Chem. Res. Toxicol. 1992,5, 816-822
816
The Enzymatic Formation and Chemical Reactivity of Quinone Methides Correlate with Alkylphenol-Induced Toxicity in Rat Hepatocytes Judy L. Bolton,? Luis G. Valerio Jr., and John A. Thompson* Division of Pharmaceutical Sciences, School of Pharmacy, Box (2238, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received June 5, 1992
The effects of o-alkyl substituents on both the cytochrome P450-catalyzed oxidation of phenols to p-quinone methides (QM’s; 4-methylene-2,5-cyclohexadien-l-ones), and on the rates of nucleophilic additions to the 4-methylene carbon of QM’s were investigated. The derivatives of 4-methylphenol studied were BHT (2,6-di-tert-butyl), BHTOH [6-tert-butyl-2-(hydroxytert-butyl)], BDMP (2-tert-butyl-6-methyl),BMP (2-tert-butyl), T M P (2,6-dimethyl),and DMP (2-methyl). QM formation was estimated to be in the range 0.17-0.70 nmol/(nmol of P4500min) in rat liver microsomes and 16-62 pmol/(106cells-min)in isolated rat hepatocytes. QM’s derived from BHT (BHT-QM), BHTOH (BHTOH-QM), BDMP (BDMP-QM), and T M P (TMP-QM) were synthesized and their rates of reaction with water and reduced glutathione (GSH) determined. BDMP-QM and TMP-QM were the most reactive, BHT-QM was consumed relatively slowly, and BHTOH-QM displayed intermediate reactivity. These variations in rate were rationalized by differences in hydrogen bonding with the carbonyl oxygen, which affects positive charge density at the site of nucleophilic attack. The loss of hepatocyte viability during incubations with BMP, BDMP, and BHTOH was preceded by GSH depletion. Pretreatment of hepatocytes with diethyl maleate exacerbated alkylphenol toxicity, and metyrapone protected the cells. These data, together with information on the formation and reactivity of QM’s, strongly support the proposal that QM’s mediate the toxicity of alkylated 4-methylphenols in rat hepatocytes.
Introduction Phenolic compounds containing a 4-alkyl substituent with at least one benzylic hydrogen may be oxidized to p-quinone methides by cytochrome P450 (1, 2). This oxidation probably occurs by two successive one-electron transfers from the aromatic T system via a phenoxy radical intermediate. Quinone methides are electrophilic, with positive charge density centered mainly on the exocyclic methylene carbon (3). Consequently, reactions of these species are characterized by nonenzymatic Michael additions of nucleophiles to form benzylic adducts as shown in Figure 1 (4). The reactivity of these compounds is decreased by substitution at the reaction site, so BHTQM1 (R3 = R4 = H) combines with nucleophiles 2- to 4-fold faster than its methylated analogues (R3 = CH3, R3 = R4 = CH3) (5). It is well-known that various electrophiles bind covalently to proteins and nucleic acids to initiate a variety of cytotoxic and/or genotoxic responses. Nevertheless, the roles of quinone methides in the metabolism and toxicity of phenolic compounds have received very little attention. One case, where quinone methide involvement
* To whom correspondence should be addressed.
+ Present address: Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6. 1 Abbreviations: P450, cytochrome P450; DMP, 2,4-dimethylphenol; BDMP, TMP, 2,4,6-trimethylphenol;BMP, 2-tert-butyl-4-methylphenol; 2-tert-4,6-dimethylphenol;BHT, 2,6-di-tert-butyl-4-methylphenol; BHTOH, 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylphenol; QM, p quinone methide derived from the parent alkylphenol (e.g., DMP-QM, 2-methyl-4-methylene-2,5-cyclohexadien-l-one); GSH, glutathione; SG, glutathione adduct of the parent alkylphenol [e.g., BMP-SG, 2-tert-butyl4-(glutathion-S-ylmethyl)phenol] ;DEM, diethyl maleate; GSSG,oxidized glutathione; FAB, fast atom bombardment.
in toxicity has been studied extensively,involves the food additive BHT (6). This alkylphenol is oxidized to BHTtogether with asecond quinone methide (discussed QM (7), bebw). Several lines of evidence indicate that one or both of these mediate the hepatotoxicity (8, 91, pulmonary toxicity (61,and enhanced tumorigenesis observed following BHT administration to rodents (10). BHT-derived radioactivity becomes covalently bound to hepatic (11) and pulmonary proteins (121, and agents that inhibit binding also diminish toxicity (12). The analog of BHT in which the 4-methyl substituent has been replaced by a trideuteriomethyl group produces smaller quantities of BHT-QM due to a kinetic isotope effect on BHToxidation; this analog also causes less pulmonary toxicity (13). 4-Methylphenols containing various substituents in the 2- and 6-positions (R1 and R2, Figure 1) have been compared with BHT for their ability to damage murine lung, presumably through quinone methide formation (14). Toxic potency decreased in the order BMP > BDMP > BHT (Figure 2), and phenols without a tert-butyl substituent, DMP and TMP, produced no effect. These data indicate that alkyl substituents substantially influence the formation rates and/or reactivities of quinone methides. Recent studies have demonstrated that microsomalP450 from murine liver hydroxylates BHT to BHTOH (Figure 2), and the potent pulmonary toxicity of the latter (15) may be due to ita further oxidation to BHTOH-QM (16). This hydroxylated quinone methide reacts substantially faster than BHT-QM with nucleophiles due to intramolecular hydrogen bonding between the hydroxyl group and the carbonyl oxygen (16). This interaction can stabilize 0 1992 American Chemical Society
Chem. Res. Toxicol., Vol. 5, No.6, 1992 817
Cytotoxic Quinone Methides OH
quinone methide
Figure
1. Oxidation of alkylphenols to quinone methides, and subsequent addition of a nucleophile (XH).
DMP
TMP
BDMP
BMP
BHT
BHTOH
Figure 2. Structures of the alkylphenols investigated.
I
I1
Figure 3. Quinone methide resonance structures. the charge-separated resonance structure shown in Figure 3, thereby increasing positive charge density at the site of nucleophilic attack. Some of the adverse effects of BHT in mice, therefore, may be due to BHTOH-QM, rather than BHT-QM. Evidence that intramolecular hydrogen bonding enhances quinone methide reactivity prompted us to investigate the possibility that interactions between the carbonyl oxygen and water molecules from the medium also enhance reactivity. The bulky, hydrophobic tertbutylgroups of BHT-QM are expected to efficiently shield the oxo group from water, so replacing these with smaller substituents should permit intermolecular hydrogen bonding. Figure 2 shows the alkylphenols investigated in this work, where the 2- and 6-substituents are hydrogen, methyl or tert-butyl. In addition to information on quinone methide reactivity, the influences of alkyl substituents on the conversion of phenols to quinone methides were determined with rat liver microsomes and isolated rat hepatocytes. The rat was chosen, because this species is deficient in the P450activity responsible for hydroxylating a tert-butyl substituent (I7),so intermolecular interactions can be differentiated from intramolecular effects. Finally, data on the formation and reactivity of each quinone methide were correlated with alkylphenol-induced cytotoxicity, which provides evidence that cell injury is mediated by these electrophiles.
Materials and Methods Materials. All chemicals were purchased from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO) unless stated
otherwise. Collagenase (type B) was obtained from Boehringer (Mannheim, FRG). [3H]GSH (gly~ine-Z-~H) was obtained from Du Pont NEN (Boston, MA) and diluted to a specific activity of 2.0 mCi/mmol. The synthesis of BHTOH (IS),BHT-QM, and BHTOH-QM were described previously (16). A similar procedure was used for preparing the other 2,6-dialkyl quinone methides (5). Briefly, 1 g of PbO2 was added to 10 mg of alkyl phenol in 10 mL of pentane and the mixture stirred for 30 min at 25 "C. The solution was filtered, acetonitrile added, and the pentane removed by evaporation. Fresh solutions of quinone methides were prepared daily and the concentrations determined by UV spectroscopy using literature values of maximum wavelengths and extinction coefficients (5). Glutathione Adducts. Glutathione adducts of quinone methides were synthesized by the following general procedure. A 10 mM solution of glutathione was prepared in 50 mL of potassium phosphate buffer (pH 7.4). The quinone methide solution was added to give a final concentration of 5.0 mM, and the solution was stirred at 25 "C for 2 h. After ether extraction, the adducts were isolated from the aqueous phase on C-18 extraction cartridges (Baker, Phillipsburg, NJ) and eluted with methanol. The eluates were concentrated and subjected to semipreparative HPLC with an Ultrasphere ODS column (10 X 250 mm, Beckman, San Ramon, CA) with a flow rate of 3.5 mL/ min and the same mobile-phase compositiondescribed below for analytical work. The glutathione adducts of BHT and BHTOH were previously synthesized and characterized (16). UV spectra were measured with a Hewlett Packard Model 1040Mdiode array detector, lH NMR spectra with a Varian VXR300 spectrometer at 300 MHz, and FAB mass spectra with a VG 7070 EQ instrument. DMP-SG lH NMR (D2O) (in addition to the glutathionylprotons) 6 1.96 (s,3 H, CH,), 3.48 (s,2 H, benzylic CH2),6.62 (d, J = 8.1 Hz, 1H, ArH), 6.86 (dd, J = 8.4 Hz, 1 H, ArH), 6.94 ( 8 , 1H, ArH); UV (CH30H) 228,278 nm; negative ion FAB-MS (glycerol) m/z 121 (15, DMP - H), 153 (100, DMP S), 325 (0.5, M - Glu), TMP-SG: lH NMR (D2O) (in addition to the glutathionyl protons) 6 1.86 (s, 6 H, 2 X CHs), 3.41 (a, 2 H, benzylic CH2), 6.66 (s,2 H, ArH); UV (CH30H) 228,280 nm; negative ion FAB-MS (dithiothreitoVdithioerythrito1,3:l) m/z 441 (22, M), 306 (100, GSH - H). BMP-SG: 'H NMR (D20) (in addition to the glutathionyl protons) 6 1.14 [s, 9 H, C(CH&], 3.49 (a, 2 H, benzylic CH2), 6.62 (d, J = 8.1 Hz, 1H, ArH), 6.89 (dd, J = 8.4 Hz, 1H, ArH), 7.07 ( ~ ,H, l ArH); UV (CHsOH) 228, 279 nm; negative ion FAB-MS (dithiothreitol/dithioerythritol, 3:l) m/z 468 (2, M), 307 (20, GSH), BDMP-SG: lH NMR (D2O) (in addition to the glutathionyl protons) 6 1.11[s, 9 H, C(CHs)S], 1.88 (s,3 H, CH3) 3.45 (e, 2 H, benzylic CHd, 6.78 (s,2 H, ArH); UV (CH30H) 228,280nm; negative ion FAB-MS (dithiothreitol/ dithioerythritol, 3:l) m / z 482 (20, M), 306 (100, GSH - H). Kinetic Experiments. Grunwald-Winstein solvolysis experiments were conducted at 25 "C in solvent mixtures of 4090% of aqueous 50 mM KzHPOd in acetonitrile (pH 7.4) (19). Reactions were conducted by adding 50 pL of a 2.3 mM solution of the quinone methide in acetonitrile (0.11 mM final concentration) to 950 pL of the reaction medium, and the first-order decay was followed by the change in absorbance at 288 nm with aHewlett Packard Mode18452 diode array UV spectrophotometer (16).All rate constants were determined in triplicate. Incubations. Male Sprague-Dawley rats (180-200 g) were obtained from Sasco Inc. (Omaha, NE). Microsomes were
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818 Chem. Res. Toxicol., Vol. 5, No. 6, 1992
prepared from rat liver and protein and P450 concentrations determined as described (18). Incubations containing 1.3 mg/ mL of microsomal protein were conducted for 10 min a t 37 “C in 50 mM phosphate buffer (pH 7.4, 500-pL total volume). Substrates were added as solutions in dimethyl sulfoxide, and [3H]GSH (specific activity of 2.0 mCi/mmol) was added in phosphate buffer to achieve final concentrations of 0.5 and 2.0 mM, respectively. An NADPH-generating system consisting of 0.4 mM NADP+, 7.5 mM glucose 6-phosphate, and 1unit/mL glucose-6-phosphate dehydrogenase was used together with 5.0 mM MgC12. For control incubations, NADP+ was omitted. The reactions were initiated by the addition of NADP+and terminated by chilling in an ice bath followed by the addition of perchloric acid (50 pL). Adduct Quantitation. The incubates were centrifuged at 13 OOO rpm for 6 min to precipitate microsomalprotein. Aliquots of the supernatant were analyzed directly by HPLC with a 4.6X 150-mmUltrasphere C-18 column (Beckman) on an LKB 2249 gradient HPLC with an LKB 2141 UV detector set a t 230 nm for the first 10 min and changed to 280 nm for the remainder of the run. The mobile phase for BDMP-SG, BHT-SG, and BHTOH-SG consisted of 40% methanol in 0.1 M NH4HzP04 at 1.0 mL/min for 5 min, followed by an increase to 90% methanol over the next 25 min. DMP-SG, TMP-SG, and BMP-SG were analyzed a t 230 nm with a more polar mobile phase consisting of 5% methanol in 0.1 M NHdHzP04 a t 1.0 mL/min for 5 min and increased to 80% CH30H over the last 30 min. HPLC retention times of the GSH adducts were as follows: DMP-SG, 17.5 min; TMP-SG, 19.3 min; BMP-SG, 26.5 min; BDMP-SG, 11.5 min; BHT-SG, 19.8 min; and BHTOH-SG, 17.2 min. For quantitation, 0.3-mL aliquots of the column effluent were collected during each run, and radioactivity was measured with a Beckman Model LS 8000 liquid scintillation counter. Concentrations of the GSH conjugates were calculated by summing the radioactivity associated with each peak and converting the data to nanomolar amounts using the specific activity of the [3HlGSH. Hepatocyte Isolation. Rat hepatocytes were isolated by collagenaseperfusion of the liver as described (20). The technique routinely yields hepatocytes of 85-95% viability. The cells were resuspended at lo6 hepatocytes/mL in Krebs-Henseleit buffer (pH 7.4) in rotating flasks at 37 “C under an atmosphere of 95% 02/5%COZ. The substrates were added in solutions of dimethyl sulfoxide (3pL/mL of incubate) to achieve concentrations in the range 0.15-0.30 mM. The control samples contained the vehicle only. Quinone methides were analyzed as their GSH conjugates. Aliquots of the cell suspensions were removed and combined with perchloric acid (50 pL/mL) to precipitate protein, and the incubates were centrifuged at 13000 rpm for 6 min. The supernatant was passed through a C-18 extraction cartridge (Baker) and washed with water, and the adducts were eluted with methanol. The methanol was concentrated to 500 pL, and 100-pL aliquots were combined with 2 pL of 0.5 mg/mL S-@bromobenzy1)glutathione (internal standard) and analyzed by HPLC as described for the microsomal incubations. Products were quantitated by the conversion of peak area ratios to picomoles using response factors determined from the analysis of radiolabeled adducts. Biochemical Assays. Hepatocyte viability was monitored by the ability of the cells to exclude Trypan blue as described (20). The effect of alkylphenols on the levels of GSH and GSSG (control ratio averaged about 16) was determined by HPLC according to the method of Reed et al. (21). Briefly, samples were treated with perchloric acid and centrifuged to precipitate protein. Aliquots of the supernatant were neutralized with sodium bicarbonate, thiolgroups were derivatized with iodoacetic acid, and the mixture was treated with Sanger’s reagent. Derivatives of GSH and GSSG were quantified by HPLC using an Ultrasil NH2columns (4.6 X 250 mm, Beckman) with detection at 350 nm.
Bolton et al. Table I. Reaction Rates of Quinone Methides with Water and Glutathione. half-life ( 8 )
TMP-QM BDMP-QM BHT-QM BHTOH-QM 26 47 3060 400 HzO GSH c5
816
The Enzymatic Formation and Chemical Reactivity of Quinone Methides Correlate with Alkylphenol-Induced Toxicity in Rat Hepatocytes Judy L. Bolton,? Luis G. Valerio Jr., and John A. Thompson* Division of Pharmaceutical Sciences, School of Pharmacy, Box (2238, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received June 5, 1992
The effects of o-alkyl substituents on both the cytochrome P450-catalyzed oxidation of phenols to p-quinone methides (QM’s; 4-methylene-2,5-cyclohexadien-l-ones), and on the rates of nucleophilic additions to the 4-methylene carbon of QM’s were investigated. The derivatives of 4-methylphenol studied were BHT (2,6-di-tert-butyl), BHTOH [6-tert-butyl-2-(hydroxytert-butyl)], BDMP (2-tert-butyl-6-methyl),BMP (2-tert-butyl), T M P (2,6-dimethyl),and DMP (2-methyl). QM formation was estimated to be in the range 0.17-0.70 nmol/(nmol of P4500min) in rat liver microsomes and 16-62 pmol/(106cells-min)in isolated rat hepatocytes. QM’s derived from BHT (BHT-QM), BHTOH (BHTOH-QM), BDMP (BDMP-QM), and T M P (TMP-QM) were synthesized and their rates of reaction with water and reduced glutathione (GSH) determined. BDMP-QM and TMP-QM were the most reactive, BHT-QM was consumed relatively slowly, and BHTOH-QM displayed intermediate reactivity. These variations in rate were rationalized by differences in hydrogen bonding with the carbonyl oxygen, which affects positive charge density at the site of nucleophilic attack. The loss of hepatocyte viability during incubations with BMP, BDMP, and BHTOH was preceded by GSH depletion. Pretreatment of hepatocytes with diethyl maleate exacerbated alkylphenol toxicity, and metyrapone protected the cells. These data, together with information on the formation and reactivity of QM’s, strongly support the proposal that QM’s mediate the toxicity of alkylated 4-methylphenols in rat hepatocytes.
Introduction Phenolic compounds containing a 4-alkyl substituent with at least one benzylic hydrogen may be oxidized to p-quinone methides by cytochrome P450 (1, 2). This oxidation probably occurs by two successive one-electron transfers from the aromatic T system via a phenoxy radical intermediate. Quinone methides are electrophilic, with positive charge density centered mainly on the exocyclic methylene carbon (3). Consequently, reactions of these species are characterized by nonenzymatic Michael additions of nucleophiles to form benzylic adducts as shown in Figure 1 (4). The reactivity of these compounds is decreased by substitution at the reaction site, so BHTQM1 (R3 = R4 = H) combines with nucleophiles 2- to 4-fold faster than its methylated analogues (R3 = CH3, R3 = R4 = CH3) (5). It is well-known that various electrophiles bind covalently to proteins and nucleic acids to initiate a variety of cytotoxic and/or genotoxic responses. Nevertheless, the roles of quinone methides in the metabolism and toxicity of phenolic compounds have received very little attention. One case, where quinone methide involvement
* To whom correspondence should be addressed.
+ Present address: Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6. 1 Abbreviations: P450, cytochrome P450; DMP, 2,4-dimethylphenol; BDMP, TMP, 2,4,6-trimethylphenol;BMP, 2-tert-butyl-4-methylphenol; 2-tert-4,6-dimethylphenol;BHT, 2,6-di-tert-butyl-4-methylphenol; BHTOH, 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylphenol; QM, p quinone methide derived from the parent alkylphenol (e.g., DMP-QM, 2-methyl-4-methylene-2,5-cyclohexadien-l-one); GSH, glutathione; SG, glutathione adduct of the parent alkylphenol [e.g., BMP-SG, 2-tert-butyl4-(glutathion-S-ylmethyl)phenol] ;DEM, diethyl maleate; GSSG,oxidized glutathione; FAB, fast atom bombardment.
in toxicity has been studied extensively,involves the food additive BHT (6). This alkylphenol is oxidized to BHTtogether with asecond quinone methide (discussed QM (7), bebw). Several lines of evidence indicate that one or both of these mediate the hepatotoxicity (8, 91, pulmonary toxicity (61,and enhanced tumorigenesis observed following BHT administration to rodents (10). BHT-derived radioactivity becomes covalently bound to hepatic (11) and pulmonary proteins (121, and agents that inhibit binding also diminish toxicity (12). The analog of BHT in which the 4-methyl substituent has been replaced by a trideuteriomethyl group produces smaller quantities of BHT-QM due to a kinetic isotope effect on BHToxidation; this analog also causes less pulmonary toxicity (13). 4-Methylphenols containing various substituents in the 2- and 6-positions (R1 and R2, Figure 1) have been compared with BHT for their ability to damage murine lung, presumably through quinone methide formation (14). Toxic potency decreased in the order BMP > BDMP > BHT (Figure 2), and phenols without a tert-butyl substituent, DMP and TMP, produced no effect. These data indicate that alkyl substituents substantially influence the formation rates and/or reactivities of quinone methides. Recent studies have demonstrated that microsomalP450 from murine liver hydroxylates BHT to BHTOH (Figure 2), and the potent pulmonary toxicity of the latter (15) may be due to ita further oxidation to BHTOH-QM (16). This hydroxylated quinone methide reacts substantially faster than BHT-QM with nucleophiles due to intramolecular hydrogen bonding between the hydroxyl group and the carbonyl oxygen (16). This interaction can stabilize 0 1992 American Chemical Society
Chem. Res. Toxicol., Vol. 5, No.6, 1992 817
Cytotoxic Quinone Methides OH
quinone methide
Figure
1. Oxidation of alkylphenols to quinone methides, and subsequent addition of a nucleophile (XH).
DMP
TMP
BDMP
BMP
BHT
BHTOH
Figure 2. Structures of the alkylphenols investigated.
I
I1
Figure 3. Quinone methide resonance structures. the charge-separated resonance structure shown in Figure 3, thereby increasing positive charge density at the site of nucleophilic attack. Some of the adverse effects of BHT in mice, therefore, may be due to BHTOH-QM, rather than BHT-QM. Evidence that intramolecular hydrogen bonding enhances quinone methide reactivity prompted us to investigate the possibility that interactions between the carbonyl oxygen and water molecules from the medium also enhance reactivity. The bulky, hydrophobic tertbutylgroups of BHT-QM are expected to efficiently shield the oxo group from water, so replacing these with smaller substituents should permit intermolecular hydrogen bonding. Figure 2 shows the alkylphenols investigated in this work, where the 2- and 6-substituents are hydrogen, methyl or tert-butyl. In addition to information on quinone methide reactivity, the influences of alkyl substituents on the conversion of phenols to quinone methides were determined with rat liver microsomes and isolated rat hepatocytes. The rat was chosen, because this species is deficient in the P450activity responsible for hydroxylating a tert-butyl substituent (I7),so intermolecular interactions can be differentiated from intramolecular effects. Finally, data on the formation and reactivity of each quinone methide were correlated with alkylphenol-induced cytotoxicity, which provides evidence that cell injury is mediated by these electrophiles.
Materials and Methods Materials. All chemicals were purchased from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO) unless stated
otherwise. Collagenase (type B) was obtained from Boehringer (Mannheim, FRG). [3H]GSH (gly~ine-Z-~H) was obtained from Du Pont NEN (Boston, MA) and diluted to a specific activity of 2.0 mCi/mmol. The synthesis of BHTOH (IS),BHT-QM, and BHTOH-QM were described previously (16). A similar procedure was used for preparing the other 2,6-dialkyl quinone methides (5). Briefly, 1 g of PbO2 was added to 10 mg of alkyl phenol in 10 mL of pentane and the mixture stirred for 30 min at 25 "C. The solution was filtered, acetonitrile added, and the pentane removed by evaporation. Fresh solutions of quinone methides were prepared daily and the concentrations determined by UV spectroscopy using literature values of maximum wavelengths and extinction coefficients (5). Glutathione Adducts. Glutathione adducts of quinone methides were synthesized by the following general procedure. A 10 mM solution of glutathione was prepared in 50 mL of potassium phosphate buffer (pH 7.4). The quinone methide solution was added to give a final concentration of 5.0 mM, and the solution was stirred at 25 "C for 2 h. After ether extraction, the adducts were isolated from the aqueous phase on C-18 extraction cartridges (Baker, Phillipsburg, NJ) and eluted with methanol. The eluates were concentrated and subjected to semipreparative HPLC with an Ultrasphere ODS column (10 X 250 mm, Beckman, San Ramon, CA) with a flow rate of 3.5 mL/ min and the same mobile-phase compositiondescribed below for analytical work. The glutathione adducts of BHT and BHTOH were previously synthesized and characterized (16). UV spectra were measured with a Hewlett Packard Model 1040Mdiode array detector, lH NMR spectra with a Varian VXR300 spectrometer at 300 MHz, and FAB mass spectra with a VG 7070 EQ instrument. DMP-SG lH NMR (D2O) (in addition to the glutathionylprotons) 6 1.96 (s,3 H, CH,), 3.48 (s,2 H, benzylic CH2),6.62 (d, J = 8.1 Hz, 1H, ArH), 6.86 (dd, J = 8.4 Hz, 1 H, ArH), 6.94 ( 8 , 1H, ArH); UV (CH30H) 228,278 nm; negative ion FAB-MS (glycerol) m/z 121 (15, DMP - H), 153 (100, DMP S), 325 (0.5, M - Glu), TMP-SG: lH NMR (D2O) (in addition to the glutathionyl protons) 6 1.86 (s, 6 H, 2 X CHs), 3.41 (a, 2 H, benzylic CH2), 6.66 (s,2 H, ArH); UV (CH30H) 228,280 nm; negative ion FAB-MS (dithiothreitoVdithioerythrito1,3:l) m/z 441 (22, M), 306 (100, GSH - H). BMP-SG: 'H NMR (D20) (in addition to the glutathionyl protons) 6 1.14 [s, 9 H, C(CH&], 3.49 (a, 2 H, benzylic CH2), 6.62 (d, J = 8.1 Hz, 1H, ArH), 6.89 (dd, J = 8.4 Hz, 1H, ArH), 7.07 ( ~ ,H, l ArH); UV (CHsOH) 228, 279 nm; negative ion FAB-MS (dithiothreitol/dithioerythritol, 3:l) m/z 468 (2, M), 307 (20, GSH), BDMP-SG: lH NMR (D2O) (in addition to the glutathionyl protons) 6 1.11[s, 9 H, C(CHs)S], 1.88 (s,3 H, CH3) 3.45 (e, 2 H, benzylic CHd, 6.78 (s,2 H, ArH); UV (CH30H) 228,280nm; negative ion FAB-MS (dithiothreitol/ dithioerythritol, 3:l) m / z 482 (20, M), 306 (100, GSH - H). Kinetic Experiments. Grunwald-Winstein solvolysis experiments were conducted at 25 "C in solvent mixtures of 4090% of aqueous 50 mM KzHPOd in acetonitrile (pH 7.4) (19). Reactions were conducted by adding 50 pL of a 2.3 mM solution of the quinone methide in acetonitrile (0.11 mM final concentration) to 950 pL of the reaction medium, and the first-order decay was followed by the change in absorbance at 288 nm with aHewlett Packard Mode18452 diode array UV spectrophotometer (16).All rate constants were determined in triplicate. Incubations. Male Sprague-Dawley rats (180-200 g) were obtained from Sasco Inc. (Omaha, NE). Microsomes were
+
818 Chem. Res. Toxicol., Vol. 5, No. 6, 1992
prepared from rat liver and protein and P450 concentrations determined as described (18). Incubations containing 1.3 mg/ mL of microsomal protein were conducted for 10 min a t 37 “C in 50 mM phosphate buffer (pH 7.4, 500-pL total volume). Substrates were added as solutions in dimethyl sulfoxide, and [3H]GSH (specific activity of 2.0 mCi/mmol) was added in phosphate buffer to achieve final concentrations of 0.5 and 2.0 mM, respectively. An NADPH-generating system consisting of 0.4 mM NADP+, 7.5 mM glucose 6-phosphate, and 1unit/mL glucose-6-phosphate dehydrogenase was used together with 5.0 mM MgC12. For control incubations, NADP+ was omitted. The reactions were initiated by the addition of NADP+and terminated by chilling in an ice bath followed by the addition of perchloric acid (50 pL). Adduct Quantitation. The incubates were centrifuged at 13 OOO rpm for 6 min to precipitate microsomalprotein. Aliquots of the supernatant were analyzed directly by HPLC with a 4.6X 150-mmUltrasphere C-18 column (Beckman) on an LKB 2249 gradient HPLC with an LKB 2141 UV detector set a t 230 nm for the first 10 min and changed to 280 nm for the remainder of the run. The mobile phase for BDMP-SG, BHT-SG, and BHTOH-SG consisted of 40% methanol in 0.1 M NH4HzP04 at 1.0 mL/min for 5 min, followed by an increase to 90% methanol over the next 25 min. DMP-SG, TMP-SG, and BMP-SG were analyzed a t 230 nm with a more polar mobile phase consisting of 5% methanol in 0.1 M NHdHzP04 a t 1.0 mL/min for 5 min and increased to 80% CH30H over the last 30 min. HPLC retention times of the GSH adducts were as follows: DMP-SG, 17.5 min; TMP-SG, 19.3 min; BMP-SG, 26.5 min; BDMP-SG, 11.5 min; BHT-SG, 19.8 min; and BHTOH-SG, 17.2 min. For quantitation, 0.3-mL aliquots of the column effluent were collected during each run, and radioactivity was measured with a Beckman Model LS 8000 liquid scintillation counter. Concentrations of the GSH conjugates were calculated by summing the radioactivity associated with each peak and converting the data to nanomolar amounts using the specific activity of the [3HlGSH. Hepatocyte Isolation. Rat hepatocytes were isolated by collagenaseperfusion of the liver as described (20). The technique routinely yields hepatocytes of 85-95% viability. The cells were resuspended at lo6 hepatocytes/mL in Krebs-Henseleit buffer (pH 7.4) in rotating flasks at 37 “C under an atmosphere of 95% 02/5%COZ. The substrates were added in solutions of dimethyl sulfoxide (3pL/mL of incubate) to achieve concentrations in the range 0.15-0.30 mM. The control samples contained the vehicle only. Quinone methides were analyzed as their GSH conjugates. Aliquots of the cell suspensions were removed and combined with perchloric acid (50 pL/mL) to precipitate protein, and the incubates were centrifuged at 13000 rpm for 6 min. The supernatant was passed through a C-18 extraction cartridge (Baker) and washed with water, and the adducts were eluted with methanol. The methanol was concentrated to 500 pL, and 100-pL aliquots were combined with 2 pL of 0.5 mg/mL S-@bromobenzy1)glutathione (internal standard) and analyzed by HPLC as described for the microsomal incubations. Products were quantitated by the conversion of peak area ratios to picomoles using response factors determined from the analysis of radiolabeled adducts. Biochemical Assays. Hepatocyte viability was monitored by the ability of the cells to exclude Trypan blue as described (20). The effect of alkylphenols on the levels of GSH and GSSG (control ratio averaged about 16) was determined by HPLC according to the method of Reed et al. (21). Briefly, samples were treated with perchloric acid and centrifuged to precipitate protein. Aliquots of the supernatant were neutralized with sodium bicarbonate, thiolgroups were derivatized with iodoacetic acid, and the mixture was treated with Sanger’s reagent. Derivatives of GSH and GSSG were quantified by HPLC using an Ultrasil NH2columns (4.6 X 250 mm, Beckman) with detection at 350 nm.
Bolton et al. Table I. Reaction Rates of Quinone Methides with Water and Glutathione. half-life ( 8 )
TMP-QM BDMP-QM BHT-QM BHTOH-QM 26 47 3060 400 HzO GSH c5
