effect of bha on longevity antioxygenic enzymes and peroxides in

The increased longevity with BHA could be attributed to its free radical quenching effect, thereby leading to the decreased peroxide levels and the increased antioxygenic e[r]

Pergamon

Experimental Gerontology, Vol 29, No 5, pp 58~591, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA All rights reserved 0531-5565/94 $6.00 + 00

0531-5565(94)E0025-W

EFFECT OF BHA ON LONGEVITY, ANTIOXYGENIC ENZYMES

AND PEROXIDES IN CALLOSOBRUCHUS MACULATUS

S MAHAJAN, S K GARG and T S LOBANA I

Department of Molecular Biology & Biochemistry, 1Department of Chemistry, Guru Nanak Dev University, Amritsar 143 005, India

A b s t r a c t - - L i f e span and antioxygenic enzyme activities of insects reared on optimal BHA concentration (1 mM) soaked seeds were higher than their respective controls However, the reproductive potential, peroxides and the level of free radicals de- clined The increased longevity with BHA could be attributed to its free radical quenching effect, thereby leading to the decreased peroxide levels and the increased antioxygenic enzyme activities of the insects

Key Words: C maculatus, longevity, antioxygenic enzymes, peroxides, BHA

I N T R O D U C T I O N

ANTIOXYGENIC ENZYMES and antioxidants are the natural defences against the delete- rious effects of oxygen and oxidative products However, despite the existence of such a system, free radicals accumulate as inorganic and lipid peroxides and their effect is accentuated in the presence of iron (Halliwell, 1981) Dietary antioxidants supplement the function of antioxygenic enzymes (Munkres and Rana, 1984) and are most effective during development (Neigdauz and Ravin, 1984) Further, the antioxidants inhibit lipid peroxidation by way of keeping the metal ions and other catalysts in the unreactive form, thereby increasing the life span (Gutteridge, 1982) Butylated hydroxyanisole (BHA) has been widely used as an antioxidant (Sims and Fioritti, 1980) Its use as food additive has been suggested as generally recognized as safe by Wattenberg (1985) However, the information regarding the effect of antioxidants on nonfeeding insects is

completely obscure C maculatus, a southern cow-pea weevil, starts its life cycle with

Correspondence to: S Mahajan, Department of Molecular Biology & Biochemistry, Amritsar 143 005, India

the laying of eggs immediately after emergence on the seeds of legumes Larvae after hatching from the eggs develop into an adult after settling and feeding inside the en- dosperm Feeding in the adult is nonexistent, thereby making its life span and repro- ductive potential dependent upon the nutrition derived during the developmental phases The present investigation was designed to study the hypothesis that free rad- icals generated during the larval phase transfer their impact to the adult organism and further that the effect of antioxidants given during the larval phase is passed on to the adult, thereby increasing the longevity of the organism by altering the physiological and biochemical parameters In view of this, the present report deals with the effect of BHA on longevity, fecundity, antioxygenic enzymes, peroxides, iron content, and free rad- icals in C maculatus

T A B L E E F F E C T OF BHA ON MEDIAN (LT5o) AND MAXIMUM (LTIoo) L I F E SPANS, A G E - I N D E P E N D E N T SUSCEPTIBILITY TO DEATH (ao) , L I F E EXPECTANCY (ex), MEAN NUMBER OF EGGS L A I D / F E M A L E AND

REPRODUCTIVE AND POSTREPRODUCTIVE PERIODS IN C m a c u l a l u s

BHA (raM)soaked Water Ethanol (10%)

Treatment Unsoaked soaked soaked O 0.25

Male

LTso (days) 6.1 ± 0.2 9.2 +- 0.6 8.3 -+ 0.03 8.6 ± t 9.9 ± 0.055

L T ] ~ (days) 10.0 -+ 0.0 t7.3 -+ 1.2 15.0 -+ 0.0 15.0 ± 1.0 16.7 -+ t

a o 1.07 -+ 0.03 - -+ 0.11 1.55 + 0.03 - -+ 0.06t - ± 0.12

e~ (days) 5.9 8,97 8.32 9.33 9.54

Female

LTso (days) 4.5 ± 0.2 8.1 +- 0.3 6.8 ± 0.2 7.9 -+ 0.2* 8.6 + 0.2+

L T i o o (days) 8.1) ± 0.0 18.7 ± 1.2 17.0 ± 0.9 20.0 -+ I.I 21.3 -+ 1.0"

a o 0.58 ± 0.05 0.98 -+ 0.30 - ± 0.06 - ± 0.21 1.08 -+ 0.20

e~ (days) 4.3 8.85 7.35 8.54 9,27

N u m b e r o f

eggs/female 47.0 ± 1.4 88.8 ± 1.6 74.4 + 1.3 49.8 -+ 1.3+ 45.1 -+ 1.9 +

Reproductive

period (days) 5.3 ± 0.3 8.3 + 0.3 8.3 ± 0.5 8.0 -+ 0.0 8.3 -+ 0.5

Post- reproductive

period (days) 2.7 ± 0.3 t0.4 + 0.3 8.7 -+ 0.5 12.0 ± 0.0+ 13.0 -+ 0.5+

BHA (raM) soaked

0.5 0.75 I 2.5 5 10

12.3 + 0.55 12.9 + 0.15 13.7 + 0.15 12.3 -+ 0.35 10.7 -+ 0.25 8.3 ± 0.2 17.3 + 0.7* 19.7 ± 1.2" 20.7 -+ 0.55 20.0 +- 0.7+ 16.3 -+ 0.9 15.0 ± 0.6

- 1.95 -+ 0,09* - ± 0.19" 1.93 -+ 0.04+ 1.72 -+ 0.06* - 1.69 + 0.07 1.79 ± O.03t

12.08 12.52 13.53 11.99 10.00 8.46

10.7 -+ 0,1+ 11.1 -+ 0.1+ 12.8 + 0.05+ 10.0 -+ 0.2~ + 8.6 ± t 7.5 -+ 0.1" 22.0 ± 0,6* 22.3 +- t 22.0 -+ 0.5+ 22.0 -+ 1.0" 18.6 ± 0.5 17.0 -+ 1.0 - ± 0.20 1.46 -+ 0.13 1.78 -+ 0.01" 1.53 -+ 0.18 - +- 0.11 1.23 -+ 0.07

11.05 I 1.41 12.69 9.73 9.11 8.14

51.i ± 4,0+ 43.3 -+ 0.08~: 33.5 -+ 5.4+ 49.8 + 6.9+ 45.9 -+ 3.45 65,3 + 2.4+[:

7.0 ± 0.8* 7.0 ± 0.0+ 7.0 ~ 0.05 7.3 ± 0.5* 7.0 -+ 0.8* 7.3 ± 0.5*

15.0 + 0.55 15.3 ± 0.0+ 16.7 -+ 0.05 14.7 ± 0.55 11.6 +- 0.8+ 9.6 -+ 0.5*

Values are Mean + S E of three experiments

EFFECT OF BHA IN CALLOSOBRUCHUS MACULATUS

MATERIALS AND METHODS

587

Fresh Vigna radiata (vern mung) seeds were soaked in different concentrations (0, 0.25, 0.5, 0.75, 1, 2.5, 5, and 10 mM) of BHA dissolved in ethanol (10%), dried in shade and were infested with pure cultures of C maculatus maintained at 28 +- I°C and 60 70% humidity Longevity and fecundity studies were carried out on insects emerg- ing from unsoaked, water, 10% ethanol (control) and BHA soaked seeds (Mahajan and Garg, 1992) Optimal concentration increasing median (LT50) and maximum (LTlo0) life spans was selected to observe its effect on catalase (Kar and Mishra, 1976), peroxidase (Kumar and Khan, 1983), glutathione reductase (Tyson et al., 1982) inorganic peroxides (HzO 2) (Bernt and Bergmeyer, 1976), lipid peroxides (MDA) (Hasan and Ali, 1981), iron content (Vogel, 1982) and free radicals (Buchvarov and Gantcheff, 1984) in whole-body homogenates of the two sexes at different intervals starting from emergence to beyond the median life span The enzymes and peroxides were determined in supernatants obtained after centrifugation The protein content was measured by Lowry et al (1951) for calculating the specific activity of the enzymes

For iron, bruchids were digested in HCI and perchloric acid for h at 420°C using Tecator 1016 digestion system The residue left after acid evaporation was dissolved in distilled water and analyzed subsequently The powdered insect samples after filling in glass capillaries (2 mm diameter) were analyzed semiquantitatively for free radicals using X-band (9.44 GHz) JES-FE3XG ESR spectrometer at a field range of 3,000 - 2,500 gauss The g value was calculated following Drago (1977) Experiments were repeated four times to confirm the reproducibility and the data were analyzed employ- ing Student's t test

T A B L E E F F E C T OF BHA (1 mM) ON SPECIFIC A C T I V I T Y OF C A T A L A S E , P E R O X I D A S E , A N D G L U T A T H I O N E R E D U C T A S E IN W H O L E BODY H O M O G E N A T E S OF A G E I N G C MACULATUS

Male Female

Age Ethanol (10%) Ethanol (10%)

Enzyme (days) soaked BHA soaked soaked BHA soaked

Catalase 64.5 ± 1.1 91.1 -+ 3.6t 67.7 ± 1.3 118.6 +- 1.7t

(ixM H202 decomposed/100 rag/ 93.3 ± 1.7" 214.8 ± 4.8"+ 75.5 -+ 7.1 131.5 -+ " t at 25°C/mg protein) 80.4 +- 4.5* 207.1 ± 5.2** 83.2 ± 3.1" 154.1 ± 3.8** 84.8 ± 1.8" 165.3 -+ 2.9"t 76.1 -+ 0.7* 141.3 ± 1.8"+ 86.5 ± 0.9* 191.9 +- 5.1"+ 78.4 ± 3.3* 146.4 +- 2.0*+

Peroxidase 3.9 ± 0.05 6.0 ± I t 5.9 ± 0.2 7.6 -+ 0.3t

(~M purpurogallin formed/ 6.5 ± 0.1" 7.0 -+ 0.2* 5.4 ± 0.1 8.0 +- 0.5* 100 mg/min at 25°C/mg protein) 7.2 +- 0.2* 6.6 -+ 0.2* 6.6 ± 0.3 7.6 ± 0.2t 7.6 +- 0.5* 6.6 ± 0.2* 5.7 ± 0.1 5.7 -+ 0.3* 6.4 ± 0.1" 6.1 ± 0.1 5.2 ± 0.06* 5.5 ± 0.09"*

Glutathione reductase 6.1 ± 0.3 10.3 ± 0.5+ 5.4 ± 0.1 9.1 ± 0.2+

(~tg G S H produced/100 mg/min 7.8 ± 0.1" 10.4 -+ 0.3t 6.5 ± 0.2* 9.6 +- 0.2+

at 30°C/rag protein) 7.5 ± 0.1" 9.6 ± 0.2t 5.9 ± 0.4 8.5 ± 0.2t

7 8.5 ± 0.3* 8.9 -+ 0.2* 5.4 ± 0.2 8.3 -+ 0.3t

9 7.5 ± 0.6 7.4 ± 0.2* 5.0 ± 0.2 7.9 ± 0.7t

V a l u e s a r e M e a n + S E o f f o u r e x p e r i m e n t s

70 60 50 n~ 4

2O

g

o

M a l e Femole M a l e

-]1 ]]]7

~]9 Age (days)

Female Male Female

Lipid peroxides Inorgonic peroxides Iron

FIG Percent decrease with respect to control in lipid peroxides, inorganic peroxides and iron content in B H A (1 mM)-treated C maculatus

R E S U L T S

Female insects emerging from unsoaked seeds were short lived than males and this decreased life span was accompanied by higher age-independent susceptibility to death

(ao) and low life expectancy (ex) Presoaking of seeds in water increased the life span of insects and a similar observation was made with 10% ethanol soaking B H A treat- ment increased the LTso and LTl0o values and e~; decreased a o, mean number of eggs laid/female as well as reproductive phase and prolonged the postreproductive period with a maximum change at mM concentration in comparison to the control cohorts (Table 1)

Age-related change in the activities of catalase, peroxidase, and glutathione reduc- tase in insects emerging from ethanol and BHA soaked seeds was observed except for glutathione reductase in females On BHA treatment, the activity of antioxygenic en- zymes generally elevated and the peroxides and iron content declined as compared to the control cohorts The percent rise was, however, more for catalase (Table 2, Fig 1) ESR spectra of and day old bruchids infesting unsoaked, ethanol and B H A (1 raM) soaked seeds revealed a single sharp peak of free radicals with a spectroscopic splitting factor (g) in the range of 2.00-2.08 and line width between 0-13 gauss The peak heights were lower in BHA-treated insects (Table 3) Some of the spectra showed

T A B L E E F F E C T OF BHA (1 raM) ON ESR PEAK H E I G H T S (cm) IN C MACULATUS

Ethanol (10%) BHA

Age Unsoaked soaked soaked

Male 5.7 5.0 2.1

5 8.5 8.5 5.4

Female 8.5 6.0 1.5

EFFECT OF BHA IN CALLOSOBRUCHUS MACULATUS 589 an additional broad peak indicative of the presence of ferric ions (Fe + 3) It was further evidenced by heating samples of one day old males infesting unsoaked seeds at 50, 100, 150, 200, and 250°C The ESR peaks clearly showed the presence of Fe + above 100°C with a g value ranging between 2.03-2.09 (Fig 2)

0.,

\ f

1 0 °

\ ~ _ ~ - - - - ~ c °

b

^, /

s' i'

t

q I ,

u

/

f ~ 200 ~

2500 3000 ]500 O0 )000 ]500 ~)00

M a g n e t i c f i e l d ( g a u s s )

DISCUSSION

Low life expectancy of female C m a c u l a t u s may be attributed to its higher metabolic rate during reproductive period to meet increased energy demands as compared to the males The longevity in C m a c u l a t u s increased with the concentrations of BHA up to I mM followed by a decline at higher doses This may be explained in terms of antiox- idative properties of B H A at lower concentrations and prooxidant effect of this antiox- idant at higher doses (Kahl et al., 1989) Further, life prolonging effect of BHA was correlated to its free-radical quenching nature and conservation of energy by way of significant reduction in the length of reproductive period and number of eggs laid/ female as reported earlier (Mahajan and Garg, 1992) The increased longevity of BHA- treated insects may also be explained in terms of decline in peroxides, iron content and free radicals (Table 3, Fig 1) However, the impact of the antioxidant by way of decreased metabolism (Emanuel and Obukhova, 1978) cannot be discounted Dubick et al (1982) reported the elimination of destructive oxidative effects of oxygen on removal of metals from the culture medium A comparison between insects emerging from water-soaked and 10% ethanol-soaked seeds reveals that alcohol as a solvent does not influence the longevity and reproductive characteristics of insects.The increase in the levels of antioxygenic enzymes with B H A could be extrapolated from the increased mRNA biosynthesis as observed by Semsei et al (1989) in diet restricted male rats The increased activity of antioxygenic enzymes and decreased iron content on BHA treat- ment support the fact that antioxidants decrease lipid peroxidation Sohal et al (1990) suggested that the maximum life of an organism is related inversely to H202 production and directly to antioxygenic defenses Further, Zs.-Nagy (1987) suggested that the decreased free radical production consequent to efficient antioxygenic enzyme system may result in less accumulation of lipid peroxides as observed in the present case In addition, the pronounced increase in catalase level is suggestive of its important role in the survival of C m a c u l a t u s Moreover, the present findings are indicative of the maintenance of strong reducing environment by declined inorganic and organic perox- ide levels (Bains et al., 1992; Sharma et al., 1992) Therefore, the increased longevity of C m a c u l a t u s emerging from BHA soaked seeds could be attributed to decreased free radicals which inflict damage to the DNA, thereby stimulating the generation of an- tioxygenic enzymes

Acknowledgments The senior author is thankful to UGC, New Delhi for SRF

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