Barbara H. Sohal

Hydrogen peroxide release by mitochondria increases during aging

The effect of aging on the release of H2O2 by mitochondria was studied in the housefly in order to elucidate the causes of previously observed age-related increase in the level of oxidative stress. Intact flight muscle mitochondria of the housefly, supplemented with alpha-glycerophosphate, produce 1-2 nmol H2O2/min per mg protein, even in the absence of respiratory inhibitors. The rate of H2O2 secretion progressively increases approximately 2-fold during aging of the fly. Neither uncoupling of oxidative phosphorylation nor mechanical damage to mitochondria during the isolation procedure appear to be responsible for the age-related increase in H2O2 production. Activities of NADH-ferricyanide reductase, succinate-ubiquinone reductase, and NADH-, succinate- and alpha-glycerophosphate-cytochrome c reductases, were approximately 2-fold higher in mitochondria from the old than those from the young flies. However, the concentration of enzymatically reducible ubiquinone remained unchanged with age. Infliction of damage by exposure of mitochondria to free radical-generating systems in vitro caused an increase in the rate of H2O2 generation. Glutaraldehyde, an intermolecular crosslinking agent, induced an increase in the rate of H2O2 generation by mitochondria. Results of this study demonstrate that aging in the housefly is associated with an increase in the rate of H2O2 generation by mitochondria probably due, at least in part, to self-inflicted damage by mitochondria. Intermolecular cross-linking in the inner mitochondrial membrane can contribute towards the increased H2O2 generation.

Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria

The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories--a regimen that increases life span by approximately 30-40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.

Mitochondrial superoxide and hydrogen peroxide generation, protein oxidative damage, and longevity in different species of flies.

The objective of this study was to further elucidate the role of oxidative stress in the aging process by determining whether or not the rates of mitochondrial superoxide anion radical and hydrogen peroxide (H2O2) production, the activity of cytochrome c oxidase, and the concentration of protein carbonyls are correlated with the life span potential of different species. A comparison was made among five different species of dipteran flies, namely, Drosophila melanogaster (fruit fly), Musca domestica (house fly), Sarcophaga bullata (flesh fly), Calliphora vicina (blow fly) and Phaenecia sericata (a species of blow flies), which range more than 2-fold in their life span potentials. The average life span potential of these species was found to be inversely correlated with the rates of mitochondrial superoxide and H2O2 production and with the level of protein carbonyls, and to be directly related to the activity of cytochrome c oxidase. The significance of these findings in context of the validity of the oxidative stress hypothesis of aging is discussed. It is inferred that longer life span potential in these insect species is associated with relatively low levels of oxidant generation and oxidative molecular damage. These results accord with our previous findings on different mammalian species.

Age-related changes in antioxidant enzymes and prooxidant generation in tissues of the rat with special reference to parameters in two insect species

The objective of this study was to explore the relationship between partially reduced oxygen species and the aging process. Effect of age on antioxidant defenses and prooxidant generation was evaluated by comparing the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, and rates of mitochondrial O-2 and H2O2 generation in the liver, heart, and brain of 3-month and 18-month-old Sprague-Dawley rats. In addition, comparisons of antioxidant defenses and mitochondrial prooxidant generation were made between short-lived insects and the rat tissues. Results indicated that antioxidant enzymes exhibit a mixed pattern of age-related alterations. In each organ of the rat examined, activities of some enzymes were up with age and of others down with age. The overall magnitude of decline in antioxidant defenses observed here and elsewhere in the literature was deemed to be unlikely to be functionally significant. In contrast, the rate of mitochondrial O-2 and H2O2 generation increased in various tissues of the rat. Antioxidant defenses in insects were comparable to tissues in the rat but rates of O-2 and H2O2 generations were notably higher. Results are interpreted to suggest that rates of prooxidant generation may be more crucial than antioxidant levels as possible longevity determinants.

Relationship between antioxidant defenses and longevity in different mammalian species

The general objective of this investigation was to examine the relationship between oxygen free radicals and the aging process. Comparisons of antioxidant defenses were made in six different mammalian species, namely, mouse, rat, guinea pig, rabbit, pig and cow, which range from 3.5 to 30 years in their maximum life span potential (MLSP). Activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase, and concentration of glutathione were measured in the liver, the heart, and the brain. SOD and catalase activities were positively correlated whereas glutathione concentration was negatively correlated with MLSP. Glutathione peroxidase activity exhibited a variable pattern: being positively correlated with MLSP in the brain and negatively correlated in the liver and the heart. In each organ, MLSP was correlated with relatively high levels of one or two of the above antioxidants and low levels of the other antioxidants, indicating the possibility of a compensatory balance among various components of the antioxidant system. No obvious pattern of a relationship was detectable between the overall level of antioxidant defenses and MLSP among the mammalian species examined. The implications of this finding concerning the role of oxidative stress in the aging process and the free radical hypothesis of aging are discussed.

Oxidative stress and aging in the Mongolian gerbil (Meriones unguiculatus).

The objective of this study was to determine if aging in the gerbil, Meriones unguiculatus, is associated with elevation in the level of oxidative stress. Studies were conducted on the brain, heart, kidney, liver and testis of young (3-6 months), adult (15 months), and old (23-25 months) animals. Oxidative damage to proteins, measured as the concentration of protein carbonyls and loss of activity of glucose-6-phosphate dehydrogenase, and to DNA, measured as the concentration of 8-hydroxydeoxyguanosine, increased with age of the animals. There was no appreciable age-related change in the activity of alkaline proteases, which preferentially degrade oxidized protein. Rates of mitochondrial superoxide anion radical and hydrogen peroxide generation also increased with age, most notably in the heart. Antioxidative defenses, measured as activities of superoxide dismutase, catalase and glutathione peroxidase and concentration of glutathione, did not exhibit a uniform pattern of age-related changes. However, when the antioxidative potential of the tissue homogenates was measured as their susceptibility to undergo protein oxidation, in response to experimentally-induced oxidative stress, using X-irradiation, tissues of the old animals were significantly more vulnerable than those of the young animals. Results of this study are interpreted to indicate: (i) that the level of molecular oxidative damage to DNA and proteins increases with age, and (ii) that the increased oxidative damage is due to both an an elevation in the rates of oxidant generation and an increase in the susceptibility of tissues to oxidative damage.

Superoxide anion radical production in different animal species.

The general objective of this study was to examine the relationship between oxygen free radicals and the aging process. The rate of superoxide anion radical (O2.-) generation was measured in liver sub-mitochondrial particles from mouse, rat, rabbit, pig and cow, and in flight muscle sub-mitochondrial particles from the housefly. The rate of O2.- generation was determined as superoxide dismutase inhibitable reduction of ferricytochrome c in the presence of antimycin A and KCN. O2.- generation was inversely related to maximum species life span potential (MLSP) (r = -0.92). A 24-fold difference in the rate of O2.- production was observed between the cow and the fly while a 6-fold difference existed among the mammals. The results are interpreted to indicate that under identical conditions, mitochondria from organisms with low MLSP have a relatively greater propensity to generate O2.-. This may be suggestive of innate differences in the molecular organization of the inner mitochondrial membrane among different species.

Effect of caloric restriction on life span of the housefly, Musca domestica

Caloric restriction (CR) has been found to extend the life spans of a wide variety of species, transcending phylogenetic boundaries. The objective of this study was to test the generality of this phenomenon, using the male housefly as an insect model in which food intake can be quantified precisely. Sucrose was found to promote a longer life span than diets additionally containing proteins and lipids. Flies were fed sucrose or a more complex diet ad libitum (AL), or in amounts ranging from 50% to 100% of the average amount consumed by young flies. CR shortened rather than prolonged the life span of houseflies, particularly flies fed sucrose only. The rate of oxygen consumption was not affected by caloric restriction or by the exclusion of proteins and lipids from the diet, and the reproductive activity of male flies remained unchanged by sucrose feeding. Thus, it is unlikely that the life‐shortening effects of CR can be explained either in terms of an adaptive response in metabolic rate or use of a suboptimal food source. Results of this study contradict the widely held view that CR has a life‐extending effect in all species.

Defining genes that govern longevity in Caenorhabditis elegans

We previously identified five regions on the chromosomal map of Caenorhabditis elegans, containing genes that help specify life span in this species, by comparing the genotypes of young and long-lived progeny from a cross between strains Bristol-N2 and Bergerac-BO [Ebert et al. (1993): Genetics 135:1003-1010]. Analyses of additional crosses, and of putative polymorphisms for the implicated genes, are necessary to clarify the roles of naturally occurring polymorphic alleles in determining longevity. We therefore carried out a second multigenerational cross, between strains Bristol-N2 and DH424 (both nonmutators at 20 degrees C), to create a different heterogeneous recombinant-inbred population. We again found strong evidence implicating multiple genes, which differ between the parental strains, in the determination of life span. Increased variance of survival, for F2 and homozygous F25 worms relative to F1 hybrids, is consistent with such alleles assorting randomly in the cross progeny. Moreover, chromosome mapping data corroborate the polygenic nature of this quantitative trait. Genotypes of young and very long-lived adult worms from a synchronous F15 population were determined by polymerase chain reaction, to identify the parental strain of origin for each of 10 polymorphic loci. Two regions, on chromosomes II and IV, each contain at least one gene with allelic differences in associated longevity. A recombinant-inbred Bergerac-BO x Bristol-N2 population, derived from the earlier cross between those strains, was exposed to an acute toxic level of hydrogen peroxide. Genotyping of H2O2-resistant worms implicated at least one of the five chromosomal regions previously identified in the same cross progeny as harboring a longevity-determining gene. Superoxide dismutase and catalase levels, determined for the three parental strains as they aged, confirm the existence of polymorphisms in the corresponding genes (or their regulatory mechanisms) inferred from the chromosome-II mapping data, and are consistent with the hypothesis that increased longevity is conferred by high levels of these enzymes late in life.