the evidence that vitamin C has radioprotective properties.

8-Oxo-7,8-Dihydro-2'-Deoxyguanosine (8-oxodG) and 8-Hydroxy-2'-Deoxyguanosine (8-OHdG) as a Potential Biomarker for Gestational Diabetes Mellitus (GDM) Development.

The growing clinical and epidemiological significance of gestational diabetes mellitus results from its constantly increasing worldwide prevalence, obesity, and overall unhealthy lifestyle among women of childbearing age. Oxidative stress seems to be the most important predictor of gestational diabetes mellitus development. Disturbances in the cell caused by oxidative stress lead to different changes in biomolecules, including DNA. The nucleobase which is most susceptible to oxidative stress is guanine. Its damage results in two main modifications: 8-hydroxy-2'-deoxyguanosineor 8-oxo-7,8-dihydro-2'-deoxyguanosine. Their significant level can indicate pathological processes during pregnancy, like gestational diabetes mellitus and probably, type 2 diabetes mellitus after pregnancy. This review provides an overview of current knowledge on the use of 8-hydroxy-2'-deoxyguanosineand/or 8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker in gestational diabetes mellitus and allows us to understand the mechanism of 8-hydroxy-2'-deoxyguanosineand/or 8-oxo-7,8-dihydro-2'-deoxyguanosine generation during this disease.

Review: immunoassays in DNA damage and instability detection.

The review includes information on the current state of knowledge of immunometric methods with emphasis on the possibility of deoxyribonucleic acid (DNA) damage detection. Beginning with basic immunoassay enzyme-linked immunosorbent assay (ELISA), this review describes methods such as tyramide signal amplification (TSA), enhanced polymer one-step staining (EPOS), and time resolved amplified cryptate emission (TRACE) as improvements of ELISA's developed over time to obtain more accurate results. In the second part of the review, surface plasmon resonance (SPR) and quantum dots (QDs) are presented as the newest outlooks in the context of immunoanalysis of biological material and molecular studies. The aim of this review is to briefly present immunoassays with emphasis on DNA damage detection; therefore, the types of methods are listed and described, types of signal indicators, basic definitions such as antigen and antibody are given. Every method is considered with an exemplary application focusing on DNA studies, DNA damage and instability detection.

Virus-directed enzyme prodrug therapy and the assessment of the cytotoxic impact of some benzimidazole derivatives.

Virus-directed enzyme prodrug therapy is one of the major strategy of increasing cytotoxicity of bioreductive agents. This research intended to examine new selected benzimidazole derivatives as a substrate for nitroreductase, the enzyme involved in nitroreduction which is responsible to the production of cytotoxic metabolites. In this way, the selectivity and strength of cytotoxicity can be raised. The effect of benzimidazoles on virus transfected cells and non-virus transfected cells A549 cell line was established by Annexin V + propidium iodide test, western blot, and polymerase chain reaction analysis of specific pro- and anti-apoptotic proteins in the corresponding gene expression and additionally nitroreductase gene expression. Our results proved the pro-apoptotic properties of all tested compounds in normoxia and hypoxia, especially according to virused A549 cells where the time of exposition was reduced from 48 to 4 h. In this shorten period of time, the strongest activity was shown by N-oxide compounds with nitro-groups. The apoptosis was confirmed by generation of BAX gene and protein and reduction of BCL2 gene and protein.