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.     

The effects of excision repair and the plasmid pKM101 on the induction of his+ revertants by chemical agents in Salmonella typhimurium

Expansion of the Ames Salmonella/microsome mutagenesis test to include plasmid pKM101-bearing, excision repair-proficient derivatives permits 1) the identification of mutagens that require both factors for activity; 2) the identification of genotoxins through the enhancement of survival by excision repair; and 3) the classification of substances according to the effects of excision repair on their mutagenesis. Class I includes substances that require excision repair to effect mutagenesis. Class II contains substances whose mutagenesis is not affected by excision repair. Class III mutagens cause premutational lesions in DNA which are readily removed by excision repair. This classification scheme is suggested as a preliminary step in making a risk estimation for a mutagen.     

The accumulation and persistence of somatic mutations in vivo

Mutant frequencies at the native Dlb-1 and the lacl transgenereach a plateau within 1 week of mutagenesis in the small intestineof mice. This indicates that these mutations are selectivelyneutral and should accumulate additively. In a test with threemutagens, one potent (ethylnitrosourea) and two weak (1, 2-dimethylhydrazineand methyl methanesulphonate), ten weekly treatments inducedten times the frequency of Dlb-1 mutations induced by a singletreatment in each case, a result that is consistent with perfectadditivity. These observations indicate that the mutation frequencyat neutral loci will be an integrated measure of the effectsof aging, endogenous mutagens, environmental mutagens and antimutagens.They suggest a simple protocol for transgenic mutation assayswhich would increase sensitivity, require fewer animals, andconform to standard toxicological practice. ¹To whom correspondence should be addressed     

Conditions for detecting the mutagenicity of divalent metals in Salmonella typhimurium.

The mutagenesis of metals in bacteria, as reported in the literature, can best be described as inconsistent. We report that cobalt chloride (Co++), ferrous sulfate (Fe++), manganese sulfate (Mn++), cadmium chloride (Cd++), and zinc chloride (Zn++) could be reproducibly detected as mutagens in Salmonella strain TA97 when preincubation exposures were made in sterile, distilled, deionized water, or in Hepes buffer in NaCl2/KCl2, rather than the standard sodium phosphate buffer. Co++ was also mutagenic under standard preincubation conditions. The individual components of Vogel-Bonner medium, i.e., potassium and ammonium phosphate, citrate, and magnesium sulfate, inhibit mutagenesis by these metals. The phosphates and the citrate probably inhibit by chelating the metals, while data are presented to suggest that Mg++ inhibition of metal mutagenesis is due to competitive inhibition for active transport via the magnesium active transport system in Salmonella. The chelator, diethyldithiocarbamate, inhibited the mutagenicity of Co++, Fe++, Zn++, and Mn++, but enhanced the mutagenicity of Cd++. The results presented show that divalent metals can be detected as mutagens in Salmonella, and that their lack of detection as mutagens is not due to an inherent insensitivity of Salmonella but to their interaction with media components and/or passive and active transport processes.     

Perspectives in mutagenesis

I would first like to thank the Environmental Mutagen Society for honoring me with its 1980 award; I also would like to take this opportunity to acknowledge all the people who have worked with me and helped me since I started my work in mutagenesis in 1953. I feel especially indebted to my wife, Martha, whose support has been absolutely essential for my career. I would now like to explore some important problems in environmental mutagenesis. Obviously, I cannot touch upon new developments in all aspects of environmental mutagenesis - a field that has become increasingly broad in recent years. As in the past, there continue to be major advances in understanding mechanisms of mutagenesis, developing short-term tests, characterizing mutations, elucidating the relationship between mutagenesis and carcinogenesis, understanding the metabolism of mutagens, and many other areas. Many of these advances are reflected in the contents of the papers, symposia, and poster sessions of this meeting. In the years that I have been involved in mutation research and environmental mutagenesis, my interests and research activity have spanned a number of these areas; and I am pleased by the recent progress that I see taking place in them. In this talk, however, I would like to direct my comments toward a rather new dimension in environmental mutagenesis in which I have become increasingly interested: The monitoring of mutations in the human population. In the monitoring of the human population there are two choices: We can monitor the progeny of an exposed population or we can consider exposed persons as populations of cells and measure the mutant frequency in individual cells in these persons. Technical developments are advancing rapidly in both of these approaches. The pioneering work of James Neel in measuring germinal effects of mutagens in the human population is well known [Neel, 1970]. Methods for measuring mutation frequencies in single cells in vivo also are beginning to be available. For instance, Strauss and Albertini [1979] are developing a system to detect HGPRT mutants in human lymphocytes. Of course, there are still difficult problems to be resolved in this system; and we do not know with certainty.     

病毒载体致突变性的实验研究

目的 :观察逆转录病毒pLXSN与腺病毒LacZ作为转基因载体所构建的转基因细胞的致突变作用 ,为转基因肿瘤细胞作为瘤苗进行临床提供安全性检测参数。方法 :将病毒与细胞共培养 ,用细胞DNA通过遗传毒理学实验技术进行体内和体外的致变性实验研究。结果 :转基因细胞DNA及培养上清液未显出致突变作用。结论 :经过修饰的病毒作为转基因载体未见其致突变作用     

Assessing human germ-cell mutagenesis in the Postgenome Era: a celebration of the legacy of William Lawson (Bill) Russell

Birth defects, de novo genetic diseases, and chromosomal abnormality syndromes occur in ～5% of all live births, and affected children suffer from a broad range of lifelong health consequences. Despite the social and medical impact of these defects, and the 8 decades of research in animal systems that have identified numerous germ‐cell mutagens, no human germ‐cell mutagen has been confirmed to date. There is now a growing consensus that the inability to detect human germ‐cell mutagens is due to technological limitations in the detection of random mutations rather than biological differences between animal and human susceptibility. A multidisciplinary workshop responding to this challenge convened at The Jackson Laboratory in Bar Harbor, Maine. The purpose of the workshop was to assess the applicability of an emerging repertoire of genomic technologies to studies of human germ‐cell mutagenesis. Workshop participants recommended large‐scale human germ‐cell mutation studies be conducted using samples from donors with high‐dose exposures, such as cancer survivors. Within this high‐risk cohort, parents and children could be evaluated for heritable changes in (a) DNA sequence and chromosomal structure, (b) repeat sequences and minisatellites, and (c) global gene expression profiles and pathways. Participants also advocated the establishment of a bio‐bank of human tissue samples from donors with well‐characterized exposure, including medical and reproductive histories. This mutational resource could support large‐scale, multiple‐endpoint studies. Additional studies could involve the examination of transgenerational effects associated with changes in imprinting and methylation patterns, nucleotide repeats, and mitochondrial DNA mutations. The further development of animal models and the integration of these with human studies are necessary to provide molecular insights into the mechanisms of germ‐cell mutations and to identify prevention strategies. Furthermore, scientific specialty groups should be convened to review and prioritize the evidence for germ‐cell mutagenicity from common environmental, occupational, medical, and lifestyle exposures. Workshop attendees agreed on the need for a full‐scale assault to address key fundamental questions in human germ‐cell environmental mutagenesis. These include, but are not limited to, the following: Do human germ‐cell mutagens exist? What are the risks to future generations? Are some parents at higher risk than others for acquiring and transmitting germ‐cell mutations? Obtaining answers to these, and other critical questions, will require strong support from relevant funding agencies, in addition to the engagement of scientists outside the fields of genomics and germ‐cell mutagenesis. Environ. Mol. Mutagen., 2007. Published 2007 Wiley‐Liss, Inc.     

Evaluation of transcriptional fusions with green fluorescent protein versus luciferase as reporters in bacterial mutagenicity tests.

A bacterial plasmid was constructed on which the regulatory region of the umuC gene of Escherichia coli was fused to the coding sequence of the green fluorescent protein gene (gfp) from the jellyfish Aequorea victoria. Escherichia coli AB1157 strains carrying the plasmid emitted fluorescence in the presence of mutagens that induce the SOS DNA repair system. Data on tests with nitrosoguanidine, methylmethane sulphonate and UV radiation (254 nm) are presented. Although fluorescent detection using this system was not as rapid or sensitive as a similar luminescent equivalent (umuC-luxAB), the gfp reporter system was more robust. Escherichia coli umu gene induction was also analysed in Salmonella typhimurium TA1537 cells following plasmid transfer and exposure to the same range of mutagens. There was no significant difference in sensitivity between the two species. These preliminary results will provide the basis for development of mutagenicity test systems useful in the testing of complex mixtures, such as environmental samples, and the investigation of physiological parameters influencing spontaneous mutagenesis in bacteria.     

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.     

Mutation measurement in mammalian cells IV: Comparison of γ-ray and chemical mutagenesis

The interaction of chemical mutagens with mammalian cells is much more complex than that of γ-irradiation because of the different ways in which chemical agents react with cell and medium components. Nevertheless, the system previously described for analysis of mutagenesis by γ-radiation appears applicable to chemical mutagenesis. The approach involves measurement of cell survival, use of caffeine to inhibit repair, analysis of mitotic index changes, and quantitation of microscopically visible structural changes in mitotic chromosomes. The behavior of a variety of chemical mutagens and nonmutagens in this system is described and compared with that of γ-irradiation. The procedure is simple and the results reasonably quantative though less so than those of γ-irradiation. The procedure can be used for environmental monitoring, analysis of mutational events, and individual and epidemiological testing. Mutational events should be classified as primary or secondary depending on whether they represent initial genomic insult, or genomic changes resulting from primary mutation followed by structural changes due to metabolic actions. While caffeine has multiple effects on the mammalian genome, when used under the conditions specified here it appears to act principally as an inhibitor of mutation repair, and so affords a measure of the role of repair in the action of different mutagens on cells in the G2 phase of the life cycle.     

Comparative analysis of CreER transgenic mice for the study of brain macrophages: A case study

Conditional mutagenesis and fate mapping have contributed considerably to our understanding of physiology and pathology. Specifically, Cre recombinase-based approaches allow the definition of cell type-specific contributions to disease development and of inter-cellular communication circuits in respective animal models. Here we compared Cx₃cr1^CreER and Sall1^CreER transgenic mice and their use to decipher the brain macrophage compartment as a showcase to discuss recent technological advances. Specifically, we highlight the need to define the accuracy of Cre recombinase expression, as well as strengths and pitfalls of these particular systems that should be taken into consideration when applying these models.     

Transgenes as screening tools to probe and manipulate the zebrafish genome.

The zebrafish, originally an object of study as an inexpensive and prolific vertebrate embryological model with a plethora of genetic tricks, has over the past decade moved to large-scale chemical mutagenesis and recently came of age as a high throughput transgenic model with a sequenced genome nearing completion. Insertional mutagenesis, gene trapping and enhancer detection are all contributing to the increasing speed with which research in this biomedical model is progressing. We review here some of the recent developments in the emerging field of zebrafish developmental genomics and transgenesis.     

Conditions for the optimal use of the L-arabinose-resistance mutagenesis test with Salmonella typhimurium

Different conditions of mutagenesis have been compared in orderto optimize the use of the L-arabinose resistance test withSalmonella typhimurium. The mutagenesis protocols compared werethe plate-incorporation, the pre-incubation and the liquid tests.Fourteen chemicals were used in the comparison: six direct-actingmutagens and eight pre-mutagens. Five concentrations of S9 (3,7.5, 10, 15 and 33% v/v) were compared in the liquid test withpre-mutagens, and three densities of bacteria (ranging from10⁸ to 10⁶ cells) were used in the comparison between the plate-incorporationand the pre-incubation mutagenesis test. In general, the liquidtest proved the most sensitive mutagenesis protocol. When carryingout this test in a mass screening of mutagens, we propose toselect the L-arabinose-resistant mutants in plates supplementedwith 0.5 mg of D-glucose, and to express the mutagenic responseas the absolute number of induced mutants. The plate-incorporationand the pre-incubation mutagenesis protocols could be consideredas alternative procedures in the case of previous negative resultswith the liquid test. Two recommendations can be finally madein order to avoid false negative results: (a) a large populationof bacteria (ranging from 10⁸ to 10⁷ cells) must be exposedto the mutagens in both the plate-incorporation and the pre-incubationmutagenesis tests, and (b) ideally, several S9 concentrations(ranging from 3 to 30% v/v) would be employed for testing inliquid samples of unknown mutagenicity. ¹To whom correspondence should be addressed     

Mammary carcinogenesis is preceded by altered epithelial cell turnover in transforming growth factor-alpha and c-myc transgenic mice

Identification of biomarkers that indicate an increased risk of breast cancer or that can be used as surrogates for evaluating treatment efficacy is paramount to successful disease prevention and intervention. An ideal biomarker would be identifiable before lesion development. To test the hypothesis that changes in cell turnover precede mammary carcinogenesis, we evaluated epithelial cell proliferation and apoptosis in mammary glands from transgenic mice engineered to develop mammary cancer due to expression in mammary epithelia of transforming growth factor α (TGF-α) or c-myc. In transgenic glands, before lesion development, epithelial cell turnover was enhanced overall compared with nontransgenic glands, indicating that aberrant cell turnover in normal epithelia may contribute to tumorigenesis. In addition, in tumor-containing glands, proliferation in normal epithelia was higher than in tumor-free transgenic glands, suggesting these cell populations influence one another. Finally, although c-myc glands displayed a uniformly high epithelial cell turnover regardless of age, cell turnover was reduced with aging in nontransgenic and TGF-α mice, indicating that some growth and death regulatory mechanisms remain intact in TGF-α epithelia. These observations support the evaluation of cell turnover as a biomarker of cancer risk and indicator of prevention/treatment efficacy in preclinical models and warrant validation in human breast cancer.     

Immuno- and genetic therapy in autoimmune diseases

Animal models of autoimmune disease have been developed that mimic some aspects of the pathophysiology of human disease. These models have increased our understanding of possible mechanisms of pathogenesis at the molecular and cellular level and have been important in the testing, development and validation of new immunotherapies. The susceptibility to develop disease in the majority of these models is polygenic as is the case in humans. The exceptions to this rule are gene knock outs and transgenic models of particular genes which, in particular genetic backgrounds, have also contributed to the understanding of single gene function and their possible contribution to pathogenesis. Gene therapy approaches that target immune functions are being developed with encouraging results, despite the polygenic nature of these diseases. Basically this novel immuno-genetic therapy harnesses the knowledge of immunology with the myriad of biotechnological breakthroughs in vector design and delivery. Autoimmune disease is the result of genetic dysregulation which could be controlled by gene therapy. Here we summarize the genetic basis of these human diseases as well as some of the best characterized murine models. We discuss the strategies for their treatment using immuno- and gene therapy.     

转基因动物模型制作进展

转基因动物是应用实验的方法将外源基因导入到早期的胚胎内,使之可以在动物染色体基因组内稳定的整合,并能遗 传给后代的一类动物遥随着转基因技术以及分子遗传学的发展,转基因动物模型逐渐应用在医学科学的各个领域遥本文对转基 因动物模型的几种常用建模方法做一综述,探讨各种不同的转基因动物模型制作方法的优劣遥最后以显微注射法为例简述制 作转基因动物的方法遥     

Finite element analysis of the spine: towards a framework of verification, validation and sensitivity analysis

A number of papers have recently emphasised the importance of verification, validation and sensitivity testing in computational studies within the field of biomechanical engineering. This review examines the methods used in the development of spinal finite element models with a view to a standardised framework of verification, validation and sensitivity analysis. The scope of this paper is restricted to models of the vertebra, the intervertebral disc and short spinal segments. In the case of single vertebral models, specimen-specific methods have been developed, which allow direct validation against experimental tests. The focus of intervertebral disc modelling has been on representing the complex material properties and further sensitivity testing is required to fully understand the relative roles of these input parameters. In order to construct complex multi-component short segment models, many geometric and material parameters are required, some of which are yet to be fully characterised. There are also major challenges in terms of short segment model validation. Throughout the review, areas of good practise are highlighted and recommendations for future development are proposed, taking a step towards more robust spinal modelling procedures, promoting acceptance from the wider biomechanics community.     

Ph stability of some mutagens produced by aqueous chlorination of organic compounds

Aqueous chlorination of many organic substances has been found to produce substantial mutagenicity in Salmonella typhimurium TA100. An effective way to decrease such mutagenicity is to raise the pH of the solutions to neutrality or higher. The effect of pH on the mutagenicity of the filtrate from the chlorination of unbleached kraft wood pulp and of certain mutagens found in such filtrates has been investigated. The decay of mutagenicity of the known mutagens has been shown to proceed by cleavage of organically bound chlorine by hydroxide ion. Caution is recommended with respect to the practice of raising the pH of solutions for mutagenesis assays.