Chem. Res. Toricol. 1991,4, 482-489
482
Rat Hepatic Microsomal Metabolism of Ethylenethiourea. Contributions of the Flavin-Containing Monooxygenase and Cytochrome P-450 Isozymes Caroline J. Decker and Daniel R. Doerge* Department of Environmental Biochemistry, University of Hawaii, Honolulu, Hawaii 96822 Received January 23, 1991
The contributions of the rat hepatic flavin-containing monooxygenase (FMO) and cytochrome P-450 isozymes (P-450) in the ethylenethiourea (ETU) mediated inactivation of P-450 isozymes and covalent binding of the compound to microsomal proteins were investigated. In vitro, ETU was found to inhibit P-450 marker activities in microsomes obtained from untreated (UT) and phenobarbital (PB),8-naphthoflavone (BNF), and dexamethasone (DEX) pretreated rats. This inhibition was dependent on the presence of NADPH and was completely abolished by coincubation with glutathione (GSH). Heat treatment of microsomes prior to ETU-mediated P-450 inactivation led to diminished loss of P-450 marker activities in microsomes obtained from UT and PB-pretreated, but not BNF- or DEX-pretreated rats, suggesting FMO involvement in the inactivation of some P-450 isozymes. Covalent binding of ['4C]ETU to microsomal proteins was found to be NADPH-dependent and enhanced with BNF or DEX pretreatment of rats. This binding was completely inhibited by coincubation with GSH. Heat treatment of microsomes and P-450 inactivation studies indicated a predominant role of FMO in the observed covalent binding. Addition of the sulfhydryl reagents dithiothreitol (DTT) or GSH after the incubation of microsomes, ['4C]ETV, and NADPH resulted in the complete release of bound ETU, suggeating the reduction of disulfide bonds between oxidized ETU and protein sulfhydryls. Microsomal heme content was not decreased following incubation of microsomes with ETU and NADPH, and P-450 appeared to be converted to P-420. Metabolism of ETU in the presence of GSH resulted in the formation of oxidized glutathione (GSSG), suggesting the initial formation of a GSH-ETU adduct and subsequent disulfide exchange reaction. These results suggest that, under normal physiological conditions, reactive metabolites from ETU generated by either FMO or P-450 are preferentially trapped by endogenous GSH and do not interact with cellular targets.
I ntroductlon ETU' is a breakdown product and metabolite of the ethylenebis[dithiocarbamate]fungicides that is toxic to both the thyroid and liver (I). Similar to many other thiocarbamides, ETU has been reported to inactivate P-460 isozymes (2),though by an unknown mechanism. ETU is also a high-affinity substrate for hog liver FMO (3). Organosulfur functional groups including sulfides, thiols, thiones, and thiocarbamides are oxidized by both the P-450 and FMO monooxygenase systems (4-8). Approximately 65% of thiobenzamide oxidation to ita S-oxide in rat liver microsomes is catalyzed by FMO and the remaining 35% by P-450 as determined by antibody inhibition studies (9). Oxidation of thioacetamide by both monooxygenases is believed to contribute to the compound's hepatotoxicity (10). Parathion and carbon disulfide are metabolized exclusively by P-450 to reactive intermediates that bind to the enzyme, causing its inactivation (11,12).Metabolic activation of the thiocarbamide methimazole by either FMO or P-450 has been proposed to produce chemical species that inactivate P-450 (1*15). Distinction between the catalytic activities of FMO and P-460 in micrmmes is difficult since both are microsomal, are found at high levels in the liver, and share requirements for the cofactors NADPH and molecular oxygen. These studies were thus initiated to aasess the effects of ETU on the liver, to elucidate the mechanism by which these effects occur, and also to determine which enzyme(s) is (are) re-
* Author to whom correspondence should be addressed. oag3-22ax/gi/2704-04a2$02.5010
sponsible for the metabolic activation of ETU to species that produce toxicity.
Experlmental Procedures Chemicals. DEX, BNF, GSH, GSSG, NADPH, EFtY, catalase, and superoxide dismutase were obtained from Sigma Chemical Co., St. Louis,Mo. AP,PCNMA, pNA, and D l T were purchaeed from Aldrich Chemical Co., Milwaukee, WI. pNA was recrystallized from hexane. All chemicals were of the highest grade obtainable. P-450 LM2 was purchased from Oxford Biomedical Research Inc., Oxford, MI. ETU, [19C]ETU,and [l'c]ETU were synthesized and purified by HPLC as described in ref 16. 1Aminobenzotriazolewas synthesized as described in ref 17. Animals and Microsome Preparation. Male 220-250-g Sprague-Dawley rata (Simonsen Labs, Gilroy, CA) were administered PB, DEX,or BNF and liver microsomes prepared as described in ref 7. Protein concentrations were determined by the method of Lowry et al. (18). Biochemical Assays. Microsomal incubations (6.0 mL) contained 1.0 or 2.0 mM ETU or EU, 1-2 mg/mL microsomal protein, 1.5 mM DTPC, and in some cases 1.0 mM NADPH, 5.0 mM GSH, 0.1 mg/mL superoxide dismutase, or 0.5 mg/mL catalase in 0.1 M phosphate buffer, pH 7.4. Reactions were initiated by the addition of NADPH (+NADPH) or buffer (NADPH) and were incubated at 37 O C for 15 min. For the heat inactivation studies, microsomes were incubated at 37 O C (in the Abbreviations: ABT,l-aminobenzotriezole;AP,aminopyrine;BNF, @-naphthoflavone;DEX, dexamethasone; DTPC, diethylenetriaminepentaacetic acid; DTT, dithiothreitol;ERY, erythromycin, ETU,ethylenethiourea; EU, 2-imidazolidone; FMO, flavin-containing monooxygenase; GSH,glutathione; GSSG, oxidized glutathione; LOD, limit of detection; PB, phenobarbital, pCNMA, p-chloro-N-methylaniline;pNA, p-nitroanisole; P-450, cytochrome P-450 isozymes.
0 1991 American Chemical Societv
Microsomal Metabolism of Ethylenethiourea
Chem. Res. Toxicol., Vol. 4, No. 4, 1991 483
Table I. Effect of EU or ETU on P-450 Enzymatic Activity in Vitro 4 NADPHn rat treatment/substrate demethylatedb BNF/PNA UT/AP PB/AP incubation system NADPH 1.88 4 0.49 EU 78.5 f 21.0 134 f 19 77.6 f 21.5 1.84 f 0.82 140 f 26 +N/-Nh 0.99 1.04 0.98 1.87 0.42 79.6 f 19.5 127 f 23 ETU + 31.3 f 12.34‘ 0.41 4 0.W 55.6 f 14.lg +N/-N 0.22 0.44 0.39 138 f 19 1.92 f 0.39 75.9 f 27.1 ETU/GSH 72.5 f 15.6 2.20 4 0.58 137 f 24 0.99 +N/-N 0.96 1.15 124 f 24 1.60 f 0.48 81.3 f 19.7 heat/ETUe 0.50 f 0.17d 80.4 f 27.d 62.0 f 20.od +N/-N 0.76 0.31 0.64
+
+
+
DEX/ERY 119 4 16 121 4 15 1.02 104 f 23 23.2 4 11.W 0.22 104 h 15 109 f 17 1.05 73.2 f 32.6 16.9 f 7.2s 0.23
aMicrosomeswere incubated with ETU or EU f NADPH and then recovered by centrifugation for the determination of P-450 enzymatic activity as described under Experimental Procedures. *nmol/(mg of protein.15 min) for AP and ERY or nmol/(mg of proteinmmin) for PNA demethylation. Microsomes were heat-inactivated. All values represent the mean f standard deviation of at least 3 individual experiments. d p < 0.05, e p < 0.005, f p < 0.002, or g p < 0.001 relative to the corresponding -NADPH value. h+NADPH/-NADPH value. P-450 contents (nmol/mg) were 0.83 f 0.28, 1.88 f 0.50, 1.39 f 0.24, and 1.50 f 0.30 for untreated, PB-pretreated, BNF-pretreated, and DEX-pretreated rats, respectively. absence of NADPH) for 1h prior to incubation with ETU. In some cases DTT (20 mM final concentration) or GSH (5.0 mM final concentration) was added after the 15-min incubation and the mixture was allowed to stand 3 h on ice. Incubations were terminated by chilling in ice. For several assays incubations were centrifuged for 30 min a t 105000g to isolate microsomes and supernatant. Pelleted microsomes were resuspended in 0.1 M phosphate buffer, pH 7.4, and protein concentration and cytochrome P-450 reductase, FMO, or P-450 enzymatic activity were determined. Demethylation of AP, ERY, and PCNMA was determined as described in ref 19. Incubations (2.0 mL) containing 1.0 mg/mL microsomal protein, 3.0 mM AP, 1.0 mM ERY, or 2.0 mM PCNMA were initiated by the addition of 1.0 mM NADPH (fiial concentration). Reactions were allowed to proceed 15min a t 37 O C and were terminated by the addition of 0.05 mL of 70% perchloric acid. pNA demethylation was determined as described in ref 20. Incubations (2.0 mL) contained 2.0 mg of microsomal protein and 0.50 mM pNA in 0.1 M phosphate buffer, pH 7.85, and were initiated by the addition of 0.5 mM NADPH (final concentration). Cytochrome c (P-450) reductase was monitored as described in ref 21. GSSG or GSH levels of microsomal incubations were measured in supernatants as described in refs 22 and 23, respectively. FMO activity was measured by determining methimazole-dependent NADPH oxidation (after subtracting endogenous rates) in incubations containing 0.5 mg/mL microsomal protein, 1.0 mM DTPC, 200 pM methimazole, 2.5 mM ABT, and 0.7 mM NADPH in 0.1 M phosphate buffer, pH 8.4 a t 33 “C,after a 10-min preincubation of the microsomes with ABT and NADPH. P-450 contributions were minimized by using a high pH and low methimazole concentration (4), as well as inclusion of the 450 suicide substrate, ABT. The P-450 content of microsomes or microsomal incubations was determined by using a Hewlett Packard 8542A diode array or Perkin Elmer Lambda 5 spectrophotometer by the method of Estabrook et al. (24).Heme concentrations of microsomal incubations were determined by the method of Omura and Sat0 (25). Incubations w i t h Purified Rabbit P-450 LM2 (IIB4). Incubations contained 0.5 nmol of P-450/mL, 0.5 nmol of P-450 reductase/mL, 2.0 mM ETU, 1 mM NADPH, and 150 pg of dilauroylphosphatidylcholine/mL in 0.1 M phosphate buffer, pH 7.4, containing 10% glycerol. Reactions were initiated with NADPH and incubated a t 22 “C for 30 min. Reactions were terminated by gassing with carbon monoxide. Covalent Binding. Covalent binding of [“CIETU (1.04 mCi/mmol) to microsomal proteins was measured in 2.0-mL incubations containing 2.0 mg/mL microsomal protein, 0.5 mM ETU (0.30MCilincubation),and 1.0 mM NADPH. ABT (3.0 mM) or GSH (5.0 mM) was included in some incubations. Microsomes recovered by centrifugation at 105OOOg after incubation with 1.0 mM [‘%]ETU and NADPH for 15 min were used in some experiments. Samples were incubated at 37 “C for 15 min, and then protein was precipitated with the addition of 15 mL of 5% HaO,
in methanol. The pellets were washed two times (3.0 mL each) with the acidic methanol, and then five times (3 mL each) with methanol alone. The resultant pellets were dissolved in 0.75 mL of 0.1 N sodium hydroxide, and 0.3-mL aliquota were added to 18 mL of Scintiverse L.C. and subjected to liquid scintillation counting. It was determined that all soluble ETU was removed by this method. Covalent binding of [“CIETU to calf thymus DNA was performed as described in ref 26. Incubations of 1.0 mL contained 1.5 mg of DNA, 2.0 mg of microsomal protein, 0.5 mM ETU (specific activity 0.96 mCi/mmol), and in some cases 1.0 mM NADPH. Metabolism of ETU. HPLC analysis (Novapak silica 4 MM, 8 X 100 mm cartridge, Waters Associates, Milford, MA) of the supernatants of microsomal incubations containing ETU was performed as previously described (27).
Results Effects of ETU on Microsomal Enzymatic Activity. The effects of ETU on P-450 activity in vitro were assessed after incubation of microsomes with ETU in the presence and absence of NADPH. Competitive inhibition of P-450 activity under assay conditions by ETU was excluded since it was determined by HPLC that ETU partitions quantitatively into the supernatant when microsomal incubations are centrifuged at 105000g. AP, ERY, and PNA dealkylation [P-450 isozyme marker activities (28-30)]waa measured by using microsomes from UT or PB-, BNF-, and DEX-pretreated rats to determine specificities in cytochrome P-450 isozymes (31) inactivated by ETU. AP is a relatively nonspecific substrate which is metabolized by many P-450 isozymes such as those of the P-450IA (BNF- or 3-methylcholanthrene-inducible),P-450IIB (PB-inducible), and P-450IIIA (steroid- or PB-inducible and constitutive in the mature male rat) subfamilies, as well as P-45OIICll (constitutive in the mature male rat). pNA is metabolized primarily by PB- (P-450IIB) and BNF-inducible (P-45OIA) isozymes, and ERY is selectively metabolized by isozymes of the P-450IIIA subfamily. Thus, these markers are useful only as broad indicators of P-450 isozyme activity. FMO is not induced by either 3MC or PB (32).It has been reported that corticosteroids such as DEX induce FMO activity, although the supporting evidence is by no means definitive (33). The highest levels of FMO-dependent oxidation of methimazole were observed in UT rat microsomes (data not shown), in accordance with published work (32). Table I demonstrates that ETU-mediated loss of P-450 isozyme activity is NADPH-dependent and ERY N- and
Decker and Doerge
484 Chem. Res. Toxicol., Vol. 4, No. 4, 1991
pNA 0-demethylation activities of microsomes prepared from DEX-pretreated and BNF-pretreated rats, respectively, are relatively more sensitive to inactivation by ETU. The oxygen analogue of ETU, EU, caused no activity loss in all in vitro system, affirming the necessity of the thione moiety of ETU for the inactivation of P-450 isozymes (2). Addition of physiological levels of GSH (Le., 5.0 mM) to incubations attenuated all ETU-mediated P-450 activity loss. This is in contrast to the effects of GSH on NADPH-dependent inactivation of P-450 by ABT where no attenuation is observed (34). The additions of catalase or superoxide dismutase alone or in combination had no effect on the NADPH-dependent ETU-mediated P-450 inactivation in microsomes of UT or DEX-pretreated rats (data not shown), thus excluding the contributions of microsomal-generated hydrogen peroxide or superoxide ion in the observed P-450 inactivation. Other microsomal enzymes (cytochrome P-450 reductase and FMO) were monitored after incubation of microsomes with ETU and NADPH, to examine the selectivity of ETU-mediated toxicity. ETU produced an NADPH-dependent 10-30% decrease in P-450 reductase activity in UT or DEX-pretreated rat microsomes [basal (ETUNADPH) activities were 234 and 99.0 nmol of cytochrome c reduced/(mg of proteinsmin), respectively]. ETU also produced a NADPH-dependent 23% decrease in the FMO-dependent oxidation of methimazole [relative to rates observed in EU + NADPH samples [5.67 f 0.78 nmol/(mg of proteinamin),n = 3, for one experiment with UT rat microsomes]]. Microsomes were incubated at 37 "C for 1 h prior to incubation with ETU to preferentially inactivate FMO (14, 15,35). This treatment was found to decrease FMO activity in UT rat microsomes by 98% [basal rate = 4.16 f 1.56 nmol/ (mg of proteinemin); rate after heat inactivation = 0.09 f 0.08, n = 3, for 3 experiments] and produced different effects on the P-450 marker activities of microsomes from rats treated with the various inducers. AP N-demethylation activity of microsomes prepared from UT or PB-pretreated rats was decreased to a smaller extent following heat inactivation relative to nontreated microsomes (Table I). ETU-mediated pNA 0-demethylation activity loss in PB-pretreated rat microsomes (68%) was also attenuated (by 43%) by microsomal heat inactivation of FMO. Heat treatment of microsomes from BNF-pretreated rats prior to inactivation by ETU had no effect on the NADPH-dependent ETU-mediated loss of PNA 0demethylase activity [PCNMA N-demethylase activity behaved similarly (data not shown)]. Heat treatment of microsomes from DEX-pretreated rats prior to inactivation by ETU resulted in a decrease in basal activity (Le., that measured from NADPH devoid incubations), although NADPH-dependent ETU-mediated inhibition of ERY N-demethylation still occurred. GSH also abolished ETU-mediated loss of PNA 0-demethylation in heat-inactivated microsomes from BNF-pretreated rats (data not shown). Chromophore Studies. ETU caused an NADPH-dependent loss of P-450 chromophore, with a concomitant increase in cytochrome P-420 (the form of P-450 lacking or possessing an altered heme iron-cysteine ligand) that was particularly apparent in microsomes of UT or BNFpretreated rats. As observed in the P-450 activity studies, addition of 5.0 mM GSH to the incubations blocked all ETU-mediated P-450 chromophore loss (Table 11). Heat treatment of microsomes produced some attenuation of chromophore loss in incubations containing microsomes from UT and PB- or BNF-pretreated rats. Incubation of
Table 11. Effect of Eu or ETU on P-450 Chromophore in Vitro & NADPH" P-450 (nmol/mg of protein) for rat treatment incubation svstem NADPH UT PB BNF DEX EU 0.98 1.77 1.18 1.50 0.91 1.77 1.03 1.26 +N/-N' 0.93 1.00 0.87 0.84 ETU 0.97 1.76 1.33 1.54 0.50 1.21 0.24 0.43 +N/-N 0.52 0.69 0.18 0.28 ETU+GSH 0.91 1.65 1.21 1.47 0.96 1.62 1.09 1.48 +N/-N 1.05 0.98 0.90 1.01 heat/ETUb 0.99 1.51 1.35 0.90 0.77 1.39 1.06 0.48 +N/-N 0.78 0.92 0.79 0.53
+ +
+
+
a P-450chromophore measured in incubations described under Experimental Procedures. Microsomes were heat-inactivated. '+NADPH/-NADPH d u e . All values represent the mean of 2 determinations for 1 experiment.
ETU with purified P-450 LM2(a rabbit PB-inducible form, homologous to P-45OIIB1 in the rat) and NADPH resulted in no loss of spectrally observable P-450. Effect of ETU on Microsomal Heme Content. Microsomal heme content was monitored to determine whether the P-450 prosthetic heme moiety is destroyed in ETU-mediated P-450 inactivation as occurs as a consequence of the action of many P-450 suicide substrates (36). Microsomal heme was not decreased in vitro for all pretreatment types following ETU-mediated inactivation of P-450 (data not shown). Comparable incubations performed with ABT, a suicide substrate known to destroy P-450 heme (34),resulted in losses of 45,49,44, and 51% of the microsomal heme content in the UT, PB, BNF, and DEX systems, respectively. Destruction of P-450 without effect on microsomal heme levels is observed during carbon disulfide (37) or diethyldithiocarbamate-mediated (38) inactivation of P-450. Covalent Binding of [W]ETU to Microsomal Proteins. The relation between covalent binding of ['QETU to microsomal proteins and P-450 loss in vitro was measured by using microsomes prepared from rata treated in the aforementioned ways (Table 111). Covalent binding of [14C]ETU to microsomal proteins was dependent on NADPH, suggesting that monooxygenase activity is required. Such binding was most pronounced in microsomes from DEX-pretreated rats, in accord with the relatively greater inactivation of DEX-inducible P-450 isozymes observed in the enzymatic activity and chromophore studies. Levels of covalent binding in UT or PB-pretreated rat microsomes were similar. Maximal covalent binding was observed at 0.5 mM ETU and at incubation times of 15 min (data not shown). GSH addition blocked all NADPH-dependent covalent binding, similar to its effects on ETU-mediated P-450 activity and chromophore loss. Heat treatment completely abolished NADPH-dependent binding in all systems except those utilizing microsomes obtained from DEX-pretreated rats. Addition of the P-450 suicide substrate ABT was used to determine the contribution of P-450 in the covalent binding of [ 14C]ETUto microsomal proteins. This addition failed to attenuate covalent binding in all microsomal systems and often enhanced binding. Incubation of ABT and NADPH with microsomes from UT and PB-, BNF-, and DEX-pretreated rats resulted in a lows of at least 65% of the corresponding P-450 enzymatic activity marker (data not shown). ABT has been shown not to affect hog liver
Microsomal Metabolism of Ethylenethiourea
Chem. Res. Toricol., Vol. 4, No. 4, 1991 486
Table 111. Covalent Binding of [lC]ETUto Microsomal Proteins in Vitro f NADPH" bound ETU (nmol/mg of protein) for rat treatment incubation system NADPH UT PB BNF DEX ETU 0.31 f 0.07 0.27 f 0.07 0.33 f 0.13 0.33 f 0.13 + 3.67 f 2 . w 3.25 f 1.8Eid 6.79 f 2.81e 8.27 i 1.981 ETU + GSH 0.16 0.09 0.09 0.07 + 0.24 f 0.17 0.18 f 0.10 0.13 f 0.06 0.39 f 0.32 heat/ ETUb 0.32 f 0.04 0.46 f 0.18 0.33 f 0.16 0.66 f 0.57 + 0.55 f 0.22 0.58 f 0.24 0.57 f 0.21 3.86 f 1.56a ETU + ABT 0.34 0.21 0.26 0.28 7.82 6.05 9.66 12.4 1.62 f 0.62 3.37 f 1.18 ETU/ETUC 1.17 f 0.28 2.96 f 0.93 + 12.1 f 2.7W 7.71 f 2.23d 6.66 f 1.24d 11.2 f 2.71#
+
'Incubations as described under Experimental Procedures. Microsomes were heat-inactivated. Microsomes incubated with NADPH and ETU and then recovered by centrifugation prior to incubation with [14C]ETU. All data are the mean f standard deviation of at least three individual experiments, except for ETU + ABT and ETU + GSH (-NADPH) where the data represents the mean of 2 determinationis for 1 experiment. d p < 0.05,( p < 0.02, f p < 0.005, or g p < 0.002 relative to the corresponding -NADPH value. P-450content (nmol/mg) was 0.85 f 0.26, 1.94 f 0.56, 1.24 f 0.38, and 1.42 f 0.38 for untreated, PB-pretreated, BNF-pretreated, and DEX-pretreated rats, respectively. Table IV. Effect of Thiols Co- and Postincubation on Covalent Binding of [l'C]ETU to Protein and P-450 Enzymatic Activity f NADPH" incubation covalent ERY Nsystem NADPH binding demethylation 0.31 f 0.10 102 f 23 ETU 8.11 i 2.26d 19.9 f 11.V ETU + DTT 0.10 112 + 0.14 102 ETU + GSH 0.10 103 f 8 0.10 109 f 22 ETU/DTTb 0.20 f 0.12 95.3 f 23.0 0.45 f 0.22 56.6 f 22.5 ETU/GSHC 0.23 105 f 19 + 0.79 32.2 f 16.0"
+
+
" Covalent binding or enzymatic activity monitored in DEXpretreated rat microsomes as in Table I11 or I, respectively. DTT or GSH added to the incubation after its termination for 3 h at 4 OC as described under Experimental Procedures. All values are the mean f standard deviation of at least 3 individual experimenta, except values without standard deviations, where the data represent the mean of determinations for 2 experiments. d p < 0.01 or e p < 0.001 relative to the corresponding -NADPH value. P-450 content (nmol/mg of protein) was covalent binding: 1.44 f 0.43; P-450 activity: 1.49 f 0.37. FMO activity (39) and in some cases actually stimulates FMO activity (40). Inactivation of P-450 with unlabeled ETU prior to measurement of covalent binding (as an alternate method to assess P-450 contributions) also failed to attenuate binding in all microsomal systems. These results were similar to those observed with ABT. The finding that covalent binding occurred after prior exposure to ETU and NADPH also indicated that all protein binding sites had not been previously saturated. NADPH-independent binding was increased in the samples containing "ETU-inactivated" microsomes compared
to those samples containing nontreated microsomes. This may be due in part to the presence of residual NADPH in the "ETU-inactivated" microsomal preparations which has sedimented from the incubation with the microsomes, prior to their incubation with ["CIETU. DNA Binding. Covalent binding of [14C]ETUto exogenous calf thymus DNA was assessed in incubations containing microsomes prepared from DEX-pretreated rats. No detectable binding of [14C]ETUto DNA (LOD = 0.10 nmol/mg of DNA) was observed in incubations that were either supplemented with or devoid of NADPH (data not shown). Reversal of ETU-Mediated Enzymatic Activity Loss and Covalent Binding to Microsomal Proteins by Treatment with Thiols. The addition of 20 mM DTT following incubation of [14C]ETUand NADPH with microsomes from DEX-pretreated rats resulted in the release of all bound radiolabel (Table IV). GSH (5.0 mM) was almost as effective as DTT, removing ca. 90% of the radiolabel from microsomal proteins. Addition of DTI' after incubation of ETU and NADPH with DEX-pretreated rat microsomes resulted in a 2-fold increase in ERY N-demethylase activity (Table IV), suggesting that the release of covalently bound species is correlated with partial restoration of enzymatic activity. Metabolism of ETU in Rat Hepatic Microsomal Systems. NMR analysis of incubation mixtures containing [ 13C]ETU, NADPH, and microsomes prepared from PB-pretreated rats resulted in the detection of no metabolites of ETU. HPLC analysis of similar incubations containing unlabeled ETU (1.0 mM) demonstrated the NADPH-dependent loss of ca. 10% of total ETU, suggesting the metabolism of ETU in these incubations to unidentified species. The metabolism of ETU in the presence of GSH was studied to provide insight into the mechanism by which this nucleophile blocks the binding
Table V. Effect of GSH Concentration on ETU-Mediated Loss of AP N-Demethylase Activity in Vitro Using UT Rat Microsomes' AP demethvlation for GSH (mM) . , incubation system 0 0.01 0.10 1.0 5.0 -NADPH 93.2 f 3.7 102 f 12 90.2 f 7.1 97.3 f 18.0 96.6 f 10.4 +NADPH 26.4 f 5.7* 29.9 f 4.gb 70.5 f 24.7 103 t 11 98.9 f 5.8 +N/-N' 0.28 0.29 0.78 1-06 1.02
" In vitro incubations containing 1.0 mM ETU as described under Experimental Procedures. AP demethylation as described in Table I. All values represent the mean f standard deviation of 3 individual experiments. b p < 0.001 relative to the corresponding -NADPH value. +NADPH/-NADPH value.
Decker and Doerge
486 Chem. Res. Toxicol., Vol. 4, No. 4, 1991 Table VI. In Vitro GSSG Formation or GSH Dillappearance incubation system
NADPH
EU
-
ETU
-
+
differen@
+
difference EU
-
ETU
-
+
difference
+
difference heat/ETUb
0
26.5 f 0.9 31.0 f 2.5 4.5 25.1 f 14.0 3.34 f 3.12 -22 11.7 f 4.4 9.43 f 6.88 -2.3 8.84 f 3.47 11.7 f 3.8 2.9
0.01 GSH (rM) 44.5 f 7.5 40.9 f 5.9 -3.6 27.1 f 17.3 2.37 f 0.86 -25 GSSG (pM) 14.0 f 2.3 14.2 f 5.7 0.20 9.81 f 5.87 27.5 f 14.6 18
-
+
difference
in the Pre.seence of ETU or EU" GSH (mM) 0.10 1.0 5.0
f NADPH
117 f 2 114 f 4 -3.0 121 f 27 26.7 f 19.4d -94 18.6 f 1.3 17.8 f 7.1 -0.80 15.1 f 4.6
53.4 f 6.4' 38
974 f 262 930 f 109 -44 1113 f 186 957 f 217 -156 39.4 f 11.3 39.2 f 7.7 -0.20 26.7 f 19.4 102 f 22e 75
4864 f 527 4888 f 469 24 4793 f 365 4498 f 615 -295 103 f 18 122 f 25 19 102 f 18 183 f 3Oe 81 139 f 39 145 f 31 6
GSH and GSSG were analyzed in supernatants derived from microsomal incubations containing UT rat liver microsomes as described under Experimental Procedures. bMicrosomes were heat-inactivated. ' p C 0.02,d p C 0.01,e p C 0.005, or f p C 0.002 relative to the corresponding -NADPH value. #(+NADPH) - (-NADPH) value. All values are the mean f standard deviation of at least 3 individual determinations. of ETU to microsomal proteins and concomitant inactivation of P-450 isozymes. The disappearance of GSH or ETU, formation of GSSG, and inactivation of P-450 isozymes were thus monitored at different G.SH concentrations by using microsomes from UT rats. GSH was found to be completely effective in blocking ETU-mediated inactivation of P-450 isozymes in UT rats at 1.0 mM (Table V). An NADPH-dependent increase in GSSG levels was observed in incubations containing ETU, but not in corresponding incubations containing EU at 5.0,1.0,and 0.10 mM GSH (Table VI). This increase was significantly higher than the autoxidation of GSH observed in the samples ([GSH] = 1.0 or 5.0 mM) lacking NADPH (2%). The use of heat-inactivated microsomes resulted in an increase in GSSG levels in the incubations lacking NADPH and blocked the NADPH-dependent increase in GSSG formation. GSH levels were decreased significantly in an NADPH-dependent manner in the presence of ETU but not EU a t 100 pM GSH, indicating GSH depletion by metabolic activation of ETU. At low ETU concentrations (20 pM), a depletion of ETU was observed in the presence of NADPH which was blocked by the addition of increasing concentrations of GSH (data not shown).
Dlscusslon Incubation of rat hepatic microsomes with [14C]ETU and NADPH in vitro results in covalent binding of radiolabel to microsomal proteins, in addition to the losses of P-450, cytochrome P-450 reductase, and FMO activities. The requirement for NADPH indicates that metabolism of ETU by microsomal monooxygenases is necessary to produce covalent binding and enzymatic activity loss. These findings indicate that several microsomal enzymes appear to be targets for electrophilic ETU metabolites, although the cytochrome P-450 isozymes are most susceptible. DNA added exogenously to microsomal incubations does not appear to be alkylated by ETU. The prosthetic heme moiety of P-450 does not appear to be the target of inactivationsince the heme content of microsomes is not decreased by ETU and NADPH treatment. Perturbation of the tertiary structure of P-450 as a result of the covalent binding of an ETU metabolite is suggested by the formation of P-420. The decrease in all P-450 marker activities monitored indicates that many isozymes
of P-450 are susceptible targets. GSH at physiological concentrations abolishes all effects of ETU (P-450 activity/chromophore loss and covalent binding to microsomal proteins). This indicates that the reactive species which produce these effects are not confined to the active site of the enzyme of activation. This observation provides explanations for the earlier observations of negligible P-450 loss when ETU is administered in vivo (2),as well as for the lack of effect of ETU on microsomal heme content, as it is unlikely that a diffusible intermediate would have access to the P-450 active site heme. A heat-sensitive factor (presumably FMO) is at least partially responsible for the inactivation of P-450 isozymes prominent in the untreated and PB-treated male rat. On the other hand, BNF- and DEX-inducible P-450 isozymes (i.e., those of the P-450IA and P-450IIIA subfamilies, respectively) appear able to catalyze their own inactivation, as heat treatment of these microsomes does not affect ETU-mediated P-450 activity loss. Since P-450 isozymes of the IIIA subfamily are also inducible by PB and found constitutivelyin the mature male rat, these P-450isozymes may in part be responsible for the metabolism of ETU to species that inactivate P-450 in microsomes from UT and PB- or BNF-pretreated animals. Both FMO and P-450 appear to oxidize ETU to diffusible reactive species because GSH abolishes all ETU-mediated activity loss in both normal and heat-inactivated BNF-pretreated rat microsomes. Covalent binding of [ 14C]ETUto microsomal proteins appears to be largely catalyzed by FMO since (1) covalent binding is prevented in microsomes from UT and BNFor PB-pretreated animals following heat inactivation of FMO and (2) inactivation of P-450 (via prior inactivation with ETU or coincubation with ABT) often enhances covalent binding. The observed enhancement in covalent binding following inactivation of P-450 may be due to the inactivation of P-450 isozymes which metabolize ETU to species not involved in covalent binding of the imidazoline ring to microsomal protein, thus depleting both ETU and NADPH. The ABT-mediated effects on covalent binding may be caused by enhancement of FMO activity (40). Higher levels of covalent binding of ETU to microsomal proteins are observed in microsomes from DEX- or BNFpretreteated rata. This may in part be due to higher levels
Microsomal Metabolism of Ethylenethiourea
of FMO in DEX-pretreated rat preparations by corticosteroid induction (33).Heat inactivation studies indicate that DEX-inducible P-450 isozymes also appear to be involved in activation of ETU to species containing the imidazoline ring which bind to microsomal proteins. P450-mediated oxidation has been described for the actiparathion (II), methimazole vation of carbon disulfide (12), (13,15), a-naphthyl isothiocyanate (411,and 6-thiopurine (42)to species that covalently bind either atomic sulfur or the carbon skeleton to microsomal proteins. Coincubation of GSH with either 6-thiopurine or a-naphthyl isothiocyanate in NADPH-supplemented microsomes caused a significant decrease in covalent binding, indicating that even in cases where P-450 is believed to mediate such binding it is also via a diffusible intermediate. For all microsomal systems examined, attenuation of P-450 activity loss by ETU and covalent binding of ETU to microsomal proteins by heat inactivation of microsomal FMO is not directly proportional (Le., in the UT system covalent binding is 100% inhibited whereas activity loss is approximately 60% inhibited). Furthermore, attenuation of [“CIETU binding to microsomal proteins is complete in BNF-pretreated rat microsomes and partial in DEX-pretreated rat microsomes without any corresponding attenuation of P-450 enzymatic activity loss in either system. This may be a consequence of the use of different concentrations of ETU in the activity studies (1-2 mM) and covalent binding studies (0.5 mM), although the concentration dependence for covalent binding demonstrated saturation at 0.5 mM. Previous studies assessing the covalent binding of methimazole to purified P-45OIIB1 were performed at 20 mM methimazole, and a K, of 18 mM was determined (13). These studies also demonstrated that ca. 70% of such covalent binding could be attributed to the sulfur atom of the molecule alone. P450-dependent metabolism of both parathion and carbon disulfide results in the binding of atomic sulfur to the P-450 apoprotein (I1,12,6). Thus, discrepancies between the attenuation of covalent binding of [WIETU and P-450 enzymatic activity loss by FMO inactivation may be caused by P-450-dependent activation of ETU to species where only atomic sulfur binds to the P-450 apoprotein, producing catalytic inactivation. Unfortunately, the lack of suitable radiolabeled precursors made it impossible to do parallel experiments with [S6S]ETU;thus these mechanisms could not be distinguished in this report. Oxidation of thiocarbamides such as ETU by FMO has been proposed to result in the formation of a sulfenic acid (4).Sulfenic acids are highly reactive electrophiles which react readily with thiols (3,4).Reaction of an ETU-derived sulfenic acid with a cysteine sulfhydryl would result in a disulfide which retains both the imidazoline ring and sulfur moiety of ETU. P-450, as mentioned, may activate ETU to species from which predominantly atomic sulfur binds, although it is also possible that P-450 catalysis produces a sulfenic acid metabolite of ETU as proposed for FMO. Thus, differences between the attenuation of [“CIETU covalent binding and activity loss may be caused by different FMO and P-450 oxidation mechanisms or affinities for ETU. Addition of DTT or GSH after ETU-mediated P-450 inactivation results in the complete release of bound ETU and partial recovery of ERY-demethylation activity in incubations containing microsomes from DEX-pretreated rats. These findings suggest the formation of disulfides between oxidized ETU metabolites and protein sulfhydryls that are reducible by thiols (Figure 1). The fact that the action of GSH is essentially equivalent to that of DTT
[FS [L>s-oH
Chem. Res. Toxicol., Vol. 4, No. 4, 1991 487 P450 or FMO
Imidazoline-2-sulfenicacid
ETU
/
?h
RSH R = G S H protein thiols Dl-l.
R’S-SR R ~ H R’ = GSH Dl-l.
(ik - S
S
R
Figure 1. Hepatic monooxygenase-mediated metabolism of ETU: effects of added thiols.
provides evidence that the disulfides are not sequestered within the structure of the protein since facile reduction occurs even with the large and ionic GSH. This finding is consistent with a mechanism in which a diffusible ETU metabolite interacts only with peripheral cysteine residues of P-450 or other microsomal proteins. Since release of all bound ETU results in only partial restoration of enzymatic activity, renaturation of the protein to its native tertiary structure is not completely reversible in vitro. Similar studies with parathion, purified P-450IIB1, and DTT (43) showed 75% release of bound [%]parathion but no restoration of either P-450 chromophore or activity. [14C]-6-Thiopurinecovalently bound to microsomal proteins is released by DTT, but not GSH (42). The observed NADPH-dependent increase in GSSG levels in the presence of ETU but not EU leads us to propose that the oxidation of GSH occurs through disulfide exchange reactions between GSH and a GSH-ETU adduct (Figure 1). However, the direct reduction of a sulfenic acid metabolite by GSH cannot be excluded at this time. The formation of GSSG even at very low GSH concentrations suggests that if a GSH-ETU adduct is formed, then further reaction with GSH must be facile. The observed NADPH-dependent depletion of GSH in the presence of ETU supports this hypothesis. The decrease in NADPHdependent GSSG formation by heat treatment of microsomes suggests an important role of FMO in this oxidation. Similar oxidation of GSH has been observed during the FMO-dependent metabolism of other thiocarbamide compounds such aa methimazole (3,441.The FMO-dependent formation of a disulfide adduct between deacetylated spironolactone and GSH has recently been characterized (a), providing direct evidence for the reaction of GSH with a sulfenic acid species derived through FMO catalysis. Evidence for the reactions in Figure 1 is also provided by the observation that NADPH-dependent ETU disappearance is blocked by coincubation with GSH. In vivo, GSSG formed is reduced to GSH through the action of glutathione reductase in an NADPH-dependent reaction (45) or is rapidly exported from the cell (46).The depletion of both GSH and NADPH stores appears to be a likely consequence of FMO-mediated oxidation upon high dose exposure to ETU in vivo with the ultimate regeneration of ETU. Such redox cycling has the potential to impair all cellular processes that require either NADPH or reduced glutathione for activity (44,46). This study provides mechanistic information regarding the hepatic microsomal monooxygenase mediated covalent binding of ETU to proteins and a potential specific target of such binding: the cytochrome P-450 isozymes. Disruption of the tertiary structure and loss of catalytic activity of P-450 isozymes appear to be a consequence of ETU bioactivation and covalent binding. The effects of
488 Chem. Res. Toricol., Vol. 4, No. 4, 1991
microsomal heat treatment and P-450inactivation on covalent binding and P-450enzymatic inactivation are consistent with a predominant role of FMO in the oxidation of ETU. The blockade and reversibility of these events by physiological concentrations of GSH provide an explanation for earlier studies in which ETU did not inactivate P-450in vivo (2)and a rationale for possible toxic effects on the liver following exposure to ETU. However, these data suggest that minimal effects of ETU on the liver are expected from consumption of the low levels typically present in food (I). Acknowledgment. This research was supported in part by Grant E504622 from the National Institutes of Health and is submitted as Journal Series No. 3527 from the Hawaii Institute of Tropical Agriculture and Human Resources. W S t W NO.ETU,96-45-7; GSH,70-18-8; GSSG, 27025-41-8; FMO, 9038-14-6; P-450, 9035-51-2; P-420, 9035-49-8; heme, 14875-96-8.
References (1) U S . Environmental Protection Agency (1989) Ethylene-bisthiocarbamates; Notice of preliminary determination to cancel certain registrations, notice of availability of technical support document and draft notice of intent to cancel. Fed. Regist. 54, 52158-52185. (2) Hunter, A. L., and Neal, R. A. (1975) Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono-sulfur-containingcompounds. Biochem. Pharmacol. 24, 2199-2205. (3) Poulaen, L. L., Hyslop, R. M., and Ziegler, D. M. (1979) SOxygenation of N-substituted thioureas catalyzed by the pig liver microsomal FAD-containting monooxygenase. Arch. Biochem. Biophys. 198, 78-88. (4) Ziegler, D. M. (1980) Microsomal flavin-containing monooxygenase: Oxygenation of nucleophilic nitrogen and sulfur compounds. In Bioactiuation of Foreign Compounds (Jacoby, W. B., Ed.) pp 201-230, Academic Press, New York. (5) Ziegler, D. M. (1988) Flavin-containing monooxygenase: Catalytic mechanism and substrate specificities. Drug Metab. Rev. 19, 1-32. (6) Neal, R. A. (1980) Microsomal metabolism of thiono-sulfur compounds: Mechanisms and toxicological significance. Reu. Biochem. Toxicol. 2, 131-171. (7) Decker, C. J., Rashed, M. S., Baillie, T. A., Maltby, D., and Correia, M. A. (1989) Oxidative metabolism of spironolactone: Evidence for the involvement of electrophilic thiosteroid species in drug-mediated destruction of rat hepatic cytochrome P-450. Biochemistry 28, 5128-5136. (8) Decker, C. J., Cashman, J. R., Sugiyama, K., Maltby, D., and Correia, M. A. Formation of glutathionyl-spironolactone disulfide by rat liver cytochromen P-450 or hog liver flavin monoxygenasee: A functional probe of two electron oxidations of the thiosteroid? (submitted for publication). (9) Tynes, R. E., and Hodgson, E. (1983) Oxidation of thiobenzamide by the FAD-containing and cytochrome P-450-dependent monooxygenases of liver and lung microsomes. Biochem. Pharmacol. 32,3419-3428. (10) Chieli, E., and Malvaldi, G. (1984) Role of the microsomal FAD-containing monooxygenase in the liver toxicity of thioacetamide 5-Oxide. Toxicology 31,41-52. (11) Kamataki, T., and Neal, R. A. (1976). Metabolism of diethyl p-nitrophenyl phosphorothionate (parathion) by a reconstituted mixed-function oxidase enzyme system: Studies of the covalent binding of the sulfur atom. Mol. Pharmacol. 12,933-944. (12) Dalvi, R. R., Poore, R. E., and Neal, R. A. (1974). Studies of the metabolism of carbon disulfide by rat liver microsomes. Life Sci. 14, 17861796. (13) Lee,P. W.,and Neal, R. A. (1978) Metabolism of methimazole by rat liver cytochrome P-450-containing monoxygenases. Drug Metab. Dispos. 6, 591-600. (14) Kedderis, G. L., and Rickert, D. E. (1983) Inhibition of the microsomal N-hydroxylation of 2-amino-6-nitrotoluene by a metabolite of methimazole. Biochem. Biophys. Res. Commun. 112, 433-438.
Decker and Doerge (15) Kedderis, G. L., and Rickert, D. E. (1985) Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase. Drug Metab. Dispos. 13, 58-61. (16) Doerge, D. R., Cooray, N. M., Yee, A. B. K., and Niemczura, W. P. (1990) Synthesis of isotopically-labelled ethylenethiourea. J. Labelled Compd. Radiopharm. 28,739-742. (17) Campbell, C. D., and Rees, C. W.(1969) Reactive intermediate. Part i. Synthesis and oxidation of I- and 2-aminobenzotriazole. J. Chem. SOC. C, 742-747. (18) Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. (19) Nash, T. (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55, 416-422. (20) Netter, K. J., and Seidel, G. (1964) An adaptively stimulated 0-demethylating system in rat liver microsomes and ita kinetic properties. J. Pharmacol. Exp. Ther. 146,61-65. (21) Masters, B. S. S., Baron, J., Taylor, W.E., Isaacson, E. L., and LoSpalluto, J. (1971) Immunochemical studies on electron transport chains involving cytochrome P-450. J. Biol. Chem. 246, 4143-4150. (22) Hill, K. E., and Burk, R. F. (1982) Effect of selenium deficiency and vitamin E deficiency on glutathione metabolism in isolated rat hepatocytes. J. Biol. Chem. 257,10668-10672. (23) Kaplowitz, N. (1977) Interaction of azathioprine and glutathione in the liver of the rat. J. Pharmacol. Exp. Ther. 200, 479-486. (24) Estabrook, R. W., Peterson, J. K., and Hildebrandt, A. (1972) The spectrophotometric measurement of turbid suspensions of cytochrome associated with drug metabolism. Methods Phar~ ~ 02,1303-350. . (25) Omura, T., and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes I. Evidence for ita hemoprotein nature. J. Biol. Chem. 239, 2370-2378. (26) Corbett, M. D., Lim, L. O., Corbett, B. R., Johnston, J. J., and Wiebkin, P. (1988) Covalent binding of N-hydroxy-N-acetyl-2to rat aminofluorene and N-hydroxy-N-glycolyl-2-aminofluorene hepatocyte DNA In vitro and cell-suspension studies. Chem. Res. Toxicol. 1, 41-46. (27) Doerge, D. R., and Takazawa, R. S. (1990) Mechanism of thyroid peroxidase inhibition by ethylenethiourea. Chem. Res. Toxicol. 3, 98-101. (28) Guengerich, F. P., Dannan, G. A., Wright, S. T., Martin, M. V., and Kaminsky, L. S. (1982) Purification and characterization of liver microsomal cytochromes P-450 Electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rata treated with phenobarbital or d-naphthoflavone. Biochemistry 21,6019-6030. (29) Guengerich, F. P. (1987) Enzymology of rat liver cytochromes P-450. In Mammalian Cytochromes P-450 (Guengerich, F. P., Ed.) Vol. I, pp 2-39, CRC Press, Boca Raton, FL. (30) Wrighton, S. A., Maurel, P., Scheutz, E. G., Watkins, P. B., Young, B., and Guzelian, P. S. (1985) Identification of the cytochrome P-450 induced by macrolide antibiotics in rat liver as the glucocorticoid responsive cytochrome P450. Biochemistry 24, 2171-2178. (31) Nebert, D. W., Adesnik, M., Coon, M. J., Estabrook, R. W., Gonzalez, F. J., Guengerich, F. P., Gunealus, I. C., Johnson, E. F., Kemper, B., Levin, W., Phillips, I. R., Sato, R., and Waterman, M. R. (1989) The P-450 gene superfamily: Recommended nomenclature. DNA 6, 1-11. (32) Sum, C. Y., and Kasper, C. B. (1982) Mixed-function amine oxidase of the rat hepatocyte nuclear envelope. Demonstration and effects of phenobarbital and 3-methylcholanthrene. Biochem. Pharmacol. 31,69-73. (33) Devereaux, T. R., and Fouta, J. R. (1975) Effect of pregnancy or treatment with certain steroids on N,N-dimethylaniline demethylation and N-oxidation by rabbit liver or lung microsomes. Drug Metab. Dispos. 3, 254-258. (34) Ortiz de Montellano, P. R., and Mathews, J. M. (1981) Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group by 1-aminobenzotriazole. Biochem. J. 195,761-764. (35) Gudzinowicz, M. J., and Neal, R. A. (1981) Differentiation of rat liver microsomal amine oxidase and cytochrome P-450 catalyzed metabolism of thioacetamide and thioacetamide S-Oxide. Toxicologist 1, 67. (36) Ortiz de Montellano, P. R. (1988) Suicide substrates for drug metabolizing enzymes: Mechanisms and biological consequences. Prog. Drug Metab. 2,99-148.
Chem. Res. Toxicol. 1991,4,489-496 (37) Dalvi, R. R., Hunter, A. L., and Neal, R. A. (1975) Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide. Chem.-Biol. Interact. 10, 347-361. (38) Miller, G. E., Zemaitis, M. A,, and Greene, F. E. (1983) Mechanisms of diethyldithiocarbabate-inducedloss of cytochrome P450 from rat liver. Biochem. Pharmacol. 16, 2433-2442. (39) Cashman, J. R. (1987) A Convenient radiometric assay for flavin-containing monooxygenase activity. Anal. Biochem. 160, 294-300. (40) Mathews, J. M., and Bend, J. R. (1986) N-Alkylaminobenzotriazoles as isozyme-selective suicide inhibitors of rabbit pulmonary microsomal cytochrome P-450. Mol. Pharmacol. 30,25-32. (41) El-Hawari, A. M., and Plaa, G. L. (1977) a-Naphthylisothiocyanate (ANIT) hepatotoxicity and irreversible binding to rat liver microsomes. Biochem. Pharmacol. 26, 1857-1866. (42) Hyslop, R. M., and Jardine, I. (1981) Metabolism of 6-thiopurines. I. Irreversible binding of a metabolite of 6-thiopurine
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to mammalian hepatic protein in vitro. J. Pharmacol. Exp. Ther. 218,621428. (43) Halpert, J., Hammond, D., and Neal, R. A. (1980) Inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion (diethyl p-nitrophenyl phosphorothionate). J. Biol. Chem. 255, 1080-1089. (44) Krieter, P. A., Ziegler, D. M., Hill, K. A., and Burk, R. F. (1984) Increased biliary GSSG efflux from rat livers perfused with thiocarbamide substrates for the flavin-containing monooxygenase. Mol. Pharmacol. 26,122-127. (45) Williams, C. H. (1976) Flavin-containing dehydrogenases. In The Enzymes (Boyer, P. D., Ed.) Vol. 13, pp 89-173, Academic Press, New York. (46) Sies,H., and Gerstenecker, C. (1972) Oxidation in the NADP system and release of GSSG from hemoglobin-free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides. FEES Lett. 27, 171-175.
On the Chemistry of the Reaction between IV-Acetylcysteine and 4-[ (4-Ethoxyphenyl)imino]-2,5-cyclohexadien-l-one, a 4-Ethoxyaniline Metabolite Formed during Peroxidase Reactions Thomas Lindqvist,**tLennart Kenne,* and Bjorn Lindekel Department of Organic Pharmaceutical Chemistry, Uppsala Biomedical Centre, Uppsala University, Box 574, S - 751 23 Uppsala, Sweden, Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden, and Swedish Pharmaceutical Society, Box 1136, S-111 81 Stockholm, Sweden Received November 13, 1990
4-Ethoxyaniline (p-phenetidine) is oxidized by peroxidases to form several products, one of which is 4-[ (4-ethoxyphenyl)imino]-2,5-cyclohexadien-l-one (1). This compound reacts with N-acetylcysteine (NAC) in methanol-phosphate buffers, generating a t least four different products. Four major products, 4- [ (4-ethoxyphenyl)amino]phenol(2), 3-(N-acetylcystein-Syl)-4-[(4-ethoxyphenyl)amino]phenol(3), 2,5-bis(N-acetylcystein-S-yl)-4-[(4-ethoxyphenyl)aminolphenol (4), and 2,5-bis(N-acetylcystein-S-yl)-4-[(4-ethoxyphenyl)imino]-2,5-cyclohexadien-1-one (5), were isolated and identified by NMR spectroscopy and mass spectrometry. The relative ratio between the formed producta depends on the pH, the concentration of NAC, and the reaction time. Compound 2, which is the reduced form of 1, was the dominating product when the reaction took place a t pH 3, whereas formation of the mono conjugate (3) was more extensive a t a neutral pH. Under alkaline conditions 2 and 3 were oxidized by 1 or 02.The oxidized form of 3 was subsequently attacked by a second molecule of NAC, generating the bis conjugate (4). Unless an excess of NAC was present, compound 4 underwent rapid oxidation to 5. Quinone imines, like 1, generating mono conjugates, which are more reactive than the quinone imines per se, are likely to inflict an increased toxic potential and an increased stress on the endogenous thiol pool, resulting in an overall greater toxicity.
Introduction 4-Ethoxyaniline (p-phenetidine), a primary metabolite of phenacetin, has been shown to be metabolized to protein arylating compounds as well as to genotoxic products in peroxidase-type reactions involving enzymes such as horseradish peroxidase (HRP)' and prostaglandin synthase (PGS) ( 1 , 2 ) . Among noted toxicological events is to be found the induction of DNA strand breaks in cultured human fibrinoblasts (2). Moreover, since PGS is present in the kidney, its involvement in the production of toxic metabolites can be instrumental to phenacetin induced nephropathy. Because of its links to anemia (3) and kidney disease (4) phenacetin has since long been banned in many countries (in the U S . since 1983). Uppsala University. University. 1Swedish Pharmaceutical Society. +
8 Stockholm
The formation of quinoid imine and diimine structures constitutes one pathway in the bioactivation of 4-ethoxyaniline effected by HRP and PGS. The oxidation product 4- [ (4-ethoxyphenyl)imino]-2,5-cyclohexadien-l-imine and ita hydrolyzed product, 4-[(4-ethoxyphenyl)imino1-2,5cyclohexadien-1-one (11, have been identified as the ultimate precursors for protein arylation (5, 6). The peroxidase-catalyzed formation of 1, as well as subsequent conversions, involves consecutive univalent steps, some of which are capable of generating free radicals. Quinones and their amino-substituted analogues, the quinone imines, are prone to undergo reactions with glutathione (GSH) as well as with other compounds con-
* Abbreviations: HRP,homeradbh peroxidme; PGS,proomglandin synthase; NAC, N-acetylcysteine; CSH, lutethione; NAPQI, N-acetylp-benzoquinone imine; TMS,tatramethy&ilane;NOE, nuclear Overhauser enhancement; COSY, correlation spectroscopy; NOESY, NOE spectroscopy; EI, electron impact; FAB,fast atom bombardment;
0893-228~/91/2704-0489$02.50/0 0 1991 American
Chemical Society
