NAD+ in Drosophila melanogaster

Mechanisms of Ageing and Development, 56 (1990)223--235 ElsevierScientificPublishers Ireland Ltd.

223

E F F E C T OF A G E ON S U P E R O X I D E DISMUTASE, CATALASE, G L U T A T H I O N E REDUCTASE, INORGANIC PEROXIDES, TBA-REACTIVE M A T E R I A L , G S H / G S S G , N A D P H / N A D P ÷ AND N A D H / N A D + IN D R O S O P H I L A M E L A N O G A S T E R

R.S. SOHAL*, L A U R E N A R N O L D and W I L L I A M C. ORR Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275 (U.S.A.)

(ReceivedJune 1lth, 1990)

SUMMARY The objective o f this study was to investigate the possible role o f oxygen free radicals in the aging process by studying the pattern o f age-related changes in a broad spectrum of antioxidant defenses and indicators o f oxidative stress in adult male Drosophila melanogaster. There were selective, but not universal, changes in the antioxidant defenses and indicators o f oxidative stress. Activities o f catalase and glutathione reductase and concentration o f reduced glutathione decreased during the latter part o f life. Superoxide dismutase activity tended to increase with age whereas glutathione peroxidase activity was undetectable. The ratios of reduced/ oxidized glutathione and N A D P H / N A D P ÷, which are widely regarded as indicators of oxidative stress, decreased in the terminal phase o f life, N A D H / N A D ÷ ratio increased in the latter part of life. Concentration of inorganic peroxides increased during the first trimester of life and remained unchanged thereafter while that of thiobarbituric acid-reactants tended to decrease during aging. The main conclusion of this study is that age-related changes in antioxidant defenses and in levels o f oxygen free radical reaction products are selective in nature and are quite variable in different species and tissues; however, the level of oxidative stress tends to increase during aging.

Key words: Free radicals; Aging; Oxyradicals; Life span; Antioxidants; Oxidative stress

*To whom all correspondenceshould be addressed. 0047-6374/90/$03.50 Printed and Publishedin Ireland

© 1990ElsevierScientificPublishers Ireland Ltd.

224

INTRODUCTION

Partially reduced oxygen species are widely hypothesized to play a causal role in the aging process of animals albeit the details of the mechanism are, at present, largely conjectural [1]. To further elucidate the nature of the putative relationship, it is relevant to know if the level of oxidative stress, i.e., the ratio of pro-oxidants to antioxidants, remains the same or is altered during aging. Such information will have a critical effect on the nature of the hypothesis explaining the mechanism by which free radicals m a y induce age-related alterations. For example, lack of agerelated alteration in the level o f oxidative stress could mean that the consequent damage remains steady during aging. Aging could thus be due, at least partly, to accumulation of age-related damage. Conversely, an increase in the level of oxidative stress with age would tend to implicate an age-related expression of genetic a n d / or epigenetic factors controlling the level of oxidative stress [2]. There are numerous reports in the literature examining the effect of age on the level of antioxidant defenses in vertebrates as well as invertebrates (for review, see Ref. 1). There are at least two notable features of these studies which have confounded the evaluation of the free radical hypothesis of aging and have also prompted this investigation. First, there is a great variability in the reported agerelated trends not only in different tissues or species but also in the same tissue of the same species (e.g., compare 3 and 4). Second, an age-related decrease in a given antioxidant is often interpreted prima facie, without direct evidence, as an indication of a corresponding increase in the vulnerability of the tissue to radical-induced damage. The objective of the present study, conducted in Drosophila melanogaster, was to satisfy the above criticism. Firstly, the levels of a broad range of antioxidants as well as indicators of oxidative stress were examined in order to elucidate an overall ageassociated trend. Secondly, to establish an age-related trend measurements were made at frequent chronological intervals rather than at 2 or 3 age groups as is often the case in studies related to aging. MATERIALS AND METHODS

Rearing of flies Studies were conducted on male Oregon R D. melanogaster. Males were separated during the first day after emergence of the adults from pupae and placed in 170-ml plastic jars, containing 50 ml of food (cornmeal-molasses-yeast-agar), at 25 °C. Flies were transferred to fresh containers every alternate day.

Biochemical assays Most of the procedures used in this study have been reported in detail previously and will be only briefly referred to here. Superoxide dismutase activity was meas-

225

ured by the direct method described by Misra and Fridovich [5] as reported previously [6l. Tissues were homogenized in 10 volumes (v/w) of 16.6 mM phosphate buffer (pH 7.8), containing 0.05 mM 3-amino-l,2,4-triazole and 0.33 mM EDTA. The homogenate was centrifuged in cold at 3000 g for 5 min, supernatant was recentrifuged at 50 000 g for 30 min and the resultant supernatant was dialyzed first for 4 h at 4°C against 50 mM potassium phosphate buffer (pH 7.6), 0.1 mM H202 and 0.05 mM aminotriazole and then redialyzed for 6 h against phosphate buffer alone. To measure SOD activity, 50/al of the homogenate was added to a reaction mixture consisting of 0.033 mM EDTA, 3.3 mM methionine, 0.3 mM dianisidine, 0.33/ag/ ml riboflavin and 0.01 mM KCN. The cuvette containing the reaction mixture was placed in a steel box illuminated with four 20 W sylvania Grolux® fluorescent tubes for 10 min. Results were calculated by subtracting the change in absorbance of the blank (lacking homogenate) from the sample and dividing A,4 by 0.012 to obtain McCord-Fridovich units of activity. The value of this constant was obtained by assaying a known concentration of the enzyme. To measure catalase activity, a 10°70 fly homogenate was made in 66 mM potassium phosphate buffer and 0.1% Triton X. Homogenate was centrifuged at 3000 g for 5 min and the supernatant was recentrifuged at 50 000 g for 30 min and the pellet was discarded. Catalase activity was measured by the method of Luck [7] by determining the length of time required for AA (0.45--0.4) for a mixture of homogenate, H202 (12.5 mM) and buffer. One unit of activity was equal to 17 s. To measure glutathione peroxidase activity homogenates were prepared as in the case of catalase. Enzyme activity was measured by the method of Paglia and Valentine [8] as described by Beutler [9]. Both the blank and the system cuvette contained 0.1 M KPO 4 buffer (pH 7.0), 2/aM EDTA, 10 units/ml glutathione reductase, 4 mM sodium azide, 200 mM NADPH and tissue homogenate. The system cuvette contained in addition 1.0 mM GSH. After l0 min preincubation at 37°C, the reaction was started by the addition of 1 mM H202 or 1.5 mM tert-butyl hydroperoxide or 1.5 mM cument hydroperoxide to the blank and system cuvette. An additional blank assay in which the buffer was substituted for the homogenate was performed in order to correct the non-enzymatic oxidation of GSH and NADPH by n202. To determine glutathione reductase activity tissue homogenates were prepared in a manner similar to that for measuring catalase activity. Enzyme activity was measured by the method described by Carlberg and Mannervik [ 10]. The reaction mixture contained 0.1 M phosphate buffer (pH 7.0), 1 mM EDTA, 100/aM NADPH and 1 mM GSSG. The reaction was started by the addition of the homogenate and the decrease in absorbance at 340 nm was noted. One unit of activity represented oxidation of 1/amol of NADPH/min. Levels of GSH and GSSG were determined according to the procedure described by Tietze [11], of inorganic peroxides by the method of Bernt and Bergmeyer [12] and of thiobarbituric acid (TBA)-reactive material according to the procedure of Ushiyama and Mihara [13], as described previously [6]. Concentrations of NADP ÷,

226 N A D P H , NAD ÷ and N A D H were determined by the spectrophotometric recycling method described by Pinder et al. [ 14]. RESULTS Survival and cross-sectional sampling The survivorship curve o f the male flies, with 100 flies per 170-ml bottle, kept at 25°C, is presented in Fig. 1. The beginning o f the dying phase, indicated by the sharp downward slope o f the survivorship curve, started around 20 days of age. The maximum life span in this population was 46 days. A profile of the age-related biochemical changes was obtained by harvesting the entire population of flies contained within a given bottle, usually ranging in age between 4 and 30 days, albeit in some cases older ages were also included. Flies younger than 4 days of age exhibit a variety o f post-emergence chemomaturation changes in enzyme activities and were therefore excluded. The reason for restricting the sampling to an age approximating the median life span has been discussed before [15]. It was pointed out that in an aging population, relatively free of accidental or disease-induced deaths, survivors progressively represent a subset undergoing a slower rate of aging. Sampling should ideally be restricted to an age when most of the population is still alive, but on the verge of death. Age-related biochemical alterations SOD activity. Activities of total SOD (cyanide-sensitive plus cyanide-insensitive) and of cyanide-insensitive SOD, measured in the homogenates of the flies, tended to

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Age (days) Fig. 2. Activities (± S.E.M.) of total (top) and cyanide-insensitive (bottom) superoxide dismutase in male D. melanogasterof various ages. Iinearly increase with age o f the flies (Fig. 2). The magnitude of the total increase from young to old age was about 50070. Catalase activity. Age-related changes in catalase activity followed a bell-shaped trend, with a 50°70 increase during the first 3 weeks of life followed by a sharp decrease during the latter part of life, whereby the level of activity was similar in the very young and the very old flies (Fig. 3). 30m o

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Glutathione peroxidase. Using

cumene hydroperoxide or tert-butyl hydroperoxide as substrates there was no detectable glutathione peroxidase activity after the rates of the control blanks were taken into account. Glutathione reductase. Glutathione reductase activity increased slightly during the first half of life followed by a gradual decline of about 30070 in the latter half of life span (Fig. 4). Glutathione. The concentration of reduced glutathione (GSH) remained quite stable for the first 3 weeks of life followed by a sharp decline in the next week. In contrast, the level of oxidized glutathione (GSSG) remained relatively unchanged during the first 2 weeks of age, but tended to increase in the next 2 weeks (Fig. 5). The ratio of G S H / G S S G , widely regarded as an indicator of the redox status of tissues, remained stable during the first part of life followed by a sharp decline in the latter part of life, indicating a shift towards a more pro-oxidizing state (Fig. 6). NADP(H) and NAD(H). Age-related changes in the levels of NADP ÷, N A D P H , NAD ÷ and N A D H are presented in Table I. The concentration of both NADP ÷ and N A P H exhibited a U-shaped age-related pattern. NAD ÷ concentration exhibited a slight upward trend with age while N A D H displayed a U-shaped pattern. The ratios of N A D P H / N A D P ÷ and N A D H / N A D ÷, shown in Fig. 7, indicate a contrasting trend; whereas N A D P H / N A D P ÷ ratio is higher in the older flies than in the young, the opposite trend is observed in the case of N A D H / N A D ÷. Inorganic peroxides. The steady state concentration of inorganic peroxides (H202) in the homogenates of flies increased about 50070 during the first trimester of life and remained relatively stable thereafter as shown in Fig. 8. TBA-reactive material. The concentration of TBA-reactive material in the H202 o r

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230 TABLE I CONCENTRATIONS OF NADPH, NADP ÷, NADH and NAD + IN WHOLE BODY HOMOGENATES OF MALE DROSOPHILA MELANOGASTER OF DIFFERENT AGES Parameter (nglmg wet wt.)

Age (days)

NAD ÷ NADH NADP* NADPH

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169 _.+ 4 40-+ 1 11 - 1 14_+ 2

162 - 3 72_+ 3 27 _+ 2 32_+ 5

Values are average _+ S.E.M. of three determinations.

h o m o g e n a t e s o f t h e h o u s e f l y e x h i b i t e d t h e p e a k level in t h e v e r y y o u n g flies f o l l o w e d b y a s h a r p d e c l i n e d u r i n g t h e first 2 w e e k s o f age a n d r e m a i n i n g r e l a t i v e l y s t a b l e t h e r e a f t e r (Fig. 9). DISCUSSION R e s u l t s o f this s t u d y i n d i c a t e t h a t a d u l t m a l e D r o s o p h i l a m e l a n o g a s t e r e x h i b i t o n l y s e l e c t i v e a n d n o t u n i v e r s a l c h a n g e s in t h e levels o f a n t i o x i d a n t s a n d m o l e c u l a r products of oxygen free radical reactions during the aging process.

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A comparison o f the results o f this study with those conducted in other species suggests that the pattern of age-related changes in various components o f the antioxidant defense system differs in different tissues and species, although there are some glaring examples of discrepancy in the values reported for the same tissue in the same species [1,3,4]. Similarly, indicators of oxidative stress are also expressed 0.021

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232 differently in different species [1,4]. Nevertheless, it appears that the level of in vivo oxidative stress tends to increase during aging in both mammals [16] and invertebrates [17] as indicted by the increased exhalation of alkanes, which are products of lipid peroxidation. Activity of SOD has been reported to increase, decrease or remain unaltered during aging in the rodent liver and brain [1,3,4]. SOD activity was previously found to decrease in the last trimester of adult life in the male housefly [18]. However, it tends to increase in Drosophila, which is in contrast to the results of Massie et al. [19] who reported the absence of an age associated change. This discrepancy may be due to the different methodology used for measuring SOD activity. There is a general accord that catalase activity tends to decline with age in insects. An approximately 40% decline in catalase activity was previously observed in the adult housefly during aging [18], which is roughly comparable to that occurring in Drosophila as reported here and by others previously [20]. The existence of selenium-dependent glutathione peroxidase in insects is a subject of controversy. Activity of this enzyme was reported to be undetectable in insects by Smith and Shrift [21] and in the housefly by us [18]. Simmons et al. [22] first reported the presence of a low level of enzyme activity in the housefly and more recently Ahmed et al. [23] have reported its presence in several insect species. However, Simmons et al. have not been able to validate their results and are now of the opinion that selenium-dependent glutathione peroxidase does not exist in the housefly [24], Notwithstanding, the level of enzyme activity reported by Ahmed et al. [2,3] is relatively very low as compared to mammalian tissues. The source of controversy appears to be the non-specific reactions between N A D P H and other components of the reaction mixture, e.g., H202 or cumene hydroperoxide or glutathione. In our opinion lack of reaction blanks accounting for such reactions can lead to the erroneous conclusion about the existence of this enzyme in insects. Since neither the gene nor mRNA or the protein for selenium-dependent glutathione peroxidase have been isolated in insects, the presence of this enzyme in this phylogenetic group is doubtful. In the second half of life, besides the age-related decline in catalase activity, the most notable alteration in the antioxidant defenses in Drosophila was the decrease in the activity of glutathione reductase and the concentration of GSH. During the same period of life the concentration of GSSG exhibited an increase. The ratio of G S H / GSSG, which is regarded as a major indicator of oxidative stress, underwent a twothirds decline in the latter half of life. This decline was accompanied by a decrease in the ratio of N A D P H / N A D P ÷. It is possible that the accumulation of GSSG and decrease in GSH concentration in the old flies is the result of an interrelated process whereby the decrease in catalase activity leads to an increased oxidation of GSH and the concomitant decline in glutathione reductase activity results in the decrease of GSH concentration and accumulation of GSSG. The decrease in N A D P H / N A D P ÷ ratio may contribute to GSSG accumulation since N A D P H is the electron donor in

233 the conversion of GSSG to GSH. The significance o f an increase in N A D H / N A D ÷ in latter part of life is unclear. Age-related indicators o f oxidative stress tend to also differ even among closely related species. For example, the housefly, which, in contrast to D. melanogaster, is a powerful and frequent flyer under laboratory conditions, exhibits a virtually linear age-related increase in the steady state concentration of inorganic peroxides and TEA-reactants in addition to declines in the ratios of G S H / G S S G , N A D P H / NADP ÷ and N A D P / N A D ÷ [25]. In contrast, in D. melanogaster, the steady-state concentration of inorganic peroxides increased only in the first trimester of life and TBA-reactive material showed a decline with age. In case of the former, it is possible that the sluggishness of D. melanogaster and the consequent lower rate of oxygen consumption may be responsible for the slower rate of mitochondrial H202production. It is worth noting that as compared to the housefly the metabolic rate of D. melanogaster is lower, the life span is 60--70°70 longer but the metabolic potential (i.e., total oxygen consumed during life) is virtually identical. Nevertheless, old D. melanogaster also tends to exhibit a higher steady state concentration of H202than young flies, which is indicative of enhanced oxidative stress. The concentration of TBA-reactive materials tended to decline with age in D. melanogaster. Levels of TBA-reactive materials have been reported, both, to go up and down with age (see Ref. 26). Although the concentration of TBA-reactive material is widely believed to be an indicator of lipid peroxidative damage, it is actually an indicator of the susceptibility or potential of tissues to undergo lipid peroxidation in vitro and not the amount of peroxidized lipids present in vivo [27]. Unfortunately this distinction is often ignored in the literature. The in vivo level of lipid peroxidation in the rat, as reflected by alkane exhalation [16], has been found to increase with age whereas the concentration of TBA-reactive material in several organs has been found to decrease with age [26]. TBA-chromogen formation is dependent upon a number of factors such as levels of polyunsaturated fatty acids, catalytic substances such as heavy metals and low molecular weight antioxidants, among others [27]. In light of such variability and inconsistency, we question the validity of the TBA test as an indicator of changes in peroxidative damage during aging. The pattern of age-related changes in the level of inorganic peroxides observed here is in sharp contrast to the approximately 4-fold increase reported by Armstrong et ai. [29] in D. melanogaster. These authors also reported a decrease in the activity of peroxidase with age in D. melanogaster. To our knowledge the validity of their assay procedure for inorganic peroxides has not been validated in biological tissues. As pointed out by Ahmed et al. [23], the methodology used by Armstrong et ai. suggests that the reported peroxidase activity is unrelated to a GSH-dependent peroxidase and is probably due to horseradish-like peroxidase. In conclusion, it is now becoming increasingly clear that the pattern of agerelated changes in antioxidant enzymes varies in different species. Some of the reported differences most probably arise from variations in procedures, sampling

234

and strains. It can be argued that lack of a uniform pattern in different species militates against the role of diminished antioxidant defenses being a significant contributor to aging. Nevertheless, in most species a selective decline in antioxidant defenses does seem to occur during aging as well as a selective increase in the level of oxidative stress indicators. The crucial point is that there tends to be an overall increase of oxidative stress with age. ACKNOWLEDGMENTS

This research was supported by the grant RO1 AG8459 f r o m N . I . H . - N . I . A . REFERENCES

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R.S. Sohal, The free radical theory of aging: a critique. In M. Rothstein (ed.), Rev&w o f Biological Research inAging, Vol. 3, Alan R. Liss, New York, 1987, pp. 385--415. R.S. Sohal and R.G. Allen, Oxidative stress as a causal factor in differentiation and aging: a unifying hypothesis. Exp. Geront., 25 (in press). G. Rao, E. Xia and A. Richardson, Effect of age on the expression of antioxidant enzymes in male Fisher F344 rats. Mech. Ageing Dev., 53 (1990) 49--60. K.A. Ansari, E. Kaplan and D. Shoeman, Age-related changes in lipid peroxidation and protective enzymes in the central nervous system. Growth Dev. Aging, 53 (1989) 117--121. H.P. Misra and I. Fridovich, The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutases. J. Biol. Chem., 247 (1972) 3170--3175. R.G. Allen, K.J, Farmer, R.K. Newton and R.S. Sohal, Effects of paraquat administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutase, catalase, glutathione reductase, inorganic peroxides and glutathione in the adult housefly. Comp. Biochem. Physiol., 78C (1984) 283--288. H. Luck, Catalase. In H. Bergmeyer (ed.), Method o f Enzymatic Analysis, Academic Press, New York, 1965, pp. 885--894. D.E. Paglia and W.N. Valentine, Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med., 70 (1967) 158--169. E. Beutler, Red cell metabolism, Grune and Stratton, New York, 1971. I. Carlberg and B. Mannervik, Glutathione reductase. Methods Enzymol., 113 (1985) 484--490. F. Tietze, Enzymatic methods for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues. Anal. Biochem., 27 (1969) 502--522. E. Bernt and H.U. Bergmeyer, Inorganic peroxides. In H.V. Bergmeyer (ed.), Methods o f Enzymatic Analysis, Vol. IV, Academic Press, New York, 1976, pp. 2246--2248. M. Ushiyama and M. Mihara, Determination of malondialdehyde precursors in tissues by thiobarbituric acid test. Anal. Biochem., 86 (i 978) 271--278. S. Pinder, J.B. Clark and A.L. Greenbaum, Estimation of nicotinic acid mononucleotide, nicotinamide mononucleotide, and diamide-NAD formed from radioactive precursors. Methods Enzymol., 58 (1977) 24--27. H. Donato, M.A. Hoselton and R.S. Sohal, An analysis of the effects of individual variation and selective mortality on population averages in aging populations. Exp. Gerontol., 14 (1979) 133-140. M. Sagai and T. lshinose, Age-related changes in lipid peroxidation as measured by ethane, ethylene, butane and pentane in respired gases of rats. Life Sci., 27 (1980) 731--738. R.S. Sohal, A. Muller, B. Koletzko and H. Sies, Effect of age and ambient temperature on npentane production in adult housefly, Musca domestica. Mech. Ageing Dev., 29 (1985) 317--326.

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