Does the oxidative stress theory of aging explain longevity differences in birds? I. Mitochondrial ROS production

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Abstract

Mitochondrial reactive oxygen species (ROS) production rates are reported to be inversely related to maximum lifespan potential (MLSP) in mammals and also to be higher in short-living mammals compared to short-living birds. The mammal–bird comparison, however, is mainly based on studies of rats and pigeons. To date, there has been no systematic examination of ROS production in birds that differ in MLSP. Here we report a comparison of mitochondrial ROS production in two short-living (quails) and three long-living bird species (parrots) that exhibit, on average, a 5-fold longevity difference. Mitochondrial ROS production was determined both in isolated mitochondria (heart, skeletal muscle and liver) as traditionally done and also in intact erythrocytes. In all four tissues, mitochondrial ROS production was similar in quails and parrots and showed no correspondence with known longevity differences. The lack of a consistent difference between quails and parrots was not due to differences in mitochondrial content as ROS production in relation to oxygen consumption (determined as the free radical leak) showed a similar pattern. These findings cast doubt on the robustness of the oxidative stress theory of aging.

Highlights

► Parrots as a group live 5–7 times longer than same-sized quails. ► To date, the reason for their long lifespan is still not known. ► Reactive oxygen species (ROS) production is associated with lifespan differences. ► Mitochondrial ROS production was measured in heart, liver, muscle and erythrocytes. ► ROS production did not correspond with the observed longevity differences.

Introduction

The oxidative stress theory of aging gained considerable support when Sohal and colleagues discovered that the rates of superoxide and hydrogen peroxide production of liver, heart and kidney mitochondria were inversely related to maximum lifespan potential (MLSP) in mammals (Sohal et al., 1990, Ku et al., 1993). Because the mammal species they studied differed significantly in size, the extent to which interspecific differences in MLSP were attributable to size effects versus differences in rates of mitochondrial formation of reactive oxygen species (ROS) was unclear. This led the group to compare mitochondrial ROS production in short-living rats (MLSP 5y) to that of similar-sized but long-living pigeons (MLSP 35y). They found mitochondrial ROS production to be significantly lower in brain, heart and kidney of pigeons, from which they concluded that pigeon tissues were under less oxidative stress than those of rats (Ku and Sohal, 1993). Subsequent studies have extended this comparison by using different tissues and substrates for the mitochondrial respiration chain, and also conclude that mitochondrial ROS production is higher in rats compared to pigeons (Barja et al., 1994, Herrero and Barja, 1997, Barja, 1998, Lambert et al., 2007, Lambert et al., 2010). Although the finding that heart mitochondria of short-living mice (MLSP 4y) have higher ROS production rates than those of long-living budgerigars (MSLP 21y) and canaries (MLSP 24y) (Herrero and Barja, 1998) further supports the oxidative stress theory of aging, Brown et al. (2009) challenge this generality by showing that liver mitochondria from mice have a lower rate of ROS production compared to longer-living house sparrows (MLSP 14y).

While previous comparative studies mainly support the oxidative stress theory of aging, the extent to which variation in mitochondrial ROS production is responsible for differences in MLSP among species is far from certain. Most conclusions are based mainly on studies comparing isolated heart mitochondria using succinate as a respiratory chain substrate. In some cases, however, different conclusions are reached when using other substrates or other tissues. For example, whereas the Barja group demonstrated a higher ROS production in pyruvate-supplemented heart mitochondria of the rat in comparison to the pigeon (Herrero and Barja, 1997), Lambert et al. (2007) did not find a relationship between heart mitochondrial ROS generation and MLSP in a variety of mammals and birds using pyruvate, but did so when providing succinate. Similarly, the relationship between MLSP and mitochondrial ROS production originally reported for liver (Barja et al., 1994) was not observed for glutamate/malate- or succinate-supplemented liver mitochondria (Brown et al., 2009).

While mammal–bird longevity differences make them well suited for examining the bases of aging, there has been surprisingly little comparative examination of ROS production rates among bird species with very different MLSP. Here we report a comparison of mitochondrial ROS production of three tissues from two short-living and three long-living bird species that exhibit, on average, 5-fold longevity differences. The two quail species (king quails and Japanese quails) and the three parrot species (budgerigars, lovebirds and cockatiels) overlap in size, but the quails are very short-living and the parrots are very long-living among bird species (Fig.1). Although traits, including MLSP, among closely related species cannot be considered to be statistically independent if phylogenetic signals strongly influence the evolution of those traits (Felsenstein, 1985), the oxidative stress theory of aging predicts convergent evolution of mechanisms responsible for longevity.

Traditionally, mitochondrial ROS generation has been determined using enzymatic detection of hydrogen peroxide production by isolated mitochondria (Barja, 2002). Extrapolation from these in vitro measurements to the in vivo situation is difficult because little is known about changes in mitochondrial superoxide production in response to physiologically relevant changes in substrate supply and energy demand in intact cells. Some of these uncertainties are alleviated by using fluorescent dyes, such as Dihydrorhodamine 123 or MitoSox Red to infer cellular and mitochondrial ROS production in intact cells (Wardman, 2007). Birds, in contrast to mammals, have nucleated erythrocytes that also possess mitochondria and other organelles; thus permitting ROS production to be analyzed in whole cells in their physiological cell environment. We have therefore determined ROS production both in intact red blood cells and in mitochondria isolated from three tissues (heart, skeletal muscle and liver) of the five bird species. ROS production of isolated mitochondria was determined under physiological substrate concentrations of succinate and pyruvate at assay temperatures representing normal bird body temperature. These measurements were undertaken in the absence and presence of the complex III inhibitor antimycin A to assess basal and maximal rates of ROS production, respectively.

Section snippets

Holding conditions

All experiments were approved by the University of Wollongong Animal Ethics Committee and were conducted in conformity with the NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Six male budgerigars (Melopsittacus undulatus) (MLSP = 21 y; average mass = 26.0 g) derived from wild-type native budgerigars were purchased from a bird breeder in Queensland, Australia. Ten Japanese quails (Coturnix japonica) (MLSP = 6 y; average mass = 221.2 g) were purchased from Kyeema

ROS production in erythrocytes

The production of hydroxyl radicals and peroxynitrate (determined over a 30 min period using Dihydrorhodamine 123) was highly variable, with king quail erythrocytes exhibiting the lowest and the budgerigar erythrocytes showing the highest fluorescence, but with no significant difference between the other species (Fig. 2A).

Although there are significant differences in erythrocyte superoxide production between individual species, there is no general pattern between short-living quails and

Discussion

The common view that mitochondrial ROS production is higher in short-living species compared to long-living species has been historically based on either bird–mammal (e.g. Ku and Sohal, 1993, Barja et al., 1994, Herrero and Barja, 1997, Lambert et al., 2007) or within-mammal comparisons (e.g. Sohal et al., 1990). To our knowledge, ours is the first study to compare mitochondrial ROS production among birds with substantial variation in MLSP. The approximately 5-fold difference in MLSP between

Acknowledgments

We gratefully acknowledge the assistance on diet formulation provided by Kirk C. Klasing and Ron Newman. This research was supported by funding from the Australian Research Council. The study was conceived and planned by all three authors; the experiments were carried out by M.K. Montgomery and the manuscript was written by all three authors.

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