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Chapter 2 METABOLISM OF AROMATIC AMINES The presence of an amine group has a strong tendency to activate the aromatic ring, resulting in a complex pattern of metabolism and ~ multiplicity of metabolites. For more than 40 years, it was assumed that 2-naphthylamine was simply metabolized to 2-amino-~-naphthylsulfate in dogs Wiley, 1938~. Subsequent studies with 14c_ labeled 2-naphthylamine indicated that although 90% of the radioactivity could be accounted for by this metabolite, seven other metabolites were also present. In addition, metabolism in the dog is much simpler than in other species. For example, acetyl metabol ites are not formed in dogs. These results illustrate the cliff iculty of conducting studies of metabolism to delineate the best test species for evaluating a new compound when there is 1 ittle or no information concerning the identity of the metabolite responsible for toxic or pharmacologic effects. The active metabolite could well be quantitatively insignif icant. In addition, despite the difference in acetylating ability between humans and dogs, the dog does appear to be a good test species for induction of bladder cancer because it develops bladder cancer from the same amines that humans do , wh lob is a more important consideration (Radomski, 1979b). However, the high cost of maintenance and long lifespan of dogs may preclude their routine use. 40
Both aromas ic amides and amides a re extend ively metabol ized by enzyme systems, pr incipally located in the liver. These enzyme systems are usually divided into two groups: phase I and phase TI (Williams, 1959 ~ . During phase I metabolism, one or more polar groups ~ such as hydroxyl ~ are introduced into the hydrophobic parent molecule, thus allowing a "handle" or position for the phase II conjugating enzymes (such as uridine diphosphoglucuronyl transferase) to attack . The con jugated products are suff iciently polar that these "detoxified chemicals can be more efficiently excreted from the cell and from the body. One of the more interesting phase I enzyme systems is a group of enzymes known collectively as the cytochrome P-450-mediated monooxygenases (Cooper et al., 1975; Gillette et al. , 1972 ; Haugen et al., 1975; Johnson, 1979; Lu and Levin, 1974; Neims et al., 1976; Thorgeirsson and Nebert, 1977~. This enzyme system is involved in a wide range of biologic activities, as it mediates the metabol ism of numerous, structurally diverse substrates. It catalyzes the metabolism of many drugs, the transformation of steroids, cholesterol, and bile acids, and the activation and detoxification of a large number of carcinogens. Among the carcinogens extensively metabolized by the cytochrome P-450-mediated monooxygenases are the aromatic amines and amides. This metabolism involves both ac t iva t ion ~ e . g ., N-hyd roxyla t ion ~ and detox i f ice t ion ~ e . g ., C-hydroxylation) . Therefore, a balance exists in each tissue between the enzymes that potentiate and those that detoxify xenobiotics. This balance is known to vary with species, sex, age. tissue, hormonal status, and exposure of the animal to certain 41
xenobiotics (Cooper et al., 1974; Gillette et al., 1972; Lu and Levin, 1974; Neims en al., 1976; lborgeirsson and Nebert, 1974) . The terminal oxide se of these monooxygenase systems is a group of hemeproteins collectively termed cytochrome P-450. The cytochromes are characterized by their spectral absorption maximum, which occurs at 450 nm when they are associated with carbon monoxide in the ir reduced state. Recent efforts have successfully resolved and characterized multiple forms of the cytochrome, and the study of these isolated forms of cytochrome P-450 provides information regarding the properties of individual cytochromes and aids investigations concerned with regulating the occurrence of each cytochrome. The term Multiple forms of cytochrome P-4SO" refers to experimentally distinguishable forms of the cytochrome that occur naturally in a single species. The significance of multiplicity to the many biologic processes in which the cytochrome has been implicated will depend largely on the difference in regulatory and functional properties of the individual forms. The most extensively pur if fed and characterized forms of the cytochromes have been those that are induced by compounds such as phenobarbital or the group of compounds designated as arylhydrocarbon(Ah)-inducers which include 3-methylcholanthrene, 6-naphthoflavone, and 2,3,7,8-tetra- chlorodibenzo-~-dioxin (TODD). me role of the different forms of cytochrome P-450 in the metabol ic processing of aromatic amines and amides has not been 42
extensively studied. The existing data, however, indicate that the f ir st step in the metabol ic activation of this class of chemicals, namely N-hydroxylation, may be, at least in some species, catalyzed by a s ingle form of cytochrome P-450 . The clearest demonstration of this selectivity in the oxidative metabolism of aromatic amines and ~ i. amides involves the metabolism of 2-acetylaminofluorene (AAF) by four purified forms of rabbit cytochrome P-450 (Johnson et al., 19801. Of these four forms, two (forms 3 and 6) are exclusively i nvo Ived in C-hyd roxyla t ion ~ i . e ., de tax i f ice ~ ion ~ , one ~ form 4 ~ i s solely responsible the N-hydroxylation ~ i.e., metabolic activation), and one ~ form 2 ) does not catalyze either C- or N-hydroxylation of the AAF molecule. Also, although the evidence is indirect, genetic studies in mice on the regulation of N-hydroxylation of AAP indicate that one or very few genes are responsible for the induction of the cytochrome P-450 form the t ca talyzes N-hydroxylation of AAF (Thorgeirsson et al., 1977~. The occur rence of each cytochrome is dependent on many factors, and the relet ive role of each cytochrome must be integrated with other processes occurring dur ing metabolism and carcinogenesis. Thus, it is difficult to predict the effect of these metabolic differences on carcinogenesis. Despite these uncertainties, metabolism of aromatic amines and amides plays an important role in understanding the carcinogenic potential of these compounds by either lifetime animal or short-term in-vitro tests. The outcome of this testing has major consequences for health, society, and the economy. Thus, there Is a need for a more clearly def ined 43
relationship between the elements of metabolism of aromatic amines and carcinogenicity. OXIDATION The primary metabolic attack on aromatic amines is usually oxidation. Two types of oxidation may occur: oxidation of the n i trogen a tom (N-ox ids t ion) and ox Ida t ion of ca rbon of the a roma t ic r i ng (Coax Ida t to n ~ . Pr ima ry a roma t ic amine s may be ox id i zed through the fol lowing stages: amp ne hydro~r~mine nitroso _IlO2 nitro There is little evidence that aromatic amines are oxidized to nitro compounds. On the other hand, the nitro compound is reducible through all the above steps to the amine. Secondary (N-alkyl aromatic amines) and tertiary amines are also Nonoxidized. Tertiary amines are oxidized to the N-oxide only. Tertiary amines may also be dea~kylated to secondary amines (e.g., dimethylaminoazobenzene to monomethylaminoazobenzene). Secondary amines may be partially N-dealkylated with the formation of hydroxylamines (Zeigler et al. , 1969 ~ . Acetamides are N-hydroxylated with the formation of hydroxamic acids (Miller _ al., 19607. Hydroxamic acids are quite stable, in contrast to the notor ious instability of arylhydroxylamines. At present, arylhydroxylamines are believed to be the proximal carcinogens in the induction of bladder cancer by 44
some aromatic amines. The esters of hydroxamic acids are believed to be the proximal carcinogenic metabolites responsible for the induction of liver cancer by the acetamides (Radomski, 1979a). Activation of the free amine group of an aromatic amine results in the metabolic hydroxylation of the aromatic ring (C-hydroxylation). The positions attacked generally correspond to the regions of highest electron density (Weisburger and Weisburger, 1958~. Thus, aniline and 1-naphthylamine are primarily hydroxylated in the 3 position and secondarily in the 2 position. 2-Naphthylamine and 4-aminobiphenyl, on the other hand, are pr imar fly hydroxylated in the 1 position and the 3 position, respectively {Radomski, 1979a). 2-Aminofluorene either teas been more extensively studied or more positions on the aromatic nucleus are fa irly ecu ivalent in electronic density, since metabolites hydroxylated in the 1, 3, 5, 7, 8, and recently the 9 positions have been detected (We isburger and We isburger, 1958 ~ . GLUCURONIDATION Perhaps the most important detoxif ication process is con jugation of metabolites of aromatic amines with glucuronic acid. As far as is known, all species are capable of this metabolic reaction. This conjugation is generally regarded as a phase II process in which the hydroxyl groups introduced by the 1 iver mixed -function oxidase system in phase I are further modified. Conjugation with glucuronic acid results in highly polar metabolites that are rapidly excreted 45
by the kidney through f titration without reabsorption or sometimes through active secretion. Glucuronides are also excreted in the bile (Handel, 1971~. The conjugation does not occur directly with glucuronic acid but requires an activated intermediate, uridine diphosphoglucuronic acid (UDPGA). The reaction is catalyzed by the enzyme glucuronyl transferase, which occurs in liver microsomes according to the following scheme (Mandel, 1971~. glucose-l-phosphate + UTP _py__p__sphory~as~ UDP-glucose + pyrophosphate UDP-glucose + 2NAD+ + H 0 _______UD_G-_____ p_ + H + 2H 2 ~ehy~rogenast UD glucuronic acid 2NAD UDP-glucuronic acid + AT-OH _---g---Ufe°nYi-+ Ar-O-glucuronic acid + ODP AT-OH = hydroxylated aromatic amine UTP = uridine triphosphate Glucuron idation occurs pr imar fly on hydroxyl groups, but may also occur with carboxyl and amine groups. The formation of highly acidic, labile N-glucuronides may or may not be enzymatic. In a newly discovered metabolic reaction, hydroxylamines formed by the N-hydroxyla t ion of aroma t ic amines are con j uga ted wi th glucuron ic acid with the formation of an N-C conjugate (Kadlubar et al., 1977; Radomski et al., 1973, 1977) . For the aromatic amines that induce bladder cancer, these con jugates may represent the carr ier form, which transports the carcinogenic metabolite (hydroxylamine) as an aglycone from the site of N-hydroxylation in the liver to the 46 .4
bladder. In the bladder, the acid pH of the ur ine and/or the presence of 6-glucuronidase in the mucosa liberates the hydroxylamine to produce its carcinogenic action (Radomski et al., 1977) . SULFATI ON A perhaps less important phase II synthetic mechanism carried out by all species of test animals is conjugation of bydroxyl groups with sulfur ic acid (sulfate) . Thus, the phenolic hydroxyl groups introduced on the aromas ic nucle i (phase I ~ are used to increase the polarity of the original, relatively hydrophobic amine. The primary amine groups may also be directly conjugated, producing N-sulfate conjugates (sulfamates) that are readily hydrolyzed in weakly acidic solutions and by ubiquitous hydrolytic enzymes (sulfatases}. Of course, these enzymes also hydrolyze O-sulfates. As with the formation of glucuronides, sulfate (SO4_' must first be activated accord ing to the following scheme (Mandel, 19 71) . At' ____ATP~ __~ acenosine-~'-~:nos~'~os~~lfa7 en i?. ? - -;.--ocho.~2~r. be sup s e AT + ,~.. __AP§~32h°s:7h°~. 3~-?hos?hoan~nosin=-~-?hosph~osulfate~pAp~) + ELF k'nase ?. ?: + Ez-, ___suito_____. R-Z-SO3w + 3-pnos?hoadenosine-5'-nhosph2~ce transferase where Z is O or NH The key enzymes in the process are sulfotranferases (sulfokinases). Several distinct enzymes that exhibit considerable substrate specif icity are known to exist. 4 7
Because the total sulfate pool is quite limited, it is readily exhausted when large amounts of exogenous chemicals are administered. Thus, sulfate con jugation becomes quantitatively less important with increasing doses. Conjugation of hydroxamic acids with sulfate may also occur. This reaction has been postulated as being responsible for the final activat ion of N-hydroxy-N, 2-fluorenylacetamine in the induction of liver cancer (Irving, 1979~. ACETYLATI ON Primary amines are acetylated to an appreciable extent by many animal species. Secondary and tertiary amines are never acetylated (Mandel, 1971~. The metabolic reaction apparently occurs in the reticuloendothelial cells of the liver, but not in the parenchymal cells (Govier, 1965~. Mucosa of the spleen and intestines are also capable of acetylation (Mandel, 19711. For exogenous compounds, acetylation occurs because of the presence in tissues of acetyl-coenzyme A, a prominent component in the Krebs cycle. Most species also contain a hydrolytic enzyme, a deacetylase, which is capable of removing the ace tyl group from acetamides. However, the ability to deacetylate is apparently not absolute; trace amounts of 2-naphthylamine were detected in the ur ine of a man who had ingested 2-naphthylacetamide (Conzelman , per sonal communication). The failure of the dog to acetylate aromatic amines 48
(Marshal, 1954 ~ may be due to the dog 's powerful deacetylating ability, the deficiency of an enzyme, or the presence of an inhibitor (Leiberman and Anaclerio, 1962~. In the rat, aromatic amines and the ir acetylated products appear to exist in biologic equ il ibra with each other (Krebs et al ., 1947; Peters and Gutn~ann, 1955 ~ . This f inding is also evident from the observation that 2-aminofluorene and A, 2-fluorenylacetamide yield very similar patterns of acetylated and nonacetylated metabolites. Almost all metabolic alterations of exogenous compounds result in increased polarity of the metabolized compounds. This property is very important to survival: in this manner, the animal is able to dispose of potentially noxious substances efficiently. Acetylation, however, usually results in compounds having decreased polarity. Acetylation of aromatic amines is carried out with the aid of the enzyme N-acetyltransferase, according to the following scheme: O N-acetyTtransferase ~ O C ?13-~-C oA + R ~N-~2 ------- ~ R-N~CH3 + Cod N-acetyltransferase appears to be present in two different genes ically determined forms, one of wh ich is more ef f icient than the other, as observed by Handel (1971) in studies with isoniaz id. The active metabolite of the carcinogenic aromatic amines for the induction of bladder cancer appears to be the hydroxylamine; thus, acetylation appears to be protective. Suggestive evidence to support this finding has also been obtained in humans (Lower, 1979 ) . 49
METABOLIC ACTIVATION Aromatic amines are thought to initiate tumor formation by modifying tissue macromolecules (Clayson and Garner, 1976; Miller, 1978 ~ . These amines can be transformed to metabol ites that can react with proteins and nucleic acids by an initial N-oxidation, followed by a second activation step. Tne reactivity of the activated metabolites renders them unstable and limits the distance through which they are likely to exert their carcinogenic properties. Conversely, it is likely that all or only the final metabolic activation step takes place in the tissue in which tumors are induced. Consequently, the capacity of tissues to carry out these metabolic events is an impor tent determinant of the susceptability of that tissue to a carcinogen. Furthermore, at a higher level of resolution, it is probable that the intracellular location of the metabolic activation event can be equally crucial. The induction of liver tumors in rats by primary aromatic amines has been associated with their conversion to reactive, toxic sulfate conjugates of their hydroxamic acid derivatives (Irving, 1977~. This pathway is apparently restricted to rat liver and is ineffective in the livers of female rats of some strains. m e hepatocarcinogenicity of the secondary amine N-methyl-4-aminoazobenzene in rats appears to result from an initial N-oxidation and a secondary conjugation with sulfate to yield a reactive metabolite (Xadlubar et al., 19761. 50
An a 1 te r na t ive mecha n i sm for the metabol ic act iva t ion of arylhydroxamic ac ids is by the formation of reactive N-acetoxyarylamines as a consequence of N,~acyltransferase (King and Allaben, 1978~. Cytosolic enzymes capable of activating N-hydroxy-AAF have been demonstrated in a wide car iety of tissues from a number of species that are susceptible to the carcinogenic ef fects of aromatic amines. Previous studies have shown that the lactating mammary glands of the rat, like rat liver, possess an N,~acyltransferase and that RNA adducts formed in this tissue are compatible with an acyltransferase-mediated mechanism of activation (King et al., 1979) . This metabolic pathway has been implicated in tumor production in two ways. Malejka-Giganti and Gutmahn (1975) demonstrated that the d irect appl ication of N-hydroxy-AAF was more tumorigenic than either N-hydroxy-2-aminofluorene or N-2-AAF. Allaben et al. (1978 ~ reported that direct application of the . N-formyl, N-acetyl, or N-propionyl der ivatives of N-hydroxy-2-aminoflurorene resulted in tumor induction that was greater than that of the N-acetyl derivative, which was also the most effective substrate with partially purified rat liver arylhydroxamic acid N,O-acyltransferase. A third type of ester if ication of N-oxidized derivatives has been repor ted in the act teat ion of 4-hydroxylaminoqu inol ine-N-oxide in incuba Lion s conta in ing ATP-ser ine a nd seryl-adenos ine- monophosphate synthetase (Tada and Tada, 1976) . The activity of this system does not appear to be of general importance in the activation of other aromatic amine derivatives. 51
Another mechanism for activating aromatic amines is the gene ra t ion of reac t ive me tabol ites v is pathways in~rol~r ing peroxidation . Bartsch et a 1., (1972 ~ descr ibed the oxidation of N-hydroxy-N-2-AAF by peroxidase and hydrogen peroxide to yield tbe reactive ester, N-acetoxy-N-2-AAF. Subsequent studies have descr ibed the generation of radicals associated with this reaction by preparations from rat mammary gland (Floyd et al., 1978) . Although adduct formation with lipids has been demonstrated in this system, reactions involving nucleic acids have apparently not been considered. A more recent war iation in this area has been the generation of reactive benzidine derivatives by an arachidonic- acid-dependent, prostaglandin synthetase system obtained from rat kidney (T. Zenser, St. Louis University, personal communication). One unique feature of these findings is that the prostaglandin synthetase activation uses the free primary amines; prior _-oxidation is not required. The relationship of this metabolic act iva t ion pa thway to the ca rc inogen ic i ty of benz id ine, howeve a, rema ins to be explored. 52
Metabolism Allaben, W.T., C.E. Weeks, N.C. Tresp, S.C. Louie, E.J. L;azear, and C.M. King. 1978. Marry tumor induction in the rat by N-acyl-~-2- f luorenyl hydroxylemines: Structure-ac tivi ty relationship. Fed. Proc. Fed . Am. Soc. Exp. Biol. 37 :1543 (Abstract No. 15030) Bar tech, H., J.A. Miller, and E.C. Miller. 1972. N-Acetoxy-N-acetylaminoarenes and nitrosoarenes. One-electron non-enzymatic and enzymatic oxidation products of various carcinogenic aromatic acethydroxamic acids. Biochim. Biophys. Acta 273:40-51. Clayson , D. B., and R.C. Garner . 1976. and related compounds. Carcinogens. ACS Monograph 173. Washington, D.C. Carcinogenic aromatic amines Pp. 366-461 in C.E. Searle, ed. Chemical American Chemical Society, Cooper, D.Y., O. Rosenthal, R. Snyder, and C. Witmer, eds. 1975. Cytochromes P-4 50 and b5. Structure, Function, and Interaction. Advances in Experi~nental Medicine and Biology, Volume 58. Plenum Publishing Corp., New York. 53
Floyd, R.A., L.M. Soong, M.A. Stuart, and D.L. Reigh. 1978. Free radicals and carcinogenesis: Some properties of the nitroxyl free radical products by covalent binding of 2-nitrosofluorene to unsaturated lipids of membranes. Arch. Biochem. Biophys. 185:450-457. Gillette, J.R., D.C. Davis, and H.A. Sasame. 19?2. Cytochrome P-450 and its role in drug metabolism. Annul Rev. Pharmacol. 12:57-84. Gorier, W.C. 1965. Reticuloendothelial cells as the site of sulfanilamide acetylation in the rabbit. J. Pharmacol. Exp. Ther. 150: 305-308 . Haugen, D.A., T.A. Ran der Hoeven, and M.J. Coon. 1975. Purified liver microsomal cytochrome P-450: Separation and characterization of multiple forms. J. Biol. Chem. 250:3567-3570. Irving, C.C. 1977. Influence of the aryl group on the reaction of glucuronides of N-arylacethydroxamic acids with polynucleotides. Cancer Res. 37:524-528. Irving, C.C. 1979. Species and tissue variations in the metabolic activation of aromatic amines. Pp. 211-227 in A.C. Griffin and C.R. Shaw, eds . Care inogens: Identif ication and Mechan isms of Action. 31st Annual Symposium on Fundamental Cancer Research, M.D. Anderson Hospital and Tumor Institute, Universities of Texas Cancer Center, Houston, 1978. Raven Press, New York . 54
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