Abstract
The biochemical facets of toxicology have always had a major role in providing insight into mechanisms. Some of the history of the development of this area is summarized, including metabolism, enzymology, and the chemistry of reactive intermediates. Knowledge in these fields has had a major impact in the areas of drug metabolism and safety assessment, which are both critical steps in the development of pharmaceuticals and the rational use of commodity chemicals. The science of toxicology has developed considerably with input from other disciplines and today is poised to emerge as a predictive science with even more dramatic impact. The challenges ahead are considerable but there is renewed excitement in the potential of the field. As in the past, further advances in the field of toxicology will require the input of knowledge from many disciplines.
I would like to begin by expressing my appreciation to the American College of Toxicology for selecting me as the 2004 recipient of the President’s Distinguished Service Award. Although I had little formal training in toxicology as such, I have spent all of my faculty career working on aspects of biochemical toxicology (Figure 1). Much of the focus and application of toxicology has changed over the last 30 years since I joined the faculty at Vanderbilt, but the importance of the field has continued to grow.
Before going into the more scientific aspects of this review, let me briefly review my background because it influences how I approach research, teaching, and my role in the field. My father came to this country from Bavaria in 1913 with his family, at the age of 8. He did not attend high school but did become a successful farmer. Work ethic and self-reliance were not spoken of much by the farmers; these qualities were simply assumed for survival. Although no one in my family had much education, I was expected to do well in school. I did a reasonably good job in my early school days, although I probably could have applied myself more. Chemistry fascinated me, as well as mathematics (or at least the math that could be used to solve problems). English and anything resembling humanities bored me and I had no experience with foreign languages; I wish I had taken English more seriously because I now spend most of my time writing one thing or another.
No one in my family had gone to college, and only my mother had gone to high school. Today that background might get you a scholarship but then it was not very unusual. Being the oldest in my family, I had to deal with all the new experiences myself. In retrospect that was good for me. Because I had done reasonably well in high school, I decided to go to college and went off to the University of Illinois, which was affordable but not particularly easy to get in. Fortunately, the University of Illinois had long had an excellent chemistry program. The high school teachers had stressed how much harder college would be, and I was scared and applied myself enough to get all A’s the first semester. I had started in food science, mainly because I had somehow received an offer of a scholarship in this area. The introductory math and chemistry classes were fine, but the thought of learning applied technology later did not appeal to me. As a sophomore I moved into nutritional science and thought this might be more interesting. I continued to do well in my coursework and easily graduated on Bronze Tablet at Illinois (top 2%).
During the summer following the second year, I was fortunate enough to be accepted in a National Science Foundation research program and worked in Prof. Harry Broquist’s lab, doing biochemistry. This was really the answer for me. Biochemistry involved the use of organic chemistry to answer questions about biology, and I never looked back. After this, everything I did was oriented toward being a biochemist. The next summer (1969) I was also able to work in Broquist’s lab.
At that time Harry Broquist had made the decision to move to Vanderbilt and suggested that I consider doing graduate work there. I applied to Vanderbilt and Duke and was accepted at both. I suppose Duke would have been fine, but I had really enjoyed working with Harry Broquist (“the Chief”), with his intellect, his bounding enthusiasm, and his experience and connections in academic and industry. Late in college I had developed potential interest in medical research and a combined MD/PhD and Vanderbilt has always had a good program. Although this was not the usual pattern, I applied to medical school and the MD/PhD program during the first fall of graduate school (1970) and was accepted. However, after seeing what the first year medical students were studying (i.e., memorizing) in anatomy and physiology I realized I would be bored silly. I decided to decline the offer and did only the PhD, graduating in 1973. The medical scientist program is fine for some people, but I cannot say that I have regretted my own decision. I have had a lot of fun and done relevant things in my own way.
As I neared completion of my PhD, I wanted to learn more enzymology as a postdoc. I received offers at Berkeley, Cornell, and Michigan, but the offer from Prof. Minor J. “Jud” Coon (Michigan) interested me. This was a leading laboratory in the rather new area of cytochrome P450 (P450) research, and I thought that this field might have potential application some day. Harry Broquist had always been a great proponent of finding an area with practical application and doing very basic work in it.
The 2 years at Michigan were interesting. I was engaged to Cheryl near the end of my doctoral studies and married a few months after I went to Ann Arbor. The projects in the lab were challenging. Fortunately Cheryl was reasonably understanding about the workload. These were the early days of P450 research, and the enzymes came from rabbit liver. The fractionations were challenging, and the techniques for handling the enzymes were not trivial. The assays were challenging in that high-performance liquid chromatography (HPLC) was just beginning and we did not have the equipment. Nevertheless, I did learn a great deal in the lab. Jud Coon was an interesting professor and I also learned much from him in addition to research. He was always busy, in large part because he was chairman of the department, but he also managed to direct a fairly big lab. I was impressed with the attention he gave to writing and recall poring through drafts of manuscripts with him; I thought he criticized everything I wrote (these were the days before word processors, by the way). I learned from the experience and hope that I, in turn, have helped my people to write better. (I think reviewing a few thousand manuscripts over the years has probably helped, too.)
I have thanked Harry Broquist and Jud Coon before (Guengerich 2004a), but I should again. These are two of the finest men I have met in science, and along with my father have been the most influential people in my life. I still have contact with both of my mentors. I hope that I am able to have the same positive influences on the people who have trained with me.
In 1974 Harry Broquist told me that the Department of Biochemistry back at Vanderbilt was interested in hiring an assistant professor working in the area of biochemical toxicology, to participate in the Center in Environmental Toxicology that had been started by Frank Blood and was now headed by Robert Neal. Would I be interested? Early in 1975 I visited Vanderbilt to give a seminar. In all honesty I was leaning toward a job in industry at the time and might well have gone there if Upjohn had not had a hiring freeze. I gave my seminar and was offered a job on the faculty.
Going back to your old department is not as easy as it might sound. Most of the staff still assume you are a student. Wherever you start on the faculty, the pace just explodes. You have been used to doing well on your projects as a postdoc, but then you find yourself suddenly in a position where you have to do everything yourself. You have to hire people, find money, fill out mindless forms, start to develop lectures for your teaching, etc. You fully realize that if you don’t do these things well and quickly, the tenure clock will pass you by.
While a graduate student, I had taken a 2-hour toxicology course at Vanderbilt. Beyond that toxicology was a pretty new experience for me. The Toxicology Center was up for renewal and we had the National Institutes of Environmental Health Sciences (NIEHS) site visit about a week and a half after I showed up. I remember that Jerry Wogan, Ernie Hodgson, and the late Leon Goldberg were on the site visit team, although I knew none of them at the time (I can’t remember the other people). I proposed a rather bold program of my own focused on characterizing the rat liver P450s and working out the chemistry of activation of aryl amines, vinyl halides, N-nitrosamines, and pyrrolizidine alkaloids. I was bold enough to also propose to extend my work to humans. Although I do not know what our score was, the NIEHS Center grant was refunded. I had no idea that less than 6 years later I would be directing the same Center, at the ripe old age of 32.
Thus, I started in chemistry and biochemistry. I am still strongly involved in these disciplines. Over the years I have learned much about toxicology and drug metabolism. I had to learn many other things along the way.
A BRIEF HISTORY OF BIOCHEMICAL TOXICOLOGY
The history of toxicology goes back to at least the time of ancient Greece (Borzelleca 2001). The aspects involving real organic chemistry and biochemistry really did not begin until the 19th century, with early determinations of structures of urinary metabolites of simple chemicals, sometimes ingested by the investigators themselves (Guengerich 1997). This work, done mainly in Germany, yielded oxidation products of benzene (Schultzen and Gräbe 1867), hippuric acids (Keller 1842), glucuronides (Jaffe 1874), mercepturic acids (Jaffe 1879), sulfates (Baumann 1876), and N-methyl (His 1887) and N-acetyl (Cohn 1893) conjugation products. The International Society for the Study of Xenobiotics has an interesting historical website: http://www.issx.org/hisintro.html. I encourage young readers to visit the library and read some of these papers from the 19th century. Chemistry was really hard then, with no chromatography or spectroscopy, and I admire these pioneers in the field.
Work on metabolism continued in the early 20th century, and monographs describing the state of the field were published by Williams (Williams 1947, 1959). Some insight into reactive intermediates began to develop. Work by Yamagiwa and Ichikawa (1915) had directly demonstrated the carcinogenicity of coal tar in a rabbit ear model. Cook et al. isolated benzo[a]pyrene in 1933 (Cook, Hewett, and Hieger 1933). Cook and deWorms (1937) and Fieser and Desreux (1938) provided evidence that the products of biotransformation had altered carcinogenicity. James Miller, with his student Jerry Mueller (Mueller and Miller 1948), demonstrated the in vitro NADPH-dependent modification of azo dyes, and the pioneering studies of James and Elizabeth Miller demonstrating covalent binding of carcinogens to proteins and, later, nucleic acids are now classic (Miller and Miller 1947; E. C. Miller 1951; J. A. Miller 1970, 1994).
As I already mentioned, knowledge about toxicity of chemicals goes back to ancient times (Borzelleca 2001) and Paracelsus described approaches to toxicity testing 500 years ago (Borzelleca 2000). Little attention was given to toxicology in the Industrial Age and the early years of the development of the chemical industry. The health effects of exposure to high levels of many of the chemicals were unappreciated, particularly if no immediate irritation was noticed. Cancers (which were not as readily characterized as today) were observed with tars, pitch, mineral oil, and aniline dyes (von Volkman 1875; Bell 1876; Manouvriez 1876; Rehn 1895). The bladder tumors observed in the aniline dye factory workers were later attributed to aniline, benzidene, 1-naphthylamine, and 2-naphthylamine (Hueper 1942).
Some examples of carcinogenicity of industrial chemicals have occurred since that time, including the lung mesotheliomas caused by asbestos (very synergistic with cigarette smoking) (Selikoff, Hammond, and Churg 1968) and the vinyl chloride–associated liver hemangiosarcomas (Creech and Johnson 1974). It is interesting to note that many of the most notable examples of mass toxicity include unintended exposures due to spills or unintended exposures due to lack of communication regarding instructions, as outlined by Lu (Lu 1996), e.g., hexachlorobenzene in wheat (Turkey 1956), tri-o-cresylphosphate in oil (Morocco 1959), methylmercury in wheat (Iraq, 1972), toxic oil syndrome (Spain 1981). Industry-related problems include polychlorinated biphenyls in Fukuoka, Japan (1968) and the Union Carbide methyl isocyanate release in Bhopal, India (1984) (Lu 1996).
Toxicity tests were done with some compounds of industrial interest, although the methods might seem unsophisticated compared to today’s methods. Other chemicals attracted attention, including those of natural origin. As early as 1938, the hormone estrone was shown to cause tumors in male mice (Lacassagne 1932). Fried horsemeat could also cause tumors, a phenomenon that is now understood in the context of heterocyclic amines (Widmark 1939). Many of the early tests involved end points such as lethality and carcinogenicity. Subsequently bioassays have become more sensitive and exquisite. The early history of testing has been reviewed by Weisburger and Williams (1981).
In some respects, exposure to chemicals was considerable in the decade or two following the end of World War II. Heavy air pollution was experienced in London in an episode in 1952, due to burning of coal, and similar high levels of air pollution were common in Eastern Europe until the end of the Communist era (1990). Carson’s indictment of pesticide contamination (Carson 1962) was a major effort in advancing the environmental movement, and testing and regulation were considerably expanded. A stimulus to more rigorous testing of new drugs was the unfortunate experience with thalidomide, which produced a large number of birth defects (Speirs 1962). The problems were not only man-made chemicals but natural products as well. A veterinary problem involving the killing of British turkeys by contaminated peanut meal from Brazil (Blount 1961) led to the discovery that aflatoxins also caused widespread human hepatocellular carcinoma in Africa, China, and other parts of the world (Busby and Wogan 1984).
The field of toxicology had historically responded to most of these situations in a rather retrospective mode. However, better understanding of mechanisms would be necessary to deal with new problems, because prospective approaches were needed to address the many new drugs and commodity chemicals that were being introduced, as well as to deal with the chemicals and natural products that people were already exposed to and make intelligent decisions regarding safety.
If we are to make enlightened choices about the safety of individual chemicals, the old postulates of Paracelsus (Borzelleca 2000) must be applied and we need to understand the basic reasons why chemicals cause toxicity, including cancer. Most of the chemicals of interest that produce acute toxicity provide little guidance. For instance, highly selective toxins such as neurotoxins act rapidly at very low doses and involve very selective receptor interactions. An example of acute toxicity from an un-specific toxin is ingestion of lye, which is simply caustic and destroys tissues on contact. The chemicals we deal with most of the time in toxicology have medium specificity and are more difficult to understand. Also, a single paradigm will probably not explain every situation.
One of the themes in explanation of toxicity and chemical carcinogenesis is covalent binding to molecules. This concept goes back to at least the early work of the Millers and findings of the persistence of azo dyes bound to protein (Miller and Miller 1947). If the Millers had not been working with colored compounds, more time would have elapsed until the phenomenon would have been discovered. The original discoveries had been of chemicals bound to protein, and these results were considered in the context of cancer (Miller 1951). This connection may seem strange today, but we must realize that the knowledge of DNA was very limited in the 1940s. Although Avery had published some seminal work on the transformation of bacteria (Avery, Macleod, and McCarty 1944), my conversations with Jud Coon and Jim Miller indicate that the prevailing view at the time was that DNA might be a structural material, somewhat akin to collagen, and that the genetics of a cell would ultimately be understood in the context of the proteins per se. The dogma changed quickly after the report of Watson and Crick (1953) and other classic work. The Millers went on to document and characterize adducts of chemical carcinogens bound to DNA (Miller, Juhl, and Miller 1966; Miller 1970). The concept of chemical damage to DNA resulted in the development of useful bacterial tests for genotoxicity. Some of the early systems did not show good correlations between mutagenicity and carcinogenicity, but the inclusion of enzymatic oxidation systems by Ames led to improved predictability (Ames et al. 1973). Although we recognize that not all carcinogens act only by genotoxic mechanisms (Figure 2), the intelligent prediction of the genotoxicity of chemicals in simple systems is one of the major achievements in mechanistic toxicology.
What about chemicals that are toxic but not carcinogenic (Figure 2)? In many respects these have been harder to understand. The early concept of covalent binding to proteins (Miller et al. 1949) was applied to toxicities other than cancer. A seminal series of papers from the laboratories of Brodie and Gillette at the National Institutes of Health (NIH) showed interesting correlations between toxicity and covalent binding of acetaminophen and bromobenzene (Jollow et al. 1973; Potter et al. 1973; Mitchell et al. 1973a, 1973b; Zampaglione et al. 1973) that have continued to influence thinking in this area today. However, the extent of covalent binding is clearly not the only issue involved in carcinogenicity or other toxicities. Good examples include 2,3,7,8-tetrachlorodibenzo[p]dioxin (Poland and Glover 1979) and the meta isomer of acetaminophen (Streeter et al. 1984; Roberts, Price, and Jollow 1990).
THE STATUS OF BIOCHEMICAL TOXICOLOGY IN 1975
As mentioned earlier, in 1975 I accepted an offer to join the faculty at Vanderbilt and leave my postdoctoral days behind. I was glad to be in a position in which I could try out some of my own ideas but also not completely knowledgeable about the general field of biochemical toxicology. When you start out, you have to find your niche. As a word of advice to young people, this is not easy. All of the obvious and easy experiments (the “low hanging fruit”) have not escaped the attention of others with more experience than you. You have to be willing to gamble. As I mentioned earlier, I proposed to purify and characterize the rat and rabbit enzymes involved in the metabolism of toxic chemicals (i.e., P450s in this case) and, if possible, extend the work to human systems. I also proposed to use a set of prototypic chemicals to define the reactive intermediates involved in the oxidation of vinyl halides, aryl amines, N-nitrosamines, and pyrrolizidine alkaloids. These were not completely unique areas of investigation and I knew of competition in most of these areas. I read the current literature very nervously in those days. I also made the decision not to put all my eggs in one basket. One “scoop” on me would not be fatal. However, I really believed I could contribute because my training gave me some advantages. Ultimately I did contribute in all of these areas and, in a sense, I am still involved in the projects I set out to study 30 years ago.
In some ways 1975 was a pivotal time. Most of the enzyme systems involved in the metabolism of the chemicals of interest were already known by this time (Guengerich 2000), although few had been very well characterized. Little was known about human enzymes except at a very descriptive level (Distlerath and Guengerich 1987). We were beginning to appreciate some of the mechanistic details of reactions involving reactive intermediates, e.g., the work of the Millers with N-hydroxy arylamines (Scribner, Miller, and Miller 1970; Kadlubar, Miller, and Miller 1976) and the rapidly developing work of Jerina and others with the diol epoxides formed from polycyclic aromatic hydrocarbons (Wood et al. 1976). Analytical chemistry was aided by the recent introduction of HPLC, which was just beginning to have an impact. Spectroscopy was not bad (and I have always relied heavily on mass and nuclear magnetic resonance [NMR] spectrometry) but certainly much more primitive than today. Scientists were beginning to work with primary cultures of hepatocytes.
An interesting development was Bartsch’s work demonstrating the apparent activation of vinyl chloride by P450s (Barbin et al. 1975), providing an explanation for the recent identification of hemangiosarcomas in the livers of workers in vinyl chloride plants (Creech and Johnson 1974). In general, there was considerable interest in the concept that covalent binding to protein would prove to be a major factor in explaining (and predicting) chemical toxicity (Jollow et al. 1973; Potter et al. 1973; Mitchell et al. 1973a, 1973b; Zampaglione et al. 1973). Also, a flurry of interest in nuclear P450s developed because of the view that reactive intermediates were simply too reactive to migrate from the cytoplasm (including the endoplasmic reticulum) to the nucleus to damage DNA (Vaught and Bresnick 1976; Fahl, Jefcoate, and Kasper 1978).
OUR CONTRIBUTIONS IN ENZYMOLOGY
Summarizing 30 years of work is not easy, and this overview certainly cannot provide my true appreciation for the effort many people put into our work, plus the unsuccessful trials that were needed to develop the systems. The work in Jud Coon’s lab had emphasized rabbit liver P450s, but I focused on rats because of their widespread use in drug metabolism and toxicology. In the next few years we were able to purify a number of the major rat liver P450s and to characterize some aspects of their roles in the metabolism of a few interesting chemicals (Guengerich 1977, 1978). By 1982 we had done a fairly extensive characterization of eight different rat liver P450s (Guengerich et al. 1982). This is the most highly cited original research paper I have published (1172 times, according to a recent check in the Institute for Scientific Information database), and I was pleased to have published this paper and seen it so well received. However, even with all the effort we put into this paper, it was the beginning of the end of work on animal P450s.
Even in the early research with rat and rabbit P450s, I had noticed some striking differences between what were supposed to be similar enzymes found in these two species. What would our work really tell us about the human P450 enzymes? I decided to go after the human P450s themselves, which at that time (late 1970s) still meant purification. Somewhat to my surprise, in 1978 I was awarded a National Cancer Institute contract I had applied for (to characterize human P450s). The early efforts were fraught with difficulty in obtaining human liver samples, which we needed in large amounts for the purifications. We very serendipitously developed a working relationship with the Nashville Transplant Agency (now Tennessee Donor Services), a private agency outside our university. This cooperation proved to be critical in our efforts over the next decade. The early work with human liver was, like the earlier work with rats and rabbits, oriented simply at getting purified proteins from liver (Wang, Mason, and Guengerich 1980; Wang et al. 1983). The reports of the debrisoquine genetic polymorphism by Smith, however, changed my thoughts (Mahgoub et al. 1977). Bob Smith’s work suggested that a single P450 could be the dominant catalyst in the metabolism of a drug, and we should be purifying enzymes based upon specific reactions, as a means of later transposing in vitro findings to relevant in vivo situations. The approach was technically much more demanding, but we were ultimately able to purify enzymes now known as P450s 2D6 (Distlerath et al. 1985), 2C9 (Shimada, Misono, and Guengerich 1986), and 3A4 (Guengerich et al. 1986). P450 1A2 came as a side product of the 2D6 work (Distlerath et al. 1985). Fortunately we can now produce all of these and other P450s in recombinant bacterial systems, and we are still characterizing various biochemical properties (Parikh, Gillam, and Guengerich 1997; Bell-Parikh and Guengerich 1999; Yun, Miller, and Guengerich 2000; Guengerich et al. 2002b). We characterized some of the reactions of the human P450s with chemicals of toxicological interest in early work (Wang et al. 1983; Wolff et al. 1985). However, a major advance was the application of a relatively high-throughput genotoxicity screen in 1988, with Tsutomu Shi-mada, which allowed us to examine many of the major carcinogens in a relatively short time (Shimada and Guengerich 1989; Shimada et al. 1989a, 1989b). Shortly after this we were also able to document the role of P450 2E1 in activating many chemicals (Guengerich, Kim, and Iwasaki 1991). The general conclusions about which human P450s are involved in carcinogen metabolism (Guengerich and Shimada 1991) have kept up reasonably well, with the addition of important information about P450 1B1 (Shimada et al. 1996, 1999; Toide et al. 2003).
Over the years we have also worked with enzymes other than P450s, including flavin-containing monooxygenase (Dannan and Guengerich 1982), epoxide hydrolase (Guengerich et al. 1979b; Wang, Meijer, and Guengerich 1982), and glutathione (GSH) transferases (Raney et al. 1992b; Johnson et al. 1997; Guengerich, McCormick, and Wheeler 2003), particularly as these enzymes are related to the metabolism of chemicals of interest.
Returning to the P450 enzymes, I had become interested in chemical mechanisms of catalysis as a postdoc. After we came to grips with some of the problems in purification of the P450s in my early days on the faculty, my colleague Tim Macdonald restimulated my interest (Macdonald et al. 1982; Guengerich and Macdonald 1984, 1990; Burka et al. 1985; Guengerich, Yun, and MacDonald 1996). Interests in chemical catalysis and kinetics have continued, particularly in the context of explaining seemingly unusual events observed in the course of studies in drug metabolism and toxicology and regarding the generation of reactive intermediates (Guengerich 2001). We have also become very interested in the mechanisms of DNA polymerases and their relevance to the blocking and misincorporation events observed with mutagens (Lowe and Guengerich 1996; Choi and Guengerich 2004; Guengerich 2005).
MECHANISMS OF ACTIVATION OF CHEMICALS
I have long been interested in organic chemistry, even in high school. To me, chemistry has offered a means of understanding the details of toxicity. In many quarters of toxicology and environmental health research, this behavior would have been discouraged in a junior faculty member. I should be grateful that Bob Neal was in charge of the program in my early days and did encourage such investigation.
I already mentioned vinyl chloride, which was our first foray into the field of vinyl halides. Some of this work began with an important collaboration at Dow Chemical with the late Phil Watanabe and Perry Gehring (Guengerich and Watanabe 1979). My prior training in enzymology had not really prepared me for considering a gas as a substrate, and some new thought was needed. We were able to establish the roles of the epoxide versus the rearrangement product, 2-chloroacetaldehyde, in binding to DNA and proteins (Guengerich, Crawford, and Watanabe 1979a; Guengerich et al. 1981; Guengerich 1992). We examined the oxidation of trichloroethylene (TCE), for which the dogma was that the epoxide “spontaneously” rearranged to chloral hydrate. Our results led us to a different view of the reaction chemistry (Miller and Guengerich 1982) and provided evidence for a stepwise mechanism of P450 oxidation of olefins (Miller and Guengerich 1982), which was further developed in our subsequent work with vinylidene chloride and the model compound trans-ethyl styrene (Liebler and Guengerich 1983) (Figure 3). We had a good grip on the nature of the reactive intermediates in the early work with vinylidene chloride (Liebler and Guengerich 1983) but the nature of the reaction with TCE remained unclear until we revisited the problem many years later with a series of heavy isotope labeling studies (and much improved mass spectrometers) (Cai and Guengerich 1999). We subsequently generalized the results to multi-halogenated olefins (Yoshioka, Krauser, and Guengerich 2002b) (Figure 4).
Another group of compounds that attracted my interest was the dihaloalkanes. Ethylene dibromide was a topic of considerable interest, with its classification as a carcinogen and withdrawal from commercial use (Sun 1984). Following the initial work by Rannug (Rannug, Sundvall, and Ramel 1978), we were able to document the role of GSH transferase (GST) in activation and the formation of crosslinked DNA-GSH adducts, in my first PNAS (Proceedings of the National Academy of Sciences of the United States of America) paper (Ozawa and Guengerich 1983). We had been interested in methylene chloride, with the Dow group, for several years and were able to successfully develop a good physiologically based pharmacokinetic model (Reitz, Mendrala, and Guengerich 1989). We were subsequently able to demonstrate the role of theta class GSTs in activating dihalomethanes to mutagens (Thier et al. 1993). More recently we have found that the same general chemistry we established for the GSH reaction is also operative with the DNA repair protein O 6-alkylguanine DNA-alkyltransferase (AGT) and is involved in crosslinking the protein to DNA and causing mutations (Liu et al. 2004). Another compound in this category is butadiene diepoxide, a reactive metabolite of 1,3-butadiene, for which the mutagenicity is enhanced by conjugation with GSH or AGT (Thier et al. 1995; Valadez et al. 2004).
I had not worked with aryl amines very much until I met Fred Kadlubar during a visit to the National Center for Toxicology Research. Over the years we have done a number of interesting chemical and biochemical studies with compounds in this group and with their relatives the heterocyclic amines (Frederick et al. 1982; Kim et al. 2004). In particular, we established the role of human P450 1A2 in activation (Butler et al. 1989) and also defined some aspects of the reaction with DNA (Humphreys, Kadlubar, and Guengerich 1992; Guengerich et al. 1999). Recently we reported the facile redox cycling of the hydroxylamine and nitroso derivatives of some heterocyclic amines (Kim et al. 2004).
N-nitrosamines have also been of interest, and we have collaborated in several investigations on the roles of P450s 2E1 and 2A6 in bioactivation (Yoo, Guengerich, and Yang 1988; Yamazaki et al. 1992a). We have worked on some other aspects of the activation of N-nitrosamines (Okazaki, Persmark, and Guengerich 1993). One observation we made that has still not been completely explained is the enhancement of genotoxicity in bacterial systems due to expression of N-acetyltransferase (Yamazaki et al. 1992b).
Although I had done some work with aflatoxin (AF)B1 early in my career (Guengerich 1979), I had not intended to do more until Tsutomu Shimada did some experiments that demonstrated a role for human P450 3A4 in the activation (Shimada and Guengerich 1989). About the same time my colleague Tom Harris and his student Steve Baertschi were able to synthesize the 8,9-epoxide (Baertschi et al. 1988), and we studied AFB1 for more than a decade, until I had finished all the biochemical experiments I was interested in (Guengerich et al. 2002a, 2002c) (Figure 5). An exciting discovery was the remarkable difference in genotoxicity of the exo and endo isomers of the epoxide (Raney et al. 1992a; Iyer et al. 1994). We were able to use stopped-flow techniques to accurately measure the half-life of AFB1 exo-8,9-epoxide as 1 s (Johnson et al. 1996). This result allowed us to establish the rate constants for conjugation of the epoxide with individual GSTs (Johnson et al. 1997) and with DNA (Johnson and Guengerich 1997). The latter study was very revealing, in that we were able to directly observe the very rapid reaction of a highly unstable reactive metabolite with DNA (Figure 6), which I had never anticipated doing when I began my work. The results we obtained say much about concepts of stability, half-lives, and reactivity in biological systems (Johnson and Guengerich 1997). I thought we were finished with our AFB1 work then but have seized some opportunities to examine reactions of AFB1 dialdehyde (Guengerich et al. 2001, 2002a, 2002c). After >10 years in the AFB1 business, we have a reasonably good view of the parameters of the major human processes involved in AFB1 reactions (Figure 5).
OTHER RESEARCH AND APPLICATIONS
My goal in life has not been so much a cause-oriented one but more of a search for adventure. That is, I never set out to cure a disease or establish a program; I have looked for interesting things to do, within the context of having enough other people be interested enough to justify resource support. As it turned out, the things that interested me most ultimately yielded the greatest practical applications even if I had not intended so. In many respects, I work in a very privileged career in that I am paid to pursue things I like to do.
The world of P450 research has been a great one to work in. First of all, the P450-related meetings seem to be held in interesting places much of the time. I also attend meetings and consult in the areas of toxicology and drug metabolism and learn of practical problems. Further, I have accepted invitations to speak at meetings on topics as seemingly diverse as physical organic chemistry, bioremediation, nutrition, endocrinology, environmental mutagenesis, microbiology, and contraceptives, all over the world. I appreciate learning the issues and approaches involved in these areas.
I have research interests outside of P450s and biochemical toxicology, too. I have already mentioned my general interests in enzyme mechanisms and bioorganic chemistry, in which I was trained and continue to work. We are opportunistic and also do other research. In the last 5+ years, we developed a system that began with the observation by Liz Gillam that bacterial cultures producing human P450 2A6 turned blue (Gillam et al. 1999, 2000) (Figure 7). The work led to thoughts about the prospects of making blue flowers (Gillam and Guengerich 2001) and many articles about us in the lay press, including National Geographic (June 2003) and many major newspapers and radio stations in the United States and other parts of the world. However, we never really followed through, in large part because of our limited background with plant science (I did get an A in Honors Botany as an undergraduate), and never made any money. However, we have been able to develop a process using recombinant human P450 2A6 mutants to generate novel indirubins with potential as protein kinase inhibitors and drugs (Guengerich et al. 2004; Wu et al. 2005). Another line of current investigation in our lab involves the characterization of P450s from Streptomyces, with the long-term goal of modifying the P450s to produce new antibiotics (Guengerich 2002).
What has our research been useful for? Good question. I do owe the taxpayers something. Where I believe we have made a difference is in the following areas (we were not the only contributors, by the way). Knowledge of the human P450s has changed the way we collectively approach problems in drug metabolism and toxicology. The real questions now are about how to predict reactions in humans, and metabolism in animals is considered in the context of how these are used as models. General knowledge about human P450 reactions is of considerable use in the consideration of screening new drug candidates. We have clearly shown the differences between human enzymes and some of the apparent animal orthologues (e.g., P450 1A2 in rats and humans) (Turesky et al. 1998). The information we have produced about P450 catalysis has been useful in explaining what might have been unexpected reaction schemes. Many of these questions and explanations have arisen with drug candidates. Some of the concepts about the chemical reactivity of particular entities have had application in consideration of certain phenomena. Finally, I believe that some of our mechanistic studies on commodity chemicals have been useful in developing scientifically better estimates of risk from exposure (Reitz, Mendrala, and Guengerich 1989; Guengerich, McCormick, and Wheeler 2003; Valadez et al. 2004), even if regulators have not acted on these.
OTHER PROFESSIONAL ACTIVITIES
I would be happy doing only research and some teaching. However, life is not that simple. One has to help create an environment that will help students and fellow faculty with similar interests. Also, one does owe the scientific community some things.
I have served as Director of the Vanderbilt Center in Molecular Toxicology for over 23 years now. As I understand, this is either close to or surpasses Bruce Ames’ record in the NIEHS center grant program. I was not looking for administrative responsibilities when I was asked to fill this role, and I have continued to decline suggestions about my interest in departmental chairmanships, etc. However, with regard to our Center, I like the area in which we have been able to develop at Vanderbilt and the faculty who have joined us (Guengerich 1999). Garnering resources has been important in our cause. I am loath to micro-manage; as long as people are doing good, relevant science and our resources are used properly, I am happy to let things proceed because I easily find other things to do.
Scientific societies are important, and over the years I have had significant roles in those involving toxicology (Society of Toxicology, American Chemical Society Division of Chemical Toxicology), drug metabolism (American Society for Pharmacology and Experimental Therapeutics, International Society for the Study of Xenobiotics), and biochemistry (American Society for Biochemistry and Molecular Biology). I believe that all of these organizations do many useful things. I regret that getting involved in committees and councils usually takes far more time than expected, and one has to place some limits on such activities.
I have also had extensive editorial experience, more than I ever expected to have. Over the years I have been on the editorial boards of at least 25 journals, in various disciplines. In particular, I have now spent over 16 years on the Editorial Board of The Journal of Biological Chemistry and over 15 years as an Associate Editor of Chemical Research in Toxicology. I have enjoyed seeing the development of this latter journal and believe that it has played an important role in the field, in establishing the relationship of chemistry and toxicology. I guess that is why you do these things, in the final analysis. (Unfortunately, any good you do as an editor is usually not rewarded when your own manuscripts get reviewed by others.) I also previously served a number of years as an associate editor of Toxicology and Applied Pharmacology, Molecular Pharmacology, and Cancer Research.
One of the necessary things I have had to do in my career is to provide NIH with advice. I have spent 12 years on study sections and 4 on an NIH council. Although I have benefited from scientific discussions with others at study sections (and meeting people), I have not enjoyed having to make some of the difficult decisions about scoring grants because I knew that they would have serious effects on people’s lives. However, someone does have to make hard choices in shaping the direction of a field. I have also been called upon for advice at NIH in other matters and tried to contribute. In the grand scheme of things I believe the NIH system has been more successful than the corresponding agencies of other governments, and I should be thankful for where I live and work because I have had many opportunities. Having said that, I do see some trends within NIH that will reduce its effectiveness in the future, and these go beyond just the availability of financial resources to the NIH and its institutes.
I have had many opportunities to work with industry during my career and have really enjoyed the interactions. The problems I have seen were intellectually stimulating and I appreciated seeing firsthand how research in my field was being applied, as well as learned much about the processes. I realized what aspects of my work were being used. The many contacts I made helped many of my students and postdocs find good jobs (Figure 8). I was able to convey good (I hope) career advice to the people in my group, and I also developed a great set of real-life examples about drugs for my medical student teaching.
STATUS OF THE FIELD
Over the past 30 years we have seen considerable improvement in our analytical techniques, particularly chromatography and spectroscopy. As a result, we know much more about the details of how chemicals are activated and how they react. Molecular biology and recombinant DNA technology have been very helpful. We know, in part because of some of our efforts, which enzymes are involved in the activation and detoxication of chemicals. As mentioned earlier in this review, we realize that reactive products can move around considerably within cells, even with a half-life of 1 s (Johnson, Harris, and Guengerich 1996; Johnson and Guengerich 1997).
A current diagrammatic view of events involved in toxicity is presented in Figure 2. Many proteins are modified; the most plentiful are not necessarily the most critical. Gross measures of covalent binding may not necessarily predict toxicity, e.g., compare acetaminophen with the meta-isomer (Streeter et al. 1984; Roberts, Price, and Jollow 1990; Qiu, Benet, and Buringame 1998). In comparing the covalent binding and reactive oxygen possibilities, it is not easy to separate these in that many of the responses are similar. In my own opinion, the toxicity of most of the chemicals under our collective interest will probably not have a single “master” target to explain the toxicity. Regulatory pathways for the maintenance of cell function are both numerous and complex. We have another issue in that low-level exposures do have protective and beneficial effects (Figure 2), e.g., Keap1-mediated events (Kwak, Wakabayashi, and Kensler 2004). Perhaps hormesis is real and more common than thought (Calabrese and Baldwin 2003).
THE FUTURE OF TOXICOLOGY
In my opinion, the field of toxicology is at a crossroads and poised to develop into a more important and more mechanistic phase. The real potential, to me, lies in the role toxicology can play in drug development (Figure 9). Establishing safety in a reliable, efficient manner is one of the most pressing needs in the pharmaceutical industry today, and I have participated in several forums in this area recently (Borman 2004). There is considerable enthusiasm for innovation in this area, and I am optimistic that progress will be made. We are at the point where chemistry, initial screening, and even bioavailability predictions are rapid, but aside from genotoxicity we still have limited capability for prediction of toxicity. The challenge is daunting, in that many drug responses and interactions remain idiosyncratic, and a very low incidence (<1/106) can still be considered a major issue. Progress will be made in industry, with help from academia in basic research. The pharmaceutical industry has the benefit of large-scale experience and pathology databases on compounds that will be nearly impossible to replicate by individual academic laboratories.
Another major issue is “environmental” chemicals, i.e., for our purposes the nonpharmaceuticals. I see a need here for modern and more enlightened approaches, and I hope that progress is made in this area. Frankly I am less optimistic about real progress here, at least in terms of application in the regulatory environment. I see the problem as political, but not in the sense that is often portrayed. Federal agencies are suspect to be influenced by misguided environmental groups, and unfortunately I have seen examples of agencies relying on such organizations for political support to enhance their own existence. As an example of the lack of common sense, I cite the activist goal of eliminating chlorine compounds in spite of the fact that hordes of these are produced in nature (Wuosmaa and Hager 1990; Gribble 2004).
I will mention six different new approaches to toxicology, or at least toxicity screening oriented towards learning about large numbers of chemicals.
The first is in silico methods. These are relatively limited. In contrast to the situation with drug targets (e.g., receptors), little information is available about what the macromolecules are for docking of chemicals associated with chronic toxicity. Thus, almost all in silico methods depend upon databases of toxicity and structural alerts (entities) present in chemicals. There is some experience with these systems at the Food and Drug Administration (FDA); the pharmaceutical industry does not find these particularly useful and utilizes these primarily to anticipate FDA responses, in a defensive strategy.
Toxicogenomics is a term that can include two parts. One is actual genomics, which involves primarily comparisons of DNA sequences to associate responses (toxicity) with particular genotypes. This approach has had some usefulness. An example is the explanation of a mouse response to ivermectin due to absence of the mdr1 gene product (Umbenhauer et al. 1997). The NIEHS has initiated an Environmental Genome Project with the long-term goal of linking outcomes of environmental exposures with (human) genotypes (Guengerich 1998).
The other heading under toxicogenomics is what should probably be called transcriptonomics, involving the analysis of levels of individual mRNA species (Nebert, Jorge-Nebert, and Vesell 2003). The assays usually involve hybridization with “chips” and measure relative levels of mRNA (versus a control experiment). The technology has developed considerably in the past few years. A challenge is to go beyond what is already known. For instance, just establishing that a new compound is another arylhydrocarbon (Ah) receptor (AhR) agonist can be done more simply. If, on the other hand, we can subdivide the responses to AhR and peroxisome proliferator-activated receptor (PPAR a ) agonists to identify responses that may be related to rodent tumors and those that are not, then we have learned something. To date, the work from in vivo systems has been far superior to in vitro (cell cultures), in that the latter are (i) highly sensitive to changes in media, etc., (ii) have specificities that may reflect each particular cell line, and (iii) may not reflect events in the animal. An issue (in vivo) is which tissues to profile, in the absence of preliminary information about where toxicity might be expected. A serious problem is the issue of temporal and dose relationships. As indicated in Figure 2, a small dose of a chemical may induce an early protective effect but be toxic at higher doses and later. Thus, single point measurements are probably unrealistic.
Proteomics, or toxicoproteomics in our case, has some of the same issues as transcriptionomics. It does have two advantages, however. First, the correlation of mRNA levels with levels of the coded proteins is low (Gygi, Rist, and Aebersold 2000) and, second, proteomics can report post-translational modifications. The dominant technology in proteomics work has been two-dimensional gel electrophoresis, but this is being superseded by complex mass spectrometry procedures (Liebler 2002).
Metabonomics involves analysis of small molecules in biological samples, usually blood and urine. Thus, this approach is usually noninvasive and, in principal, directly applicable to human studies. The current technologies in use are NMR and mass spectrometry. The focus is on changes in the balance of endogenous chemicals, as opposed to metabolites of chemicals that were consumed. The approach is to use pattern recognition methods to look for changes, even if their biochemical relationships cannot presently be discerned (Nicholson and Wilson 2003). This approach is still relatively new but has considerable potential.
Two points should be emphasized regarding all of the technologies I have mentioned: (i) They are heavily dependent on good bioinformatics, in that the data loads are enormous and the value of the work is derived from the unobvious inferences that can be made, and (ii) these approaches must be related to real pathology. It is not enough to relate various biochemical parameters to each other; ultimately we must be able to make intelligent predictions about real damage.
Let us return to the scheme of Figure 2. We already have good assays for DNA damage. We have a number of reasonable assays for interactions with receptors, at least some of the ones we know about. Efforts are being made to use more assays of covalent binding in higher-throughput and more sophisticated ways (Evans et al. 2004). I predict that the covalent binding data will be one tool we use to make decisions but not the only one, and more experience is needed. The real needs are for assays that will report useful information about events in the areas of the lower left and right hand sections of the scheme. Can we find systems that will report early damage and distinguish it from protective effects?
I expect many of the exciting advances in modern toxicology to come from the industrial sector. The interest arises because toxicology cannot be considered just a routine to satisfy regulators but a vibrant area where new insight can make a difference in getting new medicines to people and avoiding toxic responses. I do believe that academia will continue to have an important role, not only in teaching. However, I expect that the shape of academic toxicology programs will change because of the need for new innovative approaches. I have already shared some of my thoughts in another review (Guengerich 2004b), although I cannot accurately predict the future.
EPILOGUE
I have shared some of my thoughts on what I have done and tried to do in my career to date, as well as where I think we have come from, where we are, and where we should be going. These are my own views and may or may not be widely subscribed to. I am a biochemist who was attracted to interesting problems in toxicology, and I have simply tried to contribute in my own way.
I would like to thank the American College of Toxicology again for this award. It should rightfully be shared with many of the 13 graduate students and 98 postdoctoral fellows/visiting scientists who have worked with me (Figures 7, 8, 10) (I also add a very friendly and efficient administrative staff in our Center). I thank my wife Cheryl and our children (Phillip, Laura, and Anna) for their support over the years.
I consider myself in mid-career form and expect to be in this business for a long time yet. Indeed, I still work in my lab as much as I can and have never lost the excitement of getting results first-hand. I also hope that I have been able to instill the enthusiasm for research in younger people that my mentors (vide supra) did for me.