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DRUG METABOLISM REVIEWS, 22(2&3), 147-159 (1990)
CYTOCHROME P45011El: ROLES IN NITROSAMINE METABOLISM AND MECHANISMS OF REGULATION* CHUNG S. YANG,? JEONG-SOOK H. YOO, HIROYUKI ISHIZAKI, and JUNYAN HONG Department of Chemical Biology and Pharmacognosy College of Pharmacy Rutgers University Piscataway, New Jersey 088554789
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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11. ENZYMATIC BASIS FOR THE METABOLISM OF N-NITROSODIMETHYLAMINE. . . . . .
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I.
111.
IV.
ROLES OF CYTOCHROME P450IIE1 IN THE METABOLISM OF N-NITROSODIMETHYLAMINE AND OTHER CHEMICALS. .......................... A. Metabolism of NDMA by P450 Isozymes . . . . . . . . . . . . . B. Nature of the Low K,,, Form of NDMA Demethylase.. . . . C. Activation of NDMA .............................. D. Metabolism of Other Nitrosamines . . . . . . . . . . . . . . . . . . . E. Metabolism of Acetone and Other Chemicals.. . . . . . . . . .
149 149 150 151 151 152
NATURE OF THE SUBSTRATE BINDING SITE OF CYTOCHROME P450IIE1 ....................
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*Presented at the Symposium in Honor of the Retirement of Professor G. J. Mannering, June 13-14, 1988, at the University of Minnesota, Minneapolis. ?To whom correspondence should be addressed. 147 Copyright 0 1990 by Marcel Dekkcr, Inc
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V. STRUCTURE AND REGULATION OF THE P450IIE1 GENE ............................................. A. The Structure of the Gene .......................... B. Molecular Mechanisms of Regulation ................ C. Regulation by Hormones and Physiological Conditions. . .
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VI. CONCLUDING REMARKS. ..........................
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Acknowledgments.................................... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I. INTRODUCTION Many previously unrelated observations in drug metabolism, biochemical toxicology, and cancer research appeared to have converged on the functions of one specific cytochrome P450 species. Various names, such as P450LM3a, P450alc, P450et, P450ac, and P450j, have been used to describe this enzyme species. The gene sequence of this enzyme has been determined [ l ] and was given the recommended systematic name P450IIE1 [2], and this name is used herein for the protein. The present communication summarizes our work on the characterization of the properties, functions, and regulation of this enzyme.
11. ENZYMATIC BASIS FOR THE METABOLISM OF N-NITROSODIMETHYLAMINE
The early work of Magee and co-workers (reviewed in Ref. 3) demonstrated that the hepatotoxic and carcinogenic N-nitrosodimethylamine (NDMA) is activated by microsomes in an NADPH-dependent reaction. The involvement of P450 in NDMA metabolism was demonstrated by Czygan et al. [4] and others [ 5 , 6 ] .However, the role of P450 in the metabolic activation of NDMA in vivo and in vitro has been questioned because of the following observations: (1) NDMA demethylase is not induced by classical inducers such as phenobarbital and 3-methylcholanthrene. (2) The activity is not inhibited by well-recognized P450 inhibitors such as metyrapone and SKF-525A. (3) There are multiple K,,,values for NDMA demethylase, and unrealistically high &values (> 100 mM) were observed with phenobarbital-induced microsomes [7-111. We started our work in 1980 with a working hypothesis stating that the multiplicity of the K,,, values of NDMA demethylation is due to the catalytic activity of the multiple forms of P450, and that the P450 species showing the
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lowest K,,, and highest V,,, is likely to be the enzyme responsible for the activation of this carcinogen. In an effort to identify this key P450 for the metabolism of NDMA, we reexamined the kinetic parameters of NDMA demethylase and studied the induction of the low K,,, form of NDMA. With uninduced rat liver microsomes, we observed multiple K , values. In addition to K,,, values of 0.3-0.5 m M and 30 to 50 mM, which corresponded to the NDMA demethylases I and I1 described by Lai and Arcos and others [7,8], we observed a much lower K , value of 50-70 pA4 [12-151. This low K,,, form of activity was induced by pretreatment of rats with ethanol, acetone, isopropanol, pyrazole, and other chemicals as well as by fasting and diabetes [12-181. However, it was not inducible by classical P450 inducers such as phenobarbital and 3-methylcholanthrene [19]. In 1986 we purified this form of P450 from acetone-induced rat liver microsomes [20]. The name P450ac was used, because we believe acetone, which is formed endogenously,is both a natural inducer and a substrate of this enzyme. It is probably identical to P450j purified from isoniazid-induced rat liver microsomes [21] and is an ortholog of P450LM3a previously purified from rabbit liver [22, 231. Similar orthologs probably also exist in humans [24, 251, mice, hamster, guinea pigs, and other animal species [26,27]. For the convenience of descriptions,we should use the name P450IIE1 for this type of P450 in all species, although the final nomenclature should await more information for the structure of the genes in different species. This P450 species, as is documented in the next section, is the key enzyme in the metabolism of NDMA and many other environmental chemicals.
111. ROLES OF CYTOCHROME P450IIE1 IN THE METABOLISM OF KNITROSODIMETHYLAMINE AND OTHER CHEMICALS
A. Metabolism of NDMA by P450 Isozymes In a collaborative study with Drs. D. R. Koop and M. J. Coon, we have examined the ability of six purified rabbit liver P450 forms for catalyzing the metabolism of NDMA [28]. Among these isozymes, the alcohol-inducible P450LM3a (P450IIEl) was most efficient in catalyzing the demethylation and denitrosation of NDMA. Studies with purified rat liver P450 isozymes in our laboratory and in others also demonstrated that P450IIE1 (P450et, P450ac, or P450j) is more active than other forms in catalyzing the metabolism of NDMA [20, 29, 301. Other P450 forms, such as the phenobarbital-inducibleP450b or P450LM2, showed substantial activities only at high substrate concentrations, suggesting high K,,, values [28 291. The results are consistent with the concept that the multiple K, values for NDMA demethylase found in microsomes are
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due to the catalytic activity of multiple forms of P450. It was also demonstrated, in studies with both rabbit and rat P450 isozymes, that the P4SOIIEl-dependent NDMA demethylase was not effectively inhibited by the well-recognized P4SO inhibitor SKF-525A. Nevertheless, the reaction was inhibited by nonclassical inhibitors such as 2-phenylethylamine, 2-amino-1,2,4-triazole, and pyrazole [28, 291. Results from studies with these inhibitors in previous work have led to the proposal that monoamine oxidase, rather than P4S0, is responsible for NDMA metabolism [9]. Purified monoamine oxidase, however, was inactive in catalyzing the oxidation of NDMA [31]. Our work indicates that the previous results with inhibitors [9] can be interpreted solely based on the inhibition of P450.
B. Nature of the Low K,,, Form of NDMA Demethylase With the NDMA demethylase system reconstituted from both the rabbit and rat P450IIE1, K , values around 3 mM were observed [20]. This value is much higher than the low K,, value observed in liver microsomes, 50-70 fl,which is again higher than the values (10-20 phf) reported for studies using isolated liver cells, liver slices, and perfused liver [32-341. Is P4SOIIE1 responsible for the much lower K,,, values displayed in the system? Several lines of evidence indicate that this is the case: (1) Antibodies against P4SOIIE1 inhibit most, if not all, of the NDMA demethylase activity in microsomes 126, 351. (2) The apparent K,,, values determined experimentally are influenced by the assay conditions. We suspect that inhibitors are present in the isocitrate/isocitrate dehydrogenase NADPH-generating system which was used in many of o u r previous works, because a higher apparent K,, value was observed using this system in comparison to an NADPH-generating system consisting of glucose 6-phosphate and glucose-6-phosphate dehydrogenase [36]. Using the latter system, we have recently obtained an apparent K,, of 20 @4 [37], a value close to those reported for isolated liver cells, tissue slices, and perfused liver [32-341. (3) Glycerol was found to be a competitive inhibitor of NDMA demethylase [36]. This compound is present in the buffer solutions in purified P450 and NADPH:P450 reductase preparations, and is expected to increase the observed K , values in an NDMA demethylase assay system. (4) It was observed that the inclusion of cytochrome b, in a reconstituted NDMA demethylase system reduced the apparent K, value by a factor of 8 [20]. Thus, the observed higher K,, values with the reconstituted systems are due to the artificial experimental conditions used. By improving these conditions, we believe it is possible to obtain K,,, values comparable to those observed with microsomes.
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C. Activation of NDMA The thesis that NDMA demethylase is responsible for the activation of this carcinogen had been challenged because of the lack of correlation between this enzyme activity and the ability to activate NDMA to a mutagen observed in certain studies (reviewed in Ref. 38). With the insights gained from our studies on the enzyme specificity of NDMA metabolism, we were able to demonstrate the key role of NDMA demethylase in the activation of this compound in several different systems: (1) We demonstrated that P4501IE1 was more efficient than other P450 species in the activation of NDMA to a mutagen in Chinese hamster V79 cells [38]. (2) Some of the previously reported species and age differences could be interpreted based on the quantity of P450IIE1 present in the microsomes of rats and hamsters of different ages [39]. (3) Pretreatment of rats with ethanol or acetone increased NDMA-induced methylation of DNA in v i m and in vivo [ 191, and potentiated NDMA-induced hepatotoxicity [40]. The potentiating effect of these NDMA inducers on DNA methylation in vivo and hepatotoxicity, however, was observed only when high doses of NDMA (> 25 mg/kg body weight) was given to the rats [ 19,401. This result suggests that, in untreated rats, there is a sufficient amount of P450IIE1 to metabolize low doses of NDMA.
D. Metabolism of Other Nitrosamines We have reported previously that fasting or treatment with ethanol enhanced the microsomal demethylase activities with NDMA and some other nitrosamines [15]. However, this result does not mean that P450IIE1 is the enzyme responsible for the metabolic activation of all nitrosamines. Recent studies in our laboratory on the active-site mechanics indicate that the alkyl chains of nitrosamines are probably more important than the N-nitroso group in determining the affinity of binding to the active site of P450IIE1. Because of structural similarities, N-nitrosoethylmethylamine and N-nitrosodiethylamine are also preferentially metabolized by P450IIE1. It was also reported that P450IIE1 is more active than other forms in catalyzing the metabolism of N-nitrosopyrrolidine [41]. With nitrosamines with larger alkyl chains, however, the situation may be very different. For example, with N-nitrosobutylmethylamine as the substrate, the demethylation reaction was preferentially catalyzed by P4501IE1, but the debutylation reaction was more efficiently catalyzed by the phenobarbital-inducible P450IIB1 [42]. Debutylation of this compound leads to the formation of methyldiazonium, which is most likely to be the reactive species for carcinogenesis.
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/
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FlStlng Dlrbotor Acetono Kotonor lroproprnol Ethanol Benzene Ether Pyrrrole Imldrzole
N-Nltrosodlmethyl~mlne Acotono Alcohols Anlllne Enf lurane Ethers Chloroform Carbon Tetrachloride Benzene Alkanes
lronlrrld Pyrldlne
Acetamlnophen Azoxymethrnr
\
FIG. 1. Inducers (left) and substrates (right) of cytochrome P450IIE1.
E. Metabolism of Acetone and Other Chemicals An interesting observation was made by Casazza and co-workers that P450IIE1 can catalyze the oxidation of acetone to acetal and then to methylglyoxal [43, 441. This pathway can lead to the ultimate synthesis of glucose and may be an emergency gluconeogenesis pathway during fasting. P450IIE1 is also known to catalyze the metabolism of other important environmental chemicals such as diethyl ether [45], enflurane [46], acetaminophen [47], carbon tetrachloride [48], benzene [49], alcohols [50], aniline [20],p-nitrophenol [51], and other chemicals. Some of them are shown in Fig. 1.An understanding of the induction and function of P450IIE1 helps to explain, at least in part, many previously observed interactions between chemicals; for example, the potentiation of carbon tetrachloride toxicity by alcohol [52]. Many of the substrates can also serve as competitive inhibitors. This interaction may be the basis for the inhibition of nitrosamine metabolism in vivo by ethanol and diethyl ether.
IV. NATURE OF THE SUBSTRATE BINDING SITE OF CYTOCHROME P450IIE1 We have used the amino acid sequence of P450IIE1 to derive structural information concerning the conformation of the protein and the structure of the active site. The amino acid sequence alignment between P450IIE1 and other
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FIG. 2. Proposed substrate binding site of cytochrome P450IIE1.
P450s shows homologies at the substrate binding site. Based on the calculated tertiary structure of P450IIE1, the structure of P450cam as determined by X-ray crystallography, and other structural features of P450s [53, 541, a proposed arrangement of residues at the substrate binding site of P450IIE1 is shown in Fig. 2. The heme disk in P450IIE1 is buried within the protein molecule with no edge accessible to the molecular surface. The heme disk is embedded between part of the proximal (helix L, residues 439 to 441) and part of the distal (helix I, residues 295 to 303) helices running parallel to each other. The amino acid residues of part of the proximal helix L in the immediate vicinity of the heme are Gly439, Glu440, and Gly441. The amino acid sequence of the 295-303 part of the distal helix I is Asp-Leu-Phe-Phe-Ala-Gly-Thr-Glu-Thr.The residues interacting with the heme edges extend from part of the p sheet (Thr376Va1377-Phe37&Gln379-Gly38&Tyr381) and part of helix C (Arg126 Arg127). The axial thiolate ligand of heme is provided by the side chain of Cys437 and the heme is further stabilized by the propionate group interacting with Arg127 and Arg435. This interaction between propionates and two arginine residues makes the heme orient closer to helix C and twist about 30"
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clockwise, as compared to the orientation of heme in P450cam. Pyrrole ring A is open to substrate binding, but ring C is blocked by the distal helix 1. We propose that the substrate, NDMA, sits about 4 A above pyrrole ring A and is hydrogen bonded to the hydroxyl group of Tyr381. The substrate is also oriented by hydrophobic contacts with Val105 and Ile 1 14. A second hydrophobic pocket consisting of Phe298 and Ala299 may also come in contact with P450ac substrates. With N-nitrosobutylmethylamine as substrate, the N-butyl group is believed to preferentially bind to the proposed hydrophobic site, positioning the N-methyl group in the oxygenation site, the iron-oxygen complex of P450IIE1. As a consequence, demethylation is the major metabolic pathway. In the case of P450IIB1, however, such preference does not exist and debutylation also takes place at a substantial rate.
V. STRUCTURE AND REGULATION OF THE P450IIE1 GENE A. The Structure of the Gene The cDNAs for rat and human P450IIE1 were isolated and sequenced in 1986 [ 11. The coding sequence of both rat and human P4501IE1 contained 1489 base pairs. The deduced amino acid sequence for both species contained 493 amino acids with calculated molecular masses of 56,635 and 56,916 for rats and humans, respectively. Human P450IIE1 shares 75% nucleotide and 78% amino acid similarities to the orthologous rat P4501IEl. Amino acid alignment also revealed that P450IIEl is 48% similar to P450IIB1 and P450IIB2, and 54% similar to P450PB1 and P450f. Southern blot analyses of rat and human genomic DNAs verified that only a single gene shared extensive homology with P45011E1. The complete gene sequences for rat and human P450IIE1 were determined in 1988 [ 5 5 , 5 6 ] . The human gene spanned 11,413 base pairs and contained nine exons and a typical TATA box. Upstream and downstream DNAs of 2,788 and 559 base pairs were also sequenced. Significant areas of sequence similarities were observed within 140 base pairs upstream of the transcription start site in the rat and human genes.
B. Molecular Mechanisms of Regulation During the past several years, we have extensively characterized the molecular events involved in the induction of P450IIE1. The induction of NDMA demethylase activity under different conditions is accompanied by an increase in microsomal P450IIE1 level quantified by Western blot analysis, suggesting that the increase in the demethylase activity is due to the increase
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in the quantity of this enzyme rather than posttranslational modifications. The induction of P450IIE1 by acetone as well as by isopropanol, pyrazole, and 4-methylpyrazole is not accompanied by an elevation of the P450IIE1 mRNA level [ l , 57, 581. We have shown that acetone treatment may enhance the stabilization of P450IIE1 protein [59] and may also increase P450IIE1 protein synthesis (Ref. 17 and unpublished results). The role of translational control in the regulation of P450IIE1 is far from clear and requires further investigation. The induction of P450IIE1 by fasting and diabetes (chemically induced and spontaneous) is accompanied by an elevation of P450IIE1 mRNA [57,58,60]. The molecular events that lead to the elevation of P450IIE1 mRNA level are not clear. In a previous study using nuclear run-on transcriptional analysis, we failed to demonstrate an increased rate of transcription in the liver nuclei of diabetic rats, and we suggested that P450IIE1 mRNA stabilization may be a mechanism for the induction (591. We also observed previously [15] and confirmed recently that actinomycin D, an RNA synthesis inhibitor, inhibits the induction of P450IIE1 by fasting. The role of transcriptional control in the induction of this enzyme requires more in-depth examination. P450IIE1 is also regulated by development in rats: At birth, levels of P450IIE1 and its mRNA are very low or undetectable; the levels are much higher at day 4 and continue to increase, reaching a plateau at about day 30 [ l , 581. Nuclear run-on transcriptional analysis indicated that transcriptional activation is mainly responsible for the rapid increase in P450IIE1 between birth and 1 week of age [ 11. It is not known what factors, whether they are metabolites or hormones, trigger the activation of the P450IIE1 gene.
C. Regulation by Hormones and Physiological Conditions In rats, a significant sex-related difference was not observed in hepatic and renal P450IIE1 [58] suggesting that androgens and estrogens do not play a role in the regulation of this enzyme. Attempts were made to demonstrate the involvement of insulin and glucagon in the induction of P450IIE1. So far, positive results have not been obtained. In the case of diabetes, the level of P4501IE1 is affected by insulin, but this is believed to be due to the elimination of ketosis, not a direct effect [60]. In certain mouse strains, the renal NDMA demethylase activity in males is much higher than that in females [61-641. We recently demonstrated in C3H/HeJ mice that this sex-related difference is due to the different amounts of P450IIE1 present in the kidneys of the male and female mice. After treating the female mice with testosterone, the NDMA demethylase activity, P450IIE1, and P450IIE1 mRNA increased 20- to 30-fold, equal to or surpassing the male levels [27]. This system provides a unique opportunity to study the regulation of this enzyme by a hormone.
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Based on the facts that ketone bodies are produced in fasting and diabetes, and that acetone is an effective inducer, we have proposed a “ketone hypothesis” stating that acetone or other related compounds may be responsible for the induction [65]. Quantitatively, the amount of acetone produced in fasting is probably insufficient to account for the P450IIE1 induction by fasting (651. Mechanistically, we know that the elevation of P450IIE1 mRNA level in fasting and diabetes [57, 58, 601 must be caused by factors other than acetone. However, the cellular levels of acetone or other ketone bodies may play an important role in the regulation of the level of P450IIE1 under normal physiological conditions. Our recent study with dietary manipulations indicates that the rat hepatic P450IIE1 level is proportional to the “fat-to-carbohydrate ratio” in the diet and to the level of ketone bodies in the animal [66].
VI. CONCLUDING REMARKS The catalytic functions and the mechanisms of regulation of P450IIE1 have received a great deal of attention in recent years. The studies on this enzyme have enhanced our understanding of the mechanisms by which a chemical or physiological condition might affect the metabolism or toxicity of other chemicals. Because of the importance of P4501IE1 in the metabolism of many important xenobiotics, this enzyme is expected to continue to receive much attention. Many of the small organic molecules which are poor substrates for the most often studied phenobarbital-inducible P450 forms may turn out to be good or physiological substrates for P450IIEl. P450IIE1 is a constitutive enzyme that is inducible by dietary and physiological conditions as well as by many common environmental chemicals. It is a great challenge for researchers to elucidate the detailed mechanisms that modulate this enzyme. Of particular interest is whether the “constitutive” level of this enzyme is regulated by the ketone body levels as a reflection of the diet.
Acknowledgments
Supported by NIH grants ES-03938 and CA-37037. The authors thank all their collaborators who have contributed much to the work discussed herein and acknowledge the excellent secretarial work of Ms. Barbara McLouth in the preparation of this manuscript.
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M. Lee, H. Ishizaki, J. F. Brady, and C . S. Yang, Cancer Res., 49, 1479 ( 1989). J. P. Casazza, M. E. Felver, and R. L. Veech, J. Biol. Chem., 259, 231 (1984). D. R. Koop and J. P. Casazza, J , Biol. Chem., 260, 13607 (1985). J. F. Brady, M. J. Lee, M. Li, H. Ishizaki, and C. S. Yang, Mol. Pharmacol., 33, 148 (1988). E. J. Pantuck, C . B. Pantuck, and A . H. Conney,Anesthesiology, 66, 24 (1987). D. R. Koop, E. T. Morgan, G. E. Tarr, and M. J. Coon,J. Biol. Chem., 257, 8472 (1982). I. Johansson and M. Ingelman-Sundberg, FEBS Lett., 183, 265 (1985). 1. Johansson and M. Ingelman-Sundberg, Cancer Res., 48,5387 (1988). E. T. Morgan, D. R. Koop, and M. J. Coon, J. Biol. Chem., 257, 13951 (1982). D. R. Koop, Mol. Pharmacol., 29, 399 (1986). G. J. Traiger and G. L. Plaa, Toxicol. Appl. Pharmacol., 20, 105 (1971). T. L. Poulos, B. C. Finzel, and A . J. Howard, J . Mol. Biol., 195, 687 (1987). S. D. Black and M. J. Coon, Adv. Enzymol., 60, 35 (1987). M. Umeno, B. J. Song, C. Kozak, H. V. Gelboin, and F. J. Gonzalez, J. Biol. Chem., 263, 4956 (1988). M. Umeno, 0. W. McBride, C. S. Yang, H. V. Gelboin, and F. J. Gonzalez, Biochemistry, 27, 9006 (1988). J. Hong, J. Pan, F. J. Gonzalez, H. V. Gelboin, and C. S. Yang, Biochem. Biophys. Res. Commun., 142, 1077 (1987). J. Hong, J. Pan, Z. Dong, and C. S. Yang, Cancer Res., 47,5948 (1987). B. J. Song, T. Matsunaga, J. P. Hardwick, S.S. Park, R. L. Veech, C. S. Yang, H. V. Gelboin, and F. J. Gonzalez, Mol. Endocrinol., 1, 542 (1987). Z. Dong, J. Hong, Q. Ma, D. Li, J. Bullock, F. J. Gonzalez, S. S. Park, H. V. Gelboin, and C. S. Yang, Arch. Biochem. Biophys., 263,29(1988). R. L. Hawke and R. W. Welch, Mol. Pharmacol., 27, 283 (1985). F. R. Ampy and A . 0 . Williams, Life Sci., 39, 923 (1986). F. R. Ampy and A. 0 . Williams, Life Sci., 39, 931 (1986). S. Mohla, F. R. Ampy, K. J. Sanders, and W. E. Criss, Cancer Res., 41, 3821 (1981). K. W. Miller and C. S. Yang,Arch. Biochem. Biophys., 229,483 (1984). J. S. H. Yo0 and C. S. Yang, manuscript in preparation.
DRUG METABOLISM REVIEWS, 22(2&3), 147-159 (1990)
CYTOCHROME P45011El: ROLES IN NITROSAMINE METABOLISM AND MECHANISMS OF REGULATION* CHUNG S. YANG,? JEONG-SOOK H. YOO, HIROYUKI ISHIZAKI, and JUNYAN HONG Department of Chemical Biology and Pharmacognosy College of Pharmacy Rutgers University Piscataway, New Jersey 088554789
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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11. ENZYMATIC BASIS FOR THE METABOLISM OF N-NITROSODIMETHYLAMINE. . . . . .
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I.
111.
IV.
ROLES OF CYTOCHROME P450IIE1 IN THE METABOLISM OF N-NITROSODIMETHYLAMINE AND OTHER CHEMICALS. .......................... A. Metabolism of NDMA by P450 Isozymes . . . . . . . . . . . . . B. Nature of the Low K,,, Form of NDMA Demethylase.. . . . C. Activation of NDMA .............................. D. Metabolism of Other Nitrosamines . . . . . . . . . . . . . . . . . . . E. Metabolism of Acetone and Other Chemicals.. . . . . . . . . .
149 149 150 151 151 152
NATURE OF THE SUBSTRATE BINDING SITE OF CYTOCHROME P450IIE1 ....................
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*Presented at the Symposium in Honor of the Retirement of Professor G. J. Mannering, June 13-14, 1988, at the University of Minnesota, Minneapolis. ?To whom correspondence should be addressed. 147 Copyright 0 1990 by Marcel Dekkcr, Inc
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V. STRUCTURE AND REGULATION OF THE P450IIE1 GENE ............................................. A. The Structure of the Gene .......................... B. Molecular Mechanisms of Regulation ................ C. Regulation by Hormones and Physiological Conditions. . .
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VI. CONCLUDING REMARKS. ..........................
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Acknowledgments.................................... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I. INTRODUCTION Many previously unrelated observations in drug metabolism, biochemical toxicology, and cancer research appeared to have converged on the functions of one specific cytochrome P450 species. Various names, such as P450LM3a, P450alc, P450et, P450ac, and P450j, have been used to describe this enzyme species. The gene sequence of this enzyme has been determined [ l ] and was given the recommended systematic name P450IIE1 [2], and this name is used herein for the protein. The present communication summarizes our work on the characterization of the properties, functions, and regulation of this enzyme.
11. ENZYMATIC BASIS FOR THE METABOLISM OF N-NITROSODIMETHYLAMINE
The early work of Magee and co-workers (reviewed in Ref. 3) demonstrated that the hepatotoxic and carcinogenic N-nitrosodimethylamine (NDMA) is activated by microsomes in an NADPH-dependent reaction. The involvement of P450 in NDMA metabolism was demonstrated by Czygan et al. [4] and others [ 5 , 6 ] .However, the role of P450 in the metabolic activation of NDMA in vivo and in vitro has been questioned because of the following observations: (1) NDMA demethylase is not induced by classical inducers such as phenobarbital and 3-methylcholanthrene. (2) The activity is not inhibited by well-recognized P450 inhibitors such as metyrapone and SKF-525A. (3) There are multiple K,,,values for NDMA demethylase, and unrealistically high &values (> 100 mM) were observed with phenobarbital-induced microsomes [7-111. We started our work in 1980 with a working hypothesis stating that the multiplicity of the K,,, values of NDMA demethylation is due to the catalytic activity of the multiple forms of P450, and that the P450 species showing the
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lowest K,,, and highest V,,, is likely to be the enzyme responsible for the activation of this carcinogen. In an effort to identify this key P450 for the metabolism of NDMA, we reexamined the kinetic parameters of NDMA demethylase and studied the induction of the low K,,, form of NDMA. With uninduced rat liver microsomes, we observed multiple K , values. In addition to K,,, values of 0.3-0.5 m M and 30 to 50 mM, which corresponded to the NDMA demethylases I and I1 described by Lai and Arcos and others [7,8], we observed a much lower K , value of 50-70 pA4 [12-151. This low K,,, form of activity was induced by pretreatment of rats with ethanol, acetone, isopropanol, pyrazole, and other chemicals as well as by fasting and diabetes [12-181. However, it was not inducible by classical P450 inducers such as phenobarbital and 3-methylcholanthrene [19]. In 1986 we purified this form of P450 from acetone-induced rat liver microsomes [20]. The name P450ac was used, because we believe acetone, which is formed endogenously,is both a natural inducer and a substrate of this enzyme. It is probably identical to P450j purified from isoniazid-induced rat liver microsomes [21] and is an ortholog of P450LM3a previously purified from rabbit liver [22, 231. Similar orthologs probably also exist in humans [24, 251, mice, hamster, guinea pigs, and other animal species [26,27]. For the convenience of descriptions,we should use the name P450IIE1 for this type of P450 in all species, although the final nomenclature should await more information for the structure of the genes in different species. This P450 species, as is documented in the next section, is the key enzyme in the metabolism of NDMA and many other environmental chemicals.
111. ROLES OF CYTOCHROME P450IIE1 IN THE METABOLISM OF KNITROSODIMETHYLAMINE AND OTHER CHEMICALS
A. Metabolism of NDMA by P450 Isozymes In a collaborative study with Drs. D. R. Koop and M. J. Coon, we have examined the ability of six purified rabbit liver P450 forms for catalyzing the metabolism of NDMA [28]. Among these isozymes, the alcohol-inducible P450LM3a (P450IIEl) was most efficient in catalyzing the demethylation and denitrosation of NDMA. Studies with purified rat liver P450 isozymes in our laboratory and in others also demonstrated that P450IIE1 (P450et, P450ac, or P450j) is more active than other forms in catalyzing the metabolism of NDMA [20, 29, 301. Other P450 forms, such as the phenobarbital-inducibleP450b or P450LM2, showed substantial activities only at high substrate concentrations, suggesting high K,,, values [28 291. The results are consistent with the concept that the multiple K, values for NDMA demethylase found in microsomes are
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due to the catalytic activity of multiple forms of P450. It was also demonstrated, in studies with both rabbit and rat P450 isozymes, that the P4SOIIEl-dependent NDMA demethylase was not effectively inhibited by the well-recognized P4SO inhibitor SKF-525A. Nevertheless, the reaction was inhibited by nonclassical inhibitors such as 2-phenylethylamine, 2-amino-1,2,4-triazole, and pyrazole [28, 291. Results from studies with these inhibitors in previous work have led to the proposal that monoamine oxidase, rather than P4S0, is responsible for NDMA metabolism [9]. Purified monoamine oxidase, however, was inactive in catalyzing the oxidation of NDMA [31]. Our work indicates that the previous results with inhibitors [9] can be interpreted solely based on the inhibition of P450.
B. Nature of the Low K,,, Form of NDMA Demethylase With the NDMA demethylase system reconstituted from both the rabbit and rat P450IIE1, K , values around 3 mM were observed [20]. This value is much higher than the low K,, value observed in liver microsomes, 50-70 fl,which is again higher than the values (10-20 phf) reported for studies using isolated liver cells, liver slices, and perfused liver [32-341. Is P4SOIIE1 responsible for the much lower K,,, values displayed in the system? Several lines of evidence indicate that this is the case: (1) Antibodies against P4SOIIE1 inhibit most, if not all, of the NDMA demethylase activity in microsomes 126, 351. (2) The apparent K,,, values determined experimentally are influenced by the assay conditions. We suspect that inhibitors are present in the isocitrate/isocitrate dehydrogenase NADPH-generating system which was used in many of o u r previous works, because a higher apparent K,, value was observed using this system in comparison to an NADPH-generating system consisting of glucose 6-phosphate and glucose-6-phosphate dehydrogenase [36]. Using the latter system, we have recently obtained an apparent K,, of 20 @4 [37], a value close to those reported for isolated liver cells, tissue slices, and perfused liver [32-341. (3) Glycerol was found to be a competitive inhibitor of NDMA demethylase [36]. This compound is present in the buffer solutions in purified P450 and NADPH:P450 reductase preparations, and is expected to increase the observed K , values in an NDMA demethylase assay system. (4) It was observed that the inclusion of cytochrome b, in a reconstituted NDMA demethylase system reduced the apparent K, value by a factor of 8 [20]. Thus, the observed higher K,, values with the reconstituted systems are due to the artificial experimental conditions used. By improving these conditions, we believe it is possible to obtain K,,, values comparable to those observed with microsomes.
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C. Activation of NDMA The thesis that NDMA demethylase is responsible for the activation of this carcinogen had been challenged because of the lack of correlation between this enzyme activity and the ability to activate NDMA to a mutagen observed in certain studies (reviewed in Ref. 38). With the insights gained from our studies on the enzyme specificity of NDMA metabolism, we were able to demonstrate the key role of NDMA demethylase in the activation of this compound in several different systems: (1) We demonstrated that P4501IE1 was more efficient than other P450 species in the activation of NDMA to a mutagen in Chinese hamster V79 cells [38]. (2) Some of the previously reported species and age differences could be interpreted based on the quantity of P450IIE1 present in the microsomes of rats and hamsters of different ages [39]. (3) Pretreatment of rats with ethanol or acetone increased NDMA-induced methylation of DNA in v i m and in vivo [ 191, and potentiated NDMA-induced hepatotoxicity [40]. The potentiating effect of these NDMA inducers on DNA methylation in vivo and hepatotoxicity, however, was observed only when high doses of NDMA (> 25 mg/kg body weight) was given to the rats [ 19,401. This result suggests that, in untreated rats, there is a sufficient amount of P450IIE1 to metabolize low doses of NDMA.
D. Metabolism of Other Nitrosamines We have reported previously that fasting or treatment with ethanol enhanced the microsomal demethylase activities with NDMA and some other nitrosamines [15]. However, this result does not mean that P450IIE1 is the enzyme responsible for the metabolic activation of all nitrosamines. Recent studies in our laboratory on the active-site mechanics indicate that the alkyl chains of nitrosamines are probably more important than the N-nitroso group in determining the affinity of binding to the active site of P450IIE1. Because of structural similarities, N-nitrosoethylmethylamine and N-nitrosodiethylamine are also preferentially metabolized by P450IIE1. It was also reported that P450IIE1 is more active than other forms in catalyzing the metabolism of N-nitrosopyrrolidine [41]. With nitrosamines with larger alkyl chains, however, the situation may be very different. For example, with N-nitrosobutylmethylamine as the substrate, the demethylation reaction was preferentially catalyzed by P4501IE1, but the debutylation reaction was more efficiently catalyzed by the phenobarbital-inducible P450IIB1 [42]. Debutylation of this compound leads to the formation of methyldiazonium, which is most likely to be the reactive species for carcinogenesis.
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/
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FlStlng Dlrbotor Acetono Kotonor lroproprnol Ethanol Benzene Ether Pyrrrole Imldrzole
N-Nltrosodlmethyl~mlne Acotono Alcohols Anlllne Enf lurane Ethers Chloroform Carbon Tetrachloride Benzene Alkanes
lronlrrld Pyrldlne
Acetamlnophen Azoxymethrnr
\
FIG. 1. Inducers (left) and substrates (right) of cytochrome P450IIE1.
E. Metabolism of Acetone and Other Chemicals An interesting observation was made by Casazza and co-workers that P450IIE1 can catalyze the oxidation of acetone to acetal and then to methylglyoxal [43, 441. This pathway can lead to the ultimate synthesis of glucose and may be an emergency gluconeogenesis pathway during fasting. P450IIE1 is also known to catalyze the metabolism of other important environmental chemicals such as diethyl ether [45], enflurane [46], acetaminophen [47], carbon tetrachloride [48], benzene [49], alcohols [50], aniline [20],p-nitrophenol [51], and other chemicals. Some of them are shown in Fig. 1.An understanding of the induction and function of P450IIE1 helps to explain, at least in part, many previously observed interactions between chemicals; for example, the potentiation of carbon tetrachloride toxicity by alcohol [52]. Many of the substrates can also serve as competitive inhibitors. This interaction may be the basis for the inhibition of nitrosamine metabolism in vivo by ethanol and diethyl ether.
IV. NATURE OF THE SUBSTRATE BINDING SITE OF CYTOCHROME P450IIE1 We have used the amino acid sequence of P450IIE1 to derive structural information concerning the conformation of the protein and the structure of the active site. The amino acid sequence alignment between P450IIE1 and other
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FIG. 2. Proposed substrate binding site of cytochrome P450IIE1.
P450s shows homologies at the substrate binding site. Based on the calculated tertiary structure of P450IIE1, the structure of P450cam as determined by X-ray crystallography, and other structural features of P450s [53, 541, a proposed arrangement of residues at the substrate binding site of P450IIE1 is shown in Fig. 2. The heme disk in P450IIE1 is buried within the protein molecule with no edge accessible to the molecular surface. The heme disk is embedded between part of the proximal (helix L, residues 439 to 441) and part of the distal (helix I, residues 295 to 303) helices running parallel to each other. The amino acid residues of part of the proximal helix L in the immediate vicinity of the heme are Gly439, Glu440, and Gly441. The amino acid sequence of the 295-303 part of the distal helix I is Asp-Leu-Phe-Phe-Ala-Gly-Thr-Glu-Thr.The residues interacting with the heme edges extend from part of the p sheet (Thr376Va1377-Phe37&Gln379-Gly38&Tyr381) and part of helix C (Arg126 Arg127). The axial thiolate ligand of heme is provided by the side chain of Cys437 and the heme is further stabilized by the propionate group interacting with Arg127 and Arg435. This interaction between propionates and two arginine residues makes the heme orient closer to helix C and twist about 30"
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clockwise, as compared to the orientation of heme in P450cam. Pyrrole ring A is open to substrate binding, but ring C is blocked by the distal helix 1. We propose that the substrate, NDMA, sits about 4 A above pyrrole ring A and is hydrogen bonded to the hydroxyl group of Tyr381. The substrate is also oriented by hydrophobic contacts with Val105 and Ile 1 14. A second hydrophobic pocket consisting of Phe298 and Ala299 may also come in contact with P450ac substrates. With N-nitrosobutylmethylamine as substrate, the N-butyl group is believed to preferentially bind to the proposed hydrophobic site, positioning the N-methyl group in the oxygenation site, the iron-oxygen complex of P450IIE1. As a consequence, demethylation is the major metabolic pathway. In the case of P450IIB1, however, such preference does not exist and debutylation also takes place at a substantial rate.
V. STRUCTURE AND REGULATION OF THE P450IIE1 GENE A. The Structure of the Gene The cDNAs for rat and human P450IIE1 were isolated and sequenced in 1986 [ 11. The coding sequence of both rat and human P4501IE1 contained 1489 base pairs. The deduced amino acid sequence for both species contained 493 amino acids with calculated molecular masses of 56,635 and 56,916 for rats and humans, respectively. Human P450IIE1 shares 75% nucleotide and 78% amino acid similarities to the orthologous rat P4501IEl. Amino acid alignment also revealed that P450IIEl is 48% similar to P450IIB1 and P450IIB2, and 54% similar to P450PB1 and P450f. Southern blot analyses of rat and human genomic DNAs verified that only a single gene shared extensive homology with P45011E1. The complete gene sequences for rat and human P450IIE1 were determined in 1988 [ 5 5 , 5 6 ] . The human gene spanned 11,413 base pairs and contained nine exons and a typical TATA box. Upstream and downstream DNAs of 2,788 and 559 base pairs were also sequenced. Significant areas of sequence similarities were observed within 140 base pairs upstream of the transcription start site in the rat and human genes.
B. Molecular Mechanisms of Regulation During the past several years, we have extensively characterized the molecular events involved in the induction of P450IIE1. The induction of NDMA demethylase activity under different conditions is accompanied by an increase in microsomal P450IIE1 level quantified by Western blot analysis, suggesting that the increase in the demethylase activity is due to the increase
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in the quantity of this enzyme rather than posttranslational modifications. The induction of P450IIE1 by acetone as well as by isopropanol, pyrazole, and 4-methylpyrazole is not accompanied by an elevation of the P450IIE1 mRNA level [ l , 57, 581. We have shown that acetone treatment may enhance the stabilization of P450IIE1 protein [59] and may also increase P450IIE1 protein synthesis (Ref. 17 and unpublished results). The role of translational control in the regulation of P450IIE1 is far from clear and requires further investigation. The induction of P450IIE1 by fasting and diabetes (chemically induced and spontaneous) is accompanied by an elevation of P450IIE1 mRNA [57,58,60]. The molecular events that lead to the elevation of P450IIE1 mRNA level are not clear. In a previous study using nuclear run-on transcriptional analysis, we failed to demonstrate an increased rate of transcription in the liver nuclei of diabetic rats, and we suggested that P450IIE1 mRNA stabilization may be a mechanism for the induction (591. We also observed previously [15] and confirmed recently that actinomycin D, an RNA synthesis inhibitor, inhibits the induction of P450IIE1 by fasting. The role of transcriptional control in the induction of this enzyme requires more in-depth examination. P450IIE1 is also regulated by development in rats: At birth, levels of P450IIE1 and its mRNA are very low or undetectable; the levels are much higher at day 4 and continue to increase, reaching a plateau at about day 30 [ l , 581. Nuclear run-on transcriptional analysis indicated that transcriptional activation is mainly responsible for the rapid increase in P450IIE1 between birth and 1 week of age [ 11. It is not known what factors, whether they are metabolites or hormones, trigger the activation of the P450IIE1 gene.
C. Regulation by Hormones and Physiological Conditions In rats, a significant sex-related difference was not observed in hepatic and renal P450IIE1 [58] suggesting that androgens and estrogens do not play a role in the regulation of this enzyme. Attempts were made to demonstrate the involvement of insulin and glucagon in the induction of P450IIE1. So far, positive results have not been obtained. In the case of diabetes, the level of P4501IE1 is affected by insulin, but this is believed to be due to the elimination of ketosis, not a direct effect [60]. In certain mouse strains, the renal NDMA demethylase activity in males is much higher than that in females [61-641. We recently demonstrated in C3H/HeJ mice that this sex-related difference is due to the different amounts of P450IIE1 present in the kidneys of the male and female mice. After treating the female mice with testosterone, the NDMA demethylase activity, P450IIE1, and P450IIE1 mRNA increased 20- to 30-fold, equal to or surpassing the male levels [27]. This system provides a unique opportunity to study the regulation of this enzyme by a hormone.
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Based on the facts that ketone bodies are produced in fasting and diabetes, and that acetone is an effective inducer, we have proposed a “ketone hypothesis” stating that acetone or other related compounds may be responsible for the induction [65]. Quantitatively, the amount of acetone produced in fasting is probably insufficient to account for the P450IIE1 induction by fasting (651. Mechanistically, we know that the elevation of P450IIE1 mRNA level in fasting and diabetes [57, 58, 601 must be caused by factors other than acetone. However, the cellular levels of acetone or other ketone bodies may play an important role in the regulation of the level of P450IIE1 under normal physiological conditions. Our recent study with dietary manipulations indicates that the rat hepatic P450IIE1 level is proportional to the “fat-to-carbohydrate ratio” in the diet and to the level of ketone bodies in the animal [66].
VI. CONCLUDING REMARKS The catalytic functions and the mechanisms of regulation of P450IIE1 have received a great deal of attention in recent years. The studies on this enzyme have enhanced our understanding of the mechanisms by which a chemical or physiological condition might affect the metabolism or toxicity of other chemicals. Because of the importance of P4501IE1 in the metabolism of many important xenobiotics, this enzyme is expected to continue to receive much attention. Many of the small organic molecules which are poor substrates for the most often studied phenobarbital-inducible P450 forms may turn out to be good or physiological substrates for P450IIEl. P450IIE1 is a constitutive enzyme that is inducible by dietary and physiological conditions as well as by many common environmental chemicals. It is a great challenge for researchers to elucidate the detailed mechanisms that modulate this enzyme. Of particular interest is whether the “constitutive” level of this enzyme is regulated by the ketone body levels as a reflection of the diet.
Acknowledgments
Supported by NIH grants ES-03938 and CA-37037. The authors thank all their collaborators who have contributed much to the work discussed herein and acknowledge the excellent secretarial work of Ms. Barbara McLouth in the preparation of this manuscript.
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REFERENCES B. J. Song, H. V. Gelboin, S. S. Park, C. S. Yang, and F. J. Gonzalez, J. Biol. Chem., 261, 16689 (1986). D. W. Nebert, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA, 6, 1 (1987). P. N. Magee and J. M. Barnes, Adv. Cancer Res., 10, 163 (1967). P. Czygan, H. Greim, A. J. Garro, F. Hutterer, F. Schaffner, H. Popper, 0. Rosenthal, and D. Y. Cooper, Cancer Res., 33, 2983 (1973). P. D. Lotlikar, W. J. Baldy, and E. N. Dwyer, Biochem. J., 152, 705 (1975). F. P. Guengerich, G. A. Dannan, S.T. Wright, M. V. Martin, and L. S. Kaminsky, Biochemistry, 21, 6019 (1982). D. Y. Lai and J. C. Arcos, Life Sci., 27, 2149 (1980). B. G. Lake, J. C. Phillips, C. E. Heading, and S. D. Gangolli, Toxicology, 5, 297 (1976). J. C. Phillips, C. Bex, B. G. Lake, R. C. Cottrell, and S. D. Gangolli, Cancer Res., 42, 3761 (1982). J. C. Phillips, B. G. Lake, S. D. Gangolli, P. Grasso, and A. G. Lloyd, J. Natl. Cancer Inst., 58, 629 (1977). M. H. Mostafa, M. Ruchirawat, and E. K. Weisburger, Biochem. Pharmacol., 30, 2007 (1981). C. S. Yang, in Banbury Report 12: Nitrosamines and Human Cancer (P. N. Magee, ed.), pp. 487-501, Cold Spring Harbor Laboratory, New York, 1982. R. Peng, Y. Y. Tu, and C. S. Yang, Carcinogenesis, 3, 1457 (1982). C. S. Yang, Y. Y. Tu, R. X. Peng, N. A. Lorr, in Cytochrome P-450: Biochemistry, Biophysics and Environmental Implications (E. Hietanen, M. Laitinen, and 0. Hanninen, eds.), pp. 35-38, Elsevier Biomedical Press, New York, 1982. Y. Y. Tu and C. S. Yang, Cancer Res., 43, 623 (1983). Y. Y. Tu, J. Sonnenberg, K. F. Lewis, and C. S. Yang, Biochem. Biophys. Res. Commun., 103, 905 (1981). Y. Y. Tu, R. X.Peng, Z. F. Chang, and C. S. Yang, Chem.-Biol. Interact., 44, 247 (1983). R. Peng, P. Tennant, N. A. b r r , and C. S. Yang, Carcinogenesis, 4,703 (1983). J. Hong and C. S. Yang, Carcinogenesis, 6, 1805 (1985). C. Patten, S. M. Ning, A. Y. H. Lu, and C. S. Yang, Arch. Biochem. Biophys., 251, 629 (1986).
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