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Lipid peroxidation induced by some halomethanes as measured by in vivo pentane production in the rat
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
49,283-291
(1979)
Lipid Peroxidation Induced by Some Halomethanes as Measured by in Vivo Pentane Production in the Rat’ MASARU SAGA? AND AL L. TAPPEL Department
of Food Science and Technology, University of California, Davis, California 95616
Received September 29, 1978; accepted January 15, 1979 Lipid Peroxidation Induced by Some Halomethanes as Measured by in Vivo Pentane Production in the Rat. SAGAI, M., AND TAPPEL, A. L. (1979). Toxicol. Appt. Pharmacol. 49, 283-291. Pentane production in vivo in rats was used successfully as an index to support the concept that lipid peroxidation is involved in the toxicity of some halomethanes. The time-response relationships showed that the rats had maximum pentane production by 15-30 min following ip administration of carbon tetrachloride (Ccl,), bromotrichloromethane (BrCCI,), and chloroform (CHCl& BrCCI, and Ccl, caused the production of the greatest amounts of pentane, CHCll induced a smaller amount of pentane production, and dichloromethane (CH#&) did not increase pentane production over that caused by injection of the mineral oil carrier. There was a good relationship (r = 0.987; p
Peroxidation of biological membrane lipids is considered an integral part of cell damage and of many toxic processes (Tappel, 1975). Several halogenated hydrocarbons induce lipid peroxidation in viuo and in vitro (Slater, * Supported by research Grant AM-09933 from the National Institute of Arthritis, Metabolism and Digestive Diseases, U.S. Department of Health, Education and Welfare, and a grant-in-aid from The National Live Stock and Meat Board. z Present address: Division of Basic Medical Sciences, The National Institute for Environmental Studies, P.O. Yatabe-Tsukuba, Ibaraki 300-21, Japan. 283
1972). Carbon tetrachloride (Ccl,) has been thoroughly investigated as a typical halogenated hydrocarbon, and there is considerable evidence that lipid peroxidation is the principal cause of the hepatotoxicity produced by this compound (Recknagel and Glende, 1973). Slater and Sawyer (1971) reported that bromotrichloromethane (BrCCl,) is a more potent prooxidant than Ccl, and that it produces severe liver injury. It is well known that CHCl, also produces liver injury similar to that produced by Ccl,, but that it does not induce lipid per0041-008X/79/0802834%02.tW/0 Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
284
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oxidation (Klaassen and Plaa, 1969; Rey- dation than ethane in rats fed a vitamin Enolds and Yee, 1968; Cawthorne et al., deficient diet with a high linoleic acid content. 1971). In the present study, pentane measurements There is evidence that liver injury depends were made to obtain data to support the on the in vivo biotransformation of the ad- concept that lipid peroxidation can be inministered halomethanes, namely, the split- duced in vivo by some halomethanes. Conting of the covalent bond between carbon jugated dienes in rat tissues were correlated and halogen or carbon and hydrogen. The with pentane produced in vivo. The question tendency of a halomethane molecule to of what are the main organs of origin for the cleave homolytically and to initiate reactions pentane produced following treatment of rats that involve free radicals as intermediates with Ccl, and BrCCl, was examined. depends on its bond dissociation energy. For instance, the suggested order of relative METHODS activities in stimulating lipid peroxidation is Animals and treatments. Male Sprague-Dawley BrCCl, > Ccl, > CHCI, > CFCl, (Burdino et rats 7-10 weeks of age and weighing 193-283 g were al., 1973). This series corresponds inversely to the series of bond dissociation energies of housed three to a cage in hanging wire-mesh cages in a room kept at a constant temperature of 22-23°C. these compounds, which are 49, 68, 90, and They were subjected to a daily cycle of 14 hr of light 103 kcal/mol, respectively (Walling, 1957). and 10 hr of dark. The rats were fed a stock diet,3 The most probable free radical species is the and they were fasted overnight prior to the measuretrichloromethyl radical (* Ccl,), and this ment of pentane and conjugated dienes. They were injected ip with 10, 30, or 90 ~1 of BrCC1,,4 CCld,s free radical is considered to induce lipid CHC13,* or CH,Cl, 6/1OOg body wt. The haloperoxidation. methanes were administered as a 10 or 30% solution Hydrocarbon gases have long been known in mineral oil. The rats were decapitated 30 min after to appear early during the autoxidation of injection to obtain tissues for the measurement of edible fats (Frankel et al., 1961; Horvat et al., conjugated dienes. Each tissue to be examined was removed from the rat immediately, washed with cold 1964; Smouse et al., 1965). Ethane is pro- physiological saline, and weighed. duced during autoxidation of linolenic acid Analysis of pentane. Gas chromatographic analysis (Lieberman and Mapson, 1964). Riely et al. of pentane was done on an alumina column according (1974) advanced the use of hydrocarbon gas to the method described by Dillard et al. (1977). A with a flame-ionization deanalysis when they applied the method to gas chromatograph’ tector and fitted with a six-way, nut-type gas sample biological systems. They showed that ethane valve was used. The rats were placed in a holding production was characteristic of sponta- chamber (Dillard et al., 1977) to breathe hydrocarbonneously peroxidizing mouse tissues in vitro free air8 for 10 min prior to collection of the samples. and that Ccl, provoked formation of ethane One-half of the breath+air stream (120 mI/min) in vivo in mice. Since that time, other in- from each rat was collected over a IO-min period to a 600-ml sample in an activated alumina-filled vestigators (Hafeman and Hoekstra, 1977a,b; give gas sample loop immersed in liquid nitrogen-ethanol Koster et al., 1977; Lindstrom and Anders, at about - 130°C. Breath samples were collected at 15, 1978) have used ethane production to follow 30, 60, 90, and 120 min following injection of the halomethanes. The gas chromatograph was stanthe course of lipid peroxidation in vivo. Pentane was shown to be the predominant 3 Purina Rat Chow, Ralston, Purina Company, St. short chain hydrocarbon gas to arise during Louis, MO. thermal decomposition (Evans et al., 1967, 4 Eastman Kodak Company, Rochester, N.Y. 5 Mallinckrodt Chemical Works, St. Louis, MO. 1969) and iron-catalyzed decomposition 6 Aldrich Chemical Company, Inc., Milwaukee, (Dumelin and Tappel, 1977) of linoleic and arachidonic acid hydroperoxides. The work Wis. ’ Varian Associates, Palo Alto, Calif. of Dillard et al. (1977) indicates that pen8 Linde Division, Union Carbide Corporation, tane is a more sensitive index of lipid peroxi- South Plainfield, N.J.
HALOMETHANE-INDUCED
LIPID
dardized with l-ml aliquots of 0.8 ppm pentaneg in nitrogen at an electrometer setting of 4 x lo-i2 amp/ mV. The relative peak area of pentane on each chromatograph was calculated by triangulation. A comparison of the peak area obtained for the standard and for the breath sample allowed calculation of the picomol of pentane produced. The following computation was applied :
285
PEROXIDATION
0.003 M EDTA, pH 7.4, with a glass and Teflon homogenizer. A 5-ml aliquot of each homogenate was transferred to a 1%ml round-bottom centrifuge bottle and 20 ml of chloroform : methanol (2 : 1) was added. The procedure used for extraction of total lipids was that described by Hashimoto and Recknagel (1968). The recovered lipids were measured by the sulfo-phosphovanillin method (Frings and Dunn, 1970). Aliquots of the lipids were dissolved in methanol at a concentration of 1 mg/ml and were examined in a uv spectrophotometer from 220 to 300 nm. The absorbance at 237 nm was determined with small corrections being made for slight turbidity in the samples. Turbidity was determined by measurement of absorbance at 260 nm. Total conjugated dienes (Table 1) is the product of total lipids of each organ and absorbance at 237 nm/mg of lipid. Conjugated dienes were converted to a molar basis using the molecular extinction coefficient.
(pm01 pentane) (incoming flowrate of air) I (100 g body wt) (sample volume) pmol pentane/lOO g body wt/min. The amount of pentane produced during 120 min by a rat was calculated by integration of the values obtained at each of the time periods. Measurement of conjugated dienes. A 15 % homogenate of each tissue was prepared in 0.3 M sucrose9 Matheson, Newark, Calif. TABLE
1
TOTAL LIPIDS AND CONJUGATEDDIENES IN TISSUESOF RATS INJECTED WITH Ccl, OR BrCCl,”
Organ Liver
Kidney
Intestine
Spleen
Lung
Heart
Total conjugated dienes (Abm)
Compound
Total lipids bg)
Conjugated dienes (Abs,,,/mg lipid)
Control cc14 BrCCI,
167.3* 19.0b 183.3 k 50.4 186.4* 36.3
0.14+0.04 0.28 kO.01’ 0.53 f 0.06’
23.58 + 7.32 51.98 f 14.43d 97.53 k 7.89’
Control CCI, BrCCl,
36.7+ 12.0 31.8k3.5 31.5k4.2
0.11+0.03 0.19kO.04 0.27 f 0.05’
4.07 ? 2.24 5.99+ 1.07 8.38? 1.17’
5.4 5.5
Control ccl4 BrCCl,
164.2* 35.0 141.7* 18.5 128.Ok44.3
0.23 + 0.02 0.28 f 0.02d 0.26 + 0.03
37.42 + 6.77 40.38k8.18 32.82+ 8.83
8.8 -5.8
Control ccl, BrCCl,
9.8* 1.8 8.1 rt 2.0 7.6kO.4
0.13+0.02 0.18+0.01f 0.22 + 0.01’
1.23rt0.37 1.42kO.40 1.68 + 0.09
0.6 0.6
0.16f0.05 0.19+0.02 0.20+0.03
2.61 +O.Sl 2.83 _+0.32 3.39* 1.09
0.7 1.0
0.08 f 0.03 0.08 f 0.04 0.59 + 0.05e
0.73 f 0.28 0.72 f 0.47 4.51 i 1.51f
Control ccl, BrCCl, Control ccl, BrCCI,
17.1 k2.7 15.1 f0.4 16.5k3.4 9.2kO.8 8.6+ 1.0 9.2k2.2
Conjugated dienes (% of total)
84.6 94.0
-
-
-
-
-0.1
4.8
’ Rats were injected ip with 90 ,ul of test compound/l00 g body wt 30 min prior to killing. ’ Values are expressed as mean f SD. ‘p< 0.01, statistical significance between values indicated and the control values were determined by Student’s t test. dp co.05. ~p
286
SAGA1 AND TAPPEL
RESULTS Halomethane-InducedLipid Peroxidation
The time-response relationships for the appearance of expired pentane after administration of four halomethanes are shown in Fig. 1. The mean basal level of pentane for nine control rats administered only mineral oil was 0.21 kO.05 nmol of pentane/lOO g body wt/120 min. All experimental rats had increased pentane production by 15-30 min following administration of the halomethanes. The most potent prooxidant was BrCCl,, with a dose of 10 pl/lOO g causing the production of 2.25 f 0.20 nmol of pentane/lOO g/ 120 min. Ccl, given at doses of 10, 30, and 90 ,~I/100 g caused the production of 0.67 + 0.16, 2.07 kO.62, and 4.2kO.52 nmol of pentane/lOO g/120 min, respectively. There was a good dose-response relationship (r = 0.96, ~~0.01) between the amounts of Ccl, administered and the amounts of pentane produced. These values were significantly different (p
0012 0014 0016 , , _*.,?, _,^^^^,._.^..
r..r-^..
0 016
0 020
FIG. 2. Relationship between pentane production and bond dissociation energies of BrCCl,, Ccl,, and CHC&; r = 0.987, p
(0.56 + 0.04 nmol of pentane/lOO g/120 min) than did the mineral oil control. Pentane production (0.28 f 0.08 nmol/lOO g/120 min) by rats following administration of the same dose of CH,Cl, was not significantly different from the control value. The values of pentane production induced by each compound at each dosage level were determined with three rats. Relationship between Pentane Production and Halomethane Bond Dissociation Energies TIME
(MINI
FIG. 1. Time-response relationship for pentane production after ip administration of CH2ClZ, CHCl,, Ccl,, and BrCCl,. Data points and bars represent mean values f SD. There were nine rats in the control group and three rats in each of the test groups. The dose administered per 100 g was (0) 904 of CH,Cl,; (k 904 of CHCI,; (m) 304 of Ccl,; (0) 10~1 of BrCCl,; and (0) control administered mineral oil.
The relationship (r = 0.987, p< 0.001) between pentane production and bond dissociation energies of BrCCI,, Ccl,, and CHCl, is shown in Fig. 2. The pentane values used for these calculations were normalized to represent that produced following administration of 90 ~1 of halomethane/ 100 g. The value of pentane produced was also normalized to nanomoles per 100 g per
HALOMETHANE-INDUCED
LIPID
120 min per nanomole of halomethane because 9Opl of each was approximately 1 mmol. The amount of pentane produced by 90 ~1 of BrCClJlOO g was extrapolated from the dose-response curve of Ccl,. Rats administered 90 ~1 of BrCCl,/lOO g died within 120 min; therefore, actual measurements were not possible. There are several bases for this extrapolation. First, present evidence indicates that the mechanisms for the initiation of lipid peroxidation by BrCCl, and CCI, are similar. Second, the pentane produced is a linear function of the log of the CCI, dose (Sagai and Tappel, 1978a). A number of products related to the toxic action of halogenated hydrocarbons follow similar linear-log functions. An example is the accumulation of hepatic triglycerides, which are a linear function of the log of the CCI, and CHCl, doses (Klaassen and Plaa, 1969). As a check of the value obtained by extrapolation from the doseresponse curve of Ccl,, the value for pentane production was also extrapolated from the data obtained for conjugated dienes in liver following dosage with 90 ~1 of BrCCl, (Table 1, Fig. 3). A similar plot of pentane produced from 10 ,ul CCL, and BCCI, (Table
2) vs one/bond dissociation energy gives a relationship which is roughly parallel to that of Fig. 2.
LIVER CONJUGATED DIENES (pMOL/30MINl FIG. 3. Correlation betweenpentane production in vivo and formation of conjugated dienes in the liver.
Each data point representsthe mean value for three rats. Pentane was measured over a 30-min period following administration of Ccl,, BrCCl,, and CHt&; conjugated dienes were measured 30min after their administration. The dose administered/100 g was (0) 90~1 of CHCI,; (0) 10~1 of Ccl,; (0) 30~1 of Ccl,; (m) 90~1 of Ccl,; and (L) 10~1 of BrCC13; (A) 90 yl of BrCCl,. Pentane values for the liver were calculated as the product of the value for the whole body pentane and the percentage of the total conjugated dienes found in the liver (Table 1).
TABLE MOLAR RATIO BETWEEN PRODUCTION OF PENTANE DIENES IN LIVER FOLLOWING INTRAPERIMNEAL
Compound Mineral CHCI, ccl, ccl, CCI, BrCCI, BrCC!,
oil
Dose. administered per l@Jg 011)
287
PEROXIDATION
2 in Vivo AND FORMATION ADMINISTRATION
Pentane production (nmol/lOO g/ 30 min)
OF CONJUGATED OF HALOMETHANES
Total conjugated dienes (pmol/ 30 min/liver)
Molar ratio: pentane/ conjugated dienes” -
-
0.05 _+0.01 (9)b.C
0.84 f 0.26 (3)
90 10 30 90 10 90
0.16?0.02 0.23 + 0.07 0.66kO.16 1.32kO.12 0.63 rt 0.12 3.94* 0.73
0.87 1.65 1.87 1.86f0.52 1.64 3.48 +0.28
(3) (3) (3) (3) (3) (3)’
(2) (2) (2) (3)
(2) (3)
0.0062 0.0005 0.0014 0.0021 0.0014 0.0025
L1These v&es were calculated after first making corrections for control values obtained after administration of mineral oil only. b Values are expressed as mean + SD. c Values in parentheses represent the number of animals used. d This value was extrapolated from the dose-response curve for CCL.
288 Tissue Conjugated and BrCCl,
SAGA1
Dienes Induced by Ccl,
AND
TAPPEL
index of in vivo lipid peroxidation, and the measurement of conjugated dienes formed in the liver, a well-known index of in vitro lipid peroxidation induced by Ccl,, BrCCl,, and CHCl,.
Total lipids and conjugated dienes in liver, kidney, intestine, spleen, lung, and heart were determined in order to localize the principal organ responsible for the production of pentane following administration of Ccl, DISCUSSION or BrCCl, (Table 1). Total lipids in tissues Measurement of in viva pentane production from the treated animals were not significantly as an index of lipid peroxidation was first different from those in tissues of control animals. As measured by the increase in diene utilized with rats fed a vitamin E-deficient diet with a high linoleic acid content (Dillard conjugation over that in tissues from control animals, it was determined that liver ac- et al., 1977). This very sensitive index was counted for 85 and 94% of the increase in used in the present study to obtain time- and conjugated dienes measured following ad- dose-response data that lend further support to the theory that some halomethanes cause ministration of Ccl, and BrCCl,, respectively. The amount of conjugated dienes increased lipid peroxidation. The effect of vitamin E on the time- and dose-response relationships significantly also in kidney and heart followbetween Ccl, administered and pentane ing BrCCl, treatment. There was approxiproduced was reported earlier (Sagai and mately a sixfold increase in conjugated dienes in the heart following BrCCl, treat- Tappel, 1978a). The results of the pentane ment. No conjugated dienes could be de- time-response reported herein support the tected in lung. CHCl, and CH,Cl, did not findings of others (Rao and Recknagel, induce diene conjugation in any tissue when 1968 ; Klaassen and Plaa, 1969) that the maximal concentrations of conjugated diene up to 270 pl/lOO g was administered. products of lipid peroxidation induced by Molar Ratio between Pentane Produced and Ccl, in liver microsomes occurs at 15-30 Conjugated Dienes Formed min following dosage. The molar ratio between pentane proAmong the halomethanes tested, BrCCI, is duced in vivo and conjugated dienes formed the most potent prooxidant (Slater, 1972; in liver following administration of haloRecknagel et al., 1977) and Ccl, is the second genated methanes is shown in Table 2. The most potent. There is evidence (Klaassen and increase in pentane production was accom- Plaa, 1969; Reynolds and Yee, 1968; Cawpanied by an increase in conjugated dienes in thorne et al., 1971) that CHCl, produces little liver. As shown, the molar ratio was not or no lipid peroxidation. Plaa and Witschi constant, but it gradually increased with in- (1976) have pointed out, however, that failure creasing prooxidant potency and with the to detect thiobarbituric acid-reacting subdose of halogenated methane administered. stances in tissue extracts is not an indication The ratio of pentane produced ranged from that lipid peroxidation is absent. Our research 0.05 to 0.6% of the lipid peroxides measured has shown that pentane production in rats is inas conjugated dienes following injection of duced by halomethanes in the order BrCCl, > Ccl,, BrCCl,, and CHCl,. Ccl,> CHC13, with a 2.7-fold greater production being found after dosing with 90 ,ul of Correlation between Pentane Production and CHClJlOO g than after dosing with only Diene Conjugation, mineral oil. No increase in pentane proFigure 3 shows that there was a good duction was detected following administracorrelation (r = 0.96, p ~0.01) between the tion of CH,Cl,. From this study it was measurement of pentane production, a new possible to show a highly significant relation-
HALOMETHANE-INDUCED
ship between pentane production and the bond dissociation energies of these halomethanes. The data supported the concept that the -Ccl, radical induces lipid peroxidation. The fact that CH,CI, did not induce lipid peroxidation also adds support to the concept since a *Ccl, radical is not produced from CH,Cl,. The interaction of the *Ccl, radical with tissue macromolecules alters cellular integrity, which then leads to tissue necrosis. It is generally thought (Recknagel and Glende, 1973) that these active intermediates are produced by action of the hepatic microsomal P-450 system. Examination of conjugated dienes in liver kidney, intestine, spleen, lung, and heart following injection of rats with Ccl, or BrCCl, showed that liver is the principal organ in which lipid peroxidation, and hence pentane production, originates. This conclusion is based on the finding of 85% or more of the total conjugated dienes in the liver. Conjugated dienes represent lipid hydroperoxides, and pentane derives from o6-unsaturated lipid hydroperoxides. It must be emphasized that, except for lung, the amount of conjugated dienes increased in all tissues examined, indicating that a small amount of. lipid peroxidation was probably induced in these organs as well as in liver. The large increase in conjugated dienes in the heart suggests that there may be a specific effect of BrCCl, on heart. It is important to note that there was good correlation between pentane production, a new measurement of in uivo lipid peroxidation, and diene conjugation formation in liver, a known measurement of in vitro and in vivo lipid peroxidation. This correlation supports other evidence that pentane production is a good measurement of in vivo lipid peroxidation. Similar correlations were shown between ethane production and diene conjugation formation following treatment of animals with CCI, (Lindstrom and Anders, 1978), and between pentane production in vivo and formation of thiobarbituric acidreacting materials in tissues of rats fed
LIPID
PEROXIDATION
289
different amounts of dietary vitamin E and selenium (Sagai and Tappel, 1978b). Examination of the molar ratio between pentane production in z&o and formation of conjugated dienes from peroxides in liver is a useful aid to gain an understanding of the quantitative relationship between the two. The fact that the ratio with Ccl, became gradually higher as the amount of the prooxidant administered was increased shows that pentane production can be greater as the amount of peroxidation increases. At higher levels of in vivo hydroperoxide formation, the competition of the two reactions for hydroperoxide removal, mainly the glutathione peroxidase reduction and the very small amounts of hydroperoxide decomposition to pentane, favors an increased proportion of pentane production compared to conjugated dienes. Approximately 0.05-0.6 % of the lipid peroxides underwent /l-scission to form pentane. The fact that these ratios are lower than those found for iron-catalyzed decomposition of linoleate and arachidonate hydroperoxides in vitro (Dumelin and Tappel, !977) can be related to the concurrent reduction of hydroperoxides by the giutathione peroxidase system in vivo. Analysis of expired pentane is advantageous in that it is a nondestructive method that is applicable to a wide variety of studies of experimental pathology and toxicology, and it is an easy, economical, and direct measurement of products of lipid peroxidation. This method has been applied successfully in several studies to show the occurrence of in vivo lipid peroxidation. It has been used to show the effect of vitamin E (Dillard et al., 1977) or combinations of vitamin E, selenium, and polyunsaturated fats on in vivo lipid peroxidation in rats (Dillard et al., 1978); to show the effect of feeding rats a nonbiological antioxidant (Downey et al., 1978); to demonstrate that lipid peroxidation is a mechanism involved in acute ethanol toxicity (Litov et al., 1978); to show the effect of exercise, vitamin E, and ozone on lipid peroxidation in humans (Dillard et al.,
290
SAGA1 AND TAPPEL
1979) and of ozone on rats (Dumelin et al., 1978); and to study the effect of vitamin E on Ccl,-induced in uiuo lipid peroxidation in rats (Sagai and Tappel, 1978a). The method should be useful for similar applications in future research. REFERENCES BURDINO, E., GRAVELA, E., UGAZIO, G., VANNINI, V., AND CALLIGARO, A. (1973). Initiation of free radical reactions and hepatotoxicity in rats poisoned with carbon tetrachloride or bromotrichloromethane. Agents Actions 3, 244-253. CAWTHORNE, M. A., PALMER, E. D., BUNYAN, J., AND GREEN, J. (1971). In uiuo effects of carbon tetrachloride and chloroform on liver and kidney glucose-6-phosphatase in mice. Biochem. Pharmacol. 20,4941196.
DILLARD, C. J., DUMELIN, E. E., AND TAPPEL, A. L. (1977). Effect of dietary vitamin E on expiration of pentane and ethane by the rat. Lipids 12, 109-l 14. DILLARD, C. J., LITOV, R. E., SAVIN, W. M., DUMELIN, E. E., AND TAPPEL, A. L. (1978). Effects of exercise, vitamin E, and ozone on pulmonary function and lipid peroxidation. Respirat. Environ. Exercise Physiol. 45, 927-932. DILLARD, C. J., LITOV, R. E., AND TAPPEL, A. L. (1978). Effects of dietary vitamin E, selenium, and polyunsaturated fats on in vioo lipid peroxidation in the rat as measured by pentane production. Lipids 13, 396-402. DOWNEY, J. E., IRVING, D. H., AND TAPPEL, A. L. (1978). Effects of dietary antioxidants on in viva lipid peroxidation in the rat as measured by pentane production. Lipids 13, 403407. DUMELIN, E. E., AND TAPPEL, A. L. (1977). Hydrocarbon gases produced during in vitro peroxidation of polyunsaturated fatty acids and decomposition of preformed hydroperoxides. Lipids 12, 894-900. DUMELIN, E. E., DILLARD, C. J., AND TAPPEL, A. L. (1978). The effect of vitamin E and ozone on pentane and ethane expired by rats. Arch. Environ. Health 33, 129-l 34. EVANS, C. D., LIST, G. R., DOLEV, A., MCCONNELL, D. G., AND HOFFMAN, R. L. (1967). Pentane from thermal decomposition of lipoxidase-derived products. Lipids 2, 432434. EVANS, C. D., LIST, G. R., HOFFMAN, R. L., AND MOSER, H. H. (1969). Edible oil quality as measured by thermal release of pentane. J. Amer. Oil Chem. sot. 46, 501-504. FRANKEL, E. N., NOWAKOWSKA, J., AND EVANS, C. D. (1961). Formation of methyl azelaaldehydate on autoxidation of Iipides. J. Amer. Oil Chem. Sot. 38, 161-162.
FRINGS, C. S., AND DUNN, R. T. (1970). Colorimetric method for determination of total serum lipids based on the sulfo-phospho-vanillin reaction. Amer. J. Clin. Pathol. 53, 89-91. HAFEMAN, D. G., AND HOEKSTRA, W. G. (1977a). Protection against carbon tetrachloride-induced lipid peroxidation in the rat by dietary vitamin E, selenium and methionine as measured by ethane evolution. J. Nutr. 107, 656-665. HAFEMAN, D. G., AND HOEKSTRA, W. G. (1977b). Lipid peroxidation in viuo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution. J. Nutr. 107, 666-672. HASHIMOTO, S., AND RECKNAGEL, R. 0. (1968). No chemical evidence of hepatic lipid peroxidation in acute ethanol toxicity. Exp. Mol. Pathol. 8, 225242.
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LINDSTROM, T. D., AND ANDERS, M. W. (1978). Effect of agents known to alter carbon tetrachloride hepatotoxicity and cytochrome P-450 levels on carbon tetrachloride-stimulated lipid peroxidation and ethane expiration in the intact rat. Biochem. Pharmacol. 27, 563-567. LITOV, R. E., IRVING, D. H., DOWNEY, J. E., AND TAPPEL, A. L. (1978). Lipid peroxidation: A mechanism involved in acute ethanol toxicity as demonstrated by in vivo pentane production in the rat. Lipids 13, 305-307. PLAA, G. L., AND WITSCHI, H. (1976). Chemicals, drugs, and lipid peroxidation. Annu. Rev. Pharmacol. Toxicol. 16, 125-141. RAO, K. S., AND RECKNAGEL, R. 0. (1968). Early onset of lipoperoxidation in rat liver after carbon tetrachloride administration. Exp. Mol. Pathol. 9, 271-278. RECKNAGEL, R. O., AND GLENDE, E. A., JR. (1973). Carbon tetrachloride hepatotoxicity : An example of a lethal cleavage. CRC Crir. Rev. Toxicol. 2, 263297.
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LIPID PEROXIDATION
291
intact animals. Igaku No Ayumi 106, 681-683. [In Japanese]. SLATER, T. F. (1972). Free Radical Mechanisms in Tissue Znjury. Pion Limited, London. SLATER, T. F., AND SAWYER, B. C. (1971). The stimulatory effects of carbon tetrachloride and other halogen0 alkanes on peroxidative reactions in rat liver fractions in vitro. Biochem. J. 123, 805-814. SMOUSE, T. H., MOOKHERJEE, B. D., AND CHANG, S. S. (1965). Identification of dec-1-yne in the initial autoxidation products of vegetable oils. Chem. Ind. (London) 29, 1301-1303. TAPPEL, A. L. (1975). Lipid peroxidation and fluorescent molecular damage to membranes. In Pathobiology of Cell Membranes (B. F. Trump and A. U. Arstila, eds.), Vol. I, pp. 145-170. Academic Press, New York. WALLING, C. (1957). Free Radicals in Solution. Wiley, New York.
AND
APPLIED
PHARMACOLOGY
49,283-291
(1979)
Lipid Peroxidation Induced by Some Halomethanes as Measured by in Vivo Pentane Production in the Rat’ MASARU SAGA? AND AL L. TAPPEL Department
of Food Science and Technology, University of California, Davis, California 95616
Received September 29, 1978; accepted January 15, 1979 Lipid Peroxidation Induced by Some Halomethanes as Measured by in Vivo Pentane Production in the Rat. SAGAI, M., AND TAPPEL, A. L. (1979). Toxicol. Appt. Pharmacol. 49, 283-291. Pentane production in vivo in rats was used successfully as an index to support the concept that lipid peroxidation is involved in the toxicity of some halomethanes. The time-response relationships showed that the rats had maximum pentane production by 15-30 min following ip administration of carbon tetrachloride (Ccl,), bromotrichloromethane (BrCCI,), and chloroform (CHCl& BrCCI, and Ccl, caused the production of the greatest amounts of pentane, CHCll induced a smaller amount of pentane production, and dichloromethane (CH#&) did not increase pentane production over that caused by injection of the mineral oil carrier. There was a good relationship (r = 0.987; p
Peroxidation of biological membrane lipids is considered an integral part of cell damage and of many toxic processes (Tappel, 1975). Several halogenated hydrocarbons induce lipid peroxidation in viuo and in vitro (Slater, * Supported by research Grant AM-09933 from the National Institute of Arthritis, Metabolism and Digestive Diseases, U.S. Department of Health, Education and Welfare, and a grant-in-aid from The National Live Stock and Meat Board. z Present address: Division of Basic Medical Sciences, The National Institute for Environmental Studies, P.O. Yatabe-Tsukuba, Ibaraki 300-21, Japan. 283
1972). Carbon tetrachloride (Ccl,) has been thoroughly investigated as a typical halogenated hydrocarbon, and there is considerable evidence that lipid peroxidation is the principal cause of the hepatotoxicity produced by this compound (Recknagel and Glende, 1973). Slater and Sawyer (1971) reported that bromotrichloromethane (BrCCl,) is a more potent prooxidant than Ccl, and that it produces severe liver injury. It is well known that CHCl, also produces liver injury similar to that produced by Ccl,, but that it does not induce lipid per0041-008X/79/0802834%02.tW/0 Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
284
SAGA1
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TAPPEL
oxidation (Klaassen and Plaa, 1969; Rey- dation than ethane in rats fed a vitamin Enolds and Yee, 1968; Cawthorne et al., deficient diet with a high linoleic acid content. 1971). In the present study, pentane measurements There is evidence that liver injury depends were made to obtain data to support the on the in vivo biotransformation of the ad- concept that lipid peroxidation can be inministered halomethanes, namely, the split- duced in vivo by some halomethanes. Conting of the covalent bond between carbon jugated dienes in rat tissues were correlated and halogen or carbon and hydrogen. The with pentane produced in vivo. The question tendency of a halomethane molecule to of what are the main organs of origin for the cleave homolytically and to initiate reactions pentane produced following treatment of rats that involve free radicals as intermediates with Ccl, and BrCCl, was examined. depends on its bond dissociation energy. For instance, the suggested order of relative METHODS activities in stimulating lipid peroxidation is Animals and treatments. Male Sprague-Dawley BrCCl, > Ccl, > CHCI, > CFCl, (Burdino et rats 7-10 weeks of age and weighing 193-283 g were al., 1973). This series corresponds inversely to the series of bond dissociation energies of housed three to a cage in hanging wire-mesh cages in a room kept at a constant temperature of 22-23°C. these compounds, which are 49, 68, 90, and They were subjected to a daily cycle of 14 hr of light 103 kcal/mol, respectively (Walling, 1957). and 10 hr of dark. The rats were fed a stock diet,3 The most probable free radical species is the and they were fasted overnight prior to the measuretrichloromethyl radical (* Ccl,), and this ment of pentane and conjugated dienes. They were injected ip with 10, 30, or 90 ~1 of BrCC1,,4 CCld,s free radical is considered to induce lipid CHC13,* or CH,Cl, 6/1OOg body wt. The haloperoxidation. methanes were administered as a 10 or 30% solution Hydrocarbon gases have long been known in mineral oil. The rats were decapitated 30 min after to appear early during the autoxidation of injection to obtain tissues for the measurement of edible fats (Frankel et al., 1961; Horvat et al., conjugated dienes. Each tissue to be examined was removed from the rat immediately, washed with cold 1964; Smouse et al., 1965). Ethane is pro- physiological saline, and weighed. duced during autoxidation of linolenic acid Analysis of pentane. Gas chromatographic analysis (Lieberman and Mapson, 1964). Riely et al. of pentane was done on an alumina column according (1974) advanced the use of hydrocarbon gas to the method described by Dillard et al. (1977). A with a flame-ionization deanalysis when they applied the method to gas chromatograph’ tector and fitted with a six-way, nut-type gas sample biological systems. They showed that ethane valve was used. The rats were placed in a holding production was characteristic of sponta- chamber (Dillard et al., 1977) to breathe hydrocarbonneously peroxidizing mouse tissues in vitro free air8 for 10 min prior to collection of the samples. and that Ccl, provoked formation of ethane One-half of the breath+air stream (120 mI/min) in vivo in mice. Since that time, other in- from each rat was collected over a IO-min period to a 600-ml sample in an activated alumina-filled vestigators (Hafeman and Hoekstra, 1977a,b; give gas sample loop immersed in liquid nitrogen-ethanol Koster et al., 1977; Lindstrom and Anders, at about - 130°C. Breath samples were collected at 15, 1978) have used ethane production to follow 30, 60, 90, and 120 min following injection of the halomethanes. The gas chromatograph was stanthe course of lipid peroxidation in vivo. Pentane was shown to be the predominant 3 Purina Rat Chow, Ralston, Purina Company, St. short chain hydrocarbon gas to arise during Louis, MO. thermal decomposition (Evans et al., 1967, 4 Eastman Kodak Company, Rochester, N.Y. 5 Mallinckrodt Chemical Works, St. Louis, MO. 1969) and iron-catalyzed decomposition 6 Aldrich Chemical Company, Inc., Milwaukee, (Dumelin and Tappel, 1977) of linoleic and arachidonic acid hydroperoxides. The work Wis. ’ Varian Associates, Palo Alto, Calif. of Dillard et al. (1977) indicates that pen8 Linde Division, Union Carbide Corporation, tane is a more sensitive index of lipid peroxi- South Plainfield, N.J.
HALOMETHANE-INDUCED
LIPID
dardized with l-ml aliquots of 0.8 ppm pentaneg in nitrogen at an electrometer setting of 4 x lo-i2 amp/ mV. The relative peak area of pentane on each chromatograph was calculated by triangulation. A comparison of the peak area obtained for the standard and for the breath sample allowed calculation of the picomol of pentane produced. The following computation was applied :
285
PEROXIDATION
0.003 M EDTA, pH 7.4, with a glass and Teflon homogenizer. A 5-ml aliquot of each homogenate was transferred to a 1%ml round-bottom centrifuge bottle and 20 ml of chloroform : methanol (2 : 1) was added. The procedure used for extraction of total lipids was that described by Hashimoto and Recknagel (1968). The recovered lipids were measured by the sulfo-phosphovanillin method (Frings and Dunn, 1970). Aliquots of the lipids were dissolved in methanol at a concentration of 1 mg/ml and were examined in a uv spectrophotometer from 220 to 300 nm. The absorbance at 237 nm was determined with small corrections being made for slight turbidity in the samples. Turbidity was determined by measurement of absorbance at 260 nm. Total conjugated dienes (Table 1) is the product of total lipids of each organ and absorbance at 237 nm/mg of lipid. Conjugated dienes were converted to a molar basis using the molecular extinction coefficient.
(pm01 pentane) (incoming flowrate of air) I (100 g body wt) (sample volume) pmol pentane/lOO g body wt/min. The amount of pentane produced during 120 min by a rat was calculated by integration of the values obtained at each of the time periods. Measurement of conjugated dienes. A 15 % homogenate of each tissue was prepared in 0.3 M sucrose9 Matheson, Newark, Calif. TABLE
1
TOTAL LIPIDS AND CONJUGATEDDIENES IN TISSUESOF RATS INJECTED WITH Ccl, OR BrCCl,”
Organ Liver
Kidney
Intestine
Spleen
Lung
Heart
Total conjugated dienes (Abm)
Compound
Total lipids bg)
Conjugated dienes (Abs,,,/mg lipid)
Control cc14 BrCCI,
167.3* 19.0b 183.3 k 50.4 186.4* 36.3
0.14+0.04 0.28 kO.01’ 0.53 f 0.06’
23.58 + 7.32 51.98 f 14.43d 97.53 k 7.89’
Control CCI, BrCCl,
36.7+ 12.0 31.8k3.5 31.5k4.2
0.11+0.03 0.19kO.04 0.27 f 0.05’
4.07 ? 2.24 5.99+ 1.07 8.38? 1.17’
5.4 5.5
Control ccl4 BrCCl,
164.2* 35.0 141.7* 18.5 128.Ok44.3
0.23 + 0.02 0.28 f 0.02d 0.26 + 0.03
37.42 + 6.77 40.38k8.18 32.82+ 8.83
8.8 -5.8
Control ccl, BrCCl,
9.8* 1.8 8.1 rt 2.0 7.6kO.4
0.13+0.02 0.18+0.01f 0.22 + 0.01’
1.23rt0.37 1.42kO.40 1.68 + 0.09
0.6 0.6
0.16f0.05 0.19+0.02 0.20+0.03
2.61 +O.Sl 2.83 _+0.32 3.39* 1.09
0.7 1.0
0.08 f 0.03 0.08 f 0.04 0.59 + 0.05e
0.73 f 0.28 0.72 f 0.47 4.51 i 1.51f
Control ccl, BrCCl, Control ccl, BrCCI,
17.1 k2.7 15.1 f0.4 16.5k3.4 9.2kO.8 8.6+ 1.0 9.2k2.2
Conjugated dienes (% of total)
84.6 94.0
-
-
-
-
-0.1
4.8
’ Rats were injected ip with 90 ,ul of test compound/l00 g body wt 30 min prior to killing. ’ Values are expressed as mean f SD. ‘p< 0.01, statistical significance between values indicated and the control values were determined by Student’s t test. dp co.05. ~p
286
SAGA1 AND TAPPEL
RESULTS Halomethane-InducedLipid Peroxidation
The time-response relationships for the appearance of expired pentane after administration of four halomethanes are shown in Fig. 1. The mean basal level of pentane for nine control rats administered only mineral oil was 0.21 kO.05 nmol of pentane/lOO g body wt/120 min. All experimental rats had increased pentane production by 15-30 min following administration of the halomethanes. The most potent prooxidant was BrCCl,, with a dose of 10 pl/lOO g causing the production of 2.25 f 0.20 nmol of pentane/lOO g/ 120 min. Ccl, given at doses of 10, 30, and 90 ,~I/100 g caused the production of 0.67 + 0.16, 2.07 kO.62, and 4.2kO.52 nmol of pentane/lOO g/120 min, respectively. There was a good dose-response relationship (r = 0.96, ~~0.01) between the amounts of Ccl, administered and the amounts of pentane produced. These values were significantly different (p
0012 0014 0016 , , _*.,?, _,^^^^,._.^..
r..r-^..
0 016
0 020
FIG. 2. Relationship between pentane production and bond dissociation energies of BrCCl,, Ccl,, and CHC&; r = 0.987, p
(0.56 + 0.04 nmol of pentane/lOO g/120 min) than did the mineral oil control. Pentane production (0.28 f 0.08 nmol/lOO g/120 min) by rats following administration of the same dose of CH,Cl, was not significantly different from the control value. The values of pentane production induced by each compound at each dosage level were determined with three rats. Relationship between Pentane Production and Halomethane Bond Dissociation Energies TIME
(MINI
FIG. 1. Time-response relationship for pentane production after ip administration of CH2ClZ, CHCl,, Ccl,, and BrCCl,. Data points and bars represent mean values f SD. There were nine rats in the control group and three rats in each of the test groups. The dose administered per 100 g was (0) 904 of CH,Cl,; (k 904 of CHCI,; (m) 304 of Ccl,; (0) 10~1 of BrCCl,; and (0) control administered mineral oil.
The relationship (r = 0.987, p< 0.001) between pentane production and bond dissociation energies of BrCCI,, Ccl,, and CHCl, is shown in Fig. 2. The pentane values used for these calculations were normalized to represent that produced following administration of 90 ~1 of halomethane/ 100 g. The value of pentane produced was also normalized to nanomoles per 100 g per
HALOMETHANE-INDUCED
LIPID
120 min per nanomole of halomethane because 9Opl of each was approximately 1 mmol. The amount of pentane produced by 90 ~1 of BrCClJlOO g was extrapolated from the dose-response curve of Ccl,. Rats administered 90 ~1 of BrCCl,/lOO g died within 120 min; therefore, actual measurements were not possible. There are several bases for this extrapolation. First, present evidence indicates that the mechanisms for the initiation of lipid peroxidation by BrCCl, and CCI, are similar. Second, the pentane produced is a linear function of the log of the CCI, dose (Sagai and Tappel, 1978a). A number of products related to the toxic action of halogenated hydrocarbons follow similar linear-log functions. An example is the accumulation of hepatic triglycerides, which are a linear function of the log of the CCI, and CHCl, doses (Klaassen and Plaa, 1969). As a check of the value obtained by extrapolation from the doseresponse curve of Ccl,, the value for pentane production was also extrapolated from the data obtained for conjugated dienes in liver following dosage with 90 ~1 of BrCCl, (Table 1, Fig. 3). A similar plot of pentane produced from 10 ,ul CCL, and BCCI, (Table
2) vs one/bond dissociation energy gives a relationship which is roughly parallel to that of Fig. 2.
LIVER CONJUGATED DIENES (pMOL/30MINl FIG. 3. Correlation betweenpentane production in vivo and formation of conjugated dienes in the liver.
Each data point representsthe mean value for three rats. Pentane was measured over a 30-min period following administration of Ccl,, BrCCl,, and CHt&; conjugated dienes were measured 30min after their administration. The dose administered/100 g was (0) 90~1 of CHCI,; (0) 10~1 of Ccl,; (0) 30~1 of Ccl,; (m) 90~1 of Ccl,; and (L) 10~1 of BrCC13; (A) 90 yl of BrCCl,. Pentane values for the liver were calculated as the product of the value for the whole body pentane and the percentage of the total conjugated dienes found in the liver (Table 1).
TABLE MOLAR RATIO BETWEEN PRODUCTION OF PENTANE DIENES IN LIVER FOLLOWING INTRAPERIMNEAL
Compound Mineral CHCI, ccl, ccl, CCI, BrCCI, BrCC!,
oil
Dose. administered per l@Jg 011)
287
PEROXIDATION
2 in Vivo AND FORMATION ADMINISTRATION
Pentane production (nmol/lOO g/ 30 min)
OF CONJUGATED OF HALOMETHANES
Total conjugated dienes (pmol/ 30 min/liver)
Molar ratio: pentane/ conjugated dienes” -
-
0.05 _+0.01 (9)b.C
0.84 f 0.26 (3)
90 10 30 90 10 90
0.16?0.02 0.23 + 0.07 0.66kO.16 1.32kO.12 0.63 rt 0.12 3.94* 0.73
0.87 1.65 1.87 1.86f0.52 1.64 3.48 +0.28
(3) (3) (3) (3) (3) (3)’
(2) (2) (2) (3)
(2) (3)
0.0062 0.0005 0.0014 0.0021 0.0014 0.0025
L1These v&es were calculated after first making corrections for control values obtained after administration of mineral oil only. b Values are expressed as mean + SD. c Values in parentheses represent the number of animals used. d This value was extrapolated from the dose-response curve for CCL.
288 Tissue Conjugated and BrCCl,
SAGA1
Dienes Induced by Ccl,
AND
TAPPEL
index of in vivo lipid peroxidation, and the measurement of conjugated dienes formed in the liver, a well-known index of in vitro lipid peroxidation induced by Ccl,, BrCCl,, and CHCl,.
Total lipids and conjugated dienes in liver, kidney, intestine, spleen, lung, and heart were determined in order to localize the principal organ responsible for the production of pentane following administration of Ccl, DISCUSSION or BrCCl, (Table 1). Total lipids in tissues Measurement of in viva pentane production from the treated animals were not significantly as an index of lipid peroxidation was first different from those in tissues of control animals. As measured by the increase in diene utilized with rats fed a vitamin E-deficient diet with a high linoleic acid content (Dillard conjugation over that in tissues from control animals, it was determined that liver ac- et al., 1977). This very sensitive index was counted for 85 and 94% of the increase in used in the present study to obtain time- and conjugated dienes measured following ad- dose-response data that lend further support to the theory that some halomethanes cause ministration of Ccl, and BrCCl,, respectively. The amount of conjugated dienes increased lipid peroxidation. The effect of vitamin E on the time- and dose-response relationships significantly also in kidney and heart followbetween Ccl, administered and pentane ing BrCCl, treatment. There was approxiproduced was reported earlier (Sagai and mately a sixfold increase in conjugated dienes in the heart following BrCCl, treat- Tappel, 1978a). The results of the pentane ment. No conjugated dienes could be de- time-response reported herein support the tected in lung. CHCl, and CH,Cl, did not findings of others (Rao and Recknagel, induce diene conjugation in any tissue when 1968 ; Klaassen and Plaa, 1969) that the maximal concentrations of conjugated diene up to 270 pl/lOO g was administered. products of lipid peroxidation induced by Molar Ratio between Pentane Produced and Ccl, in liver microsomes occurs at 15-30 Conjugated Dienes Formed min following dosage. The molar ratio between pentane proAmong the halomethanes tested, BrCCI, is duced in vivo and conjugated dienes formed the most potent prooxidant (Slater, 1972; in liver following administration of haloRecknagel et al., 1977) and Ccl, is the second genated methanes is shown in Table 2. The most potent. There is evidence (Klaassen and increase in pentane production was accom- Plaa, 1969; Reynolds and Yee, 1968; Cawpanied by an increase in conjugated dienes in thorne et al., 1971) that CHCl, produces little liver. As shown, the molar ratio was not or no lipid peroxidation. Plaa and Witschi constant, but it gradually increased with in- (1976) have pointed out, however, that failure creasing prooxidant potency and with the to detect thiobarbituric acid-reacting subdose of halogenated methane administered. stances in tissue extracts is not an indication The ratio of pentane produced ranged from that lipid peroxidation is absent. Our research 0.05 to 0.6% of the lipid peroxides measured has shown that pentane production in rats is inas conjugated dienes following injection of duced by halomethanes in the order BrCCl, > Ccl,, BrCCl,, and CHCl,. Ccl,> CHC13, with a 2.7-fold greater production being found after dosing with 90 ,ul of Correlation between Pentane Production and CHClJlOO g than after dosing with only Diene Conjugation, mineral oil. No increase in pentane proFigure 3 shows that there was a good duction was detected following administracorrelation (r = 0.96, p ~0.01) between the tion of CH,Cl,. From this study it was measurement of pentane production, a new possible to show a highly significant relation-
HALOMETHANE-INDUCED
ship between pentane production and the bond dissociation energies of these halomethanes. The data supported the concept that the -Ccl, radical induces lipid peroxidation. The fact that CH,CI, did not induce lipid peroxidation also adds support to the concept since a *Ccl, radical is not produced from CH,Cl,. The interaction of the *Ccl, radical with tissue macromolecules alters cellular integrity, which then leads to tissue necrosis. It is generally thought (Recknagel and Glende, 1973) that these active intermediates are produced by action of the hepatic microsomal P-450 system. Examination of conjugated dienes in liver kidney, intestine, spleen, lung, and heart following injection of rats with Ccl, or BrCCl, showed that liver is the principal organ in which lipid peroxidation, and hence pentane production, originates. This conclusion is based on the finding of 85% or more of the total conjugated dienes in the liver. Conjugated dienes represent lipid hydroperoxides, and pentane derives from o6-unsaturated lipid hydroperoxides. It must be emphasized that, except for lung, the amount of conjugated dienes increased in all tissues examined, indicating that a small amount of. lipid peroxidation was probably induced in these organs as well as in liver. The large increase in conjugated dienes in the heart suggests that there may be a specific effect of BrCCl, on heart. It is important to note that there was good correlation between pentane production, a new measurement of in uivo lipid peroxidation, and diene conjugation formation in liver, a known measurement of in vitro and in vivo lipid peroxidation. This correlation supports other evidence that pentane production is a good measurement of in vivo lipid peroxidation. Similar correlations were shown between ethane production and diene conjugation formation following treatment of animals with CCI, (Lindstrom and Anders, 1978), and between pentane production in vivo and formation of thiobarbituric acidreacting materials in tissues of rats fed
LIPID
PEROXIDATION
289
different amounts of dietary vitamin E and selenium (Sagai and Tappel, 1978b). Examination of the molar ratio between pentane production in z&o and formation of conjugated dienes from peroxides in liver is a useful aid to gain an understanding of the quantitative relationship between the two. The fact that the ratio with Ccl, became gradually higher as the amount of the prooxidant administered was increased shows that pentane production can be greater as the amount of peroxidation increases. At higher levels of in vivo hydroperoxide formation, the competition of the two reactions for hydroperoxide removal, mainly the glutathione peroxidase reduction and the very small amounts of hydroperoxide decomposition to pentane, favors an increased proportion of pentane production compared to conjugated dienes. Approximately 0.05-0.6 % of the lipid peroxides underwent /l-scission to form pentane. The fact that these ratios are lower than those found for iron-catalyzed decomposition of linoleate and arachidonate hydroperoxides in vitro (Dumelin and Tappel, !977) can be related to the concurrent reduction of hydroperoxides by the giutathione peroxidase system in vivo. Analysis of expired pentane is advantageous in that it is a nondestructive method that is applicable to a wide variety of studies of experimental pathology and toxicology, and it is an easy, economical, and direct measurement of products of lipid peroxidation. This method has been applied successfully in several studies to show the occurrence of in vivo lipid peroxidation. It has been used to show the effect of vitamin E (Dillard et al., 1977) or combinations of vitamin E, selenium, and polyunsaturated fats on in vivo lipid peroxidation in rats (Dillard et al., 1978); to show the effect of feeding rats a nonbiological antioxidant (Downey et al., 1978); to demonstrate that lipid peroxidation is a mechanism involved in acute ethanol toxicity (Litov et al., 1978); to show the effect of exercise, vitamin E, and ozone on lipid peroxidation in humans (Dillard et al.,
290
SAGA1 AND TAPPEL
1979) and of ozone on rats (Dumelin et al., 1978); and to study the effect of vitamin E on Ccl,-induced in uiuo lipid peroxidation in rats (Sagai and Tappel, 1978a). The method should be useful for similar applications in future research. REFERENCES BURDINO, E., GRAVELA, E., UGAZIO, G., VANNINI, V., AND CALLIGARO, A. (1973). Initiation of free radical reactions and hepatotoxicity in rats poisoned with carbon tetrachloride or bromotrichloromethane. Agents Actions 3, 244-253. CAWTHORNE, M. A., PALMER, E. D., BUNYAN, J., AND GREEN, J. (1971). In uiuo effects of carbon tetrachloride and chloroform on liver and kidney glucose-6-phosphatase in mice. Biochem. Pharmacol. 20,4941196.
DILLARD, C. J., DUMELIN, E. E., AND TAPPEL, A. L. (1977). Effect of dietary vitamin E on expiration of pentane and ethane by the rat. Lipids 12, 109-l 14. DILLARD, C. J., LITOV, R. E., SAVIN, W. M., DUMELIN, E. E., AND TAPPEL, A. L. (1978). Effects of exercise, vitamin E, and ozone on pulmonary function and lipid peroxidation. Respirat. Environ. Exercise Physiol. 45, 927-932. DILLARD, C. J., LITOV, R. E., AND TAPPEL, A. L. (1978). Effects of dietary vitamin E, selenium, and polyunsaturated fats on in vioo lipid peroxidation in the rat as measured by pentane production. Lipids 13, 396-402. DOWNEY, J. E., IRVING, D. H., AND TAPPEL, A. L. (1978). Effects of dietary antioxidants on in viva lipid peroxidation in the rat as measured by pentane production. Lipids 13, 403407. DUMELIN, E. E., AND TAPPEL, A. L. (1977). Hydrocarbon gases produced during in vitro peroxidation of polyunsaturated fatty acids and decomposition of preformed hydroperoxides. Lipids 12, 894-900. DUMELIN, E. E., DILLARD, C. J., AND TAPPEL, A. L. (1978). The effect of vitamin E and ozone on pentane and ethane expired by rats. Arch. Environ. Health 33, 129-l 34. EVANS, C. D., LIST, G. R., DOLEV, A., MCCONNELL, D. G., AND HOFFMAN, R. L. (1967). Pentane from thermal decomposition of lipoxidase-derived products. Lipids 2, 432434. EVANS, C. D., LIST, G. R., HOFFMAN, R. L., AND MOSER, H. H. (1969). Edible oil quality as measured by thermal release of pentane. J. Amer. Oil Chem. sot. 46, 501-504. FRANKEL, E. N., NOWAKOWSKA, J., AND EVANS, C. D. (1961). Formation of methyl azelaaldehydate on autoxidation of Iipides. J. Amer. Oil Chem. Sot. 38, 161-162.
FRINGS, C. S., AND DUNN, R. T. (1970). Colorimetric method for determination of total serum lipids based on the sulfo-phospho-vanillin reaction. Amer. J. Clin. Pathol. 53, 89-91. HAFEMAN, D. G., AND HOEKSTRA, W. G. (1977a). Protection against carbon tetrachloride-induced lipid peroxidation in the rat by dietary vitamin E, selenium and methionine as measured by ethane evolution. J. Nutr. 107, 656-665. HAFEMAN, D. G., AND HOEKSTRA, W. G. (1977b). Lipid peroxidation in viuo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution. J. Nutr. 107, 666-672. HASHIMOTO, S., AND RECKNAGEL, R. 0. (1968). No chemical evidence of hepatic lipid peroxidation in acute ethanol toxicity. Exp. Mol. Pathol. 8, 225242.
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