Chem. Res. Toxicol. 1991, 4, 144-147
144
4-Aminoblphenyi-Hemoglobin Adduct Formation as an Index of in Vivo N-Oxidation by Hepatic Cytochrome P-450IA2 Introduction Sir: The measurement of covalent adducts formed with hemoglobin has received considerable attention as a marker of exposure to carcinogens and other xenobiotics (1-15). These adducts are in many instances stable enough to permit their isolation and quantification. Given the extent to which adduct formation reflects the yield of biologically reactive metabolites and to which adduct levels are exposure-related, such methods can be very useful in the critical assessment of target tissue dosimetry and of potential health risk. Adducts derived from 4-aminobiphenyl (ABP),' a urinary bladder carcinogen found in cigarette smoke, have been found in hemoglobin samples obtained from rats treated with this carcinogen (11) and also from human subjects (12,13). In examining blood samples from both cigarette smokers and nonsmokers, significantly higher levels of the hemoglobin-ABP adduct are found in smokers; however, the appreciable adduct levels in nonsmokers are suggestive of additional sources of environmental exposure to ABP (13). The hemoglobin adduct of ABP is formed by a sequence of reactions in which N-hydroxy-ABP appears to be a necessary precursor (11,14,15). The N-hydroxy derivative, which is formed in the liver in a cytochrome P-450IA2 mediated N-oxidation, is oxidized to 4-nitrosobiphenyl in the erythrocyte with subsequent addition to the P93-cysteine residue of hemoglobin (15). Since N-oxidation of the parent amine is generally regarded as the critical step in the metabolic activation of ABP to a proximate carcinogen (16,17), quantitation of the in vivo reaction product of the N-hydroxy arylamine provides a useful approach in estimating the biologically effective dose of ABP. In the present study, we assessed hemoglobin-ABP adduct formation as a reliable measure of the hepatic N-oxidation of ABP in vivo in the rat. The effect on adduct formation by 2-ethynylnaphthalene, which was recently found to be an effective mechanism-based inactivator of cytochrome P-450IA2 activity in vitro (18),was also examined. Since P-450IA2 is the predominant enzyme catalyzing the N-oxidation of ABP (19,201, inhibition of adduct formation by 2-ethynylnaphthalene should demonstrate more directly the involvement of this conversion step in forming hemoglobin adducts. Inhibitors of other enzymes and pretreatment of animals with a known inducerlhigh-affinity ligand of cytochrome P-450IA2 were also studied for their effects on adduct formation.
Materlals and Methods [2,2'-3H]ABP (94.2 mCi/mmol) was purchased from ChemSyn Science Laboratories, Lenexa, KS. The following chemicals (and commercial sources) were obtained as follows: methimidazole and chloramphenicol (Sigma Chemical Co., St. Louis, MO); isosafrole (Eastman Kodak Chemical Co., Rochester, NY); ABP (Aldrich Chemical Co., Milwaukee, WI); trioctanoin (Pfaltz & Bauer, Inc., Waterbury, CT); and NCS tissue solubilizer (Amersham Corp., Arlington Heights, IIJ. 2-Ethynylnaphthalene was obtained from Dr. W. L. Alworth, W a n e University, New Orleans, LA. All other reagents were of analytical grade. Young adult male Charles River CD rats (CD/Nctr) were used in all studies. In control experiments, animals were given ip injections of radiolabeled ABP (diluted to a specific activity of 31.4 mCi/mmol with unlabeled ABP) in trioctanoin (1.5 mL/kg) a t a dme of either 5 or 0.5 mg/kg body weight. Blood was obtained
' Abbreviation:
ABP, 4-aminobiphenyl.
T h i s article not subject to
U.S.Copyright.
Table I. Effect of 2-Ethynylnaphthalene on Hemoglobin-ABP Adduct Formation" hemoglobin-ABP adduct levels, nmol/g sample hemoglobin % inhibn 218.1 f 22.0 (n = 3) control (5 mg/kg ABP) 0 +2-EN (10 mg/kg) 154.6 f 54.2 (n = 3) 29 +2-EN (100 mg/ kg) 67 71.6 f 4.3b (n = 3) control (0.5 mg/kg ABP) 11.1 f 1.7 (n = 8) 0 +2-EN (IO mg/kg) 9.3 f 0.4b (n = 3) 16 6.9 f 0.2b (n = 3) +2-EN (30 mg/kg) 38 69 +2-EN (100 mg/kg) 3.4 f 0.26 (n = 4) Abbreviations: ABP, 4-aminobiphenyl; 2-EN, 2-ethynylnaphthalene. Significantly different ( p < 0.05) from respective control value. Table 11. Effect of Isoaafrole Pretreatment on Hemoglobin-ABP Adduct Formation" hemoglobin-ABP adduct % inhibn pretreatment levels, nmol/g hemoglobin none (control) 11.1 f 1.7 (n = 8) 0 isosafrole 0.9 f 0.16 (n = 3) 92 Abbreviation: ABP, 4-aminobiphenyl. Significantly different
J (I
< 0.05) from the control value.
via the abdominal aorta at 24 h after dosing. Analysis of the blood samples for quantitation of hemoglobin-ABP adduct levels was done according to the method developed by Green et al. (11). In order to calculate the amount of hemoglobin in each sample, the hemoglobin concentration was determined by Drabkin's method (no. 525A; Sigma). In inhibition experiments, each compound tested was injected ip, in trioctanoin (1.5 mL/kg), at 1 h prior to dosing with radiolabeled ABP. The dosing amounts are given in the tables. In animals pretreated with isodrole, the compound (150 mg in 2.5 mL of trioctanoin/kg body weight) was given by ip injections on three successive days (21). Administration of [3H]ABP followed on the fourth day. Statistical analysis of the results was conducted by using a one-way analysis of variance; subsequent comparisons between individual means was achieved by using Fisher's protected least significant difference (22). Those results that are statistically significant are indicated in the tables.
Results The effect of the mechanism-based inactivator of cytochrome P-450IA2,2-ethynylnaphthalene, on hemoglobinABP adduct formation in rats was initially examined. In control experiments, animals were dosed with [3H]ABP at levels of 5 and 0.5 mg/ kg since adduct formation had been previously demonstrated for these doses (11). The average percentages of administrated radioactivity found in the blood were 7.5 f 0.6 and 3.9 f 0.8, respectively. Quantitation of adduct levels formed at these doses yielded average values of 218.1 f 22.0 and 11.1 f 1.7 nmol/g hemoglobin, respectively. The former value is somewhat higher (approximately 2-fold) than that found by Green et al. (11) and may reflect differences in the rat strains used. As anticipated, the prior administration of the inhibitor 2-ethynylnaphthalene resulted in decreased amounts of adduct formed in animals dosed with either 5 or 0.5 mg/kg ABP (Table I). In animals treated with 0.5 mg/ kg ABP, 2-ethynylnaphthalene caused a dose-dependent decrease in the amount of adduct formed, with doses of 10, 80, and 100 mg/kg resulting in decreases of 16%, 88%, and 69%, respectively. A 100 mg/kg dose of 2-ethynylnaphthalene resulted in similar inhibition (67%) in animals treated with 5 mg/kg ABP.
Published 1991 by t h e American Chemical Society
Chem. Res. Toxicol., Vol. 4, No. 2, 1991 145
Communications Table 111. Effect of Various Inhibitors on Hemoglobin-ABP Adduct Formation’ hemoglobin-ABP adduct levels, nmol/g treatment hemoglobin 11.1 & 1.7 (n = 8) control (0.5 mg/kg ABP) +methimidazole (5 mg/kg) 11.5 f 0.9 (n = 3) +methimidazole (50 mg/kg) 11.6 f 1.2 (n = 3) 2.8 f 0.2* (n = 4) +methimidazole (50 mg/kg)/ 2-EN (100 mg/kg) +chloramphenicol (300 mg/kg) 8.9 f 0.3“ (n = 3) +chloramphenicol (300 mg/kg)/ 4.0 f 0.2* (n = 3) 2-EN (100 mg/kg)
%
inhibn 0
(-4) (-5) 75 20 64
* Abbreviations: ABP, 4-aminobiphenyl; 2-EN, 2-ethynylnaphthalene. *Significantly different ( p < 0.05) from the control value.
The effect of treating animals with the cytochrome P-450IA2 inducer and high-affinity ligand, isosafrole (23, 24), on hemoglobin-ABP adduct formation was also examined. Interestingly, this treatment resulted in a strong decrease in the amount of adduct formed (Table 11). Adduct formation in rats treated with isosafrole was found to be only 8% of that found in control animals. Additionally, the effect of other inhibitors on hemoglobin-ABP adduct formation was examined (Table 111). When chloramphenicol, a mechanism-based inactivator of the P-45011 family (25), was administered at a dose of 300 mg/kg, a decrease in adduct formation of only 20% was observed. Coadministration with 2-ethynylnaphthalene (100 mg/kg) resulted, however, in essentially no additional inhibition compared with the result found when administering 2-ethynylnaphthalene alone. Methimidazole, a strong competitive inhibitor of the flavin-containing monooxygenase (26),at a dose of either 5 or 50 mg/kg, caused no inhibition of hemoglobin-ABP adduct formation (Table 111). This result was also observed when the compound was coadministered with 2-ethynylnaphthalene (100
“3). Discussion Formation of a P93-cysteinehemoglobinadduct of ABP has been demonstrated in experimental animals (11)and in humans (12,13).Quantitation of this adduct provides an important approach for estimating exposure to the proximate carcinogenic metabolite, N-hydroxy-ABP, which is filtered from the circulation into the urinary bladder lumen where it can react with DNA and presumably initiate the neoplastic process (27). Since the formation of N-hydroxy-ABP is catalyzed predominantly by hepatic cytochrome P-450IA2, inhibition of this enzyme was utilized to provide evidence for the involvement of the Nhydroxy derivative in hemoglobin-ABP adduct formation. 2-Ethynylnaphthalene, which has been previously shown to be a strong mechanism-based inactivator of cytochrome P-450IA2 activity in vitro (181,was found to inhibit adduct formation in a dose-dependent manner, with 69% inhibition being achieved at the highest dose administered in this study. In comparison, Mico et al. (28) found that ip pretreatment of rats with varying doses of 1-aminobenzotriazole (0.3-50mg/kg), a mechanism-based inactivator of cytochrome P-450, caused the suppression of the total systemic clearance of phenacetin, with decreases of 39%-79% relative to control values. This inactivator has been shown to block the oxidative metabolism of phenacetin to acetaminophen (29)which is also primarily attributed to cytochrome P-450IA2 in the uninduced rat (30). Several other substances that are known to act in vitro as mechanism-based inactivators of cytochromes P-450
have been suggested to serve as inhibitors in vivo. This has usually been assessed by assaying cytochrome P-450 dependent monooxygenase activities of microsomal preparations from animals treated with these substances in vivo. Examples include chloramphenicol (25) and sodium 10-undecynylsulfate and 2,2-dimethyl-ll-ddecynoic acid (31).Treatment of uninduced rats with chloramphenicol (300 mg/kg) caused approximately a 60% decrease in warfarin (R)-6-and (S)-4’-hydroxylase activities. Treating phenobarbital-treated rats with this dose of chloramphenicol resulted in approximately 60% inhibition of warfarin (R)-4’-hydroxylase activity and in about 90% inhibition of warfarin (R)-7-hydroxylaseactivity. Treatment of rats with either sodium 10-undecynyl sulfate (50 acid (500 mg/kg) mg/kg) or 2,2-dimethyl-ll-dodecynoic caused approximately a 50% decrease in the lauric acid w-hydroxylase activity, whereas a dose of 50 mg/kg 2,2dimethyl-11-dodecynoic acid resulted in about 25% inhibition of this activity. Pretreatment of animals with isosafrole resulted in a decrease in the amount of adduct formed. Although isosafrole induces P-450IA2, it is also metabolized by the enzyme and forms a high-affinity complex that inhibits further catalytic activity (32).Although isosafrole is capable of forming a complex with several different cytochromes P-450, evidence has been reported suggesting that it binds largely to P-450IA2 in vivo (33). The lack of increased amounts of hemoglobin-ABP adduct in animals treated with isosafrole suggests that the net catalytic activity of the enzyme in the in vivo system is a balance between increased quantities from induction and reduced catalytic activity due to active-site inhibition. Although ABP was administered to the animals 24 h following pretreatment with isosafrole, P-450IA2 should be sufficiently inhibited in this time period since the inhibitory metabolite of isosafrole is known to remain tightly bound (34,35) and degradation of the enzyme is inhibited by pretreatment with isosafrole (33).Furthermore, prolonged elimination of (methy1enedioxy)phenyl derivatives has been suggested from studies on the metabolism of these compounds (36).In addition to the direct inhibitory effect of isosafrole, this agent may induce other enzyme systems which catalyze the transformation of N-hydroxy-ABP to derivatives that are unable to form the hemoglobin adduct. UDP-glucuronosyltransferases, for example, have been shown to be induced by various cytochrome P-450 inducing agents, including 3-methylcholanthrene, pregnenolone16a-carbonitrile, and an isoquinoline derivative (37-39). In addition to cytochrome P-450IA2, other purified P-450 enzymes have been found to catalyze the N-oxidation of ABP (19).However, their catalytic activities were much lower than that of P-450IA2. As compared with P-450IA2, the activity of P-450IA1 was about 30%; that of P-45OIICll or P-450IIA1 was 20%; and that of P450IIB1 or P-450IIB2 was only 7%-15%. Chloramphenicol, which has been shown to inhibit the P-450 enzymes, P-45OIICll and P-450IIB1, but not P-450IA2 (25),inhibited adduct formation to a small but significant extent. However, coadministering chloramphenicol with 2-ethynylnaphthalene results in no additional inhibition than that observed with 2-ethynylnaphthalene alone. An additional pathway for the N-oxidation of ABP has been demonstrated in vitro (40),where the primary arylamine is initially N-methylated. This secondary amine derivative can then undergo N-oxidation catalyzed by the microsomal flavin-containing monooxygenase. The possible contribution of this pathway in adduct formation was assessed by examining the effect of treating the animals with the
146 Chem. Res. Toxicol., Vol. 4 , No. 2, 1991
known inhibitor of the flavin-containing monooxygenase, methimidazole (26). The absence of inhibition observed with this inhibitor indicates the lack of major participation of this enzyme in ABP N-oxidation and formation of this adduct in vivo. The results found with 2-ethynylnaphthalene and with the other inhibitors suggest that the major contributing pathway for the N-oxidation of ABP in vivo is that catalyzed by cytochrome P-450IA2. However, the inhibition observed with chloramphenicol may suggest a minor participation by P-45OIICll or P-450IIB1. Additionally, the available data do not permit exclusion of some participation by P-450IA1, which is present at very low levels in uninduced animals, or P-450IIA1 since 2ethynylnaphthalene has been found to be inhibitory of P-450IA1 (18) and chloramphenicol was found not to inhibit this enzyme or P-450IIA1 (25). In summary, the results of this study contribute strong evidence for the involvement of N-oxidation of ABP in covalent hemoglobin adduct formation. This was demonstrated by the ability of 2-ethynylnaphthalene to inhibit in vivo, in a dose-dependent manner, the formation of the adduct in rats dosed with [3H]ABP. Thus, the results lend support to the usefulness of hemoglobin adduct biomonitoring in estimating the exposure of the target tissue to the carcinogen ABP. Additionally, the cytochrome P450IA2 catalyzed reaction was shown to be the predominant pathway for the N-oxidation of ABP in vivo. Registry No. ABP, 92-67-1; cytochrome P-450, 9035-51-2.
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George J. Hammons, Kenneth L. Dooley Fred F. Kadlubar* National Center for Toxicological Research (HFT-100) Jefferson, Arkansas 72079 Received October I , 1990
