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Adventures with Bruce Ames and the Ames test
Mutat Res Gen Tox En 846 (2019) 403070
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Adventures with Bruce Ames and the Ames test Lawrence J. Marnett
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Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville TN 37240-7914, United States
ARTICLE INFO
ABSTRACT
Keywords: Ames Mutagenesis Aldehydes Mechanism Sabbatical
Bruce Ames has had an enormous impact on human health by developing facile methods for the identification of mutagens. This research also provided important insights into the relationship between mutagenesis and carcinogenesis. Bruce is a highly innovative and creative individual who has followed his interests across disciplines into diverse fields of inquiry. The present author had the pleasure of spending a sabbatical in the Ames lab and utilized the Ames test in multiple aspects of his research. He describes both in this honorific to Bruce on the occasion of his 90th birthday.
1. Introduction to the Ames test My first encounter with Bruce Ames was at a seminar at the University of Michigan in the late 1970’s. I was an Assistant Professor on the Chemistry faculty at Wayne State University, and our laboratory had gotten interested in chemical carcinogenesis through our studies with the enzyme prostaglandin H synthase (PGHS, a.k.a. cyclooxygenase). PGHS catalyzes two reactions, thebis-dioxygenation of arachidonic acid to yield the hydroperoxy-endoperoxide prostaglandin G2 (PGG2) and the reduction of PGG2to the corresponding hydroxy-endoperoxide PGH2(Fig. 1). We had discovered that the peroxidase activity of PGHS oxidizes the carcinogen benzo[a]pyrene (B[a]P) during the course of hydroperoxide reduction and were testing the hypothesis that this reaction resulted in B[a]P’s metabolic activation [1]. At that time, the field of carcinogen activation pathways was exploding, but all the attention was focused on cytochrome P450-dependent pathways of oxidation. Furthermore, reports by multiple groups revealed that B[a]P metabolic activation was a three-step process in which the molecule was first oxygenated to the 7,8-oxide, which was then hydrated to B[a] P-7,8-diol. Oxidation of B[a]P-7,8-diol yields the dihydrodiolepoxide (BPDE), which was the ultimate carcinogenic form (Fig. 2). P450 enzymes had been shown to catalyze both oxidation events. I’d heard Harry Gelboin talk about the importance of the second oxidation in B[a]P activation and Don Jerina describe the stereochemistry of the individual metabolic steps as well as the critical role of stereochemistry in the reactivity of the isomeric BPDEs with DNA and their resultant mutagenicity [2,3]. That provided some introduction to mutagenicity assays and among them, the Ames test. So, when I learned of Bruce’s lecture at the U of M, I drove some of my students to Ann Arbor to listen. Bruce is a great entertainer and very engaging speaker, E-mail address: [email protected]. https://doi.org/10.1016/j.mrgentox.2019.06.006 Received 24 June 2019; Accepted 28 June 2019 Available online 04 July 2019 1383-5718/ © 2019 Elsevier B.V. All rights reserved.
so it was worth the drive. He spoke of the development of the Ames test and the importance of incorporating P450 preparations from rat liver into the incubations of organic compounds with the Salmonella tester strains to effect metabolic activation. These were Bruce’s environmental carcinogenesis days, so most of his attention was focused on man-made agents. It was an easy sell on a college campus, and the audience ate it up. I was very impressed with Bruce and with the simplicity and elegance of the Ames test. We continued our work on B[a]P and ultimately showed that PGHS oxidized it to a series of quinones (Fig. 2) [4]. As one of the first reports of the oxidation of a polycyclic aromatic hydrocarbon by an oxygenase that wasn’t a P450, this was exciting. But the relevance of quinone production to metabolic activation and DNA damage remained unclear. Years later, Ercole Cavalieri and Eleanor Rogan demonstrated that the formation of quinones from BaP was probably via the intermediacy of radical cations that could adduct the 7 position of dG and dA residues in DNA [5]. We shifted our attention to the possibility that PGHS could oxidize the proximate carcinogen, B[a]P-7,8-diol. We incubated ram seminal vesicle microsomes, which contain robust amounts of PGHS, with B[a] P-7,8-diol and added arachidonic acid to produce PGG2 in order to trigger peroxidase-mediated B[a]P-7,8-diol oxidation. However, rather than carry out extensive metabolite identification studies, we decided to swing for the fences and see if a mutagen could be detected in the incubation mixtures. If something strongly mutagenic was formed, we would then put in the effort to define the metabolic products. Thus, we decided to modify the Ames test by substituting ram seminal vesicle microsomes for rat liver microsomes. The idea was that if the enzyme in the microsomes oxidized B[a]P-7,8-diol to a diolepoxide, the epoxide would diffuse into the Salmonella, couple to DNA, and induce
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Fig. 1. Enzymatic activities of prostaglandin endoperoxide synthase (PGHS). The cyclooxygenase (COX) activity oxygenates arachidonic acid into the hydroperoxyendoperoxide, PGG2, then the peroxidase (PER) reduces the hydroperoxide of PGG2 to the alcohol of PGH2. The PER activity requires a reducing cosubstrate (AH2), which accounts for the oxidation of compounds unrelated to arachidonic acid during its conversion to PGH2.
Fig. 2. Some pathways of benzo[a]pyrene metabolism. B[a]P is oxygenated by P-450′s to arene oxides at the 1,2; 2,3; 4,5; 7,8; 9,10; and 11,12 positions. These arene oxides ring open to phenols or are hydrated by epoxide hydrolases to dihydrodiols. The example shown is for conversion of B[a]P to the 7,8-dihydrodiol (B[a]P-7,8-diol). B[a]P is also oxidized by P-450’s or PGHS to quinones. The 6,12-quinone is shown but other quinones are also formed. B[a]P7,8-diol represents the proximate mutagenic form of B[a]P. It is oxygenated to B [a]P-7,8-diol-9,10-epoxide, which represents the ultimate mutagenic form, by P-450’s or peroxyl radicals generated during the turnover of PGHS.
Fig. 3. Activation of B[a]P-7,8-diol to a potent mutagen by prostaglandin endoperoxide synthase in ram seminal vesicles. Reproduced from (6) with permission.
provided a stereochemical method with which to differentiate B[a]P7,8-diol oxidation by PGHS and P450. We also discovered that the oxidizing agent was actually a peroxyl free radical produced by lipid peroxidation [8]. Eventually, we used our stereochemical method to show that BaP-7,8-diol oxidation occurs in vivo, especially when neutrophils are recruited to sites of inflammation and activated to generate a respiratory burst [9]. This was the first report of the metabolic activation of a procarcinogen to an ultimate carcinogen by a pathway separate from a P-450-dependent oxidation. Since we had the Ames tester strains, we began using them for a range of projects in the lab. The first was to test the hypothesis that the lipid peroxidation product, malondialdehyde (MDA), was mutagenic (Fig. 4). MDA’s mutagenicity had been reported a few years earlier, but close inspection of the procedure used to isolate the chemically unstable molecule suggested some impurities might be responsible for the observed activity [10]. So, we devised three independent chemical synthesis methods to prepare MDA for biological testing. Head-to-head comparison revealed that the material prepared by all three methods yielded the same positive results in the Ames test, thereby confirming that MDA is, indeed, a mutagen [11]. Although these studies confirmed MDA’s mutagenicity, our preliminary studies yielded a hint that, as we had initially suspected, there might be a potent mutagenic impurity produced by the original method used to test MDA. That method involved the acid-catalyzed hydrolysis of tetraethoxypropane to MDA (Fig. 4). When we compared the mutagenicity of crude hydrolysates of tetraethoxypropane to the mutagenicity of purified MDA, we found that the crude hydrolysates were twice as mutagenic on a per MDA molar basis. Since MDA readily polymerizes, we passed the hydrolysates through a Sephadex LH-20 gel filtration column, hoping to separate the polymeric forms from MDA.
mutations. 1.1. Salmonella as a discovery tool We had no experience in bacteriology, but in his Ann Arbor lecture, Bruce said a high school student could do the Ames test, so we sent his lab a request for the standard set of test strains. We expected it would take a month or two to get the strains but were shocked when they arrived in the mail the week after we’d sent the request. Panic set in when we realized we had no idea what to do with the strains, but fortunately Diane Dennison, an undergraduate in the group, had some prior experience in a bacteriology lab. She ably grew the cultures up, and we were ready to incubate. Greg Reed, one of my graduate students and now at the University of Kansas, did the incubations with B[a]P7,8-diol, ram seminal vesicle microsomes, and arachidonic acid. I fully expected the experiment to be negative but was stunned when Greg walked into my office and showed me a plate with thousands of revertants (Fig. 3). I suggested a set of control experiments, but Greg, good student that he was, had already done them. They pointed definitively to the fact that when arachidonic acid was added to suspensions of ram seminal vesicles and B[a]P-7,8-diol, the latter was converted to a highly mutagenic species [6]. I still remember standing in the lab drinking in the moment with the knowledge and excitement that we’d made a significant discovery. That day, I also fully realized the power of bacterial genetics and the Ames test. We eventually discovered that the PGHS-dependent oxidation of B [a]P-7,8-diol did indeed produce a BPDE, but the stereochemistry was different than that observed when P450 was the catalyst [7]. This 2
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Fig. 4. Hydrolysis of tetraalkoxypropanes to malondialdehyde. The bis-acetal of tetraalkoxypropanes is hydrolyzed in a stepwise fashion. The first intermediate, 3,3dialkoxypropanal, is either further hydrolyzed to MDA or eliminates an alcohol to form the β-alkoxyacrolein. Crude hydrolyzates contain all these species. Although the β-alkoxyacrolein typically comprises ˜ 5% of the material, it represents the major mutagen in the crude mixture. Shown are β-ethoxyacrolein and β-methoxyacrolein. MDA is in equilibrium with its tautomer, β-hydroxyacrolein, and in aqueous solutions at physiological pH, β-hydroxyacrolein predominates.
product of the chemical synthesis was responsible for a significant portion of the mutagenicity of the crude hydrolysis solution [12]. Testing of different Salmonella strains revealed that the strain specificity of MDA and β-ethoxyacrolein mutagenicity is unusual. Neither compound is mutagenic in routine test strains such as TA98 or T100; rather, the “precursor” strain hisD3052 is most sensitive to their effects. A parental strain for TA98, hisD3052 detects frameshift mutagens. Elimination of mutagenicity of MDA or β-ethoxyacrolein by deletion of the nucleotide excision repair system, which was implemented as part of the development of TA98, is suggestive of a DNA-DNA cross-link as a premutagenic lesion. An extensive structure-activity study of the dependence of mutagenicity toward hisD3052 on chemical structure indicated that only compounds capable of undergoing two nucleophilic attacks by DNA were able to induce mutations [13]. This was also consistent with formation of an interstrand or intrastrand cross-link. However, further studies revealed that the ability of MDA to induce interstrand cross-links did not correlate with its ability to induce mutations [14]. We later showed that the primary adduct formed between MDA and DNA is the exocyclic deoxyguanosine adduct, M1dG, formation of which requires two nucleophilic attacks by the base on the aldehyde (Fig. 6) [15]. Thus, our data support the hypothesis that M1dG is the premutagenic lesion that leads to frameshift mutations in hisD3052. Fig. 5. Elution profile of mutagens produced during hydrolysis of tetraethoxypropane. The solid line depicts the elution of different compounds converted to tetraethoxypropane equivalents. The major peak corresponds to MDA. The dotted line depicts the mutagenicity of 100 μL aliquots of column fractions. The major peak does not correspond to MDA but coelutes with a sample of β-ethoxyacrolein. Reproduced from (12) with permission.
1.2. Off to Berkeley We continued using the Ames strains for various experiments in our laboratory, and publication of the results of the studies helped to get me promotion with tenure at Wayne State. A few years after achieving this
We tested each fraction for mutagenicity using the Ames test. Contrary to expectations, we found that the major mutagenic fractions did not coelute with polymeric forms of MDA nor did they coelute with MDA (Fig. 5). Rather, they eluted shortly after the major peak of MDA. When we tried to concentrate the active fractions by evaporation, the mutagenicity disappeared, suggesting that the mutagenic compound(s) was (were) either volatile or chemically unstable [12]. After some further detective work, we identified the active constituent as β-ethoxyacrolein, which is an incomplete hydrolysis product of tetraethoxypropane (Fig. 4). β-Ethoxyacrolein contains the same acrolein functional group as β-hydroxyacrolein, the tautomeric form of MDA that exists in polar solvents like water. However, ethoxy is a much better leaving group than MDA’s hydroxyl, which is deprotonated to an enolate at pH’s > 4.6. Thus, β-ethoxyacrolein is some 25-fold more mutagenic than MDA in side-by-side comparisons. Similar observations were made with the other routine precursor, tetramethoxypropane. βMethoxyacrolein is produced in ˜ 5% yield as a side product and is 35fold more mutagenic than MDA. Thus, a relatively low abundance side
Fig. 6. Structures of DNA adducts produced by malondialdehyde. MDA reacts with dG, dA, and dC to form one-to-one adducts abbreviated M1dG, M1dA, and M1dC, respectively. MDA also oligomerizes to form MDA dimers and trimers that react with dG, dA, and dC (structures not shown). MDA also reacts with duplex DNA to form interstrand crosslinks that have not been structurally characterized. 3
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Fig. 7. Pathways of reversion of hisG428 by mutations at the site of the ochre codon (forward mutation). DNA sequence analysis of spontaneous revertants of TA103 [hisG428/ pKM101]. Base pair substitution mutations were to glutamine (CAA), leucine (TTA), and lysine (AAA). Deletion revertants 14 remove the ochre codon. Reproduced from (16) with permission.
career milestone, I became eligible for my first (and, as it has turned out, only) sabbatical leave. In considering possible sites, my attention quickly focused on Bruce’s lab. I’d met Bruce at a couple of meetings and was on good terms with him, so I gave him a call and asked if he’d be willing to have me join his lab for a sabbatical from January-June, 1983. He said he wasn’t sure if he had enough space but would check and get back in touch with me. I was puzzled by his response at the time, because I had a huge amount of lab space at Wayne, and it never occurred to me that he might be space-limited. This was rather naïve on my part, but several days later, he called back and said he had space, and it would be ok for me to come. I was thrilled to hear this, and my family began preparing for the trek west. It took about a week to drive out with our belongings loaded on top of the car in an X-cargo, which acted like a misaligned sail dragging against our forward progress. We arrived in Berkeley in late December, 1982, and it was love at first sight. We were living in an apartment about two blocks from campus and about five blocks from the Biochemistry Department. It was raining but warm, which was a huge improvement over the weather we left behind in Michigan. When the rain stopped on our first Sunday morning, the clouds lifted, and we discovered we had a beautiful view of the San Francisco Bay and Golden Gate Bridge. We were surrounded by good restaurants, lived right around the corner from the University Theatre (Three Stooges Film Festival here we come), and were down the hill from Grizzly Peak and Tilden Park with its great golf course and steam trains. About two weeks after we arrived, my son asked if we ever had to go back to Michigan. I had visions of being a gentleman scientist, decompressing from the rigors of teaching large numbers of undergraduates and enjoying the good life in this garden of earthly delights. Those fantasies faded after my first day in the lab. I went to Bruce’s office, and he greeted me warmly. We discussed possible projects, and he told me to talk with people in the lab about what they were working on to find something I was interested in. We then walked into the lab, and he introduced me to some of the students and staff. They were all nice, but there were more of them than I thought the lab could hold. Then Bruce walked me to a bench that had two pieces of tape spaced 3 feet apart with my name written on the tapes. This was my space, and as I looked around the lab, I realized this was a “full space.” I immediately understood why it had taken Bruce a couple of days to see if he could accommodate my request for a sabbatical. All faculty in the Biochemistry Department at Berkeley were assigned the same amount of space in Barker Hall regardless of who they were. About half of them were members of the National Academy of Sciences, so it was hard to argue that any one faculty member deserved more space than another. Bruce shared his lab with his wife, Giovanna, who was a very accomplished scientist in her own right. In fact, Giovanna very kindly shared her office with me during my sabbatical. Several years later, she was assigned her own lab space, which was highly deserved. Anyway, in 1983, two very active research groups
with a total of ˜ 35 people coexisted in something less than 2000 sq ft. There were two labs, each with two benches, separated by a small conference room. Some people worked shifts to mitigate the density issue, and everyone was assigned the same 3 feet of bench space. I went home that first night and told my wife, Nancy, that my plans for a leisurely six months had changed abruptly because I was taking up a full space and would need to do something productive with it. She just smiled and said she’d never figured I’d be spending much time outside the lab anyway. In truth, I loved working in the lab. The group members were very friendly and supportive, and the intellectual atmosphere in the Department and elsewhere at Berkeley was fantastic. Molecular biology was in its evolutionary stage, so there was a chance to learn sequencing and cloning, which was very exciting. I wasn’t serving on committees or preparing lectures and was able to delve into a project and focus on science. 1.3. Mechanisms of hisG428 mutagenesis For my sabbatical project, I chose to work with David Levin, now at Boston University, who had just developed a multi-copy plasmid-based Salmonella strain (TA102) to detect oxidative damage and was beginning to characterize the revertants generated by treatment with peroxide and other oxidants. The pAQ1 plasmid expressed by TA102 carried the hisG428 ochre mutation (CAA→TAA) of the hisG gene. The paired dTs in the mutant codon could serve as targets for oxidant modification to form thymidine glycol - at least that was the hypothesis. DNA sequence analysis revealed that there were three different classes of revertants resulting from mutation of hisG428 – in-frame deletions that removed the ochre codon, base substitutions at the ochre codon, or mutations to generate extragenic ochre suppressors (Fig. 7). The deletion mutations were resistant to the histidine analog, thiazolealanine, which inhibited the activity of the wild-type hisG-encoded enzyme but not the enzyme resulting from the deletion revertant. In contrast, revertants resulting from mutation of the ochre suppressors were hypersensitive to inhibition by thiazolealanine, and although the base pair substitution revertants were also sensitive, they could be easily differentiated from the ochre suppressors by radial streaking. Thus, this system provided a convenient phenotypic screen for the three major classes of revertants of hisG428 [16]. Spontaneous point mutations of hisG428 increased dramatically in strains containing the pKM101 plasmid. At the time, pKM101 was thought to contain genes for error-prone repair, but later it was shown to code for the translesion DNA polymerase, pol V. Interestingly, pKM101 did not increase the frequency of in-frame deletions. Deletion of uvrB, a gene encoding an enzyme in the nucleotide-excision repair pathway, also increased the frequency of point mutations in the presence or absence of pKM101, while also suppressing spontaneous inframe deletions. TA102 carried both a uvrB deletion and pKM101, in addition to a deep rough mutation that allowed hydrophobic compounds to permeate the cell wall [16]. For comparison, we 4
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characterized a strain (TA2638) containing the hisG428 ochre mutation in a single copy on the genome. These studies led to the development of the widely used TA104 tester strain that included genomic hisG428, uvrB-, and the deep rough mutation in addition to pKM101 (see below). Note that the availability of tester strains containing a single genomic copy of hisG428 enabled us to show that the use of a multi-copy plasmid containing the mutation in TA102 substantially increased the number of spontaneous revertants and especially the percentage of deletions in the presence or absence of pKM101. The 40-fold increase in deletion revertants roughly corresponded to the number of copies of the plasmid in the strains [16]. We screened multiple chemical mutagens for their effects on the hisG428 mutations carried either on the chromosome (TA2638/TA104) or on the multi-copy plasmid (TA102). Myriad different classes of compounds induced revertant frequencies many-fold over the spontaneous reversion rate, but interestingly, none of them induced in-frame deletions on the chromosomal copy of hisG428 of TA2638. In contrast, deletions represented a significant percentage of the revertants induced on the multi-copy plasmid contained in TA102. These experiments were an early attempt to characterize spontaneous or mutagen-induced reversions at the molecular level. They illustrated that multiple mechanisms exist for processing DNA damage and that the pattern of mutations detected by reversions in Salmonella are much more complex than simple alterations at or near the site of the forward mutations. They could also include mutations at completely different loci as revealed by the detection of mutations in extragenic ochre suppressors of hisG428 [16]. 1.4. Aldehyde mutagenesis Fig. 9. Reversion of several Ames tester strains by formaldehyde. Reproduced from (17).
Bruce has always welcomed a large number of bright undergraduates to do research in his lab. In fact, the Ames test was created by undergraduates as a side project without any NIH funding. Holly Hurd was a Berkeley freshman who worked with me during my sabbatical. We assigned her a project to screen a series of aldehydes for mutagenicity in different Salmonella tester strains. Among these aldehydes were the lipid peroxidation products, MDA, 4-hydroxyalkenals, and long chain saturated and unsaturated enals. In addition, dicarbonyls such as methylglyoxal and biacetyl were tested (Fig. 8). We were particularly interested in the response of the new strains, TA102 and TA104, described above. We designed this study as a simple screen, but it turned out to be quite interesting because we were able to examine a number of different aldehydes in a range of Salmonella strains that provided insights into the genetic requirements for sensitive mutagen detection. For example, formaldehyde was the only saturated aldehyde to exhibit mutagenicity in any of the strains, but its ability to induce reversions was dramatically higher in TA102 and TA104 than in the standard tester strains, TA97, TA98, and TA100 (Fig. 9). A series of α,β-unsaturated aldehydes (e.g., acrolein, crotonaldehyde) exhibited potent mutagenicity but only in TA104; they did not revert TA102 [17]. The α,β-unsaturated aldehydes induced revertants at low concentrations, but they also displayed sharp declines in concentrationdependence due to toxicity at higher concentrations. To mitigate the toxicity, we allowed the aldehydes to incubate with the bacteria for 1 h then added a high concentration of glutathione to scavenge remaining
compound. This greatly increased the sensitivity of the assays by extending the linear portion of the concentration-dependence curves and supported our hypothesis that the chemical reactions responsible for toxicity (e.g., reaction with sulfhydryl proteins) are different than those responsible for mutagenicity (reaction with nucleic acid). Using the “glutathione chase,” we discovered that 4-hydroxypentenal is mutagenic, which was the first such demonstration for a member of the hydroxynonenal family (Fig. 10). The longer chain enals could not be detected as mutagens, possibly because of their greater toxicity, even in experiments that incorporated the glutathione chase [17]. Methylglyoxal exhibited extremely high mutagenic potency in TA104, in some cases inducing over 10,000 revertants per plate (Fig. 11). The steep linearity of the concentration-dependence curves suggested that not only is methylglyoxal highly mutagenic but that it is not very toxic to certain Salmonella strains. The high activity of methylglyoxal is particularly interesting because it is a side product of glycolysis and is ubiquitously distributed in mammalian cells and tissues. Levels of methylglyoxal are controlled by the sequential action of glyoxalases I and II, which convert methylglyoxal to lactoylglutathione, then hydrolyze lactoylglutathione to lactate and glutathione, respectively [19]. Recent work from our laboratory at Vanderbilt has shown the existence of methylglyoxal adducts to histone proteins in chromatin from multiple mouse tissues [18]. Thus, it should be considered an important candidate for the induction of endogenous mutations in mammals. The importance of nucleotide excision repair and translesion DNA polymerases to methylglyoxal mutagenicity was revealed by comparing the number of revertants induced by compound exposure in TA2659, TA2638, and TA104. All three strains contain the hisG428 mutation but in different genetic backgrounds (Fig. 12). TA2659 contains a uvrB deletion but does not contain pKM101, whereas TA2638 contains pKM101 but an intact uvrB gene. TA104 has both a uvrB deletion and
Fig. 8. Structures of some of the aldehydes tested for mutagenicity in strains containing the hisG428 ochre mutation. 5
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Fig. 10. Reversion of TA104 by 4-hydroxypentenal with or without a glutathione chase. Reproduced from (17).
Fig. 12. Methyl glyoxal reversion of the hisG428 mutation in strains containing different DNA repair backgrounds. The strains are described in the text. Reproduced from (17).
Although its genetic background supported TA104’s high sensitivity to aldehydes, an intriguing aspect of this sensitivity remained a mystery. As noted above, the forward hisG428 mutation in TA104 produces an ochre codon (TAA) from a Gln codon (CAA), and the alterations that reverse it are all point mutations or in-frame deletions of the ochre codon. Thus, one would expect high sensitivity to mutagens that target dA or dT residues. In contrast, aldehydes preferentially react with dG residues in DNA. So why is TA104 so sensitive to aldehyde mutagenesis? Our finding that the hisG428 mutation can also be “reversed” by extragenic mutations in suppressor tRNAs provides a possible explanation. Mutation of a tRNA gene that contains a dG residue in the anti-codon to produce an ochre suppressor would enable agents that modify dG to induce mutations that would enable translation of the hisG gene with sufficient efficiency to support revertant growth. Thus, although the projects I worked on during my sabbatical in Bruce’s lab seemed very disparate, in the end they dovetailed nicely and provided evidence for the versatility of TA104 for detection of a diverse range of mutagens. They have also been well-received by the community. Our PNAS paper characterizing the pathways of reversion of hisG428 has been cited 113 times, and our paper in Mutation Research on aldehyde mutagenesis has been cited 521 times. So, I feel like I used my three feet of bench space pretty well. As our time in Berkeley was coming to an end, Bruce called me into his office and asked me for a favor. It seems he had taken a new graduate student, Geoff Kramer, but had no room in the lab for him. He asked if I would be willing to share my space with Geoff since I would be leaving in about a month. Realizing that most first-year students don’t come into lab that much and that I could coordinate times for him to use the bench, I said that of course I would share my space with him. Before I could utter another sound, Bruce sprang to his feet and rushed
Fig. 11. Reversion of TA104 by a series of dicarbonyl compounds. Reproduced from (17).
pKM101. The data revealed that pKM101 is nearly essential for methyglyoxal mutagenesis and that uvrB deletion, although important, is not essential. The combination of both factors in a single genetic background produces the great sensitivity of TA104 for detection of aldehyde mutagenesis [17]. 6
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Fig. 13. Sabbatical pictures. Clockwise from upper left, Bruce Ames, Giovanna Ferro-Luzzi Ames, Dorothy Maron, David Levin (right) and Mike Christman (left). Apologies to others from the lab for whom I lack pictures.
identify essential articles. Bruce would pour over this publication religiously and then request reprints across a bewildering array of fields. He read chemistry, genetics, medicine, and anything else that looked interesting. He would then use that knowledge to cross-fertilize and thereby generate novel or unconventional ideas. This was an important lesson to me in creativity. By exposing oneself to a broad range of concepts, one increases the probability of connecting seemingly disparate observations into a new idea. Indeed, Bruce is one of the most creative scientists I’ve ever met. Even though he was still doing a lot of work on identification of mutagens when I joined his lab, he was already transitioning to oxidative DNA damage and thinking about the role of oxidative degeneration of mitochondrial enzymes in aging. While I was in the lab, Fred Jacobson, now at Genentech, discovered the alkyl hydroperoxide reductase enzymes in Salmonella. By the time of my sabbatical, Bruce had shifted the focus of his work on mutagens and carcinogens from environmental agents as causes of cancer to naturally occurring compounds and endogenous metabolic products. This reflected his willingness to integrate knowledge from across the spectrum and to look at problems in toto. As a complement to his work on mutagens, he and Lois Swirsky Gold developed the Carcinogenic Potency Project database that ranked compounds based on their mutagenicity and their estimated intake by humans. Later in his career, he focused on micronutrients for the prevention of cancer and even developed a vitamin and micronutrient-rich nutrition bar. Bruce and Giovanna were excellent and warm hosts to us during our visit. We had multiple stimulating dinners with Berkeley faculty and an unforgettable reception at his home for Linus Pauling on the occasion of his three lectures on campus. The talk was always about science. That’s what impressed me so much about the environment in Barker Hall. Not only was the faculty very accomplished, they continued doing science at a high level throughout their careers. Jesse Rabinowitz was running the department in his late 60’s, Dan Koshland became editor of Science at 65, and Horace Barker was still coming into the building that bore his name to do work in his mid-80’s. Their love of and intensity for science was a tremendous inspiration for me and still is.
into the lab. I followed him and watched as he tore off another piece of tape and bisected my 3 feet of bench space into two plots of 18 in. I had a colony counter that was 14 in. wide, so that left me 2 in. on either side to do work on. Obviously, Bruce and I were using different definitions of “share”. 1.5. Impressions of Bruce Bruce and his lab were so different from what I had anticipated based on my casual acquaintance with him in advance of my sabbatical. His public lectures are always entertaining and have a spontaneous feel to them that led me to believe that Bruce was fairly laid back and casual. That impression faded rapidly as I looked around the lab. Bruce’s office was modest, as were all offices in Barker Hall, and his lab was compact, as I have mentioned. But everything was super-organized – it had to be. In addition to parsing out space, Bruce would patrol the lab looking for detritus that was lying about and would make sure people got rid of it. Core activities were supported by technical staff, and all the HPLCs were stacked on top of each other against one wall under the watchful eye of Rick Cathcart. And every Thursday evening, Dorothy Maron would pack up all the various tester strains that had been requested that week, so she could send them out. This focus on organization not only mitigated to some degree the limited amount of space, but it also enabled the lab members to concentrate on planning and doing good experiments rather than pouring agar plates, washing dishes, making solutions, etc. Well-planned experiments were a high priority for Bruce. He made no secret of the fact that when he was young, he hated doing experiments. He would plan them meticulously in order to incorporate every conceivable control so the experiment would generate definitive results on the first try. Only then would he go into the lab. It seemed this philosophy rubbed off on some of the group members. Bruce has no shortage of interesting ideas. He reads voraciously, and the shelves in his office were lined with binders of reprints. This was in the days before the internet when we all used Current Contents to 7
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1.6. Thanks, Bruce
[5] E. Cavalieri, E. Rogan, Role of radical cations in aromatic hydrocarbon carcinogenesis, Environ. Health Perspect. 64 (1985) 69–84, https://doi.org/10.1289/ehp. 856469. [6] L.J. Marnett, G.A. Reed, D.J. Dennison, Prostaglandin synthetase dependent activation of 7,8-dihydro-7,8-dihydroxy-benzo (a) pyrene to mutagenic derivativies, Biochem. Biophys. Res. Commun. 82 (1978) 210–216. [7] A. Panthananickal, P. Weller, L.J. Marnett, Stereoselectivity of the epoxidation of 7,8-dihydrobenzo[a]pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts, J. Biol. Chem. 258 (1983) 4411–4418. [8] L.J. Marnett, M.J. Bienkowski, Hydroperoxide-dependent oxygenation of trans-7,8dihydroxy-7,8-dihydro benzo[a]pyrene by ram seminal vesicle microsomes. Source of the oxygen, Biochem. Biophys. Res. Commun. 96 (1980) 639–647. [9] C. Ji, L.J. Marnett, Oxygen radical-dependent epoxidation of (7S,8S)-dihydroxy-7,8dihydrobenzo[a]pyrene in mouse skin in vivo. Stimulation by phorbol esters and inhibition by antiinflammatory steroids, J. Biol. Chem. 267 (1992) 17842–17848. [10] L.J. Marnett, M.J. Bienkowski, M. Raban, M.A. Tuttle, Studies of the hydrolysis of 14C-labeled tetraethoxypropane to malondialdehyde, Anal. Biochem. 99 (1979) 458–463. [11] A.K. Basu, L.J. Marnett, Unequivocal demonstration that malondialdehyde is a mutagen, Carcinogenesis 4 (1983) 331–333, https://doi.org/10.1093/carcin/4.3. 331. [12] L.J. Marnett, M.A. Tuttle, Comparison of the mutagenicities of malondialdehyde and the side products formed during its chemical synthesis, Cancer Res. 40 (1980) 276–282. [13] A.K. Basu, L.J. Marnett, Molecular requirements for the mutagenicity of malondialdehyde and related acroleins, Cancer Res. 44 (1984) 2848–2854. [14] A.K. Basu, L.J. Marnett, L.J. Romano, Dissociation of malondialdehyde mutagenicity in Salmonella typhimurium from its ability to induce interstrand DNA crosslinks, Mutat. Res. 129 (1984) 39–46. [15] A.K. Basu, S.M. O’Hara, P. Valladier, K. Stone, O. Mols, L.J. Marnett, Identification of adducts formed by reaction of guanine nucleosides with malondialdehyde and structurally related aldehydes, Chem. Res. Toxicol. 1 (1988) 53–59. [16] D.E. Levin, L.J. Marnett, B.N. Ames, Spontaneous and mutagen-induced deletions: mechanistic studies in Salmonella tester strain TA102, Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 4457–4461, https://doi.org/10.1073/pnas.81.14.4457. [17] L.J. Marnett, H.K. Hurd, M.C. Hollstein, D.E. Levin, H. Esterbauer, B.N. Ames, Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104, Mutat. Res. 148 (1985) 25–34. [18] J.J. Galligan, J.A. Wepy, M.D. Streeter, P.J. Kingsley, M.M. Mitchener, O.R. Wauchope, W.N. Beavers, K.L. Rose, T. Wang, D.A. Spiegel, et al., Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) 9228–9233, https://doi.org/ 10.1073/pnas.1802901115. [19] N. Rabbani, M. Xue, P.J. Thornalley, Activity, regulation, copy number and function in the glyoxalase system, Biochem. Soc. Trans. 42 (2014) 419–424, https://doi.org/ 10.1042/BST20140008.
My sabbatical was one of the most enjoyable experiences of my scientific career. Beyond Bruce and many senior faculty, I met many talented young students and postdocs including David Levin, Mike Christman, Fred Jacobson, Betty Schwiers, Monica Hollstein and Rick Cathcart (Fig. 13). Martyn Smith had just started his career on the faculty, and we became fast friends. The environment in the lab and the community was wonderful and fun. I stayed in touch with Bruce throughout the years returning for his 60th birthday celebration, seeing him at meetings, and chatting occasionally by phone. In every single exchange, at some point in the conversation, Bruce will lower his voice, look intently at me and exclaim “I’m doing the best research of my life!” He hasn’t lost his enthusiasm for life and his joy in doing and thinking about science. He is truly an inspiration to all who have had the privilege of knowing him and interacting with him. I am fortunate to have been one of them. Thank you, Bruce. Acknowledgements Work in the Marnett laboratory on DNA damage and mutation has been supported continuously by research grant from the National Cancer Institute. Current support is provided by CA87819. We are grateful to Carol Rouzer for editorial comments References [1] L.J. Marnett, P. Wlodawer, B. Samuelsson, Co-oxygenation of organic substrates by the prostaglandin synthetase of sheep vesicular gland, J. Biol. Chem. 250 (1975) 8510–8517. [2] A.H. Conney, R.L. Chang, X.X. Cui, M. Schiltz, H. Yagi, D.M. Jerina, S.J. Wei, Dosedependent differences in the profile of mutations induced by carcinogenic (R,S,S,R) bay- and fjord-region diol epoxides of polycyclic aromatic hydrocarbons, Adv. Exp. Med. Biol. 500 (2001) 697–707. [3] H.V. Gelboin, Benzo[alpha]pyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes, Physiol. Rev. 60 (1980) 1107–1166, https://doi.org/10.1152/physrev.1980.60.4.1107. [4] L.J. Marnett, G.A. Reed, J.T. Johnson, Prostaglandin synthetase dependent benzo(a) pyrene oxidation: products of the oxication and inhibition of their formation by antioxidants, Biochem. Biophys. Res. Commun. 79 (1977) 569–576.
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Minireview
Adventures with Bruce Ames and the Ames test Lawrence J. Marnett
T
Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville TN 37240-7914, United States
ARTICLE INFO
ABSTRACT
Keywords: Ames Mutagenesis Aldehydes Mechanism Sabbatical
Bruce Ames has had an enormous impact on human health by developing facile methods for the identification of mutagens. This research also provided important insights into the relationship between mutagenesis and carcinogenesis. Bruce is a highly innovative and creative individual who has followed his interests across disciplines into diverse fields of inquiry. The present author had the pleasure of spending a sabbatical in the Ames lab and utilized the Ames test in multiple aspects of his research. He describes both in this honorific to Bruce on the occasion of his 90th birthday.
1. Introduction to the Ames test My first encounter with Bruce Ames was at a seminar at the University of Michigan in the late 1970’s. I was an Assistant Professor on the Chemistry faculty at Wayne State University, and our laboratory had gotten interested in chemical carcinogenesis through our studies with the enzyme prostaglandin H synthase (PGHS, a.k.a. cyclooxygenase). PGHS catalyzes two reactions, thebis-dioxygenation of arachidonic acid to yield the hydroperoxy-endoperoxide prostaglandin G2 (PGG2) and the reduction of PGG2to the corresponding hydroxy-endoperoxide PGH2(Fig. 1). We had discovered that the peroxidase activity of PGHS oxidizes the carcinogen benzo[a]pyrene (B[a]P) during the course of hydroperoxide reduction and were testing the hypothesis that this reaction resulted in B[a]P’s metabolic activation [1]. At that time, the field of carcinogen activation pathways was exploding, but all the attention was focused on cytochrome P450-dependent pathways of oxidation. Furthermore, reports by multiple groups revealed that B[a]P metabolic activation was a three-step process in which the molecule was first oxygenated to the 7,8-oxide, which was then hydrated to B[a] P-7,8-diol. Oxidation of B[a]P-7,8-diol yields the dihydrodiolepoxide (BPDE), which was the ultimate carcinogenic form (Fig. 2). P450 enzymes had been shown to catalyze both oxidation events. I’d heard Harry Gelboin talk about the importance of the second oxidation in B[a]P activation and Don Jerina describe the stereochemistry of the individual metabolic steps as well as the critical role of stereochemistry in the reactivity of the isomeric BPDEs with DNA and their resultant mutagenicity [2,3]. That provided some introduction to mutagenicity assays and among them, the Ames test. So, when I learned of Bruce’s lecture at the U of M, I drove some of my students to Ann Arbor to listen. Bruce is a great entertainer and very engaging speaker, E-mail address: [email protected]. https://doi.org/10.1016/j.mrgentox.2019.06.006 Received 24 June 2019; Accepted 28 June 2019 Available online 04 July 2019 1383-5718/ © 2019 Elsevier B.V. All rights reserved.
so it was worth the drive. He spoke of the development of the Ames test and the importance of incorporating P450 preparations from rat liver into the incubations of organic compounds with the Salmonella tester strains to effect metabolic activation. These were Bruce’s environmental carcinogenesis days, so most of his attention was focused on man-made agents. It was an easy sell on a college campus, and the audience ate it up. I was very impressed with Bruce and with the simplicity and elegance of the Ames test. We continued our work on B[a]P and ultimately showed that PGHS oxidized it to a series of quinones (Fig. 2) [4]. As one of the first reports of the oxidation of a polycyclic aromatic hydrocarbon by an oxygenase that wasn’t a P450, this was exciting. But the relevance of quinone production to metabolic activation and DNA damage remained unclear. Years later, Ercole Cavalieri and Eleanor Rogan demonstrated that the formation of quinones from BaP was probably via the intermediacy of radical cations that could adduct the 7 position of dG and dA residues in DNA [5]. We shifted our attention to the possibility that PGHS could oxidize the proximate carcinogen, B[a]P-7,8-diol. We incubated ram seminal vesicle microsomes, which contain robust amounts of PGHS, with B[a] P-7,8-diol and added arachidonic acid to produce PGG2 in order to trigger peroxidase-mediated B[a]P-7,8-diol oxidation. However, rather than carry out extensive metabolite identification studies, we decided to swing for the fences and see if a mutagen could be detected in the incubation mixtures. If something strongly mutagenic was formed, we would then put in the effort to define the metabolic products. Thus, we decided to modify the Ames test by substituting ram seminal vesicle microsomes for rat liver microsomes. The idea was that if the enzyme in the microsomes oxidized B[a]P-7,8-diol to a diolepoxide, the epoxide would diffuse into the Salmonella, couple to DNA, and induce
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Fig. 1. Enzymatic activities of prostaglandin endoperoxide synthase (PGHS). The cyclooxygenase (COX) activity oxygenates arachidonic acid into the hydroperoxyendoperoxide, PGG2, then the peroxidase (PER) reduces the hydroperoxide of PGG2 to the alcohol of PGH2. The PER activity requires a reducing cosubstrate (AH2), which accounts for the oxidation of compounds unrelated to arachidonic acid during its conversion to PGH2.
Fig. 2. Some pathways of benzo[a]pyrene metabolism. B[a]P is oxygenated by P-450′s to arene oxides at the 1,2; 2,3; 4,5; 7,8; 9,10; and 11,12 positions. These arene oxides ring open to phenols or are hydrated by epoxide hydrolases to dihydrodiols. The example shown is for conversion of B[a]P to the 7,8-dihydrodiol (B[a]P-7,8-diol). B[a]P is also oxidized by P-450’s or PGHS to quinones. The 6,12-quinone is shown but other quinones are also formed. B[a]P7,8-diol represents the proximate mutagenic form of B[a]P. It is oxygenated to B [a]P-7,8-diol-9,10-epoxide, which represents the ultimate mutagenic form, by P-450’s or peroxyl radicals generated during the turnover of PGHS.
Fig. 3. Activation of B[a]P-7,8-diol to a potent mutagen by prostaglandin endoperoxide synthase in ram seminal vesicles. Reproduced from (6) with permission.
provided a stereochemical method with which to differentiate B[a]P7,8-diol oxidation by PGHS and P450. We also discovered that the oxidizing agent was actually a peroxyl free radical produced by lipid peroxidation [8]. Eventually, we used our stereochemical method to show that BaP-7,8-diol oxidation occurs in vivo, especially when neutrophils are recruited to sites of inflammation and activated to generate a respiratory burst [9]. This was the first report of the metabolic activation of a procarcinogen to an ultimate carcinogen by a pathway separate from a P-450-dependent oxidation. Since we had the Ames tester strains, we began using them for a range of projects in the lab. The first was to test the hypothesis that the lipid peroxidation product, malondialdehyde (MDA), was mutagenic (Fig. 4). MDA’s mutagenicity had been reported a few years earlier, but close inspection of the procedure used to isolate the chemically unstable molecule suggested some impurities might be responsible for the observed activity [10]. So, we devised three independent chemical synthesis methods to prepare MDA for biological testing. Head-to-head comparison revealed that the material prepared by all three methods yielded the same positive results in the Ames test, thereby confirming that MDA is, indeed, a mutagen [11]. Although these studies confirmed MDA’s mutagenicity, our preliminary studies yielded a hint that, as we had initially suspected, there might be a potent mutagenic impurity produced by the original method used to test MDA. That method involved the acid-catalyzed hydrolysis of tetraethoxypropane to MDA (Fig. 4). When we compared the mutagenicity of crude hydrolysates of tetraethoxypropane to the mutagenicity of purified MDA, we found that the crude hydrolysates were twice as mutagenic on a per MDA molar basis. Since MDA readily polymerizes, we passed the hydrolysates through a Sephadex LH-20 gel filtration column, hoping to separate the polymeric forms from MDA.
mutations. 1.1. Salmonella as a discovery tool We had no experience in bacteriology, but in his Ann Arbor lecture, Bruce said a high school student could do the Ames test, so we sent his lab a request for the standard set of test strains. We expected it would take a month or two to get the strains but were shocked when they arrived in the mail the week after we’d sent the request. Panic set in when we realized we had no idea what to do with the strains, but fortunately Diane Dennison, an undergraduate in the group, had some prior experience in a bacteriology lab. She ably grew the cultures up, and we were ready to incubate. Greg Reed, one of my graduate students and now at the University of Kansas, did the incubations with B[a]P7,8-diol, ram seminal vesicle microsomes, and arachidonic acid. I fully expected the experiment to be negative but was stunned when Greg walked into my office and showed me a plate with thousands of revertants (Fig. 3). I suggested a set of control experiments, but Greg, good student that he was, had already done them. They pointed definitively to the fact that when arachidonic acid was added to suspensions of ram seminal vesicles and B[a]P-7,8-diol, the latter was converted to a highly mutagenic species [6]. I still remember standing in the lab drinking in the moment with the knowledge and excitement that we’d made a significant discovery. That day, I also fully realized the power of bacterial genetics and the Ames test. We eventually discovered that the PGHS-dependent oxidation of B [a]P-7,8-diol did indeed produce a BPDE, but the stereochemistry was different than that observed when P450 was the catalyst [7]. This 2
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Fig. 4. Hydrolysis of tetraalkoxypropanes to malondialdehyde. The bis-acetal of tetraalkoxypropanes is hydrolyzed in a stepwise fashion. The first intermediate, 3,3dialkoxypropanal, is either further hydrolyzed to MDA or eliminates an alcohol to form the β-alkoxyacrolein. Crude hydrolyzates contain all these species. Although the β-alkoxyacrolein typically comprises ˜ 5% of the material, it represents the major mutagen in the crude mixture. Shown are β-ethoxyacrolein and β-methoxyacrolein. MDA is in equilibrium with its tautomer, β-hydroxyacrolein, and in aqueous solutions at physiological pH, β-hydroxyacrolein predominates.
product of the chemical synthesis was responsible for a significant portion of the mutagenicity of the crude hydrolysis solution [12]. Testing of different Salmonella strains revealed that the strain specificity of MDA and β-ethoxyacrolein mutagenicity is unusual. Neither compound is mutagenic in routine test strains such as TA98 or T100; rather, the “precursor” strain hisD3052 is most sensitive to their effects. A parental strain for TA98, hisD3052 detects frameshift mutagens. Elimination of mutagenicity of MDA or β-ethoxyacrolein by deletion of the nucleotide excision repair system, which was implemented as part of the development of TA98, is suggestive of a DNA-DNA cross-link as a premutagenic lesion. An extensive structure-activity study of the dependence of mutagenicity toward hisD3052 on chemical structure indicated that only compounds capable of undergoing two nucleophilic attacks by DNA were able to induce mutations [13]. This was also consistent with formation of an interstrand or intrastrand cross-link. However, further studies revealed that the ability of MDA to induce interstrand cross-links did not correlate with its ability to induce mutations [14]. We later showed that the primary adduct formed between MDA and DNA is the exocyclic deoxyguanosine adduct, M1dG, formation of which requires two nucleophilic attacks by the base on the aldehyde (Fig. 6) [15]. Thus, our data support the hypothesis that M1dG is the premutagenic lesion that leads to frameshift mutations in hisD3052. Fig. 5. Elution profile of mutagens produced during hydrolysis of tetraethoxypropane. The solid line depicts the elution of different compounds converted to tetraethoxypropane equivalents. The major peak corresponds to MDA. The dotted line depicts the mutagenicity of 100 μL aliquots of column fractions. The major peak does not correspond to MDA but coelutes with a sample of β-ethoxyacrolein. Reproduced from (12) with permission.
1.2. Off to Berkeley We continued using the Ames strains for various experiments in our laboratory, and publication of the results of the studies helped to get me promotion with tenure at Wayne State. A few years after achieving this
We tested each fraction for mutagenicity using the Ames test. Contrary to expectations, we found that the major mutagenic fractions did not coelute with polymeric forms of MDA nor did they coelute with MDA (Fig. 5). Rather, they eluted shortly after the major peak of MDA. When we tried to concentrate the active fractions by evaporation, the mutagenicity disappeared, suggesting that the mutagenic compound(s) was (were) either volatile or chemically unstable [12]. After some further detective work, we identified the active constituent as β-ethoxyacrolein, which is an incomplete hydrolysis product of tetraethoxypropane (Fig. 4). β-Ethoxyacrolein contains the same acrolein functional group as β-hydroxyacrolein, the tautomeric form of MDA that exists in polar solvents like water. However, ethoxy is a much better leaving group than MDA’s hydroxyl, which is deprotonated to an enolate at pH’s > 4.6. Thus, β-ethoxyacrolein is some 25-fold more mutagenic than MDA in side-by-side comparisons. Similar observations were made with the other routine precursor, tetramethoxypropane. βMethoxyacrolein is produced in ˜ 5% yield as a side product and is 35fold more mutagenic than MDA. Thus, a relatively low abundance side
Fig. 6. Structures of DNA adducts produced by malondialdehyde. MDA reacts with dG, dA, and dC to form one-to-one adducts abbreviated M1dG, M1dA, and M1dC, respectively. MDA also oligomerizes to form MDA dimers and trimers that react with dG, dA, and dC (structures not shown). MDA also reacts with duplex DNA to form interstrand crosslinks that have not been structurally characterized. 3
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Fig. 7. Pathways of reversion of hisG428 by mutations at the site of the ochre codon (forward mutation). DNA sequence analysis of spontaneous revertants of TA103 [hisG428/ pKM101]. Base pair substitution mutations were to glutamine (CAA), leucine (TTA), and lysine (AAA). Deletion revertants 14 remove the ochre codon. Reproduced from (16) with permission.
career milestone, I became eligible for my first (and, as it has turned out, only) sabbatical leave. In considering possible sites, my attention quickly focused on Bruce’s lab. I’d met Bruce at a couple of meetings and was on good terms with him, so I gave him a call and asked if he’d be willing to have me join his lab for a sabbatical from January-June, 1983. He said he wasn’t sure if he had enough space but would check and get back in touch with me. I was puzzled by his response at the time, because I had a huge amount of lab space at Wayne, and it never occurred to me that he might be space-limited. This was rather naïve on my part, but several days later, he called back and said he had space, and it would be ok for me to come. I was thrilled to hear this, and my family began preparing for the trek west. It took about a week to drive out with our belongings loaded on top of the car in an X-cargo, which acted like a misaligned sail dragging against our forward progress. We arrived in Berkeley in late December, 1982, and it was love at first sight. We were living in an apartment about two blocks from campus and about five blocks from the Biochemistry Department. It was raining but warm, which was a huge improvement over the weather we left behind in Michigan. When the rain stopped on our first Sunday morning, the clouds lifted, and we discovered we had a beautiful view of the San Francisco Bay and Golden Gate Bridge. We were surrounded by good restaurants, lived right around the corner from the University Theatre (Three Stooges Film Festival here we come), and were down the hill from Grizzly Peak and Tilden Park with its great golf course and steam trains. About two weeks after we arrived, my son asked if we ever had to go back to Michigan. I had visions of being a gentleman scientist, decompressing from the rigors of teaching large numbers of undergraduates and enjoying the good life in this garden of earthly delights. Those fantasies faded after my first day in the lab. I went to Bruce’s office, and he greeted me warmly. We discussed possible projects, and he told me to talk with people in the lab about what they were working on to find something I was interested in. We then walked into the lab, and he introduced me to some of the students and staff. They were all nice, but there were more of them than I thought the lab could hold. Then Bruce walked me to a bench that had two pieces of tape spaced 3 feet apart with my name written on the tapes. This was my space, and as I looked around the lab, I realized this was a “full space.” I immediately understood why it had taken Bruce a couple of days to see if he could accommodate my request for a sabbatical. All faculty in the Biochemistry Department at Berkeley were assigned the same amount of space in Barker Hall regardless of who they were. About half of them were members of the National Academy of Sciences, so it was hard to argue that any one faculty member deserved more space than another. Bruce shared his lab with his wife, Giovanna, who was a very accomplished scientist in her own right. In fact, Giovanna very kindly shared her office with me during my sabbatical. Several years later, she was assigned her own lab space, which was highly deserved. Anyway, in 1983, two very active research groups
with a total of ˜ 35 people coexisted in something less than 2000 sq ft. There were two labs, each with two benches, separated by a small conference room. Some people worked shifts to mitigate the density issue, and everyone was assigned the same 3 feet of bench space. I went home that first night and told my wife, Nancy, that my plans for a leisurely six months had changed abruptly because I was taking up a full space and would need to do something productive with it. She just smiled and said she’d never figured I’d be spending much time outside the lab anyway. In truth, I loved working in the lab. The group members were very friendly and supportive, and the intellectual atmosphere in the Department and elsewhere at Berkeley was fantastic. Molecular biology was in its evolutionary stage, so there was a chance to learn sequencing and cloning, which was very exciting. I wasn’t serving on committees or preparing lectures and was able to delve into a project and focus on science. 1.3. Mechanisms of hisG428 mutagenesis For my sabbatical project, I chose to work with David Levin, now at Boston University, who had just developed a multi-copy plasmid-based Salmonella strain (TA102) to detect oxidative damage and was beginning to characterize the revertants generated by treatment with peroxide and other oxidants. The pAQ1 plasmid expressed by TA102 carried the hisG428 ochre mutation (CAA→TAA) of the hisG gene. The paired dTs in the mutant codon could serve as targets for oxidant modification to form thymidine glycol - at least that was the hypothesis. DNA sequence analysis revealed that there were three different classes of revertants resulting from mutation of hisG428 – in-frame deletions that removed the ochre codon, base substitutions at the ochre codon, or mutations to generate extragenic ochre suppressors (Fig. 7). The deletion mutations were resistant to the histidine analog, thiazolealanine, which inhibited the activity of the wild-type hisG-encoded enzyme but not the enzyme resulting from the deletion revertant. In contrast, revertants resulting from mutation of the ochre suppressors were hypersensitive to inhibition by thiazolealanine, and although the base pair substitution revertants were also sensitive, they could be easily differentiated from the ochre suppressors by radial streaking. Thus, this system provided a convenient phenotypic screen for the three major classes of revertants of hisG428 [16]. Spontaneous point mutations of hisG428 increased dramatically in strains containing the pKM101 plasmid. At the time, pKM101 was thought to contain genes for error-prone repair, but later it was shown to code for the translesion DNA polymerase, pol V. Interestingly, pKM101 did not increase the frequency of in-frame deletions. Deletion of uvrB, a gene encoding an enzyme in the nucleotide-excision repair pathway, also increased the frequency of point mutations in the presence or absence of pKM101, while also suppressing spontaneous inframe deletions. TA102 carried both a uvrB deletion and pKM101, in addition to a deep rough mutation that allowed hydrophobic compounds to permeate the cell wall [16]. For comparison, we 4
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characterized a strain (TA2638) containing the hisG428 ochre mutation in a single copy on the genome. These studies led to the development of the widely used TA104 tester strain that included genomic hisG428, uvrB-, and the deep rough mutation in addition to pKM101 (see below). Note that the availability of tester strains containing a single genomic copy of hisG428 enabled us to show that the use of a multi-copy plasmid containing the mutation in TA102 substantially increased the number of spontaneous revertants and especially the percentage of deletions in the presence or absence of pKM101. The 40-fold increase in deletion revertants roughly corresponded to the number of copies of the plasmid in the strains [16]. We screened multiple chemical mutagens for their effects on the hisG428 mutations carried either on the chromosome (TA2638/TA104) or on the multi-copy plasmid (TA102). Myriad different classes of compounds induced revertant frequencies many-fold over the spontaneous reversion rate, but interestingly, none of them induced in-frame deletions on the chromosomal copy of hisG428 of TA2638. In contrast, deletions represented a significant percentage of the revertants induced on the multi-copy plasmid contained in TA102. These experiments were an early attempt to characterize spontaneous or mutagen-induced reversions at the molecular level. They illustrated that multiple mechanisms exist for processing DNA damage and that the pattern of mutations detected by reversions in Salmonella are much more complex than simple alterations at or near the site of the forward mutations. They could also include mutations at completely different loci as revealed by the detection of mutations in extragenic ochre suppressors of hisG428 [16]. 1.4. Aldehyde mutagenesis Fig. 9. Reversion of several Ames tester strains by formaldehyde. Reproduced from (17).
Bruce has always welcomed a large number of bright undergraduates to do research in his lab. In fact, the Ames test was created by undergraduates as a side project without any NIH funding. Holly Hurd was a Berkeley freshman who worked with me during my sabbatical. We assigned her a project to screen a series of aldehydes for mutagenicity in different Salmonella tester strains. Among these aldehydes were the lipid peroxidation products, MDA, 4-hydroxyalkenals, and long chain saturated and unsaturated enals. In addition, dicarbonyls such as methylglyoxal and biacetyl were tested (Fig. 8). We were particularly interested in the response of the new strains, TA102 and TA104, described above. We designed this study as a simple screen, but it turned out to be quite interesting because we were able to examine a number of different aldehydes in a range of Salmonella strains that provided insights into the genetic requirements for sensitive mutagen detection. For example, formaldehyde was the only saturated aldehyde to exhibit mutagenicity in any of the strains, but its ability to induce reversions was dramatically higher in TA102 and TA104 than in the standard tester strains, TA97, TA98, and TA100 (Fig. 9). A series of α,β-unsaturated aldehydes (e.g., acrolein, crotonaldehyde) exhibited potent mutagenicity but only in TA104; they did not revert TA102 [17]. The α,β-unsaturated aldehydes induced revertants at low concentrations, but they also displayed sharp declines in concentrationdependence due to toxicity at higher concentrations. To mitigate the toxicity, we allowed the aldehydes to incubate with the bacteria for 1 h then added a high concentration of glutathione to scavenge remaining
compound. This greatly increased the sensitivity of the assays by extending the linear portion of the concentration-dependence curves and supported our hypothesis that the chemical reactions responsible for toxicity (e.g., reaction with sulfhydryl proteins) are different than those responsible for mutagenicity (reaction with nucleic acid). Using the “glutathione chase,” we discovered that 4-hydroxypentenal is mutagenic, which was the first such demonstration for a member of the hydroxynonenal family (Fig. 10). The longer chain enals could not be detected as mutagens, possibly because of their greater toxicity, even in experiments that incorporated the glutathione chase [17]. Methylglyoxal exhibited extremely high mutagenic potency in TA104, in some cases inducing over 10,000 revertants per plate (Fig. 11). The steep linearity of the concentration-dependence curves suggested that not only is methylglyoxal highly mutagenic but that it is not very toxic to certain Salmonella strains. The high activity of methylglyoxal is particularly interesting because it is a side product of glycolysis and is ubiquitously distributed in mammalian cells and tissues. Levels of methylglyoxal are controlled by the sequential action of glyoxalases I and II, which convert methylglyoxal to lactoylglutathione, then hydrolyze lactoylglutathione to lactate and glutathione, respectively [19]. Recent work from our laboratory at Vanderbilt has shown the existence of methylglyoxal adducts to histone proteins in chromatin from multiple mouse tissues [18]. Thus, it should be considered an important candidate for the induction of endogenous mutations in mammals. The importance of nucleotide excision repair and translesion DNA polymerases to methylglyoxal mutagenicity was revealed by comparing the number of revertants induced by compound exposure in TA2659, TA2638, and TA104. All three strains contain the hisG428 mutation but in different genetic backgrounds (Fig. 12). TA2659 contains a uvrB deletion but does not contain pKM101, whereas TA2638 contains pKM101 but an intact uvrB gene. TA104 has both a uvrB deletion and
Fig. 8. Structures of some of the aldehydes tested for mutagenicity in strains containing the hisG428 ochre mutation. 5
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Fig. 10. Reversion of TA104 by 4-hydroxypentenal with or without a glutathione chase. Reproduced from (17).
Fig. 12. Methyl glyoxal reversion of the hisG428 mutation in strains containing different DNA repair backgrounds. The strains are described in the text. Reproduced from (17).
Although its genetic background supported TA104’s high sensitivity to aldehydes, an intriguing aspect of this sensitivity remained a mystery. As noted above, the forward hisG428 mutation in TA104 produces an ochre codon (TAA) from a Gln codon (CAA), and the alterations that reverse it are all point mutations or in-frame deletions of the ochre codon. Thus, one would expect high sensitivity to mutagens that target dA or dT residues. In contrast, aldehydes preferentially react with dG residues in DNA. So why is TA104 so sensitive to aldehyde mutagenesis? Our finding that the hisG428 mutation can also be “reversed” by extragenic mutations in suppressor tRNAs provides a possible explanation. Mutation of a tRNA gene that contains a dG residue in the anti-codon to produce an ochre suppressor would enable agents that modify dG to induce mutations that would enable translation of the hisG gene with sufficient efficiency to support revertant growth. Thus, although the projects I worked on during my sabbatical in Bruce’s lab seemed very disparate, in the end they dovetailed nicely and provided evidence for the versatility of TA104 for detection of a diverse range of mutagens. They have also been well-received by the community. Our PNAS paper characterizing the pathways of reversion of hisG428 has been cited 113 times, and our paper in Mutation Research on aldehyde mutagenesis has been cited 521 times. So, I feel like I used my three feet of bench space pretty well. As our time in Berkeley was coming to an end, Bruce called me into his office and asked me for a favor. It seems he had taken a new graduate student, Geoff Kramer, but had no room in the lab for him. He asked if I would be willing to share my space with Geoff since I would be leaving in about a month. Realizing that most first-year students don’t come into lab that much and that I could coordinate times for him to use the bench, I said that of course I would share my space with him. Before I could utter another sound, Bruce sprang to his feet and rushed
Fig. 11. Reversion of TA104 by a series of dicarbonyl compounds. Reproduced from (17).
pKM101. The data revealed that pKM101 is nearly essential for methyglyoxal mutagenesis and that uvrB deletion, although important, is not essential. The combination of both factors in a single genetic background produces the great sensitivity of TA104 for detection of aldehyde mutagenesis [17]. 6
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Fig. 13. Sabbatical pictures. Clockwise from upper left, Bruce Ames, Giovanna Ferro-Luzzi Ames, Dorothy Maron, David Levin (right) and Mike Christman (left). Apologies to others from the lab for whom I lack pictures.
identify essential articles. Bruce would pour over this publication religiously and then request reprints across a bewildering array of fields. He read chemistry, genetics, medicine, and anything else that looked interesting. He would then use that knowledge to cross-fertilize and thereby generate novel or unconventional ideas. This was an important lesson to me in creativity. By exposing oneself to a broad range of concepts, one increases the probability of connecting seemingly disparate observations into a new idea. Indeed, Bruce is one of the most creative scientists I’ve ever met. Even though he was still doing a lot of work on identification of mutagens when I joined his lab, he was already transitioning to oxidative DNA damage and thinking about the role of oxidative degeneration of mitochondrial enzymes in aging. While I was in the lab, Fred Jacobson, now at Genentech, discovered the alkyl hydroperoxide reductase enzymes in Salmonella. By the time of my sabbatical, Bruce had shifted the focus of his work on mutagens and carcinogens from environmental agents as causes of cancer to naturally occurring compounds and endogenous metabolic products. This reflected his willingness to integrate knowledge from across the spectrum and to look at problems in toto. As a complement to his work on mutagens, he and Lois Swirsky Gold developed the Carcinogenic Potency Project database that ranked compounds based on their mutagenicity and their estimated intake by humans. Later in his career, he focused on micronutrients for the prevention of cancer and even developed a vitamin and micronutrient-rich nutrition bar. Bruce and Giovanna were excellent and warm hosts to us during our visit. We had multiple stimulating dinners with Berkeley faculty and an unforgettable reception at his home for Linus Pauling on the occasion of his three lectures on campus. The talk was always about science. That’s what impressed me so much about the environment in Barker Hall. Not only was the faculty very accomplished, they continued doing science at a high level throughout their careers. Jesse Rabinowitz was running the department in his late 60’s, Dan Koshland became editor of Science at 65, and Horace Barker was still coming into the building that bore his name to do work in his mid-80’s. Their love of and intensity for science was a tremendous inspiration for me and still is.
into the lab. I followed him and watched as he tore off another piece of tape and bisected my 3 feet of bench space into two plots of 18 in. I had a colony counter that was 14 in. wide, so that left me 2 in. on either side to do work on. Obviously, Bruce and I were using different definitions of “share”. 1.5. Impressions of Bruce Bruce and his lab were so different from what I had anticipated based on my casual acquaintance with him in advance of my sabbatical. His public lectures are always entertaining and have a spontaneous feel to them that led me to believe that Bruce was fairly laid back and casual. That impression faded rapidly as I looked around the lab. Bruce’s office was modest, as were all offices in Barker Hall, and his lab was compact, as I have mentioned. But everything was super-organized – it had to be. In addition to parsing out space, Bruce would patrol the lab looking for detritus that was lying about and would make sure people got rid of it. Core activities were supported by technical staff, and all the HPLCs were stacked on top of each other against one wall under the watchful eye of Rick Cathcart. And every Thursday evening, Dorothy Maron would pack up all the various tester strains that had been requested that week, so she could send them out. This focus on organization not only mitigated to some degree the limited amount of space, but it also enabled the lab members to concentrate on planning and doing good experiments rather than pouring agar plates, washing dishes, making solutions, etc. Well-planned experiments were a high priority for Bruce. He made no secret of the fact that when he was young, he hated doing experiments. He would plan them meticulously in order to incorporate every conceivable control so the experiment would generate definitive results on the first try. Only then would he go into the lab. It seemed this philosophy rubbed off on some of the group members. Bruce has no shortage of interesting ideas. He reads voraciously, and the shelves in his office were lined with binders of reprints. This was in the days before the internet when we all used Current Contents to 7
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1.6. Thanks, Bruce
[5] E. Cavalieri, E. Rogan, Role of radical cations in aromatic hydrocarbon carcinogenesis, Environ. Health Perspect. 64 (1985) 69–84, https://doi.org/10.1289/ehp. 856469. [6] L.J. Marnett, G.A. Reed, D.J. Dennison, Prostaglandin synthetase dependent activation of 7,8-dihydro-7,8-dihydroxy-benzo (a) pyrene to mutagenic derivativies, Biochem. Biophys. Res. Commun. 82 (1978) 210–216. [7] A. Panthananickal, P. Weller, L.J. Marnett, Stereoselectivity of the epoxidation of 7,8-dihydrobenzo[a]pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts, J. Biol. Chem. 258 (1983) 4411–4418. [8] L.J. Marnett, M.J. Bienkowski, Hydroperoxide-dependent oxygenation of trans-7,8dihydroxy-7,8-dihydro benzo[a]pyrene by ram seminal vesicle microsomes. Source of the oxygen, Biochem. Biophys. Res. Commun. 96 (1980) 639–647. [9] C. Ji, L.J. Marnett, Oxygen radical-dependent epoxidation of (7S,8S)-dihydroxy-7,8dihydrobenzo[a]pyrene in mouse skin in vivo. Stimulation by phorbol esters and inhibition by antiinflammatory steroids, J. Biol. Chem. 267 (1992) 17842–17848. [10] L.J. Marnett, M.J. Bienkowski, M. Raban, M.A. Tuttle, Studies of the hydrolysis of 14C-labeled tetraethoxypropane to malondialdehyde, Anal. Biochem. 99 (1979) 458–463. [11] A.K. Basu, L.J. Marnett, Unequivocal demonstration that malondialdehyde is a mutagen, Carcinogenesis 4 (1983) 331–333, https://doi.org/10.1093/carcin/4.3. 331. [12] L.J. Marnett, M.A. Tuttle, Comparison of the mutagenicities of malondialdehyde and the side products formed during its chemical synthesis, Cancer Res. 40 (1980) 276–282. [13] A.K. Basu, L.J. Marnett, Molecular requirements for the mutagenicity of malondialdehyde and related acroleins, Cancer Res. 44 (1984) 2848–2854. [14] A.K. Basu, L.J. Marnett, L.J. Romano, Dissociation of malondialdehyde mutagenicity in Salmonella typhimurium from its ability to induce interstrand DNA crosslinks, Mutat. Res. 129 (1984) 39–46. [15] A.K. Basu, S.M. O’Hara, P. Valladier, K. Stone, O. Mols, L.J. Marnett, Identification of adducts formed by reaction of guanine nucleosides with malondialdehyde and structurally related aldehydes, Chem. Res. Toxicol. 1 (1988) 53–59. [16] D.E. Levin, L.J. Marnett, B.N. Ames, Spontaneous and mutagen-induced deletions: mechanistic studies in Salmonella tester strain TA102, Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 4457–4461, https://doi.org/10.1073/pnas.81.14.4457. [17] L.J. Marnett, H.K. Hurd, M.C. Hollstein, D.E. Levin, H. Esterbauer, B.N. Ames, Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104, Mutat. Res. 148 (1985) 25–34. [18] J.J. Galligan, J.A. Wepy, M.D. Streeter, P.J. Kingsley, M.M. Mitchener, O.R. Wauchope, W.N. Beavers, K.L. Rose, T. Wang, D.A. Spiegel, et al., Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) 9228–9233, https://doi.org/ 10.1073/pnas.1802901115. [19] N. Rabbani, M. Xue, P.J. Thornalley, Activity, regulation, copy number and function in the glyoxalase system, Biochem. Soc. Trans. 42 (2014) 419–424, https://doi.org/ 10.1042/BST20140008.
My sabbatical was one of the most enjoyable experiences of my scientific career. Beyond Bruce and many senior faculty, I met many talented young students and postdocs including David Levin, Mike Christman, Fred Jacobson, Betty Schwiers, Monica Hollstein and Rick Cathcart (Fig. 13). Martyn Smith had just started his career on the faculty, and we became fast friends. The environment in the lab and the community was wonderful and fun. I stayed in touch with Bruce throughout the years returning for his 60th birthday celebration, seeing him at meetings, and chatting occasionally by phone. In every single exchange, at some point in the conversation, Bruce will lower his voice, look intently at me and exclaim “I’m doing the best research of my life!” He hasn’t lost his enthusiasm for life and his joy in doing and thinking about science. He is truly an inspiration to all who have had the privilege of knowing him and interacting with him. I am fortunate to have been one of them. Thank you, Bruce. Acknowledgements Work in the Marnett laboratory on DNA damage and mutation has been supported continuously by research grant from the National Cancer Institute. Current support is provided by CA87819. We are grateful to Carol Rouzer for editorial comments References [1] L.J. Marnett, P. Wlodawer, B. Samuelsson, Co-oxygenation of organic substrates by the prostaglandin synthetase of sheep vesicular gland, J. Biol. Chem. 250 (1975) 8510–8517. [2] A.H. Conney, R.L. Chang, X.X. Cui, M. Schiltz, H. Yagi, D.M. Jerina, S.J. Wei, Dosedependent differences in the profile of mutations induced by carcinogenic (R,S,S,R) bay- and fjord-region diol epoxides of polycyclic aromatic hydrocarbons, Adv. Exp. Med. Biol. 500 (2001) 697–707. [3] H.V. Gelboin, Benzo[alpha]pyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes, Physiol. Rev. 60 (1980) 1107–1166, https://doi.org/10.1152/physrev.1980.60.4.1107. [4] L.J. Marnett, G.A. Reed, J.T. Johnson, Prostaglandin synthetase dependent benzo(a) pyrene oxidation: products of the oxication and inhibition of their formation by antioxidants, Biochem. Biophys. Res. Commun. 79 (1977) 569–576.
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