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 MIZUTANI,*
`
`KENJI YAMAMOTO,~
`
`AND KAZUO
`
`TAJIMA~
`
`*Laboratory
`
`of Environmental
`Sakyo-ku,
`
`Prefectural
`of Food Science, Kyoto
`Department
`and Toxicology,
`Health
`606. and TDepartment
`of Chemistry,
`School
`of Pharmacy,
`Kyoto
`Hokuriku
`University.
`Kanazawa
`920-I 1, Japan
`
`University,
`
`Received
`
`December
`
`27, 1982: accepted February
`
`25, 1983
`
`in
`Isotope Effects on the Metabolism and Pulmonary Toxicity of Butylated Hydroxytoluene
`K.. AND TAJIMA,
`Mice by Deuteration of the 4-Methyl Group. MIZUTANI,
`K.
`T., YAMAMOTO,
`69, 283-290. A comparative
`test in mice for pulmonary
`(1983).
`Toxicol.
`Appl. Pharmacol.
`toxicity between butylated hydroxytoluene (2,6-di-tert.-butyl-4-methylphenol,
`BHT) and 2,6-di-
`tert.-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-cyclo-
`hexadienone (BHT-QM) was slowed by deuterating BHT in the 4-methyl group. On the other
`to 2,6-di-tert.-butyl-4-hydroxy-4-methyl-2,5-cyclohexadi-
`hand, the rate of in vitro metabolism
`enone (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 4-
`EP, 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.
`
`(2,6-di-tert.-butyl-
`Butylated hydroxytoluene
`4-methylphenol,
`BHT)
`is a widely used an-
`tioxidant. BHT has been shown to cause lung
`damage in mice (Marino and Mitchell,
`1972;
`Saheb and Witschi, 1975), which
`is charac-
`terized by early necrosis of type I alveolar cells
`and subsequent proliferation
`of type II alveo-
`lar cells (Adamson et al., 1977).
`Malkinson
`(1979)
`reported
`that BHT-in-
`duced lung damage can be prevented by ex-
`posing mice
`to cedar terpenes and that
`im-
`mature mice are nonresponsive
`to BHT. Keh-
`rer and Witschi
`(1980) demonstrated
`the
`prevention of BHT-induced
`lung damage by
`
`’ This paper was presented in part at the Ninth Sym-
`posium on Environmental Pollutants and Toxicology,
`October 14-15, 1982, Okayama, Japan.
`
`in-
`of drug metabolism
`the coadministration
`or piperonyl
`butoxide.
`hibitors SKF-525A
`BHT given to mice becomes covalently bound
`to lung
`tissue and this covalent binding
`can
`be prevented by the administration
`of SKF-
`525A (Kehrer and Witschi, 1980). These find-
`ings suggest that a reactive metabolite of BHT
`is responsible
`for the onset of lung damage
`in
`mice.
`study with
`Based upon a structure-activity
`BHT analogs, we have recently suggested that
`a quinone methide, 2,6-di-tert.-butyl-Cmeth-
`or
`ylene-2$cyclohexadienone
`(BHT-QM),
`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 ef-
`
`283
`
`0041-008X/83
`
`$3.00
`
`Copyright
`
`Q
`
`1983
`
`by
`
`Academic
`
`Press.
`
`Inc.
`
`All
`
`rights
`
`of
`
`reprc&ction
`
`in
`
`any
`
`form
`
`reserved.
`
`Par Pharm., Inc.
`Exhibit 1007
`Page 001
`
`

`
`284
`
`MIZUTANI,
`
`YAMAMOTO,
`
`AND TAJIMA
`
`fects on the metabolism and pulmonary tox-
`icity of BHT in mice by deuteration of the 4-
`methyl group.
`
`METHODS
`
`Chemicals
`
`Chemicals were purchased as follows: lithium alumi-
`num deuteride (LiA1D4, 98% isotopic purity)
`from E.
`Merck A. G., Darmstadt, West Germany; sodium boro-
`deuteride (NaBD,, 97.4% isotopic purity)
`from Com-
`missariat al’ Energie Atomique, France; BHT
`from Na-
`karai Chemicals, Ltd., Kyoto, Japan; pbromothiophenol
`from Aldrich Chemical Company, Milwaukee, Wiscon-
`sin; 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-cyclohexad-
`ienone (BHT-OH)
`(Kharasch and Joshi, 1957) and 2-
`tert.-butyl-4-ethylphenol
`(4-EP) (Mizutani
`et al., 1982)
`were synthesized according to the described methods.
`
`Svntheses
`
`(BHT-d,).
`2.6-Di-tert.-butyl-4-[~,~.~-2H~]methylphenoI
`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 ni-
`trogen for 12 hr. The excess reagent was decomposed with
`ethyl acetate and 10% sodium hydroxide, and the result-
`ing 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-2,5-
`cyclohexadienone (BHT-OH-dJ.
`According
`to the pro-
`cedure of Kharasch and Joshi (1957). BHT-d, was oxy-
`genated 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-hy-
`droxybenzoic 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-
`
`chloride. A solution of the chloride (2 mmol,
`*H$enzyl
`500 mg) in acetone (2 ml) was added to a mixture of p
`bromothiophenol
`(2 mmol, 380 mg) and potassium car-
`bonate (200 mg) in acetone (8 ml), and the mixture was
`stirred at room temperature
`for 2 hr. The mixture was
`poured into water and extracted with benzene. The or-
`ganic layer was washed with 5% sodium hydroxide and
`then with water, dried, and freed from the solvent. Col-
`umn 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).
`(4-EP-d,). Accord-
`2-tert.-Butyl-C(I,I-*HZ]ethylphenol
`ing to the procedure of Brown and White (1957), 4-hy-
`droxyacetophenone was hydrogenated with L&D4 and
`aluminum chloride
`to 4-[ l,l-2H2]ethylphenol,
`bp 64-
`66”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).
`(4-EP-dJ. 4-Hy-
`2-tert.-Butyl-4-[2,2,2-*H,]ethylphenol
`droxyphenylacetic acid was converted to methyl 4-meth-
`oxyphenylacetate, 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-meth-
`oxy[ol,a-‘H2]phenethyl bromide, bp 114- 117”C/ 11 mm
`Hg. The bromide was hydrogenated with LiAlD,
`to 4-
`methoxy[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 bed-
`ding (White Flake, Charles River Japan, Inc., Kanagawa,
`
`Par Pharm., Inc.
`Exhibit 1007
`Page 002
`
`

`
`1SOTOPE EFFECTS ON BHT METABOLlSM
`
`AND TOXICITY
`
`285
`
`Japan) and received a standard laboratory chow (Funa-
`bashi F-2, Funabashi Farms, Chiba, Japan) and water ad
`The animal room was controlled for temperature
`libitum.
`(23 & 2°C) and light cycle (12 hr).
`
`Lung
`
`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 assessed by determining wet and dry
`lung weights 4 days after the administration.
`
`In Vitro Metabolism
`
`in 3 vol of 0. I5
`The livers of mice were homogenized
`M 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.
`The incubation mixture was
`Measurement
`ofBHT-OH.
`poured into 3 ml of acetone and the mixture was cen-
`trifuged. The supematant solution was extracted with
`hexane and the extract was subjected to gas chromato-
`graphy with an electron-capture detector (GC-ECD).
`The incubation mixture
`Measurement
`of BHT-QM.
`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 super-
`natant solution was extracted with hexane. The extract
`was subjected to GC-ECD analysis after purification by
`thin-layer chromatography.
`
`In Vivo Metabolism
`
`of BHT-OH.
`BHT and BHTd, were each
`Measurement
`ip to mice at a dose of 1500 mg/kg. The
`administered
`lungs and livers were excised after 4 hr and homogenized
`in 0.1 M phosphate buffer (4 ml/g of tissue). The ho-
`mogenate was extracted with benzene and the extract was
`analyzed by GC-ECD.
`An equimolar mixture (3000
`Measurement
`ofBHT-QM.
`mg/kg) of BHT and BHT-dS was administered ip to mice.
`The lungs and livers were excised after 4 hr and homog-
`enized 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
`
`(Wakogel C-200, Wako Pure
`a silica gel dry column
`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.
`
`Instrumental
`
`Analyses
`
`GC-ECD was performed on a Shimadzu GC-3AE gas
`chromatograph
`fitted with a 2-m X 3-mm-i.d. glass col-
`umn packed with Chromosorb W containing 2% OV- I.
`The analyses of BHT-OH and BHT-SPhBr were con-
`ducted at 100 and 175”C, respectively.
`Mass spectra were obtained by using a JEOL JMS-D
`100 GC-MS spectrometer equipped with a JEOL MS-
`PD-0 I multiple ion detector. The ionizing energy was 22
`eV. The isotopic purities of BHT-dr , 4-EP-dz, and 4-EP-
`d, were determined from the parent ions in the electron-
`impact mass spectra. To determine the ratio of the abun-
`dance of BHT-SPhBr and BHT-SPhBr-ds,
`the selected
`ion monitor was focused on the ions m/e 219 for BHT-
`and the peak
`SPhBr and m/e 22 1 for BHT-SPhBr-d,,
`height ratio was measured.
`tetra-
`NMR spectra were measured in CDCI, with
`methylsilane 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. 1 A. Both compounds caused
`dose-related increases in lung/body weight ra-
`tio. 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 dose-
`related
`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 per-
`centage change in body weight of mice during
`the experiment
`(4 days) are shown in Fig. 1 C.
`A dose-dependent
`reduction
`in body weight
`
`Par Pharm., Inc.
`Exhibit 1007
`Page 003
`
`

`
`286
`
`MIZUTANI,
`
`YAMAMOTO,
`
`AND
`
`TAJIMA
`
`Isotope E#ect on in Vitro Metabolism
`
`ofBHT
`
`to determine
`A procedure was developed
`formed by in vitro
`BHT-OH
`and BHT-QM
`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 de-
`tected directly by GC-ECD.
`BHT-QM, which
`is known
`to be an unstable metabolite
`(Ta-
`jima et al., 1981) was
`trapped with p-bro-
`mothiophenol
`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% recov-
`ery, the data obtained
`through
`this method
`should be considered as semiquantitative,
`be-
`cause it appears rather likely that the resulting
`BHT-QM
`also reacts with
`cellular nucleo-
`philes, such as glutathione
`and the cysteine
`components
`of proteins. BHT-OH
`and BHT-
`SPhBr
`(derivatized
`from BHT-QM)
`were
`identified
`from
`their mass spectral
`fragmen-
`tation patterns and by comparison
`of their
`GC
`retention
`times with
`those of authentic
`samples. Typical gas chromatograms
`of BHT-
`in the in vitro
`OH and BHT-SPhBr
`formed
`experiments
`are shown
`in Fig. 2.
`
`0
`
`DOSE
`
`(mmol/kg)
`
`(0) and BHT-
`doses of BHT
`I. Effects of different
`FIG.
`d, (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
`deter-
`mined.
`Each point
`represents
`mean
`t SE of 8 to
`I8 an-
`imals.
`(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
`
`(min)
`
`20
`
`rn vitro
`of
`chromatograms
`CC-ECD
`FIG. 2. Typical
`(after
`being
`and
`(B) BHT-QM
`metabolites.
`(A) BHT-OH
`Analyses
`were performed
`on
`converted
`to BHT-SPhBr).
`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 ni-
`trogen
`and
`(B)
`175°C with an
`inlet pressure
`of 1.4 kg/
`cm* of nitrogen.
`
`Par Pharm., Inc.
`Exhibit 1007
`Page 004
`
`

`
`ISOTOPE
`
`EFFECTS
`
`ON
`
`BHT METABOLISM
`
`AND
`
`TOXICITY
`
`287
`
`the rates of in vitro
`By the above method,
`metabolism
`of BHT and BHT-d3 were com-
`pared (Table 1). Deuteration
`of BHT
`in the
`4-methyl group resulted
`in a significant
`re-
`duction
`(approximately
`40%) in
`the rate of
`metabolism
`to BHT-QM.
`On
`the contrary,
`the rate of metabolism
`to BHT-OH was sig-
`nificantly
`increased (approximately
`70%) with
`the deuterium
`substitution.
`
`Isotope Efect on in Vivo Metabolism
`
`of BHT
`
`An equimolar mixture of BHT and BHT-
`d3 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 BHT-
`QM 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
`(nmol/g
`
`formed
`liver/min)
`
`Substrate
`
`BHT-QM
`
`b
`
`BHT-OH
`
`(A)
`BHT
`BHT-ds
`(B)
`
`1.50
`0.89
`
`f 0.19
`f 0.18’
`
`Ratio
`
`(B/A)
`
`0.59
`
`k 0.06
`0.91
`1.53 + 0.25’
`
`I .68
`
`TABLE
`
`2
`
`RATIOOFDEUTEFWTEDTOUNDEUTERATED BHT-QM
`FORMED IN MICE 4 hr AFTER ip ADMINISTRATION OF
`3000 mg/kg EQUIMOLAR MIXTURE OF BHT AND BHT-
`4’
`
`Tissue
`
`Lung
`Liver
`
`BHT-QM kWdb
`
`0.66
`0.85
`
`f 0.03
`-t 0.01
`
`a Values
`b BHT-QM
`BHT-SPhBr.
`
`represent means + SE of four determinations.
`was determined
`after
`being
`converted
`
`to
`
`the relative
`to determine
`A similar attempt
`levels of deuterated and undeuterated BHT-
`OH was unsuccessful because the mass spec-
`trum of control
`tissue samples gave interfer-
`ing background peaks. Therefore, in vivo BHT-
`OH (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, BHT-
`d, 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
`
`4-EP or its
`The effects of 3.41 mmol/kg
`deuterated analogs on lung/body weight ratio
`are shown in Fig. 3A. 4-EP and 4-EP-d3 re-
`sulted
`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 mag-
`nitude of increases (approximately
`145% of
`control); 4-EP-d2, however, caused a signifi-
`cant, but only small,
`increase (113%). The
`change in dry lung weight caused by 4-EP-d,
`was significantly
`lower than that by either 4-
`EP or 4-EP-d3.
`
`a Values
`b BHT-QM
`BHT-SPhBr.
`‘Significantly
`
`represent means
`was determined
`
`f SE of
`after
`
`five determinations.
`being
`converted
`
`to
`
`different
`
`from BHT
`
`values
`
`(P < 0.05).
`
`Par Pharm., Inc.
`Exhibit 1007
`Page 005
`
`

`
`288
`
`MIZUTANI,
`
`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
`
`rig/g tissue
`
`BHT (A)
`BHT-dX (B)
`Ratio (B/A)
`
`49 * 4
`68 + 4h
`
`1.39
`
`250 k 22
`341
`* 20h
`
`1.36
`
`f 5
`162
`204 + gh
`
`1.26
`
`106 of- 5
`133
`t 9"
`
`1.25
`
`’ 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 an-
`alogs on body weight are shown in Fig. 3C.
`All three compounds were shown to have sig-
`nificant 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
`
`that
`studies have demonstrated
`Histological
`lung damage resulting
`from BHT can be well
`
`A
`
`monitored by an increase in lung weight (Wit-
`schi 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 BHT-
`d3 is less pulmonary
`toxic than BHT
`(Fig. 1).
`The rates of BHT-QM
`formation
`from BHT-
`
`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).
`
`Par Pharm., Inc.
`Exhibit 1007
`Page 006
`
`

`
`ISOTOPE EFFECTS ON BHT METABOLISM
`
`AND TOXICITY
`
`289
`
`in both the in
`dJ were slower than from BHT
`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
`et al., 1982). The deu-
`BHT-QM
`(Mizutani
`teration
`in the 4-methyl group of BHT
`is also
`expected to suppress the formation of metab-
`olites 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 BHT-
`QM,
`the in vitro and in vivo rates of BHT-
`OH
`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 ac-
`count for this observation,
`that is, deuteration
`in the 4-methyl group of BHT
`resulted in sup-
`pressed metabolic
`reactions at this site and,
`consequently, metabolism
`was most
`likely
`switched toward ring oxygenation, eventually
`leading
`to the enhanced
`formation
`of BHT-
`OH.
`It has been shown that BHT
`is metab-
`olized
`to BHT-OH
`via 4-hydroperoxy-4-
`methyl - 2,6 - di - tert - butyl - 2,5 - cyclohexadi -
`enone (BHT-OOH)
`in vitro (Shaw and Chen,
`1972; Chen and Shaw, 1974) and in vivo (Ya-
`mamoto 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.
`et al., 1982),
`In an early study (Mizutani
`we found several lung toxic alkylphenols, other
`than BHT, among which is 4-EP. To deter-
`mine
`if the activation of 4-EP to its quinone
`methide metabolite
`as suggested
`for BHT
`could be responsible
`for the pulmonary
`tox-
`icity of 4-EP, we compared
`the relative
`lung
`toxicity of 4-EP and its deuterated analogs
`
`in Fig. 3 demonstrated
`The results presented
`that 4-EP-d2 has considerably
`lower lung tox-
`icity than 4-EP, whereas 4-EP-dj has a toxic
`potency comparable
`to that of 4-EP. This re-
`sult 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 respon-
`sible 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 tech-
`nical assistance. Thanks are also due to Miss Toyoko
`Hirai and Miss Hitomi Shimomura
`for mass spectrom-
`etry measurement.
`
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