**8. Methylglyoxal and oxidative stress**

The role of MG in inducing oxidative stress is well established [17]. Several studies have helped to develop an integrated view of the multiple pathways activated by MG to increase oxidative stress (Fig. 3). The reader is referred to our earlier review on MG and oxidative stress [17]. MG increases the formation of superoxide [94, 96-99], hydrogen peroxide and peroxynitrite [94, 95, 98, 100], proinflammatory cytokines, such as interleukin 1β (IL-1β) [101], interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) [67, 101], in different cell types such as VSMCs [62, 94, 95], endothelial cells [102], rat kidney mesangial cells [97], rat hepatocytes [100], neutrophils [67, 98], platelets [99], cultured neural cells from rat hippocampus [101], cultured cortical neurons [103], and SH-SY5Y neuroblastoma cells [104].

MG has been shown to increase the activity of several prooxidant enzymes such as NADPH oxidase [94, 97] (Fig. 3), p38 MAPK [98, 102], and increase the of expression of JNK and PPAR-α [104].

Excess superoxide can react with nitric oxide (NO) to form peroxynitrite (ONOO- ) [105] (Fig. 3). Peroxynitrite is a strong oxidant and nitrating agent. Because of its oxidizing properties, peroxynitrite can damage a wide range of molecules including DNA and proteins in cells [105].

Besides directly increasing free radical production, MG can increase oxidative stress by reducing antioxidants (Fig. 3) such as GSH [104, 106, 107], glutathione peroxidase [108], glutathione reductase [60, 62, 108, 109], and manganese superoxide dismutase (MnSOD) [96], in different cells such as erythrocytes [106, 107], VSMCs [62, 96], and endothelial cells [109]. Reduced antioxidants in turn impair the detoxification of MG, increase its half-life and set up a vicious cycle to cause further oxidant damage. Glutathione peroxidase removes hydrogen peroxide with the help of GSH which in turn is converted to oxidized glutathione (GSSG). Glutathione reductase recycles GSSG to GSH [62, 110] (Fig. 3).

An increased production of ROS was also observed in monocytes treated with MGmodified albumin [111]. Thus, MG induced thrombosis and inflammation by activating monocytes, induced apoptosis of neutrophils, and caused platelet-neutrophil aggregates [112].

Aging: Drugs to Eliminate Methylglyoxal, a Reactive

stress [61, 65, 67, 68, 118].

oxidative stress [126, 132].

**9. Methylglyoxal and aging** 

peroxynitrite production [137].

direct effect on aging.

oxidative stress [73, 79, 119-123].

Glucose Metabolite, and Advanced Glycation Endproducts 689

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

An excess of MG, CEL and CML indicate carbonyl overload and are associated with

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

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]. MGinduced 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 Creactive protein, which are markers of oxidative stress, were higher in elderly people (>60 years old) compared with younger people (<45 years old) [135]. One reason why AGEs accumulate during the aging process could be due to an age-related decrease in antioxidant enzymes. Thus, Mailankot et al. [136] reported that the activity and expression of glyoxalase I protein, which is involved in MG degradation, decreased with age in the anterior epithelial cells of human lens, which causes an accumulation of MG. Similarly, an age-dependent decrease in catalase activity in the skin may be responsible for elevated MG and

However, it is doubtful whether extracellular AGEs accumulation plays a causative role in aging. On the other hand AGEs formation inside the cell, such as AGE-nucleotides in DNA, may contribute to cellular senescence [6, 60, 74, 80]. DNA integrity is an important determinant of lifespan and errors in DNA repair would lead to substitutions, deletions, insertions, and transpositions of nucleotides, with increased risk of carcinogenesis and reduced life span. Animals with a longer lifespan and more efficient DNA repair have delayed carcinogenesis [80]. In this regard MG-induced DNA damage can have a more

Studies directly implicating MG in the aging process are very few and this is one area where there is a knowledge gap. The study by Morcos et al in the worm *C. elegans* highlights the role of MG, glyoxalase I and MG-induced ROS formation in aging and life span [138]. They showed that the activity of glyoxalase I was markedly reduced with age resulting in accumulation of MG-derived adducts and oxidative stress markers, which further inhibited

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; H2O2, hydrogen peroxide; ICAM 1, intercellular adhesion molecule 1; IFNγ, interferon γ; IL1, interleukin 1; JNK, JUN Nterminal 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 69.9% 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 85.8%, 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,

**METHLYGLYOXAL**

**Mitochondrial electron transport chain**

**<sup>↓</sup>***Catalase* **<sup>↓</sup>***GSH-Px*

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; H2O2, hydrogen peroxide; ICAM 1, intercellular adhesion molecule 1; IFNγ, interferon γ; IL1, interleukin 1; JNK, JUN Nterminal 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,

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

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 69.9% 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 85.8%,

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,

respectively. Mitochondrial nitrotyrosine formation was also increased by MG [96].

complex I and complex III, and react with oxygen [23, 113-116].

**RAGE**

**•<sup>−</sup>** ↑ONOO<sup>−</sup>

**•- + 2H+ → ↑H2O2 + O2**

**↓GSH**

**2GSH + H2O2 → ↑GSSG + 2H2O**

**↓***GSH-Red*

**AGEs**

**+ Proteins**

**Sugar / glucose**

**METHLYGLYOXAL**

•-,

**Cellular injury, AGING**

↑NO **↑O2**

+

**↓***SOD*

**O2 •- + O2**

**p38 MAPK, JNK**

**NF-κB**

**2H2O2 → 2H2O + O2**

vascular cell adhesion molecule 1.

**↑iNOS**

**NADPH oxidase**

**METHLYGLYOXAL**

**IL-1, IL-6, IFNγ, ICAM I and VCAM I**

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].
