Effect of age and ambient temperature on n-pentane production in adult housefly, Musca domestica

Mechanisms of Ageing and Development, 29 (1985) 317-326

317

Elsevier ScientificPublishers Ireland Ltd.

EFFECT OF AGE AND AMBIENT TEMPERATURE ON n-PENTANE PRODUCTION IN ADULT HOUSEFLY, MUSCA DOVIES~CA

R.S. SOHAL*, ARMIN MULLER, BERT KOLETZKO÷ and HELMUT SIES lnstitut fiir Physiologische Chemic I and ÷Kinderklinik 3, Universitiit Diisseldorf (F.R. G.)

(Received October 30th, 1984)

SUMMARY The objective of this study was to examine the relationship between lipid peroxidation and aging in the male housefly. Metabolic rate of flies is known to be higher and life span shorter at elevated ambient temperature. Evolution of n-pentane and level of thiobarbituric acid (TBA) reactive material were used as indicators of lipid peroxidation, n-Pentane accumulated by houseflies in vivo and by whole body homogenates of houseflies, in response to tert-butyl hydroperoxide (1 mM), increased with age. n-Pentane accumulation in rive was markedly higher at higher ambient temperature. Furthermore, n-pentane generated by flies in vivo and by fly homogenates in vitro tended to be lower in flies raised at a lower ambient temperature. TBA-reactive material, elicited by tert.butyl hydroperoxide, was augmented in older flies, but no significant difference was found between flies aged at different ambient temperatures. Analysis of fatty acids in housefly homogenates indicated an age-associated increase in the ratio of polyunsaturated to saturated fatty acids. Key words: Aging; Free radicals; Lipid peroxidation; Insects; Lipids

INTRODUCTION It is now well established that life spans of.poikilotherms are inversely related to their metabolic rate (for review, see [1,2]). For example, average and maximum life span of common housefly can be considerably prolonged by lowering the ambient temperature or by prevention of flying activity [2]. The nature of mechanisms which govern the aging process as well as those which are responsible for the inverse relationship between *Present address and to whom correspondence should be directed." Department of Biology,Southern

Methodist University,Dallas, Texas 75275, U.S.A. 0047-6374/85/$03.30 Printed and Published in Ireland

© 1985 ElsevierScientific Publishers Ireland Ltd.

318 metabolic rate and life span are still unknown. It has, however, been postulated that oxygen free radicals, generated during normal metabolism, may be a major source of molecular damage underlying the aging process [3]. One of the consequences of free radical interactions with cellular structures can be the peroxldatlon of polyunsaturated lipids, which is detectable by the evolution of alkanes such as n-pentane and ethane from the organism and by the production of thiobarbituric acid (TBA)-reactive material in tissue samples (for review, see [4,5]). Alkane exhalation by living animals has been proposed as a sensitive indicator of in vivo lipid peroxidation [ 6 - 8 ] . Ethane and n-pentane, which are scission products of 60-3 and 60-6 polyunsaturated fatty acids, respectively, have been the most frequently used indicators. Due to the presence of relatively high amounts of linoleic acid in insects [9], n-pentane is an indicator of choice. Indeed, Young and Tappel [10] have reported the exhalation of n-pentane by honeybees. An age-associated increase in n-pentane production by rats, in vivo, has been shown by Sagai and Ishinose [11]. In the housefly, antioxidant defenses such as activities of superoxide dismutase and catalase and level of reduced glutathione or lipid soluble antioxidants tend to decrease with age; whereas the amount of inorganic peroxides increases with age [12] and also in response to elevation of metabolic rate [13]. The present study was conducted in an effort to further evaluate the possible role of oxygen free radicals in aging. Effects of age and temperature-induced alterations in metabolic rate on the production of n-pentane and TBAoreactive material are reported. MATERIALS AND METHODS

Maintenance of houseflies Following emergence from the pupae, adult flies were housed in 0.3-m 3 cages and fed a mixture of sucrose, glucose, dried milk, powdered egg yolk and corn meal ( 1 0 : 2 : 3 : 2 by vol.). Stock cultures were kept at approximately 25°C, however, in experiments dealing with the effects of altered metabolic rates, flies were transferred to other, specified, temperature-controlled rooms. To avoid complications associated with cycles of egg laying, only the male flies were used for experimental studies. Preparation of housefly homogenates Flies were immobilized by cooling, weighed and homogenized in about 6 vols. (v/w) of cold 140 mM KC1/5 mM potassium phosphate solution (pH 7.4) using a Potter-type glass and Teflon homogenizer. Homogenates were filtered through a single layer of gauze to eliminate exoskeletal debris. Protein concentration was determined by the biuret method. n-Pentane evolution n-Pentane produced by living flies and by whole body homogenates was measured by the procedure described previously [14]. For in vivo measurements, populations consist-

319 ing of 75 flies were placed in a glass chamber, with a net vol. of 820 ml, and containing 50 g of Sodasorb ® for the absorption of CO2. During the experimental period, flies were fed on sucrose and water only to exclude dietary production of n-pentane. The chamber was sealed using starch-glycerol lubricant and connected to a balloon of 100% oxygen. Flies were allowed to respire in this closed system for 24 or 48 h after which 5-ml air samples were drawn using Hamilton gas-tight syringes. Experiments for the measurement of in vitro production of n-pentane were carried out in 43-ml sealed flasks which were capped with a PVC septum impermeable to alkanes. Tissue homogenates, adjusted to a f'mal concentration of 10 mg protein/ml, were incubated for 3 h in a mixture consisting of 0.1 M phosphate buffer (pH 7.4) and 1 mM tert-butyl hydroperoxide. Five-milliliter samples of air from the flasks were withdrawn and then rapidly substituted with an equal volume of alkane-free oxygen. Air samples were analyzed by gas chromatography as described previously [14]. Calibrations were made with standard gas mixtures supplied by Messer-Griesheim (Duisburg, F.R.G.). The amount of n-pentane was calculated by using the equation of Wendel and Dumelin [7]. Results of in vivo measurements are expressed as pmoles n-pentane produced/g wet body wt of flies per 24- or 48-h periods. The in vitro measurements are expressed as pmoles n-pentane produced/lO0 mg protein of flies during 3-h periods.

Measurement of thiobarbituric acid (TBA )-reactive material The concentration of TBA-reactive material was determined in the same incubation mixtures used for the tert-butyl hydroperoxideqnduced production of n-pentane. One hundred microliters of this mixture was added to 700/zl of a solution consisting of 1.2 M trichloroacetic acid and 35 mM TBA, followed by heating for 25 min at 95°C, and cooling and centrifugation. The concentration of the chromogen in the supernatant was measured at 535-570 nm (Ae = 156 mM -1 cm -1) and expressed as nmoles of malondialdehyde/1 O0 mg protein. Extraction and measurement of fatty acids Lipids in fly homogenates containing 5 mg of protein (usually about 300/al) were extracted with 6 ml of chloroform/methanol mixture (1:1). Diheptadecanoyl.phosphatidylcholine was added as an internal standard. Following the addition of 3 ml distilled water, the chloroform layer was separated by centrifugation, transferred to another tube and dried under nitrogen. Methyl esters of fatty acids were prepared by adding 2 ml of dry HC1 in methanol (0.7 mol/1). The solution was boiled for 1 h at 85°C and excess HC1 was neutralized by the addition of 100 mg of solid Na2SO4, Na2HCO3 and Na2CO3 (2:2: 1). The supernatant was dried under nitrogen and redissolved in 200/al of hexane. One microliter of this solution was injected into Packard-Becker 417 gas chromatograph fitted with a 25 m X0.2 #m WGll glass capillary column (WGA Analytic, Dusseldorf). Temperature was programmed from 130°C to 230°C at 5°C/m. Helium was used as a carrier gas at 0.7 bar and split ratio

320 of 1:40. Peaks were identified by comparison of retention times with standard substances. Relative amounts of fatty acids were calculated with a Shimadzu CRIB chromatopac integrator. RESULTS Effect o f age on n-pentane production in vivo Age-associated studies were conducted until flies were 14-days of age which approximates the beginning of the dying phase in male flies kept at 25°C [15]. n-Pentane produced by the houseflies, in vivo, during 24-h periods was assayed in 2-, 9- and 14-day-old flies maintained at 25°C throughout life. Results presented in Fig. 1 indicate an age-dependent increase in n-pentane accumulation. An increase of approximately 1.7-fold occurred between 2 and 14 days of age. Effect o f age on tert-butyl hydroperoxide-induced n-pentane production The susceptibility of fly homogenates to undergo lipid peroxidation was determined in vitro by measuring n-pentane production elicited during tert-butyl hydroperoxide oxidative breakdown. This in vitro model system can provide information on the availability of polyunsaturated fatty acid targets in fly homogenates. Comparisons of n-pentane production by whole body homogenates of flies, in response to 1 mM tert-butyl hydroperoxide, were made in 1-, 8-, and 13-day old flies kept at 25°C. The amount ofn-pentane produced by the homogenates increased approximately 3.5-fold between 1 and 8 days, and a further 1.3-fold between 8 and 13 days of age (Fig. 2). Effect o f ambient temperature on n-pentane production The rate of oxygen consumption and life span of houseflies is influenced by environmental temperature with a Qlo of about 2 - 3 [15,16]. For the determination of n-pentane generation, 9-10-day-old flies, previously kept at 25°C, were placed in

,.f.o ~_

tOO.

o~, o_ ~ 200 C~

x

o

Ageidays)

lb

11

Fig. 1. In vivo evolution of n-pentane during 24-h periods by adult male houseflies of different ages. Procedure for the measurement is described in the Section on "Materials and Methods". Results are expressed as means + S.D. of 3-4 independent experiments.

321 ._.tO0

-:-o 0 o

i

..~.-

n," x

L.o 0 Age Idoys)

Fig. 2. tert-Butyl hydroperoxide-elicited production of n-pentane and TBA-reactive substances in homogenates of adult male houseflies of different ages. Reaction mixtures contained 10 mg fly protein/mi, 1 mM tert-butyl hydroperoxide and 0.1 M potassium phosphate buffer (pH 7.4); total time of reaction was 3 h. Results are expressed as means ± S.D. of 3 independent experiments.

temperature-controlled rooms at 20°C and 28°(:. As compared to 20°C, the accumulated amount of n-pentane produced by houseflies at 28°C was approximately 2.7-fold greater at the end of 24 h and 3.2-fold higher after 48 h (Fig. 3). In order to determine if alterations in the rate of aging have an effect on n-pentane accumulation in rive, comparisons were also made between flies kept throughout life at 18°C and 25°C (however, n-pentane measurements of both groups were made at 25°C). Results presented in Table I indicate that flies previously housed at 18°C accumulated slightly less n-pentane in vivo than those kept at 25°C. tert.Butyl hydroperoxide-induced n-pentane accumulation by whole body homogenates of 13-day-old flies was also lower in flies aged at 18°C than those at 25°C.

I°1 o~

~lOC0~

|

Q; 0 c-.o 0

0: Time(hrsl F~. 3. n-Pentane evolution by adult male houseflies in vivo. Measurements were made on 9-]O-dayold fries, kept at 20°C and 28°C for 48 h. Results are average ± range of 2 independent experiments.

322 TABLE I n-PENTANE PRODUCTION IN VIVO AND tert-BUTYL HYDROPEROXIDE-INDUCED FORMATION OF n-PENTANE AND THIOBARBITURIC ACID (TBA)-REACTIVE MATERIAL IN FLIES AGED AT TWO DIFFERENT TEMPERATURESa Group

180C 25°C

n.Pentane production (prnoles Xg body wt -~ X 24 h -I)

in vitro c (pmoles X 100 mg protein -t) X3 h -I )

328+11 355 + 4

70+ 7 87 + 16

TBA-Reactive Material c (nmoles malondiaMehyde X 100 ragprotein -t X 3 h -a)

23.6+_2.1 22.2 +_0.8

a After emergence from the pupae, flies were kept at 18°C or 25°C. Comparisons of n-pentane production in vivo were made between 11-day-old flies. The ambient temperature during the period of measurement was 25°C for both groups. Homogenates of 13-day-old flies were used for the determination of tert-butyl hydroperoxide-induced production of n-pentane and TBA-reactive material. Assay conditions are similar to those described in Fig. 2. b Results are average of 2 separate experiments +-range. c Results are means +_S.D. of 3 independent experiments.

E f f e c t o f age and a m b i e n t temperature on TBA-reactive material TBA-reactive material, elicited by 1 mM tert-butyl hydroperoxide, increased with age;

however, 8-day-old flies had higher average levels than 13-day-old flies (Fig. 2). No significant differences were found between the homogenates of 13-day-old flies aged at 18°C and 25°C (Table I). F a t t y acid composition o f flies

The degree o f unsaturation of fatty acids present in tissues can affect n.pentane production and concentration of TBA-reacting material. A comparison o f fatty acid composition was therefore made between flies of different ages, and those kept at different temperatures. As shown in Table II, the total amount of free and esterified fatty acids tended to decrease with age. In general, the relative and absolute amounts of saturated fatty acids exhibited a decline with age, whereas polyunsaturated fatty acids tended to increase with age. However, the age-associated increase in the absolute amounts of polyunsaturated fatty acids was not linear. For example, a 67% increase occurred between 2 and 8 days of age whereas only 6% augmentation was found between 8 and 13 days of age. Flies raised at 18°C contained a greater absolute amount of polyunsaturated and saturated fatty acids. DISCUSSION Results of this study indicate that aging in the housefly is associated with an increase in the accumulation of n.pentane, in vivo, most likely due to an increased generation of

323

3

3

3 Z

3

0

3

0 [-., Z

3

o ,..1

3

°. M

0 Z 0 0

o <

[...

324 the alkane. Whether an increase in the accumulated amount might be due to a decreased metabolism of the alkane [see 17] was not investigated here. Potentially, this can be due to an increase in the steady state levels of oxygen radicals or as a consequence of an increase in the amount of polyunsaturated fatty acid content of flies due to dietary intake. The latter possibility, however, is weakened by the fact that the absolute amount of polyunsaturated fatty acids between 8 and 13 days of age increases by 6% while n-pentane production rises by 30%. Furthermore, both the relative and the absolute amounts of linoleic acid (18 : 2co6), which is believed to be the main source of n-pentane, decreases between 8 and 13 days of age. It is therefore possible that previously observed age-dependent deterioration in the antioxidant defenses of flies, manifested by decrease in activities of superoxide dismutase and catalase and concentration of reduced glutathione and lipid-soluble antioxidants [12] may be at least partially responsible for the presently observed increase in n-pentane evolution. In the current belief, n-pentane evolution in vivo is an indicator of the free radicalinduced lipid peroxidation reactions. The breakdown products of lipid hydroperoxides, such as malondialdehyde, have been shown to cause deleterious changes such as crosslinking of amino group-containing molecules [see, 4,5]. The age-associated increase in the in vivo evolution of n-pentane in houseflies can be suggestive of the increased vulnerability of flies to free radical-related damage, resulting from a decrease in the efficiency of endogenous antioxidants and/or from scission products of fatty acid peroxides. Results of in vitro studies indicate that susceptibility of housefly tissues to undergo lipid peroxidation in response to experimentally-induced oxidative stress increases with age. Between 8 and 13 days of age, n-pentane evolution elicited by tert-butyl hydroperoxide increased by 30% which greatly exceeds any corresponding increase in the amount of linoleic acid or total polyunsaturated fatty acids. It is thus possible that the observed age-associated increase in tert-butyl hydroxide4nduced n-pentane accumulation may be due to the previously described decrease in lipid soluble antioxidants [12]. Results of this study also indicate that an increase in the metabolic rate of houseflies, induced by the elevation of ambient temperature, causes a pronounced increase in the in vivo rate of n-pentane evolution. On the basis of the following reason it can be suggested that this increase is more likely due to an acceleration in free radical-lipid interactions rather than any differences in the fatty acid content of flies. Adult houseflies are incapable of synthesizing polyunsaturated fatty acids and are entirely dependent on their dietary intake [9]. Flies used in this experiment were kept under identical conditions except during the 48-h experimental period when they were separated into two groups and placed at 20°C or 28°C and fed on sucrose alone. It would therefore seem that the rate of oxygen free radical peroxidation is intensified under conditions of higher metabolic activity. The finding that flies aged at 18°C accumulate less n-pentane in vivo as well as in vitro than those at 25°C also is in agreement with the existence of a relationship between metabolic rate, aging and lipid peroxidation. Regarding the decrease in TBA-reactive material elicited by tert-butyl hydroperoxide,

325 during 8 - 1 3 days of age, it should be mentioned that multiple factors are k n o w n to affect the formation of this material [18]. The exact cause of this decrease cannot be identified on the basis of this study. However, Sagai and Ishinose [11] encountered a similar trend in the serum of aging rats. TBA-reactants exhibited a tendency towards increase with age, but the value for the 32-month-old rats was lower than for 22-monthold rats. Finally, present results on fatty acid analysis emphasize the influence of dietary intake on lipid composition of the housefly. In previous studies [19,20] where flies were fed on sucrose only, the relative amount of polyunsaturated fatty acids declined with age which is in contrast to the findings of the present study where flies were fed on a more diverse diet. ACKNOWLEDGEMENTS We would like to express our deep gratitude to Mr. Harald Mikoleit and Prof. Gerhard Heide of the Zoology Institute for providing houseflies. Excellent technical assistance provided by Miss Maria Zimmer is gratefully acknowledged. This research was supported by a grant from the National Foundation for Cancer Research. REFERENCES 1 R.S. Sohal, Metabolic rate and life span. lnterdiscip. Top. Gerontol., 9 (1976) 25-40. 2 R.S. Sohal, Metabolic rate, aging and lipofuscin accumulation. In R.S. Sohal (ed.), Age l~'gments, Elsevier/North Holland, 1981, pp. 303-316. 3 D. Harman, Aging: a theory based on free radial and radiation chemistry. J. Gerontol., 11 (1956) 298-300. 4 A.L. Tappel, Lipid peroxidation and fluorescent molecular damage to membranes. In B.F. Trump and A.V. Arstila (eds.), Pathobiology of Cell Membranes, Vol. I, Academic Press, New York, pp. 145-170, 1975. 5 A. Tappel, Measurement and protection from in vivo lipid peroxidation. In W.A. Pryor (ed.), Free Radicals in Biology, Vol. IV, Academic Press, New York, 1980, pp. 1-47. 6 C. Riley, G. Cohen and M. Lieberman, Ethane evolution: a new index of lipid peroxidation. Science, 183 (1974) 208-210. 7 A. Wendel and E.E. Dumelin, Hydrocarbon exhalation. Methods Enzymol,, 77 (1981) 10-15. 8 G.D. Lawrence and G. Cohen, Ethane exhalation as an index of in vivo lipid peroxidation: concentration of ethane from a breath collection chamber. Anal, Biochem., 122 (1982) 283-290. 9 R.G. Bridges, J. Rickets and J.T. Cox, The replacement of lipid-bound choline by other bases in the phosphollpids of the housefly, Musca domestica. J. lnsect Physiol., 11 (1965) 225-236. 10 R.G. Young and A.L, Tappel, Fluorescent pigment and pentane production by lipid peroxidation in honeybees, Apis mellifera. Exp. Gerontol., 13 (1978) 457-459. 11 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. 12 R.S. Sohal, K.J. Farmer, R.G. Allen and N.R. Cohen, Effect of age on oxygen consumption, superoxide dismutase, catalase, glutathione, inorganic peroxides and chloroform-soluble antioxidants in the adult male housefly, Musca domestica. Mech. Ageing Dev., 24 (1983) 185-195. 13 R.S. Sohal, R.G. Allen, K.J. Farmer and J. Procter, Effect of physical activity on superoxide dismutase, catalase, inorganic peroxides and glutathione in the adult male housefly, Musca domestica. Mech. Ageing Dev., 26 (1984) 75-81.

326 14 A. Miiller and H. Sies, Assay of ethane and pentane from isolated organs and cells. Methods Enzymol., 105 (1984) 311-319. 15 R.S. Sohal, Oxygen consumption and life span in the adult housefly, Musca dornestica. Age, 5 (1982) 21-24. 16 S.S. Ragland and R.S. Sohal, Ambient temperature, physical activity and aging in the housefly, Musca domestica. Exp. Gerontol., 10 (1975) 279-289. 17 H. Frank, T. Hintze, D. Birnboes and H. Reemer, Monitoring lipid peroxidation by breath analysis: endogenous hydrocarbons and their metabolic elimination. Toxicol. AppL Pharmacol., 56 (1980) 337-344. 18 A.A. Barker and F. Bernheim, Lipid peroxidation: its measurement, occurrence and significance in animal tissues. Adv. Gerontol., Res., 2 (1967) 355-403. 19 R.G. Bridges and R.S. Sohal, Relationship between age-associated fluorescence and linoleic acid in the housefly, Musca domestica. Insect Biochem., 10 (1980) 557-562. 20 R.S. Sohal, H. Donato and E.R. Biehl, Effect of age and metabolic rate on lipid peroxidation in the housefly, Musca domestica L. Mech. Ageing Dev., 16 (1981) 159-167.