effect of bha on longevity antioxygenic enzymes and peroxides in

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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.

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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 1. 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. 1 0.25

Male

LTso (days) 6.1 ± 0.2 9.2 +- 0.6 8.3 -+ 0.03 8.6 ± 0 . 0 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 -+ 0 . 3 t

a o 1.07 -+ 0.03 - 1 . 6 4 -+ 0.11 1.55 + 0.03 - 2 . 0 3 -+ 0.06t - 1 . 6 9 ± 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 - 1 . 2 8 ± 0.06 - 1 . 2 9 ± 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* - 2 . 0 9 ± 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 ± 0 . 3 t 7.5 -+ 0.1"
22.0 ± 0,6* 22.3 +- 0 . 7 t 22.0 -+ 0.5+ 22.0 -+ 1.0" 18.6 ± 0.5 17.0 -+ 1.0
- 1 . 3 1 ± 0.20 1.46 -+ 0.13 1.78 -+ 0.01" 1.53 -+ 0.18 - 1 . 1 7 +- 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.

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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 4 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 2. 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 1 64.5 ± 1.1 91.1 -+ 3.6t 67.7 ± 1.3 118.6 +- 1.7t

(ixM H202 decomposed/100 rag/ 3 93.3 ± 1.7" 214.8 ± 4.8"+ 75.5 -+ 7.1 131.5 -+ 4 . 2 " t
min at 25°C/mg protein) 5 80.4 +- 4.5* 207.1 ± 5.2** 83.2 ± 3.1" 154.1 ± 3.8**
7 84.8 ± 1.8" 165.3 -+ 2.9"t 76.1 -+ 0.7* 141.3 ± 1.8"+
9 86.5 ± 0.9* 191.9 +- 5.1"+ 78.4 ± 3.3* 146.4 +- 2.0*+

Peroxidase 1 3.9 ± 0.05 6.0 ± 0 . I t 5.9 ± 0.2 7.6 -+ 0.3t

(~M purpurogallin formed/ 3 6.5 ± 0.1" 7.0 -+ 0.2* 5.4 ± 0.1 8.0 +- 0.5*
100 mg/min at 25°C/mg protein) 5 7.2 +- 0.2* 6.6 -+ 0.2* 6.6 ± 0.3 7.6 ± 0.2t
7 7.6 +- 0.5* 6.6 ± 0.2* 5.7 ± 0.1 5.7 -+ 0.3*
9 6.4 ± 0.1" 6.1 ± 0.1 5.2 ± 0.06* 5.5 ± 0.09"*

Glutathione reductase 1 6.1 ± 0.3 10.3 ± 0.5+ 5.4 ± 0.1 9.1 ± 0.2+

(~tg G S H produced/100 mg/min 3 7.8 ± 0.1" 10.4 -+ 0.3t 6.5 ± 0.2* 9.6 +- 0.2+

at 30°C/rag protein) 5 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 .

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70 
60 
50 
n~ 4 0

g

o 0

M a l e Femole M a l e

-]1

]]]7

~]9

Age (days)

Female Male Female

Lipid peroxides

Inorgonic peroxides

Iron

FIG. 1. 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 1 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 1 and 5 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 3. 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 1 5.7 5.0 2.1

5 8.5 8.5 5.4

Female 1 8.5 6.0 1.5

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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 + 3 above 100°C 
with a g value ranging between 2.03-2.09 (Fig. 2).

0.,

\ .

1 0 0 °

\ ~ _ ~ - - - - ~ c ° .

^,
/

s'
i'

t

q 
I ,

/

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 )

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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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EFFECT OF BHA IN CALLOSOBRUCHUS MACULATUS 591
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