The oxidative stress theory of aging was first proposed just more than 50 years ago and has been the focus of many studies investigating potential basic underlying mechanisms of aging. According to this theory, a deficiency in the detoxification of ROS due to reduced levels of antioxidant enzymes could lead to an accumulation of ROS and oxidative damage and thereby shorten life span. In the subsequent years, the potential role of the mitochondria in this process was highlighted and the theory extended to suggest that ROS released from the mitochondrial respiratory chain damages macromolecules, especially mitochondrial DNA (mtDNA), resulting in an accumulation of mtDNA mutations, defective mitochondrial respiration, and further increases in ROS generation and oxidative damage. To investigate the effect of reduced mitochondrial antioxidant defenses on life span, we generated mice with deficiencies in both MnSOD and Gpx-1, two primary mitochondrial ROS-scavenging enzymes. Contrary to the predicted role of mitochondrial oxidative stress in aging, in the current study we show that mice lacking both MnSOD and Gpx-1 had no reduction in life span.
Many vertebrate and invertebrate animal models have been developed to test the role of oxidative stress in aging. Although some studies in which antioxidant defenses have been increased in lower organisms such as C. elegans
have been successful in extending life span, many studies using models of altered antioxidant defenses in mammals have not been supportive of this theory. Thus, whereas deletion of MnSOD in mice, the major mitochondrial enzyme that detoxifies superoxide, is either embryonic or postnatal lethal depending on the genetic background of the strain, heterozygous (Sod2+/−
) knockout mice, which have approximately 50% of the level of MnSOD activity found in wild-type mice, develop normally and do not exhibit a reduction in life span. However, the Sod2+/−
mice do have increased oxidative damage to DNA, alterations in liver and heart mitochondrial function, and increased sensitivity to apoptosis (23
). These results seem to contradict the predictions of the oxidative stress theory of aging and have raised questions about the role of oxidative stress and damage in modulating longevity. However, the aging process per se may not be simply an issue of living or dying but rather an issue of functioning well versus functioning poorly, that is, the issue of healthy life span or healthy aging (44
). From this point of view, the increase of oxidative stress due to the deficiency of Sod2
does affect the aging process in Sod2+/−
mice as evidenced by the decrease of resistance to oxidative stress and increase of tumor incidence.
Gpx-1 is an important antioxidant enzyme that detoxifies hydrogen peroxide. Gpx-1 is located in both the cytosol and the mitochondria and thus could play an important role in detoxifying hydrogen peroxide formed by removal of superoxide in the mitochondrial matrix by MnSOD. Homozygous deletion of Gpx1
has no effect on mouse development (28
), but to date, no longevity study has been conducted with Gpx1−/−
mice. We had previously shown that cells and tissues from Gpx1−/−
mice are more sensitive to oxidative stress compared with wild-type control mice (32
). In the current study, we demonstrated that the lack of Gpx-1 does not alter life span. No differences were observed in median, mean, and maximum life span between Gpx1−/−
and wild-type mice, suggesting that Gpx-1 had no significant impact on mouse longevity. These data, again inconsistent with the oxidative stress theory of aging, also suggest that deficiency of one gene may not be enough to affect longevity.
Because the antioxidant defense system is a complex and integrated system, it is possible that deficiency of a single antioxidant enzyme may not compromise the system to a magnitude sufficient to alter longevity. To address this possibility, we generated mice with a double deficiency in MnSOD and Gpx-1. Because mice null for MnSOD are not viable, we generated mice heterozygous for Sod2
and null for Gpx1
. In a previous study, we had shown that young Sod2+/−Gpx1−/−
mice are very sensitive to ,-irradiation and paraquat treatment, and cells isolated from these mice are more susceptible to oxidative stress than those deficient in either MnSOD or Gpx-1 alone (32
). These results demonstrate that a deficiency in these two primary defense enzymes has a dramatic ability at the cellular and whole organism levels to increase sensitivity to oxidative insults.
In the present study, we examined whether deficiency of both MnSOD and Gpx-1 in mice affects aging and longevity. Our data demonstrated that Sod2+/−Gpx1−/−
mice have a life span that is not different from that of the wild-type mice, suggesting there is no additive effect on aging with deficiency of both enzymes. Our finding is similar to that of the Marklund's group, which demonstrated no life-span difference between mice with a single deletion of Cu/ZnSOD and mice with double knockout of extracellular SOD (EC-SOD) and Cu/ZnSOD (46
). In that study, deletion of EC-SOD affected neither the life span of the wild-type mice nor the life span of the Cu/ZnSOD null mice, suggesting no functional overlap between EC-SOD and Cu/ZnSOD in terms of modulating longevity. The same may be true for our study, which did not show overlapping roles between MnSOD and Gpx-1 in accelerating mouse life span.
Although we did not find a reduction in life span, we did observe a significant increase in oxidative damage in liver, skeletal muscle, and brain from Sod2+/−Gpx1−/−
mice when compared with wild-type mice. The Sod2+/−Gpx1−/−
mice also developed more pathology than wild-type and Gpx1−/−
mice, similar to our previous results in the Sod2+/−
mice in which we found increases in tumor incidence and tumor burden in the Sod2+/−
mice with age (32
). These results indicate that there is no additive effect of deleting Gpx1
in the presence of simultaneous reduction in MnSOD. It is possible that other peroxide detoxification enzymes (e.g., peroxiredoxins) may compensate for the lack of Gpx-1. Further studies will be required to address this question.
Although the sample size was somewhat small, the incidence of fatal tumors was similar among the groups. However, the overall incidence of tumors and tumor burden were significantly higher in the old (28–32 months of age) Sod2+/−Gpx1−/−
mice, when compared with age-matched wild-type control mice. The lack of increase in fatal tumors in the Sod2+/−Gpx1−/−
mice suggests that increased oxidative damage played a more important role in the early stages of carcinogenesis initiation, potentially through increased DNA oxidation that is followed by increased mutation and so forth, and may have a minimal effect on the growth and induction of tumors to become fatal. The rate of tumor growth could have a significant impact on longevity in mice because mice naturally have a high incidence of cancer (42
). In support of a role for oxidative damage in tumor progression, pathological analysis of long-lived Ames dwarf mice demonstrated that these mice have a similar incidence of cancer compared with the wild-type control but also have retarded growth of tumors, which is correlated with extended longevity (41
). Thus, the fact that the Sod2+/−Gpx1−/−
mice showed no changes in life span in spite of increased oxidative damage could be explained by the minimal effects of oxidative stress on tumor growth.
According to the oxidative stress theory of aging, a reduction in antioxidant protection, especially in mitochondria, would be predicted to have negative effect on longevity. However, our studies using both single- and double-deficient mice do not support this conclusion. In fact, we show that increased oxidative damage was not correlated to a change in life span. However, it should be noted that ROS generation and its detoxification in vivo must be maintained in a delicate balance for the animal to age normally, that is, to reach maximal life span. The effect of a partial reduction in MnSOD even in the face of a lack of Gpx-1 may not be enough to disturb oxidative homeostasis and affect longevity. Furthermore, ROS are not solely harmful to cell structure and function. They also serve a critical function as signaling molecules. Therefore, disruption of ROS homeostasis may have profound effects on multiple signaling systems that may play an important role in regulating aging and longevity, including the insulin-like growth factor–mammalian target of rapamycin axis and critical damage removal processes such as autophagy. Therefore, simple manipulation of the ROS generation/detoxification may not be enough to achieve the expected results due to the complexity of the signaling networks.
There is another important layer of complexity when studying the effects of oxidative stress on aging. Mammalian cells have developed redundant pathways for detoxifying ROS. Thus, it is possible that the experimental effect of altering one part of the defense system may be masked by unknown compensatory pathways or factors. Furthermore, the expression and distribution of different ROS detoxification enzymes vary from tissue to tissue with age. Simple deletion or overexpression of the gene in the whole animal may not be the best approach for longevity studies. The data from studies overexpressing catalase in mitochondria proved that targeted expression of genes may be a way of extending life span (47
). Thus, there may yet be other unidentified compensatory mechanisms that could reduce the effect of the loss of MnSOD and Gpx-1. Still, it is also possible that deficiency of these two enzymes may be insufficient to affect life span. In addition, it is possible that the deficiency in Sod2
might also affect functional parameters such as muscle strength or cognitive function, which may affect the health span of the mice without altering the life span.
Finally, the understanding of oxidative stress theory of aging is further complicated by some recent studies. Several investigators have reported that mildly increasing oxidative stress or increase of oxidative stress under certain genetic background after removing SODs in C. elegans
could actually increase longevity (48
). Others observed no change of longevity in mammals including mice when ROS level was reduced by overexpressing antioxidant enzymes (10
). These observations obviously contradict the prediction by the oxidative stress theory of aging. However, they are in agreement with a recently proposed hormesis theory of aging that states that mild stress-induced stimulation of protective mechanisms in cells and organisms results in biologically beneficial effects on aging (51
). As has been noted, most of the initial data supportive of the oxidative stress theory of aging are correlative observations among different species. However, the effect of oxidative stress on aging and longevity in each species may differ from each other. Moreover, in higher organisms, oxidative stress may have differential effects on various tissues during aging. Thus, whole body deletion of or overexpression of antioxidant enzymes may not achieve the expected results. Finally, whether oxidative stress is a result of the aging process or a cause of aging is still an unresolved issue.
In conclusion, although our data do not provide support for the oxidative stress theory of aging, it does not definitely rule it out either. Additional carefully designed animal models may be required to better define the full role of oxidative stress in aging, whether it is a cause or result of the aging process. Such studies could include the use of tissue- or age-specific and dose-controlled gene expression/knockout models.