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stress affect telomere length and telomerase activity which in turn affects cellular senescence [4]. Oxidative stress has been shown to damage DNA and affect life span [7-10]. A controversial view of cellular senescence is that it is an important protective mechanism against transformation of the cell into a malignant phenotype, in which case it would affect only mitotically active cells [2, 3]. The molecular mechanisms involved in cellular senescence are still being unraveled and will not be considered further in this review. The focus of this review will be on MG, a reactive dicarbonyl metabolic intermediate produced in the body, AGEs, and oxidative stress, all of which are interrelated and affect cellular as well as whole body aging. We will discuss some compounds that can scavenge MG, prevent the formation of AGEs (inhibitors) or break the existing AGEs (AGE breakers).

Aging has been attributed to a number of different causes which have been presented in the form of different theories. These theories are based broadly on two different ideas, one of which is programmed life processes (program theories, e. Biological Clock theory, Limited Number of Proliferation theory), and the other one is of errors, mainly at the DNA and gene level, in life processes (error theories, e. Disease theory, Cross-linking theory, Rate of living theory, Free radical theory). A number of theories of aging are based on the combination of these two ideas, i. program theories and error theories [11-14].

Changes at the cellular level ultimately affect the whole body. The cell is a dynamic centre of ongoing metabolic activity driven by almost constant use of oxygen. Reasonably, the metabolic activity may affect survival or the death of the cell. The ‘Rate of living theory’ implicates the role of metabolism in aging, which is based on the observation that animals with higher metabolic rates often have shorter life spans. Since the metabolic processes and oxygen consumption can also generate oxidative stress, an excess of which is deleterious for the cell, the ‘Free radical theory’ of aging has become one of the more popular theories. The free radical theory proposes a connection between the metabolic rate and aging through an increased oxidative stress generation.

Max Rubner proposed the ‘rate of living theory’ early in the 20th century [15]. He observed that larger animals, which generally have slower metabolic rates, live longer than smaller animals with faster metabolic rates [15]. Even though it is now common knowledge that metabolism is associated with the generation of free radicals, it was Commoner et al. [16] who discovered the formation of free radicals in vivo. Commoner et al. [16] found that an increase in an organism’s metabolic activity can increase the concentration of endogenous free radicals. Free radicals are atoms or molecules with an unpaired electron in an orbit, making them highly reactive. The high reactivity of free radicals makes them deleterious for cells because they react with proteins, lipids, DNA and other biomolecules, and disrupt their structure and function. Free radicals can be derived from oxygen mainly in the form of superoxide anions (O2•-) and hydroxyl radicals (·OH), which are known as reactive oxygen species (ROS). Free radicals can also be in the form of highly reactive non-radicals which do not have an unpaired electron in their orbit, such as hydrogen peroxide (H 2 O 2 ). Normally, the cells and the body have adequate antioxidant defenses which can neutralize free radicals

Aging: Drugs to Eliminate Methylglyoxal, a Reactive Glucose Metabolite, and Advanced Glycation Endproducts 683

and prevent the generation of oxidative stress resulting from an excess of free radicals [17- 22]. The formation of free radicals and the function of antioxidants have been nicely explained in reviews by Haliwell [20, 21].

The free radical theory of aging was proposed by Denham Harman in 1956 [23]. The free radical theory of aging attributes the aging process to cumulative cellular damage inflicted by the reaction of free radicals with key functional cellular and tissue constituents resulting in impaired function, disease and death [23]. The discovery of an antioxidant enzyme, superoxide dismutase (SOD) [24], which plays a key role to eliminate superoxide anion levels, provided some validity to the free radical theory, which was not initially accepted by many.

The mitochondrial respiratory chain is a major source of free radicals, mainly in the form of superoxide anions, which cause damage to the mitochondria and reduce life span [25, 26]. The damage inflicted by ROS, especially to DNA [7], rather than the metabolic rate, showed a greater correlation with life span [8]. Damage to DNA was formulated into the somatic mutation theory, which states that genetic mutations caused by an excess of free radicals could lead to accelerated aging [7, 9, 10].

The fact that increased production of ROS in the mitochondria can reduce life span was supported by several studies. Thus, Ku et al., [27] showed that the rates of mitochondrial superoxide anion and hydrogen peroxide generation were inversely correlated to maximum life span potential when they compared seven different mammalian species with different life spans ranging from 3 to 30 years. Similarly, ROS production was higher in heart mitochondria of the rat, which has a life span of about 4 yrs, than in the long-lived pigeon, which has a longer life span of 35 yrs [28]. Theoretically, therefore, if the free radical production is diminished, the life span should increase. This has been demonstrated in several species. Thus, over expression of SOD and catalase in the worm Caenorhabditis elegans (C. Elegans) through age-1 alleles, increases their oxidative defenses and life span by 65% longer on average [29, 30]. Increased activity of SOD and reduced oxidative stress in the transgenic Drosophila (Drosophila melanogaster) flies also slows the aging process and results in a longer life [31, 32]. Also, over expression of catalase in the peroxisome, the mitochondria or the nucleus in transgenic mice, reduced oxidative damage, hydrogen peroxide production, and delayed the development of cardiac pathology and cataract formation along with an average increase of 5 months in the life span [33]. The observation that long- lived animals have lower levels of antioxidant enzymes was explained as being due to a lower rate of production of oxygen radicals [34].

Interestingly, some of the studies in rodents did not produce the expected results. For example, the administration of antioxidants [35], or over expression of CuZn SOD and catalase in mice [36], or SOD in rats [37], did not increase their life spans. In rodents, one reason for the lack of additional protective effects, which are normally associated with an increase in antioxidants, could be their ability to synthesize vitamin C [38], which might already be providing the required protection. This was verified by knocking out the vitamin C synthesizing enzyme, L-gluconolactone oxidase (GLO) in mice, which then have to depend on dietary vitamin C [39]. GLO knockout mice had damaged aortic walls when they were fed a diet low in vitamin C, which underlined the importance of the constitutive antioxidant function of vitamin C in rodents [39]. Thus, studies in rodents do not provide unequivocal support for the free radical theory of aging [40].

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(lactoylglutathione lyase) and glyoxalase II (hydroxyacylglutathione hydrolase) [57-59] (Fig. 2). Reduced glutathione (GSH) plays a key role by binding MG and presenting it to glyoxalase I. Thus, adequate availability of GSH is important in keeping MG levels low in the body. For this reason enzymes involved in the synthesis and recycling of GSH, such as glutathione peroxidase and glutathione reductase are also important in the metabolism of MG [60-62].

Polyol pathway

SSAO

Glucose

F-1,6-di-P

Fructose

G-3-P

Pyruvate

Protein

Glycine, threonine

Aminoacetone

Hemithioacetal

S,D-lactoyl glutathione

Glyoxalase I

Glyoxalase II

Sorbitol

Krebs cycle

AMO

Triacylglycerol Glycerol

Fatty acid Acetone, acetol

Fig. 2. A schematic of key sources and steps of methylglyoxal (MG) formation from intermediates of glucose, protein and fat metabolism, and its degradation by the glyoxalase enzymes. Abbreviations: AGEs – advanced glycation endproducts; AMO – amine oxidase; DHAP – dihydroxacetone phosphate; FA – fatty acid; F-1-P – fructose-1-phosphate; F-1,6-di- P – fructose-1,6-diphosphate; F-6-P – fructose-6-phosphate; G-3-P – glyceraldehyde-3- phosphate; G-6-P – glucose-6-phosphate; ROS – reactive oxygen species; SSAO – semicarbazide-sensitive acetone/acetol mono-oxygenase; GSH, reduced glutathione.

Despite the efficient glyoxalase system, MG levels can increase significantly in the plasma and different organs such as the aorta and the kidneys [61, 63-66]. We have shown that MG levels are elevated in the plasma, aorta and kidney of fructose-fed Sprague-Dawley rats and spontaneously hypertensive rats (SHR) [61, 63-65]. Patients with type 1 and type 2 diabetes have 2-6 fold higher plasma levels of MG compared to healthy people [67, 68]. MG possibly plays a role in the pathogenesis of insulin resistance and type 2 diabetes as shown by several in vitro [69-71], and by our recent in vivo study in acute [66] and chronic MG-treated

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Sprague-Dawley rats [72]. Elevated MG levels are linked to the development of micro- vascular complications of diabetes such as retinopathy and nephropathy, and other conditions such as atherosclerosis and neurodegenerative diseases [73-77]. MG levels are high in the cerebrospinal fluid of patients with Alzheimer’s disease [76].

Unwanted chemical modification of physiologic constituent molecules of the body, which leads to the formation of harmful chemical entities, seems to be an unavoidable part of metabolic processes of the body. One type of modification, known as glycation, a nonenzymatic reaction, is a serious hazard of excess glucose availability in the body. The chemical interaction leading to the formation of AGEs starts when a reducing sugar condenses with the amino groups of proteins at their N terminus or on lysyl side chains (ε- amino groups) [78]. This nonenzymatic glycation involves a series of post-translational modifications. Glycation begins with the aldehyde or the ketone carbonyl group of the sugar combining with the protein to form an unstable aldimine intermediate or a Schiff base. Later on the Schiff base undergoes an Amadori rearrangement to form a stable Amadori product, a l-amino-l-deoxyfructose derivative with a stable ketoamine linkage, which can get cyclized to form a ring structure [78-80]. The Amadori product can undergo oxidation, degradation or rearrangement and form AGEs, a heterogenous group of products. Auto oxidation of glucose (Wolff pathway) [81] or of the Schiff bases (Namiki pathway) [82] can lead to formation of reactive diacrbonyls, but these pathways which are readily observed at high glucose concentrations in vitro, are not predominant in vivo [83]. The Maillard reaction, also known as the “browning reaction”, involves oxidation of the glycated product which forms a brown coloured product. Glucose, fructose and glucose-6-phosphate are all involved in glycation, albeit at different rates of reaction with glucose, the most important contributor, reacting at a comparatively slower rate than the other two [6]. Increased glucose levels, as seen in diabetic patients, causes more AGEs formation than in healthy people. These AGEs affect the normal function of several proteins and enzymes, and are responsible for aging [74, 80] and the complications of diabetes such as nephropathy and retinopathy [79]. Another way by which the glycation reaction causes damage is through the formation of reactive α-dicarbonyl compounds, such as MG, glyoxal and 3-deoxyglucosone (3-DG), when the sugar molecule undergoes fragmentation [78].

MG can also cause AGEs formation [78, 79]. In fact, MG and two other dicarbonyl metabolic intermediates, 3-DG and glyoxal, are believed to be major sources of intracellular and plasma AGEs formation [79, 84, 85], which are commonly implicated in the aging process. Any MG which is not degraded by the glyoxalase system or aldose reductase, reacts non- enzymatically with arginine or lysine residues of proteins [45] to form irreversible AGEs. This glycation is not random, but it depends on the structural configuration and (or) physical locations of the target proteins [86, 87]. The AGEs produced by the reaction between MG and arginine are hydroimidazolone Nε-(5-hydro-5-methyl-4-imidazolon-2-yl)- ornithine and argpyrimidine [88], whereas the AGE, Nε-carboxyethyllysine (CEL) [89, 90] is formed when MG reacts with lysine. Further crosslinking of these AGEs produces fluorescent products such as pentosidine and cross-line, and non-fluorescent ones such as argpyrimidine, methylglyoxal-lysine dimer (MOLD), glyoxal-lysine dimer (GOLD) and

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p38 MAPK, JNK

↑iNOS

NF-κB

Mitochondrial electron transport chain

↓SOD

↓Catalase ↓GSH-Px

↓GSH-Red

O2•- + O2•- + 2H+ → ↑H 2 O 2 + O 2

2GSH + H 2 O 2 → ↑GSSG + 2H 2 O

Cellular injury,

2H 2 O 2 → 2H 2 O + O 2

IL-1, IL-6, IFNγ, ICAM I and VCAM I

NADPH oxidase

Fig. 3. A schematic of oxidative stress pathways activated by methylglyoxal and advanced glycation endproducts and their implication in aging. Abbreviations: AGEs, advanced glycation end products; GSH-Px, glutathione peroxidase; GSH-Red, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; H 2 O 2 , hydrogen peroxide; ICAM 1, intercellular adhesion molecule 1; IFNγ, interferon γ; IL1, interleukin 1; JNK, JUN N- terminal kinase; MG, methylglyoxal; NF-κB, nuclear factor-kappaB; NO, nitric oxide; O2•-, superoxide anion; ONOO−, peroxynitrite; p38 MAPK, p38 mitogen activated protein kinase; RAGE, receptor for advanced glycation endproduct; SOD, superoxide dismutase; VCAM 1, vascular cell adhesion molecule 1.

Metabolic activity in the mitochondria is at the centre of the free radical theory of aging. Mitochondria, which are the major sites of ATP and energy production in the cell, also generate about 85% of total intracellular superoxide when electrons escape, mainly from complex I and complex III, and react with oxygen [23, 113-116].

MG increases mitochondrial superoxide production [116, 117]. Treatment of rat aortic VSMCs (A-10 cells) with MG (30 μM) significantly increased mitochondrial superoxide production by 69% compared with untreated cells. The AGEs cross-link breaker, alagebrium (50 μM), and SOD mimetic 4-hydroxy-tempo (Tempol, 500 μM) significantly decreased MG-induced mitochondrial superoxide production by 57% and 85%, respectively. Mitochondrial nitrotyrosine formation was also increased by MG [96].

In in vivo studies elevated MG levels are associated with increased oxidative stress. For example, we have shown that in 13 wk old SHR with elevated blood pressure, significantly elevated plasma and aortic MG levels are associated with increased levels of superoxide,

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and significantly reduced GSH levels, glutathione peroxidase, and glutathione reductase activities, compared with age-matched Wistar Kyoto (WKY) rats [61]. Similarly, in diabetes mellitus and hypertension, increased MG levels are associated with increased oxidative stress [61, 65, 67, 68, 118].

An excess of MG, CEL and CML indicate carbonyl overload and are associated with oxidative stress [73, 79, 119-123].

Glycated proteins and AGEs also induce oxidative stress (Fig. 3) through several mechanisms. AGEs induce production of cytokines and growth factors [124-130]. AGEs bind to the receptor for AGEs (RAGE) and scavenger receptors to induce oxidative stress in various cells including VSMCs, endothelial cells, and mononuclear phagocytes [128, 131]. In endothelial cells AGEs increase expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and increase activity of NF-κB to increase oxidative stress [126, 132].

The accumulation of AGEs in extracellular tissue proteins, such as the basement membrane and matrix proteins of blood vessels and skin, is a well known phenomenon characteristic of aging and age-related diseases. Several studies demonstrate aging associated increase in AGEs. Thus, accumulation of AGEs in the vessel walls results in a gradual loss of elasticity, which makes older subjects more susceptible to cardiovascular diseases [133, 134]. MG- induced AGEs, such as CEL and CML, increased with age in human lens and cause cataract formation [90]. In a study on 172 subjects serum levels of CML, 8-isoprostanes and C- reactive protein, which are markers of oxidative stress, were higher in elderly people (> years old) compared with younger people (