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Effect of ascorbic acid on longevity and biochemical alterations in Callosobruchus maculatus F. (Coleoptera: Bruchidae)
Archives of Gerontology and Geriatrics 18 (1994) 149-157
ELSEVIER SCIENCE IRELAND
ARCHIVES OF GERONTOLOGY AND GERIATRICS
Effect of ascorbic acid on longevity and biochemical alterations in Callosobruchus maculatus F. (Coleoptera: Bruchidae) S.K. Garg*, S. Mahajan Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143 005, India
(Received 30 September 1993; accepted 8 February 1994)
Abstract
Optimal ascorbic acid concentration (1 mM) increased the median (LTs0) and maximum (LTt00) life spans, decreased age-independent susceptibility to death (a0), reproductive period, number of eggs laid/female but prolonged the post-reproductive period in Callosobruchus maculatus. The activities of respiratory enzymes and the levels of metabolic end-products declined while the activities of antioxygenic enzymes increased. The increased longevity of insects reared on ascorbic acid soaked seeds may be interpreted in terms of conservation of energy by way of decreased reproductive potentiality and the maintenance of a homeostatic balance between pro-oxidant generation and antioxidant defences. Key words." Ascorbic acid; Callosobruchus maculatus; Respiratory enzymes; Antioxygenic enzymes; Metabolic end-products
1. Introduction
Nutrition is an important extrinsic factor involved in the generation of free radicals which cause time-related deterioration of body functions. Antioxidants are most effective if given during development (Neigdauz and Ravin, 1984) and increase the longevity of an organism (Holliday, 1989). Ascorbic acid has long been debated to be essential for health and longevity (Tappel, 1968). Non-feeding insects may pro* Corresponding author. 0167-4943/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0167-4943(94)00541-E
150
S.K Garg, S. Mahajan/ Arch. Gerontol. Geriatr. 18 (1994) 149-157
vide a variant model for the mode of action of ascorbic acid. Callosobruchus maculatus, a southern cow-pea weevil, starts its life cycle with the laying of eggs immediately after emergence on the seeds of Vigna radiata (vern. mung). The embryo settles and feeds inside the endosperm and the emerging adult leads a non-feeding life. It would, therefore, be imperative to study if the impact of free radicals generated or, as a corollary, scavenging of these radicals by antioxidants during development, is passed to the adult phase, thereby altering the physiology of the organism. The present investigation was aimed at revealing the effect of L-ascorbic acid on longevity, fecundity, respiratory (NADH-dehydrogenase, succinate dehydrogenase (SDH), NADH-oxidase and ATPase) and antioxygenic (catalase, peroxidase and glutathione reductase (GR)) enzymes and metabolic end-products (inorganic and lipid peroxides) in C. maculatus. 2. Materials and methods
Fresh V. radiata seeds were soaked in different concentrations of ascorbic acid (0, 0.1, 0.5, 1, 50, 100 and 250 mM) for 6 h, dried in shade and infested with pure cultures. Adults emerging from these seeds were used for life span and fecundity studies in three sets of experiments as per procedure described earlier (Mahajan and Garg, 1992). Ascorbic acid concentration (1 mM), most effective for longevity and fecundity of the insect, was chosen to observe its effect on NADH-dehydrogenase (King and Howard, 1967), SDH (King, 1967), NADH-oxidase (Mackler, 1967), ATPase (Kielley, 1969), catalase (Kar and Mishra, 1976), peroxidase (Kumar and Khan, 1983), GR (Tyson et al., 1982), inorganic peroxides (Bernt and Bergrneyer, 1976) and lipid peroxides (Hasan and Ali, 1981) in whole body and mitochondrial homogenates at 24 h intervals. The protein content was estimated following Lowry et al. (1951) and the specific activity of the enzymes was calculated as enzyme units/mg protein. The experiment was repeated four times to confirm reproducibility and the data were analyzed employing Student's t-test. 3. Results
The life span and life expectancy (ex) were higher and age independent susceptibility to death (a0) was lower in males infesting unsoaked seeds than those in females. Egg laying occurred until 5.3 days with a fecundity of 47 eggs/female. Ascorbic acid (0.5 and 1 mM) increased the LT50 in both sexes, whereas 50 mM concentration enhanced only the median life span of female insects. All other concentrations decreased the life span as compared to the control cohorts. The increase in LTs0 and LTl00 values at optimal (1 mM) concentration was more pronounced in females (72.8% and 35.3%) than in males (19.6% and 5.8%) and was accompanied by increased e x and post-reproductive period but decreased a0, number of eggs/ female and reproductive period (Table 1). The specific activity of NADH-dehydrogenase declined from the 4th day onwards in whole body homogenates of unsoaked males while the decline occurred until the 6th day in females. In mitochondria, its activity increased in males. SDH activity in whole body homogenates was low at most of the age intervals in males but remained
5.9
2.70 4- 0.30
10.40 ± 0.30
8.30 ± 0.30
88.80 + 1.60
-0.98 4- 0.20
8.85
18.7 ± 0.70
8.10 4- 0.20
-1.64 ± 0.06
8.97
17.30 ± 0.70
9.20 4- 0.30
soaked
Water
6.30 ± 0.50***
7.70 ± 0.30
88.80 + 1.80
-0.84 ± 0.01
6.60
14.00 4- 0.60**
6.10 ± 0.60*
- I . 0 8 4- 0.08
7.21
14.30 4- 1.70
8.00 ± 0.20*
0.1
14.80 -u 0.30***
7.70 ± 0.30
18.30 + 0.00"**
7.00 ± 0.00"*
46.10 4- 1.10"**
51.40 ± 3.30***
14.29 -1.68 ± 0.10"
9.91 -0.92 ± 0.10
14.00 ± 0.50*** 25.30 ± 0.30***
11.10 -1.91 ± 0.07*
10.04 - I . 4 4 ± 0.01"
8.90 ± 0.20*
18.30 ± 0.30
22.70 ± 0.30**
11.00 ± 0.30*
17.50 ± 0.30
1
10.40 ± 0.20*
0.5
Ascorbic acid (mM) soaked
Significantly different from control at *P < 0.05, **P < 0.01, ***P < 0.001.
Mean 4- SE of three experiments
period (days)
Post-reproductive
period (days)
5.30 ± 0.30
47.00 ± 1.40
Number of eggs
laid/female Reproductive
4.3
-0.58 ± 0.05
ao
8.00 ± 0.00
LTi0o (days)
ex (days)
4.50 4- 0.20
LTso (days)
Female
-1.07 ± 0.03
ex (days)
ao
6.1 ± 0.20
10.1 ± 0.00
LTi0o (days)
Unsoaked
LTso (days)
Male
Treatment
tive period and post-reproductive period in C. maculatus
9.00 + 0.00"*
8.00 ± 0.00
52.00 4- 1.40"**
- I . 3 6 4- 0.04
8.45
17.00 4- 0.60
9.00 ± 0.20*
- I . 4 8 -4- 0.07
9.04
14.50 ± 0.30*
9.60 ± 0.40
50
8.30 ± 0.30**
7.70 ± 0.30
68.00 ± 1.10"**
-0.90 4- 0.10
6.80
16.00 4- 0.60*
6.30 ± 0.30**
-1.37 ± 0A4
8.24
14.00 + 0.60*
8.80 ± 0.30
100
7.30 ± 0.30***
7.70 ± 0.30
38.80 4- 3.90***
-0.89 ± 0.05
6.75
15.00 4- 0.60*
6.00 ± 0.30**
-1.02 ± 0.02***
7.19
13.70 ± 0.30**
7.80 ± 0.50
250
Table 1 Effect of ascrobic acid on median (LTs0) and maximum (LTi0o) life spans, life expectancy (ex), age-independent susceptibility to death (ao), mean number o f eggs laid/female, reproduc-
.m
ATPase
50.2 40.0 43.0 42.5 37.8 32.1 42.0
56.1 58.7 54.0 38.8 41.6 41.8 40.8
± ± ± ± ± ± ±
± ± ± ± ± ± ±
1.6 2.4* 2.0* 2.2* 1.1"** 2.4*** 4.1
4.3 2.9 2.9 5.2* 3.3* 3.1" 3.2*
38.5 35.2 42.7 35.9 32.7 35.6 36.6
39.1 35.2 36.9 34.5 31.5 30.8 30.8
± ± ± ± ± ± ±
+ ± ± ± ± ± ±
1.6 2.0 1.6 1.5 1.3" 2.0 2.5
0.7 0.5** 1.6 1.5" 2.7* 2.7* 2.1"
13.9 29.1 14.7 9.7 8.4 12.1 9.4
13.7 18.7 30.1 25.8 19.8 11.2 13.2
± ± ± ± ± ± ±
+ ± ± ± ± ± ±
1.1 2.1"** 1.6 0.4* 1.0" 1.2 0.7*
0.5 1.3" 1.4'** 2.0** 0.9*** 0.6* 0.9
9.2 9.5 9.5 7.9 7.8 7.2 7.2
6.5 7.5 7.4 8.0 6.7 6.7 8.5
± ± ± ± ± ± ±
+ ± ± ± ± ± ±
0.6 1.3 0.8 0.2 0.6 0.3* 0.4*
0.6 0.4 0.7 0.5 0.3 0.4 1.4
309.9 105.8 141.0 82.2 129.9 60.9 74.1
58.6 72.9 78.5 76.1 70.0 66.9 77.4
± ± ± ± ± ± ±
+ ± ± ± ± ± ±
23.6 6.9*** 11.3"** 4.0*** 7.8*** 0.4*** 2.0***
3.7 1.6' 5.0* 3.3* 2.4* 5.0 4.1"
701.0 232.6 378.2 188.9 292.3 182.0 202.2
150.1 178.2 178.6 144.3 121.3 115.5 82.9
± ± ± ± ± ± ±
± ± ± ± ± ± ±
32.3 12.9"** 13.6"** 6.5*** 8.0*** 10.8"** 6.7***
7.1 10.0 9.9 12.7 3.9* 5.0** 7.9***
Succinate dehydrogenase
NADHdehydrogenase
NADH-oxidase
NADHdehydrogenase
Succinate dehydrogenase
Mitochondrial homogenates
Whole body h o m o g e n a t e s
70.1 69.2 59.7 40.0 37.4 30.6 34.2
17.6 41.5 22.7 28.2 18.4 18.3 15.7
± ± ± ± ± ± ±
± ± ± ± ± ± ±
3.3 5.5 5.4 1.4"** 3.4*** 3.8*** 3.1"**
0.7 l.l*** 2.8 1.4"* 2.0 1.2 1.0
NADH-oxidase
13.4 18.7 14.8 8.9 10.3 10.9 8.8
13.6 15.9 12.6 I 1.5 14.1 11.3 9.7
± ± ± ± ± ± ±
± ± ± ± ± ± ±
0.6 0.5* 0.2 1.0 0.5 0.2* 0.8**
0.4 0.7** 0.5 0.5*** 0.6** 0.6* 0.5***
ATPase
Mean + S.E. of four experiments. Specific activity = n u m b e r of enzyme units ( N A D H - d e h y d r o g e n a s e and succinate dehydrogenase: # M p o t a s s i u m ferricyanide reduced/100 rag/60 min at 30°C; N A D H - o x i d a s e : o.M N A D H oxidized/100 mg/60 min at 38°C; ATPase: tzM inorganic p h o s p h a t e liberated/100 mg/10 min at 28°C)/mg protein. Significantly different as c o m p a r e d to the value on 1st day in c o l u m n at *P < 0.05, **P < 0.01, ***P < 0.001.
1 2 3 4 5 6 7
Female
1 2 3 4 5 6 7
Male
Age (days)
Table 2 Specific activity of respiratory enzymes in ageing C. maculatus infesting unsoaked V. radiata seeds
"4
?
S,K. Garg, S. Maha]an/Arch. Gerontol. Geriatr. 18 (1994) 149-157
153
unaltered in females except for a decline on the 5th day. Both the sexes registered an age-dependent decline of SDH activity in the mitochondria, being earlier in females. Specific activity of NADH-oxidase increased during the reproductive period of ageing males emerging from unsoaked seeds while a decline was observed in females except for an increase on the 2nd day in whole body homogenates. Barring a decline on the 6th and 7th day in females, ATPase activity remained unaltered in whole body homogenates of both the sexes while in mitochondria, the level increased on the 2nd day and declined thereafter. The rise was, however, more and the decline was earlier in females than in males. Further, the activity of respiratory enzymes was higher in ageing females than in males and in mitochondria of the two sexes as compared to the whole body homogenates (Table 2). The specific activity of catalase increased on the 2nd day and declined on the 7th day in ageing males while it decreased after the 4th day in females. Higher activity of peroxidase was observed in ageing males. An elevation of GR activity was observed until the end of the reproductive period in females while the increase occurred only on the 2nd day in males. The levels of the inorganic peroxides in whole body homogenates decreased in males but increased in females with age (Table 3).
Table 3 Specific activity of ant±oxygenic enzymes, inorganic peroxide and lipid peroxide levels in whole body homogenates of ageing C. maculatus infesting unsoaked V. radiata seeds Age (days)
Catalase
Peroxidase
Glutathione reductase
Inorganic peroxide
Lipid peroxide
1
16.2 4. 0.6
4.1 4. 0.2
3.5 4. 0.2
34.8 4. 0.2
24.9 4- 0.7
2 3 4 5 6 7
18.4 17.0 16.1 15.8 14.9 12.5
4. 4. 4. 44. 4-
0.6* 0.8 0.4 0.6 0.7 0.6**
6.7 6.1 7.2 6.2 4.6 4.0
4. 4. 4444-
0.2*** 0.1"** 0.2*** 0.5** 0.4 0.3
5.0 4.0 3.4 3.8 3.4 2.8
4. 444. 44.
0.2** 0.1 0.1 0.1 0.4 0.1"
34.5 27.6 23.5 25.2 32.3 26.6
± 4. 4. 444.
1.4 0%*** 0A*** 1,4"** 1.3 1.0"**
23.7 25.6 27.6 26.2 26.9 27.4
4. 0.6 4. 0.8 4. 0.5* + 1.3 4- 1.3 -4- 2.1
16.7 15.5 15.5 14.0 11.5 11.9 11.7
4. 4. 44. 4. 44.
0.3 0.4 1.0 1.4 0.5*** 0.3*** 0.2***
6A 5,7 6.7 5.0 5.3 5.8 5.1
4. 44. 44. 4. 4.
0.2 0.1" 0.2 0.2** 0.1"* 0.3 0.2**
2.9 4.3 3.1 4.3 3.4 2.8 2.8
4. 0.04 + 0.3** 4- 0.05* -4- 0.3** 4. 0 . 1 " 4. 0.1 4. 0.08
15.1 11.8 11.3 21.4 26.2 30.6 30.6
± 4. 4. 44. 4. 4.
1.6 0.9 0.4 1.2" 1.6"* 1.3'** 0.6***
23.9 21.1 24.5 24.1 28.8 31.4 31.8
4. 444. 44. 4.
Male
Female 1 2 3 4 5 6 7
1.9 1.3 1.9 2.1 3.0 2.0* 2.0*
Mean 4. S.E. of four experiments. Specific activity = number of enzyme units (catalase:/ # M H 2 0 2 decomposed/100 mg/min at 25°C; peroxidase: #M purpurogallin formed/100 mg/min at 25°C; glutathione reductase: g g G S H p r o d u c e d / 1 0 0 mg/min at 30°C)/mg protein; inorganic peroxide: v g H 2 0 2 / 1 0 0 mg: lipid peroxide: n M M D A / 1 0 0 mg. Significantly different from the value on 1st day in column at * P < 0.05, **P < 0.01, ***P < 0.001.
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CON'~ROL
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o
o
a-g~ j-
assssssK.. ~ (N D"-4"-~r'M'M'~ I
~.~ I
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,-, p. E ~ E@ IZID ~ 3
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_LT~_g L gE_~- 4,- -~'-
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17~1
S.K Garg, S. Mahajan/Arch. Gerontol. Geriatr. 18 (1994) 149-157 100
155
B,[~2]
MALE
BO
~3
~,
~9
[~,0
Bs
6O 40
Age
~6 (d~y~)
20 0 t--
g
-20
U
-z, 0 -60 FEMALE
160 u.l
120 100 BO 6O
p
D
40 2O 0 o
•
-20 -/-,0 A
B
C
D
E
Fig. 2. Effect of ascorbic acid (1 mM) on percent change in the specific activity of antioxygenic enzymes (A, catalase; B, peroxidase; C, glutathione reductase) and levels of metabolic end-products (D, inorganic peroxide; E, lipid peroxide) in whole body homogenates of ageing C. maculatus.
Ascorbic acid (1 mM) treatment largely decreased the level of respiratory enzymes, peroxidase, metabolic end-products and increased the activity of catalase and GR as compared to the control cohorts (Figs. 1, 2). 4. Discussion
The decreased longevity of females with respect to males emerging from unsoaked seeds is attributable to their increased metabolic rate during reproductive periods, thereby leading to higher oxidative stresses. The increased life of insects reared on ascorbic acid (1 mM) soaked seeds may be due to its free radical quenching nature as reported by Rose (1990) in colonic epithelium exposed to ionizing radiations. Further, the energy conserved by way of decreased egg production and reproductive period at optimal ascorbic acid concentration could have resulted in the accumulation of reserves and made them available for the rest of life, thereby increasing Ion-
156
S.K. Garg, S. Malu(jan / Arch. Gerontol. Geriatr. 18 (1994) 149-157
gevity. The above view finds support from Begon and Mortimer (1986), who opined that a shift in the energy balance from reproductive period to the maintenance of body homeostasis contributed to the overall longevity of an organism. Age-related decline in dehydrogenase activities of insects infesting unsoaked seeds might be due to decreased reducing equivalents (Devlin, 1982). The pronounced decline in the enzyme activity in females could be due their initial high metabolic rate; thereby resulting in shorter life span as compared to that of males as already observed in Caryedon serratus (Garg et al., 1990). The increased NADHdehydrogenase activity in mitochondria of ageing males as compared to that of females is suggestive of less mitochondrial damage due to decreased oxidative stresses in the former (Garg and Mahajan, 1993). The increased NADH-oxidase activity in males could be referred to their higher catalase and decreased inorganic peroxide levels than in females. Catalase has been reported to protect NADH-oxidase in Trichomonas vaginalis (Linstead and Bradley, 1988). The unaltered lipid peroxidation during ageing in whole body homogenates might have resulted in consistent ATPase activity (Gilbert and Sawas, 1983). The decline in ATPase activity in females on the 6th and 7th day is accompanied by correspondingly increased lipid peroxidation. The increased mitochondrial ATPase activity during reproduction may be attributed to high energy demands at this time (Bains et al., 1990). Higher catalase and peroxidase activity in males may be responsible for their longer life span as compared to the females. The redox state of the cells during the reproductive period is maintained by a rise in GR activity as has also been reported by Bains et al. (1992) in Z. paravittiger. Age-related changes in inorganic peroxides are in accordance with Sharma et al. (1992), who observed higher inorganic peroxides in shorter lived males than in longer lived females of Z. paravittiger. The decreased activity of dehydrogenases with ascorbic acid might be due to the slowing down of metabolic machinery (Emanuel and Obukhova, 1978) consequent to reduced oxygen consumption (Gabibov, 1986) on antioxidant treatment. Similar to the inhibitory effect of nordihydroguaiaretic acid on mitochondriai NADHoxidase (Pardini et al., 1970), the activity of the enzyme decreased with ascorbic acid. Low ATPase activity in ascorbic acid-treated insects is similar to the observation on propyl gallate fed Z. paravittiger (Bains et al., 1990). The decline in peroxidase activity with ascorbic acid supports Sohal and Allen (1986), who suggested that organisms establish a homeostatic balance between pro-oxidant generation and antioxidant levels. Low level of pro-oxidant generated consequent to decreased dehydrogenases and their decomposition by elevated GR might have resulted in decreased inorganic and lipid peroxides in asorbic acid-treated C. maculatus. 5. References Bains, J.S., Khanna, S.C., Garg, S.K. and Sharma, S.P. (1990): Age-related analysis of adenosine triphosphatase activity as affected by propyl gallate in the Drosophilid, Zaprionus paravitti,ger. Insect Sci. Appl., 11,865-867. Bains, J.S., Sharma, S.P. and Garg, S.K. (1992): Effect of propyl gallate feeding on glutathione content in ageing Zaprionus paravittiger (Diptera). Gerontology, 38, 192-195. Begon, M. and Mortimer, M. (1986): in: Population Ecology, 2rid Edn. Sinauer Associates, USA.
S.K. Garg, S. Mahajan/Arch. Gerontol. Geriatr. 18 (1994) 149-157
157
Bernt, E. and Bergrneyer, H.U. (1976): Inorganic peroxides. In: Methods in Enzymatic Analysis, Vol. 4, pp. 2246-2248. Editor: H.U. Bergmeyer. Academic Press, New York. Devlin, T.M. (1982): Bioenergetics and oxidative metabolism. In: Textbook of Biochemistry with Clinical Correlations, pp. 255-318. John Wiley and Sons, New York. Emanuel, N.M. and Obukhova, L.K. (1978): Types of experimental delay in ageing patterns. Exp. Gerontol., 13, 25-29. Gabibov, M.M. (1986): Effect of hyperbaric oxygenation on the proton ATPase activity of mitochondria from different rat tissues. UKR Biokhim. Zh., 58, 81-83. Garg, S.K. and Mahajan, S. (1993): Effect of ascorbic acid on longevity, catalase and lipid peroxidation in Callosobruchus maculatus F. Age, 16, 87-92. Garg, S.K., Mahajan, S. and Sharma, S.P. (1990): Variations in the life span, enzymes and lipid peroxide levels in ageing Caryedon serratus (Coleoptera: Bruchidae). Gerontology, 36, 126-131. Gilbert, J.C. and Sawas, A.H. (1983): ATPase activities and lipid peroxidation in rat cerebral cortex synaptosomes. Arch. Int. Pharmacol. Ther., 263, 189-195. Hasan, M. and All, S.F. (1981): Effects of thallium, nickel and cobalt administration on lipid peroxidation in different regions of the rat brain. Toxicol. Appl. Pharmacol., 57, 8-13. Holliday, R. (1989): Food, reproduction and longevity: Is the extended life span of calorie-restricted animals an evolutionary adaptation? Bio Essay, 10, 125-127. Kar, M. and Mishra, D.B. (1976): Catalase, peroxidase and polyphenoloxidase activities during rice leaf senescence. Plant Physiol., 57, 315-319. Kielley, W.W. (1969): Mg ÷2 activated muscle ATPase. Methods Enzymol., 2, 588-591. King, T.E. (1967): Preparation of succinate dehydrogenase and reconstitution of succinate oxidase. Methods Enzymol., 10, 322-332. King, T.E. and Howard, R.L. (1967): Preparation and properties of soluble NADH-dehydrogenase from cardiac muscle. Methods Enzymol,, 10, 275-294. Kumar, K.B. and Khan, P.A. (1983): Age-related changes in catalase and peroxidase in the excised leaves of Eleusine coracana Gaetrn. CV PR 202 during senescence. Exp. Gerontol.. 18, 409-417. Linstead, D.J. and Bradley, S. (1988): The purification and properties of two soluble nicotinamide nucleotides: Acceptor oxidoreductases from Trichomonas vaginalis. Mol. Biochem. Parasitol., 27, 125-134. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951): Protein measurement with Folinphenol reagent. J. Biol. Chem., 193, 265-275. Mackler, B. (1967): DPNH-oxidase of heart muscle. Methods Enzymol., 10, 261-263. Mahajan, S. and Garg, S.K. (1992): Life prolonging effect of butylated hydroxy anisole in Callosobruchus maculatus F., (Coleoptera: Bruchidae). Arch. Gerontol. Geriatr., 15, 71-78. Neigdauz, B.M. and Ravin, V.K. (1984): The effect of physiologically active compounds on the life span of nematode, Caenorhabditis elegans. Zh. Biol., 44, 835-841. Pardini, R.S., Heidker, J.C. and Fletcher, D.C. (1970): Inhibition of mitochondrial electron transport by nordihydroguaiaretic acid (NDGA). Biochem. Pharmacol., 19, 2695-2699. Rose, R.C. (1990): Ascorbic acid metabolism in protection against free radicals: A radiation model. Biochem. Biophys. Res. Commun., 169, 430-436. Sharma, S.P., Bains, J.S. and Garg, S.K. (1992): Peroxide levels in ageing Zaprionus paravittiger on propyl gallate feeding. Age, 15, 45-46. Sohal, R.S. and Allen, R.G. (1986): Relationship between oxygen metabolism, ageing and development. Adv. Free Rad. Biol. Med., 2, 117-160. Tappel, A.L. (1968): Will antioxidant nutrients slow ageing processes? Geriatrics, 23, 97-105. Tyson, C.A., Lunan, R.D. and Stephens, R.J. (1982): Age-related differences in GSH-shuttle enzymes in NO 2 or 02 exposed rat lungs. Arch. Environ. Health, 37, 167-176.
ELSEVIER SCIENCE IRELAND
ARCHIVES OF GERONTOLOGY AND GERIATRICS
Effect of ascorbic acid on longevity and biochemical alterations in Callosobruchus maculatus F. (Coleoptera: Bruchidae) S.K. Garg*, S. Mahajan Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143 005, India
(Received 30 September 1993; accepted 8 February 1994)
Abstract
Optimal ascorbic acid concentration (1 mM) increased the median (LTs0) and maximum (LTt00) life spans, decreased age-independent susceptibility to death (a0), reproductive period, number of eggs laid/female but prolonged the post-reproductive period in Callosobruchus maculatus. The activities of respiratory enzymes and the levels of metabolic end-products declined while the activities of antioxygenic enzymes increased. The increased longevity of insects reared on ascorbic acid soaked seeds may be interpreted in terms of conservation of energy by way of decreased reproductive potentiality and the maintenance of a homeostatic balance between pro-oxidant generation and antioxidant defences. Key words." Ascorbic acid; Callosobruchus maculatus; Respiratory enzymes; Antioxygenic enzymes; Metabolic end-products
1. Introduction
Nutrition is an important extrinsic factor involved in the generation of free radicals which cause time-related deterioration of body functions. Antioxidants are most effective if given during development (Neigdauz and Ravin, 1984) and increase the longevity of an organism (Holliday, 1989). Ascorbic acid has long been debated to be essential for health and longevity (Tappel, 1968). Non-feeding insects may pro* Corresponding author. 0167-4943/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0167-4943(94)00541-E
150
S.K Garg, S. Mahajan/ Arch. Gerontol. Geriatr. 18 (1994) 149-157
vide a variant model for the mode of action of ascorbic acid. Callosobruchus maculatus, a southern cow-pea weevil, starts its life cycle with the laying of eggs immediately after emergence on the seeds of Vigna radiata (vern. mung). The embryo settles and feeds inside the endosperm and the emerging adult leads a non-feeding life. It would, therefore, be imperative to study if the impact of free radicals generated or, as a corollary, scavenging of these radicals by antioxidants during development, is passed to the adult phase, thereby altering the physiology of the organism. The present investigation was aimed at revealing the effect of L-ascorbic acid on longevity, fecundity, respiratory (NADH-dehydrogenase, succinate dehydrogenase (SDH), NADH-oxidase and ATPase) and antioxygenic (catalase, peroxidase and glutathione reductase (GR)) enzymes and metabolic end-products (inorganic and lipid peroxides) in C. maculatus. 2. Materials and methods
Fresh V. radiata seeds were soaked in different concentrations of ascorbic acid (0, 0.1, 0.5, 1, 50, 100 and 250 mM) for 6 h, dried in shade and infested with pure cultures. Adults emerging from these seeds were used for life span and fecundity studies in three sets of experiments as per procedure described earlier (Mahajan and Garg, 1992). Ascorbic acid concentration (1 mM), most effective for longevity and fecundity of the insect, was chosen to observe its effect on NADH-dehydrogenase (King and Howard, 1967), SDH (King, 1967), NADH-oxidase (Mackler, 1967), ATPase (Kielley, 1969), catalase (Kar and Mishra, 1976), peroxidase (Kumar and Khan, 1983), GR (Tyson et al., 1982), inorganic peroxides (Bernt and Bergrneyer, 1976) and lipid peroxides (Hasan and Ali, 1981) in whole body and mitochondrial homogenates at 24 h intervals. The protein content was estimated following Lowry et al. (1951) and the specific activity of the enzymes was calculated as enzyme units/mg protein. The experiment was repeated four times to confirm reproducibility and the data were analyzed employing Student's t-test. 3. Results
The life span and life expectancy (ex) were higher and age independent susceptibility to death (a0) was lower in males infesting unsoaked seeds than those in females. Egg laying occurred until 5.3 days with a fecundity of 47 eggs/female. Ascorbic acid (0.5 and 1 mM) increased the LT50 in both sexes, whereas 50 mM concentration enhanced only the median life span of female insects. All other concentrations decreased the life span as compared to the control cohorts. The increase in LTs0 and LTl00 values at optimal (1 mM) concentration was more pronounced in females (72.8% and 35.3%) than in males (19.6% and 5.8%) and was accompanied by increased e x and post-reproductive period but decreased a0, number of eggs/ female and reproductive period (Table 1). The specific activity of NADH-dehydrogenase declined from the 4th day onwards in whole body homogenates of unsoaked males while the decline occurred until the 6th day in females. In mitochondria, its activity increased in males. SDH activity in whole body homogenates was low at most of the age intervals in males but remained
5.9
2.70 4- 0.30
10.40 ± 0.30
8.30 ± 0.30
88.80 + 1.60
-0.98 4- 0.20
8.85
18.7 ± 0.70
8.10 4- 0.20
-1.64 ± 0.06
8.97
17.30 ± 0.70
9.20 4- 0.30
soaked
Water
6.30 ± 0.50***
7.70 ± 0.30
88.80 + 1.80
-0.84 ± 0.01
6.60
14.00 4- 0.60**
6.10 ± 0.60*
- I . 0 8 4- 0.08
7.21
14.30 4- 1.70
8.00 ± 0.20*
0.1
14.80 -u 0.30***
7.70 ± 0.30
18.30 + 0.00"**
7.00 ± 0.00"*
46.10 4- 1.10"**
51.40 ± 3.30***
14.29 -1.68 ± 0.10"
9.91 -0.92 ± 0.10
14.00 ± 0.50*** 25.30 ± 0.30***
11.10 -1.91 ± 0.07*
10.04 - I . 4 4 ± 0.01"
8.90 ± 0.20*
18.30 ± 0.30
22.70 ± 0.30**
11.00 ± 0.30*
17.50 ± 0.30
1
10.40 ± 0.20*
0.5
Ascorbic acid (mM) soaked
Significantly different from control at *P < 0.05, **P < 0.01, ***P < 0.001.
Mean 4- SE of three experiments
period (days)
Post-reproductive
period (days)
5.30 ± 0.30
47.00 ± 1.40
Number of eggs
laid/female Reproductive
4.3
-0.58 ± 0.05
ao
8.00 ± 0.00
LTi0o (days)
ex (days)
4.50 4- 0.20
LTso (days)
Female
-1.07 ± 0.03
ex (days)
ao
6.1 ± 0.20
10.1 ± 0.00
LTi0o (days)
Unsoaked
LTso (days)
Male
Treatment
tive period and post-reproductive period in C. maculatus
9.00 + 0.00"*
8.00 ± 0.00
52.00 4- 1.40"**
- I . 3 6 4- 0.04
8.45
17.00 4- 0.60
9.00 ± 0.20*
- I . 4 8 -4- 0.07
9.04
14.50 ± 0.30*
9.60 ± 0.40
50
8.30 ± 0.30**
7.70 ± 0.30
68.00 ± 1.10"**
-0.90 4- 0.10
6.80
16.00 4- 0.60*
6.30 ± 0.30**
-1.37 ± 0A4
8.24
14.00 + 0.60*
8.80 ± 0.30
100
7.30 ± 0.30***
7.70 ± 0.30
38.80 4- 3.90***
-0.89 ± 0.05
6.75
15.00 4- 0.60*
6.00 ± 0.30**
-1.02 ± 0.02***
7.19
13.70 ± 0.30**
7.80 ± 0.50
250
Table 1 Effect of ascrobic acid on median (LTs0) and maximum (LTi0o) life spans, life expectancy (ex), age-independent susceptibility to death (ao), mean number o f eggs laid/female, reproduc-
.m
ATPase
50.2 40.0 43.0 42.5 37.8 32.1 42.0
56.1 58.7 54.0 38.8 41.6 41.8 40.8
± ± ± ± ± ± ±
± ± ± ± ± ± ±
1.6 2.4* 2.0* 2.2* 1.1"** 2.4*** 4.1
4.3 2.9 2.9 5.2* 3.3* 3.1" 3.2*
38.5 35.2 42.7 35.9 32.7 35.6 36.6
39.1 35.2 36.9 34.5 31.5 30.8 30.8
± ± ± ± ± ± ±
+ ± ± ± ± ± ±
1.6 2.0 1.6 1.5 1.3" 2.0 2.5
0.7 0.5** 1.6 1.5" 2.7* 2.7* 2.1"
13.9 29.1 14.7 9.7 8.4 12.1 9.4
13.7 18.7 30.1 25.8 19.8 11.2 13.2
± ± ± ± ± ± ±
+ ± ± ± ± ± ±
1.1 2.1"** 1.6 0.4* 1.0" 1.2 0.7*
0.5 1.3" 1.4'** 2.0** 0.9*** 0.6* 0.9
9.2 9.5 9.5 7.9 7.8 7.2 7.2
6.5 7.5 7.4 8.0 6.7 6.7 8.5
± ± ± ± ± ± ±
+ ± ± ± ± ± ±
0.6 1.3 0.8 0.2 0.6 0.3* 0.4*
0.6 0.4 0.7 0.5 0.3 0.4 1.4
309.9 105.8 141.0 82.2 129.9 60.9 74.1
58.6 72.9 78.5 76.1 70.0 66.9 77.4
± ± ± ± ± ± ±
+ ± ± ± ± ± ±
23.6 6.9*** 11.3"** 4.0*** 7.8*** 0.4*** 2.0***
3.7 1.6' 5.0* 3.3* 2.4* 5.0 4.1"
701.0 232.6 378.2 188.9 292.3 182.0 202.2
150.1 178.2 178.6 144.3 121.3 115.5 82.9
± ± ± ± ± ± ±
± ± ± ± ± ± ±
32.3 12.9"** 13.6"** 6.5*** 8.0*** 10.8"** 6.7***
7.1 10.0 9.9 12.7 3.9* 5.0** 7.9***
Succinate dehydrogenase
NADHdehydrogenase
NADH-oxidase
NADHdehydrogenase
Succinate dehydrogenase
Mitochondrial homogenates
Whole body h o m o g e n a t e s
70.1 69.2 59.7 40.0 37.4 30.6 34.2
17.6 41.5 22.7 28.2 18.4 18.3 15.7
± ± ± ± ± ± ±
± ± ± ± ± ± ±
3.3 5.5 5.4 1.4"** 3.4*** 3.8*** 3.1"**
0.7 l.l*** 2.8 1.4"* 2.0 1.2 1.0
NADH-oxidase
13.4 18.7 14.8 8.9 10.3 10.9 8.8
13.6 15.9 12.6 I 1.5 14.1 11.3 9.7
± ± ± ± ± ± ±
± ± ± ± ± ± ±
0.6 0.5* 0.2 1.0 0.5 0.2* 0.8**
0.4 0.7** 0.5 0.5*** 0.6** 0.6* 0.5***
ATPase
Mean + S.E. of four experiments. Specific activity = n u m b e r of enzyme units ( N A D H - d e h y d r o g e n a s e and succinate dehydrogenase: # M p o t a s s i u m ferricyanide reduced/100 rag/60 min at 30°C; N A D H - o x i d a s e : o.M N A D H oxidized/100 mg/60 min at 38°C; ATPase: tzM inorganic p h o s p h a t e liberated/100 mg/10 min at 28°C)/mg protein. Significantly different as c o m p a r e d to the value on 1st day in c o l u m n at *P < 0.05, **P < 0.01, ***P < 0.001.
1 2 3 4 5 6 7
Female
1 2 3 4 5 6 7
Male
Age (days)
Table 2 Specific activity of respiratory enzymes in ageing C. maculatus infesting unsoaked V. radiata seeds
"4
?
S,K. Garg, S. Maha]an/Arch. Gerontol. Geriatr. 18 (1994) 149-157
153
unaltered in females except for a decline on the 5th day. Both the sexes registered an age-dependent decline of SDH activity in the mitochondria, being earlier in females. Specific activity of NADH-oxidase increased during the reproductive period of ageing males emerging from unsoaked seeds while a decline was observed in females except for an increase on the 2nd day in whole body homogenates. Barring a decline on the 6th and 7th day in females, ATPase activity remained unaltered in whole body homogenates of both the sexes while in mitochondria, the level increased on the 2nd day and declined thereafter. The rise was, however, more and the decline was earlier in females than in males. Further, the activity of respiratory enzymes was higher in ageing females than in males and in mitochondria of the two sexes as compared to the whole body homogenates (Table 2). The specific activity of catalase increased on the 2nd day and declined on the 7th day in ageing males while it decreased after the 4th day in females. Higher activity of peroxidase was observed in ageing males. An elevation of GR activity was observed until the end of the reproductive period in females while the increase occurred only on the 2nd day in males. The levels of the inorganic peroxides in whole body homogenates decreased in males but increased in females with age (Table 3).
Table 3 Specific activity of ant±oxygenic enzymes, inorganic peroxide and lipid peroxide levels in whole body homogenates of ageing C. maculatus infesting unsoaked V. radiata seeds Age (days)
Catalase
Peroxidase
Glutathione reductase
Inorganic peroxide
Lipid peroxide
1
16.2 4. 0.6
4.1 4. 0.2
3.5 4. 0.2
34.8 4. 0.2
24.9 4- 0.7
2 3 4 5 6 7
18.4 17.0 16.1 15.8 14.9 12.5
4. 4. 4. 44. 4-
0.6* 0.8 0.4 0.6 0.7 0.6**
6.7 6.1 7.2 6.2 4.6 4.0
4. 4. 4444-
0.2*** 0.1"** 0.2*** 0.5** 0.4 0.3
5.0 4.0 3.4 3.8 3.4 2.8
4. 444. 44.
0.2** 0.1 0.1 0.1 0.4 0.1"
34.5 27.6 23.5 25.2 32.3 26.6
± 4. 4. 444.
1.4 0%*** 0A*** 1,4"** 1.3 1.0"**
23.7 25.6 27.6 26.2 26.9 27.4
4. 0.6 4. 0.8 4. 0.5* + 1.3 4- 1.3 -4- 2.1
16.7 15.5 15.5 14.0 11.5 11.9 11.7
4. 4. 44. 4. 44.
0.3 0.4 1.0 1.4 0.5*** 0.3*** 0.2***
6A 5,7 6.7 5.0 5.3 5.8 5.1
4. 44. 44. 4. 4.
0.2 0.1" 0.2 0.2** 0.1"* 0.3 0.2**
2.9 4.3 3.1 4.3 3.4 2.8 2.8
4. 0.04 + 0.3** 4- 0.05* -4- 0.3** 4. 0 . 1 " 4. 0.1 4. 0.08
15.1 11.8 11.3 21.4 26.2 30.6 30.6
± 4. 4. 44. 4. 4.
1.6 0.9 0.4 1.2" 1.6"* 1.3'** 0.6***
23.9 21.1 24.5 24.1 28.8 31.4 31.8
4. 444. 44. 4.
Male
Female 1 2 3 4 5 6 7
1.9 1.3 1.9 2.1 3.0 2.0* 2.0*
Mean 4. S.E. of four experiments. Specific activity = number of enzyme units (catalase:/ # M H 2 0 2 decomposed/100 mg/min at 25°C; peroxidase: #M purpurogallin formed/100 mg/min at 25°C; glutathione reductase: g g G S H p r o d u c e d / 1 0 0 mg/min at 30°C)/mg protein; inorganic peroxide: v g H 2 0 2 / 1 0 0 mg: lipid peroxide: n M M D A / 1 0 0 mg. Significantly different from the value on 1st day in column at * P < 0.05, **P < 0.01, ***P < 0.001.
z~ •I.
CH&NGE
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CON'~ROL
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[o
CHANr~E o
0
6
r
o
~
o
'+ u +"
W R I o
o
o
CONTROL o
o
o
o
o
o
a-g~ j-
assssssK.. ~ (N D"-4"-~r'M'M'~ I
~.~ I
~.~ ~a
[~_
•
N
" q
I
Io
- ~ - ~ -~,- - ~ 4 - 4~-
N
o
-~- -~ -~
~.x~
e~ N
i
~z
L °
°
s~
°
~
{'r]
-6- ~ -9- d cxJy-4 k/"--d"--d
~i~:> ..~~ > . < - x x ~ . . < + >.< L r ' > . r ' - - d ' - - d ~ . . r'--._~_,
P~°I I+
.B
I°1
°
°
°
°
°
°
,-, p. E ~ E@ IZID ~ 3
+
_LT~_g L gE_~- 4,- -~'-
~ 7--
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(P661 / gl .tlt~!.t,~D '/OlUO.t,~D ll.~l"e" / uUl~ZlOl'V 'S "g.t°D "N'S
17~1
S.K Garg, S. Mahajan/Arch. Gerontol. Geriatr. 18 (1994) 149-157 100
155
B,[~2]
MALE
BO
~3
~,
~9
[~,0
Bs
6O 40
Age
~6 (d~y~)
20 0 t--
g
-20
U
-z, 0 -60 FEMALE
160 u.l
120 100 BO 6O
p
D
40 2O 0 o
•
-20 -/-,0 A
B
C
D
E
Fig. 2. Effect of ascorbic acid (1 mM) on percent change in the specific activity of antioxygenic enzymes (A, catalase; B, peroxidase; C, glutathione reductase) and levels of metabolic end-products (D, inorganic peroxide; E, lipid peroxide) in whole body homogenates of ageing C. maculatus.
Ascorbic acid (1 mM) treatment largely decreased the level of respiratory enzymes, peroxidase, metabolic end-products and increased the activity of catalase and GR as compared to the control cohorts (Figs. 1, 2). 4. Discussion
The decreased longevity of females with respect to males emerging from unsoaked seeds is attributable to their increased metabolic rate during reproductive periods, thereby leading to higher oxidative stresses. The increased life of insects reared on ascorbic acid (1 mM) soaked seeds may be due to its free radical quenching nature as reported by Rose (1990) in colonic epithelium exposed to ionizing radiations. Further, the energy conserved by way of decreased egg production and reproductive period at optimal ascorbic acid concentration could have resulted in the accumulation of reserves and made them available for the rest of life, thereby increasing Ion-
156
S.K. Garg, S. Malu(jan / Arch. Gerontol. Geriatr. 18 (1994) 149-157
gevity. The above view finds support from Begon and Mortimer (1986), who opined that a shift in the energy balance from reproductive period to the maintenance of body homeostasis contributed to the overall longevity of an organism. Age-related decline in dehydrogenase activities of insects infesting unsoaked seeds might be due to decreased reducing equivalents (Devlin, 1982). The pronounced decline in the enzyme activity in females could be due their initial high metabolic rate; thereby resulting in shorter life span as compared to that of males as already observed in Caryedon serratus (Garg et al., 1990). The increased NADHdehydrogenase activity in mitochondria of ageing males as compared to that of females is suggestive of less mitochondrial damage due to decreased oxidative stresses in the former (Garg and Mahajan, 1993). The increased NADH-oxidase activity in males could be referred to their higher catalase and decreased inorganic peroxide levels than in females. Catalase has been reported to protect NADH-oxidase in Trichomonas vaginalis (Linstead and Bradley, 1988). The unaltered lipid peroxidation during ageing in whole body homogenates might have resulted in consistent ATPase activity (Gilbert and Sawas, 1983). The decline in ATPase activity in females on the 6th and 7th day is accompanied by correspondingly increased lipid peroxidation. The increased mitochondrial ATPase activity during reproduction may be attributed to high energy demands at this time (Bains et al., 1990). Higher catalase and peroxidase activity in males may be responsible for their longer life span as compared to the females. The redox state of the cells during the reproductive period is maintained by a rise in GR activity as has also been reported by Bains et al. (1992) in Z. paravittiger. Age-related changes in inorganic peroxides are in accordance with Sharma et al. (1992), who observed higher inorganic peroxides in shorter lived males than in longer lived females of Z. paravittiger. The decreased activity of dehydrogenases with ascorbic acid might be due to the slowing down of metabolic machinery (Emanuel and Obukhova, 1978) consequent to reduced oxygen consumption (Gabibov, 1986) on antioxidant treatment. Similar to the inhibitory effect of nordihydroguaiaretic acid on mitochondriai NADHoxidase (Pardini et al., 1970), the activity of the enzyme decreased with ascorbic acid. Low ATPase activity in ascorbic acid-treated insects is similar to the observation on propyl gallate fed Z. paravittiger (Bains et al., 1990). The decline in peroxidase activity with ascorbic acid supports Sohal and Allen (1986), who suggested that organisms establish a homeostatic balance between pro-oxidant generation and antioxidant levels. Low level of pro-oxidant generated consequent to decreased dehydrogenases and their decomposition by elevated GR might have resulted in decreased inorganic and lipid peroxides in asorbic acid-treated C. maculatus. 5. References Bains, J.S., Khanna, S.C., Garg, S.K. and Sharma, S.P. (1990): Age-related analysis of adenosine triphosphatase activity as affected by propyl gallate in the Drosophilid, Zaprionus paravitti,ger. Insect Sci. Appl., 11,865-867. Bains, J.S., Sharma, S.P. and Garg, S.K. (1992): Effect of propyl gallate feeding on glutathione content in ageing Zaprionus paravittiger (Diptera). Gerontology, 38, 192-195. Begon, M. and Mortimer, M. (1986): in: Population Ecology, 2rid Edn. Sinauer Associates, USA.
S.K. Garg, S. Mahajan/Arch. Gerontol. Geriatr. 18 (1994) 149-157
157
Bernt, E. and Bergrneyer, H.U. (1976): Inorganic peroxides. In: Methods in Enzymatic Analysis, Vol. 4, pp. 2246-2248. Editor: H.U. Bergmeyer. Academic Press, New York. Devlin, T.M. (1982): Bioenergetics and oxidative metabolism. In: Textbook of Biochemistry with Clinical Correlations, pp. 255-318. John Wiley and Sons, New York. Emanuel, N.M. and Obukhova, L.K. (1978): Types of experimental delay in ageing patterns. Exp. Gerontol., 13, 25-29. Gabibov, M.M. (1986): Effect of hyperbaric oxygenation on the proton ATPase activity of mitochondria from different rat tissues. UKR Biokhim. Zh., 58, 81-83. Garg, S.K. and Mahajan, S. (1993): Effect of ascorbic acid on longevity, catalase and lipid peroxidation in Callosobruchus maculatus F. Age, 16, 87-92. Garg, S.K., Mahajan, S. and Sharma, S.P. (1990): Variations in the life span, enzymes and lipid peroxide levels in ageing Caryedon serratus (Coleoptera: Bruchidae). Gerontology, 36, 126-131. Gilbert, J.C. and Sawas, A.H. (1983): ATPase activities and lipid peroxidation in rat cerebral cortex synaptosomes. Arch. Int. Pharmacol. Ther., 263, 189-195. Hasan, M. and All, S.F. (1981): Effects of thallium, nickel and cobalt administration on lipid peroxidation in different regions of the rat brain. Toxicol. Appl. Pharmacol., 57, 8-13. Holliday, R. (1989): Food, reproduction and longevity: Is the extended life span of calorie-restricted animals an evolutionary adaptation? Bio Essay, 10, 125-127. Kar, M. and Mishra, D.B. (1976): Catalase, peroxidase and polyphenoloxidase activities during rice leaf senescence. Plant Physiol., 57, 315-319. Kielley, W.W. (1969): Mg ÷2 activated muscle ATPase. Methods Enzymol., 2, 588-591. King, T.E. (1967): Preparation of succinate dehydrogenase and reconstitution of succinate oxidase. Methods Enzymol., 10, 322-332. King, T.E. and Howard, R.L. (1967): Preparation and properties of soluble NADH-dehydrogenase from cardiac muscle. Methods Enzymol,, 10, 275-294. Kumar, K.B. and Khan, P.A. (1983): Age-related changes in catalase and peroxidase in the excised leaves of Eleusine coracana Gaetrn. CV PR 202 during senescence. Exp. Gerontol.. 18, 409-417. Linstead, D.J. and Bradley, S. (1988): The purification and properties of two soluble nicotinamide nucleotides: Acceptor oxidoreductases from Trichomonas vaginalis. Mol. Biochem. Parasitol., 27, 125-134. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951): Protein measurement with Folinphenol reagent. J. Biol. Chem., 193, 265-275. Mackler, B. (1967): DPNH-oxidase of heart muscle. Methods Enzymol., 10, 261-263. Mahajan, S. and Garg, S.K. (1992): Life prolonging effect of butylated hydroxy anisole in Callosobruchus maculatus F., (Coleoptera: Bruchidae). Arch. Gerontol. Geriatr., 15, 71-78. Neigdauz, B.M. and Ravin, V.K. (1984): The effect of physiologically active compounds on the life span of nematode, Caenorhabditis elegans. Zh. Biol., 44, 835-841. Pardini, R.S., Heidker, J.C. and Fletcher, D.C. (1970): Inhibition of mitochondrial electron transport by nordihydroguaiaretic acid (NDGA). Biochem. Pharmacol., 19, 2695-2699. Rose, R.C. (1990): Ascorbic acid metabolism in protection against free radicals: A radiation model. Biochem. Biophys. Res. Commun., 169, 430-436. Sharma, S.P., Bains, J.S. and Garg, S.K. (1992): Peroxide levels in ageing Zaprionus paravittiger on propyl gallate feeding. Age, 15, 45-46. Sohal, R.S. and Allen, R.G. (1986): Relationship between oxygen metabolism, ageing and development. Adv. Free Rad. Biol. Med., 2, 117-160. Tappel, A.L. (1968): Will antioxidant nutrients slow ageing processes? Geriatrics, 23, 97-105. Tyson, C.A., Lunan, R.D. and Stephens, R.J. (1982): Age-related differences in GSH-shuttle enzymes in NO 2 or 02 exposed rat lungs. Arch. Environ. Health, 37, 167-176.