Aging increases the load of oxidative damage in DNA, lipids, and proteins, yeastto humans (Sohal and Wendruch, 1996; Finch, 1990). Free radicals (ROS, reactive oxygen species) generated by mitochondria are a major source of oxidative‘bystander’ damage. Other damage comes from extracellular ROS generated by macrophages, as is prominent in atheromas. Additionally, DNA, lipids, and proteinsbecome glycated in an oxidizing process that is chemically driven by glucose and other sugars in tissue fluids. These advanced glycation endproducts (AGEs), while not initiated by free radicals, can generate ROS in further complexreactions and by activating macrophages (RAGE pathway, receptor of AGE), discussed below. In the following discussions of adverse effects of ROS, we must be mindful that ROS are essential in functions of the brain, heart, and many other organs that employ ROS in signaling processes. As examples from this large field, in the brain superoxide modulates synaptic plasticity (Hu et al, 2006), whereas in the heart nitric oxide modulates contractility (Massion et al, 2005). Intracellular ROS is mainly derived from normal mitochondrial respiration (Barja, 2004; Wallace, 2005). The respiratory chain releases electrons that form the superoxide anion (O2 ·-) by single electron reduction of O2 (Fig. 1.11). Enzymes of free radical homeostasis include catalase; two types of supraoxide dismutase (SOD)—Cu/ZnSOD and MnSOD; and glutathione peroxidase. Superoxide is enzymatically converted by superoxide dismutase (SOD) into H2O2, which is then catalytically degraded by transition metals to the highly reactive hydroxyl radicals. These reactions are limited by the enzymatic degradation of H2O2 by catalase, or by glutathione peroxidase. H2O2 diffuses freely across cell membranes, unlike superoxide.
FIGURE 1.11 Free radicals and anti-oxidant homeostasis. Molecular oxygen (O2) is reduced by loss of electrons to form superoxide (O2-•, 1e) or hydrogen peroxide (H2O2,2e-). Superoxide spontaneously reacts with nitric oxide (NO). Most H2O2 forms spontaneously, or from the dismutation of O2-• by SOD (superoxide dismutase) and is used in cell signaling. H2O2 is degraded by intracellular catalase (CAT), extracellular glutathione peroxidase (Gpx) or thiols. Adapted from (Cai, 2005)
Pathways of hydrogen peroxide metabolism. Molecular oxygen (O2) ireduced by loss of electrons to form superoxide (O2 -, 1e-) or hydrogen peroxide (H2O2, 2e-). Superoxide spontaneously reacts with nitric oxide (NO) to form peroxynitrite radicals (ONOO-). Most H2O2 forms spontaneously, or from the dismutation of O2 – by SOD and is used in cell signaling. H2O2 is degraded by intracellular catalase (CAT), extracellular glutathione peroxidase (Gpx), or thiols. (Adapted from Cai, 2005.)
ROS are strongly associated with mitochondrial DNA damage (deletions,
rearrangements, and point mutations). The age-related increase of damaged mitochondriaDNA (Wallace, 2005; Chomyn and Attardi, 2003) has become a centerpiece in the molecular pathophysiology of aging (Brookes et al, 1998; deGrey, 2005; Harper et al, 2004; Van Remmen and Richardson, 2001). Mitochondrialdysfunctions are found in many disorders of aging, e.g., Alzheimer disease,atherosclerosis, atrial fibrillation, diabetes, deafness, muscle atrophy, retinal degeneration. However, cause and effect are not well resolved in these long term processes of cell degeneration. Mitochondrial production of ROS increases withage in rat liver and muscle (Bevilacqua et al, 2005; Hagopian et al, 2005; Harper et al, 2004). ‘Proton leak’ across the inner mitochondrial membrane regulatesmitochondrial ROS production with high sensitivity and increases during aging (Brookes et al, 1998; Brookes, 2004; Hagopian et al, 2005; Harper et al 2004). Mitochondrial oxidative damage to DNA and proteins is often attributed to endogenously generated mitochondrial ROS. Because proton leak increases with oxidative damage, progressive mitochondrial impairments of various typesmay arise during aging through subcellular bystander damage, which propagates cell oxidative damage (Brookes et al, 1998; deGrey, 2005; Harper et al, 2004).
According to the oxidant stress theory of aging, life span should be influencedby levels of enzymes or anti-oxidants that produce or remove free radicals (ROS, NOS) (Bokov et al, 2004; Sohal and Weindruch, 1996; Stuart and Brown, 2005). The role of ROS is being tested in transgenic flies and mice by varyingthe levels of catalase and SOD that remove ROS (Landis and Tower, 2005; Mele et al, 2006).In flies, transgenic overexpression of mitochondrial Cu/ZnSOD increased life span by >35%, while catalase overexpression did not increase life span, reviewedby Landis and Tower (2005). Mice with partial deficits of MnSOD (heterozygote knockout, Sod2+ /-) lived slightly longer (Van Remmen et al, 2003). Althoughthe 2.5% difference was not statistically significant, the survival curves showlittle overlap. This careful study also showed that SOD2 deficiency increased DNAoxidative damage (8-OH dG) and tumor incidence several- fold—e.g., lymphomas 61% versus 22%. The lack of SOD2 deficiency on skin collagen glycooxidation is discussed below and in Section 1.4.2.
From these results and more systematic species comparisons (Kapahi et al, 1991), I suggest that anti-oxidant mechanisms may be related to the levels of molecular turnover and repair. Flies may show these stronger effects on thelife span than rodents because adult flies have no somatic cell replacement and, probably, less protein turnover, which, in mammals, removes oxidative damagedmolecules. Long-lived organisms may have needed to evolve more effective
repair processes (see Section 1.2.8).
Transgenic overexpression of catalase in mitochondria (mCAT) in miceincreases life span by 20% (5 months) and delays important pathology (Schriner et al, 2005). This study is exemplary for its genetic design, detailedhistopathology, and animal care (husbandry), even reporting the infection rate in sentinel mice. Mortality accelerations were right-shifted by increased mCAT, but without change in slope, implying that aging was delayed. Tissue changesare consistent with the Gompertz interpretation that aging is delayed. At middle age, cardiac pathology was decreased (fibrosis, calcification,arteriolosclerosis), which are common causes of congestive heart failure in human aging. In skeletal muscle, DNA oxidation (8-OHdG) and mitochondrial deletions were decreased. These findings directly link decreased mitochondrial ROS to heart pathology, which is recognized as of inflammatory origin (Query II). In a mouse model of accelerated atherosclerosis (apoE- knockout, apoE-/- with extreme hypercholesterolemia on standard diets), the systemic overexpression of catalase decreased aortic atherosclerosis (lesion area) by 66% and decreased F2-isoprostanes (lipid oxidation product) in plasma by 45% (Yang et al, 2004). Aortic lesion size correlated strongly with aortic isoprostane levels, again consistent with the importance of oxidized damage in inflammation. Cu/Zn-SOD had smaller effects on aortic lesions or lipid oxidation, specifically implicating hydrogen peroxide.
The hyperglycemia of diabetes is associated with another source of oxidative damage through glycation, which has not been well integrated into the free radical theory of aging. Glucose and other reducing sugars can oxidize and crosslink proteins by spontaneous and complex chemical reactions with lysine and arginine sidegroups yielding ‘advanced glycation endproducts’ (AGEs) that include highly reactive carbonyls (ketones and aldehydes) (Biemel et al,2002; Monnier et al, 2005; Stadtman and Levine, 2003). Carbonyls also form by many other free radical reactions (Stadtman and Levine, 2003). Other targets of glycation are lipids (ALE, advanced lipid endproducts) (Baynes, 2003) and DNA
AGE adducts accumulate progressively during aging in extracellular matrix proteins as cross-links that reduce vascular and skin elasticity (Hamlin and Kohn, 1971; Monnier et al, 2005). Aortic stiffening causes progressive increases in systolic blood pressure (Fig. 1.6B) and pulse wave velocity (De Angelis et al, 2004) that are underway early in adult life. The formation of atheromas is superimposed on these slow arterial aging processes. Diabetes accelerates these vascular and lens changes, implying the importance of glucose and other blood sugars in damage to long-lived proteins during aging (Cerami, 1985). Conversely, AGE formation is slowed by diet restriction, which lowers blood glucose (Chapter 3). The lack of SOD2 deficiency on skin collagen glyco-oxidation (carboxymethyl lysine and pentosidine) in the study of van Remmen et al. (2003), discussed(Bucala et al, 1984).previously, points to the role of blood glucose, rather than extracellular ROS in glyco-oxidation (see below). As discussed below, AGE adducts participate in oxidative stress and inflammation.
Pentosidine was the first chemically characterized AGE cross-link identified in
tissues (Sell and Monnier, 1989). Skin collagen pentosidine accumulates progressively during aging in many species, and the accumulations are accelerated byhyperglycemia and diabetes (Sell and Monnier, 1990). However, pentosidine accumulations are dwarfed by glucosepane, a recently characterized AGE thatis 50-fold higher than pentosidine in skin collagen (Biemel et al, 2002; Monnieret al, 2005; Sell et al, 2005). Over the life span, glucosepane is added to about 1% of skin collagen arginine and lysine residues. This is equivalent to cross linking of every five collagen molecules of normal individuals and every other collagen molecule in diabetics. Lens proteins accumulate far less glucosepane than skin collagen (Biemel et al, 2002). We do not yet know the specificcontribution of glucosepane and diverse minor glycation products to cross-linking in skin and vascular stiffening.
Besides pentosidine and glucosepane, more than 20 other adducts derive fromglucose, pentose, and ascorbate. AGEs form readily in test-tube reactions of proteins with glucose or other reducing sugars through Amadoriand Maillardchemistry. The resulting brownish, autofluorescent mixtures are models for brunescent cataracts and other in vivo sites of AGEs (Cerami, 1985; Monnier etal, 2005). The aorta also accumulates fluorescent AGEs (Fig. 1.6D). Oxygen levels are critical to AGE chemistry and may degrade Amadori products (Ahmed,1986). Glucosepane formation, however, forms directly from reactions that do not depend on oxygen, and is influenced by competing reactions; e.g., in thelens, the lower glucosepane may be due to high levels of methylglyoxal (Sell et al, 2005). Tissues also differ in enzymatic removal of AGEs (deglycatingamidoriases)(Brown et al, 2005).
Of critical importance to inflammation, AGE adducts activate scavenger receptors‘RAGE’ (receptors for AGE) on macrophages and many other cells that stimulate the production of ROS via NAD(P)H oxidases (gp91phox et al.) and electron transport (Fan and Watanabe, 2003; Schmitt et al, 2006). RAGEs are also activated by the amyloid ß-peptide of Alzheimer disease and by AGEs presentin cooked foods (Lin, 2003; Uribarri et al, 2003) (Chapter 2). AGEs and RAGEs appear to mediate feed-forward loops of oxidative stress and inflammationthat increase bystander molecular damage in atherosclerosis, Alzheimer, and other chronic inflammatory diseases (Lu et al, 2004; Ramasamy et al, 2005) (Queries II and III).
RAGE activation also releases cytokines (e.g., IL-6 ) and leukocyte adhesion factors (e.g., MCP-1 and VCAM-1). Feedback loops induce RAGE by TNFa through production of ROS, mediated by NFkappaB (Mukherjee et al, 2005). RAGE signaling pathways utilize familiar workhorses in inflammation and oxidative stress,including the transcription factor NFkappaB and PI3K (Dukic-Stefanovic et al,2003; Xu and Kyriakis, 2003). Moreover, PI3K interfaces with other signaling systems implicated in longevity (Fig. 1.3A). Lastly, RAGE activation may stimulate feed-forward ‘vicious cycles’ by autoinduction in the same cell (Basta et al,2005; Feng et al, 2005; Wautier et al, 2001). RAGE-dependent processes are a major focus in atherosclerosis, particularly inflammation of arterial endothelia by AGE during diabetes (Feng et al, 2005; Naka et al, 2004; Ramasamy et al, 2005) (Section 1.5.1). RAGE-dependent processes are also implicated in Alzheimer disease and cancer. These observations are consistent with Query II that inflammation causes further bystander damage and Query III that nutrition influences bystander damage by AGE production from hyperglycemia and by AGE present in cooked food.
The molecular life span (turnover or half-life, t1/2) is a major determinant of
accumulated damage, as exemplified by AGE accumulation in arterial elastin (Fig. 1.6D). In arteries and lungs, elastin may be almost as old as the individual, asevaluated by two independent measures: D-aspartate (Powell et al, 1992; Shapiro et al, 1991), which accumulates linearly through spontaneous racemization (Fig. 1.6D) (Helfman and Bada, 1975) and by ‘bomb-pulse’ 14C radiolabeling1 (Shapiro et al, 1991). Human aortic elastin and cartilage collagen have t1/2 >100 y, while skin collagen is 15 y. With lab tracer labeling, rodent elastin has t1/2of months to years (references in Martyn et al, 1995; Shapiro et al, 1991). Elastin progressivelyaccumulates glyco-oxidation (AGE) (Fig. 1.6D), at the same rate as collagen, when corrected for turnover (Verzijl et al, 2000). Damage to arterial elastin and collagen contributes to the loss of elasticity and stiffening that cause the increase of blood pressure during aging (Fig. 1.6B) (Section 1.6.3, below). In Alzheimer disease, senile plaque amyloid and neurofibrillary tangles also include very long-lived proteins (also bomb-pulse 14C) (Lovell et al, 2002). Other very longlived proteins accumulate D-aspartate in tooth dentine, eye lens, and in brain white matter myelin. The accumulating oxidative damage to these life-long molecules is associated with creeping dysfunctions in arteries, skin, and eye lens; the role in myelin dysfunction is not known.
In contrast, molecules with short life spans of days to weeks have less oxidativedamage. Diabetics accumulate glycated hemoglobin A1c, for example, which turns over at the erythrocyte t1/2 of about 120 d. Erythrocyte turnover scaleswith body size across species (M0.18) (Finch, 1990, p. 289). The t1/2 of many proteinsis allometrically related to body size and may be a crucial determinant of the rates of damage accumulated during aging across species. Moreover, the ratesof basal metabolism correlate with molecular turnover in species comparisons of mammals. The insulin-like signaling pathways (Fig. 1.3) may mediate many of these fundamental energy relationships.
Aging slows the turnover of many shorter-lived proteins (Finch, 1990, pp. 370–373; Goto et al, 2001)—e.g., bulk proteins in worm (Reznick and Gershor, 1979) and in mouse liver (Lavie et al, 1982; Reznick et al, 1981). The causes of slowed turnover during aging are not known and could include impaired proteasomal degradation, as in aging rodents (Goto et al, 2001). These metabolic level aging processes thus tend to accelerate the accumulation of oxidized damage. The effectiveness of diet restriction in slowing aging may be due in part to the accelerated protein turnover and decreased oxidative load (Chapter 3).
The balance of reduction:oxidation (‘redox’) in glutathione and other keyhomeostatic regulators (Fig. 1.11) is shifted to a more oxidized state (GSSG and protein-SSG) in blood, liver, and other tissues (Lang et al, 1989; Lang et al,1990; Rebrin et al, 2003; Rebrin and Sohal, 2004), and in whole aging flies (Rebrin et al,2004). Glutathione, the major redox couple, is at much higher levels than otherredox links involving cysteine, thioredoxin, NAD, etc. (Sies, 1999). Inhealthy aging humans, blood GSH remains relatively stable; however, with cardiovasculardisease, diabetes, or kidney disorders, blood GSH tends to drop below the normal range (Lang et al, 2000). Similar redox shifts occur during chronic infections;e.g., in HIV patients, blood GSH decreases in proportion to the viral load(Sbrana et al, 2004). Conversely, redox shifts are opposed by diet restriction (Chapter 3, Fig. 3.14). A component of the GSH shifts of aging could also be a response to low-grade infections, which would also be consistent with the increase of CRP, IL-6, and other acute phase reactants in aging populations (see below and Chapter 2). It is important to resolve the contributions to the oxidized load of aging from three sources: (I) endogenous mitochondrial free radicals and other tissue processes; (II) interactions with the commensal gut and skin flora; and (III) specific infections.
The naked mole rat (Heterocephalus glaber) is adding surprising findings to these debates. H. glaber is the most longevous rodent (at least 28 y), yet has similarbody weights of lab mice (30–80 g) (Andziak et al., 2006; Andziak and Buffenstein, 2006). In comparison with lab mice (CB6F1) at 10% of the lifespan(24 m vs. 4 m), H. glaber had indicators of greater oxidative stress than in lab mice, e.g., 10-fold more urinary isoprostanes, 35% higher myocardial isoprostanes,and 25% lower hepatic GSH:GSSG ratios. While it might be concluded that sustained oxidative stress is not incompatable with extraordinarylongevity, much remains to be learned about other aspects of metabolism in theseremarkabl animals. Its membrane lipids differ from other mammals by much lower levels of unsaturated fatty acids in muscle and brain,particularly docosahexaenoic acid (DHA or 22:6 n-3), which is highly susceptible to peroxidation(Hurlbert et al., 2006). The oxidizability of membranes (peroxidation index) fits well with the inverse allometry on lifespan.
These varying results across species suggest that anti-oxidant mechanismsvary within and between phyla. Besides differences in membrane composition and antioxidants, I suggest the importance of molecular turnover and repair. Flies may show these stronger oxidative effects on the life span than rodents because adult flieshave no somatic cell replacement, except in the gut, and, probably, less protein turnover, while mammals have extensive molecular turnover, which removes oxidative damaged molecules. Long-lived organisms may have needed to evolve more effective repair processes (see Section 1.2.8).