Many theories regarding aging and mortality hypothesize that the human body eventually succumbs to the accumulation of damage over time as a result of long-term exposure to a variety of environmental factors that are reactive with organic biomolecules. These theories note that while repair and turnover mechanisms exist to restore or replace many classes of damaged molecules, these mechanisms are less than perfect. Hence, some damage inevitably leaks through—damage that will accumulate over time, particularly in long-lived cell populations that experience little, if any, turnover (Table 58–2). Ironically, many of the agents that are most damaging to proteins, DNA, and other biomolecules are also essential for terrestrial life: water, oxygen, and sunlight.
TABLE 58–2Time Required for All of the Average Cells of This Type to Be Replaced ||Download (.pdf) TABLE 58–2 Time Required for All of the Average Cells of This Type to Be Replaced
|Tissue or Cell Type ||Turnover |
|Intestinal epithelium ||34 ha |
|Epidermis ||39 db |
|Leukocyte ||<1 yc |
|Adipocytes ||9.8 yc |
|Intercostal skeletal muscle ||15.2 yc |
|Cardiomyocytes ||≥100 yc |
Hydrolytic Reactions Can Damage Proteins & Nucleotides
Water is a relatively weak nucleophile. However, because of its ubiquity and high concentration (>55 M, see Chapter 2), even this weak nucleophile will react with susceptible targets inside the cell. In proteins, hydrolysis of peptide bonds leads to cleavage of the polypeptide chain. The amide bonds most frequently targeted by water are those found on the side chains of the amino acids asparagine and glutamine, presumably because they are more exposed, on average, to solvent than the amide bonds in the protein’s backbone. Hydrolysis leads to the replacement of the neutral amide group with an acidic carboxylic acid group, forming aspartate and glutamate, respectively (Figure 58–1A and B). This change introduces both a negative charge and a potential proton donor or acceptor to the affected region of the protein. As the protein population within a living organism is subject to continual turnover, in most cases the chemically modified protein will be degraded and replaced by a newly synthesized protein.
Examples of hydrolytic damage to biological macromolecules. Shown are a few of the ways in which water can react with and chemically alter proteins and DNA: (A) Net substitution of aspartic acid via hydrolytic deamidation of the neutral side chain of asparagine. (B) Net substitution of glutamic acid via hydrolytic deamidation of the neutral side chain of glutamine. (C) Net mutation of cytosine to uracil by water. (D) Formation of an abasic site in DNA via hydrolytic cleavage of a ribose-base bond.
Of perhaps greater potential biological consequence are the reactions of the nucleotide bases in DNA with water. The amino groups projecting from the heterocyclic aromatic rings of the nucleotide bases cytosine, adenine, and guanine are each susceptible to hydrolytic attack. In each case, the amino group is replaced by a carbonyl to form uracil, hypoxanthine, and xanthine, respectively (Figure 58–1C). If the affected base is located in the cell’s DNA, the net result is a mutation that, if left unrepaired, can potentially perturb gene expression or generate a dysfunctional gene product. The bond between the nucleotide base and the deoxyribose moiety in DNA is also vulnerable to hydrolysis. In this instance the base is completely eliminated, leaving a gap in the sequence (Figure 58–1D) which, if left unrepaired, can lead to either a substitution or a frame-shift mutation (see Chapter 37).
Many other bonds within biological macromolecules constitute potential targets for random chemical hydrolysis. Included in this list are the ester bonds that bind fatty acids to their cognate glycerolipids, the glucosidic bonds that link the monosaccharide units of carbohydrates, and the phosphodiester bonds that hold polynucleotides together and link the head groups of phospholipids to their diacylglycerol partners. However, these reactions appear to take place too infrequently (eg hydrolysis of phosphodiester bonds within the backbone of DNA and RNA) or to generate insufficiently perturbing products to manifest significant biological consequences.
Respiration Generates Reactive Oxygen Species
Numerous biological processes require enzyme-catalyzed oxidation of organic molecules by molecular oxygen (O2). These processes include the hydroxylation of proline and lysine side chains in collagen (see Chapter 5), the detoxification of xenobiotics by cytochrome P450 (see Chapter 47), the degradation of purine nucleotides to uric acid (see Chapter 33), the reoxidation of the prosthetic groups in the flavin-containing enzymes that catalyze oxidative decarboxylation (eg, the pyruvate dehydrogenase complex, see Chapter 17) and other redox reactions (eg, amino acid oxidases, see Chapter 28), and the generation of the chemiosmotic gradient in mitochondria by the electron transport chain (see Chapter 13). Redox enzymes frequently employ prosthetic groups such as flavin nucleotides, iron-sulfur centers, or heme-bound metal ions (see Chapters 12 and 13) to assist in the difficult task of generating and stabilizing the highly reactive free radical and oxyanion intermediates formed during these processes. The electron transport chain employs specialized carriers such as ubiquinone and cytochromes to safely transport individual, unpaired electrons among and within its various multiprotein complexes.
Occasionally, these highly reactive intermediates escape into the cell in the form of ROS such as superoxide and hydrogen peroxide (Figure 58–2A). By virtue of its structural and functional complexity and extremely high level of electron flux, “leakage” from the electron transport chain constitutes by far and away the major source of ROS in most mammalian cells. In addition, many mammalian cells synthesize and release the second messenger nitric oxide (NO•), which contains an unpaired electron, to promote vasodilation and muscle relaxation in the cardiovascular system (see Chapter 51).
Reactive oxygen species (ROS) are toxic by-products of life in an aerobic environment. (A) Many types of ROS are encountered in living cells. (B) Generation of hydroxyl radical via the Fenton reaction. (C) Generation of hydroxyl radical by the Haber-Weiss reaction.
Reactive Oxygen Species Are Chemically Prolific
The extremely high reactivity of ROS makes them extremely dangerous. ROS can react with and chemically alter virtually any organic compound, including proteins, nucleic acids, and lipids. Some reactions lead to the cleavage of covalent bonds. ROS also display a strong tendency to form adducts—a term referring to the product formed when two compounds combine together—with nucleotide bases, polyunsaturated fatty acids, and other biological compounds possessing multiple double bonds (Figure 58–3). Adducts formed with nucleotide bases can be especially dangerous because of their potential, if uncorrected, to cause misreads during replication that introduce mutations into DNA.
ROS react directly and indirectly with a wide range of biological molecules. (A) Peroxidation of unsaturated lipids generates reactive products such as malondialdehyde and 4-hydroxynonenal. (B) Guanine can be directly oxidized by ROS to produce 8-oxoguanine or form an adduct, M1dG, with the ROS product malondialdehyde. (C) Common reactions of proteins with ROS, including oxidation of amino acid side chains and cleavage of peptide bonds. Oxygen atoms derived from ROS are marked in red. Carbon atoms derived from malondialdehyde in M1dG are colored blue. The complete chemical name for M1dG is 3-(2-Deoxy-d-erythro-pentofuranosyl)pyrimido(1,2-α)purin-10(3H)-one.
The ease with which oxygen evokes the chemical changes that turn household butter rancid is a testament to the reactivity of unsaturated fats, those containing one or more carbon-carbon double bond (see Chapter 23) with ROS. Lipid peroxidation can lead to the formation of cross-linked lipid-lipid and lipid-protein adducts and a loss of membrane fluidity and integrity. Loss of membrane integrity, in turn, can—in the case of the mitochondria—undermine the efficiency with which the electron transport chain converts reducing equivalents to ATP, leading to greater production of deleterious ROS. Loss of mitochondrial membrane integrity can also trigger apoptosis, the programmed death of a cell.
Chain Reactions Multiply the Destructiveness of ROS
The destructiveness inherent in the high reactivity of many of ROS, particularly free radicals, is exacerbated by their capacity to participate in chain reactions in which the product of the reaction between the free radical and some biomolecule is a damaged biomolecule and another species containing a highly reactive unpaired electron. The chain will terminate when a free radical is able to acquire another lone electron to form a relatively innocuous electron pair without generating a new unpaired electron as a by-product. Such is the case when one free radical encounters another. The two “odd” electrons combine to form a pair. Alternatively, the ROS may be eliminated by one of the cell’s suite of dedicated antioxidant enzymes (see Chapters 12 and 53).
The reactivity, and hence destructiveness, of individual ROS varies. Hydrogen peroxide, for example, is less reactive than superoxide, which in turn is less reactive than hydroxyl radical (OH•). Unfortunately, two pathways exist in living organisms by which highly toxic hydroxyl radical can be generated from less destructive ROS. If ferric iron is present, for example, the Fenton reaction can transform hydrogen peroxide into hydroxyl radicals (Figure 58–2B). The ferrous (+3) iron, in turn, can be reduced back to the ferric (+2) state by other hydrogen peroxide molecules, permitting the iron to act catalytically to produce additional hydroxyl radicals. Hydroxyl radical can also be generated when superoxide and hydrogen peroxide disproportionate, a process called the Haber-Weiss reaction (Figure 58–2C).
Free Radicals & the Mitochondrial Theory of Aging
In 1956, Denham Harmon proposed the so-called free radical theory of aging. It had been reported that the toxicity of hyperbaric oxygen treatment and radiation could be explained by a factor common to both, the generation of ROS. This report dovetailed nicely with Harmon’s own observation that lifespan was inversely related to metabolic rate and, by extrapolation, respiration. He therefore postulated that the cumulative damage was caused by the continual and inescapable production of ROS.
In more recent years, the proponents of the free radical theory of aging have focused attention on the mitochondria. Not only is the mitochondria host to the major source of ROS in the cell, the electron transport chain, but oxidative damage to the components of this pathway could lead to increased leakage of hydrogen peroxide, superoxide, etc, into the cytoplasm. Damage to the mitochondria would be likely to adversely affect the efficiency with which it performs its most important function, the synthesis of ATP. A significant slowing in the rate of ATP synthesis could readily lead to the types of wholesale declines in physiological function that occur in aging.
A second contributor to the proposed self-perpetuating cycle of mitochondrial redox damage is the fact that several components of the electron transport chain are encoded by the mitochondrion’s indigenous genome. The mitochondrial genome is a much reduced, vestigial remnant of the genome of the ancient bacterium that was the precursor of the current organelle. Through a process called endosymbiosis, primitive eukaryotes became dependent upon surrounding bacteria to provide certain materials, and vice versa. Eventually, the smaller bacterium was absorbed by and lived within the interior of its eukaryotic host. Over time most, but not all, of the genes contained in the bacterial genome were either eliminated as superfluous to the needs of the new fusion organism or were transferred into the host cell’s nuclear DNA. At present, the genome of the human mitochondrion encodes a small and a large ribosomal RNA, 22 tRNAs, and certain polypeptide subunits for complexes I, III, and IV of the electron transport chain as well as the F1, F0 ATPase (Table 58–3). The mitochondrial genome lacks the surveillance and repair mechanisms that help maintain the integrity of nuclear DNA. Hence, mutations induced by adducts or reaction with ROS, and any functional defects resulting from these mutations, become a permanent feature of each individual mitochondrion’s genome, which will continue to accumulate mutations with time.
TABLE 58–3Genes Encoded by the Genome of Human Mitochondria ||Download (.pdf) TABLE 58–3 Genes Encoded by the Genome of Human Mitochondria
|rRNA ||12S, 16S rRNA |
|tRNA ||22 tRNAs (2 for Leu and Ser) |
|Subunits of NADH-ubiquinone oxidoreductase (Complex I, >40 total) ||ND 1-6, ND 4L |
|Subunits of ubiquinol-cytochrome c oxidoreductase (Complex III, 11 total) ||Cytochrome b |
|Subunits of cytochrome oxidase (Complex IV, 13 total) ||COX I, COX II, COX III |
|Subunits of the F1, F0 ATPase (ATP synthase, 12 total) ||ATPase 6, ATPase 8 |
While the mitochondrial hypothesis is no longer viewed as providing a unifying explanation for all of the changes that are associated with human aging and its comorbidities, it likely is an important contributor. Powerful circumstantial evidence for this is provided by the central role played by this organelle in the sensor-response pathways that trigger apoptosis.
Mitochondria Are Key Participants in Apoptosis
Apoptosis imbues higher organisms with the ability to selectively eliminate cells that are rendered superfluous by developmental changes, such as those that take place on a continual basis during embryogenesis, or which have been damaged beyond repair. During developmental tissue remodeling, the apoptotic cell death program is triggered by receptor-mediated signals. In the case of damaged cells, any one of several interior indicators may serve as trigger: ROS, viral dsRNA, DNA damage, and heat shock. These signals induce the opening of the permeability transition pore complex embedded in the mitochondrial outer membrane, through which molecules of the small (≈12.5 kDa) electron carrier protein cytochrome c then escape into the cytoplasm. Here, cytochrome c serves as the core for nucleating a multiprotein complex, the apoptosome, that initiates a cascade of proteolytic activation events targeting the proenzyme forms of a set of cysteine proteases known as caspases. The terminal caspases, numbers 3 and 7, break down structural proteins in the cytoplasm and chromatin proteins in the nucleus; events that lead to the death of the affected cell and its elimination by phagocytosis. Needless to say, the presence of an intrinsic, receptor-mediated cell death pathway offers the hope that we can eliminate harmful cells, such as cancer, by selectively activating their apoptotic pathway.
Ultraviolet Radiation Can Be Extremely Damaging
The term ultraviolet (UV) radiation refers to those wavelengths of light that lie immediately beyond the blue or short wavelength end of the visible spectrum. While the human eye cannot detect these particular wavelengths of light, they are strongly absorbed by organic compounds possessing aromatic rings or multiple, conjugated double bonds such as the nucleotide bases of DNA and RNA; the aromatic side chains of the amino acids phenylalanine, tyrosine, and tryptophan; polyunsaturated fatty acids; heme groups; and cofactors and coenzymes such as flavins, cyanocobalamine, etc. Absorption of this short wavelength, high-energy light can cause the rupture of covalent bonds in proteins, DNA, and RNA; the formation of thymine dimers in DNA (Figure 58–4); cross-linking of proteins; and the generation of free radicals including ROS. While UV radiation does not penetrate beyond the first few layers of skin cells, the high efficiency of absorption leads to the rapid accumulation of damage among the limited population of skin cells that are impacted. Because the nucleotide bases of DNA and RNA are particularly effective at absorbing UV radiation, it is highly mutagenic. Prolonged exposure to intense sunlight can lead to the accumulation of multiple DNA lesions that can overwhelm a cell’s intrinsic repair capacity. It is thus relatively common for persons whose work or lifestyle involves prolonged exposure to sunlight to manifest aberrant skin tissue, in the form of both moles and cancerous myelomas. Many of the latter can proliferate and spread with great rapidity, necessitating careful surveillance and rapid medical intervention.
Formation of a thymine dimer following excitation by UV light. When consecutive thymine bases are stacked one above the other in a DNA double helix, absorption of UV light can lead to the formation of a cyclobutane ring (red, not to scale) covalently linking the two bases together to form a thymine dimer.
Protein Glycation Often Leads to the Formation of Damaging Cross-links
When amino groups such as those found on the side chain of lysine or some of the nucleotide bases are exposed to a reducing sugar such as glucose, a reversible adduct is slowly generated through the formation of a Schiff’s base between the aldehyde or ketone group of the sugar and the amine. Over time, the glycated protein undergoes a series of rearrangements to form Amadori products, which contain a conjugated carbon-carbon double bond that can react with the amino group on a neighboring protein (Figure 58–5). The net result is the formation of a covalent crosslink between two proteins or other biological macromolecules that can, in turn, undergo further glycation and crosslink to yet another macromolecule. These cross-linked aggregates are sometimes called advanced glycation endproducts or AGEs.
Protein glycation can lead to the formation of protein-protein cross-links. Shown are the sequence of reactions that generate the Amadori product on the surface of the protein marked in green, and the subsequent formation of a protein–protein crosslink via an amino group on the surface of a second, red, protein.
The physiological impact of protein glycation can be especially pronounced when long-lived proteins such as collagen or β-crystallins are involved. Their persistence provides the opportunity for multiple glycation and cross-linking events to occur. The progressive cross-linking of the collagen network in vascular endothelial cells leads to the progressive loss of elasticity and thickening of the basement membrane in blood vessels, promoting plaque formation. The overall result is a progressive increase in the heart’s workload. In the eye, the accumulation of aggregated proteins compromises the opacity of the lens and eventually manifests itself in the form of cataracts. Impairment of glucose homeostasis renders diabetics particularly susceptible to the formation of advanced glycation end products. In fact, the glycation of hemoglobin and serum albumin are used as biomarkers for the diagnosis of diabetes and the assessment of its treatment.