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Inhibitory actions of butylated hydroxytoluene on isolated ileal, atrial, and perfused heart preparations
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
49, 45-52 (1979)
Inhibitory Actions of Butylated Hydroxytoluene on Isolated flea& Atrial, and Perfused Heart Preparations SHAYNE C. GAD,~ STEVEN W. LESLIE,~ AND DANIEL ACOSTA~ Chemical Hygiene Fellowship, Carnegie-Mellon Institute of Research, Pittsburgh, Pennsylvania 15213, and Department of Pharmacology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712
Received August I, 1978; accepted November 8, 1978
Inhibitory Actions of Butylated Hydroxytoluene on Isolated Ileal, Atrial, and Perfused Heart Preparations. GAD, S. C., LESLIE, S. W., AND ACOSTA, D. (1979). Toxicol. Appl. Pharmacol. 49, 45-52. Butylated hydroxytoluene (BHT), in concentrations of 500, 100, and 10 mg/liter (but not 1 mg/liter), significantly depressed methacholine-induced contractions of isolated rat and rabbit ileal preparations. BHT, in concentrations of 500, 100, 10, and 1 mg/liter, significantly (p < 0.05) depressed the frequency and amplitude (force) of contractions of isolated atria1 preparations as compared to controls. Control rat and rabbit atria1 beating rates were monitored continuously for 120 min (although isolated control atria1 preparations continue to beat spontaneously for several hours). BHT at 500 mg/liter terminated spontaneous contractions of isolated rabbit atria after 20 min and after 72 min when tested on rat atria. Although concentrations below 500 mg/liter did not terminate atria1 beating activity, they did produce a pronounced concentration-dependent depression of rate and force of atria1 contractions. The most pronounced depression of atria1 contractions produced by BHT were always seen within the first 30 min (out of a period up to 120 min) of exposure but the depression persisted throughout the exposure period. Similar significant dose-related depressions were seen using the same concentrations in intact perfused rat heart preparations. Additionally, perfused hearts treated with BHT (in concentrations from 1 to 500 mg/liter) showed significant dose-related increases in creatine phosphokinase leakage into the perfusion solutions.
Antioxidants are commonly used in food products to prevent (by a free radical mechanism) the autoxidation of fats and oils, a process which begins with the fixation of a molecule of oxygen to an unsaturated fat chain (Johnson, 1971). The broad distribution and the large consumption of food products containing antioxidants leaves vir-
tually no human being in the U.S. unexposed to these agents. Earlier studies showed that sodium bisulfite, an antioxidant “generally regarded as safe” by the Food and Drug Administration, depressed the contractility of isolated rat ileum and atrium (Gad et al., 1977) and that butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) had significant cytotoxic effects on cultured rat heart cells (Leslie et al., 1978). The present investigation examined the implications of toxicity seen in these earlier studies.
I To whom all correspondence should be addressed. 2 Department of Pharmacology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712. 45
All
0041-008X/79/070045~8s02.~/0 Copyright 0 1979 by Academic Press. Inc. rights of reproduction in any form reserved. Printed in Great Britain
46
GAD,
LESLIE,
METHODS Animals. Male, Sprague-Dawley rats (250-350 g) and male, white New Zealand, rabbits (2-2.5 kg) were used as source animals for tissue preparations. They were fed Purina Lab Chow and given access to water ad libitum. Rats and rabbits were sacrificed by cervical dislocation immediately before removal of tissue. Isolated ileai assays. Rat ileal segments (3 cm long) were maintained in Tyrode’s solution at a constant 37°C and aerated with laboratory air. Assays were performed after 1 hr of equilibration with the segments suspended between two hooks in a 50-ml Magnus bath (Offermeier and Ariens, 1966). One gram of tension was placed across the longitudinal muscle of the ileal segments, with contractions measured by a Grass FT03 force transducer and recorded by a Grass 5D polygraph. Test solutions were prepared by dissolving appropriate quantities of BHT in dimethylformamide (DMF). The DMF carrier-solution was then dissolved in Tyrode’s solution, with all final DMF concentrations in Tyrode’s solution being 0.05% (v:v). The final BHT concentrations used were 500, 100, 10, and 1 mg/liter (0.05, 0.01, 0.001, and 0.0001%). Control assays, using both pure Tyrode’s and Tyrode’s containing 0.05% DMF, were conducted. Initially the bath contained only Tyrode’s solution. Methacholine was added in cumulative concentrations to the bath until a maximal response was obtained. Thus, a control concentration-response profile was obtained. The segments were then washed with pure Tyrode’s. After a 5-min equilibration with Tyrode’s solution containing BHT, another cumulative methacholine concentration-response profile was determined. The tissue was again washed and allowed to equilibrate for 5 min with pure Tyrode’s, when a recovery cumulative concentration-response profile was produced. Each assay was repeated four times, each time with a new ileal segment. Isolated atria1 assays. Atria excised from rats (right one) and rabbits (both) were maintained in a LockRinger’s solution at 37”C, perfused with 95 % O2 and 5% CO,. Assays were performed with the atria suspended between two hooks in a 50-ml Magnus bath, with 1 g of fixed diastolic load imposed. on each. After a 5-min equilibration, contractions were measured for 2 hr by a Grass FT03 force transducer and recorded by a Grass 5D polygraph. Test solutions were prepared as described for the ileal assays. Isolated perfused rat heart assays. A modified Langendorff apparatus (Aronson and Serlick, 1976) was used to perfuse intact rat hearts. The system was so constructed that the test solution flowed from a reservoir where the solution was equilibrated with
AND
ACOSTA
95% 0,/5x CO* in a self-leveling chamber. A parallel system contained control solution. The leveling chambers maintained the height of the fluid column at 74 cm throughout the experimental period, so that perfusion pressure was constant. A small flow of perfusion medium dripping over the surface of the heart kept the exterior moist. KrebsRinger (K-R) bicarbonate buffer solution (Aronson and Serlick, 1976) at 37°C was used. BHT solutions were prepared as described for the ileal assay. Isometric systolic tension was measured by attaching a Palmer clip to the apex of the heart and connecting it by a Nylon suture over two pulleys to a Grass FT03 force transducer. A fixed diastolic load of 5 g was imposed on each heart by adjusting the position of the transducer. A Grass Model 5D polygraph was used to record the output of the force transducer. Aliquots (1 ml) were drawn from the perfusate at 5-min intervals throughout the assay, starting with a sample 5 min prior to exposure, so that a colorimetric assay (Swanson and Wilkinson, 1972) could be used to determine creatine phosphokinase (CPK). Assays consisted of equilibration of the hearts with a control buffer solution for 30 min, after which the hearts were perfused from a parallel system for 2 hr. Four assays were performed at each separate concentration. Control experiments were performed using pure Krebs-Ringer bicarbonate solution and K-R buffer with 0.05 % DMF. Statistical methods. The mean and SD of each group was computed, and the data were analyzed for significant differences by analysis of variance followed by Scheffe’s test (Scheffe, 1959).
RESULTS The inhibitory effects of BHT on methacholine-induced contractions of the isolated rat ileum are shown in Fig. 1. BHT significantly (p < 0.05) reduced methacholineinduced contractions in all concentrations tested; namely, 0.05, 0.01, and 0.001%. At 0.05% DMF did not significantly alter methacholine-induced contractions as compared to controls. Recovery concentration-responses of the ileum to methacholine after BHT removal (Fig. 2) show that 0.001% BHT produced very little residual depression during the recovery concentration-response, but 0.05 % BHT markedly depressed contractions during the recovery period.
INHIBITORY
ACTIONS
47
OF BHT
FIG. 1. Inhibition of methacholine-induced ileal contractions by BHT. Each data point represents the mean+ SD of four experiments using isolated rat ilea. SD are not shown for some data points; where this occurs, they are smaller than the data point symbols. BHT significantly (p < 0.05) depressed ileal contractions, as compared to controls, at all concentrations used. 0, Control; 0, DMF; A, 0.001% BHT; I-J, 0.01% BHT; n , 0.05% BHT.
01
2
4 Uothmohollno
6 Comoontratlon
16 t x10-’
32
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FIG. 2, Recovery concentration-response to methacholine subsequent to BHT removal from the ileal tissue bath and equilibration for 5 min. After this time, methacholine concentration-response profiles were again obtained to determine residual inhibitory effects from BHT exposure. Each data point represents the mean f SD of four experiments using isolated rat ilea. SD are not shown for some data points; where this occurs, they are smaller than the data points. Prior exposure to BHT, at all concentration levels, resulted in a significant depression in contraction of ileal segments during the recovery concentration-response to methacholine. 0, Control; 0, DMF; A, 0.001% BHT; H, 0.01% BHT; q ,0.05 % BHT.
GAD, LESLIE, AND ACOSTA
RABBIT
RAT
FIG. 3. BHT-induced depression of the duration of spontaneous atria1 beating. Each error bar represents the mean? SD of four experiments. Since BHT is only slightly soluble in water, it was dissolved in 0.05 ‘A dimethylformamide (DMF). DMF (0.05 %) alone did not alter atria1 beating activity as compared to controls.
At the concentration of 0.05x, BHT stopped beating activity in rabbit and rat atria after approximately 20 and 72 min (Fig. 3), respectively. At concentrations of 0.1 and 0.5% it terminated rat atria1 beating activity after 38 and 4.5 min, respectively. BHT concentrations below 0.05% (500 mg/ liter) did not totally stop atria1 beating, but did markedly depress the frequency and amplitude of atria1 contractions. Dimethylformamide (DMF) at 0.05% did not alter atria1 beating activity. BHT at a 0.05 % concentration totally stopped rabbit atria1 beating after 20 min (Fig. 4), while in lower concentrations (0.01, 0.001, and 0.0001%) it significantly (p < 0.05) depressed the frequency of atria1 beating in a dose-related manner. In all cases, the depression was most dramatic in the first 30 min of BHT exposure, but persisted throughout the 2-hr test. At concentrations of 0.01, 0.001, and
0.0001 %, BHT also significantly (~~0.05) depressed the amplitude of atria1 contractions in the rat and rabbit (rabbit data are shown in Fig. 5), with the most dramatic amplitude depressant effect being seen within 30 min but persisting for 2 hr. The BHT effects seen in the perfused rat heart parallel those seen in the rat and rabbit atria1 preparations. While DMF controls showed no significant differences in beating rates, force of contraction, or CPK leakage rates, all concentrations of BHT tested (0.05, 0.01, 0.001, and 0.0001%) significantly (p< 0.05) depressed the beating rates within 10 min. Similarly, all concentrations tested significantly depressed force of contractions. BHT at a 0.05% concentration stopped spontaneous beating of intact perfused hearts totally within 90 min. The results of the CPK assays performed on l-ml aliquots of perfusate from the isolated perfused rat hearts may be found in
INHIBITORY
ACTIONS
49
OF BHT
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FIG. 4. Inhibition of the frequency of atria1 beating activity by BHT. Each data point represents the mean?SD of four experiments using isolated rabbit atria. SD are not shown for some data points in this figure; where this occurs, SD are smaller than the data points. BHT significantly depressed (p < 0.05) frequency of atria1 contractions as compared to controls, at all time periods and with all concentrations shown in this figure. 0, Control; 0, DMF; x , 0.0001% BHT; A, 0.001% BHT; 0, 0.01% BHT; n , 0.05 % BHT.
FIG. 5. Depression of the amplitude of rabbit atria1 contractions by BHT. Each data point represents the mean + SD of four experiments using isolated rabbit atria. SD are not shown for some data points; where this occurs, they are smaller than the data points. BHT significantly (piO.05) depressed the amplitude of atria1 contractions as compared to controls, at all time periods and with all concentrations shown in this figure. 0, Control; 0, DMF; x, 0.0001% BHT; A, 0.001% BHT; 0, 0.01% BHT; n , 0.05 % BHT.
50
GAD,
LESLIE,
AND
TABLE EFFECT OF BHT
Perfusion time” (mm) 0 10 20 30 40 50 60 70 80 90 100 110 120
CONCENTRATION ISOLATED CPK
Control 220.4 4jIO.6 5kO.5 5+0.7 4+0.5 6kO.6 7kO.9 9+0.7 11 kO.8 lo+ 1.0 11kl.l 12+ 1.1 12kl.l
’ All BHT concentrations b Duration of perfusion ’ Significantly different
0.05 % DMF 2kO.2 4+0.6 4kO.5 6+0.7 7+0.9 7kO.7 9kl.O 1121.0 12+ 1.1 llkO.9 lOk1.2 12+ 1.1 1151.1
leakage
ACOSTA 1
ON LEAKAGE OF CPK INTO PERFUSATE BY THE PERFUSED RAT HEART’ (mIU
CPK
released/ml
0.05 % BHT
0.01%
BHT
3+ 0.5 22lk 14.2’ 281+ 63.9’ 342 k 58.7’ 401 f 54.6’ 453&51.1” 501* 47.3’ 526541.9’ 563 + 37.2’ 5525 34.2c 527+_26.1’ 536 + 24.2’ 512+ 18.7’
2& 0.1 236k63.1” 253 + 59.7’ 275f41.9’ 296+_ 37.8’ 3125 34.1’ 319C29.7’ 329 + 19.9’ 340& 17.6’ 343 + 12.4’ 347kll.l’ 349& 9.9’ 3535 5.8’
coronary 0.001%
flow) BHT
4+ 0.3 97+ 32.1’ 99 3~ 29.8” 104k27.7’ 109 + 23.8’ 116+21.1’ 121 f 19.7’ 128& 16.1’ 133 + 14.3’ 137 f 12.7’ 141 f 9.6’ 148+ 7.9’ 154+_ 4.5’
0.0001%
BHT
3+ 0.3 39*27.4c 42 + 23.6” 46k21.9’ 49+ 19.1’ 53 f 17.4’ 54* 15.3’ 57 + 12.7” 59 + 10.5c 6lk 8.2’ 64+ 6.3’ 65+_ 5.1’ 65+ 3.9’
are in addition to 0.05 % DMF. after initial 30-min equilibration period. from controls at the p c 0.05 level.
Table 1. The perfusate from control and 0.05% DMF-treated hearts showed only a very low CPK activity. CPK activity was significantly elevated in a concentrationrelated manner in all BHT-treated heart perfusates.
However, at 0.1% BHT with 10% lard, female rats had increased serum cholesterol concentrations, but male rats did not. At 0.5 % male rats had increases in serum cholesterol concentrations, while female rats showed increases in serum cholesterol, mucoprotein, and phospholipid levels. Gaunt et al. DISCUSSION (1965) demonstrated that when BHT was The results of the present investigation administered at a dose of 5000 mg/kg, liver show that BHT, in concentrations as low as phospholipids and cholesterol remained con0.0001% (which according to the NTIS stant, but serum cholesterol and phos(1973) review are within the range of human pholipid concentrations rose to high levels. exposure), significantly depressed the freBHT is lipid-soluble and is efficiently absorbed from the gastrointestinal tract quency and amplitude of contractions of isolated atria and heart. This was accom- (Daniel and Gage, 1965). The rate of absorppanied in the isolated perfused heart with a tion and the extent of BHT deposition in dose-dependent increase in leakage of CPK. adipose tissue may be sex dependent. The In addition, BHT depressed gastrointestinal female rat absorbed BHT more readily than contractions in a nonspecific manner. These the male, resulting in greater fat deposition in results are of potential importance as BHT is the female rats (Tye et al., 1965). BHT decommonly used in a wide variety of food posits in fat reached a concentration of about products consumed by the American public. 30 ppm for males and 45 ppm for females. Previous studies have shown a wide variety During a 7-week period of ingestion, BHT of parameters to be affected by BHT. Day et concentrations did not rise beyond these al. (1959) reported that 0.0001% BHT pro- figures (Daniel and Gage, 1965). Rats duced no changes in blood lipids in rats. rapidly excreted metabolites of BHT into the
INHIBITORY
ACTIONS
bile, with 95% of a single intravenous dose being eliminated by this route in about 6 hr. Excretion into the urine and feces occurred slowly (80 to 90% in 4 days). This delay is thought to occur because of enterohepatic circulation of one or more metabolites. The half-life of BHT deposited in the body fat is 7-10 days (Daniel and Gage, 1965; Ladomery et al., 1967). BHT is also reported to have pronounced proliferative, ultrastructural, and enzymatic effects on the rat liver in vivo and in vitro (Gilbert and Golberg, 1965; Lane and Lieber, 1967; Botham et a/., 1969; Conning and McElligott, 1970; West and Redgrave, 1974). In man, about 75% of a single dose was reported to be excreted in the urine. The major metabolites appeared within 24 hr of dosing. The remainder of the dose was excreted more slowly in the feces. The slow phase of BHT excretion had a half-life of about 5 days (Daniel et al., 1967, 1968; Holder et al., 1970). BHT has been reported to damage (a) lung tissue, both in vivo and in vitro (Adamson et al., 1977; Hirai et al., 1977; Omaye et al., 1977; Nakagawa et al., 1978), (b) heart cells in vitro (Leslie et al., 1978), and (c) endothelial cells of the circulatory system throughout the body (Takahasi and Hiraga, 1978; Witschi and Lock, 1978). The results of the current investigations have provided the first evidence that BHT directly depresses contractility of isolated atria, heart, and ileum and that cellular damage (as measured by elevated CPK leakage) occurs in the isolated heart during this process. The effects of inhibitory actions seen in ileum may or may not be related to the toxic effects seen in atrium and heart preparations. This investigation suggests that BHT may affect cardiovascular function. These actions of BHT might be of particular concern in human patients suffering from congestive heart failure or other heart diseases. Additional studies examining the chronic effects of low BHT concentrations on the compromised heart should be performed.
51
OF BHT
ADAMSON,
REFERENCES BOWDEN, D. H.,
I. Y.,
WITSCHI, H.
(1977).
BOTHAM, C. M., LITCHFIELD,
M. H.,
M. G.,
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Lung injury induced by butylated hydroxytoluene. Lab. Invest. 36, 26-32. ARONSON, C. E., AND SERLICK, E. R. (1976). Effects of prolonged perfusion time on the isolated perfused rat heart. Toxicot. Appl. Pharmacol. 38,479488. CONNING,
D. M., JOHAYES, AND MCELLIGOTT,
M.
H.,
T. F. (1969). Effects of butylated hydroxytoluene on enzyme activity and ultra-structure of rat hepatotocytes. Food Cosmet. ToxicoI. 8, l-8.
CONNING,
D.
M.,
AND MCELLIGOTT,
T.
F. (1970).
Quantitation of the proliferative response of smooth endoplasmic reticulum to dietary butylated hydroxytoluene and its toxicological significance. Proc. Eur. Sot. Drug Toxicity 11, 203-212. DANIEL, J. W., AND GAGE, J. C. (1965). The absorption and excretion of butylated hydroxytoluene (BHT) in the rat. Food Cosmet. Toxicol. 3, 405415. DANIEL, J. W., GAGE, J. C., JONES, D. I., AND STEVENS, M. A. (1967). Excretion of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) by man. Food Cosmet. Toxicoi. 5, 415-479. DANIEL,
J. W., GAGE,
J. C., AND JONES, D. I. (1968).
The metabolism of 3,5-ditert-butyl-4-hydroxytoluene in the rat and in man. Biochem. J. 106, 783-792. DAY, A. J., JOHNSON, A. R., O’HALLQRAN, AND SCHWARTZ, C. J. (1959). Effect of
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the antioxidant butylated hydroxytoluene on serum lipid and glycoprotein levels in the rat. Aust. J. Exp. Biol, 31, 295-306. GAD, S. C., LESLIE, S. W., BROWN, R. G., AND SMITH, R. V. (1977). Inhibitory effects of dithiothreitol and sodium bis-sulfite on isolated rat ileum and atrium. Life Sci. 20, 657-664. GAUNT, I. F., GILBERT, D., AND MARTIN, D. (1965). Liver response tests. V. Effect of dietary restriction on a short-term feeding study with butylated hydroxytoluene (BHT). Food Cosmet. Toxicol. 3, 445-456. GILBERT, D., AND GOLBERG, L. (1965). Liver weight and microsomal processing (drug metabolizing) enzymes in rats treated with butylated hydroxytoluene or butylated hydroxyanisole. Biochem. J. 97,28P-29P. HIRAI, K. I., WITSCHI, H., AND CBti, M. G. (1977). Electron microscopy of butylated hydroxytolueneinduced lung damage in mice. Exp. Mol. Pathoi. 27, 295-308. HOLDER, G. M., RYAN, A. J., WATSON, T. R., AND WEIBE, L. I. (1970). The metabolism of butylated hydroxytoluene, (3,5-di-t-butyl-4-hydroxytoluene) in man. J. Pharm. Pharmacol. 22, 375-376.
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GAD, LESLIE, AND ACOSTA
JOHNSON, F. G. (1971). A critical review of the safety of phenolic antioxidants in foods. CRC Crit. Rev. Food Technol. 2,267-3&l. LADOMERY, L. G., RYAN, A. J., AND WRIGHT, S. E. (1967). The excretion of butylated hydroxytoluene14C in the rat. J. Pharm. Pharmacol. 19, 383-396. LANE, B. P., AND LIEBER, C. S. (1967). Effects of butylated hydroxytoluene on the ultra-structure of rat hepatocytes. Lab. Invest. 16, 342-346. LESLIE, S. W., GAD, S. C., AND ACOS~A, D. (1978). Cytotoxicity of butylated hydroxytoluene and butylated hydroxyanisole in cultured heart cells. Toxicology 10, 28 l-289. NAKAGAWA, Y., IKAWA, M., AND HIRAGA, K. (1978). Biological fate of butylated hydroxytoluene (BHT): Subcellular distribution of BHT in the rat liver. Chem. Pharm. Bull. 26, 374-378. Nns(1973). Evaluation of the Health Aspects of Butylated Hydroxytoluene as a Food Ingredient, publication No. PB-259-917. National Technical Information Service, Springfield, Va. OFFERMEIR,T., AND ARIENS, E. J. (1966). Serotonin. I. Receptors involved in its action. Arch. Int. Pharmacodyn. Ther. 164, 192-215.
OMAYE, S. T., REDDY, K. A., AND CROSS, C. E. (1977). Effect of butylated hydroxytoluene and other antioxidants on mouse lung metabolism. J. Tox. Environ. Health 3, 829-836. SCHEFFE, H. A. (1959). The Analysis of Variance. Wiley, New York. SWANSON, J. R., and WILKINSON, J. H. (1972). Measurements of creatine kinase activity in serum. In Standard Methods of Clinical Chemistry 7, pp. 33-42. Academic Press, New York. TAKAHASI, O., AND HIRAGA, K. (1978). Dose-response study of hemorrhagic death by dietary butylated hydroxytoluene (BHT) in male rats. Toxicof. Appl. Pharmacol. 42, 399-406. TYE, R., ENGLE, J. D., AND RAPIEN, J. (1965). Summary of toxicological data. Disposition of butylated hydroxytoluene in the rat. Food Cosmet. Toxicol. 3,547-551. WEST, C. E., AND REDGRAVE, T. G. (1974). Reservations on the use of polyunsaturated fats in human nutrition. Search 5, 9&94. WITSCHI, H., AND LOCK, S. (1978). Toxicity of butylated hydroxytoluene in mouse following oral administration. Toxicology 9, 137-146.
AND
APPLIED
PHARMACOLOGY
49, 45-52 (1979)
Inhibitory Actions of Butylated Hydroxytoluene on Isolated flea& Atrial, and Perfused Heart Preparations SHAYNE C. GAD,~ STEVEN W. LESLIE,~ AND DANIEL ACOSTA~ Chemical Hygiene Fellowship, Carnegie-Mellon Institute of Research, Pittsburgh, Pennsylvania 15213, and Department of Pharmacology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712
Received August I, 1978; accepted November 8, 1978
Inhibitory Actions of Butylated Hydroxytoluene on Isolated Ileal, Atrial, and Perfused Heart Preparations. GAD, S. C., LESLIE, S. W., AND ACOSTA, D. (1979). Toxicol. Appl. Pharmacol. 49, 45-52. Butylated hydroxytoluene (BHT), in concentrations of 500, 100, and 10 mg/liter (but not 1 mg/liter), significantly depressed methacholine-induced contractions of isolated rat and rabbit ileal preparations. BHT, in concentrations of 500, 100, 10, and 1 mg/liter, significantly (p < 0.05) depressed the frequency and amplitude (force) of contractions of isolated atria1 preparations as compared to controls. Control rat and rabbit atria1 beating rates were monitored continuously for 120 min (although isolated control atria1 preparations continue to beat spontaneously for several hours). BHT at 500 mg/liter terminated spontaneous contractions of isolated rabbit atria after 20 min and after 72 min when tested on rat atria. Although concentrations below 500 mg/liter did not terminate atria1 beating activity, they did produce a pronounced concentration-dependent depression of rate and force of atria1 contractions. The most pronounced depression of atria1 contractions produced by BHT were always seen within the first 30 min (out of a period up to 120 min) of exposure but the depression persisted throughout the exposure period. Similar significant dose-related depressions were seen using the same concentrations in intact perfused rat heart preparations. Additionally, perfused hearts treated with BHT (in concentrations from 1 to 500 mg/liter) showed significant dose-related increases in creatine phosphokinase leakage into the perfusion solutions.
Antioxidants are commonly used in food products to prevent (by a free radical mechanism) the autoxidation of fats and oils, a process which begins with the fixation of a molecule of oxygen to an unsaturated fat chain (Johnson, 1971). The broad distribution and the large consumption of food products containing antioxidants leaves vir-
tually no human being in the U.S. unexposed to these agents. Earlier studies showed that sodium bisulfite, an antioxidant “generally regarded as safe” by the Food and Drug Administration, depressed the contractility of isolated rat ileum and atrium (Gad et al., 1977) and that butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) had significant cytotoxic effects on cultured rat heart cells (Leslie et al., 1978). The present investigation examined the implications of toxicity seen in these earlier studies.
I To whom all correspondence should be addressed. 2 Department of Pharmacology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712. 45
All
0041-008X/79/070045~8s02.~/0 Copyright 0 1979 by Academic Press. Inc. rights of reproduction in any form reserved. Printed in Great Britain
46
GAD,
LESLIE,
METHODS Animals. Male, Sprague-Dawley rats (250-350 g) and male, white New Zealand, rabbits (2-2.5 kg) were used as source animals for tissue preparations. They were fed Purina Lab Chow and given access to water ad libitum. Rats and rabbits were sacrificed by cervical dislocation immediately before removal of tissue. Isolated ileai assays. Rat ileal segments (3 cm long) were maintained in Tyrode’s solution at a constant 37°C and aerated with laboratory air. Assays were performed after 1 hr of equilibration with the segments suspended between two hooks in a 50-ml Magnus bath (Offermeier and Ariens, 1966). One gram of tension was placed across the longitudinal muscle of the ileal segments, with contractions measured by a Grass FT03 force transducer and recorded by a Grass 5D polygraph. Test solutions were prepared by dissolving appropriate quantities of BHT in dimethylformamide (DMF). The DMF carrier-solution was then dissolved in Tyrode’s solution, with all final DMF concentrations in Tyrode’s solution being 0.05% (v:v). The final BHT concentrations used were 500, 100, 10, and 1 mg/liter (0.05, 0.01, 0.001, and 0.0001%). Control assays, using both pure Tyrode’s and Tyrode’s containing 0.05% DMF, were conducted. Initially the bath contained only Tyrode’s solution. Methacholine was added in cumulative concentrations to the bath until a maximal response was obtained. Thus, a control concentration-response profile was obtained. The segments were then washed with pure Tyrode’s. After a 5-min equilibration with Tyrode’s solution containing BHT, another cumulative methacholine concentration-response profile was determined. The tissue was again washed and allowed to equilibrate for 5 min with pure Tyrode’s, when a recovery cumulative concentration-response profile was produced. Each assay was repeated four times, each time with a new ileal segment. Isolated atria1 assays. Atria excised from rats (right one) and rabbits (both) were maintained in a LockRinger’s solution at 37”C, perfused with 95 % O2 and 5% CO,. Assays were performed with the atria suspended between two hooks in a 50-ml Magnus bath, with 1 g of fixed diastolic load imposed. on each. After a 5-min equilibration, contractions were measured for 2 hr by a Grass FT03 force transducer and recorded by a Grass 5D polygraph. Test solutions were prepared as described for the ileal assays. Isolated perfused rat heart assays. A modified Langendorff apparatus (Aronson and Serlick, 1976) was used to perfuse intact rat hearts. The system was so constructed that the test solution flowed from a reservoir where the solution was equilibrated with
AND
ACOSTA
95% 0,/5x CO* in a self-leveling chamber. A parallel system contained control solution. The leveling chambers maintained the height of the fluid column at 74 cm throughout the experimental period, so that perfusion pressure was constant. A small flow of perfusion medium dripping over the surface of the heart kept the exterior moist. KrebsRinger (K-R) bicarbonate buffer solution (Aronson and Serlick, 1976) at 37°C was used. BHT solutions were prepared as described for the ileal assay. Isometric systolic tension was measured by attaching a Palmer clip to the apex of the heart and connecting it by a Nylon suture over two pulleys to a Grass FT03 force transducer. A fixed diastolic load of 5 g was imposed on each heart by adjusting the position of the transducer. A Grass Model 5D polygraph was used to record the output of the force transducer. Aliquots (1 ml) were drawn from the perfusate at 5-min intervals throughout the assay, starting with a sample 5 min prior to exposure, so that a colorimetric assay (Swanson and Wilkinson, 1972) could be used to determine creatine phosphokinase (CPK). Assays consisted of equilibration of the hearts with a control buffer solution for 30 min, after which the hearts were perfused from a parallel system for 2 hr. Four assays were performed at each separate concentration. Control experiments were performed using pure Krebs-Ringer bicarbonate solution and K-R buffer with 0.05 % DMF. Statistical methods. The mean and SD of each group was computed, and the data were analyzed for significant differences by analysis of variance followed by Scheffe’s test (Scheffe, 1959).
RESULTS The inhibitory effects of BHT on methacholine-induced contractions of the isolated rat ileum are shown in Fig. 1. BHT significantly (p < 0.05) reduced methacholineinduced contractions in all concentrations tested; namely, 0.05, 0.01, and 0.001%. At 0.05% DMF did not significantly alter methacholine-induced contractions as compared to controls. Recovery concentration-responses of the ileum to methacholine after BHT removal (Fig. 2) show that 0.001% BHT produced very little residual depression during the recovery concentration-response, but 0.05 % BHT markedly depressed contractions during the recovery period.
INHIBITORY
ACTIONS
47
OF BHT
FIG. 1. Inhibition of methacholine-induced ileal contractions by BHT. Each data point represents the mean+ SD of four experiments using isolated rat ilea. SD are not shown for some data points; where this occurs, they are smaller than the data point symbols. BHT significantly (p < 0.05) depressed ileal contractions, as compared to controls, at all concentrations used. 0, Control; 0, DMF; A, 0.001% BHT; I-J, 0.01% BHT; n , 0.05% BHT.
01
2
4 Uothmohollno
6 Comoontratlon
16 t x10-’
32
W)
FIG. 2, Recovery concentration-response to methacholine subsequent to BHT removal from the ileal tissue bath and equilibration for 5 min. After this time, methacholine concentration-response profiles were again obtained to determine residual inhibitory effects from BHT exposure. Each data point represents the mean f SD of four experiments using isolated rat ilea. SD are not shown for some data points; where this occurs, they are smaller than the data points. Prior exposure to BHT, at all concentration levels, resulted in a significant depression in contraction of ileal segments during the recovery concentration-response to methacholine. 0, Control; 0, DMF; A, 0.001% BHT; H, 0.01% BHT; q ,0.05 % BHT.
GAD, LESLIE, AND ACOSTA
RABBIT
RAT
FIG. 3. BHT-induced depression of the duration of spontaneous atria1 beating. Each error bar represents the mean? SD of four experiments. Since BHT is only slightly soluble in water, it was dissolved in 0.05 ‘A dimethylformamide (DMF). DMF (0.05 %) alone did not alter atria1 beating activity as compared to controls.
At the concentration of 0.05x, BHT stopped beating activity in rabbit and rat atria after approximately 20 and 72 min (Fig. 3), respectively. At concentrations of 0.1 and 0.5% it terminated rat atria1 beating activity after 38 and 4.5 min, respectively. BHT concentrations below 0.05% (500 mg/ liter) did not totally stop atria1 beating, but did markedly depress the frequency and amplitude of atria1 contractions. Dimethylformamide (DMF) at 0.05% did not alter atria1 beating activity. BHT at a 0.05 % concentration totally stopped rabbit atria1 beating after 20 min (Fig. 4), while in lower concentrations (0.01, 0.001, and 0.0001%) it significantly (p < 0.05) depressed the frequency of atria1 beating in a dose-related manner. In all cases, the depression was most dramatic in the first 30 min of BHT exposure, but persisted throughout the 2-hr test. At concentrations of 0.01, 0.001, and
0.0001 %, BHT also significantly (~~0.05) depressed the amplitude of atria1 contractions in the rat and rabbit (rabbit data are shown in Fig. 5), with the most dramatic amplitude depressant effect being seen within 30 min but persisting for 2 hr. The BHT effects seen in the perfused rat heart parallel those seen in the rat and rabbit atria1 preparations. While DMF controls showed no significant differences in beating rates, force of contraction, or CPK leakage rates, all concentrations of BHT tested (0.05, 0.01, 0.001, and 0.0001%) significantly (p< 0.05) depressed the beating rates within 10 min. Similarly, all concentrations tested significantly depressed force of contractions. BHT at a 0.05% concentration stopped spontaneous beating of intact perfused hearts totally within 90 min. The results of the CPK assays performed on l-ml aliquots of perfusate from the isolated perfused rat hearts may be found in
INHIBITORY
ACTIONS
49
OF BHT
....................(“..a ...I..... ‘. . ’iy--h ;x ‘Al . hl. I ; t. l * \ %.
‘h
----------.---.o----------------,~
\ I
----...
\
i
.[<----wz
i I I I iI I
jl:
I
1 ‘\ \
,! s
l6
\ \\
. 30
00
00
1:
FIG. 4. Inhibition of the frequency of atria1 beating activity by BHT. Each data point represents the mean?SD of four experiments using isolated rabbit atria. SD are not shown for some data points in this figure; where this occurs, SD are smaller than the data points. BHT significantly depressed (p < 0.05) frequency of atria1 contractions as compared to controls, at all time periods and with all concentrations shown in this figure. 0, Control; 0, DMF; x , 0.0001% BHT; A, 0.001% BHT; 0, 0.01% BHT; n , 0.05 % BHT.
FIG. 5. Depression of the amplitude of rabbit atria1 contractions by BHT. Each data point represents the mean + SD of four experiments using isolated rabbit atria. SD are not shown for some data points; where this occurs, they are smaller than the data points. BHT significantly (piO.05) depressed the amplitude of atria1 contractions as compared to controls, at all time periods and with all concentrations shown in this figure. 0, Control; 0, DMF; x, 0.0001% BHT; A, 0.001% BHT; 0, 0.01% BHT; n , 0.05 % BHT.
50
GAD,
LESLIE,
AND
TABLE EFFECT OF BHT
Perfusion time” (mm) 0 10 20 30 40 50 60 70 80 90 100 110 120
CONCENTRATION ISOLATED CPK
Control 220.4 4jIO.6 5kO.5 5+0.7 4+0.5 6kO.6 7kO.9 9+0.7 11 kO.8 lo+ 1.0 11kl.l 12+ 1.1 12kl.l
’ All BHT concentrations b Duration of perfusion ’ Significantly different
0.05 % DMF 2kO.2 4+0.6 4kO.5 6+0.7 7+0.9 7kO.7 9kl.O 1121.0 12+ 1.1 llkO.9 lOk1.2 12+ 1.1 1151.1
leakage
ACOSTA 1
ON LEAKAGE OF CPK INTO PERFUSATE BY THE PERFUSED RAT HEART’ (mIU
CPK
released/ml
0.05 % BHT
0.01%
BHT
3+ 0.5 22lk 14.2’ 281+ 63.9’ 342 k 58.7’ 401 f 54.6’ 453&51.1” 501* 47.3’ 526541.9’ 563 + 37.2’ 5525 34.2c 527+_26.1’ 536 + 24.2’ 512+ 18.7’
2& 0.1 236k63.1” 253 + 59.7’ 275f41.9’ 296+_ 37.8’ 3125 34.1’ 319C29.7’ 329 + 19.9’ 340& 17.6’ 343 + 12.4’ 347kll.l’ 349& 9.9’ 3535 5.8’
coronary 0.001%
flow) BHT
4+ 0.3 97+ 32.1’ 99 3~ 29.8” 104k27.7’ 109 + 23.8’ 116+21.1’ 121 f 19.7’ 128& 16.1’ 133 + 14.3’ 137 f 12.7’ 141 f 9.6’ 148+ 7.9’ 154+_ 4.5’
0.0001%
BHT
3+ 0.3 39*27.4c 42 + 23.6” 46k21.9’ 49+ 19.1’ 53 f 17.4’ 54* 15.3’ 57 + 12.7” 59 + 10.5c 6lk 8.2’ 64+ 6.3’ 65+_ 5.1’ 65+ 3.9’
are in addition to 0.05 % DMF. after initial 30-min equilibration period. from controls at the p c 0.05 level.
Table 1. The perfusate from control and 0.05% DMF-treated hearts showed only a very low CPK activity. CPK activity was significantly elevated in a concentrationrelated manner in all BHT-treated heart perfusates.
However, at 0.1% BHT with 10% lard, female rats had increased serum cholesterol concentrations, but male rats did not. At 0.5 % male rats had increases in serum cholesterol concentrations, while female rats showed increases in serum cholesterol, mucoprotein, and phospholipid levels. Gaunt et al. DISCUSSION (1965) demonstrated that when BHT was The results of the present investigation administered at a dose of 5000 mg/kg, liver show that BHT, in concentrations as low as phospholipids and cholesterol remained con0.0001% (which according to the NTIS stant, but serum cholesterol and phos(1973) review are within the range of human pholipid concentrations rose to high levels. exposure), significantly depressed the freBHT is lipid-soluble and is efficiently absorbed from the gastrointestinal tract quency and amplitude of contractions of isolated atria and heart. This was accom- (Daniel and Gage, 1965). The rate of absorppanied in the isolated perfused heart with a tion and the extent of BHT deposition in dose-dependent increase in leakage of CPK. adipose tissue may be sex dependent. The In addition, BHT depressed gastrointestinal female rat absorbed BHT more readily than contractions in a nonspecific manner. These the male, resulting in greater fat deposition in results are of potential importance as BHT is the female rats (Tye et al., 1965). BHT decommonly used in a wide variety of food posits in fat reached a concentration of about products consumed by the American public. 30 ppm for males and 45 ppm for females. Previous studies have shown a wide variety During a 7-week period of ingestion, BHT of parameters to be affected by BHT. Day et concentrations did not rise beyond these al. (1959) reported that 0.0001% BHT pro- figures (Daniel and Gage, 1965). Rats duced no changes in blood lipids in rats. rapidly excreted metabolites of BHT into the
INHIBITORY
ACTIONS
bile, with 95% of a single intravenous dose being eliminated by this route in about 6 hr. Excretion into the urine and feces occurred slowly (80 to 90% in 4 days). This delay is thought to occur because of enterohepatic circulation of one or more metabolites. The half-life of BHT deposited in the body fat is 7-10 days (Daniel and Gage, 1965; Ladomery et al., 1967). BHT is also reported to have pronounced proliferative, ultrastructural, and enzymatic effects on the rat liver in vivo and in vitro (Gilbert and Golberg, 1965; Lane and Lieber, 1967; Botham et a/., 1969; Conning and McElligott, 1970; West and Redgrave, 1974). In man, about 75% of a single dose was reported to be excreted in the urine. The major metabolites appeared within 24 hr of dosing. The remainder of the dose was excreted more slowly in the feces. The slow phase of BHT excretion had a half-life of about 5 days (Daniel et al., 1967, 1968; Holder et al., 1970). BHT has been reported to damage (a) lung tissue, both in vivo and in vitro (Adamson et al., 1977; Hirai et al., 1977; Omaye et al., 1977; Nakagawa et al., 1978), (b) heart cells in vitro (Leslie et al., 1978), and (c) endothelial cells of the circulatory system throughout the body (Takahasi and Hiraga, 1978; Witschi and Lock, 1978). The results of the current investigations have provided the first evidence that BHT directly depresses contractility of isolated atria, heart, and ileum and that cellular damage (as measured by elevated CPK leakage) occurs in the isolated heart during this process. The effects of inhibitory actions seen in ileum may or may not be related to the toxic effects seen in atrium and heart preparations. This investigation suggests that BHT may affect cardiovascular function. These actions of BHT might be of particular concern in human patients suffering from congestive heart failure or other heart diseases. Additional studies examining the chronic effects of low BHT concentrations on the compromised heart should be performed.
51
OF BHT
ADAMSON,
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Lung injury induced by butylated hydroxytoluene. Lab. Invest. 36, 26-32. ARONSON, C. E., AND SERLICK, E. R. (1976). Effects of prolonged perfusion time on the isolated perfused rat heart. Toxicot. Appl. Pharmacol. 38,479488. CONNING,
D. M., JOHAYES, AND MCELLIGOTT,
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T. F. (1969). Effects of butylated hydroxytoluene on enzyme activity and ultra-structure of rat hepatotocytes. Food Cosmet. ToxicoI. 8, l-8.
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Quantitation of the proliferative response of smooth endoplasmic reticulum to dietary butylated hydroxytoluene and its toxicological significance. Proc. Eur. Sot. Drug Toxicity 11, 203-212. DANIEL, J. W., AND GAGE, J. C. (1965). The absorption and excretion of butylated hydroxytoluene (BHT) in the rat. Food Cosmet. Toxicol. 3, 405415. DANIEL, J. W., GAGE, J. C., JONES, D. I., AND STEVENS, M. A. (1967). Excretion of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) by man. Food Cosmet. Toxicoi. 5, 415-479. DANIEL,
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The metabolism of 3,5-ditert-butyl-4-hydroxytoluene in the rat and in man. Biochem. J. 106, 783-792. DAY, A. J., JOHNSON, A. R., O’HALLQRAN, AND SCHWARTZ, C. J. (1959). Effect of
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the antioxidant butylated hydroxytoluene on serum lipid and glycoprotein levels in the rat. Aust. J. Exp. Biol, 31, 295-306. GAD, S. C., LESLIE, S. W., BROWN, R. G., AND SMITH, R. V. (1977). Inhibitory effects of dithiothreitol and sodium bis-sulfite on isolated rat ileum and atrium. Life Sci. 20, 657-664. GAUNT, I. F., GILBERT, D., AND MARTIN, D. (1965). Liver response tests. V. Effect of dietary restriction on a short-term feeding study with butylated hydroxytoluene (BHT). Food Cosmet. Toxicol. 3, 445-456. GILBERT, D., AND GOLBERG, L. (1965). Liver weight and microsomal processing (drug metabolizing) enzymes in rats treated with butylated hydroxytoluene or butylated hydroxyanisole. Biochem. J. 97,28P-29P. HIRAI, K. I., WITSCHI, H., AND CBti, M. G. (1977). Electron microscopy of butylated hydroxytolueneinduced lung damage in mice. Exp. Mol. Pathoi. 27, 295-308. HOLDER, G. M., RYAN, A. J., WATSON, T. R., AND WEIBE, L. I. (1970). The metabolism of butylated hydroxytoluene, (3,5-di-t-butyl-4-hydroxytoluene) in man. J. Pharm. Pharmacol. 22, 375-376.
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JOHNSON, F. G. (1971). A critical review of the safety of phenolic antioxidants in foods. CRC Crit. Rev. Food Technol. 2,267-3&l. LADOMERY, L. G., RYAN, A. J., AND WRIGHT, S. E. (1967). The excretion of butylated hydroxytoluene14C in the rat. J. Pharm. Pharmacol. 19, 383-396. LANE, B. P., AND LIEBER, C. S. (1967). Effects of butylated hydroxytoluene on the ultra-structure of rat hepatocytes. Lab. Invest. 16, 342-346. LESLIE, S. W., GAD, S. C., AND ACOS~A, D. (1978). Cytotoxicity of butylated hydroxytoluene and butylated hydroxyanisole in cultured heart cells. Toxicology 10, 28 l-289. NAKAGAWA, Y., IKAWA, M., AND HIRAGA, K. (1978). Biological fate of butylated hydroxytoluene (BHT): Subcellular distribution of BHT in the rat liver. Chem. Pharm. Bull. 26, 374-378. Nns(1973). Evaluation of the Health Aspects of Butylated Hydroxytoluene as a Food Ingredient, publication No. PB-259-917. National Technical Information Service, Springfield, Va. OFFERMEIR,T., AND ARIENS, E. J. (1966). Serotonin. I. Receptors involved in its action. Arch. Int. Pharmacodyn. Ther. 164, 192-215.
OMAYE, S. T., REDDY, K. A., AND CROSS, C. E. (1977). Effect of butylated hydroxytoluene and other antioxidants on mouse lung metabolism. J. Tox. Environ. Health 3, 829-836. SCHEFFE, H. A. (1959). The Analysis of Variance. Wiley, New York. SWANSON, J. R., and WILKINSON, J. H. (1972). Measurements of creatine kinase activity in serum. In Standard Methods of Clinical Chemistry 7, pp. 33-42. Academic Press, New York. TAKAHASI, O., AND HIRAGA, K. (1978). Dose-response study of hemorrhagic death by dietary butylated hydroxytoluene (BHT) in male rats. Toxicof. Appl. Pharmacol. 42, 399-406. TYE, R., ENGLE, J. D., AND RAPIEN, J. (1965). Summary of toxicological data. Disposition of butylated hydroxytoluene in the rat. Food Cosmet. Toxicol. 3,547-551. WEST, C. E., AND REDGRAVE, T. G. (1974). Reservations on the use of polyunsaturated fats in human nutrition. Search 5, 9&94. WITSCHI, H., AND LOCK, S. (1978). Toxicity of butylated hydroxytoluene in mouse following oral administration. Toxicology 9, 137-146.