Memories of a friend and colleague - Takashi Sugimura.

Takashi Sugimura, M.D., Honorary President of the National Cancer Center in Tokyo, and former President of The Japan Academy, is regarded by many as a pre-eminent contributor to the field of environmental genotoxicology. His pioneering spirit led to many key discoveries over a long and distinguished scientific career, including the first preclinical models for gastric cancer, identification of novel mutagens from cooked food, and the development of fundamental concepts in environmental chemical carcinogenesis. With his passing on September 6, 2020, many will reflect on the loss of an astute and engaging "Scientific Giant," who with warmth and good humor maintained lasting friendships both at home and abroad, beyond his many important scientific contributions.

The mutagenesis moonshot: The propitious beginnings of the environmental mutagenesis and genomics society.

A mutagenesis moonshot addressing the influence of the environment on our genetic wellbeing was launched just 2 months before astronauts landed on the moon. Its impetus included the discovery that X-rays (Muller HJ. [1927]: Science 64:84-87) and chemicals (Auerbach and Robson. [1946]: Nature 157:302) were germ-cell mutagens, the introduction of a growing number of untested chemicals into the environment after World War II, and an increasing awareness of the role of environmental pollution on human health. Due to mounting concern from influential scientists that germ-cell mutagens might be ubiquitous in the environment, Alexander Hollaender and colleagues founded in 1969 the Environmental Mutagen Society (EMS), now the Environmental Mutagenesis and Genomics Society (EMGS); Frits Sobels founded the European EMS in 1970. As Fred de Serres noted, such societies were necessary because protecting populations from environmental mutagens could not be addressed by existing scientific societies, and new multidisciplinary alliances were required to spearhead this movement. The nascent EMS gathered policy makers and scientists from government, industry, and academia who became advocates for laws requiring genetic toxicity testing of pesticides and drugs and helped implement those laws. They created an electronic database of the mutagenesis literature; established a peer-reviewed journal; promoted basic and applied research in DNA repair and mutagenesis; and established training programs that expanded the science worldwide. Despite these successes, one objective remains unfulfilled: identification of human germ-cell mutagens. After 50 years, the voyage continues, and a vibrant EMGS is needed to bring the mission to its intended target of protecting populations from genetic hazards. Environ. Mol. Mutagen. 61:8-24, 2020. © 2019 Wiley Periodicals, Inc.

Adventures with Bruce Ames and the Ames test.

Bruce Ames has had an enormous impact on human health by developing facile methods for the identification of mutagens. This research also provided important insights into the relationship between mutagenesis and carcinogenesis. Bruce is a highly innovative and creative individual who has followed his interests across disciplines into diverse fields of inquiry. The present author had the pleasure of spending a sabbatical in the Ames lab and utilized the Ames test in multiple aspects of his research. He describes both in this honorific to Bruce on the occasion of his 90th birthday.

Isolation of plasmid pKM101 in the Stocker laboratory.

pKM101 is a mutagenesis-enhancing resistance transfer plasmid (R plasmid) that was introduced into several tester strains used in the Salmonella/microsome mutation assay (Ames test). Plasmid pKM101 has contributed substantially to the effectiveness of the Ames assay, which is used on a world-wide basis to detect mutagens and is required by many government regulatory agencies for approval to market new drugs and other chemical agents. Widely used since 1975, the Ames test is still regarded as one of the most sensitive genetic toxicity assays and a useful short-term test for predicting carcinogenicity in animals. Plasmid pKM101, which is a deletion derivative of plasmid R46 (also referred to as R-Brighton after its origin of isolation in Brighton, England), has also been used to elucidate molecular mechanisms of mutagenesis. It was isolated in the laboratory of Professor Bruce A.D. Stocker at Stanford University as part of my doctoral research with 20 R plasmids. Professor Stocker's phenomenal insight into the genetics of Salmonella typhimurium and plasmid behavior was a major factor that led to the isolation of pKM101. This paper includes a tribute to Bruce Stocker, together with a summary of my research with mutagenesis-enhancing R plasmids and a brief discussion of the molecular mechanisms involved in pKM101 plasmid-mediated bacterial mutagenesis.

History of the science of mutagenesis from a personal perspective.

A career in the study of mutagenesis spanning 50 years is a gift few scientists have been bestowed. My tenure in the field started in 1953, the year the structure of DNA became known (Watson and Crick [1953]: Nature 171:737). Before that time, it was suspected that DNA was the genetic material based on the research of Oswald T. Avery (Avery et al. [1944]: J Exp Med 79:137), but many scientists still believed that proteins or polysaccharides could be the genetic material. The present article describes a lifetime of personal experience in the field of chemical mutagenesis. The methods used to treat viruses with chemical mutagens were well developed in the 1950s. Here I review the early use of nitrous acid and hydroxylamine as mutagens in eukaryotes, the development of methods for the metabolic activation of mutagens by microsomal preparations, and the selection of a mutant tester set for the qualitative characterization of the mutagenic activity of chemicals. These studies provided critical background information that was used by Bruce Ames in the development of his Salmonella/microsome assay, widely known as the Ames test (Ames et al. [1973]: Proc Nat Acad Sci USA 70:2281-2285). This article also describes how a set of diagnostic chemical mutagens was selected and used to identify the molecular nature of gene mutations. Today, DNA sequencing has replaced the use of diagnostic mutagens, but studies of this kind formed the foundation of modern mutation research. They also helped set the stage for the organization of the Environmental Mutagen Society and the Environmental Mutagen Information Center, which are described. The article ends with the development of mammalian single-cell mutation assays, the first system for studying in vivo mutagenesis using recoverable vectors in transgenic animals, other mutation assays in intact mammals, and my thoughts on the critically important area of germ cell mutagenesis. This narrative is not a complete autobiographical account, in that I have selected only those experiences that I feel are important for the history of the field and the edification of today's students. I hope I have shown that science not only is a valuable pursuit but can also be fun, stimulating, and satisfying. A good sense of humor and the knowledge that many discoveries come by serendipity are essential.

History and rationale of genetic toxicity testing: an impersonal, and sometimes personal, view.

Genetic toxicity testing is a necessary and pivotal component of product development and registration. This article traces the historical development and evolution of genetic toxicity testing, and the rationale for such testing, and identifies some of the individuals who played key roles in this process. The evolution of the present test batteries and some of the research and rationales behind the decisions to accept or reject tests are described.

Origins of genetic toxicology and the Environmental Mutagen Society.

A brief history of events that contributed to the establishment of genetic toxicology as a distinct research area and influenced the formation of the Environmental Mutagen Society is presented.

Mechanisms of mutagenesis.

Our understanding of mechanisms of mutation, which has expanded greatly in the past few decades, served as the original impetus to the formation of the Environmental Mutagen Society. The advances in genetics and chemistry that have conditioned our present degree of knowledge are here catalogued and the future is predicted.

Concern for environmental mutagens: some personal reminiscences.

This article is a personal, anecdotal account of the early days of concern for environmental mutagenesis. The history of the original National Academy of Sciences Committee on the Biological Effects of Atomic Radiations (BEAR) and its early controversies are reviewed, along with the initial establishment of principles for discovering potential chemical mutagens. Although it emphasizes the great advances in cellular and molecular understanding over the past 35 years, the article ends with a pessimistic assessment of any possibility of quantitative assessment of mutational impacts on future generations.

Reminiscences of a mouse specific-locus test addict.

This paper describes some of the historical events surrounding the development of and achievements with the mouse specific-locus test in radiation and chemical mutagenesis. Some ongoing and future contributions of the test to research in molecular genetics are also mentioned.

Development of bacterial mutagenicity tests: a view from afar.

A brief (and subjective) history of the progressive development of bacterial mutagenicity assays with Salmonella typhimurium and Escherichia coli is presented, with emphasis on the need to view such assays as being capable of detecting genetically active substances rather than being dedicated to the detection of carcinogenic chemicals. The role of mutation-enhancing plasmids in the improvement of Salmonella tester strains and the need for batteries of tests to allow the detection of genetic endpoints that cannot be detected with bacteria (e.g., aneuploidy) are also discussed.

Evolution of social concerns and environmental policies for chemical mutagens.

The founding of the Environmental Mutagen Society 20 years ago coincided with the beginning of general social concern about exposure to chemical mutagens. Initially, this concern focused on the potential of chemicals to induce heritable genetic damage in humans. Within a few years, however, mutagenicity tests came to be regarded primarily as short-term tests for carcinogenicity. Serious doubts have recently been cast upon the relationship between mutagenicity and carcinogenicity, and, as a result the real utility of mutagenicity tests is being questioned. Justification for the continued use of these tests will require 1) more detailed mechanistic knowledge concerning the role of genetic changes in the development of cancer and 2) an improved ability to relate the results of mutagenicity tests to the potential for inducing heritable genetic effects in humans.

Concepts and models for DNA repair: from Escherichia coli to mammalian cells.

Much of our early understanding of the mechanisms of excision-repair and its roles in maintaining genome integrity and cellular viability was derived from studies with bacteria. In fact, the discoveries of damage excision and repair replication were made in ultraviolet (UV)-irradiated Escherichia coli. Recent advances in recombinant DNA technology have helped to further our understanding of the manner in which mammalian cells deal with damage in their complex genomes. These include the discovery that expressed genes may be preferentially repaired and, furthermore, that the transcribed DNA strand, for some types of damage, is selectively repaired within an active gene. The latter finding has now been documented in E. coli as well, so it is likely that it is of widespread importance as a mechanism to ensure the expression of active genes in otherwise damaged cells. It is certain that studies with bacterial systems as models will continue to have an important impact on the development of the field of mammalian DNA repair.

Ultraviolet mutagenesis and the SOS response in Escherichia coli: a personal perspective.

The study of ultraviolet (UV) mutagenesis in Escherichia coli began with the assumption that genes were likely to be changed at the instant of photon absorption. Over many decades, it became clear that postirradiation cellular activities, including enzymatic DNA repair of UV photo products and error-prone modes of tolerating unrepaired DNA lesions can exert profound influences on the mutagenic outcome of irradiation. Current study focusses on the molecular details of radiation-induced translesion DNA replication as the final event in UV mutagenesis.

Reciprocal relationship between mouse germ-cell mutagenesis and basic genetics: from early beginnings to future opportunities.

The scientific foundations for several mammalian germ-line mutagenesis tests in common use today were laid in the 1930s, 1940s, and early 1950s. Subsequent developments in the field have had multiple objectives: detection of mutagenicity of environmental agents (which has led to the development of numerous methodologies), identification of biological and physical factors that affect mutation yield, analysis of the structural nature of the genetic alterations, and assessment of the organismic effects of various types of mutations. Mutagenesis studies have made numerous contributions to basic genetics by generating mutant types that led to elucidation of sex-determining mechanisms in mammals; formulation of the single-active-, or inactive-, X-chromosome hypothesis; correlation of genetic and cytological maps; discovery of genetic "imprinting" phenomena; study of developmental pathways and cell lineages, etc. Particularly useful are sets of complexly overlapping deletions that have been recovered in radiation mutagenesis studies, propagated in breeding stocks, and genetically analyzed; these have constituted prerequisites for molecular genetic studies aimed at development of the DNA structure-function relationships for important genomic regions. Mutagenesis experiments have also served to identify mutagens that are particularly effective in inducing specific types of genetic lesions desired for basic studies. Reciprocally, basic genetics has contributed to the development of mutagenesis tests and has enhanced the value of the specific-locus test by adding to its quantitative capabilities the capability for qualitatively characterizing the actions of mutagens.

Early years of the Salmonella mutagen tester strains: lessons from hycanthone.

The 1960s witnessed detailed studies on the genetic properties of a large number of histidine-requiring mutants of Salmonella typhimurium. The early 1970s saw development of selected strains, the Ames strains, for use in rapid, cheap, sensitive, and manipulable tests of chemicals and chemical mixtures for genotoxic activities. Our contribution during this latter period was an investigation into the mutagenicity of hycanthone and some of its analogues. Some lessons that this study provided are enumerated. Hycanthone is definitely a liver carcinogen in rodents predisposed by hepatic hyperplasia. Between 1969 and 1975, an estimated total of 100 kg of hycanthone was injected into some 1,000,000 humans with liver hyperplasia caused by infections with parasites. It may now be possible to assess directly the long-term impacts of hycanthone in man.

Evolution of testing strategies for genetic toxicity.

Shortly following the inception of genetic toxicology as a distinct discipline within toxicology, questions arose regarding the type and number of tests needed to classify a chemical as a mutagenic hazard or as a potential carcinogen. To some degree the discipline separated into two sub-specialties, (1) genetic risk assessment and (2) cancer prediction since data from experimental oncology also supports the existence of a genotoxic step in tumor initiation. The issue of which and how many tests continued to be debated, but is now focused more tightly around two independent phenomena. Tier or sequential testing was initially proposed as a logical and cost-effective method, but was discarded on the basis that the lower tier tests appeared to have too many false responses to force or exclude further testing of the test agent. Matrix (battery) testing was proposed for screening on the hypothesis that combinations of endpoints and multiple phylogenetic target organisms were needed to achieve satisfactory predictability. As the results from short-term test 'validation' studies for carcinogen prediction and evaluations of EPA's Gene-Tox data accumulated, it became obvious that qualitative differences remained between predictive and definitive tests and by assembling different combinations of short-term assays investigators did not appear to resolve the lack of concordance. Recent trends in genetic toxicology testing have focused on mathematical models for test selection, and standardized systems for multi-test data assessment.