Introduction

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

      I am very honored at being chosen as the recipient of the 1996 Bernard B. Brodie Award in Drug Metabolism and to join the ranks of the previous recipients of the award (Gillette, 1979; Coon, 1981; Jerina, 1983; Mannering, 1986; Nebert, 1986; Levin, 1990; Zeigler, 1991; Guengerich, 1993; Ortiz de Montellano, 1995). I wish to thank the American Society for Pharmacology and Experimental Therapeutics and Dow Elanco for their recognition of the research done in my laboratory. I am also very grateful to my colleagues and collaborators for their participation and contributions to the research conducted in my laboratory. It has been a privilege to work with so many dedicated and talented scientists over the years in exploring the scientific new frontiers and meeting the many challenges we faced. I would particularly like to express my sincere gratitude to my mentors, Professor Minor J. Coon at the University of Michigan and Professor Allan Conney at Rutgers University, for the opportunity to work on some very fascinating and challenging research projects and for setting high scientific and moral standards to follow. Both Jud and Allan made monumental contributions to our field; they are my role models.

	A Vibrant Environment

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

My entry into the area of cytochrome P450 and drug metabolism research was totally accidental. It all started when I joined Jud Coon's laboratory as a postdoctoral fellow after I completed my graduate study in biochemistry at the University of North Carolina in Chapel Hill. At that time, the major research effort in Jud's laboratory was the investigation of the fatty acid and hydrocarbon -hydroxylase system in Pseudomonas oleovorans by Julian (Bill) Peterson, Deb Basu, and Eva McKenna. Larry Cottam and Paul Hollenberg worked on the pyruvate kinase project. The liver microsomal -hydroxylase system was a new, small-effort project. Later, Henry Strobel and Ann Autor joined me in pursuing this work. Since 1971, the microsomal cytochrome P450 system has been the major research focus in Jud's laboratory. I am pleased to note that Bill Peterson, Paul Hollenberg, Henry Strobel, and many of Jud's former graduate students and postdoctoral fellows are all now making major contributions to the cytochrome P450 and drug-metabolism field; this is quite a compliment to Jud's mentorship.

It was a very exciting time when I joined Jud's laboratory. Peterson had made tremendous progress in resolving the bacterial -hydroxylase system into three components: a flavoprotein, a nonheme iron protein rubredoxin, and the -hydroxylase (Peterson et al., 1966). Interestingly, cytochrome P450 is not a component of this multicomponent enzyme system. Because of this progress, Jud suggested that I initiate an investigation into the liver microsomal fatty acid -hydroxylase system, perhaps using Peterson's approach. So we set up our goal to solubilize, identify, and purify the components necessary for fatty acid -hydroxylation. Little did we know that it would eventually take more than 10 years by many dedicated scientists to accomplish this task. Since the bacterial system contained multiple components, we assumed that the microsomal system would also contain several components. Therefore, we decided that we would always combine fractions for assay during purification. This was an important decision since at that time, many investigators in the field appeared to be concentrating more on the solubilization and purification of cytochrome P450 alone. Our approach would ensure that we would not miss any component essential for -hydroxylation. At the beginning of this investigation neither Jud nor I had any experience in dealing with the membrane-bound enzyme system. In addition, we had no knowledge about cytochrome P450 and drug metabolism. I also had no publications up to that point. However, this lack of knowledge and experience did not dampen my enthusiasm in our research efforts since exciting and new findings were generated by Peterson and others from the bacterial hydroxylase system almost on a daily basis, and we anticipated that similar excitement could be generated from the microsomal hydroxylase work.

	Searching for Functional Components

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

My first attempt to solubilize the mammalian -hydroxylase system was to extract the rabbit liver microsomal suspension by diluted Tris buffer containing EDTA, a procedure used successfully by Peterson to release rubredoxin from the bacterial cells (Peterson et al., 1966). After centrifugation, all -hydroxylase activity was recovered in the microsomal fraction, and no functional components were solubilized from the microsomal membrane by this method. This and other initial attempts proved to be unsuccessful, clearly indicating to us that the bacterial and mammalian -hydroxylases are two very different enzyme systems.

For almost a year, I carried out daily experiments, systematically examining various factors (pH, ionic strength, buffers, detergents, sonication, etc.) that affect solubilization of the fatty acid -hydroxylase activity from rabbit liver microsomes. A variety of agents (e.g. EDTA, dithiothreiotal, citrate, sucrose, and glycerol) were found to increase the recovery of the -hydroxylase activity in the supernatant fraction, although the mechanism by which these agents enhanced activity recovery was totally unclear to me at that time. By including many of these components in the solubilization mixture, recovery of activity in the soluble fraction increased from 5% in the beginning to approximately 50%, probably an underestimation due to the inhibitory effect of deoxycholate in the -hydroxylase assay. We know today that some of the components used in the initial solubilization method are not needed for cytochrome P450 solubilization, but they were included at that time to increase the activity recovery after solubilization, even by just a few per cent for some of the components. I repeated the solubilization experiment several times before I showed the data to Jud. I was hoping that the solubilization results were good enough for publication, but Jud thought that it would be a much better paper if we could include resolution and reconstitution results. This was a good lesson to me: don't just publish any paper if you can publish a good paper.

Resolution of the solubilized preparations proved to be as difficult as the initial step. At first I thought that difficulties in fractionation originated from the presence of detergent. But I soon found out that all enzyme activities would end up in the particulate fraction by centrifugation when detergent was removed from the preparations. A series of experiments was conducted to determine the minimum concentration of detergent needed to keep enzyme activity in solution. Detergent was then incorporated into every fractionation step, followed by reconstitution of the -hydroxylase activity. This strategy paid off handsomely since, at that time, incorporating detergent in cytochrome P450 fractionation was not a common practice. Near the end of 1967, the solubilized -hydroxylase system was resolved by column chromatography into three fractions. Although there was considerable cross-contamination in each fraction, maximum -hydroxylase activity could be reconstituted by combining these three fractions. The brown color fraction contained cytochrome P450, assayed for the first time in our laboratory by a CO-difference spectrum. The yellow color fraction contained NADPH-cytochrome P450 reductase, assayed by the NADPH-dependent cytochrome c reduction. We contacted the late Henry Kamin at Duke University for help, and Bettie Sue Masters, then a member of Henry's laboratory, promptly and generously supplied us with a sample of purified NADPH-cytochrome c reductase. Surprisingly, this reductase, which was purified from protease-solubilized microsomes, could not substitute for our reductase fraction in supporting -hydroxylation, providing the first indication that detergent-solubilized and protease-solubilized reductase exhibited different catalytic functions. The third fraction required for -hydroxylation was pink in color. Initially we thought that this fraction contained a nonheme iron protein like rubredoxin. However, this hope faded quickly as I found that the active component in this fraction was stable at 100°C, unstable by ashing at high temperature (which ruled out the involvement of metals), and extractable by organic solvent. All of these results pointed to the involvement of lipid in -hydroxylation. A manuscript describing the initial work on solubilization, resolution, and reconstitution was promptly accepted for publication. It was my first paper (Lu and Coon, 1968) and, fortunately, also a citation classic (Lu, 1992). This reconstituted system was further characterized (Lu et al., 1969a, 1969b; Lu et al., 1970), and the heat-stable factor was later identified by my good friend Henry Strobel as phosphatidylcholine (Strobel et al., 1970). With this reconstituted system, we could, for the first time, characterize the catalytic function of cytochrome P450 and thus paved the way for eventual cytochrome P450 purification and the demonstration of multiple P450s.

	From -Hydroxylation to Drug Metabolism

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

Although the first successful solubilization, resolution, and reconstitution of the liver microsomal cytochrome P450 system generated much excitement in our own and other laboratories, there was also skepticism about our studies (e.g. low activity of the reconstituted system, low specific content of the solubilized cytochrome P450, enzyme components not pure, number of components required for activity unknown). One often-asked question was whether the reconstituted -hydroxylase system could also metabolize drugs.

Among the most popular substrates in the field of cytochrome P450 and drug metabolism at that time were ethylmorphine and aniline, the so-called type I and type II substrates. Because of my limited knowledge about cytochrome P450, I tried benzphetamine instead as my first drug substrate for the reconstituted system. I learned about benzphetamine at one of the Federation Meetings in Atlantic City, and this turned out to be a very lucky choice. Benzphetamine proved to be one of the best drug substrates (Lu et al., 1969b), whereas the metabolism of ethylmorphine and aniline by the reconstituted system was only marginal. We know today that these three drugs are metabolized by different cytochrome P450s and that the enzyme preparations available at that time may not contain all cytochrome P450 isoforms.

As I read more papers, I was fascinated by the literature and elegant contributions by many of the pioneers of the cytochrome P450 and drug-metabolism field. This was the field full of action and excitement involving biochemistry (heme and flavoproteins, enzyme mechanism involving multiple components, lipid-protein interactions, electron transport), pharmacology (in vivo and in vitro drug metabolism, enzyme induction and inhibition, drug action) and toxicology (metabolism-mediated toxicity; mechanism of cytotoxicity, mutagenicity, and carcinogenicity). There was no doubt in my mind that I should devote my research career to this wonderful scientific field.

	From One Cytochrome P450 to Many Cytochrome P450s

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

After my postdoctoral training, I had the good fortune to join the laboratory of Allan Conney, then at Hoffmann-LaRoche. At that time, there was an intense debate among investigators as to whether there is one or more than one cytochrome P450. This question is central to our understanding of cytochrome P450 and its role in drug metabolism, cytotoxicity, mutagenicity, and carcinogenicity. Therefore, the challenge to the investigators at that time was whether one could separate, purify, and characterize individual cytochrome P450s if multiple isoforms exist.

Previous studies from the laboratories of Conney, Mannering, and others (Gillette et al., 1969) had demonstrated that liver microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated rats have different substrate specificities in the metabolism of drugs and steroids. We thought that as a first step to answer the question about cytochrome P450 multiplicity, the reconstituted system could be used to examine the substrate specificity of cytochrome P450s isolated from different sources, even though the cytochrome P450 preparations were not pure. Susan West, who joined my laboratory shortly after my arrival at Hoffmann-LaRoche, participated in these studies from the very beginning. Susan was resourceful, hard-working, and dependable. She made many important contributions in this and other research projects. We isolated the three components from rats treated with either phenobarbital or 3-methylcholanthrene and assayed benzphetamine N-demethylation and benzo(a)pyrene hydroxylation in the reconstituted system. Benzphetamine and benzo(a)pyrene were selected as substrates since the metabolism of these two compounds was preferentially induced in rats by phenobarbital and 3-methylcholanthrene treatment, respectively. As expected, cytochrome P450 from phenobarbital-treated rats metabolized benzphetamine rapidly and benzo(a)pyrene poorly, whereas P450 from 3-methylcholanthrene-treated rats metabolized benzo(a)pyrene rapidly but benzphetamine poorly (Lu et al., 1971; Lu et al., 1972a). Subsequent studies (Lu et al., 1973) indicated that substrate specificities of cytochrome P450 from untreated rats differed from the cytochrome P450 from induced animals. These results demonstrated that substrate specificity of liver microsomes from rats treated with different inducers is determined primarily by the cytochrome P450 component, rather than by the reductase or the lipid.

Encouraged by these results, which pointed to the presence of multiple cytochrome P450s, we initiated the purification of cytochrome P450 in rats. In a very productive collaboration with Wayne Levine, Dene Ryan, and Paul Thomas, a number of cytochrome P450 forms were purified and characterized (Ryan et al., 1975; Thomas et al., 1976). Joseph Kawalek and M. T. Huang also purified several cytochrome P450s from rabbits (Kawalek et al., 1975) and mice (Huang et al., 1976) for metabolism, toxicity, and mechanistic studies. These studies, along with those from the laboratory of Coon, Guengerich, Johnson, Philpot, and Sato, paved the way for the discovery of many more cytochrome P450s.

	The Lipid Dream

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

Since detergent was used for solubilization and resolution, the question was raised as to whether the requirement of lipid for catalytic activity in the reconstituted system is due to its reversal of detergent inhibition on cytochrome P450 function. To address this question, Mary Vore extracted lyophilized liver microsomes with organic solvents (acetone and butanol) to remove the lipids. Under strictly anhydrous conditions, virtually all neutral lipids and greater than 80% of the phospholipids were removed, while recovery of cytochrome P450 and reductase was better than 80% (Vore et al., 1974a, 1974b). The lipid-depleted microsomes had greatly reduced activities for metabolism, but activity could be restored by the addition of lipids. These results indicate that the requirement of lipid for metabolism activity is unrelated to the presence of detergent in the reconstituted system. Subsequent studies showed that the lipid component can be replaced by certain detergents (Lu et al., 1974) and that lipid may function by enhancing the interaction between NADPH-cytochrome P450 reductase and cytochrome P450 (Miwa and Lu, 1981).

I was hoping that with the lipid-depleted microsomes, I could extract cytochrome P450 from the microsomes without the use of any detergent. This dream was never fulfilled. Despite extensive effort, cytochrome P450 could not be released from the membrane unless a detergent was used.

	A New Inducer

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

In 1971, Arpad Somogyi, a postdoctoral fellow with Allan Conney, demonstrated that 7,12-dimethylbenz(a)anthracene-induced adrenal necrosis in female rats could be inhibited by steroids capable of inducing aryl hydrocarbon hydroxylase (Somogyi et al., 1971). Pregnenolone-16-carbonitrile (PCN)³ was among the most potent agents being tested. Arpad suggested that together we characterize the induction of cytochrome P450 in rat by PCN, a hormonally inactive steroid. We found that the specificity of the inductive effect of PCN on the oxidative metabolism of benzphetamine, ethylmorphine, and benzo(a)pyrene differs from the specificity of either phenobarbital or 3-methylcholanthrene (Lu et al., 1972b). These results suggest that PCN is a new type of cytochrome P450 inducer. Susan West subsequently purified the cytochrome P450 from PCN-treated rats. Although electrophoretically pure, this cytochrome P450 preparation exhibited little or no catalytic activity toward a number of substrates when reconstituted with the reductase and lipid. Various attempts were made to enhance activities of the reconstituted system, but all efforts failed. As a result, we made the unwise decision to drop this project. Today we know that PCN-induced cytochrome P450 belongs to the CYP3A subfamily, and reconstitution of this subfamily of cytochrome P450 has always been a problem. It also turns out that this is one of the most important human cytochrome P450s in the metabolism of many important therapeutic agents.

	Deuterium Isotope Effect and Metabolic Switching

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

In 1975, Gerald Miwa joined my laboratory as a postdoctoral fellow after completing his graduate study with Arthur Cho at the University of California, Los Angeles. Because of Gerald's interest and strong background in mechanistic organic chemistry, we initiated a series of studies to investigate the mechanism of cytochrome P450-catalyzed reactions, particularly the use of a deuterium isotope as a probe. My long-term collaboration with Gerald has been scientifically and personally very rewarding.

One of the most interesting studies carried out in this series was the oxidative O-deethylation of 7-ethoxycoumarin by rat cytochrome P450s 1A1 and 2B1 (Miwa et al., 1984a; Harada et al., 1984). Substitution of the hydrogens on the -carbon of the ethoxy side chain of 7-ethoxycoumarin resulted in an observed deuterium isotope effect of 2 for P450 1A1 and 4 for 2B1. The intrinsic isotope effects for the O-deethylation of 7-ethoxycoumarin were 13-14 for both enzymes, indicating the masking of the expression of the intrinsic isotope effect by other rate factors. Careful analysis of the stoichiometric data by Nobuhiro Harada and Gerald Miwa indicated that deuterated 7-ethoxycoumarin has no effect on rat cytochrome P450 1A1-dependent NADPH oxidation, oxygen and 7-ethoxycoumarin consumption, and H₂O₂ formation. Thus, despite a very large intrinsic isotope effect and a significant observed isotope effect on the O-deethylation of 7-ethoxycoumarin, the oxidase and the overall monooxygenase activities of rat 1A1 are not altered. Furthermore, the decreased rate in O-deethylation was associated with a pronounced increase in 6-hydroxylation on the aromatic ring. These results suggest that 7-ethoxycoumarin can bind to the active site of rat 1A1 in at least two different orientations and that the active oxygen species, once formed, is committed to catalysis. Therefore, cytochrome P450 is capable of switching to an alternate metabolic site of the drug substrate when the preferred site of metabolism is slowed down or blocked by deuterium or other substituents.

	Bound Residues in Food-Producing Animals: A Regulatory Challenge

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

In 1978, I joined Merck Research Laboratories and established the Biochemical Toxicology Group in the Department of Animal and Exploratory Drug Metabolism. Turning my attention from basic cytochrome P450 research to drug development, I became interested in probing the possibility of using basic metabolism knowledge to develop safer and more efficacious drugs. The major objective of the group at that time was to evaluate the toxicological significance of covalently bound drug residues, a critical issue in the development of veterinary medicine in the 1970s.

Bound residues in food-producing animals are generated primarily from the metabolic activation of drugs to electrophilic metabolites that covalently bind to tissue macromolecules. The safety assessment of bound residues derived from toxic veterinary drugs presents a unique and difficult challenge to scientists involved both in product development and regulatory decision-making. The chemical nature of bound residues is generally unknown because of their extremely low levels in the tissues of food-producing animals as well as the complex nature of the in vivo metabolic activation leading to covalent binding of drug residues to numerous macromolecules. Furthermore, because drug residues are immobilized on macromolecules, most of the in vivo and in vitro methods used in toxicity evaluations are not suitable for covalently bound residues. Because of the paucity of scientific information, very little progress was made in regulatory policies for many years.

	Analytical vs. Mechanistic Approach

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

Ronidazole (fig. 1), a substituted 5-nitroimidazole used for the treatment of swine dysentery, was selected as the model compound for our study. In swine, ronidazole is extensively metabolized. After several days, a great portion of the total radioactivity in liver and muscle is protein-bound, and no parent compound can be found. Since ronidazole is mutagenic in Ames' test and carcinogenic in rodents, meat containing bound residues cannot be approved by the Food and Drug Administration (FDA) for human consumption unless the safety of these bound residues can be established. Two approaches can be used to study tissue-bound residues. In the analytical approach, one attempts to isolate all tissue-bound residues and determine the structure and toxicity of these residues. This is the classic approach taken by many other laboratories but proved to be too difficult technically to answer the regulatory questions. Alternatively, one can use the mechanistic approach to define the metabolic pathway and chemical and biochemical mechanisms leading to covalent binding, along with the use of structure-activity relationship and model compounds to define the toxicity potential of bound residues. After extensive discussions with Gerald Miwa, Susan West, and Peter Wislocki, we decided to take the mechanistic approach, starting with an in vitro covalent-binding study in rat liver microsomes, followed by in vivo bound-residue studies in rats and swine (Lu et al., 1988; Lu et al., 1990; Wislocki and Lu, 1990). In all of these studies, the support of a group of talented radiosynthesis chemists (Robert Ellsworth, Greg Gatto, and Avery Rosegay) was invaluable, and the contributions by Gerald Miwa and Peter Wislocki were outstanding.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Structure of ronidazole.

	Covalent Binding: In Vitro vs. In Vivo

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

In a series of studies in rat-liver microsomes (table 1), we established that covalent binding of ronidazole involves nitro reduction and binding of reactive species to cysteine residues on proteins through the 2-methylene position (West et al., 1982a, 1982b; Miwa et al., 1982; Wislocki et al., 1984a; Miwa et al., 1984b; Alvaro et al., 1992). A critical step in this mechanistic approach was to demonstrate that ronidazole covalent adducts (i.e., bound residues) generated from in vitro studies were identical to those obtained in vivo in rats and in swine, the target animals (table 2). Extensive characterization of the covalent adducts generated in vitro made it possible to make this correlation in vivo since one can focus on specific parameters in the more complex in vivo studies.

	Mutagenicity of Metabolites and Bound Residues

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

Since many nitroimidazoles are mutagenic and carcinogenic, the ultimate goal of our study was to determine if bound residues derived from ronidazole (i.e., covalent adducts) are also mutagenic or carcinogenic. Our strategy was to establish a structure-mutagenicity relationship and to ask the question of whether ronidazole covalent adducts still retain the structural features necessary for mutagenicity and whether cysteine adducts, the ultimate hydrolysis products from protein-bound residues, are still mutagenic.

During ronidazole activation, 95% of the reactive intermediate formed is converted to water-soluble breakdown products that are nonmutagenic while the other 5% is protein-bound (Wislocki et al., 1984b; Lu et al., 1988; Wislocki and Lu, 1990). The following observations support the conclusion that ronidazole protein adducts are not mutagenic: (1) the structural features essential for the mutagenic activity of ronidazole no longer exist in the protein adduct; (2) the ronidazole cysteine adduct is not mutagenic nor can it be activated to a mutagenic product; and (3) the enzymatic hydrolysis of ronidazole-bound proteins fails to generate any mutagenic products. Thus, despite the fact that ronidazole is genotoxic, bound residues generated from ronidazole are not.

	Regulatory Solution

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

In July 1994, The Center for Veterinary Medicine of the FDA issued the "Guideline for the Human Food Safety Evaluation of Bound Residues Derived from Carcinogenic New Animal Drugs." The end of the document contains the following statement: "FDA has written this guideline after considering the views of numerous scientists. In particular, FDA has relied upon the paper entitled `Development of a Unified Approach to Evaluate the Toxicological Potential of Bound Residues' [Lu et al., 1990]. Sponsors are directed to this paper for an excellent overview of the bound residue issue. In addition, FDA suggests that sponsors consider addressing the safety of the bound residue of their own drugs refer to the paper entitled `Toxicological Significance of Covalently Bound Drug Residues' [Lu et al., 1988]. This latter paper and references therein describe the extensive work that was conducted in attempting to elucidate the toxicological potential of the found residues of ronidazole."

The bound-residue project has been a very complex and challenging one. I was very fortunate to have a group of talented and dedicated colleagues (Raul Alvaro, Edward Bagan, Karen Fiorentini, Gerald Miwa, John Walsh, Regina Wang, Susan West, and Peter Wislocki) working together. My colleagues and I are very proud of our achievements in contributing to the regulatory guideline.

	Many Fruitful Collaborations

Top Introduction A vibrant environment Searching for functional... From -hydroxylation to drug... From one cytochrome p450... The lipid dream A new inducer Deuterium isotope effect and... Bound residues in food-... Analytical vs. mechanistic... Covalent binding: in vitro... Mutagenicity of metabolites and... Regulatory solution Many fruitful collaborations References

One of the most satisfying experiences in my research career has been the collaboration with many talented scientists. These include the metabolism of ivermectin with Lee Chiu (Chiu et al., 1987), the structure and function of quinone oxidoreductase with Chung S. Yang (Ma et al., 1992), the metabolism of polycyclic aromatic hydrocarbons with Shen K. Yang and Peter Fu (Yang et al., 1982), the catalytic mechanism of glutathione S-transferase with William Atkins (Atkins et al., 1993), the metabolism of carcinogens by cytochrome P450 and epoxide hydrolase with Wayne Levin and Don Jerina (Lu et al., 1977), and the study of glutathione S-transferase with Regina Wang (Wang et al., 1992), and Su Huskey (Huskey et al., 1991). To support drug discovery and development, I collaborated with Regina Wang and Deborah Newton to evaluate the role of CYP3A in lovastatin metabolism (Wang et al., 1991), the selectivity of cytochrome P450 inhibitors (Newton et al., 1995) and the substrate interaction with CYP3A4 (Wang et al., 1997), and with Jiunn Lin on many strategic metabolism issues (Lin and Lu, 1997a, 1997b). Finally, my long-term collaboration with Cecil Pickett on glutathione S-transferase (Pickett and Lu, 1989) has been extremely productive and rewarding. It has been a privilege to work with so many talented individuals.