Oxidant-Based Cytotoxic Agents During Aging

1. Introduction

Aging is accompanied by a gradual decline of physiological functions and physical activity and is associated with increased predisposition to different health-threatening diseases. Despite the existence of about 300 theories and hypothesis about the reasons of aging, there does not exist any comprehensive description explaining all miraculous facets of the aging process [1]. These theories on aging can be roughly divided into three main categories: i) theories related to deviations in description of genetic information, ii) theories favoring any problems in hormone metabolism, and iii) theories based on accumulation of waste products in cells and the organism [2]. Although numerous facts are provided in favors of all these theories, it remains unknown how these different approaches can be synergized in a unifying theory.

In attempt to create a more complex trial of description of the aging process, I proposed that aging is an inevitable consequence of growth limitation during the development of complex multicellular organisms [2,3]. On the one hand, in a growing organism, cells are dividing permanently, increasing thus the mass of the biological material. In order to supply adequately all cells of this organism with energy and food, differentiation and functional specification of cells and formation of specific tissue types and organs are mandatory. This further increases the complexity of the developing organism. On the other hand, each kind of biological material is devoid to numerous chemical and physical impacts that can disturb the integrity of this highly ordered living matter and impairs thus physiological processes in the growing organism. Despite the existence of numerous immediately acting and inducible protective mechanisms in living matter, only a sufficient high growth rate can ensure functionally intact organisms. After reaching an optimum size for each organism, the general growth rate declines more and more. As a result, destructive elements can’t be compensated as efficiently as before [3]. There is also a gradual shift in energy balance in elderly organisms from organism-specific processes like active movement, sensing the environment, food intake, or physical and mental work towards the predominance of processes counterbalancing any deviations from normal physiological functioning on systemic and also cellular level [2]. With increasing age, more and more energy equivalents are required to maintain essential physiological parameters in cells and tissues of the organism.

In organisms, important protective functions are realized by the immune and associated systems like acute-phase, complement, coagulation, and contact systems [4,5]. The activity of these systems is directed to ensure the maintenance of basic homeostatic parameters in cells, tissues, and the whole organism. The steady increase of any kind of destructions in elderly persons enhances the activity of these protective systems and is associated with an increase of permanent inflammatory states and immune dysfunctions. This condition, which is known as inflammaging [6], represents a key risk factor for the increased appearance of life-threatening chronic diseases and adverse health outcomes [7].

During inflammatory response several host-derived cytotoxic agents are generated and released from activated immune cells and undergoing tissue cells [8]. These agents contribute to destruction and elimination of foreign microorganisms and unwanted cells, but can also disturb the integrity of healthy cells and tissues. Cytotoxic agents are usually well controlled by corresponding antagonizing principles to avoid any damage of unperturbed cells in close neighborhood to inflammatory sites. Disturbances in the balance between cytotoxic agents and protecting mechanisms favor chronic inflammatory states and disease progression [8]. Otherwise, overexpression of antagonizing principles as found for example in late-stage tumor cells promotes also markedly the disease process [9].

In this review, the interplay between oxidant-based host-derived cytotoxic agents and protective mechanisms is analyzed in elderly persons. The question will be addressed how the increased appearance of inflammatory states in aged individuals affects this balance and contributes to pathogenesis of life-threatening diseases.

2. Key Concepts About Inflammation in Aged Individuals

2.1. The Concept of Inflammaging

The concept of inflammaging was developed from the network hypothesis of aging [10,11], which is based on the idea that in elderly individuals the ability of different anti-stress systems is limited to counterbalance any destructive deviations in cells and organs of elderly organisms. A key point of inflammaging is that aging is associated with an increased activity of both the innate and activated immune responses in elderly persons [12]. Indeed, with advanced age, the serum level of a variety of pro-inflammatory mediators increases as shown for IL-6, IL-15, IL-8 [12,13,14], and coagulation factors like fibrinogen and von Willebrand factor [15,16]. In total, this increase indicates the presence of a permanent inflammatory state that can be identified as a low-grade chronic inflammation [17].

According to the inflammaging hypothesis, the balance between pro-inflammatory and anti-anti-inflammatory agents is disturbed in aged individuals [12]. This balance is crucial for resistance against any kind of diseases. Important anti-inflammatory agents in elderly persons are TGF-β, cortisol, IL-10, and lipoxins [12,18,19].

Without going into details, the concept of inflammaging summarizes key features characteristic of the chronic inflammatory process in aged persons [7,12,17,20,21,22,23,24]. To get an answer why an inflammation persists under these conditions, it is necessary to analyze how molecular patterns and host-derived cytotoxic agents contribute to the development of long-lasting inflammatory states.

2.2. Molecular Patterns and Host-Derived Cytotoxic Agents in Inflammation

During inflammation, the activation of the immune system and associated protective mechanisms is mandatory to eliminate pathogens, transformed and damaged cells and to induce repair processes. In humans, inflammatory response is initiated by the presence of molecular patterns, which activate immune cascades via interaction with pattern recognition receptors (PRRs) [25]. These molecules result from pathogens (pathogen-associated molecular patterns (PAMPs)) and undergoing immune and non-immune cells of the host (damage-associated molecular pattern, DAMPs) [26,27]. Important DAMPs are heat-shock proteins, high-mobility group box 1 protein, fragments of hyaluronan, uric acid, heparan sulfate, free heme, and tumor DNA [28,29,30,31,32,33]. An acute inflammation is terminated when the number of molecular patterns becomes beneath an unrecognizable level. In other words, inflammation becomes resolved when the pathogen load is considerably reduced and the damage of host cells is substantially limited.

Concerning major mediators and pathways, an acute inflammation can be subdivided into two main phases, initiation and propagation of inflammation as well as resolution of inflammation. During the first phase of an acute inflammation, as a result of the presence of molecular patterns several immune cells are recruited to inflammatory sites and activated, whereby pro-inflammatory cytokines like IL-1, IL-6, IL-8, IL-15, tumor-necrosis factor α (TNF-α) are involved in attraction of neutrophils, monocytes, macrophages, T-cells, and others [34,35,36]. In addition, several acute-phase proteins are released into circulation, at which C-reactive protein (CRP) and serum amyloid A (SAA) serve as important biomarkers of the course of inflammation [34,37,38]. At inflamed loci, immune cells combat against pathogens, and remove damaged cells and cell debris. In macrophages (here subtype M1) and other immune cells, glycolysis is the preferred pathway for ATP production [39,40,41,42]. In the second phase of an acute inflammation, immune cells are suppressed. There is now a dominance of repair processes and synthesis of novel extracellular matrix components. These processes are driven by resolving mediators like transforming growth factor β (TGF-β), IL-10, vascular endothelial growth factor (VEGF), and lipoxins and directed to restore the former tissue homeostasis [43,44,45,46,47,48,49]. The number of myeloid-derived suppressor cells (MDSCs) increases markedly [50,51]. The macrophage subtype M2, which actively promotes oxidative phosphorylation, dominates now [52,53]. Key events, mediators, and pathways of both main phases of an acute inflammation are summarized in Table 1.

By which mechanisms, resolution of inflammation is initiated at inflammatory sites remains puzzling. Under discussion are the enhanced formation of specific resolving mediators like TGF-β, IL-10, VEGF, and lipoxins, which drive accumulated immune cells into an anti-inflammatory and immunosuppressive state and promote tissue repair processes [43,44,45,46,47,48,49]. Another aspect concerns the prevailing dominance of apoptotic cell death processes over the uncontrolled release of cytotoxic agents from necrotic tissues and activated immune cells at inflammatory loci. In addition, release of lactate from activated pro-inflammatory macrophages (M1 type) can induce in neighbored yet unperturbed invading macrophages a metabolic switch towards the M2 type [54,55,56,57]. Lactate serves as fuel in these cells to drive mitochondrial energy metabolism.

In order to better understand how an acute inflammatory response becomes persistent, it is necessary to analyze the role of different host-derived cytotoxic agents in these processes [8]. Molecular patterns are able to initiate inflammatory events. Recruited and activated immune cells are directed to inactivate and kill pathogens and transformed cells, and to destroy and remove damaged cell material. In these activities, immune cells apply different cytotoxic agents, which can also damage yet unperturbed cells at inflammatory sites. Healthy cells and tissues are equipped with numerous antagonizing principles, which inactivate immediately cytotoxic agents and limit thus their unwanted destructive actions. Cytotoxic products of activated neutrophils and their antagonizing principles are listed in Table 2. Other major sources for cytotoxic agents are other immune cells, defective mitochondria, blood hemorrhages, and damaged muscles. In addition to cytotoxic agents listed in Table 2, other important cytotoxic agents are peroxynitrite, free heme, lipid hydroperoxides, mast cell proteases, angiotensin, bradykinin, and matrix metalloproteases [8].

The interplay between cytotoxic agents and antagonizing principles highly determines the further fate of an inflammation [8]. The inflammation becomes resolved when antagonizing principles properly control the action of cytotoxic agents and deactivate them. Any disturbance in the balance between cytotoxic agents and antagonizing principles can result in chronic inflammatory states due to the inability of protective mechanisms to eliminate or inactivate efficiently host-derived cytotoxic agents [8]. As a result of this inability, novel DAMPs and antigens are released by the action of cytotoxic agents and the inflammatory process continues. A scheme about the interplay between molecular patterns and cytotoxic agents in persistence of chronic inflammatory states is given in Figure 1.

In aged people, low-grade chronic inflammation dominates. It is evident that the terms pro-inflammatory and anti-inflammatory agents used in the concept of inflammaging [12] correspond to the predominating mediators in the two main phases of an acute inflammation. During initiation and propagation of inflammation pro-inflammatory agents play an important role and anti-inflammatory mediators are typical of the resolution of inflammation. In analyzing inflammatory processes in aged individuals, the main focus has been directed on the behavior of different pro- and anti-inflammatory markers in association with selected disease processes. Considering the imbalance between cytotoxic agents and antagonizing principles in the development of different age-related disease scenarios, in this review the attention is focused on the role of reactive species, the loss of control over the sequestration of transition metal ions, and diminution and exhaustion of haptoglobin and hemopexin. Oxidant-related cytotoxic agents have a high potential to disturb metabolic processes within cells during aging and to contribute thus to development of disease scenarios associated in aged people.

In analyzing molecular mechanisms of age-related metabolic alterations and conditions for the development of diseases, changes in energy metabolism provide the fundamental clue to better understand the disturbed interplay between oxidant-based cytotoxic agents and antagonizing principles in aged individuals.

3. Peculiarities of the Energy Metabolism and Oxidative Stress in Aged Individuals

3.1. Energy Metabolism Under Normoxic Conditions

In humans and higher animals, adenosine triphosphate (ATP) is the master molecule to provide energy equivalents for numerous energy-intensive processes such as muscle contraction, kinase activities, active transport, intra- and extracellular signaling and others [87,88,89]. In human cells, ATP can be generated both in the cytosol by anerobic glycolysis and most of all within mitochondria by oxidative phosphorylation. Consumption of 1 mole glucose during dioxygen-independent cytosolic glycolysis yields 2 moles ATP. In mitochondria, production of ATP is linked to the tricarboxylic acid (TCA) cycle (Krebs cycle) and the electron transport chain through mitochondrial complexes I-IV. During these processes a pH gradient and an electrical potential are generated across the inner mitochondrial membrane, which are essential for proper functionality of ATP synthase. These metabolic pathways highly depend on the presence of dioxygen and yield about 30-36 moles ATP per mole initial substrate [90].

Several pathways can supply substrates that enter the TCA cycle for subsequent ATP production. Degradation of glucose by glycolysis yields pyruvate and further acetyl-CoA, which enters the TCA cycle. The latter substance is also produced during β-oxidation of long-chain fatty acids. Which type of fuel molecules is utilized for ATP production in cells depends on cell function and physiological context [91]. In cardiac muscle cells, and adipose tissue, β-oxidation of fatty acids predominates to produce ATP. Otherwise, glucose is the preferred fuel molecule in cells of the central nervous system. Degradation of free amino acids contribute to about 10-15 % ATP generated via oxidative phosphorylation [92]. After deamination, the remaining carbon skeleton of amino acids is converted into intermediates, which can enter the TCA cycle at several places.

Although mitochondrial oxidative phosphorylation is more efficient than cytosolic glycolysis concerning the amount of ATP generated per mole initial substrate, glycolysis predominates as main process of energy production in some immune cells (e. g. in activated neutrophils [42], activated macrophages (M1 type) [40], maturating dendritic cells [41], T_h1, T_h2, T_h17 lymphocytes [39,93,94], muscle fibers type IIB [95], and under certain (patho)physiological conditions (e. g. starvation, prolonged exercise). ATP formation by glycolysis is more rapid than within mitochondria. Moreover, glycolysis can be further intensified by up-regulation of the involved enzymes and fast supply of additional glucose from glycogen stores [96].

Starvation, prolonged intense exercise, some diets, dioxygen deprivation, or disease conditions can deplete glycogen stores in liver and muscles [96,97]. As a result, ketone bodies are formed and the de novo synthesis of glucose is accelerated in the liver. Both mechanisms contribute to ATP production. Ketone bodies (acetoacetic acid, acetone, and β-hydroxybutyrate) are derived from fatty acids in the liver and converted after their transport to tissues into acetyl-CoA, which enters the TCA cycle [98]. In the liver and to some extent also in the kidneys, glucose can be generated by gluconeogenesis, whereby lactate, glycerol, glutamine, and alanine are the starting sources for this pathway [92,99]. By these mechanisms, ATP maintenance is ensured to some extent even under short-term and mild disturbances of energy supply and different (patho)physiological conditions are bypassed. Major pathways of cellular energy metabolism are schematically depicted in Figure 2.

3.2. Deviations in Energy Metabolism in Aged Individuals

In aged individuals, production of energy equivalents by oxidative phosphorylation can be highly disturbed by dioxygen deficiency, the presence of dysfunctional mitochondria, and a lower number of these organelles [100,101,102]. Under these conditions, the TCA cycle and mitochondrial electron transport chain are unable to generate high amounts of ATP. During aging, it may occur that glycolysis is highly up-regulated with lactate as final product. Deficient mitochondrial processes can lead to enhanced formation of superoxide anion radicals (O₂^∙-) and other reactive species like hydrogen peroxide (H₂O₂), Fe²⁺, hydroxyl radicals (HO^∙), and peroxynitrite (ONOO^-), which all are derived from reactions of superoxide anion radicals. Important alterations in energy metabolism in aged individuals are also highlighted in Figure 2.

Several aspects are under discussion contributing to reduced capacity of mitochondria for ATP production in aged individuals. i) Mitochondrial DNA is more prone to damage than nuclear DNA [103]. The lack of histones within mitochondria and a less efficient nucleic acid repair machinery are responsible for the higher mutation rate of mitochondrial DNA [104]. ii) Enhanced production of mitochondria-derived reactive species is another key mediator disturbing enzymes and proteins involved in oxidative phosphorylation. Hypoxic conditions are a major contributor to the increased generation of mitochondria-derived reactive species. iii) In skeletal muscle, the number of mitochondria declines gradually with increasing age [105]. iv) The rate of apoptosis of cells with defective mitochondria increases too with increasing age [106].

Age-related alterations in mitochondria are also reflected by the decline of NAD⁺ [107,108] and α-ketoglutarate [109,110]. NAD⁺ is involved in many enzymatic reactions within mitochondria. This decline impairs, for example, the activity of sirtuins, which play an important role in the maintenance of mitochondrial homeostasis [111,112,113,114]. Decreased level of α-ketoglutarate affects the epigenetic landscape in several tissues [110,115,116].

Regarding molecular mechanisms of energy metabolism in mitochondria, accelerated aging is mainly favored by activation of the mTOR pathway and suppression of autophagy by increased formation of aspartate from oxalacetate or increased yield of pyruvate, which is further converted to acetate [117]. These mechanisms are supported by high caloric food and sedentary lifestyle. Further factors contributing to accelerated aging are formation of advanced glycation end products due to enhanced glycolysis rate, impaired repair of DNA hypermethylation induced by increased succinate levels, and reduced maintenance of the stem cell pool by increased level of α-ketoglutarate [117]. Several alterations in the activity of TCA cycle enzymes are responsible for the aforementioned processes. Oxidative stress is associated with decreased activity of succinate dehydrogenase, α-ketoglutarate dehydrogenase, and aconitase and favors the conversion of oxalacetate and glutamate by aspartate aminotransferase into α-ketoglutarate and aspartate. Hence, enhanced levels of aspartate, succinate, and α-ketoglutarate result [117]. Aconitase contains a 4Fe-4S-cluster at active site. Under stress conditions, superoxide anion radicals are known to release Fe²⁺ from this iron-sulfur cluster [118,119].

Decreased energy expenditure by oxidative phosphorylation can be compensated by increased anaerobic glycolysis to yield pyruvate, which is further converted by lactate dehydrogenase (LDH) to lactate. This reaction sequence is activated under hypoxic conditions and in cases of high energy demand [120,121]. As the LDH-driven conversion of pyruvate to lactate is accompanied by reduction of NADH, the resulting NAD⁺ stabilizes the cytoplasmic redox status [122,123]. Lactate is secreted from cells together with H⁺. Once released, lactate is disposed by other cells within the tissue or transported via bloodstream mainly to liver, brain, heart, and working skeletal muscle, where it is taken up and served as fuel [124,125,126] or is used for gluconeogenesis as in the liver [127]. Furthermore, lactate promotes release of Mg²⁺ from endoplasmic reticulum, favors gene expression by interaction with histones, and inhibits lipolysis via ligand binding to the G-protein GPR81 [128,129].

Lactate is an anti-inflammatory agent. At sites with enhanced lactate levels, monocytes and macrophages are reprogramed into an anti-inflammatory M2-type [56,57]. In human monocytes and macrophages, extracellular lactate shifts the energy metabolism from glycolysis to oxidative phosphorylation. Whereas short-term lactate exposure has limited effects on cytokine production, long-term exposure reprograms immune cells to anti-inflammatory responses [57]. Lactate inhibits monocyte migration and cytokine release [130,131].

When glycolysis prevails for ATP production, mitochondria serve mainly as biosynthetic organelles to yield initial components for synthesis of lipids, nucleotides, and non-essential amino acids [132]. The uptake of glutamine increases markedly under these conditions. The stepwise conversion of glutamine into α-ketoglutarate provides the basis for synthesis of biological molecules independent of glucose metabolism. For example, α-ketoglutarate is converted via the intermediate formation of citrate into acetyl-Co-A, which is necessary for lipogenesis [133].

The aforementioned deviations in energy metabolism in aged individuals affect general physiological processes, induce numerous adaptations to altered cellular metabolic routes, and promote inflammatory reactions by release of DAMPs from defective cells. In particular, oxidative stress and oxidants as cytotoxic agents play an increasing role under these conditions.

3.3. Responses to Hypoxia and Oxidative Stress

In order to better understand the real nature of inflammatory processes in individuals of advanced age, it is necessary to focus on basic mechanisms how cell and tissue homeostasis can be maintained over long time under conditions of increased impacts. In elderly persons, these impacts result predominantly from deficits in dioxygen and nutrient’s supply. Hypoxia is a key factor affecting metabolic routes in elderly persons [134,135,136]. Without considering external reasons for generalized hypoxia such as dioxygen deficiency in the breathing gas (e. g. stay of people at high altitude), at advanced age hypoxic conditions are often the consequence of a diminished blood flow, lung problems, or the reduced capacity of blood cells to carry dioxygen. In addition, health problems and enhanced blood pressure values are other major players in formation of hypoxic conditions.

In the affected tissue regions, hypoxia causes activation and stabilization of cytosolic hypoxia-inducible factor 1α (HIF-1α), which up-regulates glycolysis, down-regulates mitochondrial oxidative phosphorylation, and favors the enhanced formation of reactive species in dysfunctional mitochondria [137,138,139]. In highly vascularized tissues, HIF-1α is replaced by HIF-2α during prolonged hypoxia [140,141].

HIF-1α is a master regulator of glycolysis in muscle and many other cells. Cytosolic HIF-1α is usually tagged by prolyl hydroxylase for proteasome degradation [142,143]. Essential cofactors for active prolyl hydroxylase are O₂, Fe²⁺, and α-ketoglutarate [144]. This proteasomal degradation of HIF-1α is lost under hypoxic conditions due to lack of O₂ [137]. In addition, HIF-1α is known to up-regulate transcription of several enzymes promoting glycolysis such as hexokinase II (HK2), pyruvate kinase M2 (PKM2), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase-1 (PDHK1). The latter enzyme is a negative regulator of pyruvate dehydrogenase (PDH) [138]. As a result, pyruvate is mainly metabolized to lactate, but not to acetyl-CoA in hypoxic cells.

In addition to stabilization of cellular redox processes, long-lasting adaptations to hypoxia mainly concern the over-expression of proteins antagonizing oxidative stress. Activation of the stress-sensitive transcription factor Nrf2 protects cells against damaging reactions of reactive species and related oxygen-based cytotoxic agents [145,146,147]. Under stress conditions, superoxide dismutase, catalase, glutathione peroxidase and other protecting antioxidative proteins can be up-regulated. This counter-regulates at least partially or more efficiently stress-related oxidant-based cytotoxic agents. Several potential scenarios determine the further fate of affected cells and tissues. First, cell metabolism can be stabilized by up-regulation of protective antioxidant systems and cells survive as found in many cancers. Second, processes of autophagy and mitophagy are enhanced to remove damaged cell material and defective organelles. Third, last but not least, cells are driven into apoptosis or necrosis. These scenarios have fundamental consequences for the (patho)physiological features of hypoxic areas.

Enhanced expression of hypoxia-induced factors (HIFs) causes numerous gene-induced adaptations to stabilize the cellular redox status, to resist against oxidative stress, and to reprogram cell metabolism. Enhanced intracellular values of reactive species and other oxidant-based cytotoxic agents activate the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) that plays a key role in enhanced synthesis of antioxidant proteins [145,146]. Under normal physiological conditions Nrf2 is complexed with the by Kelch like-ECH-associated protein 1 (KEAP1) and Cullin 3, which cause ubiquitination and degradation of Nrf2 [147]. Cellular stress releases Nrf2 from this complex and promotes nuclear transcriptional processes by Nrf2. Genes activated by Nrf2 code proteins, which are involved in carbohydrate metabolism including NADPH generation, redox regulation and antioxidant defense systems like glutathione and thioredoxin mediated reactions, heme detoxification, iron metabolism, lipid metabolism, proteasomal degradation, autophagy, apoptosis, and biotransformation and detoxification [148].

3.4. Redox Regulation and Antioxidative Defense in Aged Individuals

Many physiological functions of cells depend on the presence of a reducing milieu. A high cytoplasmic level of NADPH is mandatory to ensure redox homeostasis in cells. NADPH is the major electron source within cells [123]. It is oxidized to NADH⁺ by glutathione reductase [149] and thioredoxin reductase [150,151] keeping both glutathione (GSH) and thioredoxin (Trx) in their reduced states. In quiescent cells, the ratio [NADPH]/[NADP⁺] is approximately 100:1 [123]. Two enzymes of the pentose phosphate pathway that runs parallel to glycolysis, namely glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, reduce NADP⁺ to NADPH [152]. An overview about basic mechanisms of cellular redox homeostasis and potential alterations under stress situations is given in Figure 3. In contrast, in the redox couple NAD⁺/NADH, the oxidized form predominates giving values between 1:10 and 1:1000 for the ratio [NADH]/[NAD⁺] [123]. Interconversions between members of both nicotinamide-based redox couples are controlled by NADH kinases and NADPH phosphatases [153]. By these reactions and the presence of numerous cytosolic binding sites for NADH and NADP⁺ [123] both redox couples are kept far from equilibrium. These conditions allow to fulfil them key physiological functions in maintenance of redox homeostasis.

With advanced age, the increasing significance of glycolysis has several consequences for the redox status. In addition, cytoplasmic and mitochondrial stress increases too. The higher demand of NADPH can be compensated by enhancement of the pentose phosphate pathway. The enhanced formation of lactate from pyruvate is coupled to reduction of NADH to NAD⁺. The latter species is a cofactor for numerous enzymes including sirtuins, which are important modulators of mitochondrial homeostasis and aging [111,112,113,114]. Both reduced formation of novel NAD⁺ and higher activity of NAD⁺ consuming enzymes contribute to age-dependent decline of NAD⁺ [102,107,108]. Reduced level of NAD⁺ is associated with defects in oxidative phosphorylation [154,155,156,157,158].

The glutathione system is involved in removal of lipid hydroperoxides, peroxynitrite, and hydrogen peroxide by glutathione peroxidase-mediated reactions, reduction of disulfides, and reduction of glutaredoxin [159]. Glutathione reductase converts oxidized glutathione (GS-SG) to the reduced form (GSH). In addition, GSH can be supplied by de novo synthetic processes. In elderly individuals, disturbance of components of redox metabolism highly depends on the intactness of immune functions. Thus, in this cohort, improved immune functions correlate with higher values of glutathione peroxidase, glutathione reductase, GSH, and GSH/GS-SG ratio [160]. Impaired immune functions are associated with lower levels of these parameters. Investigation of large groups of healthy humans revealed a gradual decline of the GSH content with increasing age in blood and blood cells [161,162,163]. A gradual decrease of GPx activity is reported in female persons after age 65 [164]. In brain of aged persons, impaired glutathione is associated with different neurological disorders [165,166].

The thioredoxin system is another major contributor to redox homeostasis in cells. Reduced thioredoxin (Trx) is not only an essential factor to reduce cellular oxidized proteins such as oxidized peroxiredoxins, ribonucleotide 5’-diphosphatase, and methionine sulfoxide [159]. It exerts also an important regulatory role as cofactor for several enzymes and transcription factors. In other words, Trx is regarded as a biomarker for age-related diseases, oxidative stress, inflammation, and cellular senescence [167]. For example, reduced Trx forms complexes with apoptosis-signal-regulating protein 1 (ASK1) and phosphates and tensin homolog (PTEN) keeping these binding partners in an inactive state [168].

Enhanced cellular damage, redox imbalance, and disease progression are linked to diminished Trx and activation of the thioredoxin-interacting protein (Txnip) [169] and activation of proteins like ASK1 and PTEN, which both are involved in apoptosis induction. In cells, Txnip forms a complex with reduced Trx via formation of a disulphide bond [170]. Oxidative stress as well as Trx deficiency leads to decomposition of this inhibitory complex and allows Txnip to interact with other target molecules. Txnip activates the (NOD)-like receptor protein-3 (NRLP3) inflammasome complex via interaction of Txnip with the proapoptotic protein ASK1 [171,172]. The inflammasome complex contributes to mitochondrial stress-induced apoptosis via expression of inflammatory cytokines. In addition, Txnip binds to phosphatase and tensin homolog (PTEN) and favors thus apoptosis induction through regeneration of PIP₂ [168]. Txnip suppresses also glycolysis and promotes oxidative phosphorylation. Glucose transporters 1 and 4 are up-regulated by Txnip [157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173]. The expression of Txnip in blood precursor cells implies a role of Txnip during hematopoiesis [176].

Txnip is up-regulated during aging in primary human cells [177]. This up-regulation is associated with increased oxidative stress, DNA damage, and reduced lifespan [177]. In diabetes patients, Txnip is up-regulated too [178,179,180]. Txnip is also implicated in the pathogenesis of Alzheimer disease and Morbus Parkinson [181,182,183]. In brain samples of patients with Alzheimer disease, less neuronal Txnip was observed in comparison to unperturbed brains. However, an increased number of microglia cells was found in these samples, which were associated with Txnip-positive plaques [184].

Otherwise, Txnip is a negative regulator of cell proliferation. Up-regulated Txnip inhibits cyclin A and several others factors promoting cell cycle [169]. Cancer cell growth is associated with decreased Txnip, increased glycolytic flux, and enhancement of proliferative processes [185,186].

In addition to the aforementioned components of the glutathione and thioredoxin systems, further proteins and enzymes contribute to defense against oxidative stress. In drosophila and some other invertebrate models, over-expression of superoxide dismutase (SOD) has been associated with protection against reactive species and extension of lifespan [187,188]. Otherwise, SOD activity is dispensable for normal animal aging but is useful in stress situations [189]. In humans, a potential relationship between SOD and aging remains unclear. In older women, higher level of plasma SOD activity was linked to lower overall mortality, but not in older men [190]. SODs are essential to catalyze the dismutation of superoxide anion radicals, which mainly result from dysfunctional mitochondria, activated immune cells, and other sources, into dioxygen and hydrogen peroxide. In this way, SODs prevent side reactions of superoxide anion radicals with nitrogen oxide to yield the powerful oxidant peroxynitrite [191,192] and with iron-sulfur clusters of some mitochondrial proteins resulting in release of catalytically active Fe²⁺ [118,119].

Hydrogen peroxide is inactivated by glutathione peroxidases, catalase, and peroxiredoxins. Catalase malfunction or deficiency is hypothesized to play a role in the pathogenesis of age-related diseases such as diabetes mellitus, hypertension, neurodegenerative disorders, vitiligo, and some others [193]. Age-related alterations in functions of peroxiredoxins are mainly obtained from experiments with mice [194]. Peroxiredoxin 3 (Prx3), the mitochondrial form of peroxiredoxins, is involved in maintenance of mitochondrial redox homeostasis [195,196]. As the expression of Prx3 declines with age mitochondrial functions and homeostasis are impaired [197]. Prx1 contributes to dampen telomere shortening, a key age-dependent process [198,199,200].

Iron is an essential cofactor of many proteins. Within mitochondria, iron is part of many proteins in form of iron-sulfur-clusters and as central ion in the heme group of heme proteins. As free iron ions have a high potential to catalyze damaging reactions in cells, all aspects of iron transport, utilization, and metabolism are usually well controlled [201,202]. In cells, transferrin receptor (iron uptake), ferritin (iron storage), and ferroportin (iron export) are major players to ensure iron homeostasis and utilization [66,67,68,69]. In aged individuals, increased ferritin level is associated with the presence of a chronic inflammatory state [203]. Otherwise, lower ferritin levels are found in patients with iron deficiency [203].

Similar conclusions concerning transport, utilization, and metabolism can be drawn for copper ions. In blood, copper is mostly bound and transported by ceruloplasmin followed by serum albumin [204,205]. The copper-containing ceruloplasmin detoxifies Fe²⁺ by oxidation to Fe³⁺ and contributes thus to maintenance of iron homeostasis [206]. A glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin [207] is present in astrocytes, oligodendrocytes, hepatocytes, macrophages, and epithelial cells in pancreas and retina [208,209] and is part of the iron efflux machinery [210]. GPI-anchored ceruloplasmin oxidizes intracellular Fe²⁺ to Fe³⁺, which is excreted by ferroportin and further safely transferred to transferrin [211]. Patients deficient in ceruloplasmin develop massive iron deposition in brain, liver, and other organs and exhibit neurological symptoms and motor deficits [212,213,214]. Similarly, ceruloplasmin knockout mice are characterized by iron accumulation in astrocytes, ferritin expression, and neurodegeneration in the central nervous system [207,215].

The uncontrolled increase of the free labile pool of iron ions can lead to ferroptosis. In this kind of cells death, enhanced levels of free iron ions catalyze the oxidation of lipid hydroperoxides, which has fatal consequences on the barrier function of biological membranes. These mechanisms are promoted by glutathione deficiency and decreased activity of glutathione peroxidase 4 [216,217]. The latter enzyme is together with its cofactor glutathione responsible for detoxification of lipid hydroperoxides [62]. Another player in removal of lipid hydroperoxides and prevention of ferroptosis is peroxiredoxin 6 [218,219].

In plasma, haptoglobin binds hemoglobin after its release from red blood cells and myoglobin resulting from damaged muscles [220,221]. The haptoglobin-heme protein complex is removed from circulation by spleen and liver macrophages [222,223]. Both released hemoglobin and myoglobin can liberate the powerful oxidant ferriprotoporphyrin IX, also known as free heme [224]. Hemopexin inactivates free heme by forming a high-affinity complex with free heme and clearing this complex in the liver [225]. In elder humans, blood level of haptoglobin is elevated [226]. Another study revealed enhanced serum values for the haptoglobin-hemoglobin complex with age [227]. These data indicate an enhanced leakage of red blood cells and maybe problems with the clearance of the haptoglobin-heme protein complex in aged individuals.

In cell cytoplasm, hemoxygenase 1 (HO-1) degrades free heme to biliverdin, carbon monoxide, and Fe²⁺. The role of HO-1-derived Fe²⁺ is controversially discussed [228,229]. Low amounts of Fe²⁺ can be stored in ferritin and induce synthesis of additional ferritin [230]. Higher levels of Fe²⁺ and overload of ferritin with iron can promote ferroptosis [231]. There is a decline of HO-1 expression with increasing age in macular and peripheral retinal pigment epithelium [229,232].

3.5. Activation of Proteolytic Systems in Aged Individuals

Normally, processes of protein synthesis and degradation are in equilibrium in living tissues. With increasing age, however, degradative mechanisms prevail. This is mainly caused by disturbances in energy metabolism, dioxygen and nutrient deficiencies, and increased defects in cellular constituents and organelles in association with stress situations.

There are several levels how cell organelles and cells respond to stress situations. Mitochondrial unfolded protein response (UPR) is activated when the number of unfolded proteins is increased beyond a certain threshold. In this pathway, genes are activated favoring the translation of proteins involved in detoxification of reactive species, supporting the correct folding, and removing misfolded proteins [102]. In mouse models, positive effects of enhanced levels of NAD⁺ have been demonstrated on improvement of UPR and mitochondrial homeostasis [154,233]. As UPR links mitochondrial and nuclear genomes, dysregulation of this pathway is reported in several age-related human diseases like sarcopenia, and Morbus Alzheimer [234,235,236,237].

In stress situations, processes of mitochondrial membrane dynamics, known as mitochondrial fusion and fission, allow to dilute and segregate damaged organelles in order to ensure homeostasis and to survive [238,239]. The dynamin-related protein 1 plays an important role in fission regulation [102]. In mouse models, reduced activity of this protein is impaired with increasing age [240,241].

In cells, defective proteins are recognized and degraded by the ubiquitin-proteasome system [242,243]. With increasing age misfolded and damaged proteins accumulate [244]. During aging induction of chaperones is impaired [245] and proteasomal activity is declining as evidenced in different cells and tissues [246,247,248]. For example, age-related decreased activities of 20S and 26S proteasome complexes are reported [249,250,251].

Damaged cell organelles, dysfunctional cytoplasmic and structural components, protein aggregates, and sometimes invading pathogens are preferentially cleared by autophagic mechanisms by means of lysosomes [252,253]. The selective removal of damaged mitochondria by autophagy is known as mitophagy. Both ubiquitin-dependent and -independent pathways of mitophagy are differentiated [102]. In mice and humans, a decline of mitophagy has been observed in several tissues upon aging [254,255,256,257,258].

Defective mitochondria are linked to immune activation via the release of DAMPs. Putative mitochondrial DAMPs are mitochondrial DNA, cardiolipin, and N-formyl peptides resulting from translation of mitochondrial-encoded proteins [259]. There is a gradual increase of mitochondrial DNA in blood in elderly persons [260]. Mitochondria-derived DAMPs are involved in NLRP3 inflammasome activation, and consequently in release of pro-inflammatory cytokines like IL-1β and IL-18 and apoptosis induction [261,262,263].

Both the ubiquitin-proteasome system and autophagy are intensified in cachectic patients [264]. Cachexia is associated with progressive skeletal muscle wasting and substantial weight loss as a consequence of an underlying illness and is an important comorbidity in cancer patients [265,266,267] as well as in individuals of advanced age [268,269].

3.6. Inflammaging and Necrotic Cell Death

Inflammaging is characterized by increasing number of factors secreted from senescent cells. These factors are summarized as senescence-associated secretory cell phenotype. They include pro-inflammatory cytokines, chemokines, growth factors, proteases, extracellular matrix components and some others [270]. Pathophysiological consequences of inflammaging are very complex. On the cellular level, increased oxidative stress is assumed to be a crucial factor for accumulation of numerous structural and functional defects and initiation of novel inflammatory events [271,272,273]. This interplay between oxidative stress and inflammation during aging is also associated with the increased death of dysfunctional cells. In addition to apoptotic cell death, which occurs usually without further disturbances to neighbored cells and tissues, different forms of necrosis increase with age. These forms include ferroptosis, necroptosis, pyroptosis, and others [270]. During necrotic cell death, both cytotoxic agents as well as different DAMPs are released. These agents contribute to further tissue damage and trigger novel inflammatory events.

A few examples will underline the pathophysiological significance of necrotic cell death in aged persons. In aged liver, increase of necroptosis, a regulated form of programmed necrosis, contributes to chronic liver inflammation and development of liver cirrhosis [274]. The risk for pyroptosis increases in aged cells, as these cells are more susceptible to external stimuli that can cause cell damage [275]. Cytokine release and inflammasome activation by pyroptosis has been linked to development of atherosclerosis and neurodegenerative diseases [276,277]. Iron-dependent lipid peroxidation leads to ferroptotic cell death. This mechanism is discussed to contribute to pathogenesis of neurodegenerative diseases, autoimmune disorders, and cardiovascular diseases [278,279].

Increasing immunosuppression is another key feature of inflammaging. During resolution of inflammation, a transient immunosuppression allows to down-regulate all inflammatory processes including apoptosis of immune cells, to induce de novo formation of matrix and tissue components, and to restore the normal tissue homeostasis [43,44]. Under chronic conditions as given in inflammaging, immunosuppressive states can be pronounced more stronger and takes longer. Both enhanced expression of host-derived cytotoxic agents and decline, exhaustion, or inactivation of the corresponding antagonizing principles favor the chronic inflammatory process [8]. Under these conditions, different comorbidities are developing in persons of advanced age. Moreover, long-lasting immunosuppression dampens general immune functions and favors infections with commensal and mutualistic pathogens [280,281,282,283,284]. In the worst case, organ failure, sepsis and septic shock are developing as may occur in immunocompromised individuals [285,286].

4. Age-Related Alterations in the Protection Against Oxidant-Based Cytotoxic Agents in Selected Disease Scenarios

4.1. Diseases of the Cardiovascular System

Disturbances in blood vessel and heart function are very common in elder persons. Most of all, physiological functions in aged individuals are affected by increased blood pressure, hypoxia, alterations in blood vessel elasticity, processes of intravascular hemolysis, internal hemorrhages, and inflammatory processes in vessel walls. Common health problems are atherosclerotic vascular diseases including coronary artery disease, myocardial infarction, cerebrovascular disease, and stroke. Further serious blood flow disturbances are caused by tissue damage due to ischemic and reperfusion events, thrombosis, reduced wound healing, and vasculitis.

Accumulation of cholesterol-rich low-density lipoproteins in damaged endothelium is a hallmark of atherosclerosis. Lipid oxidation, plaque formation, attraction of immune cells, alterations of endothelial cells, and development of foam cells are further characteristics of this disease [287,288,289,290,291,292]. As cholesterol crystals and numerous DAMPs are present in the plaques, novel inflammatory cascades are continuously activated contributing to the chronic inflammatory process.

Increasing age is considered as a risk factor for the development of atherosclerotic vascular diseases [293,294,295,296]. Molecular details of the pathogenesis of atherosclerosis are not fully understood. Nevertheless, it is evident that oxidative processes play an important role in disease progression. In addition to cholesterol and lipid oxidation, the involvement of myeloperoxidase, a heme protein released form recruited neutrophils, is discussed in plaque formation. This enzyme is found in atherosclerotic plaques [297,298], produces the powerful oxidant hypochlorous acid and catalyzes numerous other oxidative processes [299]. The potential participation of free heme presents another route discussed in atherosclerotic plaque formation [300]. As free heme is known to easily penetrate into hydrophobic areas of proteins, membranes, and lipoproteins, it contributes to endothelial damage and lipoprotein oxidation during atherosclerosis [300,301].

Production of nitric oxide (NO) by NO synthases in the vessel wall is crucial for vessel relaxation and regulation of the blood flow. Decreased bioavailability of NO is observed by enhanced processes of intravascular hemolysis from red blood cells or rhabdomyolysis from damaged muscles [221,302]. Both released hemoglobin and myoglobin are known to be rapidly oxidized by NO [303,304,305]. In addition, myeloperoxidase, which can be attached to the inflamed endothelium, interacts with NO and diminishes thus the bioavailability of NO [306].

Another complication in cardiovascular diseases is the formation of internal hemorrhages. These bleedings are associated with uncontrolled hypertension, problems in blood clotting, traumata, aneurysms, loss of blood vessel elasticity, reduced arterial compliance with increasing age, and weakening of collagen linkages to the vessel wall [307,308,309]. In newly formed hemorrhages, there is a loss of glucose, dioxygen, and other nutrients with time. Concomitantly, intravascular hemolysis of red blood cells increases and protecting haptoglobin and hemopexin are exhausting. The resulting cytotoxic free heme can induce numerous damaging reactions at adjacent tissue areas [301,310,311].

Thrombotic complications can result from rupture and erosion of atherosclerotic plaques and from deep vein thrombotic events. With increasing age, the shifted balance between coagulation proteins and anticoagulant factors increases the risk for development of venous thrombosis [312,313,314].

4.2. Diabetes Mellitus

Majority of people over 65 years have prediabetes or diabetes, whereby diabetes type 2 predominates [315]. Diabetes is associated with many serious disease complications like vascular problems, neuropathy, nephropathy, and retinopathy [316]. Hyperglycemia leads to dysfunctions of red blood cells, endothelial cells, and other cells. These cells are known to accumulate glucose in an uncontrolled fashion independent of the presence of insulin. As a result, glycation of proteins increases in these cells beyond the level found in healthy individuals and different advanced glycation end products accumulate [317,318].

In red blood cells, glycation of hemoglobin as measured by the HbA1c value serves as a long-term marker for hyperglycemia [319,320]. In these cells, part of glucose is converted into sorbitol, which cannot leave the cells [321]. Accumulated sorbitol contributes to increased stiffness and diminished deformability of red blood cells [322,323,324]. Hence, these cells become osmotically instable and intravascular hemolysis increases [323]. Vascular endothelial cells develop an inflammatory phenotype on hyperglycemia [325,326]. These alterations diminish the blood flow by reduced bioavailability of nitric oxide, decreased vasorelaxation, impaired fibrinolytic activities, changes in the cellular cytoskeleton, and thickening of the basal lamina [326,327,328]. In addition, release of the von Willebrand factor from endothelial cells exposed to high levels of glucose can initiate thrombolytic events and adverse cardiovascular complications [329,330,331].

4.3. Cancer

With advanced age, the incidence increases for the development of different kinds of cancers. Which concrete conditions contribute to cancer development in a given patient remains puzzling. Key factors for cancer development in aged individuals are the increasing hypoxia in tissues and underlying problems in immune defense due to the presence of a low-stage chronic inflammation.

In patients with advanced cancers, host-own protective mechanisms are triggered in a way that they support cancer growth and metastasis formation rather than recognition and elimination of cancer cells by the immune system [332,333]. In cancer cells, antioxidative proteins and mechanisms stabilizing cellular redox homeostasis are highly up-regulated [9]. By these adaptations, cancer cells tolerate enhanced values of reactive species. This allows them also to survive even therapeutic approaches like radio- and chemotherapy. Moreover, in the tumor microenvironment (TME), invading immune cells are in a permanent state of immunosuppression [334,335,336,337] that correspond to down-regulated immune functions during the resolution phase of an acute inflammation [9]. Cancer cells release several factors such as tumor-necrosis factor β, IL-10, vascular endothelial growth factor, and components contained in secreted exosomes, which promote immunosuppression and support resolution of inflammation [338].

High up-regulation of glycolysis with lactate as final product is a metabolic hallmark in cancer cells. Anaerobic glycolysis supplies the necessary energy equivalents in these cells instead of the dioxygen-dependent mitochondrial oxidative phosphorylation. Secreted lactate can be used in the TME as fuel by invading macrophages to trigger them into an anti-inflammatory subtype M2 [56,339]. Enhanced expression of carbonic anhydrases on the external surface of cancer cells and up-regulation of several transport proteins in the plasma membrane acidifies the pH value in TME and decreases the cytoplasmic proton concentration [340,341,342,343]. The resulting mild alkalization in cancer cell cytosol is typical of proliferating cells. In other words, enhancement of glycolysis and subsequent metabolic alterations support cancer growth, metastasis, and invasion [344,345].

4.4. Neuro-Degenerative Diseases

Neuronal plaque deposits determine the morphological picture of neuro-degenerative diseases like Alzheimer’s disease, Parkinson’s disease, and others. These diseases often appear in persons of advanced age. The accumulation of fibrillated proteins coincides with a progressive loss of mental abilities due to the loss of functional neurons and synaptic linkages.

In Alzheimer’s disease, large extracellular deposits of amyloid β and hyperphosphorylated τ protein accumulates [346,347]. However, molecular details supporting fibrillation are largely unknown. Disturbances in endosomal-lysosomal and autophagic pathways contribute to processing of peptide fragments into insoluble fibrils. During fibril formation, soluble proteins with dominating α-helices are converted into insoluble proteins with preferred β-sheets [348,349]. This conversion is favored by acidic pH values as found in endosomes. Pathogenesis of Alzheimer’s disease is linked to the presence of very low pH values in endosomes [350]. This hyper-acidification enhances the formation of amyloid β fibrils and affects also the clearance of these fibrils.

Postmortem measurements revealed a slight decrease of pH values in the brain and cerebrospinal fluid of humans and mice with increasing age [351]. In Alzheimer’s patients, pH values of brain and cerebrospinal fluid were lower compared to controls without this pathology [351,352]. Moreover, lower brain pH values were associated with a more severe course of the disease [352].

Disturbances in the iron metabolism are also discussed in the pathogenesis of neuro-degenerative disorders. Enhanced values of brain iron are observed in several disorders named neurodegeneration with brain iron accumulation [353]. Iron accumulation within mitochondria contributes to oxidative stress, mitochondrial dysfunction, and neurodegeneration in Alzheimer’s disease, Parkinson’s disease and others [354]. Unbalanced iron ions are able to oxidize lipids and other cell constituents that leads in consequence to cell death by ferroptosis [355].

In Morbus Wilson, excessive accumulation of copper ions in liver, brain, and other organs is associated with serious damaging reactions [356]. In Alzheimer’s disease, increased values of free copper have been observed, which are not bound to ceruloplasmin [357,358].

Dysfunctions in heme metabolism are another source for neurotoxic events. In intracerebral and subarachnoid hemorrhages, the powerful oxidant free heme can result from released hemoglobin of defective red blood cells [359,360] after exhaustion of the protecting proteins haptoglobin and hemopexin [222,223,225]. The flat and hydrophobic free heme inserts easily into biological membranes, lipoproteins and hydrophobic areas of proteins. At these loci, it favors oxidative processes and contributes thus to development of atherosclerotic lesions [300], lipid peroxidation [301], neurodegenerative processes [360], and induction of inflammatory events [32,361]. Amyloid β forms a complex with free heme, which promotes the formation of amyloid β fibrils [362]. Enhanced levels of haptoglobin in serum of Alzheimer’s patients versus control support the hypothesis that neuroinflammation and heme-induced stress contribute to pathogenesis of Alzheimer’s disease [363]. Contrariwise, hemopexin level is lower in the cerebrospinal fluid in patients with Alzheimer’s disease [364]. The different behavior between haptoglobin and hemopexin is maybe caused by the fact that human haptoglobin is an acute phase protein unlike human hemopexin [365,366].

5. Conclusions

Our organism is equipped with powerful protective systems to resist a wide variety of external and internal impacts, to repair any damage, and to restore and maintain cell and tissue homeostasis. In many physiological processes, during activation of immune cells, and under stress situations, an enhanced formation of host-derived cytotoxic agents can occur. To avoid any damage by these agents, ready-to-use antagonizing principles exist, which immediately inactivate and eliminate these agents. Serious problems arise when cytotoxic agents are very strong expressed and act over a long time. Under these conditions, protection is limited and cells and tissues are progressively damaged.

During aging, there is a gradual decline in production of energy equivalents, and supply of tissues with dioxygen and nutrients. On this background, a chronic inflammatory state is developing in aged persons. In this persistent inflammation, known as inflammaging, activated immune cells, and pro-inflammatory mediators are present and coincides with a state of immunosuppression. In turn, oxidant-based cytotoxic agents affect considerably the cell and tissue integrity in aged individuals and contribute to the development of age-related diseases. The balance between cytotoxic agents and antagonizing principles is crucial for the further fate of an inflammation. In stressed and hypoxic tissues, there is an up-regulation of components of cellular redox homeostasis and anti-oxidative proteins. These adaptations have, however, long-lasting consequences for the distribution of energy substrates in relation to other physiological processes. More and more energy substrates are needed to stabilize protective mechanisms. These energy substrates are lacking for other physiological processes like active movement, mental processes, sensing the environment, and creating reserves for immunological defense.

It is very hard to predict, how the imbalance between cytotoxic agents and protective mechanisms will affect the health status in a given person during aging. As protection against different oxidants and other cytotoxic agents plays an important role in stabilizing immune responses and preventing disease progression, a thorough analysis of the status of protective mechanisms is highly mandatory for aged individuals.