DNA Damage: Disease Relevance
DNA is permanently being attacked by a broad spectrum of endogenous and exogenous factors that generate aberrant DNA structures. If left unresolved, these lesions can result in cellular senescence and/or apoptosis as well as genomic destabilization. Genomic instability is characterized by an up-regulated rate of genomic mutations. The mechanisms causing genomic instability comprise inherited or acquired defects in the DNA damage repair (DDR) system, and, more frequently, in DNA replication, DNA DSBR, cell cycle control, or chromosome segregation 105. Due to the degree of genetic interruption, genomic instability can be divided into three classes: i) nucleotide instability resulting from the modification of single or a small number of nucleotides; ii) microsatellite instability based on irregularities culminating in the expansion or condensation of short nucleotide repeats; and iii) chromosomal instability (CIN) affecting chromosome number and structure as well as nuclear architecture 106-108. Genomic instability can be considered as being a hallmark of ageing and several pathologies, including immunodeficiency, degenerative diseases, and cancer (see Table 1 for an overview).
Table 1: Genomic instability associated with diseases and pathologies
|Disease||Clinical Presentation||Mutated DDR Genes and Their Corresponding Proteins|
|Ataxia-oculomotor apraxia 1||Cerebellar atrophy, ataxia, sensorimotor axonal neuropathy||APTX (aprataxin)|
|Ataxia telangiectasia||Neurodegeneration, immunodeficiency, premature aging, radiation sensitivity, cancer||ATM (ataxia telangiectasia mutated)|
|Ataxia-telangiectasia-like disorder||Cerebellar degeneration, radiation sensitivity||MRE11A (double-strand break repair protein Mre11A), ATM|
|Baller-Gerold syndrome||Premature fusion of the skull bones and malformations of facial, forearm, and hand bones||RECQL4 (RecQ protein-like 4)|
|Bloom syndrome||Immunodeficiency, premature ageing, cancer||BLM (Bloom syndrome protein)|
|Cancer||Uncontrolled cell proliferation, metastasis||CHEK2 (serine/threonine-protein kinase Chk2 isoform), BRCA1 (breast cancer type 1 susceptibility protein), BRCA2 (breast cancer type 2 susceptibility protein), RAD51 (DNA repair protein RAD51), TP53 (cellular tumor antigen p53 isoform), MLH3 (DNA mismatch repair protein Mlh3), MLH1, MSH2, MSH6, MUTYH (A/G-specific adenine DNA glycosylase), PMS1, PMS2, ALKBH3 (alpha-ketoglutarate-dependent dioxygenase alkB), etc.|
|Cellular ageing||Declining ability to respond to mitotic signals and increased homeostatic imbalances||Several proteins involved in DNA repair|
|Cockayne‘s syndrome||Dwarfism, mental retardation, UV light sensitivity||CSA (Cockayne syndrome WD repeat protein CSA), CSB|
|Fanconi anemia||Congenital abnormalities, bone-marrow failure, cancer||FANCM (Fanconi anemia group M protein), FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL|
|LIG4 syndrome||Immunodeficiency and developmental and growth delay||LIG4 (DNA ligase 4)|
|Nijmegen breakage syndrome||Microcephaly and mental retardation, immunodeficiency, radiation sensitivity, cancer||NBN (nibrin)|
|Mitochondrial chronic progressive external ophthalmoplegia||Weakness of the external eye muscles and exercise intolerance, cataracts, hearing loss, hypogonadism||POLG1 (mitochondrial DNA polymerase gamma, catalytic subunit)|
|Rothmund-Thompson syndrome||Immunodeficiency, premature ageing, cancer||RECQL4|
|Seckel syndrome||Growth retardation, microcephaly with mental retardation, ‘bird-headed’ facial appearance||ATR (ATM and Rad3 related)|
|Severe combined immunodeficiency with microcephaly||Microcephaly, growth retardation, sensitivity to ionizing radiation||NHEJ1 (Non-homologous end joining factor 1)|
|Spinocerebellar ataxia||Cerebellar ataxia, axonal neuropathy, muscular atrophy||TDP1 (Tyrosyl-DNA phosphodiesterase 1)|
|Trichothiodystrophy||Hair abnormality, mental, and growth retardation||XPB (Xeroderma pigmentosum group B-complementing protein), XPD|
|Werner’s syndrome||Immunodeficiency, cancer||WRN (Werner syndrome ATP-dependent helicase)|
|Xeroderma pigmentosa||UV light sensitivity, skin ageing, skin cancer||XPA (Xeroderma pigmentosum group A-complementing protein), POLH (DNA polymerase eta), DDB1/2 (DNA damage binding protein 1 and 2); ERCC3 (excision-repair cross-complementing 2), ERCC3, ERCC4, ERCC5 encoding XPD, XPB, XPF, and XPG, respectively|
Table was reproduced, with modifications, from Georgoulis et al. (2017) 109. License at https://creativecommons.org/licenses/by/4.0/
Role of DNA Damage in Ageing
Accumulation of DNA damage has been associated with the mechanism of ageing and the onset of age-related disorders including diabetes and cancer 110. It has long been accepted that particularly mitochondrial dysfunction plays a crucial role in normal ageing, contributing to the development of numerous ageing-associated diseases. As reviewed by Sun et al. (2016), mitochondrial dysfunction, increased frequency of mtDNA mutations, and alterations in the biogenesis of mitochondria are characteristic features of ageing 111. Emerging data suggest that mtDNA mutations are most likely due to an imperfect replication process, rather than due to ROS-mediated oxidative mtDNA lesions, implying that mtDNA polymerase γ (Pol γ) might be causative for the majority of mtDNA mutations 112. However, a mitochondrial dysfunction disturbs the redox balance and thus significantly up-regulates ROS production, which in turn causes oxidative stress and concomitant damage of DNA, a characteristic feature of inflammation, vigorous exercise, and ageing 113, 114.
As outlined before, accumulation of oxidative DNA lesions can lead to cellular senescence which has been found to increase with advancing age and, thus, contributing to age-related pathologies 115-117. Amongst the numerous signaling molecules in the DDR pathway, the tumor suppressor p53 has been identified as being a critical factor in ageing and age-related disorders whose activity increases with age 118. Mechanistically, telomeres, repeating DNA sequences located to chromosome ends, are known to play a central role in maintaining genomic integrity 119. Telomeres are successively shortened during semiconservative DNA replication, which can lead to telomere dysfunction and activation of the p53-dependent DNA damage repair/response (DDR) 120. Thus, preservation of functional telomeres is critical for preventing genomic destabilization. In order to protect chromosome ends, resembling DNA double-strand breaks from homologous recombination (HR) and non-homologous end joining (NHEJ), telomeres associate with numerous single- and double-strand telomere adapter molecules and assist in the formation of a certain chromosomal architecture which protects the chromosome ends from being recognized as DSB 121. Usually, telomeric DNA is replicated and preserved by the ribonucleoprotein complex telomerase containing the catalytic protein component telomerase reverse transcriptase (TERT) which acts as the sole instrument in preserving telomere length 122. A reduction in the activity of telomerase promotes telomere erosion, thereby impacting ageing and ageing-associated diseases such as cancer 123. Shortened and thus unprotected telomeres induce the DDR by recruiting DSBR components aiming at repairing the exposed termini. The inappropriate action of the NHEJ or HR machinery culminates in deleterious nucleolytic degradation, recombination, and chromosomal fusions 124. Examples of DSBR components gathered by short telomeres comprise ATM, 53BP1, γ-H2AX, and Mre11, termed telomere dysfunction-induced foci (TIF), that are prone to NHEJ-mediated end-to-end fusion 125-127. In this context, telomere dysfunction has been found not only to facilitate cellular senescence, but also to impair mitochondrial biogenesis and functions as well as to up-regulate ROS production 128.
The failure of telomerase in maintaining telomere length has been reported to promote premature ageing via p53 activation 129, 130. It is noteworthy that telomerase-deficient mice have shorter lifespans with early onset of ageing phenotypes 131, 132. Similarly, premature replicative senescence also causes early onset of senescence in Ku80-deficient mice devoid of functional NHEJ 133. Interestingly, the profound ageing process in these animal models can be attenuated by the depletion of p53, thereby also highlighting the key role of p53 in DDR-mediated premature ageing 134, 135. Comparable results have been obtained in H2AX-deficient mice that are defined by, inter alia, premature senescence and genomic instability 136. From these observations, it can be hypothesized that DNA damage-induced p53 and H2AX activation obviously plays a crucial role in the progress of ageing phenotypes.
It is generally acknowledged that aged cells harbor increased amounts of oxidatively damaged DNA 137. One of the most common oxidative DNA lesions in aged cells is represented by 8-oxo-dG 138. In humans, urinary 8-oxo-dG levels were found to gradually increase with age as a consequence of an up-regulated ROS production and reduced activity of 8-oxoguanine DNA glycosylase (OGG1) at advanced age 139, 140. It is noteworthy that levels of the latter remain relatively constant over a lifetime 141, while, however, the acetylated variant significantly decreases in the elderly 141, 142. An age-dependent down-regulation of mitochondrial OGG1 could also be demonstrated in mice 143. OGG1, which is primarily involved in the removal of 8-oxo-dG, and NEIL3 were recently identified to affect differentiation mechanisms and cellular senescence in neural stem cells 144.
Role of oxidative stress and inflammation in age-related disorders
Emerging evidence suggests that individuals at advanced age become more susceptible towards ageing-associated disorders, including heart disease, cancer, respiratory disease, and diabetes. Ageing cells are characterized by an arrest in normal cell division, termed cellular senescence 145. Cellular senescence is associated with the production of pro-inflammatory cytokines, leading to chronic inflammation irrespective of the activation of immune cells 146. According to Zuo et al. (2019), this process of chronic weak systemic inflammation in conjunction with ageing is designated as “inflammaging” 147. Inflammation up-regulates ROS levels causative for the induction of oxidative stress which in turn activates pro-inflammatory signaling pathways, impacting the pathogenesis of numerous ageing-related disorders 148. Apart from an enhanced ROS production, ageing is associated with an impaired anti-oxidant defense. This is given by the fact that ROS and inflammaging exert reciprocal stimulatory actions on each other. ROS have been noted to activate nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasomes that have been implicated in the pathogenesis of cardiovascular disease (CVD) and cancer 149.
A further intriguing feature of ageing relates to immunosenescence, a senescence-induced impairment of the immune system. Such oxidative stress-induced immune senescence is characterized by a serious age-dependent impairment of innate and adaptive immune responses such as, e.g., chronic inflammatory state, alterations in lymphocyte subsets, and reduced proliferative responses. Recently, De Cecco and colleagues provide evidence for a type-I interferon (IFN-I)-mediated maintenance of the senescence-associated secretory phenotype which promotes age-associated production of various inflammatory mediators 150. It is of note that elderly individuals show an increased reactivity to autoantigens, loss of tolerance, and systemic inflammation, whereas they simultaneously suffer from degenerative diseases that in turn enhance the risk of evolving autoimmune diseases 151. Epigenetic changes and the up-regulated production of inflammatory mediators make older individuals more susceptible to metabolic, neurodegenerative, and infectious diseases as well as autoimmune disorders and cancer 152.
Oxidative stress represents a critical factor in the pathogenesis of cardiovascular diseases (CVD). The risk of CVD which comprises numerous disorders such as atherosclerosis, arrhythmia, atrial fibrillation, heart failure, myocardial infarction, and stroke, increases with age and intensifies by the rise of life expectancy 153, 154. As age has an direct impact on the cardiovascular status, age-dependent impairment of vascular function can be considered as being one of the hallmarks of CVD 147, 153.
Oxidative stress has been found to impact CVD pathogenesis by aggravating atherosclerosis. With respect to low density lipoprotein (LDL), ROS-mediated LDL oxidation induces the activation of the endothelium and the initiation of immune responses. Inflammatory cells are then recruited to the arterial intima followed by phagocytosis of oxidized LDL and the release of ROS and pro-inflammatory mediators that in turn oxidize LDL, thereby promoting atherosclerosis 147, 155, 156. Concomitant endothelial dysfunction is attributable to the up-regulated generation of mtROS and mtRNS that cause subsequent cellular damage 156. For more detailed information on the molecular mechanisms contributing to CVD progression, please see review by Zuo et al. (2019) 156.
Chronic weak systemic inflammation in conjunction with ageing serves as a central player in CVD. As demonstrated by Olivieri and co-workers, the tissues surrounding the cardiovascular system are chronically inflamed in aged individuals leading to an up-regulated formation of ROS, a phenomenon termed age-associated chronic vascular age 157. Furthermore, senescence of endothelial and cardiac cells observed over normal ageing appears to be enhanced in age-related disorders such as CVD contributing to the formation of atherosclerotic plaques 157. microRNAs obviously play a central role in inflammaging, since they trigger proliferation of dysfunctional mitochondria and thereby facilitate inflammaging and CVD susceptibility 158, 159. Oxidative stress in the ageing vasculature also leads to epigenetic alterations in gene transcription culminating in aberrant molecular pathways through the process of chromatin remodelling 160. The age-dependent accumulation of dysfunctional cells is a common feature of CVD and caused by telomere-dependent senescence 157. The transcription factor NF-κB obviously plays a key role in inflammation-associated vessel dysfunction as its activity has been reported to increase with age 161.
Immunosenescence represents a further risk factor for CVD since senescent macrophages have been identified to directly contribute to the formation of atherosclerotic plaques 157. In addition, the age-dependent activation of the innate immune system by oxidative stress has been described to boost inflammation and the initiation and progression of atherosclerosis, respectively 162. It is noteworthy that the telomere length is shortened in senescent leukocytes and endothelial cells and thus assists in promoting CVD development 163.
Systemic lupus erythematosus (SLE)
Systemic lupus erythematosus (SLE) represents a chronic systemic autoimmune disease characterized by autoantibody production, complement activation, and immune complex deposition 164. Data raised by Grieves et al. in 2015 revealed that the increased frequency of polymorphisms in genes encoding certain DDR factors, such as the exonuclease TREX1, might be causative for an aberrant DDR pathway as a characteristic feature of the SLE pathogenesis 165. Accordingly, autoantibodies against certain DDR molecules could be identified in a proportion of SLE patients 166. Animal studies further underlined the central role of defective DDR mechanisms in the pathogenesis of SLE 167, 168. Meanwhile, several gene polymorphisms associated with a defective DDR system and the development and progression of SLE have been identified and include, amongst others, the BER key enzymes X-ray repair cross-complementing protein 1 (XRCC1) and DNA polymerase β (Pol β) as well as poly(ADP-ribose) polymerae 1 (PARP-1; reviewed by Mireles-Canales et al., 2018) 169. It is of note that the activity of PARP-1 after UV radiation was found as being significantly lower in SLE patients compared to healthy controls, implying that PARP-1 is implicated in the susceptibility for SLE outcome 170. The group of Petros Sfikakis previously identified defects in the NER and DSBR in peripheral blood mononuclear cells (PBMCs) from SLE patients as a consequence of a down-regulation of diverse BER and DSBR genes 171, 172. Further studies revealed that neutrophils from SLE patients show increased DNA damage, defective repair of oxidative DNA damage, and increased apoptosis rates 173, 174.
Autoantibodies against DNA repair enzymes are characteristic features of SLE and comprise antibodies against Ku70 and Ku80 involved in NHEJ and DNA-PK regulating the DDR as well as DNA ligase IV (LIG4) and XRCC4 175. Fell and colleagues found a correlation between anti-Ku positive sera from SLE patients and the presence of antibodies against the DNA repair proteins DNA-PK, PARP, Mre11, and Werner protein, respectively, in half of the patients with SLE thus providing further evidence for an impact of abnormal DSB repair on the manifestation of SLE 176. A positive correlation could also be demonstrated for anti-dsDNA antibodies (hallmark of SLE) and the levels of autoantibodies directed against apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), high mobility group box 1 (HMGB1), vaccinia-related kinase 1 (VRK1), aurora kinase A (AURKA), peptidyl arginine deiminase 4 (PADI4), and signal recognition particle 19 (SRP19) in patients with SLE 177. This correlation underlines the possibly pivotal role of certain antibodies in the SLE pathogenesis. Moreover, various apoptosis-associated genes have been noted as being up-regulated in PBMCs from SLE patients and comprise genes encoding protein phosphatase 1 regulatory subunit 15A (PPP1R15A), cyclin-dependent kinase inhibitor 1A (CDKN1A), BRCA1-associated RING domain protein 1 (BARD1), RAD21, RAD9 checkpoint clamp component A (RAD9A); DNA-PK, catalytic subunit (PRKDC); calcium and integrin-binding protein 1 (CIB1), breast cancer type 1 susceptibility protein (BRCA1), Abelson tyrosine-protein kinase 1 (ABL1), checkpoint kinase 2 (CHEK2), and Bcl-2-binding component 3 (BBC3) 171. From these observations it can be proposed that SLE patients bear a lower DNA repair capacity, leading to the accumulation of DNA lesions and the concomitant induction of the apoptotic signaling pathway. In this context, several animal studies revealed an association between SLE pathogenesis and defective apoptosis. Mice deficient of the T cell immunoglobulin mucin 4 (Tim-4), a phosphatidylserine receptor which facilitates macrophage-mediated phagocytosis of apoptotic fragments, have been reported to develop autoantibodies to dsDNA 178. Autoantibodies to both, dsDNA and ssDNA could also be detected in mice lacking the intracellular receptor of the membrane tyrosine kinase c-Mer. These mice show an impaired clearance of infused apoptotic cells and develop a lupus-like autoimmunity phenotype 179. Together with the finding that PBMCs from SLE patients harbor an increased expression of the Tim-4 mRNA 180, Tim-4 can be considered as being a crucial factor in the elimination of dead cells.
Rheumatoid arthritis (RA) is a chronic autoimmune disease classified by inflammation and joint destruction affecting functionality and being associated with increased morbidity and mortality 181-183. It is generally accepted that oxidative DNA damage and aberrations of the DDR machinery are critical features in the pathogenesis of RA 184. With respect to oxidative damage, the blood of RA patients has been characterized to exert an elevated rate of ROS production in lymphocytes, accompanied by an increase in lipid peroxidation, DNA damage, and protein oxidation 185. In addition, immune cells isolated from the synovial fluid of RA patients have been shown to produce high amounts of ROS and nitric oxide (NO) contributing to the oxidative burst as part of the inflammatory process 186. Several investigations further revealed increased levels of DNA lesions in PBMCs or granulocytes from patients with RA compared to healthy individuals 187-189. In these studies, increased levels of oxidative stress have been shown to positively correlate with the degree of endogenous DNA damage in RA blood cells. PBMCs, CD4+ CD45RA+ T cells, and granulocytes from RA patients generate higher amounts of 8-oxo-dG and abasic sites in DNA, respectively 189-191. 8-oxo-dG together with extracellular mtDNA could also be identified in the synovial fluid from RA patients, but not in controls, correlating positively with the presence of rheumatoid factor 192. These data imply a possible contribution of endogenous nucleic acid compounds to joint inflammation by stimulating joint immune cells to produce pro-inflammatory molecules. It is worth mentioning that early investigations revealed an up-regulated expression of the tumor suppressor p53 and the presence of tissue-specific p53 mutations in the synovium of RA patients 193, 194. p53 serves the function of a key DNA repair component and regulator of apoptosis. p53 mutations are typically located to the lining region of the synovium which mainly consists of fibroblast-like synoviocytes (FLS), playing a central role in destructive joint inflammation 195.
Emerging evidence suggests an association of failures in the DDR system with elevated senescence and apoptosis in RA patients 190, 191. Interestingly, T cells from RA patients have been noted as being highly susceptible to ionizing radiation (IR) together with a reduced capacity to repair DNA lesions. These T cells are characterized by a reduced basal expression of DSB sensors, such as ATM and the oligomeric MRN (Mre11-Rad50-Nbs1) complex that did not increase upon exposure to IR 191. In this context, the DNA repair nuclease Mr11a obviously plays a pivotal role in protecting mtDNA since its absence in T cells from RA patients suppressed mitochondrial oxygen utilization and ATP formation, mediated discharge of mtDNA into the cytosol, and caused tissue inflammation, caspase-1 activation, and subsequent apoptosis induction 191, 196. Souliotis and co-workers recently emphasized defects in the global genomic nucleotide excision repair (GG-NER) pathway in PBMCs from RA patients that could be reversed by a 3-month treatment with anti-rheumatic drugs 189. Owing to the above mentioned observations, the assemblage of DNA lesions as the result of a defective DDR system and intensified DNA damaging is obviously critically implicated in the pathogenesis of RA.
Diabetes mellitus (DM) is as a chronic metabolic disease which exists in multiple subtypes characterized by high-glucose levels. The main subtypes are represented by type 1 diabetes (T1DM) and type 2 diabetes mellitus (T2DM), respectively. T2DM is characterized by pre- and postprandial hyperglycemia, combined with relative insulin insufficiency as the result of inadequate insulin secretion and low insulin sensitivity 197. In contrast, T1DM represents an autoimmune disorder in which the insulin-producing β-cells in the pancreas are destroyed, thereby blocking the body’s capacity to adequately regulate blood glucose levels 198. Recent evidence suggests an association of oxidative stress with the diabetes pathogenesis as high levels of oxidative DNA damage correlates with an increase of ROS production in rodents and humans with diabetes. Oxidative stress is directly affected by glucose fluctuations that are crucially involved in the pathogenesis of diabetes 199. In obesity and diabetes, increased serum levels of 8-oxo-dG indicative for oxidatively damaged DNA have been observed correlating with the body mass index of diabetic individuals 200. A past study identified a significantly up-regulated mean urinary excretion rate of 8-iso-PGF2α (F2-isoprostane 8-iso-prostaglandin F2α), a key biomarker of oxidative stress, in patients with T2DM compared to controls 199. Also, progressive telomere shortening has been shown as being associated with obesity and diabetes 201. With respect to T2DM, individuals harboring atherosclerotic plaques present greater shortening of telomere length in comparison to those without plaques 202. As oxidative stress has been known to promote telomere shortening, it is plausible that the diabetic state aggravates the per se constrained glucose homeostasis through boosting telomere failure 203. In this context, telomere dysfunction has been found not only to facilitate senescence of adipocytes, but also to impair mitochondrial biogenesis and functions as well as to up-regulate ROS production 128. Of note, telomere dysfunction induces the activation of p53 thereby reducing the expression of the transcriptional cofactors peroxisome proliferator-activated receptor-γ co-activator 1 alpha (PGC)-1α and -1β, triggering the expression of various genes involved in mitochondrial function and glucose metabolism 128. In mice, shortened telomeres might be causative for attenuated insulin secretion which drives glucose intolerance 204. Summarizing the above, age-associated telomere shortening might contribute to the attenuation of glucose homeostasis by inducing tissue inflammation and interfering with cell metabolism.
Apart from adipocyte senescence, senescence of pancreatic β-cells obviously contributes to the pathogenesis of diabetes 205. It has been shown previously that the increase in glucose metabolism by β-cells induces the formation of DSB and activation of p53, culminating in β-cell failure in T2DM mice 206. A p53 polymorphism at position 72 (Arg72Pro) has been observed to affect insulin resistance in patients with T2DM 207. Saxena et al. convincingly reported on a strong association of T2DM with a single-nucleotide polymorphism of a non-coding region near CDKN2A and CDKN2B, coding for cyclin-dependent kinase inhibitor 2A and 2B 208. Together with the observation that variations of genes involved in DNA damage and repair, e.g., checkpoint kinase 2 (CHEK2), have been implicated in the onset of T2DM 209, one can ascertain that the DDR system is crucially involved in human glucose metabolism.
An important alteration associated with the onset of diabetes is vascular ageing 210. Vascular ageing relates to the enlargement of vessels, thickening, stiffness, and compromised endothelial barrier strength from which all have been associated with pro-inflammatory mediators. In this context, the senescence-associated secretory phenotype (SASP) has been proposed as the main origin of inflammaging in both, T2DM and ageing 211. As demonstrated by Prattichizzo and colleagues, SASP genes coding for pro-inflammatory IL-1α, IL-1β, IL-6, and TNF-α are constantly activated in the diabetic body 210. Activated endothelial cells and immune cells are the main sources of pro-inflammatory factors in diabetic patients in conjunction with epigenetic modifications. The presence of these pro-inflammatory molecules down-regulates insulin expression and boosts the recruitment of macrophages to the pancreas, ultimately leading to β-cell apoptosis in inflammation- and obesity-induced insulin resistance 212.
As mentioned before, hyperglycemia has been found to induce the formation of ROS. In particular the superoxide radical O2•− has been shown as being up-regulated in both, in vitro and in vivo studies of diabetes 213. O2•− is highly reactive and can be converted into H2O2 via disproportionation catalyzed by mitochondrial superoxide dismutase (SOD). H2O2 attacks DNA, thereby attenuating cellular functions or inducing cell death. H2O2 represents the most prominent mediator in the formation of 8-oxo-dG as it facilitates the release of HO• through the Fenton reaction 86. O2•− generation due to high glucose levels in diabetes additionally triggers multiple pathways, including increased polyol formation and hexosamine pathway flux as well as activation of protein kinase C 213. Over-expression of SOD was shown to protect insulin-secreting RINm5F cells against cytokine-mediated toxicity most likely due to the blockage of ROS production 214. In line with this observation, up-regulated expression of glutathione peroxidase (GPx) was noted to protect insulin-producing INS-1 cells against ROS and RNS assault 215. Beneficial effects of a GPx over-expression were also obtained in isolated human pancreatic islets in which GPx up-regulation prevented the adverse effects of ribose-induced oxidative stress by enhancing GPx activity and stimulating insulin gene and protein expression 216. This anti-oxidant strategy might thus be helpful in reducing oxidative DNA damage as occurring in diabetic patients.
Genomic instability can be considered as being one of the hallmarks of cancer as tumor cells are characterized by unlimited proliferation, chromosomal translocations, and aneuploidy as a result of mutagenic DNA damage 217. Mutations in DNA repair genes are the causative factors in the pathogenesis of several hereditary malignancies, thereby outlining their impact in tumorigenesis 218-220. For instance, xeroderma pigmentosum (XP) is a rare autosomal recessive genodermatosis that manifests due to mutations in nucleotide excision repair (NER) leading to the accumulation of unrepaired DNA lesions and consequently predisposing to skin cancer 221-223. A total of eight distinct mutations have been identified to associate with diverse subtypes and clinical presentations of XP. These mutations affect genes encoding DNA damage binding protein 1 (DDB1) and 2 (DDB2); excision-repair cross-complementing 2 (ERCC2), ERCC3, ERCC4, ERCC5 as part of the TFIIH complex encoding XPD, XPB, XPF, XPG; as well as POLH coding for DNA polymerase eta 221 (Table 1). Any patient with XP will present with skin alterations secondary to extreme sun sensitivity. Patients may also present with oral, ophthalmologic, and/or neurologic manifestations of the disorder.
Similarly, germ line mutations in the DNA repair gene ataxia telangiectasia mutated (ATM) can lead to increased sensitivity towards ionizing radiation, humoral and cellular immunodeﬁciency, chromosomal instability, and cancer predisposition 223-226. The ATM protein is crucially involved in mediating the cellular response to radiation-induced DNA damage 227. Patients with the disease of ataxia telangiectasia undergo serious and desastrous responses to ionizing radiotherapy 225 and harbor a massively increased risk for cancer development most likely due to the failure in detecting DNA DSB which leads to genetic instability and consequently to enhanced susceptibility towards cancer development. Meanwhile, several ATM gene polymorphisms causative for a defective DDR system have been associated with the development and progression of certain cancers such as colorectal 228, prostate 229, breast 230 and lung cancer 231, 232. Likewise, germ line mutations in the breast cancer susceptibility gene 1 and 2 (BRCA1 and BRCA2), which participate in the repair of DNA DSB through homologous recombination (HR), have been estimated to contribute to 5–10% of all breast carcinomas and approximately 13% of all ovarian cancer cases 233, 234.
Characteristic features of cancer cells are their unlimited proliferative potential and the resultant higher energy requirements. Consequently, cancer cells produce elevated amounts of ROS that play a pivotal role in tumor initiation and progression 235. Up-regulated amounts of mtROS stimulate carcinogenesis via induction of mtDNA mutations and stimulation of oncogenic cell signaling pathways through activation of NF-κB and signal transducer and activator of transcription 3 (STAT3) 236-239. These mutagenic DNA lesions comprise, amongst others, 8-oxo-7,8-dihydro-2’-deoxyguanine (8-oxo-dG) and 8-nitroguanine that can be found especially in inflamed tumor tissues where they act as biomarkers of oxidative stress 240, 241. A breast cancer case-control study recently revealed that 8-oxo-dG is located to the coding strand of exon 5 of the tumor suppressor gene TP53, a region which has a high mutation prevalence. Moreover, the location of this specific oxidative DNA lesion has significantly been associated with the pathological stage and the histological grade of tumors 242. A growing body of incidence suggests that mtDNA mutations in turn can result in an elevated formation of ROS by virtue of interruptions in the electron transport chain, driving the oxidative burst seen in cancer cells 243.
Chronic inflammation has emerged as a further hallmark of cancer. The persistence of chronic inﬂammation in the tumor microenvironment stimulates tumor growth through the engagement of inflammatory cells and uncontrolled tissue repair 244. Inflammatory cells are able to produce a broad spectrum of inflammatory mediators such as cytokines, chemokines, proteases and prostanoids as well as ROS and RNS. Leukocytes represent the primary source of RNS and ROS serving as chemical triggers in inflammation-driven tumorigenesis 245. All of them co-operate in maintaining and augmenting the inflammatory cascade. On the one hand, inflammatory mediators inhibit DNA repair mechanisms culminating in microsatellite instability (MSI) as an expression of genomic destabilization caused by mutations or epigenetic alterations of members of the mismatch repair (MMR) family (Figure 7) 246. In this regard, the transcription factor HIF-1 plays a crucial role in inflammation-driven carcinogenesis as it is induced by various inflammatory mediators including cytokines, prostanoids, ROS, and RNS 247, 248. HIF-1α, the oxygen-regulated subunit of HIF-1, has been noted to affect genomic stability by suppressing the MMR proteins MSH-2 and MSH-6, thereby diminishing MutSα levels responsible for the detection of base mismatches. HIF-1α removes the transcriptional activator c-Myc from Sp1 binding to block MutSα expression in a p53-dependent manner 249. In addition, ROS such as H2O2 have the capacity to inhibit members of the MMR family at the protein level 250.
Figure 7: Genomic destabilization of cancer cells caused by inflammatory mediators. For details see text. Image was reproduced from Multhoff and Radons (2012) 104. License at https://creativecommons.org/licenses/by-nc/3.0/.
On the other hand, inflammatory mediators can cause chromosomal instability leading to abnormal chromosomal segregation and aneuploidy. These molecules induce the formation of DNA DSB 251, 252, trigger the action of mitotic checkpoint molecules 253, 254 and deregulate HR of DNA DSBR 246 leading to random genetic diversification of cancer cells caused by microsatellite and chromosomal instability. Cancer cells bearing an optimum combination of activated oncoproteins and inactivated oncosuppressors will be evolving the cancerous phenotype 103. It is worth mentioning that genomic instability is also driven by hypoxia, a common feature of most advanced solid tumors. Hypoxia triggers genomic destabilization by suppressing DSBR via transcriptional, translational, and epigenetic regulation of central repair molecules including DNA-PKcs (DNA-PK catalytic subunit), Ku70/80, BRCA1, and RAD51 255. As outlined before, oxidative stress is known to suppress MMR by repressing the expression of MutS 250. Notably, patients with colorectal tumors characterized by MMR deﬁciency bear enhanced inﬁltration of T cells and higher microsatellite instability implying a profund interaction between the MMR and immune cells 256. Oxidative stress in the context of chronic inflammation has also been found to block BER by stimulating the inﬂammasome 149, 256. The NLRP3 inflammasome is able to inhibit chemotherapeutic agents and promote tumorigenesis 257. Although the NLRP3 inflammasome functions as a putative oncoprotein in non-small cell lung cancer genomic analyses, however, recent data indicate that it also serves as a tumor suppressor 258-260 thereby underlining the ambivalent role of the NLRP3 inflammasome in tumorigenesis.
Emerging evidence indicates a crucial role of oxidative stress and inflammation in the pathogenesis of various neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Oxidative stress in the nervous system has been demonstrated to increase with age accompanied by a diminished regenerative capacity and functionality of nerves 261, 262. In the intact ageing nerves, persistent inflammation coincides with amplified macrophage infiltration as well as up-regulated levels of CC chemokine ligand 11 (CCL11) and monocyte chemoattractant protein 1 261. According to Buttner et al. (2018), up-regulation of CCL11 suppresses the differentiation of Schwann cells with increasing age, thereby attenuating the regenerative capacity of ageing nerves 261. Levels of pro-inflammatory TNF-α, IP-10, and IL-8 in the cerebrospinal fluid (CSF) have been shown to increase linearly with age, consistent with a shift from a Th1 to a non-Th1 cytokine milieu particularly in multiple sclerosis and AD 263. AD is characterized classically by two hallmark pathologies: β-amyloid plaque deposition and neurofibrillary tangles of hyper-phosphorylated tau 264, 265.
Oxidative stress mediated by ROS is crucially involved in the pathogenesis of AD contributing to neuronal cell death. Numerous investigations during the past years revealed an increased oxidative DNA damage in AD brains as evidenced by the detection of 8-oxo-G and 8-oxo-G DNA glycosylase 1 (OGG1) 266. More recent studies detected elevated levels of 8-oxo-G and reduced levels of the DNA repair enzyme OGG1 in the serum of AD patients 267. Weimann et al. used an isotope-dilution UPLC-MS/MS method with high specificity and sensitivity for the quantification of 8-oxo-G and 8-oxo-dG in CSF and found up-regulated levels of these oxidative DNA damage biomarkers in neurodegenerative diseases as well 268. Elevated ROS generation seen in AD brains takes part in the aggregation of Aβ and hyper-phosphorylation of Tau by inducing the JNK and p38 MAPK signaling pathways 269. Aβ plaques not only interfere with the Ca2+ balance in the ER but also boost the production of ROS which attack various cellular macromolecules such as DNA, lipids and proteins thereby inducing apoptosis-mediated death of neurons 270. Interestingly, Aβ itself is able to induce the expression of several pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α and IFN-γ by glial cells that, in turn, intensify the Aβ production 271-274. In brain specimens from AD patients, elevated expression of pro-inflammatory IL-18 could be detected by Sutinen and co-workers who described an increase of IL-18-mediated expression of proteins involved in the processing of the amyloid precursor protein (APP), consequently leading to an increase in APP levels and Aβ synthesis 275. These pro-inflammatory mediators are known to activate several transcription factors such as STAT3 and NF-κB. The latter has been found as being highly activated in the brain of AD patients 276, 277. NF-κB activation is mediated by oxidative stress, hyper-activated microglia, and down-regulated SIRT1 expression, collectively resulting in the expression of pro-inflammatory genes 276.
As already discussed, oxidative stress is also implicated in the pathogenesis of PD contributing to dopamine cell degeneration 278. Oxidative damage to macromolecules has been observed in PD, and toxic products of oxidative damage, i.e., 4-hydroxynonenal (HNE), can react with proteins to impair cell viability. In particular HNE has been found to inhibit the 26S proteasome which, in turn, up-regulates free radical production and oxidative stress 278. Also, Nakabeppu et al. have shown a significant increase in 8-oxo-G in mtDNA as well as an elevated expression of MTH1, OGG1, and MUTYH in nigrostriatal dopaminergic neurons of PD patients, implying that the formation of these lesions might be implicated in dopamine neuron depletion 279. A clinical study conducted in 44 patients with PD revealed elevated levels of 8-OH-G and 8-OH-dG in CSF compared to controls 280. The same study identified significantly higher CSF values of 8-OH-dG in PD patients without dementia, while patients with dementia harbored reduced levels 8-OH-G in CSF compared to controls. From these observations the authors speculate that the determination of 8-OH-dG in CSF might serve as an “early-stage marker”, whereas the decrease of 8-OH-G in CSF might reflect the degree of neurodegeneration during disease progression.
Inhibition of complex I in the mitochondrial respiratory chain and excessive generation of mtROS in the substantia nigra pars compacta are held responsible for most of the neuronal cell depletion in PD because dopaminergic neurons are specifically sensitive towards the deleterious effects of oxidative stress 281-283. New evidence emerges demonstrating that various PD-related mutations, affecting leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1), and parkin, have been implicated in mitochondrial dysfunction and the breakdown of redox equilibrium in neuron cells 269. It is worth noting that activation of LRRK2 has been correlated with elevated oxidative stress and intensified inflammatory response, thereby promoting disease progression 284. A major source of ROS, RNS, matrix-degrading metalloproteinases (MMP) and pro-inflammatory mediators is found in activated microglia which in turn can be activated by Toll-like receptor (TLR) signaling or pro-inflammatoty cytokines 285, 286. Microglia- and inflammation-derived pro-inflammatory cytokines were reported to facilitate the permeabilization of the blood brain barrier in PD individuals thereby enabling the infiltration of T cells and further leukocytes into the CNS and thus impacting microglia activation and pathogenesis of neurodegeneration 285-287. Similar to AD, oxidative stress in the context of inflammation has been reported to activate the inflammasome also in aged mice leading to neuronal cell death 288. These findings indicate that the inflammasome contributes to oxidative stress and cell damage in the ageing brain prior to the onset of PD 288. Considering the manifold implications of oxidative stress in neurodegenerative disorders, the regulation of cellular ROS levels may represent a potential tool to aggravate neurodegeneration and impair associated symptoms 289.
Inflammaging is also critically involved in the pathogenesis of digestive diseases such as inflammatory bowel diseases (IBD) 290 and chronic obstructive pulmonary disease (COPD) 291. Interested readers will find detailed information in the relevant literature. For an overview, see reviews by Zuo et al. (2019) 147 and Liu et al. (2018) 269.