Twenty Years of Reflections in Mutation Research

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

Contents lists available at ScienceDirect

Mutation Research-Reviews in Mutation Research journal homepage: www.elsevier.com/locate/mutrev

Reflections in Mutation Research

Twenty Years of Reflections in Mutation Research☆ George R. Hoffmann

⁎

T

Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610, USA

ARTICLE INFO

ABSTRACT

Keywords: Reflections History of science Mutagenesis Genetic toxicology Environmental mutagenesis

Reflections is a component of Mutation Research Reviews devoted to historical and philosophical themes pertaining to the subject of mutation. Reflections was initiated in 1999 and has included a broad array of topics centered on mutation research, but overlapping other scientific fields and touching upon history, sociology, politics, philosophy and ethics. This commentary offers an editor's reflections on the 44 papers in the Reflections series, including the people who contributed to the series and the topics that they discussed.

1. Establishment of Mutation Research Reviews and Reflections The journal Mutation Research was founded in 1964 by Professor F.H. Sobels at the University of Leiden [1]. Professor Sobels, known to his many friends as "Frits," established the reviews section of the journal a decade later [2]. The reviews section flourished, and through the years it operated under several similar titles, initially Mutation Research: Reviews in Genetic Toxicology and most recently Mutation Research Reviews. After Prof. Sobels' death in 1993, Frederick J. de Serres and John S. Wassom succeeded him as the managing editors of the reviews section, serving from 1994 until Fred de Serres' retirement in 1998. The present editors, David M. DeMarini and Michael D. Waters, assumed the editorship at that time. Reflections was established shortly after the appointment of DeMarini and Waters. As I recall the history, David DeMarini telephoned me to discuss his idea for a new feature in Mutation Research Reviews in the style of the "Perspectives" feature in the Genetics Society of America's journal Genetics, edited by James F. Crow and William F. Dove. Perspectives was then a decade old and was well established as an appealing and extremely valuable component of Genetics [3]. David wondered what I thought of his idea. I liked it. He suggested that it would be a good idea to have two editors in different countries, befitting the international character of Mutation Research. He asked whether I would be interested in serving as coeditor. I was certainly interested, but the thought of undertaking a feature modeled on Perspectives in Genetics was daunting, as it set a very high standard. One of the first people that I spoke to about this idea was James Crow, whom I had come to know through the National Academy of Sciences'

Committee on Chemical Environmental Mutagens. He was encouraging, and I moved closer to taking on the new project. David and I discussed the particulars of how Reflections could be organized and managed. His plan was to give the coeditors complete autonomy in running Reflections. I asked whether he had someone in mind as the second editor, and he did. It was Donald G. MacPhee at LaTrobe University in Australia. I was delighted, and no further discussion was needed from my perspective. Fortunately, Donald agreed, and we were soon in regular email contact about Reflections. 2. Management of Reflections We intended for Reflections to be comprised largely of invited papers from people who had made major contributions to the field of mutagenesis or related subjects. In extending invitations, we suggested topics that struck us as fitting, based on the prospective author's work, but we made it clear that we were receptive to alternatives. We would also be receptive to spontaneously submitted manuscripts, and several such unsolicited submissions became valuable contributions to the series. We decided at the outset that all Reflections articles would be subject to careful peer review. We made a fine distinction, however, from conventional peer review, so we referred to those who commented on the manuscripts as "Readers" rather than "Reviewers." Readers were asked to offer suggestions for the author's consideration. The main difference from a formal review is that we gave Reflections authors great latitude in expressing their viewpoints. This process fits the purpose of Reflections, as we specifically wanted the personal perspectives of people who had contributed much to the field. At the same time, if

This article is part of the Reflections in Mutation Research series. To suggest topics and authors for Reflections, readers should contact the series editor, G.R. Hoffmann ([email protected]). ⁎ Corresponding author. E-mail address: [email protected]. ☆

https://doi.org/10.1016/j.mrrev.2019.05.004 Accepted 25 April 2019 Available online 23 May 2019 1383-5742/ © 2019 Published by Elsevier B.V.

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

errors were identified, we expected them to be corrected, and if substantive criticisms were raised, we expected that the authors would reconcile them. Though less structured than formal peer review, this style has served us and the authors well, and we have never heard a complaint from a reader who thought that criticisms were not properly considered. Running Reflections with autonomy meant that Donald and I sent accepted manuscripts directly to issue managers or production editors at Elsevier. This spared authors the administrative process after their paper was complete, and it gave us the ability to work collaboratively with the publisher on special considerations in the formatting or style of the articles. Proofs were sent to the authors and to us, and the authors, of course, would approve the final version. The name and purposes of Reflections suggest some bias toward older authors who look back on a history. This bias has not been extreme, however, and there have been exceptions where the reflections of a young scientist fit the purposes of Reflections perfectly. In a few cases, the misfortunes of time meant that accepted invitations could not be completed, and I would like to say a word of appreciation for some valued colleagues who fall into this category: Tom Cebula, Mary Lyon, Barry Margolin, Bill Morgan, Jim Parry, Herb Rosenkranz, Jack Von Borstel and John Wassom.

Principle in population genetics, who had noted in 1912 an age effect in the occurrence of genetic diseases. This was a perfect start for Reflections, in that Jim traced his topic to the early days of genetics, linked it to a central question in modern mutation research –the origin of new mutations – and speculated about its current importance. 3.3. Bernard Strauss on frameshift mutagenesis and mismatch repair DNA repair already comprised a thriving subdiscipline of mutation research when Reflections began, and we wanted it to be represented early in the series. We were in luck when Bernard S. Strauss, who had been active in the field of DNA damage, mutagenesis and repair since around 1960, accepted Donald MacPhee's invitation to contribute. He wrote his reflections on the growing understanding of frameshift mutagenesis and genomic instability in eukaryotic cells, drawing together repetitive sequences, slippage, and mismatch repair [8]. The article begins with comments on differences between the genomes of eukaryotes and bacteria, notably including extensive repetitive sequences and introns in eukaryotes. He reviews early studies elucidating the nature of base-pair substitutions and frameshift mutations, and he discusses models of frameshift mutagenesis, including the widely known Streisinger slippage model [9,10]. While slippage remains an important mechanism, it is not the only mechanism, and he points out that frameshifts also arise, even at repeated sequences, by mechanisms not associated with slippage. He notes the association between frameshift mutagenesis and replication, and he relates deficiencies in mismatch repair to genetic instability. He suggests that the attributes of eukaryotic genomes necessitated highly effective surveillance to protect against frameshifts and that the eukaryotic mismatch repair system, having differences from that in prokaryotes, is a key element in that surveillance. Professor Strauss is still professionally active, and readers may like to see a commentary that he wrote recently on the doublestrandedness of DNA and the history of DNA repair studies [11].

3. The first Reflections articles 3.1. An introductory essay To initiate the Reflections series in Mutation Research Reviews, Donald and I submitted an introductory essay on our plans for the new series [4]. We included a table of papers that we saw as being seminal works in the formative years of the field of mutation research. We set the pattern that all submissions to Reflections would be peer reviewed, and we made no exception here. I had heard from several of Herman Brockman's students at Illinois State University that when they submitted drafts of papers to him for review, their manuscript was "Brockmanized." So, we chose Herman Brockman as a reviewer of our paper, along with John Wassom and Bill Healy, and we received three valuable reviews, including good suggestions for the table. I might add that the manuscript had been properly "Brockmanized."

3.4. Zhores Medvedev on Lysenkoism in the Soviet Union The Lysenko period in the Soviet Union, extending from the 1930s until 1964, was a bizarre and troubled time in the history of science. As Trofim Lysenko gained influence with the Soviet government and promoted views incompatible with modern genetics and evolutionary biology, legitimate geneticists and others who argued against the antiscientific trend were removed from their positions and sometimes imprisoned. The biologist Zhores Medvedev courageously spoke out against this trend and later managed to get a book manuscript to I. Michael Lerner, a geneticist at the University of California, Berkeley. The manuscript told the history of Lysenkoism in a direct way that was not permitted in the Soviet Union even after the fall of Lysenko. Lerner translated Medvedev's manuscript from Russian, and it was published in 1969 as The Rise and Fall of T.D. Lysenko [12]. The book received strong praise from the eminent evolutionary biologist Theodosius Dobzhansky, both for its courage and its thorough documentation of the history [13]. In 1998, Donald MacPhee and I were able to contact Dr. Medvedev, who was now living in England, and we invited him to write an article for Reflections. We expressed interest in the recovery from Lysenkoism and lingering effects, but we gave him the latitude to define his specific topic. We quickly received a courteous response that he was reluctant to accept our invitation. A short time later, I received another letter from him, in which he explained that he found it increasingly difficult to write text in English, and he asked whether we could consider a manuscript in Russian. I immediately thought of Charles ("Chuck") Severens, a former Russian professor and my colleague at Holy Cross. Chuck asked to see the text before committing himself to translating it. We took the gamble and agreed to Dr. Medvedev's proposal. When the manuscript arrived, Chuck looked it over and, fortunately, agreed. He wanted nothing more than a footnote indicating "Translated from Russian by Charles Severens." Dr. Medvedev's English turned out to be

3.2. James Crow on mutation in human germ cells Reflections had an auspicious beginning when James F. Crow accepted my invitation to write the first Reflections article after the introductory essay. Professor Crow, known to his friends worldwide as "Jim," was one of the truly great geneticists of the twentieth century and made many contributions to theoretical population genetics and applied topics in human genetics, including risks associated with ionizing radiation and chemical mutagens. He joined the faculty of the University of Wisconsin in 1948 and worked there for 64 years, until his death at age 95 in 2012. He is remembered not only for his scientific achievements but also for his fine qualities as a person and teacher, many contributions to the local, national and international scientific community, skill as a musician and extraordinary generosity of spirit [5,6]. In Jim's case, we did not suggest a topic, as we knew that whatever he chose would be good. As was typical of him, a well-written manuscript arrived on time, and it dealt with a historical theme that was, at the same time, completely relevant to major questions in modern mutation research and genetic toxicology. Jim entitled his article "Spontaneous mutation in man" [7] and began with a clever introduction, noting that using "man" generically to refer to the species Homo sapiens was no longer quite acceptable. However, for him, it was okay, because he was making the point that the problem is indeed "man," in that mutations disproportionately arise in the continuing meiotic divisions of male meiosis. He then traced the age effect in mutagenesis to Wilhelm Weinberg, now most familiar because of the Hardy-Weinberg 107

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

very good, and he identified a few fine differences in meaning from what he had intended. He and Chuck easily worked this out. When Dr. Medvedev's manuscript on Lysenko and Stalin was published [14], Donald MacPhee and I preceded the article with an "editorial," which was actually a brief biography and tribute to Zhores Medvedev, including his photograph in his apartment in the U.K. [15]. Dr. Medvedev died in November 2018, and his obituaries give a good account of his remarkable life [16,17].

Society (EMGS). He offered a sociological analysis of how the EMS, founded in 1969, had functioned at the boundary between environmental science and environmental politics, and he explored how it thrived in the early 1970s despite a variety of institutional challenges. His reflections bring sociological theory to bear on the historical development of genetic toxicology [24]. A book by Scott Frickel, now a professor of sociology at Brown University, gives a complete account of his sociological study of genetic toxicology and the EMS [25].

3.5. Geoffrey Grigg on the "Grigg effect" and the history of reverse mutation assays

4.2. Elliot Volkin on the discovery of mRNA

Those who think of the classical Ames test as the "good old days" of environmental mutagenesis should enjoy reading Geoff Grigg's reflections on questions surrounding the methods and first applications of reverse mutation assays [18]. His studies of back mutations from auxotrophy to prototrophy in microorganisms, principally Neurospora crassa, include a critical paper in 1952 demonstrating that large numbers of auxotrophs in the background can suppress the expression and detection of new revertants [19]. This discovery, which is now well known but came as a surprise to many, became known as "the Grigg Effect." It was our good fortune that Donald MacPhee had come to know Geoff Grigg in Australia, and Donald's invitation to write for Reflections was readily accepted. In his Reflections paper [18], Geoff Grigg describes the discovery of the Grigg Effect and the ability to detect it by means of reconstruction experiments that simulate the conditions of the mutation experiment, including cell concentration, selective medium and growth conditions. He points out that after he reported the effect, he discovered that a similar phenomenon had been reported earlier in bacteria, and he credits Ryan and Schneider for their discovery in E. coli [20]. Grigg also explores other sources of error in back-mutation experiments, and he raises the question whether the Grigg Effect fundamentally limits the applicability of reverse mutation assays. He concludes that it does not if one is careful to use appropriate strains and have proper controls to detect it, and history has borne this out. Besides the "Grigg Effect," diverse other discoveries and applications bear his imprint, as nicely summarized in a tribute by Robin Holliday and colleagues [21].

John Wassom, the director of the Environmental Mutagen Information Center (EMIC) in Oak Ridge, was deeply interested in the history of environmental mutagenesis, and he contributed historical reviews on the subject to the EMS's journal Environmental and Molecular Mutagenesis [26,27]. He also took an interest in our plans for Reflections and wrote to me that Elliot Volkin, who was then retired from the research staff at Oak Ridge National Laboratory (ORNL), still came to ORNL regularly and that John had "adopted" him at EMIC. They discussed the history of genetics and mutation research often, and John thought that Dr. Volkin might be interested in writing an article for Reflections. John wrote, "He is a jewel and an encyclopedia of knowledge. I think an article by him on the events leading up to and the actual discovery of mRNA would be of great interest to the MR readership." As usual, John's advice was good. I contacted Dr. Volkin and invited him to write a paper for Reflections, and he agreed. Working with him was a real pleasure. I was struck by his modesty in discussing his studies, working in collaboration with Lazarus Astrachan, that were so close to one of the premier achievements of the "classical phase" of molecular biology – the discovery of mRNA. His Reflections paper is a perspective on this discovery [28]. Those interested in an independent appraisal of the substantial importance of the experiments of Elliot Volkin and Lazarus Astrachan in the history of molecular biology might refer to The Eighth Day of Creation, a highly regarded history by Horace Freeland Judson [29] and the autobiography of the Nobel laureate François Jacob, who viewed their research as a contemporary researcher pursuing closely related problems [30].

3.6. Robin Holliday on somatic mutations and aging

4.3. F. Peter Guengerich on metabolism and carcinogenesis

Another Reflections article from "Down Under" followed shortly after that of Geoff Grigg from another distinguished colleague and friend of Donald MacPhee – Robin Holliday. Given the diversity of his scientific contributions, notably including recombinational mechanisms (and the eponymous Holliday junction), there were many possible topics on which he might have written. His choice was his longstanding interest in the biology of aging. His Reflections article is a theoretical discussion of the many ways in which genetic and epigenetic alterations can contribute to the aging process and the difficulty of sorting out their relative importance [22]. Those interested in the breadth of scholarly works of this noted polymath, ranging from molecular biology and aging to sculpture, will appreciate a tribute to Robin Holliday by Leonard Hayflick [23].

F. Peter ("Fred") Guengerich is a member of the Department of Biochemistry at Vanderbilt University School of Medicine with a remarkable record of research on carcinogen metabolism. Yet, when I invited him to write some reflections on the growing understanding of the role of metabolism in carcinogenesis and mutagenesis, he seemed surprised. He first joked that he was too young to write reflections, and then he came to his point directly: Professor James Miller, though at advanced age, was professionally active, and I should really ask him to write it. As Professor Miller's health was fragile, Fred graciously agreed to write the article, and he gave us a great paper on "forging the links" between metabolism and carcinogenesis [31]. James Miller died in late 2000 as Fred's manuscript was being completed, and Fred dedicated the paper to the memory of James and Elizabeth Miller. This Reflections paper thus became the first of several dedicated to mentors and leaders in the field. I would refer readers who would like more information on the lives and scientific legacy of James and Elizabeth Miller to a tribute by Fred Kadlubar [32]. Fred Guengerich starts his reflections with the history of epidemiologic associations of tumors with carcinogens. He carries the story forward into the 20th century by addressing animal studies, endogenous carcinogens, mutagenesis, and research on the metabolism of carcinogens in the 1930s and 1940s, which includes the early studies of the Millers. He reviews the cytochrome P450 monooxygenases and other enzymes of xenobiotic metabolism, the metabolic activation of procarcinogens into electrophiles, detoxication reactions, enzyme

4. Reflections through the years 4.1. Scott Frickel on sociological analysis of the Environmental Mutagen Society We had been thinking of Reflections as being centered on historical and philosophical themes when interesting presentations at scientific meetings led us to think about an occasional sociological or political commentary. The first of these was by Scott Frickel, a sociologist who studied the growth and mission of the Environmental Mutagen Society (EMS), now renamed the Environmental Mutagenesis and Genomics 108

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

induction, and polymorphisms. He notes how the bacterial umu assay, which measures the induction of the umu operon as part of the SOS response to DNA damage, was used to monitor genotoxicity. This simple assay was useful in guiding his attempts to identify genotoxic metabolites and his work on the purification of human P450 enzymes. The elucidation of the roles of different P450 enzymes was also aided by the development of transgenic microbial and mammalian-cell systems that express human P450's. A review of "Phase 1" reactions and the "Phase 2" enzymes that catalyze conjugation reactions is followed by a detailed look at a specific case that illustrates broad principles – metabolism of the mycotoxin aflatoxin B1. Fred then addresses the evolution of xenobiotic-metabolizing enzymes and provides perspective on conceptual advances in the understanding of carcinogen metabolism [31]. These insights and his concluding remarks are well-worth reading for specialists and nonspecialists alike.

interests evolved from physics to biophysics, and he describes his early work "shedding light" on simple biological systems. He found new ways to use UV action spectra to elucidate the properties of proteins, nucleic acids and viruses. After joining the Biology Division of ORNL in 1960, he extended this work to the formation of cyclobutane pyrimidine dimers in DNA by UV and their detrimental effects in bacteria. This was followed by his pioneering work on the elimination of dimers by photorepair, and ultimately the discovery of excision repair in 1963-1964. His interests soon expanded to include repair-deficient mutants, mutagenesis and carcinogenesis. In 1974, Dick moved to Brookhaven National Laboratory, where he spent the remainder of his career. His creative research continued, but his focus expanded to complex multicellular systems, including fish. The Amazon molly permitted informative comparisons with mammalian cells, because fish have an active photorepair system, which nonmarsupial mammals lack. Dick began with basic biophysics and through his professional life increasingly concerned himself with the implications of his field for carcinogenesis, genetic effects, aging and health. I would refer readers to an in memoriam tribute to Dick Setlow by Phil Hanawalt and colleagues [36].

4.4. Richard Albertini on mutations in human cells in vivo Clear positive selection techniques for forward mutations led to a few genes being predominant in early mutation tests in mammalian cells. Among these is the X-linked HPRT gene encoding hypoxanthineguanine phosphoribosyltransferase. Mutants that cannot convert the purine base analog 6-thioguanine or 8-azaguanine to its nucleotide form do not experience its toxicity and are selected by their growth in medium containing the analog. A single mutational event is sufficient to confer the mutant phenotype in wild-type cells because there is only one copy of this X-linked gene in cells from males and because females are functionally hemizygous due to X-inactivation. For this reason, HPRT was a prime candidate for detecting point mutations in human cells in vivo. Richard ("Dick") Albertini led the way in developing HPRT into a functional in vivo mutation system in human peripheral T-lymphocytes. An elevation in mutant frequencies was readily detected in such exposed populations as patients who had received mutagenic chemotherapy. An astute observer of human immunology and physiology, Dick was able to uncover mechanistic complexities in the induction, proliferation, frequencies and molecular mechanisms underlying the mutants detected in vivo. In his Reflections article [33] he describes this history and some of the surprises along the way. Being a clinician, as well as a researcher, he ends his reflections with speculation about the possibility of long-term transmissible effects of HPRT mutations in patients receiving therapy with purine analogs.

4.6. Edward J. Calabrese on hormesis Dose-response relationships had received much attention throughout the history of mutation research, and various models with and without thresholds have been considered. A linear model with no threshold was, and to a great extent remains, a leading model for genetic effects of ionizing radiation and direct-acting chemical mutagens. Edward ("Ed") Calabrese, a toxicologist at the University of Massachusetts, has been a leading advocate of an alternative model based on hormesis, which is a process whereby effects at low doses are opposite to those caused by high doses of the same agent for the same biological endpoint. The notion that hormesis is broadly applicable to biological processes is considered eccentric by many, especially in suggesting that low doses of radiation and toxicants may be beneficial. However, the idea was receiving growing attention, so we invited Ed Calabrese to offer his reflections on the subject. We anticipated that it would be controversial and so-noted in our introductory editorial [37]. Ed Calabrese, open to suggestions and to contrary views of reviewers, enjoyed the debate and refined his text, holding to his views while exercising restraint about overstatement. He offered a fascinating view of a changing landscape with respect to thinking about hormesis [38].

4.5. Richard B. Setlow on UV light, DNA damage and repair

4.7. John Savage on complex chromosomal rearrangements

Richard B. ("Dick") Setlow is remembered for his important discoveries on the biological effects of ultraviolet light (UV), characterization of pyrimidine dimers, and the process of nucleotide excision repair. In a Festschrift celebrating his eightieth birthday in 2001, he was described as "the Father of DNA Repair" [34]. I clearly remember the first time I met Dick Setlow. As a graduate student at the University of Tennessee, I did my graduate research at ORNL. Around 1970 was a good time to be at ORNL because the Biology Division had flourished under the leadership of Alexander Hollaender as its director from 19461966. When I arrived in Oak Ridge, my fellow graduate student, Mike Shelby, told me that Dr. Hollaender met with Oak Ridge researchers, visitors and students on Sunday mornings by the railroad trestle in Oliver Springs for hikes in the nearby Cumberland Mountains. The Cumberlands were of interest for their scenic beauty and natural history, and also for the abundant fossils of Carboniferous plants found in disturbed sites near strip mines. On my first such hike, I found myself talking to Dick Setlow and hearing first-hand about the groundbreaking research in his lab on effects of UV and the repair of DNA damage. I was therefore pleased many years later when he accepted my invitation to write an article for Reflections [35]. Dick's Reflections paper begins with his days as a physics student and, later, a faculty member at Yale University from 1941 to 1960. His

The use of fluorescence in situ hybridization (FISH) to study chromosomal aberrations had become common in the 1990's, and John Savage was devising means to classify the richer array of alterations that could be seen with FISH than had been readily detectable by conventional staining. "Reflections and Meditations" in the title of his Reflections paper [39] is exactly right, as he thought deeply about chromosome structure and the mechanisms underlying chromosomal alterations. The previously unseen array of aberrations now apparent with FISH led him to write "these strange, unexpected patterns required new descriptive methods, and two complementary scoring schemes were developed, 'S&S' and 'PAINT'" [39]. The initials of John Savage and his colleague Paul Simpson became the name of the "S&S" nomenclature system, which they devised to classify complex aberrations on the basis of the possible interchanges formed by chromosome breaks [40,41]. His Reflections paper elucidates the underlying theory and its history [39]. I will return to the "PAINT" nomenclature system when commenting on a later Reflections paper by Jim Tucker. In 2002, we were still receiving printed manuscripts by conventional mail when John sent me his manuscript. The stationery that he used for his cover letter had a unique heading – his home address in Reading, UK, to the left of which were his initials – JS – large and in two colors. The "morphology" of the initials was that of chromosomes 109

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

stained by FISH – an isochromatid / chromatid interchange.

pharmacology and toxicology at the University of Bologna, he would work together with Marion Nestle, a professor of nutrition, food studies and public health at New York University, to give a shared perspective. We were pleased to have such distinguished scholars of nutrition and food toxicology seek out the young Reflections feature to present their views of this complex and important problem. In their Reflections article [43], they explore prospects for chemoprevention and express skepticism about oversimplification in the growing enthusiasm for farreaching benefits conferred by single dietary supplements or phytochemicals. Their article offers reasoned warning about risks of simple solutions to complex problems, and they call for integrative studies of the complexity of diet and public health.

4.8. Akio Awa on cytogenetic analysis of atomic bomb survivors Donald MacPhee took a temporary leave from his academic position at LaTrobe University to serve from 1999 to 2004 as Chief of the Department of Radiobiology at the Radiation Effects Research Foundation (RERF), in Hiroshima, Japan. Early in his stay, he invited Akio Awa to write a paper for Reflections on the extensive cytogenetic studies of survivors of the atomic bombings of Hiroshima and Nagasaki. From 1968 to 1993, Dr. Awa ran the cytogenetics program of ABCC/ RERF (Atomic Bomb Casualty Commission, established in 1947 and replaced by the Radiation Effects Research Foundation in 1975), and his article gives a personal account of his experience in this unique and important effort [42]. Dr. Awa begins with the history of cytogenetics, noting that the human chromosome number was not correctly identified until the 1950s, and with the technical challenges that characterized the early period at ABCC/RERF. He describes technical advances and efforts to identify not only the easily observed unstable chromosome aberrations (i.e., asymmetrical exchanges – dicentrics, acentric fragments, and rings), but also the stable aberrations (i.e., symmetrical exchanges – reciprocal translocations and inversions) that would persist in blood long after the exposure in 1945. Dosimetry was a complex problem for several reasons, including distance from the epicenter, shielding in different locations, differences between the two bombs in relative proportions of gamma rays and neutrons, and even the atmospheric humidity. Characterizing the cytogenetic damage was a formidable challenge in the days before chromosome banding and FISH. Despite the difficulty, they measured higher frequencies of unstable and stable chromosome aberrations in exposed survivors than in controls. Lower frequencies of unstable than stable aberrations reflected the loss of cells bearing dicentrics, fragments and rings. An important goal was the use of aberration frequencies to estimate doses received by individual survivors, and Dr. Awa chose to pursue the more difficult, but necessary, quantification of stable aberrations. The quality control effort was meticulous. The frequency of cells with aberrations increased with exposure, and the data permitted an evaluation of not only dose-response relationships, but also differences between Hiroshima and Nagasaki and ratios of stable and unstable aberrations (initially a theoretical 1:1) reflecting loss of the latter over time. They observed a few individuals who had clones of many cells with identical aberrations, probably derived from aberrant lymphopoietic stem cells [42]. Through the years, methodology was updated to include chromosome banding and, later, FISH. These methods led to somewhat higher frequencies of aberrations detected, and FISH improved the ease of scoring, but they supported the conclusions that Awa and colleagues had reached through their excellent work with the earlier technology. As teeth of survivors became available, the enamel was analyzed by electron spin resonance (ESR) to estimate gamma-ray doses received, and these estimates were consistent with the cytogenetic dosimetry. Dr. Awa summarizes results with conventional staining, banding, FISH and ESR in Section 3 of his reflections, and I call your attention to his personal commentary at the end of this section. He met with many children and parent groups, and he offers his perspective on the ethical issues surrounding the fears of the descendants of survivors and the need to explain the science and its uncertainties [42]. The fine character of Akio Awa is reflected in his Reflections article, and his acknowledgments include "the citizens of Hiroshima and Nagasaki."

4.10. W. Gary Flamm on mutation research in relation to regulatory policy In his Reflections article, Gary Flamm recalls the field of environmental mutagenesis seeming to "explode into existence like some cosmic big bang" [44]. The time was roughly 1970; the EMS had recently been founded; and EMIC, which was later assimilated into the National Library of Medicine's TOXNET databases, was being established by Heinrich Malling and John Wassom in Oak Ridge. The Food and Drug Administration in Washington, DC, was a center of activity for research on mutagenesis, and Dr. Flamm was its Branch Chief of Genetic Toxicology from 1972 to 1974. His reflections give a perspective on these specific organizations but, even more so, on the integration of environmental mutagenesis into the field of toxicology and work at the interface of basic science and policy that led to the development of regulatory standards in this field [44]. A tribute by Gary's former colleagues gives a clear sense of his remarkable accomplishments working at the intersection of research, governmental agencies, industry and scientific societies [45]. 4.11. Ernest H.Y. Chu on mammalian somatic cell mutagenesis The next two Reflections articles came from Ernest H.Y. Chu and Heinrich Malling, two colleagues in the Biology Division of ORNL in the late 1960s and early '70 s. They were good friends, conferring with each other often, while "Ernie" worked on the detection of point mutations in mammalian cells and Heinrich worked on the analysis of mutagenesis, principally but not exclusively in Neurospora crassa. In his reflections [46], Ernie traces the unequivocal demonstration of experimentally induced gene mutations in mammalian cells to three laboratories working independently of one another in the 1960's. He then describes his own scientific development, leading to his lab being one of the three. His research on the induction of azaguanine-resistant mutants in V79 Chinese hamster cells was groundbreaking work, contributing to the foundation of the widely used hprt assays in mammalian cells both in vitro and in vivo. His descriptions give a sense of the atmosphere of mutation research at the time and his excitement at new insights that arose in discussion with colleagues, notably including his talks with Heinrich Malling at the blackboard in Heinrich's tiny office. The way in which he credits Heinrich for a specific insight that contributed to his research also quietly reflects Ernie Chu's well-deserved reputation as one of the great gentlemen of our field. 4.12. Heinrich Malling on metabolic activation in mutagenesis Heinrich Malling was responsible for one of the seminal findings in the history of environmental mutagenesis and genetic toxicology testing – the use of an exogenous metabolic activation system based on a mammalian tissue homogenate to activate promutagens into mutagens in in vitro mutation assays. It is not an exaggeration to say that his work led to methodological changes that revolutionized the testing of chemicals for mutagenic activity. Heinrich begins his Reflections paper [47] by citing W.J. Burdette's argument in the 1950's that mutagenicity does not correlate with carcinogenicity. An important consideration in

4.9. Moreno Paolini and Marion Nestle on diet, cancer and public health We were pleasantly surprised to receive a detailed proposal from Moreno Paolini for a Reflections article on chemoprevention as a strategy to minimize risks of dietary carcinogens. A professor of 110

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

this argument was that such clear carcinogens as nitrosamines and polycyclic hydrocarbons gave negative results in tests for mutagenicity. The lack of correlation was later explained by the lack of mammalian metabolic activation in the mutation assays. Heinrich reviews the history of metabolic-activation studies that resolved this problem [47]. The early stages were the isolation of reactive carcinogen metabolites by James and Elizabeth Miller in 1960, host-mediated assays in the late 1960s, and his own studies on in vitro chemical activation in 1966. In a host-mediated assay, an indicator organism (e.g., bacteria or Neurospora) was injected into an animal (e.g., rat or mouse) that was then treated with a test chemical. The microorganism was later recovered from the animal to measure mutations that had been induced by the chemical and those metabolites that reached the target organism in the animal. A major breakthrough came in 1971 with Heinrich's reporting the metabolic activation of the promutagen dimethylnitrosamine into a mutagen by a tissue homogenate from mouse liver [48]. This was followed by Bruce Ames's assimilation of liver homogenates into the newly developed Ames test in 1973 [49]. Heinrich carries this history forward in a discussion of transgenic mutation assays in mammals in vivo, of which he was one of the early developers. He also reviews bacteria and mammalian cell lines engineered to express human cytochrome P450 enzymes and the detection of induced mutations in endogenous mammalian genes in vivo [47]. On a personal note, Heinrich was my major professor as a graduate student, and I surely wanted to include him in the Reflections series. I invited him at the outset of Reflections, and he delayed a bit. When I reminded him, I was pleased to receive the response that I wanted: "Yes! I should write the reflections paper, and I have a bad conscience that I have not done it yet." His explanation was that he always liked to look forward rather than back, and this was true. Heinrich was always focused on the future. His Reflections paper was completed in 2004 [47], and it was well worth the wait. Those interested in reading of the life and work of Heinrich Malling are referred to tributes written in his memory [50,51].

studies. Thus, he points the way to a trend that has been dominant in toxicology and risk assessment in recent years. 4.15. Kristien Mortelmans on plasmid pKM101 in mutation assays Anyone who has worked with the Ames assay, more formally called the Salmonella/mammalian-microsome mutagenicity test, knows that several of the key strains rely on plasmids that markedly enhance the sensitivity of the bacteria to mutagenesis. Among these, the most known and widely used is plasmid pKM101. What may be less known nowadays is that the "KM" designates Kristien Mortelmans, and she wrote a fascinating Reflections article on how pKM101 came to have its important role in the most widely used bacterial mutation assay [55]. Kristien describes not only the history of pKM101 but also its mechanisms of action, the Ames test more broadly, and the atmosphere of the bacterial genetics laboratory of Bruce Stocker, where she was a graduate student in the early 1970s. Kristien isolated pKM101 as a deletion derivative of the resistance-transfer plasmid R46 (also called RBrighton). The strain constructions involved in the origin of pKM101 were far more elaborate than one might have guessed by saying simply that pKM101 is a deletion derivative of R46. Plasmid pKM101 has greatly enhanced the effectiveness of the Ames tester strains, including the well-known strains TA100, TA98, TA97, TA102 and TA104, and it has also been incorporated into other strains of Salmonella and E. coli for studies of mutagenesis. While substantively commenting on the bacterial genetics, Kristien also includes a tribute to her mentor, Bruce A.D. Stocker. It includes a sense of the laboratory of the times and photographs of Professor Stocker, the vials that he used for his large Salmonella culture collection, and the prong replicator that he designed to screen colonies for altered antibiotic resistance. Thus, this became the first of several Reflections articles centered around a tribute to a fine mentor. 4.16. Mary Esther Gaulden, John Jagger and Virginia White on the legacy of Alexander Hollaender

4.13. Fred Sherman on molecular analysis of mutagenesis in yeast

As a broad, international field, environmental mutagenesis has several founders. In the United States, Alexander Hollaender (1898–1986) stands out as the leader of the first generation of scientists in the field. Dr. Hollaender became the director of the newly organized Biology Division of ORNL in 1946. He would develop it into a leading research institution and a center of activity in the new field of environmental mutagenesis. His influence extended far beyond ORNL, as he is the acknowledged founder of the Environmental Mutagen Society, which was formed in 1969. When I was thinking about a prospective author to write on Dr. Hollaender's legacy, Mary Esther Gaulden immediately came to mind. Mary Esther met Dr. Hollaender in 1942, was a Senior Radiation Biologist at ORNL from 1949 to 1960, and remained associated with him for the next 26 years while she was a professor of radiology at the University of Texas Southwestern Medical Center at Dallas. Mary Esther accepted my invitation and began work on it promptly. Known for her great productivity and generous service, I thought that this would proceed quickly to a fine article. It did indeed become a fine article [56], but the process became complicated. Mary Esther submitted a beautiful draft, with substantive details and many personal reflections, but the story was incomplete when our correspondence lagged. Mary Esther was apologetic about this, but the situation was largely out of her hands, as she was suffering from declining health. It seemed likely that the story would not be completed when her husband John Jagger and her friend Virginia P. White stepped in to complete the story. John was a biophysicist, photobiologist, and highly regarded teacher at the University of Texas at Dallas. He knew Dr. Hollaender since 1955 and joined the staff of ORNL in 1956. The Oak Ridge period was eventful in the lives of Mary Esther and John in many respects, and the Reflections article [56] includes a photograph of their wedding in 1956 at the home of Alexander and Henrietta

Fred Sherman was a brilliant geneticist who designed the iso-1-cytochrome c (cyc1) mutation system in yeast. He was able to classify mutations at the molecular level by sequencing. While this may seem ordinary to a modern reader, you need to remember that he and his colleague John Stewart did this work in the 1960s and 1970s, before the era of automated DNA sequencing. What they sequenced was the iso-1-cytochrome c protein, and from the amino acid sequences they deduced the nucleic acid sequence changes in the cyc1 gene. While few would be tempted to do such work today when DNA sequencing is fast and commonplace, it was an important part of the foundation for understanding mutagenesis at an increasingly molecular level. In his Reflections article [52], Fred tells the story with an interesting, appealing style. Besides his brilliance in science, Fred will be remembered by many as a man with a great sense of humor and far-reaching creativity [53]. 4.14. R.J. Preston on extrapolations in risk assessment R. J. "Julian" Preston had an illustrious career in cytogenetics and radiation biology, which increasingly moved him into the area of risk assessment, working for years at the Chemical Industry Institute of Toxicology (later renamed the Hamner Institutes) and more recently at the U.S. Environmental Protection Agency prior to his retirement. Not surprisingly, his reflections are devoted to a hazard, but in this case it is neither chemical nor radiation but rather, the hazard of extrapolations. Julian entitled his Reflections essay "Extrapolations are the Achilles Heel of Risk Assessment" [54], and he comments on extrapolations among species, among doses and among life stages, with an emphasis on carcinogen risk assessment and a call for greater reliance on mechanistic 111

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

Hollaender in Oak Ridge. John tells the story in his reflections with great style. Virginia White had been an administrator at Fisk University in Nashville and joined the staff of ORNL in 1955, ultimately becoming the Assistant to the Director of the Biology Division, where she managed such matters as personnel, budgeting, purchasing and publications. Virginia includes interesting observations about the location and times that extend those of Mary Esther and John. In the end, this is "Reflections in Three Parts" that complement one another perfectly.

undergone a single mitotic division were easily recognized because they had two nuclei. Micronuclei – small chromatin-containing bodies – were counted only in binucleated cells, thereby normalizing the frequency to those cells that had undergone a single mitotic division. Dose-dependent increases in micronucleus frequencies after mutagen exposure were quantified, and the scoring of large numbers of cells was much faster and required less skill than classical metaphase analysis [59,60]. Within a few years, Fenech and Morley added kinetochore staining to the assay to distinguish micronuclei containing whole chromosomes from those containing fragments [61]. In the years that followed, the assay became widely used around the world, and Michael Fenech attentively cultivated it, unraveled its mechanisms and ingeniously expanded its possible applications through his research at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Adelaide. His Reflections article offers an appealing blend of scientific advances and personal memories [62]. Michael traces his scientific development to his native Malta, where he became interested in marine ecology, an interest that extended to what is now called ecotoxicology. His pursuit of advanced studies brought him to Australia, which he considered unlikely at the time but proved fortuitous as his scientific life blossomed "Down Under," and it is now over 35 years later. He gives much credit to Alexander ("Alec") Morley, his mentor at Flinders University Medical School, where his experiences launched him on an illustrious research career combined with a substantial commitment to environmental mutagenesis, nutrition and public health. Michael describes the origin of the cytokinesis-block micronucleus assay – his "eureka moment" – with great style and then moves to the developments that led to worldwide use of the assay and its refinement and broadening applications. He credits earlier studies of the inhibitory action of cytochalasin mycotoxins [63] and of micronuclei without the cytokinesis block [64]. He then describes the great advances of the later "Cytome" version of his CBMN assay, supported by photographs and drawings, including binucleated and multinucleated cells, micronuclei, nucleoplasmic bridges, nuclear buds, and necrotic cells, giving distinguishable evidence of chromosome breakage, aneuploidy, dicentric chromosomes, gene amplification, mitotic inhibition, necrosis, and apoptosis. His "Reflections from Down Under" on his "Lifetime passion for micronucleus cytome assays" is a great story told well [62].

4.17. A.T. Natarajan on the origin of the journal Mutation Research The article on the life and legacy of Alexander Hollaender, a founder of our field, was followed by our shortest, yet very fitting, Reflections article on another foundational event. A.T. ("Nat") Natarajan submitted a reflection on the origin of the journal Mutation Research [57]. It is a brief commentary with a photograph from an international symposium on DNA repair, which Nat thought may have been the first ever on this topic. It was held in Leiden, The Netherlands, in August 1962. At the meeting, attended by such luminaries of our field as Charlotte Auerbach, Hermann Muller, Evelyn Witkin and Tikvah Alper, Professor F.H. ("Frits") Sobels proposed the establishment of a new journal, and the inaugural issue of Mutation Research appeared in January 1964. Nat comments that he was early in his scientific career at the time, and he found the conference inspirational. A half-century after the conference, in 2012, Nat would be writing reflections on his own illustrious cytogenetics career. 4.18. K. Sankaranarayanan and John Wassom on radiation risk assessment K. Sankaranarayanan ("Sankar") studied radiation-induced genetic damage for decades and combined his scientific expertise with public spiritedness through service to international radiation protection organizations and scientific journals. He has served on committees and task forces for the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR), which was established in 1955, and for the International Commission on Radiological Protection (ICRP), which was formed 1928. He wrote chapters of UNSCEAR reports from 1970 to 2001. In 2008, he teamed up with John Wassom to write reflections on the history of radiation protection [58]. This was a good combination of skills, as John was the Director of EMIC and had access to all published articles in the field. The emphasis of their article is on genetic (i.e., transmissible) effects of radiation and the work of UNSCEAR and ICRP, but they also consider carcinogenic effects and the work of the U.S. National Academy of Sciences' Committee on Biological Effects of Atomic Radiation (BEAR) and its more recent successor, the Committee on Biological Effects of Ionizing Radiation (BEIR). Their analysis spans the history of radiation protection with substantive, well-documented discussion. Its tables define the specialized terminology of the field; list the reports of UNSCEAR, ICRP and BEAR/BEIR; identify major developments in the early decades of radiation protection; and compile estimates of genetic disease frequencies, genetic risks, and dose limits. The detailed scientific content is nicely complemented by personal reflections on the events and the people who played an important role.

4.20. Richard Stevens on circadian disruption and cancer incidence I had the good fortune to meet Richard Stevens, an epidemiologist from the University of Connecticut School of Medicine, at the meeting of the European Environmental Mutagen Society in Cavtat, Croatia, where he made a presentation on "Breast cancer and circadian disruption from electric lighting." I enjoyed the presentation, discussed it with him, and invited him to submit an article for Reflections. He agreed. I knew that I was about to read something interesting and unconventional when I received his manuscript, entitled "Electric light causes cancer? Surely you’re joking, Mr. Stevens," adapting the title from a popular book by the Nobel laureate physicist Richard Feynman [65] and starting with a fitting Feynman quotation. The manuscript did not disappoint. Richard describes the association between exposure to light at night and cancer incidence [66]. His text shows the power of epidemiologic studies in revealing an unanticipated association and leading to an understanding of the association when combined with mechanistic studies. I won't comment further, so as not to detract from his compelling story [66], except to note that the International Agency for Research on Cancer (IARC) now classifies "shiftwork that involves circadian disruption" in its Group 2A, designating "Probably carcinogenic to humans" [67].

4.19. Michael Fenech on cytokinesis-block micronucleus assays The development of the cytokinesis-block micronucleus (CBMN) assay in human lymphocytes radically changed the cytogenetic detection of chromosomal damage induced by radiation and chemicals. The CBMN assay was first reported in the 1980s by Michael Fenech and Alexander Morley of Flinders University in South Australia [59,60]. In this new method, cytochalasin B was added to cultures of mitogen-stimulated lymphocytes to block cytokinesis after enough time had elapsed for the cells to be entering their first post-stimulation mitotic division. At an appropriate time, slides were made, and cells that had

5. Reflections on Donald MacPhee Working with Donald MacPhee ranks high among the pleasures of 112

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

being an editor of Reflections. When we were invited to be co-editors, I had met him several times at scientific meetings, and we had on a few occasions exchanged bacterial strains through the mail. I held him in high regard, knowing him to be a first-rate microbial geneticist whose expertise extended to radiation biology and radiation protection, as well as mutational mechanisms, DNA repair and chemical mutagenesis. He was also a deep thinker, a skilled writer and an enthusiastic contributor to the vitality of the scientific community. I soon came to appreciate how these qualities were complemented by dedication, sense of humor and generosity of spirit. Our co-editing of Reflections kept us in regular contact, while he was in the School of Microbiology at LaTrobe University in Melbourne, Australia, or at the Radiation Effects Research Foundation in Hiroshima, Japan, from 1999 to 2004 during a reorganization in the Radiobiology Department. Our collaboration on Reflections soon grew from a cordial professional relationship into a real friendship, forged through electrons zipping around the world in frequent email messages. In the early days, we often added a few stray comments on the weather or whatever was going on in the world. This evolved into discussions of many things, and we came to know each other's families (whom we had never met), adventures and misadventures, health problems, pleasures and problems of work, Massachusetts ice and snow storms or Australian brush fires, and whatever else may have been on our minds at the time. While email had become the norm, it still impressed those of us coming from the era of typewriters, carbon paper, and awaiting conventional mail, and so we enjoyed joking remarks about the impending Y2K disaster, when there were unnecessary worries about the entire cyber world collapsing at the turn of the millennium, or the curiosity of time zones in such remarks as "G'day, I was pleased to receive the email that you sent me tomorrow." The saddest day in the history of Reflections came with Donald's death on September 16, 2009 [68,69]. I think often about our 11 years of regular correspondence, and I am glad to have had the chance for us to meet at the time of the International Conference on Environmental Mutagens in Shizuoka, Japan, in 2001. A fine scholar and a true gentleman, it is no wonder that Donald MacPhee is missed by friends and colleagues around the world.

considered the distinguished Neurospora geneticist David Perkins of Stanford University as an important mentor early in his career. His experience with Neurospora was enriched by a postdoctoral fellowship with Fred de Serres at the National Institute of Environmental Health Sciences (NIEHS) in North Carolina, where I came to know him as a friend in the 1970s. Hirokazu never abandoned Neurospora, but he adopted the methodology of molecular biology early, making major contributions that led to his being recognized with the Metzenberg Award for his lifetime achievements in Neurospora research, given by the Neurospora research community at the Fungal Genetics Conference in Asilomar, California, in 2009. His Reflections article combines a personal perspective with a review of the early history of Neurospora research, the transition to molecular analysis, and an emphasis on current understanding of DNA repair, recombination, and homologous integration in Neurospora [71]. His research over many years at Saitama University in Japan is an important component of this history, and his account of it includes detailed tables. He concludes with thoughts on promising new directions for research on DNA repair and mutagen sensitivity in Neurospora. 6.3. Carmel Mothersill and Colin Seymour on unforeseen discoveries in radiation biology Carmel Mothersill is perhaps best known for her creative research on nontargeted effects of ionizing radiation, especially bystander effects, in which cells that did not experience the direct energy deposition are affected by signals received directly or indirectly from irradiated cells. A Reflections article that she wrote together with her husband and longtime research collaborator Colin Seymour comments on diverse aspects of radiation biology [72]. They focus on unforeseen discoveries and lingering uncertainties that led to changes in longstanding views about effects of radiation, which had been derived from target theory. The changes stem from findings on nonlinear dose responses, dose-rate effects, indirect effects of radiation mediated by radicals, inducible DNA repair, adaptive responses, and bystander effects. An incomplete understanding of such phenomena remains an obstacle to defining the best practices for radioprotection and radiotherapy. Besides a fascinating commentary on unresolved questions in radiation biology, Carmel and Colin include a tribute to the pioneering radiobiologist Tikvah Alper (1909–1995), and Carmel gives us a view of her own original abstract paintings on the theme of bystander effects of radiation [72].

6. The second decade of Reflections 6.1. Martin Marinus on DNA methylation and mutator genes in bacteria Martin Marinus worked for decades on DNA methylation and mutators in E. coli – studies that have contributed significantly to a modern understanding of mutagenesis and DNA repair. His Reflections article gives a personal perspective on this history, including his discovery of the first DNA adenine methyltransferase (dam) mutants in E. coli in the early 1970s and his many years of research at the University of Massachusetts Medical School elucidating mechanisms underlying bacterial DNA methylation, its functions, and associated mutator phenotypes [70]. Later research revealed the role of 6-methyladenine in strand discrimination in mismatch repair in E. coli. The content of Martin's commentary is broad, including an overview of DNA methylation mutants, mismatch repair, effects on recombination and mutator phenotypes. He ends his commentary with the interesting observation that the frequency of mutators in pathogenic or commensal strains of E. coli is much higher than that in standard laboratory strains that are not grown under the intense selective pressures of new habitat colonization [70].

6.4. A.T. Natarajan on a lifetime of studies in cytogenetics A.T. Natarajan returned to Reflections a few years after his initial commentary [57] to reflect on his long career in cytogenetics [73]. It is a great personal story. Nat begins autobiographically with his early years in India. His research career began in 1948, and he offers a fascinating look at a time and place when the challenges that a young scientist encountered were very different from those in modern India today or in prosperous countries then. He began in botany, because that was the most practical choice, but from the beginning he longed to get into genetics and work with chromosomes. He recounts his leaving India to pursue opportunities in the U.S. and Sweden, followed by an interlude in India, then Vienna, and ultimately at Leiden University in the Netherlands, where he spent the rest of his professional life. He recalls specific events with great style and humor, but he never loses his focus on science. Nat summarizes some of his major scientific accomplishments and the revolutionary changes that occurred in cytogenetics during his career, including the molecular analysis of alterations and FISH. Applied research was important to Nat, and this is reflected in his research contributions in radiation cytogenetics and chemical mutagenesis, complemented by his contribution to social and political efforts to minimize the impact of radiation accidents and chemical exposures. He comments with appreciation on his mentors along the way, including

6.2. Hirokazu Inoue on DNA repair and homologous integration in Neurospora Hirokazu Inoue was introduced to the fungus Neurospora crassa as a model organism in 1969, when he became a graduate student of Tatsuo Ishikawa at the University of Tokyo. In addition to Dr. Ishikawa, he 113

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

M.S. Swaminathan, who is known as the Father of India's Green Revolution, Lars Ehrenberg in Sweden, and later F.H. "Frits" Sobels in Leiden. A gentle man with great intellect and wit, he describes how he valued the many friends who enriched his professional and personal life. This was reciprocal, as Nat was highly regarded and is missed by many former students and colleagues around the world. For those who would like to read more of Nat's life and scientific contributions, I would refer you to a beautiful tribute by Awadhesh Jha of the University of Plymouth, who knew Nat as his mentor both in India and later in Leiden [74], and to a special issue of Mutation Research in Nat's honor, nicely introduced by Adayabalam Balajee and colleagues [75].

his advocacy for radiation safety; his distinction among gene mutations, changes in chromosome structure, and changes in chromosome number; his recognition of genetic interactions and environmental influences on gene expression; his explanation for the rarity of polyploidy in animals relative to plants; and his views on various social and political controversies, including eugenics and Lysenkoism. 6.7. Elof Axel Carlson on the introduction of Drosophila into mutation research Shortly after writing his reflections on H.J. Muller [79], including Muller's experience in Morgan's Fly Lab, Prof. Carlson began to write some reflections on the fly. How did Drosophila come to its central position in twentieth century genetics? Elof weaves the history of Drosophila's arriving in the Fly Lab at Columbia into a fascinating story with surprising elements, such as studies in Indiana's limestone caves and committee meetings involving mirror tricks at Indiana University, complete with photographs dating from 1902 to roughly 1914 [80]. Fruit flies were chosen early for studies of experimental evolution, and the route to Morgan's lab involved William Castle at Harvard University, Frank Lutz at Cold Spring Harbor, and a critical role played by researchers at Indiana University. The Indiana biologists Carl Eigenmann, William Moenkhaus, and Fernandus Payne were interested in the evolution of blind cave fish and other blind cave fauna. Moenkhaus introduced fruit fly research to Indiana University, and Payne was intrigued by the prospect of using them to study experimental evolution. It is likely that Castle, who was conducting studies with Drosophila at Harvard, influenced Morgan to consider using flies. The actual source of the flies, however, was probably Fernandus Payne. Payne moved from Indiana to Columbia University for graduate work, and he used flies there for his studies of experimental evolution and for teaching. Elof Carlson had met Payne at Indiana University in 1957 and later interviewed him when he was writing his biography of H.J. Muller. Payne told Elof that he caught his flies on a windowsill at Columbia, and when Morgan later asked him for some, he obliged. This led eventually to Morgan's isolation of the famous white-eye mutation and to the beginnings of classical genetic analysis, involving such luminaries among Morgan's students as Alfred Sturtevant, Calvin Bridges and H.J. Muller.

6.5. Urbain Weyemi and Corinne Dupuy on reactive oxygen species, NOX4 and DNA damage Rather than reflecting on events of years gone by, our next Reflections authors, Urbain Weyemi and Corinne Dupuy, reflected on the implications of a new area of investigation – the role of a specific oxidase in the generation of reactive oxygen species (ROS) involved in cellular oxidative stress [76]. The NADPH oxidase NOX4 is localized in the perinuclear region of cells, and the ROS that it generates when activated are implicated in genetic damage and genomic instability but also play a role in normal redox processes, signal transduction and repair. Two informative figures nicely illustrate their hypothesis: one introducing the generation of ROS by NOX enzymes, and the other presenting the hypothesized actions of NOX4 in modulating oxidative stress. Weyemi and Dupuy suggest that dysregulation of NOX4 is implicated in inflammation, cancer, and such other disorders as diabetes, fibrosis and kidney dysfunction. They speculate that NOX4 (and perhaps other NOX enzymes) can be considered as a potential target for therapeutic applications [76]. At the end of 2018, a PubMed search for "NOX4" yielded 2101 citations, including 1527 for "NOX4 and reactive oxygen species," reflecting the fact that NOX4 remains a topic of intense interest. 6.6. Elof Axel Carlson on the legacy of Hermann J. Muller Hermann J. Muller is one of the giants of 20th century genetics and mutation research. The history of Muller's life and work was told beautifully by Elof Axel Carlson in a biography written in 1981 [77]. When Professor Sobels established the journal Mutation Research [1], he commented that he was "particularly honored" that the first paper in the new journal was by the distinguished geneticist H.J. Muller. In this paper on the relationship between recombination and the accumulation of mutations in populations, Muller discussed what later came to be known as "Muller's Ratchet" [78]. Given this background, I contacted Dr. Carlson, who is now a Visiting Scholar at the University of Indiana, where he continues his work with the Muller archives, in hopes that he would consider submitting an article to Reflections. I was delighted to find that he was receptive to my invitation, and that began an enjoyable association with him that has spanned three Reflections articles. His first was on H.J. Muller's contributions to mutation research and includes personal reflections on having been a student of H.J. Muller in the 1950s [79]. The article offers both a substantive commentary on Muller's farreaching scientific achievements and a nice personal touch, including a photograph of several pages of his own class notes when he took Muller's course, called "Mutation and the Gene,’’ at Indiana University in 1955. Elof's Reflections article comments on many aspects of H.J. Muller: his personality and qualities as a teacher and mentor; his early, deep understanding of the role of the gene in life and evolutionary theory; his formative years in the fabled "Fly Lab" of Thomas Hunt Morgan at Columbia University; his extraordinary ability to design genetic stocks to solve important problems experimentally; his discovery of x-ray mutagenesis in 1927, followed by his Nobel Prize in 1946; his commitment to understanding dose-response relationships;

6.8. Lutz Müller and Elmar Gocke on photomutagenesis The history of photochemical mutagenesis has had its ups and downs, and the story is nicely told by Lutz Müller and Elmar Gocke who were in the thick of the action at Hoffmann-La Roche, Ltd., in Basel, Switzerland [81]. They called their reflections "The Rise and Fall of Photomutagenesis." Concerns about possible phototoxicity associated with exposure to sunscreens, cosmetics, and pharmaceuticals included photomutagenic, photocarcinogenic, and photoallergenic effects. Substantial efforts were made to develop simple screening methods for photomutagenicity, especially using bacterial reversion assays and mammalian cell cytogenetics. Despite the initial enthusiasm, it became increasingly evident that photomutagenesis entails complexity with interactions at several levels and technical obstacles that were hard to resolve. This led to a realization that simple screening assays would not suffice and that a more laborious case-by-case evaluation was needed. The Reflections paper includes interesting anecdotes, such as Elmar's observation that holding an Ames-test plate of strain TA100 in the sunlight outside the lab for 15 s led to a doubling of the revertant frequency. In another, Elmar noticed that a puzzling, apparently irreproducible, positive response to compound "RO19-8022" in strain TA102 occurred only when the test was performed in the afternoon. It turned out that the cause was the increased ambient light when the "sun came around the building." Compound RO19-8022 turned out to be highly phototoxic and photomutagenic; it was terminated as a drug candidate but was made available for research purposes. In commenting on the "Rise and Fall," Müller and Gocke stated that 114

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

"the Fall" applies to hopes for a simple solution to a complex problem and not to the value of basic research on photomutagenesis or to the goal of avoiding phototoxicity and photocarcinogenesis. To support the conclusion that photomutagenicity screening is not practical in safety assessment, they draw upon collaborative European studies; problems of evaluating UVB, UVA or a mixture of wavelengths; phototoxicity of fluoroquinolone antibiotics; micronucleus and comet assays in skin; and the problem of "pseudo-photoclastogens" – apparent photoclastogens that do not even absorb light. In 2012, the International Conference on Harmonisation (ICH) guideline stated that "Testing of photogenotoxicity is not recommended as a part of the standard photosafety testing programme,’’ but a phototoxicity assay would remain in place.

the Mouse House for a general audience, and she comments on other aspects of her experience in Oak Ridge. Those who know Lee as a mouse geneticist may be unaware that she and Bill became involved in the environmental movement in the 1960's, working to protect Tennessee rivers and wilderness. They founded Tennessee Citizens for Wilderness Planning (TCWP) in 1966, and she is still the editor of the TCWP newsletter in 2019. TCWP has had remarkable influence in protecting the Obed River and the Big South Fork of the Cumberland River and in many other conservation projects. It is a tribute to Lee and Bill that they have had such a profound impact in two very different areas.

6.9. Liane B. Russell on genetics research in the "Mouse House" in Oak Ridge

Delbert ("Del") Shankel's Reflections article is a tribute to Charlotte Auerbach (1899–1994), who obtained the first unequivocal evidence of chemical mutagenesis. Del describes her scientific contributions, which go far beyond the discovery of chemical mutagenesis in the 1940s, and his narrative is enriched by his memories of her based on a sabbatical that he spent with her in Edinburgh in 1967 [84]. Del gives both a detailed biographical account of Charlotte Auerbach, called "Lotte" by her friends, and a sense of her vibrant personality and humanitarian values. Sadly, Del Shankel died in July 2018. Del was soft-spoken and not inclined to boast of his accomplishments. I knew him for years as a microbiologist and an EMS colleague working on antimutagens before I realized the scale of his service and leadership over 50 years at the University of Kansas. Beginning as an Assistant Professor of Microbiology in 1959, he became Professor of Microbiology, Chairman of the Department of Microbiology, and a Fellow of the American Academy of Microbiology. He later served as Assistant Dean, Associate Dean, Acting Dean of the College, Executive Vice Chancellor, and Acting Chancellor. Whenever the University of Kansas called upon him, Del served with distinction, and in 1995 he was named as the 15th Chancellor of the University. Those who know him may want to see a tribute to him from the University of Kansas Alumni Association, which includes his picture standing by the Delbert M. Shankel Structural Biology Center, named in his honor [85]. I have a specific memory from working with Del on his Reflections paper. He had an original photograph of Charlotte Auerbach, and he was unsure about how to prepare it for publication, so I offered to take care of it if he would send the photo to me. I suggested sending it registered and insured. A few days later, the photo arrived by regular mail. Del said that he mailed his first letter in ordinary U.S. mail when he was 5 years old in 1932, has been doing so ever since, and never lost a single item.

6.10. Delbert Shankel in remembrance of Charlotte Auerbach

One of the pleasures of being the editor of Reflections was working with Liane ("Lee") Russell as she wrote her reflections on the history of the mouse genetics facility at ORNL, known as the Mouse House [82]. The rationale for its establishment in 1946 was the need to understand the effects of ionizing radiation in the context of the bombings of Hiroshima and Nagasaki, nuclear testing, and prospects for peacetime uses of atomic energy. When Alexander Hollaender was developing the Biology Division of ORNL, he recruited William L. ("Bill") Russell (1910–2003), a mammalian geneticist at the Jackson Laboratory in Maine, to develop and direct the program. Liane Brauch ("Lee") Russell, Bill's wife and also a mammalian geneticist, would be an independent researcher at ORNL. She became the director of the Mouse House upon Bill's retirement in 1975. In fact, Bill and Lee worked as a team both before and after his official retirement. In her Reflections article [82], Lee gives a thorough review of the research in the Mouse House, a fascinating historical account, and her personal perspective on the times, people and events. There are photographs of Bill and Lee in the 1950s, other Mouse-House people, and the specialized equipment of the time. There are also cartoons of mice with personalities reflecting their genetic attributes, drawn by the animal-care supervisor Louis Wickham. Lee praises her young technicians and comments on rewarding international collaborations and visiting dignitaries. She includes such interesting anecdotes as that of a congressman getting stuck between floors on a lab visit to ORNL, and Bill driving mice to Nevada to oversee their exposure in one of the last above-ground nuclear bomb tests. The Mouse House is probably most known for studies of germ-cell mutagenesis, the mouse specific-locus test, and the use of mouse data for estimating risks of ionizing radiation. However, the contributions were much broader than that. Over six decades, the Mouse House generated a wealth of knowledge on mammalian genetics, molecular biology, cytogenetics, reproductive biology and teratology. Lee summarizes an impressive list of accomplishments spanning such diverse topics as germ-cell development and stage sensitivity, dosimetry, spontaneous mutations in the perigametic period, the supermutagen Nethyl-N-nitrosourea (ENU), DNA repair in vivo, fine structure genomic mapping, molecular analysis of mutations, radiation-induced teratogenesis, critical periods in embryonic development, homeotic shifts, mammalian sex determination, heritable translocations, dominant lethals, aneuploidy, mosaicism, and mouse models for human disorders [82]. Lee's story ends with changing priorities in funding, the gradual decline of the Mouse House and, ultimately, the closing of the facility. This is told with some sadness, but also with an appreciation for its remarkable legacy. I would like to point out an interview that Lee gave in Oak Ridge in April 2018 for "Voices of the Manhattan Project." The website [83] shows Lee on camera responding to questions about many aspects of her life, including her family history, their having to leave her native Austria because of its annexation by Nazi Germany in her teen years, and the events that brought her to Oak Ridge. She describes the work of

6.11. James D. Tucker on FISH and chromosome painting James D. ("Jim") Tucker played a leading role in the development of whole-chromosome painting by means of fluorescence in situ hybridization (FISH). In a Reflections article [86], he reviews key elements of his scientific work and describes the obstacles that he encountered and how they were resolved. He offers a rich personal commentary on mentors, students, the upheavals and uncertainties that young scientists often face, and the struggles and pleasures of a research career, viewed through his experience at Lawrence Livermore National Laboratory (LLNL) in California and later at Wayne State University in Detroit. Jim begins his story with thoughts on the seminal role that a key paper can play in stimulating the interests of a young scientist and suggesting a path for research. In his case the paper was by Mary Lou Pardue and Joseph Gall on the first localization of nucleic acids within metaphase chromosomes by in situ hybridization using radioactive label in the days before FISH [87]. Mentors were another source of inspiration, and he acknowledges Joginder Nath at West Virginia University and Tong-man Ong at the National Institute for Occupational Safety and Health in this context. A dedicated teacher himself in later years, he comments on his hopes for students. 115

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

Jim had his first experience in molecular cytogenetics at LLNL, leading to his pioneering work on chromosome painting. His text contains 7 figures of fluorescent human and mouse chromosomes illustrating specific advances in the methodology, one photo of fluorescent kinetochores in the CBMN assay, and one of a coffee cup. Why a coffee cup? I'll let you read Jim's Reflections article for the story. Early in its development, chromosome painting worked well with human chromosome 4, and it permitted the detection of translocations that were not readily detected with conventional staining. Simultaneously staining chromosomes 1, 2 and 4 greatly increased the portion of the genome covered. By 1998, Jim and his colleagues were simultaneously painting chromosomes 1, 2 and 4 in one color and chromosomes 3, 5, and 6 in another. Ultimately, painting probes would be available for the entire genome [86]. The aim now was to use chromosome painting to explore basic scientific problems and applications. The development of painting probes for mouse and rat chromosomes took a high priority, as methods in model organisms would be useful for comparative studies and controlled exposures. Jim comments on the use of chromosome painting to study chemical mutagens (e.g., heterocyclic amines in foods), radiation-exposed cleanup workers at Chernobyl, and effects of age on spontaneous aberration frequencies. He explores diverse topics where painting has been useful, including complex aberrations, the persistence of translocations in vivo and their decline over time, the distribution of breakpoints in chromosomes, clones bearing translocations in vivo, deviations from the theoretical 1:1 ratio of translocations to dicentrics, and the use of translocations in radiation dosimetry [86]. The ability to see many more aberrations with FISH than with conventional staining, including small slivers of color in the unpainted chromosomes, led to controversy about nomenclature for classifying the aberrations. Jim describes a meeting of nine cytogeneticists in 1993 devising a nomenclature system called PAINT (Protocol for Aberration Identification and Nomenclature Terminology) [88]. What may have appeared to be competitive nomenclature systems, "PAINT" and "S&S," were resolved by two of their principal proponents. Jim Tucker and John Savage explained the utility, strengths, weaknesses, and somewhat different purposes of Savage's S&S system and the PAINT system in which Jim had a central role [89].

most recent BEIR Committee. They also comment on computational modeling as it relates to LNT. They conclude that computational modeling is a promising approach for resolving important questions and strengthening risk assessment in the years ahead [90]. 6.13. Krishna Dronamraju on J.B.S. Haldane as a scholar and a mentor We are privileged to have personal reflections on the great geneticist, evolutionary biologist, mathematician, statistician and general scholar J.B.S. Haldane (1892–1964), written by a distinguished geneticist and historian of science who knew him personally – Krishna Dronamraju. Dr. Dronamraju was a student when Haldane moved to India in 1957. He wrote to Haldane expressing interest in working with him, and he became Haldane's student at the Indian Statistical Institute in Calcutta. He admired Haldane greatly and received his Ph.D. as Haldane's student in 1964, in the same year that Haldane died. Through the years, he has written several books on Haldane's life and work, the first in 1968 [91] and the most recent in 2017 [92]. Dr. Dronamraju begins his Reflections article with comments on Haldane's brilliance, his colorful personality and his remarkable scientific productivity, including being one of the founders of population genetics [93]. He notes Haldane's observations on interspecific crosses in animals that came to be known as "Haldane's Rule" and Haldane's hypothesis about the origin of life dating to 1929. In discussing Haldane's life, he includes interesting stories of his childhood in a scientific family, his preference for a simple way of living, his adoption of an Indian life style, and his great generosity. His interest in mutation developed early, and it included the first estimate of a mutation rate for a human gene – the sex-linked hemophilia gene – in 1932. Haldane explored many topics related to mutation and evolution, including relationships between mutation and selection, the evolutionary selection of mutation rates, genetic load, the impact of mutation, effects of ionizing radiation, and the relationship between frequencies of genetic polymorphisms and diseases. Much of evolutionary genetics bears Haldane's imprint, including relationships among mutation, natural selection, inbreeding, linkage, population distributions, and environmental factors. Dr. Dronamraju ends his reflections with thoughts about the last years of Haldane's life in India, where he became a citizen, guided students toward research on Indian plants and animals, and adopted an Indian perspective on many things, including evolution. It is a fine tribute to the extraordinary person that Dronamraju knew well, and to the geneticist / evolutionary biologist who left an indelible mark on our field.

6.12. K. Sankaranarayanan and H. Nikjoo on computational modeling of radiation risks K. Sankaranarayanan ("Sankar") had long been interested in prospects for strengthening radiation risk assessment. After retiring from Leiden University in 1998, he became a visiting scientist at the Karolinska Institute in Stockholm where he joined Hooshang Nikjoo, a professor in the Radiation Biophysics Group, to pursue this subject. In their Reflections article [90], Sankar and Hooshang assert that the time has come for strategies that draw more substantively on genomic information, mechanistic studies on radiation effects, and sophisticated computational modeling. They focus on germ-cell effects, and the paper begins with a history of radiation protection, supported by a table of estimates of genetic risks of ionizing radiation and a glossary of technical terminology and abbreviations. They identify uncertainties that remain despite decades of effort, including questions about the use of a doubling-dose method in risk assessment, insufficient information on germ-cell radiosensitivity in human females, and the absence of evidence on radiation-induced genetic disease. Sankar and Hooshang review computational modeling related to genomic data, mechanisms of mutagenesis, responses to DNA damage, and repair mechanisms. They point out a critical need for modeling studies of deletions. To relate recent findings to historical radiation protection, they briefly review the linear no-threshold (LNT) model that has been the longstanding default assumption in genetic risk assessment. They point out alternative views, but they endorse the continued use of LNT, as recommended by ICRP and the U.S. National Academy's

6.14. Narendra Singh on the comet assay The comet assay has had a large impact on genetic toxicology over several decades. It has the advantage of being a simple, direct indicator of DNA damage that can be measured in diverse tissues and in many different organisms, not restricted to defined genotypes as are many assays. I thought of Ray Tice, a longtime friend and colleague, as a possible author for Reflections on the assay, given his extensive work with it. Ray advised me that the person who was the creative force behind the assay and should write the article is his colleague Narendra P. Singh. I invited Dr. Singh, and he was receptive. His article covers not only the development of the comet assay but also gives a view of his early life and scientific development in India, followed by his years as a young scientist living abroad, and work with the comet assay over three decades [94]. Singh describes his experience at King George’s Medical College at Lucknow, India, with fond memories, having been there as a student, then a graduate student, and finally a faculty member. He gives a sense of the limitations of the times through anecdotes about isolating phytohemagglutinin from beans to culture lymphocytes, bringing a pressure cooker from home to use as an autoclave, and suffering through power outages. He also recalls hours in the library learning about DNA 116

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

and chromosomes, along with his wife, who copied journal articles by hand to help him. Singh's interest from the outset was to study aging by means of cytogenetics and molecular biology. Ultimately, he sought opportunities abroad, and this brought him to Ohio State University (OSU). He describes the adventure of this trip with funny stories, such as carrying a suitcase of cashews and raisins, as a cautious vegetarian not knowing what to expect. He was pleased with OSU, supportive mentors and a "kindly grandmother" who rented him a room, but there were also many trips to the immigration office in Cincinnati seeking approval to stay in the U.S. He later moved to various institutions, with critical periods at the National Institutes of Health (NIH) and the University of Washington. Singh acknowledges a book chapter by B. Rydberg and Karl-Johan Johanson for ideas leading him to what would become the comet assay and also a chance meeting with Ray Tice at a scientific conference as the origin of their fruitful collaboration. A critical opportunity offered to him at the NIH Institute on Aging (NIA) was training with Peter Cerutti in Lausanne, Switzerland, where he learned to do alkaline elution of DNA. He describes Cerutti as an inspiring teacher, and in a funny story he notes that everything there was perfect, except that at a dinner, he unknowingly ate caviar and was aghast when he learned a few minutes later what it was. Back at NIA in 1986, everything was in place for his development of the comet assay. Thinking back to Rydberg and Johanson, he had recognized three elements of the problem – isolation of living cells, embedding of cells, and lysis of cells – and the idea came to him to electrophorese the cells in order to move small DNA fragments. He describes the problems encountered (e.g., getting rid of RNA and finding good stains) and how they were resolved. It turns out that Ostling and Johanson had independently decided on electrophoresis and published a paper using similar methods a few years earlier [95]. Singh identified a shortcoming of the assay as described, and he modified it by using alkaline electrophoresis. He describes his excitement at first observing "comets" and running to tell his colleagues Mike McCoy, Ray Tice and Ed Schneider, who became his coauthors in publishing the method that is the basis for today's comet assay [96]. The name of the method also evolved from “microelectrophoresis” [95], to "microgel electrophoresis" [96], to "single-cell gel electrophoresis," and ultimately the "comet assay," a name introduced by Peggy Olive and colleagues based on the appearance of the affected cells and their DNA when viewed through a microscope. Singh comments that the name "comet assay" has "rightly stuck for the last 25 years." Singh devotes the remainder of his reflections to his subsequent studies and applications of the assay [94]. These include the influence of aging on DNA damage, detection of exposure to low doses of ionizing radiation, effects of radiofrequency radiation, genotoxicity of chemicals, apoptosis, DNA damage in various cell types, and refinements of equipment and computer analysis. With A.T. Natarajan, he combined the comet assay with FISH for visualizing specific gene sequences and centromeres. He notes that the comet assay is now used internationally for such varied purposes as human biomonitoring of DNA damage, ecotoxicological applications in diverse species, and genotoxicology assessment.

in editorial policies. He briefly traces the history of ad hominem arguments in science to antiquity, discusses Isaac Newton and Robert Hooke in the 17th to 18th century, and concentrates on the time after the historic work of Mendel and Darwin in the 19th century. While Darwin preferred to stay clear of disputes, his detractors and defenders did not. After the rediscovery of Mendel's work in 1900, the disputes in the new field of genetics became intense, and Elof gives a vivid account of the antagonism between William Bateson, who tended to think of Mendelian heredity in terms of discontinuous phenotypes, and the biometric school of Francis Galton, Karl Pearson and Raphael Weldon. These disputes spilled over to T.H. Morgan's fly lab, including H.J. Muller, and ultimately, they were largely reconciled through the merging of classical Mendelian genetics and evolutionary biology. Other cases in Elof's review are the very consequential attacks of T.D. Lysenko on the agricultural geneticist N.I. Vavilov in the Soviet Union and the less consequential, yet illustrative, public disdain of Erwin Chargaff for James Watson and Francis Crick in the early days of molecular biology. With this background, Elof takes up the resurgence of ad hominem attacks in the case of H.J. Muller, and he comments on the degrading effect of such polemics on scientific discourse and truth [97]. He cites the attacks by E.J. Calabrese on Muller and others involved in National Academy of Sciences studies of ionizing radiation, and he offers an evidence-based defense of their reputations, but there is still concern that clarity can be lost in the repetitive attacks. He makes recommendations on how the structure of scientific literature offers the hope of avoiding personal attacks and the distortion of historical events. They include a role for journal editors and reviewers, instructions to authors, and more referrals of such manuscripts to journals that focus on (and fairly review) the history, sociology and philosophy of science. In his Reflections article on "scientific feuds, polemics, and ad hominem arguments," Elof Carlson makes important points about the integrity of the scientific process that, in my view, deserve careful consideration. 6.16. Errol Zeiger on the history of genetic toxicology testing The last three decades of the 20th century were the heyday of largescale systematic screening of chemicals for mutagenicity. The testing program of the U.S. National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS) was central to this effort, and Errol Zeiger was central to the NTP program. His involvement in genetic toxicology testing and evaluation preceded the NTP program. In his Reflections article [99], he gives a fine historical review of this period, combined with personal anecdotes. Errol begins with the circumstances by which he came to the field that would become genetic toxicology. In 1968, he was looking for part-time laboratory work while continuing his studies toward a Ph.D. By a circuitous route, described with some funny elements, he was hired by Marvin Legator at the Food and Drug Administration (FDA) in 1969 to work on the metabolic activation of chemicals into mutagens in Salmonella in the host-mediated assay. He was able to continue his graduate work for his Ph.D. at the FDA. At that time, Marvin was involved in organizational work as part of the group that formed the EMS in 1969, led by Alexander Hollaender. This group was urging the FDA and other agencies to take up the testing of food additives, drugs, pesticides and other chemicals for mutagenicity. Errol worked with the key players in this movement, including Gary Flamm, Heinrich Malling and Fred de Serres. Gary succeeded Marvin as FDA Branch Chief, and in 1976 Heinrich and Fred recruited Errol to NIEHS, where he ran the NIEHS (later NTP) genetic toxicology testing program. He also came to know Bruce Ames, who was developing the Ames test and sent him the Salmonella strains. In 1971 Marvin Legator persuaded the FDA to award two contracts to test food additives for mutagenicity in vitro and in vivo, and Errol was responsible for test protocols for the host-mediated assay, Salmonella spot tests (which preceded the Ames assay) and yeast recombination tests. In the next few years, Ames and colleagues reported on the Salmonella/mammalian-microsome mutagenicity test, and Errol

6.15. Elof Axel Carlson on ad hominem arguments in the scientific literature An issue of publication policy and ethics that occasionally arises in science is the use of vitriolic arguments and ad hominem attacks to promote or denigrate viewpoints. Elof Carlson has been deeply concerned about personal attacks being made against H.J. Muller because of Muller's historic influence in the process of radiation risk assessment. This led Elof to reflect on ad hominem attacks in the scientific literature, both with respect to current and historical instances [97]. While working on his book, The Gene: A Critical History [98], Elof had the impression that disputes among respected geneticists were especially intense shortly after the turn of the 20th century. Then, around 1919, the vitriolic arguments seemed to disappear, perhaps owing to changes 117

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

oversaw bacterial mutagenicity testing at FDA. An interagency advisory committee was formed in 1975 with members from FDA, NIEHS and the National Cancer Institute (NCI), chaired by Virginia Dunkel of NCI, to establish a validation program for mutagenicity tests. Contracts were awarded to perform testing in Salmonella and in mouse lymphoma cells, the protocols for which were later refined and adopted for international test guidelines. A little-remembered event had a big impact: Bruce Ames and colleagues reported the mutagenicity of hair dye components. This created concern in some and alarm in others, including congressmen, and recommendations were made at NIH budget hearings for a major mutagenicity testing program. Funding and staffing requests were approved for a large program centered at NIEHS. Errol designed and managed the program and describes in detail how it evolved and grew, beginning with Salmonella, chromosome aberrations and sister chromatid exchanges (SCE) in cultured mammalian cells, and the mouse lymphoma TK mutation assay. Staff members were recruited to the NIEHS, and Errol describes the intricate planning and staffing needed to make the massive effort work. He also gives a sense of the conditions at the time – analyzing, managing, organizing and maintaining the program and database in the days before the computer capabilities that we now consider routine. As results were obtained, the new information led to changes in assays, protocols, and contractors doing the laboratory work. The data handling evolved to take advantage of newer computer technology, and the information and test results from the program were made publicly available and published in Environmental Mutagenesis [99]. Quality control was critical, and it included coded samples, appropriate positive and negative controls, monitoring of strains, and checks on intra- and inter-laboratory reproducibility and on the frequency of mislabeling of chemical samples. The NIEHS project officers were not only administrators but also researchers having first-hand experience with the assays and methods. A highly qualified statistician, Barry Margolin, provided the expertise to evaluate statistical methods. The number of chemicals for systematic screening gradually declined in the 1990s, and the emphasis shifted to comparative studies, concordance with carcinogenicity data, and prospects for alternative assays and methods. Errol retired from NIEHS in 2000 but remains active in genetic toxicology and gives a sense of the current program at NIEHS. He ends his reflections with thoughts on the role of chance in determining the course of events, and satisfaction that he had the good fortune to be "in the right place at the right time."

Adams's comedy and science fiction series “Hitchhiker’s Guide to the Galaxy.” 6.18. Micheline Kirsch-Volders and colleagues on European collaborative efforts on aneuploidy The most recent Reflections article reviews a large European program on aneuploidy, including both the science and implications for public policy [101]. This article differs from its predecessors in being an effort by 6 authors in 4 countries who reflect on an international collaborative effort that has had a lasting legacy and influence. Micheline Kirsch-Volders (Belgium), Francesca Pacchierotti (Italy), E.M. ("Liz") Parry (UK), Antonella Russo (Italy), Ursula Eichenlaub-Ritter (Germany) and Ilse-Dore Adler (Germany) worked intensively on bringing together a text that is simultaneously a scientific review, a history, and a reflective commentary fully documented with literature citations, tables and figures. The organizational structure that they describe ran from 1981 through 2004. This was a period of remarkable accomplishment with respect to the understanding of mechanisms underlying aneuploidy and their implications for human health. Among the topics discussed are historical studies on abnormal chromosome numbers, mechanisms and consequences of aneuploidy in somatic cells and germ cells, cellular and molecular targets for aneuploidy induction, the role of aneuploidy in cancer, relationships to apoptosis and chromothripsis, aneugenic chemicals, assays for aneuploidy, adaptation of micronucleus assays to detect aneuploidy, applications of FISH, environmental influences on aneuploidy, metabolic activation of aneugens, dose-response relationships, the likelihood of thresholds, data requirements for effective regulation, and efforts to formulate regulatory guidelines. As in any area of science, there are still many unresolved questions, but the perspectives of the authors provide valuable suggestions for charting the route ahead so as to protect public health. The authors dedicated their Reflections article to the memory of J.M. ("Jim") Parry of Swansea University who inspired and coordinated many of the studies discussed and who was a major presence in the genetic toxicology community. He was a strong advocate for the inclusion of aneuploidy as an endpoint of concern, along with gene mutations and chromosomal aberrations. Like the authors, I remember Jim for his incisive thinking, productivity, dynamic personality, and sense of humor. I feel confident that he would be complimented to have this group of authors dedicate their efforts to his memory. I would refer readers to a remembrance of Jim in the journal Mutagenesis, of which he was the founding editor [102].

6.17. Jeffrey Miller on unexpected interactions between mutagens

7. Final reflections on Reflections

Reflections articles have taken us to many disciplines and have spanned the globe. In a new twist, Jeffrey H. Miller's article carries us to a parallel universe [100]. After a distinguished career in bacterial genetics and mutagenesis, Jeffrey was nonetheless taken aback by results that emerged from his studies of mutagen-induced mutation spectra. One might have anticipated that combined treatments with different mutagens would give responses that were additive, or perhaps antagonistic or synergistic, as interactions of these types are known to occur in toxicology, including mutagenesis. When determining mutation spectra after a combined treatment, one might expect to see molecular mechanisms that reflect the two individual agents, perhaps augmented or diminished by each other. Jeffrey's point is that some combinations of treatments with base- and nucleoside-analogs give unique mutation spectra and hotspots of mutagenesis with no obvious relatedness to the spectra of the individual agents, as though the mutagenesis were happening in another universe. He documents his argument with data on mutation spectra induced by the purine analog 2-aminopurine and the cytidine analog zebularine in the rpoB gene of E. coli. He speculates on mechanisms, and he notes that there is now evidence that such interactions also occur with compounds other than base analogs. Jeffrey develops this analogy in an amusing way, referring back to Douglas

Since its outset in 1999, the Reflections series in Mutation Research Reviews has been centered on historical themes related to mutation, while simultaneously offering insight into current mutation research. In doing so, the focus has frequently been on the genetics and molecular biology that are at the heart of mutation research, but we have viewed mutation broadly, incorporating toxicological, radiological, evolutionary, medical, statistical, and public policy aspects of the field. As such, there have been interesting extensions into sociology, philosophy, politics, ethics and, of course, the history of science. I was pleased to learn that the publisher, Elsevier, would like to prepare a compilation of all the Reflections articles in time for the 50th anniversary of the Environmental Mutagenesis and Genomics Society (EMGS), founded in 1969 as the Environmental Mutagen Society (EMS). We are also looking ahead to the International Conference on Environmental Mutagens (ICEM) that will take place in Ottawa in 2021. For me, Reflections has been a pleasure. I have enjoyed a great partnership with Donald MacPhee, my coeditor for the first 10 years, and the interaction with a fine group of scientists who have contributed much to our field. I am indebted to many excellent "readers" (i.e., reviewers), who offered a great service to me and to the authors. I also 118

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann

acknowledge the editors of Mutation Research Reviews, David DeMarini and Mike Waters, for giving Donald and me autonomy in running Reflections and for giving us their continuing support whenever needed. Finally, I thank the superb scientists and authors who made Reflections what it is.

Society, Environ. Mol. Mutagen. 51 (#8–9) (2010) 746–760. [28] E. Volkin, The discovery of mRNA, Mutat. Res. 488 (2001) 87–91. [29] H.F. Judson, The Eighth Day of Creation: Makers of the Revolution in Biology, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1996. [30] F. Jacob, The Statue Within: An Autobiography, Basic Books, New York, 1988. [31] F.P. Guengerich, Forging the links between metabolism and carcinogenesis, Mutat. Res. 488 (2001) 195–209. [32] F.F. Kadlubar, In memoriam: James A. Miller (1915-2000), Chem. Res. Toxicol. 14 (2001) 335–337. [33] R.J. Albertini, HPRT mutations in humans: biomarkers for mechanistic studies, Mutat. Res. 489 (2001) 1–16. [34] T. Cebula, P. Hanawalt, L. Thompson, J. von Borstel, Of repair and reflections: A Richard B. Setlow Festschrift, Environ. Mol. Mutagen. 38 (2001) 88. [35] R.B. Setlow, Shedding light on proteins, nucleic acids, cells, humans and fish, Mutat. Res. 511 (2002) 1–14. [36] P. Hanawalt, A. Grollman, S. Mitra, A tribute in memory of Richard B. (Dick) Setlow (1921-2015), DNA Repair (Amst.) 33 (2015) 111–114. [37] G.R. Hoffmann, D.G. MacPhee, Editorial: reflections of Edward Calabrese on hormesis, Mutat. Res. 511 (2002) 179. [38] E.J. Calabrese, Hormesis: changing view of the dose response, a personal account of the history and current status, Mutat. Res. 511 (2002) 181–189. [39] J.R.K. Savage, Reflections and meditations upon complex chromosomal exchanges, Mutat. Res. 512 (2002) 93–109. [40] J.R.K. Savage, P. Simpson, On the scoring of FISH-"painted" chromosome-type exchange aberrations, Mutat. Res. 307 (#1) (1994) 345–353. [41] J.R.K. Savage, P.J. Simpson, FISH "painting" patterns resulting from complex exchanges, Mutat. Res. 312 (#1) (1994) 51–60. [42] A. Awa, Mutation research at ABCC/RERF: cytogenetic studies of atomic-bomb exposed populations, Mutat. Res. 543 (2003) 1–15. [43] M. Paolini, M. Nestle, Pitfalls of enzyme-based molecular anticancer dietary manipulations: food for thought, Mutat. Res. 543 (2003) 181–189. [44] W.G. Flamm, Observations at the interface of mutation research and regulatory policy, Mutat. Res. 544 (2003) 1–7. [45] S. Green, D.D. Levy, R.J. Lorentzen, E. Zeiger, In memoriam: William Gary Flamm (1935–2017), Environ. Mol. Mutagen. 59 (2018) 100–102. [46] E.H.Y. Chu, Early days of mammalian somatic cell genetics: the beginning of experimental mutagenesis, Mutat. Res. 566 (2004) 1–8. [47] H.V. Malling, Incorporation of mammalian metabolism into mutagenicity testing, Mutat. Res. 566 (2004) 183–189. [48] H.V. Malling, Dimethylnitrosamine: formation of mutagenic compounds by interaction with mouse liver microsomes, Mutat. Res. 13 (1971) 425–429. [49] B.N. Ames, W.E. Durston, E. Yamasaki, F.D. Lee, Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. U. S. A. 70 (1973) 2281–2285. [50] D.A. Casciano, J.B. Bishop, D.M. DeMarini, E. Zeiger, G.R. Hoffmann, Obituary: Heinrich Valdemar Malling (1931–2016), Mutat. Res. 769 (2016) 63–66. [51] G.R. Hoffmann, M.D. Shelby, E. Zeiger, Remembering Heinrich Malling, Environ. Mol. Mutagen. 57 (2016) 575–578. [52] F. Sherman, The importance of mutation, then and now: studies with yeast cytochrome c, Mutat. Res. 589 (2005) 1–16. [53] G.R. Fink, In Memoriam: Fred Sherman – the first yeast molecular biologist, Genetics 196 (2014) 363–364. [54] R.J. Preston, Extrapolations are the Achilles heel of risk assessment, Mutat. Res. 589 (2005) 153–157. [55] K. Mortelmans, Isolation of plasmid pKM101 in the Stocker laboratory, Mutat. Res. 612 (2006) 151–164. [56] M.E. Gaulden, J. Jagger, V.P. White, Personal reflections on the life and legacy of Alexander Hollaender, Mutat. Res. 635 (2007) 1–16. [57] A.T. Natarajan, Mutation Research: the origin, Mutat. Res. 635 (2007) 79–80. [58] K. Sankaranarayanan, J.S. Wassom, Reflections on the impact of advances in the assessment of genetic risks of exposure to ionizing radiation on international radiation protection recommendations between the mid-1950s and the present, Mutat. Res. 658 (2008) 1–27. [59] M. Fenech, A.A. Morley, Measurement of micronuclei in lymphocytes, Mutat. Res. 147 (1985) 29–36. [60] M. Fenech, A.A. Morley, Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation, Mutat. Res. 161 (1986) 193–198. [61] M. Fenech, A.A. Morley, Kinetochore detection in micronuclei: an alternative method for measuring chromosome loss, Mutagenesis 4 (1989) 98–104. [62] M. Fenech, A lifetime passion for micronucleus cytome assays – reflections from Down Under, Mutat. Res. 681 (2009) 111–117. [63] S.B. Carter, Effects of cytochalasins on mammalian cells, Nature 213 (1967) 261–264. [64] P.I. Countryman, J.A. Heddle, The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes, Mutat. Res. 41 (1976) 321–332. [65] R.P. Feynman, Surely you’re joking, Mr. Feynman!, W. W. Norton & Company, New York, 1985. [66] R.G. Stevens, Electric light causes cancer? Surely you’re joking, Mr. Stevens, Mutat. Res. 682 (2009) 1–6. [67] IARC, (International Agency for Research on Cancer), IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Agents Classified by the IARC Monographs Vols. 1–123 (2019) (Accessed 8 December 2018), https:// monographs.iarc.fr/list-of-classifications-volumes/.

Conflicts of interest None. Acknowledgements I thank Malcolm Lippert, Mike Shelby, and Errol Zeiger for helpful comments on this manuscript. I thank the Elsevier journal managers who worked with Reflections manuscripts through the years and Associate Publisher Hélène Hodak for her support in bringing this twentieth-anniversary collection of Reflections papers to fruition. References [1] F.H. Sobels, Preface, Mutat. Res. 1 (1964) 1. [2] F.H. Sobels, The new section of Mutation Research – Reviews in Genetic Toxicology, Mutat. Res. 32 (1975) 1–2. [3] J.F. Crow, W.F. Dove, Birds’ eye view: a decade of perspectives, Genetics 148 (1998) 1405–1407. [4] G.R. Hoffmann, D.G. MacPhee, Reflections in mutation research: an introductory essay, Mutat. Res. 436 (1999) 123–130. [5] D. Hartl, James F. Crow and the art of teaching and mentoring, Genetics 189 (2011) 1129–1133. [6] D. Gianola, G.J.M. Rosa, D.B. Allison, Humble thanks to a gentle giant (an obituary for James F. Crow), Front. Genet. 3 (2012) 93, https://doi.org/10.3389/fgene. 2012.00093. [7] J.F. Crow, Spontaneous mutation in man, Mutat. Res. 437 (1999) 5–9. [8] B.S. Strauss, Frameshift mutation, microsatellites, and mismatch repair, Mutat. Res. 437 (1999) 195–203. [9] G. Streisinger, J.E. Owen, Mechanisms of spontaneous and induced frameshift mutation in bacteriophage T4, Genetics 109 (1985) 633–659. [10] G. Streisinger, Y. Okada, J. Emrich, J. Newton, A. Tsugita, E. Terzaghi, M. Inouye, Frameshift mutations and the genetic code, Cold Spring Harb. Symp. Quant. Biol. 31 (1966) 77–84. [11] B.S. Strauss, Why is DNA double stranded? The discovery of DNA excision repair mechanisms, Genetics 209 (2018) 357–366. [12] Z.A. Medvedev, The Rise and Fall of T.D. Lysenko (Translation and Foreword by I. Michael Lerner), Columbia University Press, New York, 1969. [13] T. Dobzhansky, Soviet Biology and the Powers That Were: Book Review of the Rise and Fall of T. D. Lysenko by Zhores A. Medvedev (Translated From the Russian by I. Michael Lerner, With the Editorial Assistance of Lucy G. Lawrence), Columbia University Press, New York, 1969 Science 164, #3887 (1969), pp. 1507–1509. [14] Z.A. Medvedev, Lysenko and Stalin: commemorating the 50th anniversary of the August 1948 LAAAS Conference and the 100th anniversary of T.D. Lysenko’s birth, September 29, 1898, Mutat. Res. 462 (2000) 3–11. [15] G.R. Hoffmann, D.G. MacPhee, Editorial: reflections of Zhores Medvedev, Mutat. Res. 462 (2000) 1–2. [16] R.D. McFadden, Zhores Medvedev, 93, Dissident Scientist Who Felt Moscow’s Boot, Is Dead, The New York Times, 2018 Nov. 16 (https://www.nytimes.com/ 2018/11/16/obituaries/zhores-medvedev-dead.html). [17] J. Steele, Zhores Medvedev Obituary, February 23 The Guardian, 2018, (https:// www.theguardian.com/world/2018/nov/23/zhores-medvedev-obituary). [18] G.W. Grigg, The origins of the back-mutation assay method: a personal recollection, Mutat. Res. 463 (2000) 1–12. [19] G.W. Grigg, Back mutation assay method in micro-organisms, Nature 169 (1952) 98–100. [20] F.J. Ryan, L.K. Schneider, The consequences of mutation during the growth of biochemical mutants of Escherichia coli. IV. The mechanism of inhibition of histidine-independent bacteria by histidineless bacteria, J. Bacteriol. 58 (#2) (1949) 201–213. [21] R. Holliday, M. Sleigh, G. Winter, In memoriam: Geoffrey W. Grigg (1926–2008), Genetics 185 (#1) (2010) 1–3. [22] R. Holliday, Somatic mutations and ageing, Mutat. Res. 463 (2000) 173–178. [23] L. Hayflick, Obituary: Robin Holliday (1932–2014), Biogerontology 15 (#3) (2014) 213–215. [24] S. Frickel, The Environmental Mutagen Society and the emergence of genetic toxicology: a sociological perspective, Mutat. Res. 488 (2001) 1–8. [25] S. Frickel, Chemical Consequences: Environmental Mutagens, Scientist Activism, and the Rise of Genetic Toxicology, Rutgers University Press, New Brunswick, NJ, 2004. [26] J.S. Wassom, Origins of genetic toxicology and the Environmental Mutagen Society, Environ. Mol. Mutagen. 14 (Suppl. 16) (1989) 1–6. [27] J.S. Wassom, H.V. Malling, K. Sankaranarayanan, P.Y. Lu, Reflections on the origins and evolution of genetic toxicology and the Environmental Mutagen

119

Mutation Research-Reviews in Mutation Research 780 (2019) 106–120

G.R. Hoffmann [68] D.M. DeMarini, M.D. Waters, G.R. Hoffmann, A tribute to Donald G. MacPhee, Mutat. Res. 705 (2010) 1. [69] P. Fisher, Donald Graham MacPhee: in memoriam, Mutat. Res. 705 (2010) 2. [70] M.G. Marinus, DNA methylation and mutator genes in Escherichia coli K-12, Mutat. Res. 705 (2010) 71–76. [71] H. Inoue, Exploring the processes of DNA repair and homologous integration in Neurospora, Mutat. Res. 728 (2011) 1–11. [72] C. Mothersill, C. Seymour, Changing paradigms in radiobiology, Mutat. Res. 750 (2012) 85–95. [73] A.T. Natarajan, Reflections on a lifetime in cytogenetics, Mutat. Res. 751 (2012) 1–6. [74] A.N. Jha, Professor Adayapalam Tyagarajan Natarajan (1928-2017): a tribute, Mutagenesis 32 (2017) 545–546. [75] A.S. Balajee, F. Palitti, J. Surrallés, M.P. Hande, In Memory of Professor Adayapalam T Natarajan, Mutat. Res. 836 (Pt A) (2018) 1–2. [76] U. Weyemi, C. Dupuy, The emerging role of ROS-generating NADPH oxidase NOX4 in DNA-damage responses, Mutat. Res. 751 (2012) 77–81. [77] E.A. Carlson, Genes, Radiation, and Society: The Life and Work of H.J. Muller, Cornell University Press, Ithaca, 1981. [78] H.J. Muller, The relation of recombination to mutational advance, Mutat. Res. 1 (1964) 2–9. [79] E.A. Carlson, H.J. Muller’s contributions to mutation research, Mutat. Res. 752 (2013) 1–5. [80] E.A. Carlson, How fruit flies came to launch the chromosome theory of heredity, Mutat. Res. 753 (2013) 1–6. [81] L. Müller, E. Gocke, The rise and fall of photomutagenesis, Mutat. Res. 752 (2013) 67–71. [82] L.B. Russell, The Mouse House: a brief history of the ORNL mouse genetics program, 1947-2009, Mutat. Res. 753 (2013) 69–90. [83] L.B. Russell, "Liane Russell’s Interview," Voices of the Manhattan Project, April 25, 2018, Atomic Heritage Foundation, Oak Ridge, TN, 2018 Accessed Dec. 10, 2018 https://www.manhattanprojectvoices.org/oral-histories/liane-russells-interview. [84] D.M. Shankel, Memories of a friend and mentor – Charlotte Auerbach, Mutat. Res. 761 (2014) 1–5. [85] University of Kansas Alumni Association, Remembering Chancellor Shankel, Alumni News, 2018 July 12 (http://www.kualumni.org/news/rememberingchancellor-shankel/ ). [86] J.D. Tucker, Reflections on the development and application of FISH whole chromosome painting, Mutat. Res. 763 (2015) 2–14.

[87] M.L. Pardue, J.G. Gall, Chromosomal localization of mouse satellite DNA, Science 168 (1970) 1356–1358. [88] J.D. Tucker, W.F. Morgan, A.A. Awa, M. Bauchinger, D. Blakey, M.N. Cornforth, L.G. Littlefield, A.T. Natarajan, C. Shasserre, PAINT: a proposed nomenclature for structural aberrations detected by whole chromosome painting, Mutat. Res. 347 (#1) (1995) 21–24. [89] J.R. Savage, J.D. Tucker, Nomenclature systems for FISH-painted chromosome aberrations, Mutat. Res. 366 (#2) (1996) 153–161. [90] K. Sankaranarayanan, H. Nikjoo, Genome-based, mechanism-driven computational modeling of genetic risks of ionizing radiation: the next frontier in genetic risk estimation? Mutat. Res. 764 (2015) 1–15. [91] K. Dronamraju, Haldane and Modern Biology, The Johns Hopkins University Press, Baltimore, 1968. [92] K. Dronamraju, Popularizing Science: The Life and Work of JBS Haldane, Oxford University Press, New York, 2017. [93] K. Dronamraju, J.B.S. Haldane as I knew him, with a brief account of his contribution to mutation research, Mutat. Res. 765 (2015) 1–6. [94] N.P. Singh, The comet assay: reflections on its development, evolution and applications, Mutat. Res. 767 (2016) 23–30. [95] O. Ostling, K.J. Johanson, Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells, Biochem. Biophys. Res. Commun. 123 (1984) 291–298. [96] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191. [97] E.A. Carlson, Scientific feuds, polemics, and ad hominem arguments in basic and special-interest genetics, Mutat. Res. 771 (2017) 128–133. [98] E.A. Carlson, The Gene: A Critical History, W.B. Saunders, Philadelphia, 1966. [99] E. Zeiger, Reflections on a career and on the history of genetic toxicity testing in the National Toxicology Program, Mutat. Res. 773 (2017) 282–292. [100] J.H. Miller, Mutagenesis: interactions with a parallel universe, Mutat. Res. 776 (2018) 78–81. [101] M. Kirsch-Volders, F. Pacchierotti, E.M. Parry, A. Russo, U. Eichenlaub-Ritter, I.D. Adler. Risks of aneuploidy induction from chemical exposure: twenty years of collaborative research in Europe from basic science to regulatory implications, Mutat. Res. 779 (2019) 126–147. [102] R. Waters, M. Kirsch-Volders, Obituary: James M. Parry (1940–2010), Mutagenesis 26 (#1) (2011) 1–2.

120