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Isotope effects on the metabolism and pulmonary toxicity of butylated hydroxytoluene in mice by deuteration of the 4-methyl group
TOXICOLOGYANDAPPLIED
PHARMACOLOGY
69,283-290(1983)
Isotope Effects on the Metabolism and Pulmonary Toxicity of Butylated Hydroxytoluene in Mice by Deuteration of the It-Methyl Group’ TAMIO *Laboratory
MIZUTANI,*
of Environmental Sakyo-ku,
KENJI YAMAMOTO,~
AND KAZUO
TAJIMA~
Health and Toxicology, Department of Food Science, Kyoto Prefectural Kyoto 606. and TDepartment of Chemistry, School of Pharmacy, Hokuriku University. Kanazawa 920-I 1, Japan
Received
December
27, 1982: accepted
February
University,
25, 1983
Isotope Effects on the Metabolism and Pulmonary Toxicity of Butylated Hydroxytoluene in Mice by Deuteration of the 4-Methyl Group. MIZUTANI, T., YAMAMOTO, K.. AND TAJIMA, K. (1983). Toxicol. Appl. Pharmacol. 69, 283-290. A comparative test in mice for pulmonary toxicity between butylated hydroxytoluene (2,6-di-tert.-butyl-4-methylphenol, BHT) and 2,6-ditert.-butyl-4-(a,cY,c+*H3]methylphenol (BHT-d,) showed a significantly lower toxic potency of the latter. The rate of in vitro BHT metabolism to 2,6-di-tert.-butyl-4-methylene-2,5-cyclohexadienone (BHT-QM) was slowed by deuterating BHT in the 4-methyl group. On the other hand, the rate of in vitro metabolism to 2,6-di-tert.-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-OH) was increased with the deuteration. A similar isotope effect of the deuterium substitution on the in vivo metabolic rates of BHT was observed. These observations support the concept that the lung damage caused by BHT is mediated by BHT-QM. The pulmonary toxicity of 2-tert.-butyl-4-ethylphenol (4-EP) and their deuterated analogs was also compared. 2-tert.-Butyl-4-[ 1,1-*H2]ethylphenol (4-EP-d2) showed a significantly lower toxic potency than 4EP, whereas 2-tert.-butyl-4-[2,2,2-2H~]ethylphenol (4-EP-dl) showed a toxic potency comparable to that of 4-EP. This result is consistent with the hypothesis that a quinone methide metabolite is responsible for the onset of lung damage produced by 4-EP as well as BHT.
Butylated hydroxytoluene (2,6-di-tert.-butyl4-methylphenol, BHT) is a widely used antioxidant. BHT has been shown to cause lung damage in mice (Marino and Mitchell, 1972; Saheb and Witschi, 1975), which is characterized by early necrosis of type I alveolar cells and subsequent proliferation of type II alveolar cells (Adamson et al., 1977). Malkinson (1979) reported that BHT-induced lung damage can be prevented by exposing mice to cedar terpenes and that immature mice are nonresponsive to BHT. Kehrer and Witschi (1980) demonstrated the prevention of BHT-induced lung damage by ’ This paper was presented in part at the Ninth Symposium on Environmental Pollutants and Toxicology, October 14-15, 1982, Okayama, Japan. 283
the coadministration of drug metabolism inhibitors SKF-525A or piperonyl butoxide. BHT given to mice becomes covalently bound to lung tissue and this covalent binding can be prevented by the administration of SKF525A (Kehrer and Witschi, 1980). These findings suggest that a reactive metabolite of BHT is responsible for the onset of lung damage in mice. Based upon a structure-activity study with BHT analogs, we have recently suggested that a quinone methide, 2,6-di-tert.-butyl-Cmethylene-2$cyclohexadienone (BHT-QM), or closely related metabolites may play a role in producing lung damage in mice dosed with BHT (Mizutani et al., 1982). The present work was undertaken to add positive support for this suggestion and deals with the isotope ef0041-008X/83 Copyright All
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fects on the metabolism and pulmonary toxicity of BHT in mice by deuteration of the 4methyl group. METHODS Chemicals Chemicals were purchased as follows: lithium aluminum deuteride (LiA1D4, 98% isotopic purity) from E. Merck A. G., Darmstadt, West Germany; sodium borodeuteride (NaBD,, 97.4% isotopic purity) from Commissariat al’ Energie Atomique, France; BHT from Nakarai Chemicals, Ltd., Kyoto, Japan; pbromothiophenol from Aldrich Chemical Company, Milwaukee, Wisconsin; NADP from Kohjin Company Ltd., Tokyo, Japan; glucose 6-phosphate from Sigma Chemical Company, St. Louis, Missouri. All other reagents were of the highest purity available. 2,6-Di-tert.-butyI-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-OH) (Kharasch and Joshi, 1957) and 2tert.-butyl-4-ethylphenol (4-EP) (Mizutani et al., 1982) were synthesized according to the described methods.
Svntheses 2.6-Di-tert.-butyl-4-[~,~.~-2H~]methylphenoI (BHT-d,). 3.5-Di-tert.-butyl-4-hydroxybenzoic acid (Yohe et al., 1956) was converted to the methyl ester, mp; 166-167°C. The ester (0.03 mol, 7.9 g) dissolved in ether (100 ml) was added to a solution of LiAID, (0.09 mol, 3.8 g) in ether (200 ml), and the mixture was refluxed under nitrogen for 12 hr. The excessreagent was decomposed with ethyl acetate and 10% sodium hydroxide, and the resulting precipitate was filtered off. The organic layer was washed with water, dried, and freed from the solvent. Column chromatography on silica gel gave BHT-dS, mp 68-69°C; MS m/e 223 (M+, 32%), 208 (lOO), 180 (S), 148 (I l), isotopic purity 98%; NMR (CDCI,) 6 7.05 (s, 2 H), 5.04 (s, I H), 1.44 (s, 18 H). 2.6-Di-tert.-butyl-4-h.vdroxy-4-[a,cu,cu-*H,lmethyl-2,5cyclohexadienone (BHT-OH-dJ. According to the procedure of Kharasch and Joshi (1957). BHT-d, was oxygenated to BHT-OH-d,, mp 1 IO-1 12’C; MS m/e 239 (M+. 21%), 221 (21), 183 (loo), 168 (97), isotopic purity 97%; NMR (CDC&) 6 6.66 (s, 2 H). 1.92 (s, 1H). 1.21 (s. 18 H). 2.6 - Di- tert. - butyl- I- (4’- bromophenylthio[cY.o -‘HJ] methyophenoi (BHT-SPhBr-dJ. 3,5-Di-tert.-butyl-4-hydroxybenzoic acid (Yohe et al., 1956) was converted to the acid chloride, which was hydrogenated with NaBD4 in dioxane to 3,5-di-tert.-butyl-4-hydroxy[cu,ol-2H2lbl alcohol, mp 142-143°C. Treatment of the alcohol with thionyl chloride yielded 3,5-di-tert.-butyl-4-hydroxy[a,a-
AND TAJIMA
*H$enzyl chloride. A solution of the chloride (2 mmol, 500 mg) in acetone (2 ml) was added to a mixture of p bromothiophenol (2 mmol, 380 mg) and potassium carbonate(200mg) in acetone(8 ml), andthemixturewas stirred at room temperature for 2 hr. The mixture was poured into water and extracted with benzene. The organic layer was washed with 5% sodium hydroxide and then with water, dried, and freed from the solvent. Column chromatography on silica gel gave BHT-SPhBrd*, mp 95-96°C; MS m/e 408 (M+, 1.5%), 22 1 (IOO), 205 (7); NMR (CD(&) 6 7.48, 7.25 (q, J = 8 Hz, 4 H), 7.11 (s, 2 H). 5.23 (s, 1 H), 1.40 (s, 18 H), isotopic purity 96%. 2,6 - Di - tert. - butyl - 4 - (4’ - bromophenylthiomethyl) phenol (BHT-SPhBr). This compound was prepared by the same method as described above for BHT-SPhBrd, with 3,5di-tert.-butyl-4-hydroxybenzyl chloride (Chasar and Westfahl, 1977) and pbromothiophenol as starting materials. BHT-SPhBr, mp 93-95°C; MS m/e 406 (M+, 1.2%). 219 (IOO), 203 (6); NMR (CDCI,) 6 7.48, 7.25 (q, J = 8 Hz, 4 H), 7.1 1 (s, 2 H), 5.22 (s, I H), 4.08 (s, 2 H), 1.40 (s, 18 H). 2-tert.-Butyl-C(I,I-*HZ]ethylphenol (4-EP-d,). According to the procedure of Brown and White (1957), 4-hydroxyacetophenone was hydrogenated with L&D4 and aluminum chloride to 4-[ l,l-2H2]ethylphenol, bp 6466”C/4 mm Hg. The ethylphenol was alkylated with tert.butyl chloride as described previously (Mizutani et al., 1982). 4-EP-d2, bp I IO-1 13”C/14 mm Hg; MS m/e 180 (M+, 32%), 165 (IOO), 137 (27). isotopic purity 95%; NMR (CDCI,) 6 7. I5 (d, J = 2 Hz, 1 H), 6.97 (d of d, J = 2, 8 Hz, 1 H), 6.61 (d, J = 8 Hz, I H), 4.72 (s, I H), 1.41 (s, 9 H), 1.20 (s, 3 H). 2-tert.-Butyl-4-[2,2,2-*H,]ethylphenol (4-EP-dJ. 4-Hydroxyphenylacetic acid was converted to methyl 4-methoxyphenylacetate, which was hydrogenated with LiAlD4 to 4-methoxy[a,cu-‘H2]phenethyl alcohol, bp 125- 126”C/ 10 mm Hg. Treatment of the alcohol with phosphorous tribromide in carbon tetrachloride yielded 4-methoxy[ol,a-‘H2]phenethyl bromide, bp 114- 117”C/ 11 mm Hg. The bromide was hydrogenated with LiAlD, to 4methoxy[2,2,2-ZHA]ethylbenzene by refluxing for 10 hr in tetrahydrofuran. Demethylation with boron tribromide in dichloromethane yielded 4-[2,2.2-‘H3]ethylpheno1, which was treated with tert.-butyl chloride as descibed above. 4-EP-d,, bp I 12-l 14”C/14 mm Hg; MS m/e 18 1 (M+, 32%), 166 (IOO), 138 (35), isotopic purity 97%; NMR (CD’&) 6 7.13 (d, / = 2 Hz, I H), 6.97 (d of d, J = 2. 8 Hz. 1 H), 6.63 (d. J = 8 Hz, 1 H). 4.71 (s, I H), 2.58 (s, 2 H). 1.41 (s, 9 H). .4nimals Male ddY mice (Shizuoka Agricultural Cooperative Association for Laboratory Animals. Shizuoka, Japan), 8 weeks of age, were used. Mice were housed in plastic cages on a wood chip bedding (White Flake, Charles River Japan, Inc., Kanagawa,
1SOTOPE EFFECTS ON BHT METABOLlSM Japan) and received a standard laboratory chow (Funabashi F-2, Funabashi Farms, Chiba, Japan) and water ad libitum. The animal room was controlled for temperature (23 & 2°C) and light cycle (12 hr). Lung
AND TOXICITY
285
a silica gel dry column (Wakogel C-200, Wako Pure Chemical Industries, Ltd., Osaka, Japan; lo-cm X &mm i.d.). Elution with hexane-benzene (4: I) gave fractions 1 (8 ml) and 2 (IO ml). Fraction 2 was analyzed by selected ion monitoring to determine the ratio of the abundance of BHT-SPhBr and BHT-SPhBr-d2.
Toxicity
BHT, BHT-d,, 4-EP, 4-EP-d* , and 4-EP-dj were each dissolved in olive oil and administered ip to mice. Control animals were treated with the vehicle alone. Lung damage was assessedby determining wet and dry lung weights 4 days after the administration. In Vitro Metabolism
The livers of mice were homogenized in 3 vol of 0. I5 KCl, and the homogenate was centrifuged for 20 min at 9000 g. Each incubation mixture contained 2 ml of the 9000 g supematant fraction, 0.5 mM NADP, 5 mM glucose 6-phosphate, and 2 mM MgC12 in a total volume of 5 ml of 0.1 M phosphate buffer (pH 7.4). Either BHT or BHT-ds (2.5 pmol in 50 ~1 ethanol) was added to the incubation mixture, and the mixture was shaken at 37°C for IO min in air. Measurement ofBHT-OH. The incubation mixture was poured into 3 ml of acetone and the mixture was centrifuged. The supematant solution was extracted with hexane and the extract was subjected to gas chromatography with an electron-capture detector (GC-ECD). Measurement of BHT-QM. The incubation mixture was poured into 3 ml of acetone containing 25 rmol of p-bromothiophenol. After standing for 30 min at room temperature, the mixture was centrifuged and the supernatant solution was extracted with hexane. The extract was subjected to GC-ECD analysis after purification by thin-layer chromatography. M
In Vivo Metabolism Measurement of BHT-OH. BHT and BHTd, were each administered ip to mice at a dose of 1500 mg/kg. The lungs and livers were excised after 4 hr and homogenized in 0.1 M phosphate buffer (4 ml/g of tissue). The homogenate was extracted with benzene and the extract was analyzed by GC-ECD. Measurement ofBHT-QM. An equimolar mixture (3000 mg/kg) of BHT and BHT-dS was administered ip to mice. The lungs and livers were excised after 4 hr and homogenized in 0.1 M phosphate buffer (4 ml/g of tissue). A solution of 5 mM pbromothiophenol in dioxane (1 ml/ g of tissue) was added to the homogenate and the mixture was shaken for 20 min at room temperature. The reaction mixture was extracted with benzene. The extract was dried and evaporated to dryness under a stream of nitrogen. The residue dissolved in hexane was chromatographed on
Instrumental
Analyses
GC-ECD was performed on a Shimadzu GC-3AE gas chromatograph fitted with a 2-m X 3-mm-i.d. glass column packed with Chromosorb W containing 2% OV- I. The analyses of BHT-OH and BHT-SPhBr were conducted at 100 and 175”C, respectively. Mass spectra were obtained by using a JEOL JMS-D 100 GC-MS spectrometer equipped with a JEOL MSPD-0 I multiple ion detector. The ionizing energy was 22 eV. The isotopic purities of BHT-dr , 4-EP-dz, and 4-EPd, were determined from the parent ions in the electronimpact mass spectra. To determine the ratio of the abundance of BHT-SPhBr and BHT-SPhBr-ds, the selected ion monitor was focused on the ions m/e 219 for BHTSPhBr and m/e 22 1 for BHT-SPhBr-d,, and the peak height ratio was measured. NMR spectra were measured in CDCI, with tetramethylsilane as an internal standard on a JEOL JMN MH-100 100 MHz spectrometer. The isotopic purity of BHT-SPhBr-dz was determined by measuring the residual methylene resonance at d 4.08.
RESULTS Isotope E#ect on Pulmonary
Toxicity of BHT
The effects of 1.39, 1.67, and 2.00 mmol/ kg BHT or BHT-ds on lung/body weight ratio are shown in Fig. 1A. Both compounds caused dose-related increases in lung/body weight ratio. At each dose, however, BHT-ds produced a significantly lower increase in lung/body weight ratio than did BHT. Similarly, BHT and BHT-d3 caused doserelated increases in dry lung weight, and the increase caused by BHT-ds was consistently lower than that caused by BHT (Fig. I B). The differences were statistically significant except at the highest dose level, 2.00 mmol/kg. The effects of BHT and BHT-d3 on the percentage change in body weight of mice during the experiment (4 days) are shown in Fig. 1C. A dose-dependent reduction in body weight
286
MIZUTANI,
YAMAMOTO,
AND
TAJIMA
Isotope E#ect on in Vitro Metabolism
0
DOSE
ofBHT
A procedure was developed to determine BHT-OH and BHT-QM formed by in vitro or in vivo metabolism of BHT. ECD exhibited a sensitive response toward BHT-OH possibly because of its conjugated structure. Therefore, BHT-OH at nanogram levels could be detected directly by GC-ECD. BHT-QM, which is known to be an unstable metabolite (Tajima et al., 1981) was trapped with p-bromothiophenol and analyzed as BHT-SPhBr by GC-ECD. There was no isotope effect on this trapping reaction. Although the trapping reaction resulted in a more than 80% recovery, the data obtained through this method should be considered as semiquantitative, because it appears rather likely that the resulting BHT-QM also reacts with cellular nucleophiles, such as glutathione and the cysteine components of proteins. BHT-OH and BHTSPhBr (derivatized from BHT-QM) were identified from their mass spectral fragmentation patterns and by comparison of their GC retention times with those of authentic samples. Typical gas chromatograms of BHTOH and BHT-SPhBr formed in the in vitro experiments are shown in Fig. 2.
(mmol/kg)
FIG. I. Effects of different doses of BHT (0) and BHTd, (0) on (A) lung/body weight ratio, (B) dry lung weight, and (C) body weight change during the experiment. Mice were injected ip with an olive oil solution of each agent at doses of 1.39, 1.67. and 2.00 mmol/kg. Control mice were given the vehicle alone. Mice were killed 4 days after injection, and the lung and body weights were determined. Each point represents mean t SE of 8 to I8 animals. (a) and(b) Indicate values are significantly different from corresponding BHT values (P < 0.0 I and I’ i 0.05, respectively).
was found for both BHT and BHT-d3. At each dose, mice receiving BHT lost more weight than did mice treated with BHT-d3, and the differences were statistically significant except at the highest dose level.
0
10 TIME
20 (min)
FIG. 2. Typical CC-ECD chromatograms of rn vitro metabolites. (A) BHT-OH and (B) BHT-QM (after being converted to BHT-SPhBr). Analyses were performed on a 2-m X 3-mm-i.d. glass column of 2% OV-I operated at (A) 100°C with an inlet pressure of I.0 kg/cm* of nitrogen and (B) 175°C with an inlet pressure of 1.4 kg/ cm* of nitrogen.
ISOTOPE
EFFECTS
ON
BHT
METABOLISM
AND
287
TOXICITY TABLE
2
By the above method, the rates of in vitro metabolism of BHT and BHT-d3 were compared (Table 1). Deuteration of BHT in the 4-methyl group resulted in a significant reduction (approximately 40%) in the rate of metabolism to BHT-QM. On the contrary, the rate of metabolism to BHT-OH was significantly increased (approximately 70%) with the deuterium substitution.
4’
Isotope Efect on in Vivo Metabolism
a Values represent means + SE of four determinations. b BHT-QM was determined after being converted BHT-SPhBr.
of BHT
An equimolar mixture of BHT and BHTd3 was administered to mice, and the ratio of deuterated to undeuterated BHT-QM levels in the lung and liver was determined by mass spectrometry with selected ion monitoring. In the lung the amount of BHT-QM per gram of tissue was approximately two times greater than that in the liver 4 hr after dosing. As shown in Table 2, the ratios of deuterated to undeuterated BHT-QM in the lung and liver were 0.66 and 0.85, respectively, indicating that BHT-d3 was metabolized in vivo to BHTQM at a lower rate than BHT. Although the isotope effect seen in the liver was somewhat small, the effect of the deuteration observed in the lung was comparable to that in the in vitro study.
TABLE
I
RELATIVE RATESOF IN VITRO METABOLISM OF BHT AND BHT-d,’ Metabolite formed (nmol/g liver/min) Substrate BHT (A) BHT-ds (B) Ratio
(B/A)
BHT-QM
b
BHT-OH
1.50 f 0.19 0.89 f 0.18’
0.91 k 0.06 1.53 + 0.25’
0.59
I .68
a Values represent means f SE of five determinations. b BHT-QM was determined after being converted to BHT-SPhBr. ‘Significantly different from BHT values (P < 0.05).
RATIOOFDEUTEFWTEDTOUNDEUTERATED BHT-QM FORMED IN MICE 4 hr AFTER ip ADMINISTRATION OF 3000 mg/kg EQUIMOLAR MIXTURE OF BHT AND BHT-
Tissue
BHT-QM
Lung Liver
kWdb
0.66 f 0.03 0.85 -t 0.01
to
A similar attempt to determine the relative levels of deuterated and undeuterated BHTOH was unsuccessful because the mass spectrum of control tissue samples gave interfering background peaks. Therefore, in vivo BHTOH (BHT-OH-d3) levels were determined by GC-ECD after the separate administration of BHT and BHT-dS. A comparison of the tissue levels of BHT-OH and BHT-OH-d3 after the administration of BHT or BHT-d3 is presented in Table 3. In both the lung and liver, BHTd, resulted in a significantly higher level of BHT-OH than did BHT in agreement with the in vitro results. isotope Efleci on Pulmonary
Toxicity ofl-EP
The effects of 3.41 mmol/kg 4-EP or its deuterated analogs on lung/body weight ratio are shown in Fig. 3A. 4-EP and 4-EP-d3 resulted in significant increases in lung/body weight ratio to 16 1 and 172%, respectively, of the control. 4-EP-d2, on the contrary, did not cause any significant change in lung/body weight ratio. With the effects on dry lung weight, a similar pattern was observed (Fig. 3B), that is, 4-EP and 4-EP-d3 resulted in about the same magnitude of increases (approximately 145% of control); 4-EP-d2, however, caused a significant, but only small, increase (113%). The change in dry lung weight caused by 4-EP-d, was significantly lower than that by either 4EP or 4-EP-d3.
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MIZUTANI,
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AND TAJIMA
TABLE 3 BHT-OH FORMED IN MICE 4 hr AFTER ip ADMINISTRATION OF 1500 mg/kg BHT OR BHT-d,” BHT-OH Lung
Liver
rig/total organ
rig/g tissue
rig/total organ
49 * 4 68 + 4h
250 k 22 341 * 20h
162 f 5 204 + gh
106 of- 5 133 t 9"
1.39
1.36
1.26
1.25
BHT (A) BHT-dX (B) Ratio (B/A)
rig/g
tissue
’ Values represent means f SE of four determinations. b Significantly different from BHT values (P < 0.05).
The effects of 4-EP and its deuterated analogs on body weight are shown in Fig. 3C. All three compounds were shown to have significant effects upon growth as measured by body weight change. 4-EP-d2 but not 4-EP-d3 resulted in a significantly lower inhibition in body weight gain than did 4-EP. DISCUSSION Histological studies have demonstrated that lung damage resulting from BHT can be well
monitored by an increase in lung weight (Witschi and Saheb, 1974; Saheb and Witschi, 1975). Moreover, it has been reported that lung damage caused by BHT or its analogs was always accompanied by a marked body weight loss (Malkinson, 1979; Kawano et al., 1981; Mizutani et al., 1982). In this study, therefore, changes in wet and dry lung weights and in body weight were used as indices of toxicity. The present study clearly showed that BHTd3 is less pulmonary toxic than BHT (Fig. 1). The rates of BHT-QM formation from BHT-
A
m
CONTROL
0
4-EP
m
4-EP-d2
881
4-EP-d3
FIG. 3. Effects of 4-EP, 4-EP-d2, and 4-EP-d, on (A) lung/body weight ratio, (B) dry lung weight, and (C) body weight change during the experiment. Mice were injected ip with an olive oil solution of each agent at a dosage of 3.41 mmol/kg. Control mice were given the vehicle alone. Mice were killed 4 days after injection, and the lung and body weights were determined. Means + SE of four to seven animals are plotted as percentage of control. (a) Indicates values are significantly different from control (P < 0.05); (b) indicates values are significantly different from 4-EP values (P < 0.05).
ISOTOPE EFFECTS ON BHT METABOLISM
dJ were slower than from BHT in both the in vitro and the in viva studies (Tables 1 and 2). These findings support the concept that the lung damage caused by BHT is mediated by BHT-QM (Mizutani et al., 1982). The deuteration in the 4-methyl group of BHT is also expected to suppress the formation of metabolites such as BHT-alcohol, BHT-aldehyde, and BHT-acid. This may suggest the possibility that these metabolites are responsible for the toxic effect of BHT. However, none of these metabolites was shown to be toxic (Malkinson, 1979). In contrast to what was observed with BHTQM, the in vitro and in vivo rates of BHTOH formation were significantly increased with deuterium labeling of BHT (Tables 1 and 3). The concept “metabolic switching” raised by Horning et al. (1979) can conceivably account for this observation, that is, deuteration in the 4-methyl group of BHT resulted in suppressed metabolic reactions at this site and, consequently, metabolism was most likely switched toward ring oxygenation, eventually leading to the enhanced formation of BHTOH. It has been shown that BHT is metabolized to BHT-OH via 4-hydroperoxy-4methyl - 2,6 - di - tert - butyl - 2,5 - cyclohexadi enone (BHT-OOH) in vitro (Shaw and Chen, 1972; Chen and Shaw, 1974) and in vivo (Yamamoto et al., 1979). Possible participation of this metabolic pathway in BHT toxicity has been implicated, since BHT-OOH is a highly reactive compound (Shaw and Chen, 1972; Kehrer and Witschi, 1980). However, this seems rather unlikely because BHTd3 resulted in suppressed pulmonary toxicity even though the formation of BHT-OH was considerably increased with the deuterium substitution of BHT. In an early study (Mizutani et al., 1982), we found several lung toxic alkylphenols, other than BHT, among which is 4-EP. To determine if the activation of 4-EP to its quinone methide metabolite as suggested for BHT could be responsible for the pulmonary toxicity of 4-EP, we compared the relative lung toxicity of 4-EP and its deuterated analogs
289
AND TOXICITY
The results presented in Fig. 3 demonstrated that 4-EP-d2 has considerably lower lung toxicity than 4-EP, whereas 4-EP-dj has a toxic potency comparable to that of 4-EP. This result implies that the cleavage of the C-H bond of the benzylic methylene group of 4-EP is critical in the process producing lung damage, thus supporting the hypothesis (Mizutani et al., 1982) that a quinone methide is responsible for the onset of lung damage produced by 4-EP as well as by BHT. ACKNOWLEDGMENTS The authors wish to thank Miss Nobuko Kitano, Mrs. Hitomi Fujimaki, and Miss Yuko Koyama for their technical assistance. Thanks are also due to Miss Toyoko Hirai and Miss Hitomi Shimomura for mass spectrometry measurement.
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(E. R. Klein and P. D. Klein, eds.), pp. 379384. Academic Press, New York. KAWANO, S., NAKAO, T., ANDHIRAGA, K. (1981). Strain differences in butylated hydroxytoluene-induced deaths in male mice. Toxicol. Appl. Pharmacol. 61,475-479. KEHRER, J. P., AND WITSCHI, H. (1980). Effects of drug metabolism inhibitors on butylated hydroxytoluene-induced pulmonary toxicity in mice. Toxicol. Appl. Pharmucol. 53, 333-342. KHARASCH, M. S., AND JOSHI, B. S. (1957). Reactions of hindered phenols. II. Base-catalysed oxidations of hindered phenols. J. Org. Chem. 22, 1439-1443.
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MALKINSON, A. M. (1979). Prevention of butylated hydroxytoluene-induced lung damage in mice by cedar terpene administration. Toxicol. Appl. Pharmacol. 49, 55 I-560. MARINO, A. A., AND MITCHELL, J. T. (1972). Lung damage in mice following intraperitoneal injection of butylated hydroxytoluene. Proc. Sot. Exp. Biol. Med. 140, 122-125. MIZUTANI, T., ISHIDA, I., YAMAMOTO, K., AND TAJIMA, K. (1982). Pulmonary toxicity of butylated hydroxytoluene and related alkylphenols: Structural requirements for toxic potency in mice. Toxicol. Appl. Pharmacol. 62, 273-28 1. SAHEB, W., AND WITSCHI, H. (1975). Lung growth in mice after a single dose of butylated hydroxytoluene. Toxicol. Appl. Pharmacol. 33, 309-3 19. SHAW, Y.-S., AND CHEN, C. (1972). Ring hydroxylation
AND TAJIMA
of di-l-butylhydroxytoluene by rat liver microsomal preparations. Biochem. J. 128, 1285- 129 1. TAJIMA, K., YAMAMOTO, K., AND MIZUTANI, T. (1981). Biotransformation of butylated hydroxytoluene (BHT) to BHT-quinone methide in rats. Chem. Pharm. Bull. 29, 3738-3741. YAMAMOTO, K., TAJIMA, K., ANDMIZUTANI, T. (1979). Identification of new metabolites of butylated hydroxytoluene (BHT) in rats. J. Pharm. Dyn. 2, 164-168. YOHE, G. R., DUNBAR, J. E., PEDROTTI, R. L., SCHEIDT, F. M., LEE, F. G. H., AND SMITH, E. C. (1956). The oxidation of 2,6di-terf-butyl-4-methylphenol. J. Org. Chem. 21, 1289-1292. WICHI, H., AND SAHEB,W. (1974). Stimulation of DNA synthesis in mouse lung following intraperitoneal injection of butylated hydroxytoluene. Proc. Sot. Exp. Biol. Med. 147, 690-693.
PHARMACOLOGY
69,283-290(1983)
Isotope Effects on the Metabolism and Pulmonary Toxicity of Butylated Hydroxytoluene in Mice by Deuteration of the It-Methyl Group’ TAMIO *Laboratory
MIZUTANI,*
of Environmental Sakyo-ku,
KENJI YAMAMOTO,~
AND KAZUO
TAJIMA~
Health and Toxicology, Department of Food Science, Kyoto Prefectural Kyoto 606. and TDepartment of Chemistry, School of Pharmacy, Hokuriku University. Kanazawa 920-I 1, Japan
Received
December
27, 1982: accepted
February
University,
25, 1983
Isotope Effects on the Metabolism and Pulmonary Toxicity of Butylated Hydroxytoluene in Mice by Deuteration of the 4-Methyl Group. MIZUTANI, T., YAMAMOTO, K.. AND TAJIMA, K. (1983). Toxicol. Appl. Pharmacol. 69, 283-290. A comparative test in mice for pulmonary toxicity between butylated hydroxytoluene (2,6-di-tert.-butyl-4-methylphenol, BHT) and 2,6-ditert.-butyl-4-(a,cY,c+*H3]methylphenol (BHT-d,) showed a significantly lower toxic potency of the latter. The rate of in vitro BHT metabolism to 2,6-di-tert.-butyl-4-methylene-2,5-cyclohexadienone (BHT-QM) was slowed by deuterating BHT in the 4-methyl group. On the other hand, the rate of in vitro metabolism to 2,6-di-tert.-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-OH) was increased with the deuteration. A similar isotope effect of the deuterium substitution on the in vivo metabolic rates of BHT was observed. These observations support the concept that the lung damage caused by BHT is mediated by BHT-QM. The pulmonary toxicity of 2-tert.-butyl-4-ethylphenol (4-EP) and their deuterated analogs was also compared. 2-tert.-Butyl-4-[ 1,1-*H2]ethylphenol (4-EP-d2) showed a significantly lower toxic potency than 4EP, whereas 2-tert.-butyl-4-[2,2,2-2H~]ethylphenol (4-EP-dl) showed a toxic potency comparable to that of 4-EP. This result is consistent with the hypothesis that a quinone methide metabolite is responsible for the onset of lung damage produced by 4-EP as well as BHT.
Butylated hydroxytoluene (2,6-di-tert.-butyl4-methylphenol, BHT) is a widely used antioxidant. BHT has been shown to cause lung damage in mice (Marino and Mitchell, 1972; Saheb and Witschi, 1975), which is characterized by early necrosis of type I alveolar cells and subsequent proliferation of type II alveolar cells (Adamson et al., 1977). Malkinson (1979) reported that BHT-induced lung damage can be prevented by exposing mice to cedar terpenes and that immature mice are nonresponsive to BHT. Kehrer and Witschi (1980) demonstrated the prevention of BHT-induced lung damage by ’ This paper was presented in part at the Ninth Symposium on Environmental Pollutants and Toxicology, October 14-15, 1982, Okayama, Japan. 283
the coadministration of drug metabolism inhibitors SKF-525A or piperonyl butoxide. BHT given to mice becomes covalently bound to lung tissue and this covalent binding can be prevented by the administration of SKF525A (Kehrer and Witschi, 1980). These findings suggest that a reactive metabolite of BHT is responsible for the onset of lung damage in mice. Based upon a structure-activity study with BHT analogs, we have recently suggested that a quinone methide, 2,6-di-tert.-butyl-Cmethylene-2$cyclohexadienone (BHT-QM), or closely related metabolites may play a role in producing lung damage in mice dosed with BHT (Mizutani et al., 1982). The present work was undertaken to add positive support for this suggestion and deals with the isotope ef0041-008X/83 Copyright All
rights
Q
1983
of reprc&ction
$3.00 by
Academic in any
Press.
Inc.
form
reserved.
284
MIZUTANI,
YAMAMOTO,
fects on the metabolism and pulmonary toxicity of BHT in mice by deuteration of the 4methyl group. METHODS Chemicals Chemicals were purchased as follows: lithium aluminum deuteride (LiA1D4, 98% isotopic purity) from E. Merck A. G., Darmstadt, West Germany; sodium borodeuteride (NaBD,, 97.4% isotopic purity) from Commissariat al’ Energie Atomique, France; BHT from Nakarai Chemicals, Ltd., Kyoto, Japan; pbromothiophenol from Aldrich Chemical Company, Milwaukee, Wisconsin; NADP from Kohjin Company Ltd., Tokyo, Japan; glucose 6-phosphate from Sigma Chemical Company, St. Louis, Missouri. All other reagents were of the highest purity available. 2,6-Di-tert.-butyI-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-OH) (Kharasch and Joshi, 1957) and 2tert.-butyl-4-ethylphenol (4-EP) (Mizutani et al., 1982) were synthesized according to the described methods.
Svntheses 2.6-Di-tert.-butyl-4-[~,~.~-2H~]methylphenoI (BHT-d,). 3.5-Di-tert.-butyl-4-hydroxybenzoic acid (Yohe et al., 1956) was converted to the methyl ester, mp; 166-167°C. The ester (0.03 mol, 7.9 g) dissolved in ether (100 ml) was added to a solution of LiAID, (0.09 mol, 3.8 g) in ether (200 ml), and the mixture was refluxed under nitrogen for 12 hr. The excessreagent was decomposed with ethyl acetate and 10% sodium hydroxide, and the resulting precipitate was filtered off. The organic layer was washed with water, dried, and freed from the solvent. Column chromatography on silica gel gave BHT-dS, mp 68-69°C; MS m/e 223 (M+, 32%), 208 (lOO), 180 (S), 148 (I l), isotopic purity 98%; NMR (CDCI,) 6 7.05 (s, 2 H), 5.04 (s, I H), 1.44 (s, 18 H). 2.6-Di-tert.-butyl-4-h.vdroxy-4-[a,cu,cu-*H,lmethyl-2,5cyclohexadienone (BHT-OH-dJ. According to the procedure of Kharasch and Joshi (1957). BHT-d, was oxygenated to BHT-OH-d,, mp 1 IO-1 12’C; MS m/e 239 (M+. 21%), 221 (21), 183 (loo), 168 (97), isotopic purity 97%; NMR (CDC&) 6 6.66 (s, 2 H). 1.92 (s, 1H). 1.21 (s. 18 H). 2.6 - Di- tert. - butyl- I- (4’- bromophenylthio[cY.o -‘HJ] methyophenoi (BHT-SPhBr-dJ. 3,5-Di-tert.-butyl-4-hydroxybenzoic acid (Yohe et al., 1956) was converted to the acid chloride, which was hydrogenated with NaBD4 in dioxane to 3,5-di-tert.-butyl-4-hydroxy[cu,ol-2H2lbl alcohol, mp 142-143°C. Treatment of the alcohol with thionyl chloride yielded 3,5-di-tert.-butyl-4-hydroxy[a,a-
AND TAJIMA
*H$enzyl chloride. A solution of the chloride (2 mmol, 500 mg) in acetone (2 ml) was added to a mixture of p bromothiophenol (2 mmol, 380 mg) and potassium carbonate(200mg) in acetone(8 ml), andthemixturewas stirred at room temperature for 2 hr. The mixture was poured into water and extracted with benzene. The organic layer was washed with 5% sodium hydroxide and then with water, dried, and freed from the solvent. Column chromatography on silica gel gave BHT-SPhBrd*, mp 95-96°C; MS m/e 408 (M+, 1.5%), 22 1 (IOO), 205 (7); NMR (CD(&) 6 7.48, 7.25 (q, J = 8 Hz, 4 H), 7.11 (s, 2 H). 5.23 (s, 1 H), 1.40 (s, 18 H), isotopic purity 96%. 2,6 - Di - tert. - butyl - 4 - (4’ - bromophenylthiomethyl) phenol (BHT-SPhBr). This compound was prepared by the same method as described above for BHT-SPhBrd, with 3,5di-tert.-butyl-4-hydroxybenzyl chloride (Chasar and Westfahl, 1977) and pbromothiophenol as starting materials. BHT-SPhBr, mp 93-95°C; MS m/e 406 (M+, 1.2%). 219 (IOO), 203 (6); NMR (CDCI,) 6 7.48, 7.25 (q, J = 8 Hz, 4 H), 7.1 1 (s, 2 H), 5.22 (s, I H), 4.08 (s, 2 H), 1.40 (s, 18 H). 2-tert.-Butyl-C(I,I-*HZ]ethylphenol (4-EP-d,). According to the procedure of Brown and White (1957), 4-hydroxyacetophenone was hydrogenated with L&D4 and aluminum chloride to 4-[ l,l-2H2]ethylphenol, bp 6466”C/4 mm Hg. The ethylphenol was alkylated with tert.butyl chloride as described previously (Mizutani et al., 1982). 4-EP-d2, bp I IO-1 13”C/14 mm Hg; MS m/e 180 (M+, 32%), 165 (IOO), 137 (27). isotopic purity 95%; NMR (CDCI,) 6 7. I5 (d, J = 2 Hz, 1 H), 6.97 (d of d, J = 2, 8 Hz, 1 H), 6.61 (d, J = 8 Hz, I H), 4.72 (s, I H), 1.41 (s, 9 H), 1.20 (s, 3 H). 2-tert.-Butyl-4-[2,2,2-*H,]ethylphenol (4-EP-dJ. 4-Hydroxyphenylacetic acid was converted to methyl 4-methoxyphenylacetate, which was hydrogenated with LiAlD4 to 4-methoxy[a,cu-‘H2]phenethyl alcohol, bp 125- 126”C/ 10 mm Hg. Treatment of the alcohol with phosphorous tribromide in carbon tetrachloride yielded 4-methoxy[ol,a-‘H2]phenethyl bromide, bp 114- 117”C/ 11 mm Hg. The bromide was hydrogenated with LiAlD, to 4methoxy[2,2,2-ZHA]ethylbenzene by refluxing for 10 hr in tetrahydrofuran. Demethylation with boron tribromide in dichloromethane yielded 4-[2,2.2-‘H3]ethylpheno1, which was treated with tert.-butyl chloride as descibed above. 4-EP-d,, bp I 12-l 14”C/14 mm Hg; MS m/e 18 1 (M+, 32%), 166 (IOO), 138 (35), isotopic purity 97%; NMR (CD’&) 6 7.13 (d, / = 2 Hz, I H), 6.97 (d of d, J = 2. 8 Hz. 1 H), 6.63 (d. J = 8 Hz, 1 H). 4.71 (s, I H), 2.58 (s, 2 H). 1.41 (s, 9 H). .4nimals Male ddY mice (Shizuoka Agricultural Cooperative Association for Laboratory Animals. Shizuoka, Japan), 8 weeks of age, were used. Mice were housed in plastic cages on a wood chip bedding (White Flake, Charles River Japan, Inc., Kanagawa,
1SOTOPE EFFECTS ON BHT METABOLlSM Japan) and received a standard laboratory chow (Funabashi F-2, Funabashi Farms, Chiba, Japan) and water ad libitum. The animal room was controlled for temperature (23 & 2°C) and light cycle (12 hr). Lung
AND TOXICITY
285
a silica gel dry column (Wakogel C-200, Wako Pure Chemical Industries, Ltd., Osaka, Japan; lo-cm X &mm i.d.). Elution with hexane-benzene (4: I) gave fractions 1 (8 ml) and 2 (IO ml). Fraction 2 was analyzed by selected ion monitoring to determine the ratio of the abundance of BHT-SPhBr and BHT-SPhBr-d2.
Toxicity
BHT, BHT-d,, 4-EP, 4-EP-d* , and 4-EP-dj were each dissolved in olive oil and administered ip to mice. Control animals were treated with the vehicle alone. Lung damage was assessedby determining wet and dry lung weights 4 days after the administration. In Vitro Metabolism
The livers of mice were homogenized in 3 vol of 0. I5 KCl, and the homogenate was centrifuged for 20 min at 9000 g. Each incubation mixture contained 2 ml of the 9000 g supematant fraction, 0.5 mM NADP, 5 mM glucose 6-phosphate, and 2 mM MgC12 in a total volume of 5 ml of 0.1 M phosphate buffer (pH 7.4). Either BHT or BHT-ds (2.5 pmol in 50 ~1 ethanol) was added to the incubation mixture, and the mixture was shaken at 37°C for IO min in air. Measurement ofBHT-OH. The incubation mixture was poured into 3 ml of acetone and the mixture was centrifuged. The supematant solution was extracted with hexane and the extract was subjected to gas chromatography with an electron-capture detector (GC-ECD). Measurement of BHT-QM. The incubation mixture was poured into 3 ml of acetone containing 25 rmol of p-bromothiophenol. After standing for 30 min at room temperature, the mixture was centrifuged and the supernatant solution was extracted with hexane. The extract was subjected to GC-ECD analysis after purification by thin-layer chromatography. M
In Vivo Metabolism Measurement of BHT-OH. BHT and BHTd, were each administered ip to mice at a dose of 1500 mg/kg. The lungs and livers were excised after 4 hr and homogenized in 0.1 M phosphate buffer (4 ml/g of tissue). The homogenate was extracted with benzene and the extract was analyzed by GC-ECD. Measurement ofBHT-QM. An equimolar mixture (3000 mg/kg) of BHT and BHT-dS was administered ip to mice. The lungs and livers were excised after 4 hr and homogenized in 0.1 M phosphate buffer (4 ml/g of tissue). A solution of 5 mM pbromothiophenol in dioxane (1 ml/ g of tissue) was added to the homogenate and the mixture was shaken for 20 min at room temperature. The reaction mixture was extracted with benzene. The extract was dried and evaporated to dryness under a stream of nitrogen. The residue dissolved in hexane was chromatographed on
Instrumental
Analyses
GC-ECD was performed on a Shimadzu GC-3AE gas chromatograph fitted with a 2-m X 3-mm-i.d. glass column packed with Chromosorb W containing 2% OV- I. The analyses of BHT-OH and BHT-SPhBr were conducted at 100 and 175”C, respectively. Mass spectra were obtained by using a JEOL JMS-D 100 GC-MS spectrometer equipped with a JEOL MSPD-0 I multiple ion detector. The ionizing energy was 22 eV. The isotopic purities of BHT-dr , 4-EP-dz, and 4-EPd, were determined from the parent ions in the electronimpact mass spectra. To determine the ratio of the abundance of BHT-SPhBr and BHT-SPhBr-ds, the selected ion monitor was focused on the ions m/e 219 for BHTSPhBr and m/e 22 1 for BHT-SPhBr-d,, and the peak height ratio was measured. NMR spectra were measured in CDCI, with tetramethylsilane as an internal standard on a JEOL JMN MH-100 100 MHz spectrometer. The isotopic purity of BHT-SPhBr-dz was determined by measuring the residual methylene resonance at d 4.08.
RESULTS Isotope E#ect on Pulmonary
Toxicity of BHT
The effects of 1.39, 1.67, and 2.00 mmol/ kg BHT or BHT-ds on lung/body weight ratio are shown in Fig. 1A. Both compounds caused dose-related increases in lung/body weight ratio. At each dose, however, BHT-ds produced a significantly lower increase in lung/body weight ratio than did BHT. Similarly, BHT and BHT-d3 caused doserelated increases in dry lung weight, and the increase caused by BHT-ds was consistently lower than that caused by BHT (Fig. I B). The differences were statistically significant except at the highest dose level, 2.00 mmol/kg. The effects of BHT and BHT-d3 on the percentage change in body weight of mice during the experiment (4 days) are shown in Fig. 1C. A dose-dependent reduction in body weight
286
MIZUTANI,
YAMAMOTO,
AND
TAJIMA
Isotope E#ect on in Vitro Metabolism
0
DOSE
ofBHT
A procedure was developed to determine BHT-OH and BHT-QM formed by in vitro or in vivo metabolism of BHT. ECD exhibited a sensitive response toward BHT-OH possibly because of its conjugated structure. Therefore, BHT-OH at nanogram levels could be detected directly by GC-ECD. BHT-QM, which is known to be an unstable metabolite (Tajima et al., 1981) was trapped with p-bromothiophenol and analyzed as BHT-SPhBr by GC-ECD. There was no isotope effect on this trapping reaction. Although the trapping reaction resulted in a more than 80% recovery, the data obtained through this method should be considered as semiquantitative, because it appears rather likely that the resulting BHT-QM also reacts with cellular nucleophiles, such as glutathione and the cysteine components of proteins. BHT-OH and BHTSPhBr (derivatized from BHT-QM) were identified from their mass spectral fragmentation patterns and by comparison of their GC retention times with those of authentic samples. Typical gas chromatograms of BHTOH and BHT-SPhBr formed in the in vitro experiments are shown in Fig. 2.
(mmol/kg)
FIG. I. Effects of different doses of BHT (0) and BHTd, (0) on (A) lung/body weight ratio, (B) dry lung weight, and (C) body weight change during the experiment. Mice were injected ip with an olive oil solution of each agent at doses of 1.39, 1.67. and 2.00 mmol/kg. Control mice were given the vehicle alone. Mice were killed 4 days after injection, and the lung and body weights were determined. Each point represents mean t SE of 8 to I8 animals. (a) and(b) Indicate values are significantly different from corresponding BHT values (P < 0.0 I and I’ i 0.05, respectively).
was found for both BHT and BHT-d3. At each dose, mice receiving BHT lost more weight than did mice treated with BHT-d3, and the differences were statistically significant except at the highest dose level.
0
10 TIME
20 (min)
FIG. 2. Typical CC-ECD chromatograms of rn vitro metabolites. (A) BHT-OH and (B) BHT-QM (after being converted to BHT-SPhBr). Analyses were performed on a 2-m X 3-mm-i.d. glass column of 2% OV-I operated at (A) 100°C with an inlet pressure of I.0 kg/cm* of nitrogen and (B) 175°C with an inlet pressure of 1.4 kg/ cm* of nitrogen.
ISOTOPE
EFFECTS
ON
BHT
METABOLISM
AND
287
TOXICITY TABLE
2
By the above method, the rates of in vitro metabolism of BHT and BHT-d3 were compared (Table 1). Deuteration of BHT in the 4-methyl group resulted in a significant reduction (approximately 40%) in the rate of metabolism to BHT-QM. On the contrary, the rate of metabolism to BHT-OH was significantly increased (approximately 70%) with the deuterium substitution.
4’
Isotope Efect on in Vivo Metabolism
a Values represent means + SE of four determinations. b BHT-QM was determined after being converted BHT-SPhBr.
of BHT
An equimolar mixture of BHT and BHTd3 was administered to mice, and the ratio of deuterated to undeuterated BHT-QM levels in the lung and liver was determined by mass spectrometry with selected ion monitoring. In the lung the amount of BHT-QM per gram of tissue was approximately two times greater than that in the liver 4 hr after dosing. As shown in Table 2, the ratios of deuterated to undeuterated BHT-QM in the lung and liver were 0.66 and 0.85, respectively, indicating that BHT-d3 was metabolized in vivo to BHTQM at a lower rate than BHT. Although the isotope effect seen in the liver was somewhat small, the effect of the deuteration observed in the lung was comparable to that in the in vitro study.
TABLE
I
RELATIVE RATESOF IN VITRO METABOLISM OF BHT AND BHT-d,’ Metabolite formed (nmol/g liver/min) Substrate BHT (A) BHT-ds (B) Ratio
(B/A)
BHT-QM
b
BHT-OH
1.50 f 0.19 0.89 f 0.18’
0.91 k 0.06 1.53 + 0.25’
0.59
I .68
a Values represent means f SE of five determinations. b BHT-QM was determined after being converted to BHT-SPhBr. ‘Significantly different from BHT values (P < 0.05).
RATIOOFDEUTEFWTEDTOUNDEUTERATED BHT-QM FORMED IN MICE 4 hr AFTER ip ADMINISTRATION OF 3000 mg/kg EQUIMOLAR MIXTURE OF BHT AND BHT-
Tissue
BHT-QM
Lung Liver
kWdb
0.66 f 0.03 0.85 -t 0.01
to
A similar attempt to determine the relative levels of deuterated and undeuterated BHTOH was unsuccessful because the mass spectrum of control tissue samples gave interfering background peaks. Therefore, in vivo BHTOH (BHT-OH-d3) levels were determined by GC-ECD after the separate administration of BHT and BHT-dS. A comparison of the tissue levels of BHT-OH and BHT-OH-d3 after the administration of BHT or BHT-d3 is presented in Table 3. In both the lung and liver, BHTd, resulted in a significantly higher level of BHT-OH than did BHT in agreement with the in vitro results. isotope Efleci on Pulmonary
Toxicity ofl-EP
The effects of 3.41 mmol/kg 4-EP or its deuterated analogs on lung/body weight ratio are shown in Fig. 3A. 4-EP and 4-EP-d3 resulted in significant increases in lung/body weight ratio to 16 1 and 172%, respectively, of the control. 4-EP-d2, on the contrary, did not cause any significant change in lung/body weight ratio. With the effects on dry lung weight, a similar pattern was observed (Fig. 3B), that is, 4-EP and 4-EP-d3 resulted in about the same magnitude of increases (approximately 145% of control); 4-EP-d2, however, caused a significant, but only small, increase (113%). The change in dry lung weight caused by 4-EP-d, was significantly lower than that by either 4EP or 4-EP-d3.
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YAMAMOTO,
AND TAJIMA
TABLE 3 BHT-OH FORMED IN MICE 4 hr AFTER ip ADMINISTRATION OF 1500 mg/kg BHT OR BHT-d,” BHT-OH Lung
Liver
rig/total organ
rig/g tissue
rig/total organ
49 * 4 68 + 4h
250 k 22 341 * 20h
162 f 5 204 + gh
106 of- 5 133 t 9"
1.39
1.36
1.26
1.25
BHT (A) BHT-dX (B) Ratio (B/A)
rig/g
tissue
’ Values represent means f SE of four determinations. b Significantly different from BHT values (P < 0.05).
The effects of 4-EP and its deuterated analogs on body weight are shown in Fig. 3C. All three compounds were shown to have significant effects upon growth as measured by body weight change. 4-EP-d2 but not 4-EP-d3 resulted in a significantly lower inhibition in body weight gain than did 4-EP. DISCUSSION Histological studies have demonstrated that lung damage resulting from BHT can be well
monitored by an increase in lung weight (Witschi and Saheb, 1974; Saheb and Witschi, 1975). Moreover, it has been reported that lung damage caused by BHT or its analogs was always accompanied by a marked body weight loss (Malkinson, 1979; Kawano et al., 1981; Mizutani et al., 1982). In this study, therefore, changes in wet and dry lung weights and in body weight were used as indices of toxicity. The present study clearly showed that BHTd3 is less pulmonary toxic than BHT (Fig. 1). The rates of BHT-QM formation from BHT-
A
m
CONTROL
0
4-EP
m
4-EP-d2
881
4-EP-d3
FIG. 3. Effects of 4-EP, 4-EP-d2, and 4-EP-d, on (A) lung/body weight ratio, (B) dry lung weight, and (C) body weight change during the experiment. Mice were injected ip with an olive oil solution of each agent at a dosage of 3.41 mmol/kg. Control mice were given the vehicle alone. Mice were killed 4 days after injection, and the lung and body weights were determined. Means + SE of four to seven animals are plotted as percentage of control. (a) Indicates values are significantly different from control (P < 0.05); (b) indicates values are significantly different from 4-EP values (P < 0.05).
ISOTOPE EFFECTS ON BHT METABOLISM
dJ were slower than from BHT in both the in vitro and the in viva studies (Tables 1 and 2). These findings support the concept that the lung damage caused by BHT is mediated by BHT-QM (Mizutani et al., 1982). The deuteration in the 4-methyl group of BHT is also expected to suppress the formation of metabolites such as BHT-alcohol, BHT-aldehyde, and BHT-acid. This may suggest the possibility that these metabolites are responsible for the toxic effect of BHT. However, none of these metabolites was shown to be toxic (Malkinson, 1979). In contrast to what was observed with BHTQM, the in vitro and in vivo rates of BHTOH formation were significantly increased with deuterium labeling of BHT (Tables 1 and 3). The concept “metabolic switching” raised by Horning et al. (1979) can conceivably account for this observation, that is, deuteration in the 4-methyl group of BHT resulted in suppressed metabolic reactions at this site and, consequently, metabolism was most likely switched toward ring oxygenation, eventually leading to the enhanced formation of BHTOH. It has been shown that BHT is metabolized to BHT-OH via 4-hydroperoxy-4methyl - 2,6 - di - tert - butyl - 2,5 - cyclohexadi enone (BHT-OOH) in vitro (Shaw and Chen, 1972; Chen and Shaw, 1974) and in vivo (Yamamoto et al., 1979). Possible participation of this metabolic pathway in BHT toxicity has been implicated, since BHT-OOH is a highly reactive compound (Shaw and Chen, 1972; Kehrer and Witschi, 1980). However, this seems rather unlikely because BHTd3 resulted in suppressed pulmonary toxicity even though the formation of BHT-OH was considerably increased with the deuterium substitution of BHT. In an early study (Mizutani et al., 1982), we found several lung toxic alkylphenols, other than BHT, among which is 4-EP. To determine if the activation of 4-EP to its quinone methide metabolite as suggested for BHT could be responsible for the pulmonary toxicity of 4-EP, we compared the relative lung toxicity of 4-EP and its deuterated analogs
289
AND TOXICITY
The results presented in Fig. 3 demonstrated that 4-EP-d2 has considerably lower lung toxicity than 4-EP, whereas 4-EP-dj has a toxic potency comparable to that of 4-EP. This result implies that the cleavage of the C-H bond of the benzylic methylene group of 4-EP is critical in the process producing lung damage, thus supporting the hypothesis (Mizutani et al., 1982) that a quinone methide is responsible for the onset of lung damage produced by 4-EP as well as by BHT. ACKNOWLEDGMENTS The authors wish to thank Miss Nobuko Kitano, Mrs. Hitomi Fujimaki, and Miss Yuko Koyama for their technical assistance. Thanks are also due to Miss Toyoko Hirai and Miss Hitomi Shimomura for mass spectrometry measurement.
REFERENCES ADAMSON, I. Y. R., BOWDEN, D. H., C~TB, M. G., AND WITSCHI, H. (1977). Lung injury induced by butylated hydroxytoluene. Cytodynamic and biochemical studies in mice. Lab. Invest. 36, 26-32. BROWN, B. R., AND WHITE, A. M. S. (1957). Hydrogenolysis of aromatic carbonyl compounds and alcohols with aluminum chloride and lithium aluminum hydride. J. Chem. Sot. 3755-3757. CHASAR, D. W., AND WESTFAHL, J. C. (1977). 2,6-D& terf-butyl-4,4-bis(3,5-di-tert-butyl-4-hydroxybenzyl)2,5-cyclohexadienone. A new reaction product ofa hindered phenol. J. Org. Chem. 42, 2177-2179. CHEN, C., AND SHAW, Y.-S. (1974). Cyclic metabolic pathway of a butylated hydroxytoluene by rat liver microsomal fractions. Biochem. J. 144, 497-50 1. HORNING, M. G., NOWLIN, J., THENOT, J.-P., AND BOUWSMA, 0. J. (1979). Effect of deuterium substitution on the rate of caffeine metabolism. In Stable Isotopes, ference
Proceedings
of the Third
International
Con-
(E. R. Klein and P. D. Klein, eds.), pp. 379384. Academic Press, New York. KAWANO, S., NAKAO, T., ANDHIRAGA, K. (1981). Strain differences in butylated hydroxytoluene-induced deaths in male mice. Toxicol. Appl. Pharmacol. 61,475-479. KEHRER, J. P., AND WITSCHI, H. (1980). Effects of drug metabolism inhibitors on butylated hydroxytoluene-induced pulmonary toxicity in mice. Toxicol. Appl. Pharmucol. 53, 333-342. KHARASCH, M. S., AND JOSHI, B. S. (1957). Reactions of hindered phenols. II. Base-catalysed oxidations of hindered phenols. J. Org. Chem. 22, 1439-1443.
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