Pharmacogenetics of the Arylamine N-acetyltransferases: a Symposium in Honor of Wendell

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics presented at the joint meeting of the American Society for Biochemistry and Molecular Biology and the American Society for Pharmacology and Experimental Therapeutics, June 4–8, Boston, Massachusetts. The presentations focused on the pharmacogenetics of the NAT1 and NAT2 arylamine N-acetyltransferases, including developmental regulation, structure-function relationships, and their possible role in susceptibility to breast, colon, and pancreatic cancers. The symposium honored Wendell W. Weber for over 35 years of leadership and scientific advancement in pharmacogenetics and was highlighted by his overview of the historical development of the field. N-Acetyltransferase Pharmacogenetics: From the Beginning (Wendell W. Weber) So much has happened lately in biology and in pharmacogenetics that it is easy to lose sight of the earlier accomplishments that brought us to the present. The acetylator trait was one of the first human hereditary traits affecting human drug response to be identified, and the observations that led to its discovery contain some of the elements of a good medical detective story. Discovery of this trait followed directly on the heels of the introduction of isoniazid as an antituberculosis drug to clinical practice nearly five decades ago. At first, the identity of the drug was shrouded in secrecy, but in February 1952, hospitalized patients, hopelessly ill with tuberculosis, began to experience dramatic improvements from the drug. News of these events were recounted almost daily in the press (Kaempffert, 1952). Within a week of starting isoniazid, treated patients regained weight, strength, and appetite, and their fever disappeared. After a few weeks of treatment, tubercle bacilli could no longer be found in their saliva and within 8 months, favorable X-ray changes were seen in more than half the patients. But despite its remarkable therapeutic effectiveness, a high proportion (3.5–17%) of patients convalescing on the drug, complained of numbness and tingling in the fingers and toes, as well as other signs of progressive damage to the nervous system. As the occurrence of these devastating side effects threatened to stop the use of isoniazid, efforts to find the cause intensified, leading to discovery of this human metabolic trait (reviewed in Weber, 1987; Vatsis and Weber, 1997). Hughes and colleagues (1954) solved the puzzle by deducing from animal studies that certain other drugs, which resembled isoniazid chemically, such as sulfonamides, underwent acetylation before the body could excrete them. They found that acetyl-isoniazid was the main urinary metabolite of humans, and that persons typically fell into either low or high excretor groups, depending on their capacity to acetylate the drug. Following this clue, they showed that patients who excreted the largest amount of unchanged isoniazid and the least amount of acetyl-isoniazid were most likely to suffer neurological damage from the drug. Further studies of twins and families revealed blood concentrations of the drug distributed into two (or three) genetically determined subgroups (Price Evans et al., 1960). This led to the proposal that persons with low blood levels be classified as “rapid” inactivators and those with high blood levels as “slow” inactivators of isoniazid. After biochemical studies, the genetic variability (polymorphism) in drug levels was attributed to differences in N-acetyltransferase (NAT) activity, and the term “inactivators” was supplanted by “acetylators” (reviewed in Vatsis and Weber, 1994). Discovery of acetylation polymorphism was followed by many genetic studies of the mode of inheritance of NAT activity and the frequency of slow acetylation in different ethno-geographic populations, which now has involved more than 10,000 people. In the present This work was supported in part by Grants CA-34627 (D.W.H.), ES10047, ES09812, and Arizona Disease Control Research Commission (C.A.M). 1 Abbreviations used are: NAT, N-acetyltransferase; NAT1, N-acetyltransferase 1; NAT2, N-acetyltransferase 2; 4-ABP, 4-aminobiphenyl; GD, gestational day; ND, neonatal day; PABA, p-aminobenzoic acid; PABA-Glu, p-aminobenzoyl glutamate; PAS, p-aminosalicylic acid; SMZ, sulfamethazine; AFMU/1X, 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine; OR, odds ratio; CI, confidence interval. Send reprint requests to: David W. Hein, Department of Pharmacology and Toxicology, University of Louisville Helath Science Center, Louisville, KY 40292. E-mail: [email protected] 2 N-Acetyltransferase proteins and genes are printed in regular type (NAT) and italics (NAT), respectively. 0090-9556/00/2812-1425–1432$03.00/0 DRUG METABOLISM AND DISPOSITION Vol. 28, No. 12 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 180/868130 DMD 28:1425–1432, 2000 Printed in U.S.A. 1425 at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from article, I want to show how knowledge of the acetylator trait advanced from the pioneering steps taken by a few investigators in the 1950s to a much better understanding of the trait through works of many others using modern strategies of human genetics and molecular epidemiology. In 1962, I read a description of the acetylator trait in Werner Kalow’s (1962) new book on pharmacogenetics while I was an NIH fellow in human genetics at the Galton Laboratory, University College, London. Little was known of the cause of this trait at that time, and when the opportunity arose a year or so later at New York University Medical School in Pharmacology to try to find the cause of person-to-person differences in acetylation, I jumped at it. The time was right for such studies. The identification of the DNA double helix, the visualization and enumeration of human chromosomes, the recognition of protein polymorphism as an important biological phenomenon, and the discovery of heritable patterns of drug response that had occurred in the 1950s encouraged pharmacologists to take a more genetic approach to their research. Then too, the genetic basis of two other pharmacogenetic traits—“primaquine sensitivity” and “succinylcholine sensitivity”—had been examined. Primaquine sensitivity had been shown to be a sex-linked trait due to G6PD deficiency while succinylcholine sensitivity was found to be an autosomal recessive trait due to an atypical form of serum cholinesterase. However, these traits were both expressed and could be studied in the peripheral blood. The isoniazid acetylation polymorphism, on the other hand, is expressed mainly in the liver and gut, and the inaccessibility of these tissues necessitated an appropriate animal model before we could begin a comprehensive study of the human condition. Fortunately, Frymoyer and Jacox (1963a,b) reported that the variable capacity of rabbits to acetylate certain sulfonamides was genetically determined, and that “slow acetylation” was inherited in a manner like that for humans. Thus, using the rabbit model, we set out to answer the question: what causes slow acetylation? First, to be on solid enzymological grounds, we studied the enzymatic mechanism of drug acetylation. We found that this reaction proceeds according to a “ping-pong Bi-Bi” mechanism (Weber and Cohen, 1967, 1968). In this mechanism the enzyme swings back and forth between free and acetylated forms as it catalyzes the acetylation of drugs. In the course of this investigation, we also found we needed a stable source of rapid and slow acetylator rabbits and thus developed a colony of rapid and slow acetylator rabbits, which we kept for more than 15 years for use in our studies. During the 1970s, we continued to characterize the rabbit acetylator trait and initiated studies on human acetylator polymorphism. We also began to explore other animal models for hereditary acetylation polymorphisms, including inbred strains of mice, hamsters, and rats (reviewed in Weber and Hein, 1985; Weber, 1987). During the next two decades, we gathered a great deal of fascinating information about the metabolic, genetic, toxicological, developmental, and molecular genetic aspects of these traits. In mice for example, using C57BL/6J (B6) mice as representative of rapid acetylation and A/J (A) mice as representing slow acetylation, we showed that the NAT polymorphism in liver also occurs in kidney, urinary bladder, blood, and other tissues. We also developed two congenic acetylator mouse lines derived from B6 and A mice, one on the B6 genetic background and the other on the A background (Mattano et al., 1988) A little later, the hamster was also used to develop congenic hamster lines (Hein, 1991). These congenic lines have been powerful aids to clarify the role of acetylation polymorphism in toxicology studies, particularly those involving carcinogenesis, and the mouse and hamster models are both in use today (reviewed in Levy et al., 1992; Hein et al., 1997). As we moved through the 1980s, more than a hundred human traits of pharmacogenetic interest were discovered and characterized, but the field was shaped mainly by the study of drug-metabolizing enzyme polymorphisms like the acetylation polymorphism. The main purpose of those studies was to determine whether susceptibility of people to drugs, foods, and other exogenous chemicals, was altered by a particular polymorphism, one gene at a time. Associations between acetylator phenotype and various drug-induced disorders that were sought, revealed new insights into the causes of isoniazid hepatitis, drug-induced lupus erythematosus, sulfasalazine side effects, and toxicity from sulfonamides (reviewed in Weber, 1987). Important associations were also observed between acetylator phenotypes and occupationally induced bladder cancer, and colon cancer induced by smoking and food mutagens that occur in cooked meats. The evidence that has accumulated suggests that NAT activity in combination with other genetically determined traits is a significant risk factor for certain cancerous disorders, but this is a complex problem still under active investigation (reviewed in Weber and Hein, 1985; Hein, 1988; Kadlubar et al., 1992; Vatsis and Weber, 1997; Hein et al., 2000a; Levy and Weber, 2000). The emergence of recombinant DNA technology during the 1980s brought the genetic analysis of protein polymorphisms within reach of many investigators, and the pace of pharmacogenetic research at all levels has increased enormously within the last 10 years. In 1987, after we reported the sequences of hepatic NAT peptides of liver NAT from homozygous rapid acetylator rabbits (Andres et al., 1987), our understanding of the molecular basis of acetylation also advanced very rapidly. In quick succession, two functional human loci, NAT1 and NAT2 were identified and characterized for humans (Blum et al., 1990), and mapped to the short arm of human chromosome 8 (reviewed in Vatsis and Weber, 1994; Grant et al., 1997; Vatsis and Weber, 1997). The human isoniazid N-acetylation polymorphism was then attributed to variation at the NAT2 locus. A systematic survey of NAT1 genotypes in Caucasians showed NAT1 to be a polymorphic locus, but the role of NAT1 in susceptibility to unwanted effects of exogenous chemicals remains to be established (Vatsis and Weber, 1994). Currently, 25 human NAT1 and 27 human NAT2 alleles have been identified (Vatsis et al., 1995; Hein et al., 2000 a,b). Most molecular genetic studies of acetylation polymorphism in humans and other species have concentrated on defects within the coding region so there are large gaps regarding effects of development, nutritional state, and hormonal factors on NAT expression. Recently, some progress has been made on these topics (Estrada-Rodgers et al., 1998b; Mitchell et al., 1999; Estrada et al., 2000) but limitation of space precludes their consideration in this article. During the last 40 years, we have witnessed the transformation of pharmacogenetics from a cottage industry that involved a handful of academic investigators in the 1950s to a worldwide phenomenon that has attracted the attention of clinical scientists and the pharmaceutical industry. Improved patient care through customized therapy and discovery of new drugs are now within reach of these efforts. I often think, nowadays, how the discovery of human biotransformation of exogenous chemicals by pioneering physiological chemists, of the laws of heredity by Mendel, and of the theories of the existence of drug receptors postulated by Langley and Ehrlich, created the starting point some 100 to 150 years ago for understanding the peculiarities of human drug response (Weber, 1997). From those discoveries, Archibald Garrod predicted the role of the genetic material in the chemical individuality of humans, and suggested that substances in foods, drugs, and exhalations of animals or plants produce effects in some people wholly out of proportion to any that they bring about in average individuals—effects that might vary from slight or temporary 1426 HEIN ET AL. at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from discomfort to morbid syndromes, which amount to severe and fatal illnesses (Garrod, 1931; Scriver and Childs, 1989). As one of the first human hereditary traits affecting drug response to be discovered, the human acetylation polymorphism occupies a position of singular importance in the history of pharmacogenetics and in the future impact of the field on the practice of medicine. There is, I believe, no better example to teach us how a broad spectrum of individual responses to exogenous chemicals, including drugs, can arise from a single, genetically determined, metabolic theme, and to demonstrate how a better understanding of such traits can guide us in devising strategies to prevent human illness of environmental origin. Developmental Regulation of the Arylamine N-Acetyltransferases (Charlene A. McQueen) Prenatal exposure to xenobiotics is modulated by maternal absorption, distribution, biotransformation, and excretion. The genotypes of maternal biotransformation enzymes and environmental factors will affect the chemical nature and concentration of xenobiotics reaching the placenta where additional enzymatic reactions may occur. Biotransformation enzymes expressed by the fetus can result in further activation or detoxification of xenobiotics. The capacity to acetylate aromatic amines has been associated with the likelihood of toxicity in adults. Although it is reasonable to assume that a similar relationship exists at earlier stages of life, the contribution of fetal N-acetyltransferases to the developmental toxicity of aromatic amines is less clear. During the prenatal and neonatal periods, both cigarette smoke and breast milk may serve as sources of aromatic amines. For example, babies born to mothers who smoke have higher levels of 4-aminobiphenyl (4-ABP)-hemoglobin adducts than offspring of nonsmoking mothers (Coghlin et al., 1991; PinoriniGodly and Myers, 1996). Exposure of neonatal mice to 4-ABP one day after birth was sufficient to induce liver carcinomas at 12 months (Dooley et al., 1992), and in utero exposure of Balb/c mice to 4-ABP at gestational day (GD) 18 resulted in the formation of fetal 4-ABP DNA adducts (Lu et al., 1986). Further investigation of the formation of 4-ABP-DNA adducts was performed in C57BL/6 mice utilizing an adduct-specific antibody (Al-Atrash et al., 1995). 4-ABP-DNA adducts were present in maternal and fetal tissue from C57BL/6 mice at GD 15 and 18, 24 h after a single oral dose of 120 mg of 4-ABP/kg (McQueen et al., 2000). No interor intralitter variation was noted at either GD. Comparison of the relative fluorescent intensities of the antibody used to detect the 4-ABP-DNA adducts revealed no differences between GD 15 and 18 in either maternal or fetal tissue. Significantly higher (P . .05) average fluorescence was seen in maternal liver compared with fetal sections. For hemoglobin or DNA adducts to be formed, 4-ABP must undergo biotransformation to genotoxic products. Detection of 4-ABPhemoglobin and DNA adducts in fetal tissue indicates that reactive products are formed but not whether this biotransformation is maternal, placental, or fetal in origin. In adult liver, N-acetyltransferases are involved in the biotransformation of aromatic amines. N-Hydroxylation followed by NAT-catalyzed O-acetylation is considered a major route of activation of 4-ABP while N-acetylation of the parent amine is thought to be a detoxification step. Investigation of the expression of NAT1 and NAT2 in C57BL/6 mice showed that these genes were expressed before birth (Mitchell et al., 1999). Reverse transcriptase-polymerase chain reaction was used to detect NAT1 and NAT2 mRNAs at GD 10, 15 and 18. At GD 10, the middle of the second trimester, both genes were expressed in the conceptal/placental complex. At GD 15, the middle of the third trimester, and at GD 18, just before birth, placental expression of both genes was confirmed. The GD 15 fetus and GD 18 extrahepatic fetal tissue had measurable NAT1 and NAT2 mRNAs. However, only NAT2 was expressed in liver at GD 18 (Table 1). The lack of expression of NAT1 continued until neonatal day (ND) 3, the latest time analyzed. Recently, it has been shown that NAT2 mRNA was present in embryonic stem cells (Payton et al., 1999). The NAT2 protein has also been detected in CD1 mice by immunochemical analyses at GD 9.5, 11.5, and 13.5 (Stanley et al., 1998). These studies demonstrate that NAT genes are transcribed and translated in preimplantation embryonic stem cells as well as during the second and third trimesters of pregnancy. Fetal and placental N-acetyltransferase activity has been evaluated using selective and nonselective substrates. Acetylation of p-aminobenzoic acid (PABA) and sulfamethazine have been detected in human placenta while PABA NAT activity was found in fetal hepatic and extrahepatic tissue (Pacifici et al., 1986; Derewlany et al., 1994; Smelt et al., 1998). Since 4-ABP-DNA adducts were present in fetal tissue, there was particular interest in determining 4-ABP NAT activity. Fetal tissue from C57BL/6 mice at GD 10, 15, and 18 had detectable 4-ABP NAT activity (McQueen et al., 2000). This activity increased from GD 10 to GD 18 then remained constant through ND 4. Hepatic 4-ABP NAT activity was lower at ND 4 than in adult tissue. Increasing PABA NAT activity has been observed in CD1 mice in the first 25 days after birth (Estrada et al., 2000). These studies clearly show that functional NATs are present before birth, suggesting that biotransformation of aromatic amines by the mother and the fetus can contribute to the potential fetal toxicity of aromatic amines. Additionally, the early and continued expression of murine NAT2 during gestation suggests the possibility of acetylation of endogenous substrates is required during embryonic development. The folate breakdown product, PABA glutamate (PABA-Glu) is a specific substrate for human NAT1 and murine NAT2 (Ward et al., 1995; Estrada-Rodgers et al., 1998a). Folic acid is required for normal neural tube development, and it has been proposed that acetylation of PABA-Glu may be involved in regulation of folate (Stanley et al., 1998; Payton et al., 1999). The expression of murine NAT2 in preimplantation embryonic stem cells and developing neural tissue would allow this to occur during gestation (Stanley et al., 1998; Payton et al., 1999). Thus, prenatal NATs have the potential to influence fetal aromatic amine toxicity and to play an essential role in normal embryonic development. Structure/Function Analyses of the Human Arylamine N-Acetyltransferases (Geoffrey H. Goodfellow and Denis M. Grant) The overall goal of the studies described in this section is to determine the structural features of human NAT1 and NAT2 that impart their distinct catalytic specificities for acceptor amine substrates. The human arylamine N-acetyltransferases (EC 2.3.1.5) catalyze a two-step substituted-enzyme (“ping-pong”) kinetic mechanism TABLE 1 Prenatal expression of murine NAT1 Data from Mitchell et al., 1999. Tissue Gestational Day