2 F. Sherman / Mutation Research 589 (2005) 1–16

Mutation Research 589 (2005) 1–16 Contents 1. My first encounter with yeast research ...... 2

The importance of mutation, then and now: 2. Identification of CYC1 ...... 3

Mutation Research 589 (2005) 1–16 ☆ 3. Importance of cytochrome c ...... 4 studies with yeast cytochrome c www.elsevier.com/locate/reviewsmr Community address: www.elsevier.com/locate/mutres Fred Sherman * Reflections in Mutation Research 4. CYC1 encodes iso-1-cytochrome c ...... 4 5. Isolation and characterization of cyc1 mutants...... 5 Department ofThe Biochemistry importance and Biophysics, of University mutation, of Rochester then Medical andSchool, now:Box 712, Rochester, studies NY 14642, USA Accepted 26 July 2004 with yeast cytochrome c$ 6. Generating altered genes and proteins, then and now ...... 6 Available online 23 September 2004 7. Deducing DNA sequences from protein sequences...... 8 Fred Sherman* 8. ‘‘Site-directed’’ mutagenesis in early times ...... 10 Department of Biochemistry and Biophysics, University of Rochester Medical School, Box 712, Rochester, NY 14642, USA 9. Cloning CYC1 and transcriptional regulation...... 10 Accepted 26 July 2004 Available online 23 September 2004 10. Subsequent and current studies ...... 11

11. A final reflection ...... 14 Abstract Acknowledgements ...... 14 The development of a genetic system based on the CYC1 gene was initiated over 40 years ago, primarily because of the anticipated ease of sequencing of the corresponding encoded protein, iso-1-cytochrome c from Saccharomyces cerevisiae. The References ...... 14 success of the iso-cytochrome c system was dependent on the early development of methods for detecting and selecting cyc1 defective mutants and CYC1 functional revertants, and of methods for fine-structure genetic mapping using deletions and single- site mutations. The nonsense codons TAA and TAG, and the initiation codon ATG, were determined from the amino acid 1. My first encounter with yeast research emphasized to me that the existence of bacteriophages alterations of iso-1-cytochromes c from intragenic revertants; this represented the first assignments of such codons in a eukaryotic organism. The types of desired sequences were expanded by selecting recombinants from cyc1 cyc1 nonfunctional was one of the major advantages for using E. coli, and mutants or CYC1 CYC1 functional mutants, permitting the early determination of the rules of translation,� which differed from I began using yeast, Saccharomyces cerevisiae, as it was suggested that I find phages acting similarly on � those of prokaryotes by use of the most 50 AUG codon for initiation of translation. The sequence of 44 base pairs of CYC1 was an experimental organism 50 years ago, when I first yeast. My immediate response was ‘‘where do I find determined with altered iso-1-cytochromes c from revertants of frameshift and initiation mutants, allowing the early cloning of the joined the biophysics graduate program at Berkeley in them?’’ It was explained to me that the natural habitat gene. A method was developed for transforming yeast directly with synthetic oligonucleotides, resulting in the convenient 1954. Soon after my arrival, the director of the program of yeast was on the surface of fruits and that tropical production of CYC1 mutants with defined sequences. At this point in time, Sherman and colleagues have published approximately discussed a possible research project involving yeast. birds eat fruit. Therefore, a likely source of yeast 240 papers on or using the iso-cytochrome c system, dealing with such diverse topics as translation, informational suppressors, In this regard, one of the main interests of biophysicists phages was tropical bird feces. At that moment a transcription and transcription termination, recombination, ectopic recombination, mutagen specificity, regulation by Ty1 at that time was target theory and the effects of ionizing thought flashed through my mind, ‘‘I am going to be elements, evolution of duplicated chromosomal segments, structure–function relationships of cytochrome c, protein stability radiation. This was a time when physicists overly sent to the jungles of the Amazon or the Congo and degradation, biosynthesis and mitochondrial import of cytochrome c, mitochondrial proteases, co- and post-translational interpreted survival curves and before DNA repair was to collect tropical bird feces.’’ ‘‘Where do I go to modifications, and mRNA degradation. Current work on degradation of proteins in mitochondria, on degradation of mRNA in the considered. Carl C. Lindegren had already described collect tropical bird feces?’’ I asked. The answer was nucleus, and on N-terminal acetylation stems from properties of CYC1 mutants isolated in early screens more than a decade ago. the heterothallic life cycle, and Robert K. Mortimer logical but disappointing, ‘‘The local San Francisco # 2004 Elsevier B.V. All rights reserved. was busy making a polyploid series of yeast, haploid Fleishhacker Zoo across the Bay Bridge.’’ My next Keywords: Cytochrome c; Nonsense codons; Inititation codon; Protein stability and degradation; N-terminal acetylation; mRNA degradation through hexaploid, a series which appeared to be useful question was ‘‘how do I get inside the cages?’’ I was for exploring target theory [1]. I was informed that reminded that I am now a graduate student and have to any new fundamental information on yeast would be be resourceful and independent. of considerable importance, since it was the up-and- I called the Zoo and asked to speak to the Director, $ This article is part of the Reflection in Mutation Research series. To suggest topics and authors for Reflection, readers should contact the series editors G.R. Hoffmann ([email protected]) or D.G. MacPhee ([email protected]). coming microorganism of the future. a Ph.D. in zoology. I explained to the Director that I * Tel.: +1 585 275 6647; fax: +1 585 275 6007. This was the time when Escherichia coli was was a graduate student at the University of Calfornia, E-mail address: [email protected]. clearly the model system for molecular biology. It was and that I would like to collect fresh tropical bird

1383-5742/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2004.07.001

200 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 201 F. Sherman / Mutation Research 589 (2005) 1–16 3 4 F. Sherman / Mutation Research 589 (2005) 1–16 feces. There was pause, then a hesitant response, ‘‘Oh, chronize cells by heat shock. I attempted to deduce the the RAD7 and OSM1 loci as well as the CYC1 locus encompassing the heme group was already sequenced you mean bird shit—yeah, that’s OK.’’ number of genetic determinants in a cell by [5–7]. Some 5 years later, a second deletion, cyc1-237, [13], and entire cytochromes c from various species The next day I arrived at the Zoo fully equipped mathematically modeling the kinetics of rÀ induction with the same seemingly pleiotropic set of phenotypes were being sequenced in several laboratories. Thus, with vials, spatulas, a notebook and my roommate, a after growth at elevated temperatures. After receiving was unexpectedly uncovered among meiotic segre- investigating cytochrome c appeared to be an ideal pre-law student who was responsible for recording the my degree, I investigated recombination in yeast as a gants. The origin of this type of deletion was not project, especially because yeast had the advantage of species of the birds and the time of collection of the Post-doctoral Fellow at Seattle with the late Herschell understood until Liebman et al. [8] noted that certain being a eukaryotic microorganism with a well-defined specimens. We were both adorned with official- Roman. Subsequently, I entered a second post- laboratory strains spontaneously gave rise to high genetic system. The advantages of using yeast looking white lab coats. After entering an aviary, a doctoral position in the laboratory of the late Boris frequencies of deletions encompassing the CYC1, cytochrome c became even more evident a few years crowd gathered in front of the cage and it soon became Ephrussi at Gif-sur-Yvette, France, where I initiated a OSM1 and RAD7 genes and that the deletions were later when Narita et al. [14] announced the complete clear to them what our purpose was. Apparently, we research program in 1960 that led to the discovery of flanked by Ty1 elements [6,9]. Thus, cyc1-1, the first amino acid sequence of iso-1-cytochrome c. were more interesting than the animals. As soon as a the CYC1 gene. cytochrome c deficient mutant, was uncovered because bird defecated, several members of the crowd pointed of the rare occurrence of a spontaneous deletion in a and shouted ‘‘There’s one, there’s one.’’ strain containing an unrelated pet mutation. 4. CYC1 encodes iso-1-cytochrome c After returning to the University and refrigerating 2. Identification of CYC1 the samples, I asked my mentor ‘‘what do I do next?’’ I Because the cyc1-1 mutant contained a minor form was told that phages are viruses and that the samples After arriving in Boris Ephrussi’s laboratory, Piotr 3. Importance of cytochrome c of a chromatographically distinct cytochrome c,I are full of microorganisms. The simplest way to P. Slonimski and I began a study of pet mutants, i.e., initiated experiments soon after arriving at the separate the viruses is by filtration with a membrane those mutants that have mutations of nuclear genes Finding a cytochrome c yeast mutant was of University of Rochester early in 1960 that led to just developed by Millipore. It was also explained that and that are unable to grow on media having considerable importance in 1960. At that time, when the finding of two forms of cytochrome c in yeast, iso- the phages should be detected by plaques on a lawn of nonfermentable carbon sources as a sole energy DNA sequencing was in the realm of science fiction, 1-cytochrome c and iso-2-cytochrome c (Fig. 1) [4].A yeast cells. I immediately prepared filtrates, which I source (NfsÀ) [2,3]. These PET nuclear genes encode all information on gene structure was inferred from similar study was carried out at Gif-sur-Yvette by mixed with a yeast cell suspension and spread on the essential components of mitochondria that are mutationally altered proteins, and a major effort was Slonimski et al. [15], who suggested the intriguing, but surface of a nutrient plate. My career as a yeast required for aerobic metabolism, but are distinct from needed to decipher the genetic code by the analysis of fallacious, hypothesis that the apo form of the minor biologist began! the r+ determinant that was eventually shown to amino acid replacements and mutagenic specificity. species, iso-2-cytochrome c, was the repressor of iso- I examined the plates every few hours for 2 days. correspond to mitochondrial DNA. I started this study Early in 1960, only three proteins were amenable to 1-cytochrome c. On the second day, the lawn of cells had clearly grown, by requesting mutants that were already demonstrated, mutational analysis, tryptophan synthetase from E. In addition, a major effort was in progress to devise and to my astonishment there was an approximately or suspected, to have NfsÀ nuclear defects. For this coli [10], lysozyme from bacteriophage T4 [11] and methods to detect cytochrome c deficient mutants (see one millimeter clear spot on one of the plates. I ran purpose, I contacted David Pittman, Maurice Ogur, the TMV coat protein [12]. Also, cytochrome c was below). The first systematic screen, involving spectro- screaming to my advisor, ‘‘I got a plaque! I got a Donald C. Hawthorne and Robert K. Mortimer, who one of the few proteins that could be easily purified. Its scopic examination of a large number of strains, plaque!’’ I was quickly advised that this was not constituted the majority of yeast geneticists at that low molecular weight allowed easy diagnosis of resulted in uncovering cyc1-2, the second mutation at enough and that I must demonstrate multiplication of time. One of the strains, 662.8, obtained from M. altered sequences by peptide mapping and amino acid the CYC1 locus, as well as mutations of the CYC2 and the phage by preparing a similar lawn of cells with Ogur, turned out to be of considerable importance. The compositional analysis. The 12 amino acid segment CYC3 loci [16]. In order to establish that CYC1 material from the clear spot. This was done with great strain consisted of a mixture of haploid and diploid enthusiasm; waiting for the results was truly an cells and, as expected, did not grow on nonfermentable exciting but stressful experience. This time, after the substrates (NfsÀ). Genetic analysis revealed that the second day, I observed four clear spots. However, I strain contained two mutations, pet4-1, which now examined the surface of the plates with a low- prevented growth on nonfermentable substrates, and power microscope. The clear spots were caused by cyc1-1, which still allowed growth on nonfermentable imperfections of the agar surface and uneven substrates but caused a 95% diminution of cytochrome spreading of the cells. I changed my advisor and c. Thus, the cyc1-1 mutation was uncovered only project, and elected to work with Robert K. Mortimer, because it was fortuitously in the same strain as pet4 a new faculty member who was just starting his career and because cytochrome spectra of the meiotic in radiation biology of yeast using genetics as his segregants were examined. At the time, it was very major approach. puzzling why the cyc1-1 mutation was in the 662.8 Under the guidance of Professor Mortimer, I strain. Furthermore, the cyc1-1 mutation also con- Fig. 1. The two forms of cytochrome c in S. cerevisiae, iso-1-cytochrome c and iso-2-cytochrome c, are encoded by CYC1 and CYC7, respectively. iso-1-cytochrome c and iso-2-cytochrome c, which are 80% identical, normally comprise 95% and 5% of total cytochrome c, studied the induction of rÀ mitochondrial mutants ferred sensitivity to UV-light and hypertonic media respectively, in aerobically-grown, derepressed cells [4]. CYC1 and CYC7 are located in two regions, denoted COR and ARC, respectively, which by elevated temperature. The induction of rÀ mutants [4], phenotypes eventually shown to be due to deletion are ancestrally related to each other by a duplication, a transposition, and a single rearrangement, followed by divergence. Four of the parologous was inadvertantly uncovered after attempts to syn- of an approximately 12 kb segment that encompassed genes are shown in the figure [68].

202 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 203 F. Sherman / Mutation Research 589 (2005) 1–16 5 6 F. Sherman / Mutation Research 589 (2005) 1–16 encodes iso-1-cytochrome c and was not a regulatory required a short exposure to a H2O2 solution, followed almost unknown in yeast. Fortunately, we were able to colonies after 5–7 days of incubation, whereas gene, it was necessary to demonstrate that a mutant by exposure to a benzidine solution. Because the develop a novel procedure for generating deletions at colonies of the second usually arose after 10 days. allele encodes an altered form of iso-1-cytochrome c transfer and removal of solutions disrupted the the CYC1 locus. Deletions were recovered from The first class consisted of intragenic Arevertants with with a change in the primary sequence. Because colonies, a search was made for a method that would crosses that contained extensive dissimilarities of normal or altered iso-1-cytochromes c, together with cyc1-1 was a deletion and did not produce intragenic fix the colonies but still allow effective contact with the sequences in homologous regions of two CYC1 alleles. the low amount of iso-2-cytochrome c characteristic of revertants, the cyc1-2 mutant was critical for establish- solutions. After testing numerous agents and treat- These alleles encoded iso-1-cytochromes c that were normal strains. In contrast, most revertants of the ing in 1966 that the CYC1 gene encodes the primary ments, we discovered that gently spraying the surface functional but that contained two different sequences in second type contained only iso-2-cytochrome c, structure of iso-1-cytochrome c [17]. An intragenic of Petri plates with ordinary hair spray was effective. the dispensable amino-terminal region of the protein. usually in amounts higher than normal. Thus, a large revertant, CYC1-2-A, was shown to have a Q21Y Conditions were worked out with a low-priced brand of The diploids were sporulated, plated on chlorolactate number of altered iso-1-cytochromes c were uncov- replacement within the heme peptide. hair spray, which we used for several years. However, medium, and cyc1 mutants deficient in iso-1-cyto- ered in early studies by analyzing a series of mutations It is also of interest to note that the CYC3 gene, one day this brand was no longer available from our chrome c were selected from the meiotic progeny. Over of the type: CYC1+ cyc1-x CYC1-x-y, where uncovered in the initial screen, was shown 23 years usual vender. I wrote to the company asking who the 25% of these cyc1 mutants contained deletions of CYC1+ denotes the wild-type! gene! that encodes iso-1- later to encode heme lyase, the enzyme catalyzing the local distributors were. I received a letter informing me various lengths, from those covering adjacent codons cytochrome c, cyc1-x denotes mutations that causes covalent attachment of the heme group to the iso- that their product was no longer available in Rochester. to those encompassing the entire CYC1 locus and deficiency or nonfunction of iso-1-cytochrome c, and cytochromes c [18]. Subsequently, the CYC2 gene was The company also told me that they had sent copies of flanking genes. We uncovered a total of 60 different CYC1-x-y denotes intragenic reversions that restore at cloned, sequenced and shown to encode a mitochon- my letter to all local wholesalers as evidence that their deletion lengths among the 104 deletions obtained by least partial activity and that give rise to either the drial protein required for normal mitochondrial import hair spray was in great demand. Fortunately, other this procedure [25]. Although it is still unclear exactly normal or an altered iso-1-cytochrome c (Fig. 2). Over of cytochrome c [19], although its mode of action is more expensive brands were equally effective. how these deletions arose, the mechanism may be 500 cyc1-x mutants were isolated and characterized, still not fully understood [20–22]. Eventually we developed an even more expedient related to heteroallelic mispairing. Nevertheless, the and over 100 different iso-1-cytochrome c sequences procedure for isolating CycÀ mutants that depended deletions we isolated proved to be invaluable for were obtained from CYC1-x-y revertants in our early on either the absence of, or lack of function of, mapping point mutations, especially after calibration studies (Fig. 3) [26,27]. 5. Isolation and characterization of cyc1 mutants cytochrome c [24]. This method was based on the with sites defined by amino acid replacement of iso-1- In more recent times, numerous altered iso-1- finding that mutants that are partially deficient in cytochrome c from intragenic revertants. Years later, cytochromes c have also been generated using In the early 1960s, studies of gene structure and cytochrome c but that still contain approximately 5% DNA sequencing revealed that the sites of cyc1 point standard methods of site-directed mutagenesis, a gene expression were highly dependent on the isolation of the normal level are defective in the utilization of mutations were generally within a codon or two of the procedure which relies on single-stranded E. coli and characterization of a large number of mutants. As lactate but are still able to utilize other nonfermentable sites estimated by genetic mapping. vectors containing the target sequence and a short mentioned above, the first method for the detection of substrates such as glycerol or ethanol. These partially synthetic oligonucleotide containing the desired cytochrome c deficient mutants was developed over 40 deficient mutants unable to utilize lactate are resistant alterations. Various other procedures, some using years ago [16]. This method involved low-temperature to the toxic action of the analogue chlorolactate. Thus, 6. Generating altered genes and proteins, then PCR, have also become part of the standard repertoire ( 196 8C) spectroscopic examination of large num- chlorolactate medium, which contains chlorolactate and now for producing specific mutations. In 1988, we bersÀ of strains on the surface of nutrient agar plates. and the nonfermentable carbon source glycerol, can be described a more convenient procedure for producing Colonies derived from mutagenized cells were used to enrich for mutants partially defective in Two major classes of revertants were distinguished specific alterations of genomic DNA by transforming inoculated onto square plastic Petri dishes, with each cytochrome c. As expected, and fortunately for us, the after high densities of cyc1 cells were plated on yeast directly with synthetic oligonucleotides [28–31]. dish containing 36 strains. After incubation and growth major class of cytochrome c deficient mutants arising synthetic medium containing lactate as the sole carbon This procedure is easily carried out by transforming a of the strains, the dishes were frozen in liquid nitrogen. on chlorolactate medium consisted of cyc1 mutants, source [23]. The first class usually formed visible defective cyc1 mutant and selecting for revertants that The dishes that did not explode were placed on a rack which lacked iso-1-cytochrome c but retained the under a simple spectroscope, and they were moved by normal low-level amount of iso-2-cytochrome c. hand to center each strain in the light path. Although The determination of the sites of the mutations in altered absorption spectra were just barely perceptible, the CYC1 gene was critical for their characterization. I was eventually able to examine over 2000 strains At that time – before DNA sequencing – genetic a day after the method was perfected. However, I mapping provided the only means of estimating the was hardly able to see anything the next day. More relative positions of point mutations without having to importantly, the critical single mutant described resort to protein sequencing. Furthermore, the only above as cyc1-2 was isolated by this technique after reliable mapping scheme required a combination of Fig. 2. Isolation of cyc1 mutants and intragenic revertants. CYC1+ denotes the wild-type gene that encodes iso-1-cytochrome c; cyc1-x denotes approximately 14,800 strains had been examined. deletion mapping and two point crosses. Deletion mutations that causes deficiency or nonfunction of iso-1-cytochrome c; and CYC1-x-y denotes intragenic reversions that restore at least partial activity and that give rise to either the normal or altered iso-1-cytochrome c. The cyc1-x mutants were isolated by (i) the spectroscopic A few years later, we developed a more expedient mapping established the order unambiguously, examination procedure; (ii) the benzidine staining procedure; and (iii) the chlorolactate selection procedure. Prior to 1980, the sequences of procedure that relied on the staining of colonies with whereas two point crosses established identity of altered proteins were used to deduce the changes in DNA, whereas after 1980, the sequences of the altered DNAs were used to deduce the benzidine reagents [23]. The staining of colonies sites. However, deletions were extremely rare, being changes in the protein.

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are at least partially functional. The oligonucleotide commonly used mutagens were reported, but the bulk used for transformation contains a sequence that of such studies were disappointing and thus were corrects the defect and produces additional alterations never published. While the procedure with synthetic at nearby sites. This technique is ideally suited for oligonucleotides is a form of DNA transformation, I producing a large number of specific alterations that cannot help but secretly look upon this process as a change a completely nonfunctional allele to a form dream come true, providing as it does the ultimate that is at least partially functional. The selection specific and controllable mutagen. procedure used with cyc1 mutants allows recovery of altered iso-1-cytochromes c with activity ranging from normal to less than 1% of the normal activity [28,29]. 7. Deducing DNA sequences from protein By using cyc1 mutants with various alterations along sequences the gene, all 20 amino acid replacements could be conveniently generated at almost any site by simply Of critical importance in the early studies was transforming the strain with sets of oligonucleotides. collaboration with John W. Stewart in which DNA For example, all possible amino acid residues were sequences were deduced from the amino acid conveniently introduced at various sites [32], includ- alterations in revertant proteins [38,39]. During the ing adjacent to the initiator methionine residue, for the course of experiments that spanned more than two systematic investigation of N-terminal processing of decades, John Stewart analyzed over 3000 samples of iso-1-cytochrome c [33]. Also, transformation directly iso-1-cytochromes c. These early studies covered with degenerate oligonucleotides, followed by DNA diverse topics including nonsense codons and sup- sequencing of the pertinent PCR amplified region, has pressors, initiation of translation, mutagenesis, recom- been used to produce iso-1-cytochromes c with all 20 bination and structure–function relationships of iso-1- replacements at various sites [32,34,35]. Complex and cytochrome c. For example, the nucleotide sequences multiple amino acid replacement have also been of chain terminating codons were deduced in the early conveniently produced by oligonucleotide transfor- 1970s from the finding that almost all of the revertant mation [36,37]. proteins contained single replacements of amino acids In a sense, transformation directly with oligonu- whose codons differed from TAA or TAG by single cleotides amounts to the achievement of an ultimate bases (Fig. 4) [40–42]. Furthermore, these defined goal – discovering specific mutagens. In early studies, TAA and TAG cyc1 mutations allowed the determina- many mutagens were investigated with the hope of tion of amino acids inserted by a wide range of introducing specific base-pair changes or at least suppressors, including those corresponding to altered controlling the distribution of base-pair changes. The tRNAs or ribosomal subunits, and those influenced by results obtained with only a few of the more the prion c+ [43–46].

Fig. 4. The amino acid sequences and the corresponding mRNA sequences related to the formation and reversion of the cyc1-9 and cyc1-179 Fig. 3. Composite of amino acid sequences of eukaryotic cytochromes c and of mutationally altered iso-1-cytochromes c (modified from [26,27]). nonsense mutants. The cyc1-9 and cyc1-179 revertants contained replacements of all amino acids having codons that differ by one base from, The normal iso-1-cytochrome c sequence is presented as a continuous sequence. The other residues found in 96 other species are presented above the respectively, UAA and UAG codons, except for the lack of cyc1-179 revertants containing lysine, which is found at the corresponding site in normal iso-1-cytochrome c sequence; the amino acid replacements in the mutationally altered forms of iso-1-cytochrome c are listed below the line. normal iso-1-cytochrome c. A total of 92 cyc1-9 revertants and 52 cyc1-179 revertants were analyzed. A broad range of mutational changes were The positions of the amino-terminal residues in the phylogenetic and mutant series are indicated, respectively, by arrows above and below the sequ- obtained by deriving the revertants spontaneously and by induction with the mutagens ultraviolet light, X-rays, polonium-210 a-particles, ethyl ences; D denotes deletion of the particular amino acid. Amino acid replacements abolishing function but still allowing detectable levels are indicated methanesulfonate, diethyl sulfate, methyl methanesulfonate, 1-nitrosoimidazolidone-2, N-methyl-N0-nitro-N-nitrosoguanidine, and nitrous acid. in bold; whereas amino acid replacements at positions 19, 22, and 23 causing the complete absence of iso-1-cytochrome c are indicated in bold-italics. Not shown are the two cyc1-9 and four cyc1-179 revertants that arose by multiple base changes (adapted from [42]).

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Fig. 6. The amino acid sequence of the amino terminal region of iso-1-cytochrome c, and the corresponding sequence of the 44 nucleotides that was deduced from frameshift and initiator mutants [52,53]. Synthetic probe denotes the 15-mer [57] used to clone the CYC1 gene by hybridization [60].

One of the major highlights of these early studies in vivo. Using techniques similar to those developed was the identification of the ATG initiator codon by for selecting cyc1 mutants, we were able to obtain using mutationally altered iso-1-cytochromes c. In our cyc1 recombinants in vivo with desired sequences by first report in 1971 [47], 9 out of 210 cyc1 mutants crossing certain CYC1 mutants that contained altered were shown to be deficient in iso-1-cytochrome c due iso-1-cytochromes c and then plating the sporulated to alterations of the ATG codon that is required for cross on chlorolactate medium. The resistant colonies initiation of protein synthesis. Structural analysis of 64 were analyzed genetically for cyc1 defects, and the revertant proteins from 17 of these cyc1 initiator sites of the lesions were determined by fine-structure mutants indicated that some of the reverse mutations mapping with defined cyc1 tester strains. Likewise, introduced initiator codons at new sites as illustrated CYC1 recombinants could be constructed from two in Fig. 5A for cyc1-13. Each of the cyc1 mutants gave cyc1 mutants by crossing, sporulating, and plating on rise to revertant iso-1-cytochromes c that had one of lactate medium. The initial cyc1 mutants and CYC1 the following amino terminal additions: Met-Ile-; revertants served as a resource for designing the Met-Leu-; Met-Arg-; Met-Lys-; and Met-Val-. These desired sequences. This approach using mutation and results could be explained by the mutational pathways recombination was especially useful for generating presented in Fig. 5A, which illustrates the formation single and multiple ATG triplets in the 50 region of the and reversion of cyc1-13. Further rules governing the gene [48,49]. For example, sequences containing initiation of translation were deduced from specific various combinations of ATG triplets at positions 1 sequences that were generated by recombination and 4 and TAA triplets at position 2 were generated by between cyc1 mutations in vivo; this was done long the steps outlined in Fig. 5. Some of the basic before the existence of site-directed mutagenesis (see conclusions derived from these studies were that below) [48,49]. initiation occurred only at ATG codons, that transla- Early in 1970, we attempted to isolate altered forms tion can occur at any site within at least a 37 nucleotide of revertant proteins that could be used to deduce the region, that translation initiates only at the most 50 DNA sequence of the CYC1 gene. The sequence of 44 ATG codon, and that translation does reinitiate after a base-pairs at the 50 translated region of the gene, terminating codon (Fig. 5) [49,50]. However, some 15 shown in Fig. 6, was actually deduced from altered years later it was established that initiation of iso-1-cytochromes c from frameshift and initiator translation could only occur in a restricted region of revertants [52,53]. the CYC1 mRNA [51].

8. ‘‘Site-directed’’ mutagenesis in early times 9. Cloning CYC1 and transcriptional regulation

Fig. 5. Mutational events leading from the normal gene CYC1+ to (A) the initiator mutant cyc1-13, and (B) the frameshift mutant cyc1-183, and In our early studies, we employed a variety of The development of recombinant DNA procedures the mutational events giving rise to intragenic revertants with altered iso-1-cytochromes c. (C–F) Specific cyc1 and CYC1 meiotic recombinants mutagens in order to recover all possible single base- in the mid-1970s obviously superseded the require- were obtained from the indicated crosses. The amino acid residues and codons that differ from the normal are shown in italics. The methionine pair changes, and in the hope of obtaining certain ment for altered protein sequences in the deductive residues shown in parentheses are excised from the iso-1-cytochromes c. The formation of the cyc1-13 mutation and revertants containing the specific changes. While mutagenic treatments alone determination of DNA sequences. However, prior to long (CYC1-13-A) and short (CYC1-13-S) forms of iso-1-cytochrome c is shown in (A). These and other results from Stewart et al. [47] indicated mainly gave rise to single base-pair changes that could the reports of Struhl et al. [54] and especially Hinnen that ATG is the initiation codon; protein synthesis can initiate at several sites without any obvious ribosomal binding site, and a methionine not be predicted (apart from G C A T transitions), et al. [55], the only practical procedure for identifying aminopeptidase cleaves amino-terminal residues of methionine from some but not all termini. Recombination was used to generate sequences � ! � with ATG triplets at positions 1 and 4, and TAA triplets at position 2, as shown in (G). These results, as well as other results summarized in specific sequences could be constructed by elaborate a DNA yeast clone was by hybridization to nucleic Sherman and Stewart [49], established some of the basic properties of translation (see text). and systematic mutational and recombinational steps acid probes. Other than the tRNA and rRNA genes, the

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CYC1 gene was the only yeast gene with at least a studies even superficially, I would like to mention a partially known DNA sequence. With knowledge of few areas where CYC1 mutants played an important the 44 bp sequence encoding the N-terminal region role subsequent to the 1980s. (Fig. 6), Szostak et al. [56,57] and Montgomery et al. In recent years, we have been identifying and [58] synthesized oligonucleotides 13 and 15 residues characterizing mitochondrial protein degradation long (shown in Fig. 6) that were used to identify the systems by investigating mutant forms of iso-1- clone containing the CYC1 gene [58–60]. The cytochrome c that are rapidly degraded and by availability of the sequence of the CYC1 gene, and obtaining and examining mutations in other genes soon afterwards the CYC7 gene [61], stimulated that diminish the degradation. Although cytochrome c investigations of transcriptional regulation in numer- is remarkably stable, altered iso-1-cytochromes c and ous laboratories, including those of Michael Smith, iso-2-cytochromes c with many different amino acid Lenny Guarente, Ben Hall and Richard Zitomer, as replacements are present at diminished levels due to well as in my laboratory. Detailed analysis of the degradation, and a subclass of the labile forms are promoter region, primarily by Guarente and co- significantly protected from degradation by the workers, revealed upstream activation sites, UAS1 and presence of cytochromes a a3 and c1, the physiological UAS2 [62]. These upstream regions were instrumental partners of cytochrome c [69Á –73]. For example, iso-1- in the identification and characterization of proteins cytochrome c with a G41E replacement is found at the required for CYC1 transcription, including Hap1p, normal 100% level in r+ strains, but is completely or which activates UAS1, and Hap2p and Hap3p, which nearly completely absent in rÀ strains, which lack activate UAS2 [9]. Furthermore, the roles of the cytochromes a a3 and c1 [72]. The pathway respon- multiple TATA elements (a and b) and their rules for sible for degradingÁ such iso-1-cytochromes c has been transcription initiation were investigated in detail [63]. designated the RDD (r dependent degradation) pathway. On the other hand, altered iso-1-cyto- chromes c and iso-2-cytochromes c that are present 10. Subsequent and current studies at approximately the same diminished level in both r+ and rÀ strains can be due to defects in import or heme Since the first report of CYC1 in 1964 [3], I have attachment, as well as to enhanced degradation. The published approximately 240 papers using the iso- degradation pathway responsible for such mutants that cytochrome c system for investigating such diverse are found in similarly diminished amounts in r+ and topics as translation, informational suppressors, rÀ strains has been denoted the labile dependent transcription and transcription termination, recombi- degradation (LDD) pathway. Futhermore, Chen et al. nation, mutagen specificity, regulation by Ty1 [37] recently described another pathway, the amphi- Fig. 7. The steps leading from transcription of CYC1 to the formation of an altered form of iso-1-cytochrome c with an abnormal Ac-Ser- elements, evolution of cytochrome c and of duplicated pathic dependent degradation (ADD) pathway, which terminus, illustrating N-terminal acetylation. (Wild-type iso-1 contains an N-terminal threonine residue that is unacetylated.) Processes that have chromosomal segments, structure–function relation- acts on iso-1-cytochromes c that have amphipathic been investigated include the requirements of the UAS1 and UAS2 elements and two TATA elements (a and b) for initiation of transcription at I ships of cytochrome c, protein stability and degrada- structures at the dispensible N-terminal region. By sites; 30-end formation; mRNA degradation in both the nucleus (DRN) and the cytosol (NMD), especially of the abnormal cyc1-512 mRNAs; translation initiation; and termination. During translation of the nascent apo-iso-1-cytochrome c, the N-terminal methionine is excised by tion, biosynthesis and mitochondrial import of isolating and characterizing suppressors of an RDD methionine aminopeptidase, and the penultimate residue is acetylated. After completion of translation, lysine 77 of apo-iso-1-cytochrome c is cytochrome c, mitochondrial proteases, co- and iso-1-cytochrome c, Wei and Sherman [73] recently trimethylated by the a specific methyltransferase, Ctm1p. Apo-iso-1-cytochrome c can be degraded by the ubiquitin-dependent degradation post-translational modifications, and mRNA degrada- identified Sue1p, a mitochondrial protein that is pathway, especially if import is impaired. apo-iso-1-Cytochrome c is imported in mitochondria by insertion in the outer face of the outer tion. All steps involved in the biosynthesis, regulation, responsible in part for the degradation of RDD, LDD membrane, and by translocation across the outer membrane, requiring interaction with the translocases in the outer membrane (TOM) complex. and degradation of cytochrome c have been investi- and ADD iso-1-cytochromes c. On the other hand, the Subsequently apo-iso-1-cytochrome c binds to cytochrome c heme lyase, which is encoded by the CYC3 gene, and which covalently attaches the heme moiety to the apo-cytochrome c within mitochondria. Attachment of heme produces a conformational change, trapping holo-iso-1- gated (Fig. 7), and many general processes have been degradation of only ADD iso-1-cytochromes c was cytochrome c in the intermembrane space and promoting binding to the outside of the inner membrane, where it carries out its physiological studied with the iso-cytochrome c system. Some of the suppressed by deletions of any of the genes encoding functions. Subsequently, holo-iso-1-cytochrome c can be degraded especially when it corresponds to certain mutationally altered forms. phenomena first reported to occur in S. cerevisiae were the membrane-associated mitochondrial proteases uncovered with the iso-cytochrome c system and YME1, AFG3, or RCA1 [37]. It was suggested that A transcription termination region was initially The cyc1-512 mutant contained abnormally long include, for example, chromosomal translocations the amphipathic structures caused a stronger associa- identified in 1982 by Zaret and Sherman [74] using the transcripts that appeared to be partially retained in the [64], ectopic recombination [65,66], and evolutionary tion with the mitochondrial inner membrane, and cyc1-512 mutant that was isolated in one of the early nucleus and degraded. Intragenic revertants and the divergence of duplicated regions [67,68]. While it is consequently with the proteases that are associated mutant screens and that was found to contain a 38 bp systematic introduction of defined sequences led to the beyond the scope of this review to cover all these with the inner membrane. deletion that mapped outside of the translated region. identification of 30-end forming signals [75,76].

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11. A final reflection [7] L. Melnick, F. Sherman, Nucleotide sequence of the COR region: a cluster of six genes in the yeast Saccharomyces cerevisiae, Gene 87 (1990) 157–166. Our basic philosophy is still to use the iso- [8] S.W. Liebman, A. Singh, F. Sherman, A mutator affecting the cytochrome c system for addressing diverse problems, region of the iso-1-cytochrome c gene in yeast, Genetics 92 and to rely on the treasure house of mutants and (1979) 783–802. techniques that have been developed over the decades. [9] S.W. Liebman, P. Shalit, S. Picologlous, Ty elements are It is satisfying to know that the use of this beautiful involved in the formation of deletions in DEL1 strains of Saccharomyces cerevisiae, Cell 26 (1981) 401–409. iso-cytochromes c system has not been exhausted, [10] C. Yanofsky, D.H. Helinski, B.D. Maling, The effects of even though it started over 40 years ago. mutation on the composition, properties of the A protein of It is an understatement to claim that such major Escherichia coli tryptophan synthetase, Cold Spring Symp. technical breakthroughs as recombinant DNA proce- Quant. Biol. 26 (1962) 11–23. dures and genomic approaches have revolutionized [11] G. Streisinger, Y. Okada, J. Emrich, J. Newton, A. Tsugita, E. Terzaghi, M. Inouye, Frameshift mutations and the genetic genetics and molecular biology; the way we do code, Cold Spring Symp. Quant. Biol. 31 (1967) 77–84. science today is certainly very different from the way [12] A. Tsugita, H. Fraenkel-Conrat, Contributions from TMV Fig. 8. Examples of four types of altered iso-1-cytochromes c processed differently at the N-terminus. The altered iso-1-cytochromes c were it was done 40 years ago. At every point in time, many studies to the problem of genetic information transfer and created by transforming the cyc1-31 strain with synthetic oligonucleotides and selecting for functional transformants. Amino acid sequences of if not most scientists are likely to be struck by the coding, in: J.H. Taylor (Ed.), Molecular Genetics, Part I, the N-terminal region of the iso-1-cytochromes c are presented along with the corresponding DNA sequences of CYC1 alleles. Nucleotides of the primitiveness of scientific techniques just a few years Academic Press, New York, 1963, pp. 477–520. [13] H. Tuppy, U¨ ber die Artspezifta¨t der Proteinstrukur, in: A. transformants that differ from the cyc1-31 sequence are designated in green. The penultimate residues are denoted in red. Cleaved N-terminal back. Nonetheless, mutations are just as important for methionine residues are shown in parentheses. The cyc1-31 mutant completely lacks iso-1-cytochrome c because of the frameshift and TAA Neuberger (Ed.), Symposium on Protein Structure, Wiley, nonsense mutations, shown in blue. Altered iso-1-cytochromes c with four types of amino termini are illustrated, without (0) and with (+) unraveling the mysteries of biological processes now New York, 1958, pp. 66–67. cleavage of the N-terminal methionine and without (0) and with (+) N-terminal acetylation (adapted from [33,80,85]). as they were four decades ago. One way of thinking is [14] K. Narita, K. Titani, Y. Yaoi, H. Murakami, The complete that it is much easier to make and analyze the desired amino acid sequence in baker’s yeast cytochrome c, Biochim. Furthermore, extragenic suppressors of cyc1-512, sequences can be generated by transformation with mutations now than it was way back then. Biophy. Acta 77 (1963) 688–690. [15] P.P. Slonimski, R. Acher, G. Pe´re´, A. Sels, M. Somlo, E´ le´ments including cbc1 and rrp6, led to a novel mRNA synthetic oligonucleotides, the iso-1-cytochrome c du syste´me respiratoire et leur re´gulation: cytochromes et iso- degradation system, designated degradation of mRNA system has been used for the systematic investigation Acknowledgment cytochromes, Colloq. Int. CNRS 124 (1965) 435–461. in the nucleus (DRN), that acts on mRNAs partially of N-terminal processing [33,78–82]. These studies [16] F. Sherman, Mutants of yeast deficient in cytochrome c, retained in the nucleus [77]. By using microarray with altered forms of iso-1-cytochromes c were Genetics 49 (1964) 39–48. The work reported in this review and carried out by procedures to determine the half-lives of approxi- critical for deciphering the amino acid requirements [17] F. Sherman, J.W. Stewart, E. Margoliash, J. Parker, W. Camp- the author was supported for 42 years by the U.S. bell, The structural gene for yeast cytochrome c, Proc. Natl. mately 6000 mRNAs from cbc1-D and rrp6-D strains, for the two N-terminal processes, methionine cleavage Public Health Service research grant RO1 GM12702. Acad. Sci. U.S.A. 55 (1966) 1498–1504. L. Kuai and F. Sherman (unpublished) determined that and acetylation, as well as for identifying the substrate [18] M.E. Dumont, J.F. Ernst, F. Sherman, Identification, sequence certain wild-type mRNAs, such as SKS1, were specificities for each of the three N-terminal acetyl- of the gene encoding cytochrome c heme lyase in the yeast particularly susceptible to DRN. Thus, DRN may transfereases, NatA, NatB and NatC [81–86]. References Saccharomyces cerevisiae, EMBO J. 6 (1987) 235–241. play a role in at least partially determining the steady- Polevoda and Sherman [85] presented a compre- [19] M.E. Dumont, J.B. Schlichter, T.S. Cardillo, M.K. Hayes, F. Sherman, CYC2 encodes a factor involved in mitochondrial [1] R.K. Mortimer, Some recollections on forty years of research state levels of a small subset of wild-type mRNAs, in hensive analysis of N-terminal sequences for more import of yeast cytochrome c, Mol. Cell. Biol. 13 (1993) 6442– in yeast genetics, in: M.N. Hall, P. Linder (Eds.), The Early much the same way as DRN does for mutant cyc1-512 than 450 yeast proteins, over 300 mammalian proteins, 6451. Days of Yeast Genetics, Cold Spring Harbor Laboratory Press, mRNA. and numerous eubacteral and archaeal proteins. The [20] D.A. Pearce, T.S. Cardillo, F. Sherman, Cyc2p is required for Cold Spring Harbor, New York, 1993, pp. 173–185. maintaining ionic stability and efficient cytochrome c import Since the first report of N-terminal alterations of 450 yeast proteins included 78 mutationally altered [2] F. Sherman, Respiration-deficient mutants of yeast. I. Genet- and mitochondrial function in Saccharomyces cerevisiae, mutant forms of iso-1-cytochrome c in 1985 [78], the iso-1-cytochromes c and examples from mutationally ics, Genetics 48 (1963) 375–385. FEBS Lett. 439 (1998) 307–311. [3] F. Sherman, P.P. Slonimski, Respiration-deficient mutants iso-cytochrome c system has been recognized as altered b-galactosidases, abundant proteins, riboso- [21] N.S. Sa´nchez, D.A. Pearce, T.S. Cardillo, S. Uribe, F. Sherman, of yeast II. Biochemistry, Biochim. Biophys. Acta 90 having played a major role in investigations of the mal proteins, and 19 S and 20 S proteasome subunits. Requirements of Cyc2p and the porin, Por1p, for ionic stability (1964) 1–15. co-translational processing of eukarotyic proteins. In Although numerous sources of yeast proteins are and mitochondrial integrity in Saccharomyces cerevisiae, [4] F. Sherman, H. Taber, W. Campbell, Genetic determination of Arch. Biochem. Biophys. 392 (2001) 326–332. normal yeast, the N-terminal methionine of iso-1- available for analysis, N-terminal acetylation is iso-cytochromes c in yeast, J. Mol. Biol. 13 (1965) 21–39. [22] D.G. Bernard, S.T. Gabilly, G. Dujardin, S. Merchant, P.P. cytochrome c is cleaved and the newly exposed most conveniently assayed with the iso-cytochrome [5] A. Singh, F. Sherman, Deletions of the iso-1-cytochrome c and Hamel, Overlapping specificities of the mitochondrial cyto- adjacent genes of yeast: discovery of the OSM1 gene control- threonine residue is not acetylated. However, in the c system. For example, CYC1-853 encodes an altered chrome c and c heme lyases, J. Biol. Chem. 278 (2003) ling osmotic sensitivity, Genetics 89 (1978) 653–665. 1 course of numerous studies spanning three decades, iso-1-cytochrome c having an N-terminus of Met-Glu- 49732–49742. many mutant forms of iso-1-cytochrome c were found Phe-Leu-Ala-, which is normally acetylated but not in [6] J.I. Stiles, L.R. Friedman, F. Sherman, Studies on transposable [23] F. Sherman, J.W. Stewart, J.H. Parker, E. Inhaber, N.A. Ship- elements in yeast. II. Deletions, duplications and transpositions to have their N-termini processed in different ways, as nat3-D or mdm20-D strains. Because cytochrome c man, G.J. Putterman, R.L. Gardisky, E. Margoliash, The of the COR segment that encompasses the structural gene of mutational alteration of the primary structure of yeast iso-1- illustrated in Fig. 8. Because of the dispensability of has a small mass, acetylation can be conveniently yeast iso-1-cytochrome c, Cold Spring Symp. Quant. Biol. 45 cytochrome c, J. Biol. Chem. 243 (1968) 5446–5456. the N-terminal region, and the ease with which altered determined by MALDI-TOF mass spectrometry [87]. (1981) 602–607.

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[24] F. Sherman, J.W. Stewart, M. Jackson, R.A. Gilmore, J.H. by amino-acid replacements in iso-1-cytochrome c, J. Mol. (Eds.), Molecular and Environmental Aspects of Mutagenesis, [72] D.A. Pearce, F. Sherman, Degradation of yeast cytochrome c Parker, Mutants of yeast defective in iso-1-cytochrome c, Biol. 68 (1972) 83–96. C.C. Thomas Publisher Inc, Springfield, Illinois, 1974 , pp. dependent and independent on its physiological partners, Arch. Genetics 77 (1974) 255–284. [41] J.W. Stewart, F. Sherman, Demonstration of UAG as a non- 102–127. Biochem. Biophys. 352 (1998) 85–96. [25] F. Sherman, M. Jackson, S.W. Liebman, A.M. Schweingruber, sense codon in baker’s yeast by amino-acid replacements in [54] K. Struhl, J.R. Cameron, R.W. Davis, Functional genetic [73] J. Wei, F. Sherman, Sue1p is required for degradation of labile J.W. Stewart, A deletion map of cyc1 mutants and its corre- iso-1-cytochrome c, J. Mol. Biol. 68 (1972) 429–443. expression of eukaryotic DNA in Escherichia coli, Proc. Natl. forms of altered cytochromes c in yeast mitochondria, J. Biol. spondence to mutationally altered iso-1-cytochromes c of [42] F. Sherman, B. Ono, J.W. Stewart, Use of the iso-1-cytochrome Acad. Sci. U.S.A. 73 (1976) 1471–1475. Chem. 279 (2004) 30449–30458. yeast, Genetics 81 (1975) 51–73. c system for investigating nonsense mutants and suppressor in [55] A. Hinnen, J.B. Hicks, G.R. Fink, Transformation of yeast, [74] K.S. Zaret, F. Sherman, DNA sequence required for efficient [26] D.M. Hampsey, G. Das, F. Sherman, Yeast iso-1-cytochrome c: yeast, in: J.E. Cellis, J.D. Smith (Eds.), Nonsense Mutations Proc. Natl. Acad. Sci. U.S.A. 75 (1978) 1929–1933. transcription termination in yeast, Cell 28 (1982) 563–573. genetic analysis of structure–function relationships, FEBS and tRNA Suppressors, Academic Press, London, 1979, pp. [56] J.W. Szostak, J.I. Stiles, C.P. Bahl, R. Wu, Specific binding of a [75] P. Russo, W.-Z. Li, D.M. Hampsey, K.S. Zaret, F. Sherman, Lett. 231 (1988) 275–283. 133–153. synthetic oligonucleotide to yeast cytochrome c mRNA, Nat- Distinct cis-acting signals enhance 30 endpoint formation of [27] X. Chen, Investigation of mitochondrial protein degradation [43] R.A. Gilmore, J.W. Stewart, F. Sherman, Amino acid replace- ure 265 (1977) 61–63. CYC1 mRNA in the yeast Saccharomyces cerevisiae, EMBO J. using mutationally altered forms of yeast cytochrome c, Ph.D. ments resulting from super-suppression of a nonsense mutant [57] J.W. Szostak, J.I. Stiles, B.K. Tye, P. Chiu, F. Sherman, R. Wu, 10 (1991) 563–571. Thesis, Universty of Rochester, Rochester, NY 2003. of yeast, Biochim. Biophys. Acta 161 (1968) 270–272. Hybridization with synthetic oligonucleotides, Methods Enzy- [76] Z. Guo, F. Sherman, 30 End forming signals of yeast mRNA, [28] R.P. Moerschell, S. Tsunasawa, F. Sherman, Transformation of [44] P.W. Piper, M. Wasserstein, F. Engbaek, K. Kaltoft, J.E. Celis, mol. 68 (1979) 419–428. Trends Biochem. Sci. 21 (1996) 477–481. yeast with synthetic oligonucleotides, Proc. Natl. Acad. Sci. J. Zeuthen, S. Liebman, F. Sherman, Nonsense suppressors of [58] D.L. Montgomery, B.D. Hall, S. Gillam, M. Smith, Identifica- [77] B. Das, J.S. Butler, F. Sherman, Degradation of normal mRNA U.S.A. 85 (1988) 524–528. Saccharomyces cerevisiae can be generated by mutation of the tion, isolation of the yeast cytochrome c gene, Cell 14 (1978) in the nucleus of Saccharomyces cerevisiae, Mol. Cell. Biol. 23 [29] R.P. Moerschell, G. Das, F. Sherman, Transformation of yeast tyrosine tRNA anticodon, Nature 262 (1976) 757–761. 673–680. (2003) 5502–5515. directly with synthetic oligonucleotides, Methods Enzymol. [45] F. Sherman, M. Strathern, Suppression in the yeast Sacchar- [59] M. Smith, D.W. Leung, S. Gillam, R. Astell, D.L. Montgom- [78] S. Tsunasawa, J.W. Stewart, F. Sherman, Amino-terminal 194 (1991) 362–369. omyces cerevisiae, in: J.N. Jones, E.W. Broach, J.R. Stoppani ery, B.D. Hall, Sequence of the gene for iso-1-cytochrome c in processing of mutant forms of yeast iso-1-cytochrome c: the [30] T. Yamamoto, R.P. Moerschell, L.P. Wakem, D. Ferguson, F. (Eds.), Molecular Biology of the Yeast Saccharomyces: Meta- Saccharomyces cerevisiae, Cell 16 (1979) 753–761. specificities of methionine aminopeptides and acetyltransfer- Sherman, Parameters affecting the frequencies of transforma- bolism and Gene Expression, Cold Spring Harbor Laboratory [60] J.I. Stiles, J.W. Szostak, A.T. Young, R. Wu, S. Consaul, F. ase, J. Biol. Chem. 260 (1985) 5382–5391. tion, co-transformation with synthetic oligonucleotides in Press, Cold Spring Harbor, New York, 1982, pp. 463–486. Sherman, DNA sequence of a mutation in the leader region of [79] F. Sherman, J.W. Stewart, S. Tsunasawa, Methionine or not yeast, Yeast 8 (1992) 935–948. [46] S.W. Liebman, F. Sherman, Cytoplasmic inheritance, in: P. the yeast iso-1-cytochrome c mRNA, Cell 25 (1981) 277–284. methionine at the beginning of a protein, BioEssays 3 (1985) [31] T. Yamamoto, R.P. Moerschell, L.P. Wakem, S. Komar-Pani- Linder, D. Shore, M. Hall (Eds.), Landmark Papers in Yeast [61] D.L. Montgomery, D.W. Leung, M. Smith, P. Shalit, G. Faye, 27–31. cucci, F. Sherman, Strand-specificity in the transformation of Biology, Cold Spring Harbor Laboratory Press, Cold Spring B.D. Hall, Isolation, sequence of the gene coding for iso-2- [80] F. Sherman, R.P. Moerschell, S. Tsunasawa, R. Sternglanz, F. yeast with synthetic oligonucleotides, Genetics 131 (1992) Harbor, New York, 2004, in press. cytochrome c, Proc. Natl. Acad. Sci. U.S.A. 77 (1980) 541– Imahori, N-terminal acetylation of mutationally altered form 811–819. [47] J.W. Stewart, F. Sherman, N.A. Shipman, M. Jackson, Identi- 545. of iso-1-cytochromes c in normal and nat1� strains deficient in [32] D.A. Pearce, F. Sherman, Degradation of yeast cytochrome c fication and mutational relocation of the AUG codon initiating [62] L. Guarente, Regulatory proteins in yeast, Ann. Rev. Genet. 21 the major N-terminal acetyl transferase of the yeast Sacchar- dependent and independent on its physiological partners, Arch. translation of iso-1-cytochrome c in yeast, J. Biol. Chem. 246 (1987) 425–452. omyces cerevisiae, in: K. Sakiyama, F. Sherman (Eds.), Meth- Biochem. Biophys. 352 (1998) 85–96. (1971) 7429–7445. [63] W.-Z. Li, F. Sherman, Two types of TATA elements for the ods in Protein Sequence Analysis, Plenum Publ., New York, [33] R.P. Moerschell, Y. Hosokawa, S. Tsunasawa, F. Sherman, The [48] F. Sherman,J.W.Stewart,Theuseofiso-1-cytochromecmutants CYC1 gene of the yeast Saccharomyces cerevisiae, Mol. Cell. 1993, pp. 173–181. specificities of yeast methionine aminopeptidase, acetylation of yeast for elucidating the nucleotide sequences that govern Biol. 11 (1991) 666–676. [81] B. Polevoda, J. Norbeck, H. Takakura, A. Blomberg, F. Sher- of amino-terminal methionine in vivo: processing of altered initiation oftranslation, in:G. Bernardi,F.Gros (Eds.), Organiza- [64] F. Sherman, C. Helms, A chromosomal translocation causing man, Identification and specificities of N-terminal acetyltrans- iso-1-cytochromes c created by oligonucleotide transforma- tion, Expression of the Eukaryotic Genome; Biochemical overproduction of iso-2-cytochrome c in yeast, Genetics 88 ferases from Saccharomyces cerevisiae, EMBO J. 18 (1999) tion, J. Biol. Chem. 265 (1990) 19638–19643. Mechanisms of Differentiation in Prokaryotes, Eukaryotes, (1978) 689–707. 6155–6168. [34] L.I. Linske-O’Connell, F. Sherman, G. McLendon, Stabilizing vol. 38, Proceedings of the 10th FEBS Meetings, North-Hol- [65] J.F. Ernst, J.W. Stewart, F. Sherman, The cyc1-11 mutation in [82] B. Polevoda, F. Sherman, NatC Na-terminal acetyltransferase amino acid replacements at position 52 in yeast iso-1-cyto- land/American Elseiver, New York, 1975, pp. 175–191. yeast reverts by recombination with a nonallelic gene: com- of yeast contains three subunits, Mak3p, Mak10p and Mak31p, chrome c, Biochemistry 34 (1995) 7094–7102. [49] F. Sherman,J.W.Stewart,Mutationsalteringinitiationoftransla- posite genes determining the iso-cytochromes c, Proc. Natl. J. Biol. Chem. 276 (2001) 20154–20159. [35] L.I. Linske-O’Connell, F. Sherman, G. McLendon, Site spe- tionofyeastiso-1-cytochromec; contrast betweentheeukaryotic Acad. Sci. U.S.A. 78 (1981) 6334–6338. [83] J.R. Mullen, P.S. Kayne, R.P. Moerschell, S. Tsunasawa, M. cific combinations of stabilizing and destabilizing replace- and prokaryotic process, in: J.N. Strathern, E.W. Jones, J.R. [66] J.F. Ernst, J.W. Stewart, F. Sherman, Formation of composite Gribskov, M. Colavito-Shepanski, M. Grunstein, F. Sherman, ments in yeast cytochrome c: in vivo and in vitro effects, Broach (Eds.), Molecular Biology of Yeast Saccharomyces: iso-cytochromes c by recombination between nonallelic genes R. Sternglanz, Identification and characterization of genes and Biochemistry 34 (1995) 7103–7112. Metabolism and Gene Expression, Cold Spring Harbor Labora- of yeast, J. Mol. Biol. 161 (1982) 373–394. mutants for an N-terminal acetyltransferase from yeast, EMBO [36] J.S. Fetrow, T.S. Cardillo, F. Sherman, Deletions and replace- tory Press, Cold Spring Harbor, New York, 1982, pp. 301–333. [67] F. Sherman, J.I. Stiles, L.R. Friedman, J.F. Ernst, G.L. J. (1989) 8. ments of omega loops in yeast iso-1-cytochrome c, PRO- [50] F. Sherman, J.W. Stewart, A.M. Schweingruber, Mutants of McKnight, Divergent and concerted evolution of the two [84] J.C. Tercero, R.B. Wickner, MAK3 encodes an N-acetyltrans-

TEINS: Structure Funct. Genet. 6 (1989) 372–381. yeast initiating translation of iso-1-cytochrome c within a regions encompassing the iso-1-cytochrome c and iso-2-cyto- ferase whose modification of the L-A gag NH2 terminus is [37] X. Chen, R.P. Moerschell, D. Pearce, D.D. Ramanan, F. Sher- region spanning 37 nucleotides, Cell 20 (1980) 215–222. chrome c genes of yeast, Stadler Symp. 14 (1983) 111–131. necessary for virus particle assembly, J. Biol. Chem. 267 man, Enhanced mitochondrial degradation of yeast cyto- [51] D.-F. Yun, F. Sherman, Initiation of translation can occur only [68] L. Melnick, F. Sherman, The gene clusters COR and ARC in (1992) 20277–20281. chrome c with amphipathic structures, Curr. Genet., in press. in a restricted region of the CYC1 mRNA of Saccharomyces the yeast Saccharomyces cerevisiae share a common ancestry, [85] B. Polevoda, F. Sherman, N-terminal acetyltransferases and [38] F. Sherman, J.W. Stewart, Genetics and biosynthesis of cyto- cerevisiae, Mol. Cell. Biol. 15 (1995) 1021–1033. J. Mol. Biol. 233 (1993) 371–388. sequence requirements for N-terminal acetylation of eukar- chrome c, Ann. Rev. Genetics 5 (1971) 257–296. [52] F. Sherman, J.W. Stewart, Mutations at the end of the iso-1- [69] J.A. Downie, J.W. Stewart, F. Sherman, Yeast mutants defective yotic proteins, J. Mol. Biol. 325 (2003) 595–622. [39] F. Sherman, J.W. Stewart, The genetic control of yeast iso-1 cytochrome c gene of yeast, in: J.K. Pollak, J.W. Lee (Eds.), in iso-2-cytochrome c, J. Mol. Biol. 117 (1977) 369–386. [86] B. Polevoda, F. Sherman, Composition and function of eukar- and iso-2-cytochrome c after 15 years, in: M. Bacila, B.L. The Biochemistry of Gene Expression in Higher Organisms, [70] D.A. Pearce, F. Sherman, Diminished degradation of yeast yotic N-terminal acetyltransferase subunits, Biochem. Bio- Horecker, A.O.M. Stoppani (Eds.), Biochemistry of Genetics Australian, New Zealand Book Co. PTY. Ltd., Sydney, 1973, cytochrome c by interactions with its physiological partners, phys. Res. Commun. 308 (2003) 1–11. of Yeast. Pure, Applied Aspects, Academic Press, New York, pp. 56–86. Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 3735–3739. [87] B. Polevoda, T.S. Cardillo, T.C. Doyle, G.S. Bedi, F. Sherman, 1978, pp. 273–316. [53] J.W. Stewart, F. Sherman, Yeast frameshift mutations identi- [71] D.A. Pearce, F. Sherman, Enhanced stability in vivo of a Nat3p and Mdm20p are required for function of yeast NatB [40] J.W. Stewart, F. Sherman, M. Jackson, F.L. Thomas, N. Ship- fied by sequence changes in iso-1-cytochrome c, in: L. Pra- thermodynamically stable mutant form of yeast iso-1-cyto- Na-terminal acetyltransferase and of actin and tropomyosin, J. man, Demonstration of the UAA ochre codon in bakers yeast kash, F. Sherman, M.W. Miller, C.W. Lawrence, H.W. Taber chrome c, Mol. Gen. Genetics 249 (1995) 155–161. Biol. Chem. 278 (2003) 30686–30697.

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Mutation Research 589 (2005) 153–157 challenges, some of which are similar, others of which assumed that humans will be a factor of 10 more are unique. sensitive than rodents. Thus, the use of such defaults is I have become increasingly concerned about the designed to be protective of the health of the public. validity of the various ways in which extrapolation Although defaults of 10 are generally used, defaults Extrapolations are the Achilles Heel approaches are applied because of my involvement of 3 can be used when the data allow. Inevitably the Mutation Research☆ 589 (2005) 153–157 with the risk assessment process as an employee of the use of defaults such as these will lead to increases of Risk Assessment www.elsevier.com/locate/reviewsmr U.S. Environmental Protection Agency. Because my in the uncertainty of estimates of risk values at low R.J. Preston * Community address: www.elsevier.com/locate/mutres involvement has been weighted quite heavily towards doses. For this reason, I describe these extrapolations Reflections in Mutation Research the cancer risk assessment guidelines, these will form and their attendant default values as the ‘‘Achilles § the framework for this Reflections article. Heel of Risk Assessment.’’ A significant challenge EnvironmentalExtrapolations Carcinogenesis Division, are U.S. the Environmental Achilles Protection Heel Agency, of RiskResearch AssessmentTriangle Park, NC 27711, USA As background information, the cancer risk for the enhancement of cancer risk assessment is to Accepted 1 March 2005 assessment paradigm upon which this article is based develop ways of obtaining mechanistic data that R.J. Preston * is that developed by the National Academy of will help to delineate the key events in tumor Environmental Carcinogenesis Division, U.S. Environmental Protection Agency, Sciences’ National Research Council (NRC), as most development or other types of data that will lead to Research Triangle Park, NC 27711, USA recently described in its 1994 report Science and a reduction in the use of default approaches or the use Judgment in Risk Assessment [1]. The components of of more reliable defaults. This will, in turn, result in a Accepted 1 March 2005 this paradigm are: Toxicity Assessment (incorporating reduction in the uncertainty that is inherent in current Hazard Identification and Dose–Response Assess- risk estimates. ment); Exposure Assessment; and Risk Characteriza- There is realistic optimism that progress is being Abstract tion. This whole risk assessment process, as so made and will lead to an acceleration in the defined, contains a fair degree of uncertainty largely elucidation of underlying mechanisms of cancer This Reflections article considers the problems associated with the various extrapolations that are required for the estimation of human cancer risks from exposure to environmental carcinogens at low doses. These include extrapolation between species because of the available data sets. There is an ongoing formation and the impact of environmental carcino- (particularly rodent to human), from responses at high doses to those at low doses, and among different stages of life. Reductions need to reduce uncertainty in risk assessment practices gens. I would like to provide my perspectives on how in uncertainty in risk estimates are closely coupled to the ability to conduct reliable extrapolations. The best way forward appears and in the human exposure limits established via the we might enhance our ability to conduct the various to be the use of data on mechanisms of carcinogenesis to develop bioindicators of responses related to the pathway to tumor risk assessment process. Much of this uncertainty extrapolations inherent in the cancer risk assessment formation. Such an approach is proposed based on the phenotypes represented by the six acquired characteristics forming the results from the incorporation of ‘‘default options’’ process. Hanahan–Weinberg model for carcinogenesis (The Hallmarks of Cancer). In addition, approaches can be established that use the into the risk assessment process when important data Hanahan–Weinberg model as the basis for the collection and/or analysis of microarray or similar data. The reduction in reliance are simply not available. These defaults generally on default options and safety factors in the risk assessment process is a real possibility. account for the extrapolations that are necessary to 2. Parallelograms and pragmatism # 2005 Elsevier B.V. All rights reserved. predict cancer or other adverse effects in humans from the available experimental data. An approach that has been widely used to assist Keywords: Carcinogens; Carcinogenesis; Extrapolations; Risk assessment; Genomics In the absence of adequate information to the interspecies extrapolation for a variety of endpoints contrary, the primary default in the assessment of a (including tumors) involves one version or another of a cancer risk to humans exposed to low (environmental) parallelogram. In its original form, the parallelogram 1. Introduction and ideals from the past, the progress in the present, and the doses, on the basis of data from high-dose experi- was applied to the estimation of genetic risk from potential solutions in the future when new methods ments, is that ‘‘the linearized multistage procedure radiation and chemical exposures using somatic and As I thought about the area upon which I might and models will be available. I realized further that an will be used’’ [2]. Additional defaults are incorporated germ cell data in rodents and humans. The original reflect, I tried to decide whether to choose one from issue that has both intrigued and frustrated me through into the cancer risk assessment process when citation for this approach is usually ascribed to Sobels the past, the present or the future. It became clear that the years is how we can best utilize experimental data appropriate data are not available; these include in his truly excellent paper on comparative mutagen- the need might well be for all three because I generally that, based on the nature of the assay, have to be interspecies extrapolations (most frequently rodent to esis [4]. However, I feel that it may not be consider issues from the point of view of a benefit collected within defined limits for predicting what human), early life stage sensitivity [3], high-to-low inappropriate in a Reflections article to point out that might be going on outside such limits. The approach dose extrapolations, and extrapolations for dose to my colleagues and I had already reported in Nature on used, of course, is extrapolation with all its hydra- target tissues. There are occasions when default the utility of the parallelogram approach in estimating § This article is part of the Reflections in Mutation Research headed forms and which is seemingly just as options may have to be used to take account of other the genetic risk of radiation exposures for humans [5] series. To suggest topics and authors for Reflections, readers should menacing. The most commonly used extrapolations uncertainties in the available data. These defaults are in a paper which was published some two years before contact the series editors, G.R. Hoffmann (ghoffmann@holycross. edu) or D.G. MacPhee ([email protected]). are those extending beyond the tested dose ranges, best guess and conservative estimates of the probable the oft-quoted paper by Sobels appeared in print. A * Tel.: +1 919 541 0276; fax: +1 919 541 1525. across species, among tissues and organs, and from extrapolation relationships. For example, if tumor data much more important point for the present purposes is E-mail address: [email protected]. observations in vitro to those in vivo. All of these carry are available for rodents but not humans, it is generally that the Sobels paper [4] contains an informative

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216 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 217 R.J. Preston / Mutation Research 589 (2005) 153–157 155 156 R.J. Preston / Mutation Research 589 (2005) 153–157 discussion of ‘‘Extrapolations problems.’’ It is striking demonstrated by the BBDR model developed for the Human Relevance Framework (HRF) is the develop- reasonable to propose that a suitable model will also that for most thorny issues the problems remain cancer risk assessment for formaldehyde [6,7]. In this ment of a set of key events whereby specific classes of apply across different etiologies, including the range largely the same, although it is clear that the particular case, the key elements of the model were: chemicals, with particular modes-of-action, can of environmental chemicals. The six characteristics approaches that can be used to address them are (1) use of a three-dimensional computer reconstruc- induce tumors. This ILSI effort has been expanded that lead to unrestricted cell growth are self- likely to be expanded and become more informative as tion of the rat nasal passages and the prediction of to incorporate noncarcinogenic endpoints into the sufficiency in growth signals, insensitivity to anti- technology and knowledge advance. A quotation from regional dosimetry for formaldehyde using computa- HRF, and the results of their findings will be reported growth signals, evading apoptosis, limitless replicative Sobels [4] highlights these points: tional fluid dynamics; (2) use of formaldehyde flux in the near future. This approach will provide valuable potential, sustained angiogenesis and tissue invasion/ predicted in rat nasal mucosa to link the formation of information of a qualitative nature on the relevance of metastasis. It appears that these characteristics do not ‘‘For the induction of mutation, one cannot rely on DNA–protein crosslinks to cytolethality and to laboratory animal data to human cancer risk and also have to be acquired in any particular order. experimental data with the intact mammal, because regenerative cell proliferation within the same nasal on the shape of dose–response curves. However, The great value of a model of this type is that it is extrapolation requires quantification in terms of dose– region; (3) use of a two-stage clonal growth model because of the clear role of specific host factors that based on phenotype, and any particular phenotype can effect curves. The absence of fast tests for gene (reviewed in [8]) to link DNA–protein crosslinks and can significantly influence tumor outcomes, the key- result from many different genotypic alterations. Thus, mutations in mammals defies work in the low dose cytotoxicity/regenerative cell proliferation to tumor events approach will probably not enhance the the Hanahan–Weinberg model does not depend on any range, and since all kinds of intracellular factors may formation. The initial model incorporated parameters prediction of human tumor frequencies—this will specific set of genetic alterations being responsible for a modify the shape of the dose–effect curves, simple for tumor, cellular and molecular data for the rat and require additional computational approaches. A particular tumor type across species but instead depends linear extrapolation from values obtained at high linked them to the rat dosimetry model. An extension broader approach that utilizes quantitative bioindica- on a set of functional changes. Thus, it stands in contrast concentrations introduces an element of considerable of the rat model to predict human tumor data tors of response would seem to hold the real promise to the familiar multistage model for colon carcinogen- uncertainty.’’ assimilated human dosimetric calculations and either for quantitative risk assessments [11]. esis that was originally proposed by Fearon and This same evaluation applies to cancer risk human data itself or estimated human parameters into Vogelstein [14] and which is built on a sequence of assessment, particularly because the accumulation of the BBDR. In this way, the impacts of extrapolations acquired genetic changes. One outcome of the a set of specific mutations or mutation types in a single between species (rat to human), across tissues and 4. Predictions and promise Hanahan–Weinberg proposal is that it ought to be cell lineage forms the basis for cell transformation in from high to low dose were minimized because of the possible to develop bioindicators that are predictive of tumor formation. The use of a parallelogram approach availability of a large amount of pertinent data. Herein The more that is learned about the mechanisms of the six acquired characteristics and can therefore be for estimating human cancer risk is pragmatic at best lie the challenges, clearly discerning the extent of tumor formation and about the impact of environ- used as quantitative molecular markers to predict and relies upon animal and human data being directly available data, their relevance to risk predictions and mental exposures, the more complex things seem to frequencies of tumors. It might be that a particular comparable in both a quantitative and qualitative sense. the additional data needs. It is entirely possible to become. The interplay among genomic instability, cell bioindicator will prove to be of value across species, It is clear that this is not the case, hence the use of the develop models and estimate parameters with almost signaling changes, alterations in cell cycle regulation, tissues or dose ranges, for example, although it is types of default options discussed above. no experimental data being available, but clearly the chromatin remodeling and the many other cellular equally possible that it will not. In either case, BBDR uncertainty in the risk estimates developed will be in changes that may be of relevance is indeed complex. models can be developed using these kinds of direct proportion to the accuracy of the parameters How specific cellular phenotypes and/or genotypes are informative bioindicators for setting model parameters. 3. Models and mechanisms involved. In this context, it is important to appreciate selected to continue along the path to cell transforma- How might informative bioindicators be developed? that there is an optimal degree of confidence in the tion remains the subject of much research and One approach is to conduct studies at the whole genome A more viable approach than the parallelogram, data. Outside the optimal range there is likely to be on discussion (see for example [12]). Clearly, since level (e.g., monitoring gene expression or proteins) on and one that can be greatly enhanced by the growth in the one side an unacceptable level of uncertainty, and cancer is a tissue response involving interactions tumor samples, or on preneoplastic cell samples from knowledge of mechanisms affecting dose to target on the other, the prospect that efforts to significantly among clones of genetically and phenotypically control and treated animals. Microarrays, for example, tissues and cancer endpoints, is the use of biologically reduce the uncertainty would require the expenditure altered cells, the extracellular environment and could be produced in the form of six defined regions, based dose–response (BBDR) models. The aim of a of substantial resources for rather little return. surrounding normal cell populations, the best with each region representing one of the six acquired BBDR model for a specific chemical is to utilize So back to extrapolations in the context of which approaches to understanding carcinogenesis are likely characteristics of the Hanahan–Weinberg model. In this available data for the various parameters in a model data are relevant. As an example, let us consider the to be those that are systems-based. One way to begin to way, changes in gene expression for a region could be that links dose and response. These data include tumor circumstances under which rodent tumor data are address a systems approach that will aid in the considered in terms of their potential impact upon the frequencies for humans and rodents, exposure data for pertinent to human tumor risk assessment. The processes of extrapolation might be to develop models particular acquired characteristic that the gene set humans and rodents, and cellular and molecular data International Life Sciences Institute (ILSI) recently that are neither species specific nor tumor specific. represents. Alternatively, computational analysis could for humans and rodents. At the same time, and most initiated an effort to establish a generalized process for This has been achieved in the model developed by be conducted by considering the 6 acquired character- importantly, a BBDR model can be used to identify evaluating the human relevance of a carcinogenic Hanahan and Weinberg [13] that proposes a set of six istics as constituting clusters of genes. In this way, the significant uncertainties in the various available data mode-of-action established in animals (mainly acquired characteristics that are necessary for any bioindicators would be informative with respect to the sets. This in turn can lead to studies being designed to rodents) together with a consideration of the relevance normal cell to become a metastatic cancer. A critical cancer process itself. This does not imply that any address, and potentially reduce, these uncertainties. of specific chemically induced animal tumors to component of this model is that it is independent of particular bioindicator would be identical across An example of how this overall process can work is human risk assessment [9,10]. The basis for the ILSI species, tumor and cell type. In the same vein, it seems species, tumor type or chemical inducer, but rather a

218 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 219 R.J. Preston / Mutation Research 589 (2005) 153–157 157 proportional relationship among bioindicators, for any References Mutation Research 612 (2006) 151–164 phenotypic endpoint, could be established based upon the relative frequencies of the phenotypic endpoints. [1] NRC (National Research Council), Science and Judgment in Again, in many ways this is a systems approach to Risk Assessment, Committee on Risk Assessment of Hazar- dous Air Pollutants, Commission on Life Sciences, National Isolation of plasmid pKM101 in cancer risk assessment. Academy Press, Washington, DC, 1994. It is feasible that similar methods could be [2] U.S. EPA (Environmental Protection Agency), Guidelines for Mutation Research 612☆ (2006) 151–164 developed such that related approaches could be used Carcinogen Risk Assessment, EPA/630/R-00/004, EPA, the Stocker laboratory www.elsevier.com/locate/reviewsmr for assessing risk irrespective of the adverse health Washington, DC, 1986. Kristien Mortelmans * Community address: www.elsevier.com/locate/mutres outcome under consideration. Clearly the initial need [3] U.S. EPA, Draft Final Guidelines for Carcinogen Risk Assess- Reflections in Mutation Research ment, 2003, EPA/630/P-03/001A, http://cfpub2.epa.gov/ncea/ will be to better understand the key events or acquired § raf/recordisplay.cfm?deid=55445. SRI International,Isolation Biosciences of plasmid Division, Microbiology pKM101 Program, in 333 the Ravenswood Stocker Avenue, laboratory Menlo Park, characteristics that underlie noncancer adverse health [4] F.H. Sobels, Some problems associated with the testing for outcomes. This is the aim of the proposal by the U.S. environmental mutagens and a perspective for studies in CA 94025-3493, United States Kristien Mortelmans * EPA to harmonize risk assessment approaches across ‘‘comparative mutagenesis’’,Mutat.Res.46(1977)245– Accepted 14 March 2006 cancer and noncancer endpoints. The result would 260. SRI International, Biosciences Division, Microbiology Program, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493, United States [5] J.G. Brewen, R.J. Preston, N. Gengozian, Analysis of X-ray- Accepted 14 March 2006 ultimately be the assessment of total detriment from induced chromosomal translocations in human and marmoset exposure to an environmental chemical (or mixture of spermatogonial stem cells, Nature 253 (1975) 468–470. chemicals). Maybe this is a distant goal, but perhaps [6] R.B. Conolly, J.S. Kimbell, D. Janszen, P.M. Schlosser, D. not given the rapid progress in systems approaches to Kalisak, J. Preston, F.J. Miller, Biologically motivated com- Abstract putational modeling of formaldehyde carcinogenicity in the biological assessments. pKM101 is a mutagenesis-enhancing resistance transfer plasmid (R plasmid) that was introduced into several tester strains used F344 rat, Toxicol. Sci. 75 (2003) 432–447. in the Salmonella/microsome mutation assay (Ames test). Plasmid pKM101 has contributed substantially to the effectiveness of the The promise of eradicating the many-headed hydra [7] R.B. Conolly, J.S. Kimbell, D. Janszen, P.M. Schlosser, D. of extrapolations in risk assessment is real. The time- Kalisak, J. Preston, F.J. Miller, Human respiratory tract cancer Ames assay, which is used on a world-wide basis to detect mutagens and is required by many government regulatory agencies for scale for completing this is ripe for debate as are the risks of inhaled formaldehyde: dose–response predictions approval to market new drugs and other chemical agents. Widely used since 1975, the Ames test is still regarded as one of the most specific approaches that will be viable. Thus, while derived from biologically-motivated computational modeling 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 this article is a reflection, it is one that has its roots of a combined rodent and human data set, Toxicol. Sci. 82 (2004) 279–296. used to elucidate molecular mechanisms of mutagenesis. It was isolated in the laboratory of Professor Bruce A.D. Stocker at firmly planted in the realm of ‘‘when’’, not ‘‘if’’. [8] S. Moolgavkar, D. Krewski, M. Schwarz, Mechanisms of Stanford University as part of my doctoral research with 20 R plasmids. Professor Stocker’s phenomenal insight into the genetics of carcinogens and biologically based models for estimation Salmonella typhimurium and plasmid behavior was a major factor that led to the isolation of pKM101. This paper includes a tribute and prediction of risk, IARC Sci. Publ. 131 (1999) 179–237. to Bruce Stocker, together with a summary of my research with mutagenesis-enhancing R plasmids and a brief discussion of the Acknowledgements [9] S.M. Cohen, M.E. Meek, J.E. Klaunig, D.E. Patton, P.A. molecular mechanisms involved in pKM101 plasmid-mediated bacterial mutagenesis. Fenner-Crisp, The human relevance of information on carci- # 2006 Elsevier B.V. All rights reserved. nogenic modes of action: overview, Crit. Rev. Toxicol. 33 I am very pleased to thank Dr. Doug Wolf and Dr. (2003) 581–589. Keywords: R plasmids; pKM101; Ames test; Salmonella/microsome assay; Bruce A.D. Stocker; umuDC; mucAB; Bacterial mutagenesis Hal Zenick for their informative review of this article. [10] S.M. Cohen, J. Klaunig, M.E. Meek, R.N. Hill, T. Pastoor, L. I also thank Carolyn Fowler for her help in formatting Lehman-McKeem, J. Bucher, D.G. Longfellow, J. Seed, V. and preparation of the manuscript. I have enjoyed the Dellarco, P. Fenner-Crisp, D.E. Patton, Evaluating the human many opportunities spent with colleagues reflecting on relevance of chemically induced animal tumors, Toxicol. Sci. 1. Introduction tests to screen chemicals for mutagenicity. The test is 78 (2004) 181–186. required for approval to market new drugs and other the past, present and future of the study of cellular and [11] R.J. Preston, Mechanistic data and cancer risk assessment: the molecular mechanisms of adverse health outcomes. need for quantitative molecular endpoints, Environ. Mol. Plasmid pKM101 has played a major role in the chemical agents by many government regulatory However, these colleagues need not worry, I do not Mutagen 45 (2005) 214–221. success of the Salmonella/microsome mutagenicity agencies all over the world, and it has figured hold them responsible for the views expressed in this [12] R.A. Gatenby, T.L. Vincent, An evolutionary model of carci- assay (Ames test), which detects mutagens on the basis prominently in international guidelines [2]. The test is Reflections in Mutation Research article. Also, I need nogenesis, Cancer Res. 63 (2003) 6212–6220. of their ability to induce reverse mutations in histidine still regarded as one of the most sensitive genetic toxicity [13] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 to note that, although this article has been reviewed in genes, and in elucidating molecular mechanisms of assays for predicting carcinogenicity in animals [3,4]. (2000) 57–70. mutagenesis. Since 1975, when Bruce Ames and co- I am deeply honored that I was invited to prepare an accordance with EPA guidelines, it does not necessa- [14] E.R. Fearon, B. Vogelstein, A genetic model for colorectal workers at the University of California in Berkeley article for the Reflections Series of Mutation Research. rily reflect EPA policy. tumorigenesis, Cell 61 (1990) 759–767. published their often-cited methods paper [1], the Ames Although many plasmids might have made the Ames test has remained one of the most widely used short-term test a more sensitive detector of chemical mutagens, it is gratifying that my graduate work in Bruce Stocker’s laboratory at Stanford University led to the isolation of § This article is part of the Reflections in Mutation Research series. plasmid pKM101, which was later included in some of To suggest topics and authors for Reflections, readers should contact the Salmonella tester strains, namely strains TA100 and the series editors, G.R. Hoffmann ([email protected]) or TA98 [1,5], strain TA97 [6] and strains TA102 and D.G. MacPhee ([email protected]). * Tel.: +1 650 859 4312; fax: +1 650 859 2889. TA104 [7]. In this paper, I will present a tribute to Bruce E-mail address: [email protected]. Stocker, a short overview of bacterial plasmids, a

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220 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 221 152 K. Mortelmans / Mutation Research 612 (2006) 151–164 K. Mortelmans / Mutation Research 612 (2006) 151–164 153 summary of my research efforts with mutagenesis- Bruce was a kind person and was remembered in the cannot grow in an animal or human. These vaccines are conditions plasmids may integrate into the chromo- enhancing R plasmids and the isolation of plasmid memorial service at the Stanford Faculty Club as a now used in livestock. Bruce also created a ‘‘piggy- some, and by doing so lose their independence in DNA pKM101, and a discussion of the mechanisms involved devoted scientist who pursued science without concern back’’ vaccination regime by genetically modifying replication. Plasmids or phages that integrate in the in pKM101 plasmid-mediated bacterial mutagenesis. for personal fame or money. Salmonella flagella with partial replicas of a component chromosome are referred to as episomes. Plasmids are In the early days of his career, Bruce contributed of a disease-causing agent [15–17]. usually found in bacteria, but they have also been found 2. A tribute to Bruce A.D. Stocker, M.D. greatly to the understanding of abortive transduction, Bruce had a phenomenal ability to cite referenced in eukaryotic organisms such Saccharomyces cerevi- which is a form of unstable genetic inheritance whereby papers, some of them having been published decades siae. Their size varies from 1 to 400 kbp and there may the transduced DNA remains extrachromosomal and earlier. A wonderful example of this is the help I be from one copy of the plasmid (for large plasmids) to only one daughter cell receives the transduced gene. received in the early 1990s when I consulted Bruce for a hundreds of copies of the same plasmid in a single When transducing phage P22 was used to study flagellar possible explanation for why there were no revertant bacterium. Many plasmids are self-transmissible, a motility genes in Salmonella typhimurium, transduc- colonies appearing on the agar plates when the Ames characteristic mediated by the presence of transfer (tra) tants were isolated by inoculating the infected non- assay is performed under strictly anaerobic conditions genes. The following subsections provide a short motile recipient in soft gelatin agar. Motile transduc- [18]. It took Bruce only a few minutes to cite two papers overview of some plasmid properties. Several excellent tants moved away from the non-motile population on bacterial mutagenesis with histidine-requiring E. coli reviews of plasmids [22–24] provide more comprehen- giving rise to a ‘‘flare’’ growth. In addition, a number of strains published in 1949 by Ryan and Schneider sive information. linear trails of colonies was observed in the soft gelatin [19,20]. Within a week Bruce sent me hard copies of agar plates. It was concluded that the motility gene does these articles which he had copied at Lane Library at the 3.1. Classification of plasmids according to } not integrate in the chromosome and does not replicate Stanford Medical School. function as an extrachromosomal element. The trail phenomenon Bruce was also a great teacher who always was explained as follows: when a cell divides, one reviewed my research with interest and enthusiasm. Plasmids serve many functions and have been daughter cell remains motile and moves away from its He never failed to offer new ideas from which I could divided accordingly into five major classes: non-motile sister cell which produces a colony in the freely choose. During the three and one-half years I soft agar. This process of unilinear inheritance gives rise worked in the Stocker laboratory, I spent a consider- (1) Fertility (F) plasmids, which carry genes for their to a trail of colonies consisting of non-motile cells until able amount of time dissecting plasmid R46. This own transfer. Some of these genes code for pili on the gene is lost [8]. In an elegant way Bruce confirmed work led to the isolation of the mutagenesis-enhancing the cell surface. Pili can be considered as transfer the unilinear inheritance hypothesis using single cell plasmid pKM101, a deletion derivative of plasmid tubes for plasmid DNA and consist of thin, long and studies of transduced populations [9,10]. R46 that was incorporated into several Salmonella hollow protein tubes that have sticky receptors on Bruce also performed pioneering work on the genetic tester strains used in the Salmonella/microsome their ends that firmly attach to ligands on the cell analysis of the biosynthesis of the lipopolysaccharide mutagenicity assay [1,5–7,21]. The pKM101 plasmid walls of recipient cells. The F plasmid can exist in (LPS) layer of S. typhimurium. Studies were conducted was one of two significant contributions from the three different states: in the F+ state, the F plasmid This section, and indeed the whole article, is a tribute on the permeability of smooth and different types of Stocker laboratory that made the Ames test more exists in an autonomous extrachromosomal state. In to the late Professor Bruce A.D. Stocker (Bruce), who rough strains, including rfa strains, of S. typhimurium to a sensitive to chemical mutagens. An earlier contribu- the high frequency recombination (Hfr) state, the F passed away on 30 August 2004 at his home in Palo wide range of antibiotics. Penicillins were found to be tion was Salmonella phage C21, which Bruce Stocker plasmid is integrated into the chromosome and may Alto, CA, at the age of 87 years. Bruce was my major inactive against smooth (wild-type LPS) strains, and it had generously donated to the Ames laboratory in the initiate the transfer of chromosomal genes under professor and mentor at Stanford University where I was concluded that the lack of activity of some penicillins early 1970s for the selection of the Salmonella rough certain conditions. In the F prime (F0) state, the was a doctoral student from 1971 to 1975. He remained against Gram-negative enteric bacilli may result from rfa tester strains. Because the Stocker laboratory plasmid exists as an extrachromosomal element but active as a researcher at Stanford until shortly before his their inability to penetrate the normal LPS layer [11,12]. played a significant role in the development and contains sections of the chromosomal DNA death. His mentorship did not end when I left Stanford. I Other antibiotics such as bacitracin, vancomycin, success of the Ames Salmonella mutagenicity assay, it covalently attached to it. occasionally called or visited him for many years to get erythromycin, polymyxin and novobiocin exhibited seems more than appropriate to dedicate this article to (2) Colicin plasmids, which contain genes for the his advice on Salmonella genetics and bacterial different levels of antimicrobial activity depending on Bruce. production of proteins, referred to as colicins, that mutagenesis (SRI International, in Menlo Park, CA, the different types of rough lesions in the LPS mutants. It can kill other bacteria (see below for additional is only 3 miles from the Stanford Campus). From 1997 was the presence or absence of single sugars (e.g., 3. Bacterial plasmids information on colicin plasmids). to 2004, shortly before his death, Bruce and I met nearly galactose or glucose) from the polysaccharide of the LPS (3) Resistance or R plasmids, which contain genes weekly late on Friday afternoon in the cafeteria at the that greatly influenced permeability to some of the Plasmids are non-essential, independent and extra- whose products confer resistance to one or more Stanford University Medical Center for coffee and a antibiotics [12,13]. The above-described work is of direct chromosomal DNA elements that may be added to or antibiotics (see below for additional information on friendly scientific chat. As Emeritus professor in the relevance to the rfa Salmonella mutants that were later lost from bacteria without affecting their viability. They R plasmids). Department of Microbiology and Immunology, Bruce produced in the Ames laboratory, because a complete are circular double-stranded DNA molecules that can (4) Degradative plasmids, which carry genes that planned to continue working for a long time to come. A LPS layer was shown to be a barrier to the penetration of exist in three different physical states: (1) ‘‘super- allow the host bacterium to degrade organic material few days before he passed away, he bought a brand new certain bulky mutagenic chemicals into the cells [14]. coiled’’ (covalently closed) as intact DNA with both (e.g., toluene). computer so he could surf the web to find the latest Bruce Stocker greatly contributed to the movement strands uncut, (2) ‘‘nicked-open circular’’, with one cut (5) Virulence plasmids, which carry genes that turn the updates on the genetics of his favored enteric to develop replacements for the killed-bacterial-type in one strand of the DNA and (3) ‘‘linearized’’ with both host bacterium into a pathogen (e.g., plasmids pX01 organisms, Salmonella, Shigella and Escherichia coli. vaccines. He engineered strains of live Salmonella that strands of the DNA cut at one site. Under some and pX02 in fully virulent B. anthracis).

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3.2. Plasmid incompatibility groups mutations resulting in temperature sensitivity for R In the late 1960s, it was discovered that transposons plasmid maintenance have been reported [30,31]. The may play a role in the spread of certain genes such as Naturally occurring plasmids have been classified into maintenance of the small ColE2 plasmid is dependent those responsible for resistance to antibiotics. Such groups according to their compatibility. Plasmids on the DNA polymerase I activity of the host [32]. genes on transposons can undergo recombination with belonging to the same compatibility group cannot stably plasmids thereby giving rise to multiple drug resistance coexist in the same host and are therefore termed 3.5. Colicin plasmids plasmids [36–38]. incompatible. However, plasmids in one compatibility Single or multiple antibiotic resistance mediated by group can coexist stably with plasmids in any other group Some strains of the family Enterobacteriaceae (e.g., R plasmids is currently prevalent all over the world. and are therefore termed compatible. At least 30 plasmid Salmonella, Shigella and E. coli) carry plasmids that Resistant strains often emerge within a few weeks to a incompatibility groups have been identified, with no code for a range of bactericidal agents (colicins) few months after the introduction of a novel anti- upper limit in sight. Incompatibility may occur between believed to be protein-like in nature because they are bacterial drug. R plasmid-mediated resistance against two plasmids that are nearly isogenic (e.g., F0-lac and F0- susceptible to proteolytic enzymes. Many of these antibiotics of the neomycin–kanamycin group was gal) or between pairs that are less closely related (e.g., plasmids are self-transmissible on an interspecies and reported in 1963 [39] and plasmid-mediated resistance ColV2-K94 and F; F and ColV3-K30; ColB2-K77 and intraspecies level. Many different types of colicins can against the penicillins was discovered in 1965 [40]. 222/R) [25]. Plasmid incompatibility does not result from be readily distinguished experimentally by their host However, antibiotic resistant bacteria have also been DNA restriction, since incompatibility also occurs when susceptibility and their diffusibility, which is usually found in places with no known use of antibiotics such as attempts are made to transfer incompatible plasmids measured in terms of the diameter of the zone of growth Borneo [41]. The self-transmissible nature of many R Fig. 1. Vials in which Bruce Stocker’s extensive Salmonella typhi- between isogenic donors and recipients. inhibition. Host susceptibility is determined by unique plasmids is responsible for the transfer of their murium strain collection was preserved on Dorset egg agar slants. receptors on the bacterial cell wall to which the colicins respective resistance determinants among members of + 3.3. fi� and fi plasmids attach. Different capital letters are used to designate the family Enterobacteriaceae including Yersinia pestis S. typhimurium LT2 strains as well as plasmids to study different colicins, such as B1,E2, I, K and V [25]. and in some instances to more distantly related genera plasmid-mediated mutagenesis. Indeed, Bruce had a Naturally occurring R plasmids have been divided Salmonella strains are naturally resistant to colicins, such as Vibrio cholerae and Serratia marcescens [42]. very extensive collection of S. typhimurium LT2 into two broad types according to their interaction with whether or not they carry colicin plasmids. Also, S. cultures, which he had brought with him from the the F plasmid of E. coli K-12. R plasmids that inhibit the typhimurium LT2 strains are inherently non-colicino- 3.7. Mutagenesis-enhancing plasmids Lister Institute in London when he joined the Faculty of fertility of the F plasmid are designated fi+; those that do genic, but they will readily accept colicin plasmids via Medical Microbiology at Stanford in 1966. These not are called fi� [26]. Meynell and Datta [27] proposed conjugation by mixed growth with E. coli, Shigella or Colicin plasmid ColI was the first plasmid shown to cultures were preserved on Dorset egg agar slants in that the fi+ R plasmids interfere with the formation of F other Salmonella strains that carry a colicin plasmid. enhance survival after ultraviolet light (UV) exposure as small bijou vials (see Fig. 1), all of which were kept at pili by determining the production of a repressor well as the frequency of UV-induced mutations in S. room temperature in an old unlocked wooden cabinet in substance. This substance not only represses the 3.6. Resistance transfer factors (R plasmids) typhimurium LT2 [43,44]. The first R plasmid reported to the hallway of the Medical Microbiology Department. production of pili specified by the R plasmid, but also enhance chemical mutagenesis was R205, also referred to Bruce was proud that his approach to storage never represses the constitutive production of the F-type pili The presence of R plasmids in microbial host cells is asR-UtrechtaftertheoriginofitsisolationinUtrecht,The failed to revive any of these cultures. Some of these determined by the F plasmid. one of the major causes of antibiotic resistance, Netherlands. This finding, published by MacPhee [45], Salmonella cultures carried colicin plasmids or R factor currently a serious public health concern. Many R led Ames and co-workers to systematically evaluate a plasmids. 3.4. Plasmid replication plasmids carry resistance determinants to one or number of R plasmids for their effect on chemical While most of my work was ultimately performed multiple antibiotics. For instance, plasmid R46, the mutagenesis. Plasmid pKM101, isolated in Bruce Stock- with plasmid R46, 19 other plasmids were used for Plasmids are grouped into two distinct classes based parent plasmid of pKM101, confers resistance to er’s laboratory, was reported by McCann et al. to be better evaluation of their effect on UV-survival, as well as on on the number of copies that are present in the host cell. ampicillin, streptomycin, sulfonamide and tetracycline. compared to all of the other plasmids that they evaluated spontaneous and UV-induced mutagenesis. Table 1 lists Some plasmids replicate at the same rate as the The first R plasmids were discovered in Japan in the mid for their ability to enhance MMS-induced mutagenesis these plasmids and includes a reference to the source of chromosomal DNA, and therefore their DNA replication 1940s, shortly after World War II, when sulfonamide [21]. MacPhee had begun working with plasmid R205 the following plasmids: Drabble and Stocker [46] for is under ‘‘stringent’’ control by the host cell. At most two drugs were used for the treatment of bacillary dysentery when he was a postdoctoral fellow in Bruce Stocker’s plasmids R46 (R-Brighton), R-South Africa, R-Peru, R- copies of such plasmids are present in the cell at any time caused by Shigella strains. Initially these antimicrobial laboratory in the Medical Microbiology Department at Singapore, R45 (R-Enfield), R48 (R-Bradford), R205 [28]. Other plasmids replicate independently of the host drugs proved very effective in treating the disease. Stanford University from 1967 tolate 1968, while I joined (R-Utrecht), R6 (R-Munich) and R6-Tc; donations to chromosome andareunder ‘‘relaxed-regulation’’control. However, by 1949 Shigella strains emerged that were the laboratory as a Ph.D. student in 1971. My reflections the Stocker laboratory by Datta [47] for plasmids N3, Ten or more copies of the plasmid can be present in a cell resistant to the sulfonamides. When effective new on research in the Stocker laboratory follow. R447a, R205 (second R205/R-Utrecht), R269 and [29]. When plasmid DNA replication is under relaxed- antibiotics were introduced (e.g., streptomycin, chlor- R390. There are no references for the plasmids obtained regulation control, plasmid replication continues when amphenicol and tetracycline), bacteria resistant to these 4. Salmonella culture collections available at from Dr. Marjorie Bissett at the California Public the host cells are in stationary phase. Up to 100 copies of antibiotics soon emerged. In 1953, Shigella strains Stanford from 1971 to 1975 Health Department in Berkeley. The plasmids refer- some plasmids can thus be generated in stationary-phase resistant to streptomycin or tetracycline were isolated enced by Drabble and Stocker [46] were initially cells. Although plasmids in general are considered self- and the first multiple drug resistant pathogenic bacteria 4.1. Stocker culture collection obtained from clinical bacterial cultures and were replicating extrachromosomal genetic elements, some seem to have appeared in 1955 [33,34]. In 1960, Akiba subsequently transferred into S. typhimurium strains. plasmids are dependent on host functions for their et al. [35] demonstrated the self-transmissible nature of The Stocker laboratory had available for my research Bruce had also collected many Salmonella phages maintenance and replication. For instance, chromosomal multiple drug resistant R plasmids in vitro in Shigella. a large collection of auxotrophic and multi-auxotrophic that had been isolated from London sewage by various

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Table 1 5. Mutagenesis work with plasmid R46 Plasmids evaluated for effects on UV-survival and spontaneous and UV-induced mutagenesis

+ R plasmids Host strain of plasmid prior to Character (drug resistances; fiÀ or fi , References Of the many plasmids available, I chose plasmid R46 conjugation into hisG46 incompatibility group; host restriction– more or less at random to initiate my work. Plasmid R46 modification specificity, if known) is an fiÀ plasmid that belongs to the compatibility group

R-Brighton (R46) S. typhimurium LT2, SL1137 Amp, Sm, Sul, Tc; fiÀ,N [30] N; its size is 50 kbp. Eventually I also worked with the  R-Enfield (R45) S. typhimurium LT2, SL1138 Amp, Sm, Tc; fiÀ,N [30] other 19 plasmids listed in Table 1. R-Bradford (R48) S. typhimurium LT2, SL1141 Amp, Sm, Sul, Tc; fiÀ,N [30] Plasmid R46 was first tested in a tryptophan- R-Utrecht (R205) S. typhimurium LT2, SL1142 Amp, Sm, Tc; fiÀ,N [30] requiring mutant SL1156 (trpD1) from the Stocker R-Peru S. typhimurium LT2, SL1131 Chl, Sm, Sul, Tc; fi+ [30] R-South Africa S. typhimurium LT2 SL1128 Sm, Sul, Tc; fi+ [30] collection. This mutant was selected because its R-Singapore S. typhimurium LT2 SL1134 Amp, Chl, Sm, Sul, Tc; fi+ [30] frequency of reversion to prototrophy after UV- R-Munich (R6) S. typhimurium LT2 SL1145 Chl, K, Sm, Sul, Tc; fi+ [30] irradiation is substantially increased when it carries a R6-Tc S. typhimurium LT2 SL1148 Tc Unpublished UV-protective ColI plasmid [43,44]. Fig. 2 clearly N3 Shigella flexneri Sm, Sul, Tc; N; hspII [31] shows the UV-protective effect of plasmid R46 [49]. In R447a Proteus morganii Amp, Chl, Sm, Sul, Tc; N; hspII [31] Fig. 2. UV-protective effect of plasmid R46 on strain SL1156, a trpD R205 S. typhimurium LT2 Amp, Sul, Tc; fiÀ,N [31] addition to providing UV-protection, plasmid R46 also R269 Shigella sonnei Amp, Sm, Tc; N; hspII [31] enhanced UV-induced mutagenesis in SL1156. UV- mutant of Salmonella typhimurium. R390 Proteus rettgeri Amp, Chl, Sm, Sul, Tc; N; hspII [31] irradiation at 200 erg/mm2/s yielded 1880 tryptophan R3916CPH Salmonella anatum Amp, Sm, Tc (source: human) Unpublished revertants per 108 survivors of SL1156 (R46) versus 174 of restriction sites and no restriction enzymes available R9095CPH Salmonella saint paul K, Ne, Sm, Sul, Tc (source: chicken) Unpublished revertants for the strain not carrying the plasmid. for cutting up the plasmid, physical means were used in R3729CPH Salmonella anatum Amp, K, Ne, Sm, Sul, Tc (source: calf) Unpublished R11576CPH Salmonella newport Amp, Ne, K, Sm, Tc (source: human) Unpublished Plasmid R46 was next evaluated in Salmonella LT2 attempts to segregate the mutator effect from the other R7842CPH Salmonella typhimurium Sm, Sul, Tc (source: bovine) Unpublished hisG46 from the Ames laboratory. The enhancement of two properties. The end point used in the segregation R11687CPH Salmonella heidelberg Amp, Sm, Sul, Tc (source: human) Unpublished survival and mutagenesis after UV-irradiation occurred experiments involved loss of resistance to one or more much as they had in the trpD1 strain, but an unexpected antibiotics. Thousands of clones were tested for increase in spontaneous mutagenesis was also observed, antibiotic resistance. A simple but very effective device of his UK colleagues and that he used for studying made the cells permeable to many chemicals to which as shown in Table 3. This was the first reported was used to screen colonies for changes in antibiotic defects in the LPS layer of Salmonella. I worked with they had previously been impermeable and hence observation that plasmids have the ability to enhance resistance. The device, referred to as a prong replicator, a set of 12 of these phages whenever phage typing of were likely to give false-negative results in Ames spontaneous mutagenesis in S. typhimurium LT2, a designed by Bruce Stocker, consisted of 25 equally Salmonella strains was required. Bruce donated one assays. property that is referred to as a ‘‘mutator effect’’. This spaced prongs with 2 additional prongs reserved for of them, phage C21, to the Ames laboratory for the observation was a milestone and became a turning point controls, as shown in Fig. 3. The prong replicator is first selection of the rfa mutants. The deep rough (rfa) 4.2. Ames culture collection in my research, which was thereafter primarily focused used to make slight indentations in the agar of the derivatives of the tester strains were isolated in the on determining whether the spontaneous mutator effect master plate. Colonies are then transferred to each Ames laboratory by selection for bacteria resistant to Also available for my research were several of plasmid R46 could be separated from two other marked spot. After overnight incubation, the prong C21 phage from Salmonella strains that were initially ‘‘ancestral’’ histidine-dependent S. typhimurium LT2 properties, namely enhancement of UV-survival and replicator is used to replicate the 25 + 2 control colonies sensitive to C21 because they had already had their strains, which the Ames laboratory in Berkeley had enhancement of UV-mutagenesis. to selective agar plates. gal-chl-bio-uvrB chromosomal region deleted [14]. kindly provided to the Stocker laboratory. By the early The following treatments were initially used with C21 resistant colonies which appeared on confluent- 1970s, Dr. Ames had already selected S. typhimurium 6. Search for incomplete forms of plasmid R46 cultures of hisG46 (R46) in attempts to obtain lysis nutrient agar plates were picked and purified. LT2 tester strains with unique mutations acting as hot incomplete forms of R46 that display some, but not The introduction of the rfa mutation into the tester spots for reverse mutagenesis, including most notably 6.1. Use of chemical/physical means all, properties associated with the plasmid: adding strains enhanced the sensitivity of the Ames assay as hisG46, hisC207, hisD3052 and hisC3076, for use in his sodium dodecyl sulfate (SDS); maintaining cultures in the defect in the LPS layer allowed easier access to then-developing mutagenicity test system [48]. Table 2 It took a long and tedious effort to produce the refrigerator for up to 2 months; vigorous shaking of the cells by certain test chemicals; however, it also lists these strains and their genotypes. incomplete (i.e., shortened) forms of plasmid R46 cultures over extended periods of time; and repeated and ultimately to produce pKM101. With no knowledge subculturing in broth containing SDS. Conjugation

Table 2 Table 3 Strains of the Salmonella/microsome assay developed by Bruce Ames and co-workers Effect of plasmid R46 on UV-survival and spontaneous and UV-induced reversion in S. typhimurium hisG46 Histidine mutation in Salmonella strains and derivative strains with LPS or repair Additional mutations Strain %Survival Spontaneous UV-induced defects (UV-dose: 60 ergs/mm2) revertant revertant colonies LPS Repair colonies/plate per 108 survivors hisG46 hisC207 hisC3076 hisD3052 Wild-type Wild-type his46 40 3 60 a TA1950 TA1951 TA1952 TA1534 Wild-type (chl-uvrB-bio)D hisG46 (R46) 85 37 182 TA1530 TA1531 TA1532 TA1964 gal (chl-uvrB-bio) D D a The enhanced spontaneous mutation frequency seen in the presence of plasmid R46 prompted the search for an incomplete form of R46, which TA1535 TA1536 TA1537 TA1538 rfa (chl-uvrB-bio) D ultimately led to the isolation of plasmid pKM101.

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important because the one transductant that retained novel incomplete forms of the plasmid in conjugation also tested Dr. Cohen’s plasmid, pSC101, for its resistance to both ampicillin and streptomycin carried experiments between SL3810 (donor of transduced potential mutagenesis-enhancing effect and found it the R46 derivative that was to become the precursor of plasmids) and hisG46 (recipient). As described above, was not able to enhance chemical mutagenesis [21]. plasmid pKM101. the transduced plasmids were initially transferred from the transductants into SL3810 via conjugation. Plasmid- 9. Molecular studies with plasmid pKM101 6.3. Additional experiments with R46 transductants containing derivatives of SL3810 then served as donors in and the emergence of pKM101 conjugation experiments with Salmonella hisG46. It was 9.1. Characterization during the conjugal transfer between SL3810 and hisG46 6.3.1. Conjugal transfer of complete and that the ‘‘final’’ incomplete forms were obtained. As a postdoctoral fellow in Bruce Ames’ laboratory incomplete transduced forms of R46 into SL3810 Many experiments were performed in attempts to in the mid 1970s, Graham Walker used molecular Plasmid pKM101 emerged only after many more answer the following questions: Is plasmid R46 techniques to elucidate the mechanisms involved in experiments had been performed with certain of the inherently unstable in SL3810? Is a previously plasmid-mediated enhanced mutagenesis and protec- transduced plasmids mentioned above. Some of these transduced plasmid unstable in SL3810? Was an tion. Most of his work in the Ames laboratory was done experiments involved conjugal transfer of the complete anomalous clone of LT2 hisG46 used as recipient in with plasmid pKM101 in E. coli, and he continued these Fig. 3. Prong replicator designed by Bruce Stocker to screen colonies and incomplete transduced plasmids to an intermediary conjugation experiments with SL3810? None of these studies when he moved to Massachussetts Institute of for altered antibiotic resistance. recipient, SL3810 (=LT2 pyrE135, rfa-738), which was experiments provided clues to explain the mechanism or Technology, Cambridge, MA, where he remains as unable to adsorb phage P22 because of its rfa character. mechanisms that led to the production of the various professor of Biology. A few highlights from the Walker experiments were also performed (crosses between an Without this step, any liberated phage from the incomplete forms of R46, including plasmid pKM101. studies are described below. R46 donor strain and a suitable recipient), with transduced donor might transfer plasmid DNA into a Plasmid pKM101 is a 35.4 kb plasmid belonging to selection on medium containing only one of the four genetically inappropriate (wild-type for rfa) recipient. 8. Assigning names to the incomplete forms of the N incompatibility group [53]. It lacks a 13.8 kb antibiotics to which R46 confers resistance. The In these experiments the majority of the transduced R46 region of plasmid R46, a region that carries the marker predominant results of these experiments can be plasmids were able to self-transmit via conjugation to conferring resistance to streptomycin, sulfonamide and summarized as follows: either no loss of antibiotic SL3810 and the pattern of antibiotic resistance did not Many interesting stories have been told about how tetracycline. Plasmid pKM101 retained resistance to resistance (and presumably no loss of the plasmid) or a change. the name pKM101 came about. I was once told that the ampicillin and codes for two proteins analogous to the complete loss of all resistance to all antibiotics ‘‘101’’ must have been selected to refer to a local cellular UmuC and UmuD proteins of E. coli which are (presumably due to plasmid loss). However, a few 6.3.2. Conjugal transfer of transduced R46 from freeway, ‘‘Bayshore Freeway 101’’, which runs into San involved in DNA repair and mutagenesis. It has been colonies were found that had only lost their resistance to SL3810 into Salmonella hisG46 Francisco. The true story is as follows. When the Ames shown that E. coli mutants lacking these proteins are tetracycline. The next series of experiments involved conjugal laboratory was ready to publish the R factor paper [21], non-mutable by UV [54]. When the umuC DNA region transfer of 25 transduced plasmids from SL3810 into a one of the authors, Neil Spingarn, called me at Stanford was first cloned, analysis revealed the presence of two 6.2. Transduction of plasmid R46 into standard genetic background, Salmonella hisG46, to wondering how they should name the plasmid. I genes coding for the gene products UmuC and UmuD, hisG46(P22.sie)strA permit testing of the transduced plasmids for their approached Dr. Esther Lederberg, an Adjunct professor with molecular weights of 45,000 and 16,000, ability to enhance spontaneous mutagenesis, UV- in the Medical Microbiology Department who was in respectively [55]. When pKM101 was introduced into Transduction with phage mutant P22.C2 HT 13/4, survival and UV-induced mutagenesis. The following charge of the Plasmid Reference Center at Stanford. At umuC mutants of E. coli, DNA repair and mutagenesis which causes a high frequency of transduction, was next transduced plasmids housed in SL3810 were chosen for that time Dr. Lederberg was working in Dr. Stanley functions were restored [56]. From these studies it was used in attempts to obtain segregants of R46 based on these conjugation experiments: 15 of the transduced Cohen’s laboratory in the Genetics Department. She inferred that pKM101 carries genes analogous to the earlier work reported by Drabble and Stocker [46].A plasmids that had retained resistance to all 4 antibiotics, recommended that I name the plasmid as follows: p for chromosomal umuC region in E. coli. P22 lysogenic Salmonella strain, hisG46(P22.sie)strA 9 that had lost resistance to tetracycline and 1 that had plasmid, followed by my initials KM, followed by the The plasmid umuC-like region analogous to the was used as the recipient because the transducing phage retained resistance to ampicillin and streptomycin. number 101. This was after all how Dr. Cohen had cellular umuDC genes has been located on plasmid was a virulent C2 (clear plaque) form of P22. The When the hisG46 recipient exconjugants were tested for named his plasmid, pSC101, which had been used in the pKM101 and is referred to as the muc region transductants were selected on plates supplemented their respective antibiotic resistance patterns, a new set first recombinant DNA experiments [52]. The number (mutagenesis, UVand chemical). Its two protein products with ampicillin, sulfonamide or tetracycline. Strepto- of six incomplete forms of R46 were discovered and it ‘‘101’’ had been selected instead of ‘‘1’’ to ensure that are encoded by two genes, mucA and mucB, whose gene mycin could not be used on the selective agar plates was from this set that pKM101 emerged. Table 4 sufficient computer storage space would be available in products have molecular weights of 16,000 and 45,000, because the recipient in the transduction experiments summarizes the antibiotic resistance patterns of all the the future if a researcher generated many new plasmids respectively. These two gene products are required for the carried the chromosomal strA marker (streptomycin incomplete forms of R46 that had been obtained in the that would alter the data entry space by as many as 2 plasmid to exert its mutagenic and protective effects. resistance; now called rpsL). When 100 colonies of each conjugation experiment. Further analysis of the various spaces (that is, if at least 100 new plasmids were Several other plasmids with properties similar to those of of the transductants from the different selection plates incomplete forms of R46 revealed that all plasmids generated). Today, this rationale is unthinkable, but in pKM101 have been found to contain DNA regions were analyzed for antibiotic resistance (except for except those that had retained only streptomycin 1973 computers were just starting to be used for data analogous to the muc region of plasmid pKM101 [57]. streptomycin resistance), 272 clones had retained their 3 resistance were able to enhance UV-survival and both entry, computer space was limited and data entries were Like the cellular umuC and umuD genes, the plasmid muc testable antibiotic markers, 27 had lost their tetracycline spontaneous and UV-induced mutagenesis, as well as not readily amenable to changes in formatting. genes are believed to be directly regulated by the recA marker and 1 had retained only the ampicillin resistance retaining the ability to self-transmit. The plasmids that Following this example, all the other incomplete forms and lexA gene products [58,59]. Additional information marker. Further analysis indicated that all plasmids had had retained only streptomycin resistance were no of plasmid R46 that are listed in Table 4 were given a on the molecular studies with plasmid pKM101 is retained their streptomycin resistance. This result is longer able to self-transmit via conjugation and had lost unique plasmid number. Note that the Ames laboratory available from a number of sources [60–67].

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Table 4 stemmed from the paper by MacPhee [45] reporting that 9.2. Mutational specificity 10. Novel uses in research with plasmid pKM101 Plasmid pKM101 and other incomplete forms of plasmid R46 plasmid R205 markedly enhanced chemical mutagen- Plasmid number Antibiotic resistance markers esis in Salmonella hisG46. When Ames laboratory staff Fowler et al. evaluated the spontaneous and UV- In recent studies plasmid pKM101 has been used to (antibiotic resistance) on transduced plasmid in SL3810 evaluated the plasmids from the Stocker laboratory, they induced mutational specificity of pKM101 in E. coli in study the mechanisms of conjugative DNA transfer in E. prior to conjugation found that plasmid pKM101 enhanced MMS-induced an attempt to determine whether one or several coli [76]. Plasmid pKM101 is also being used to study pKM101 (Amp) Amp, Sm mutagenesis to a greater extent than any of the others mechanisms were involved in spontaneous and UV- type IV secretion systems in Agrobacterium tumefa- pKM105 (Amp) Amp, Sm that they had tested [21]. Therefore, plasmid pKM101 induced plasmid-mediated mutagenesis. By analyzing ciens [77–79]. Type IV secretion systems mediate pKM106 (Amp) Amp, Sm was used in several of the major Salmonella tester reversion patterns of defined trpA alleles, it was shown conjugative plasmid transfer as well as the translocation pKM103 (Amp, Sul) Amp, Sm, Sul pKM107 (Amp, Sul) Amp, Sm, Sul, Tc strains: first in TA98 and TA100 [1,5] and later in TA97 that in both instances pKM101 enhanced mutations at of virulence factors from various Gram-negative pKM108 (Amp, Sul) Amp, Sm, Sul [6] and TA102 and TA104 [7]. Table 5, adapted from the A:T base pairs, especially transversions, suggesting a bacteria in eukaryotic host cells. pKM109 (Amp, Sul) Amp, Sm, Sul R factor paper by McCann et al. [21], compares the similar mechanism [68,69]. Two other mutagenesis- pKM110 (Amp, Sm) Amp, Sm results obtained with chemical mutagens in strains enhancing plasmids, R269 and R390, were reported to 11. Plasmid R46 behavior in host cells with pKM104 (Amp, Sm, Sul) Amp, Sm, Sul TA1535, TA1538 and their pKM101 derivatives. The have the same spontaneous and UV-induced muta- different genetic background pKM111 (Amp, Sm, Sul) Amp, Sm, Sul pKM112 (Amp, Sm, Sul) Amp, Sm, Sul, Tc results show that plasmid pKM101 enhances the tional specificity as plasmid pKM101 [70]. An PKM113 (Amp, Sm, Sul) Amp, Sm, Sul mutagenicity of a wide range of mutagens and also enhancement of transversion events over transition My work included many more studies with plasmid pKM114 (Sm) Amp, Sm, Sul, Tc allows for the detection of chemical mutagens events was also observed in studies designed to R46 in Salmonella strains that were deficient in DNA pKM115 (Sm) Amp, Sm, Sul, Tc previously not detected by Salmonella strains evaluate the influence of both the uvrB gene involved repair. Themutatoreffect,enhancedUV-mutagenesis and pKM116 (Sm) Amp, Sm, Sul TA1535 and TA1538. A good example of one such in excision DNA repair and pKM101 on the spectrum enhanced UV-survival were abolished in recA mutants, pKM117 (Sm) Amp, Sm pKM102 (Amp, Sul) Amp, Sul, Tc mutagen is furylfuramide (AF-2), which is highly of spontaneous, UV- and g-ray-induced base substitu- butnotinpolAanduvrBmutants.Thisresultwasexpected pKM119 (Amp, Sul) Amp, Sul, Tc mutagenic in strain TA100. AF-2 is a nitrofuran food tions in S. typhimurium hisG46 [71]. In another study for the uvrB mutants, since most of the Salmonella tester pKM120 (Amp, Sul) Amp, Sul, Tc additive that was widely used in Japan before it was Gordon et al. [72] evaluated the influence of pKM101 strains carry a deletion through the uvrB region. The pKM121 (Amp, Sul) Amp, Sm, Sul, Tc banned in September 1974. The carcinogenic nitrofur- on spontaneous mutational specificity at the DNA spontaneous mutator effect associated with plasmid R46 ans were first detected as mutagens in the E. coli B/r sequence level with 198 lacI mutations generated in E. was studied in various missense mutants, as well as in WP2 (tryptophan reversion) assay [51]. Interestingly, coli strains carrying pKM101. The plasmid did not ochre, amber and UGA (stop codon) mutants. The the ability to protect against UV or to enhance UV- Table 5 shows that plasmid pKM101 may actually show the mutator effect on the spontaneous forward plasmid was found to enhance the spontaneous reversion + induced mutagenesis, but they still conferred an reduce the mutagenicity of some chemical mutagens, as mutation frequency of lacI to lacIÀ. The pKM101 rate of some, but not all of the missense mutants, and of all enhanced mutator effect [49,50]. it does in the case of diethyl sulfate in strain TA100. mutational spectrum yielded an almost identical lacIÀ amber, ochre and UGA mutants tested. However, I used distribution to that seen in an E. coli strain deficient in different non-isogenic strains of S. typhimurium, each 6.3.3. Evaluation of incomplete transduced R46 7. Mechanism(s) involved in the generation of DNA polymerase I activity [73]. Base substitutions carrying a different missense mutation, in these experi- plasmids by the Ames laboratory plasmid pKM101 and other incomplete forms of from A:T to C:G (transversions) were most often ments and so differences in genetic background may well Representatives of the different incomplete forms of R46 observed in the presence of pKM101, confirming the have contributed to differences in results. A similar plasmid R46 were sent to the Ames laboratory in results of Fowler et al. [68] and Eisenstadt et al. [71]. proviso applies to the S. typhimurium strains carrying Berkeley in response to a request for plasmids to screen It is still not clear how conjugation of transduced R46 Plasmid pKM101 was also evaluated in a uvrB amber, ochre and UGA mutations; they too were non- for their effect on chemical mutagenesis. The rationale plasmids (either complete or incomplete) came to yield background to study its effect on the spectrum of isogenic. The mutagenicity of some of the many spontaneous frameshift mutations at the hisD3052 frameshift mutants evaluated was also enhanced in the allele in S. typhimurium [74]. The authors reported presence of plasmid R46 [49,80]. Table 5 Induced revertants per plate (spontaneous revertants subtracted) in strains TA1535 and TA1538 with and without pKM101 that the effect of pKM101 was similar in both wild- type and uvrB deletion strains, with an increase in 12. Summary results for an additional 19 Mutagen (mg/plate) TA1535 TA100 (TA1535 TA1538 TA98 (TA1538 percentage of hotspot mutations. Both the uvrB plasmids evaluated in hisG46 with pKM101) with pKM101) deletion mutation and pKM101 were required for Methyl methanesulfonate 570 5 3244 0 5 the production of complex mutations that involved Table 6 summarizes the results obtained when 19 Ethyl methanesulfonate 5000 220 406 2 13 4-Nitroquinoline oxide 0.5 118 7640 339 641 misincorporation and slippage at the hisD3052 frame- additional plasmids were evaluated for a mutator effect N-Methyl-N0-nitro-N-nitrosoguanidine 2 1511 18701 0 22 shift allele at the TGA stop codon. Fowler [75] is and for their effects on UV-survival and UV-induced Furylfuramide (AF-2) 0.02 0 1674 0 169 currently using plasmid pKM101 in an undergraduate mutagenesis in LT2 hisG46. The following 11 plasmids Niridazole 0.2 3 1636 180 468 program at San Jose Sate University, San Jose, CA, enhanced UV-survival and UV-induced mutagenesis and Benzyl chloride 2000 12 230 0 20 entitled Research by Undergraduates using Molecular exhibited the mutator effect to about the same extent as 2-Nitrosofluorene 0.5 0 462 3936 3841 a Biology Applications (RUMBA). The program, plasmid R46: R45, R48, R205, R6, N3, R447a and R205 Aflatoxin B1 0.1 0 2260 80 1940 Sterigmatocystina 0.1 2 282 4 144 funded by the National Science Foundation, is (Datta plasmid), R3916CPH, R9095CPH and Benzopyrenea 5 7 2398 196 685 attempting to characterize the enhanced survival that R7842CPH (this last plasmid was not tested for 2-Aminoanthracenea 10 333 8835 7725 6801 pKM101 provides to starving E. coli cells and to enhancement of UV-mutagenesis). Six of the plasmids Diethyl sulfate 5000 14762 2123 0 9 determine how it may be related to the increase in (R-Peru, R-South Africa, R-Singapore, R3729CPH, a Activated by Aroclor-induced rat liver S-9. starvation mutagenesis. R11576CPH and R11687CPH) did not exhibit any of

230 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 231 162 K. Mortelmans / Mutation Research 612 (2006) 151–164 K. Mortelmans / Mutation Research 612 (2006) 151–164 163

Table 6 [2] K. Mortelmans, E. Zeiger, The Salmonella/microsome muta- [21] J. McCann, N.E. Spingarn, J. Kobori, B.N. Ames, Detection of [44] S. Howarth, Increase in frequency of ultraviolet-induced muta- Variation among R plasmids in effects on the spontaneous mutation genicity assay, Mutat. Res. 455 (2000) 29–60. carcinogens as mutagens: bacterial tester strains with R factor tion brought about by the colicin factor ColI in Salmonella frequency and on UV-induced killing and mutagenesis [3] E. Zeiger, Carcinogenicity of mutagens: predictive capability of plasmids, Proc. Natl. Acad. Sci. U.S.A. 72 (1975) 979–983. typhimurium, Mutat. Res. 3 (1966) 129–134. the Salmonella mutagenesis assay for rodent carcinogenicity, [22] R.P. Novick, Plasmid incompatibility, Microbiol. Rev. 51 (1987) [45] D.G. MacPhee, Salmonella tyhimurium hisG46 (R-Utrecht): R plasmid in Mutator UV-protective UV-mutagenesis Cancer Res. 47 (1987) 1287–1296. 381–395. possible use in screening mutagens and carcinogens, Appl. hisG46 effect effect effect [4] E. Zeiger, J.K. Haseman, M.D. Shelby, B.H. Margolin, R.W. [23] S. Mitsuhashi, Epidemiology and genetics of R factors, Ann. N. Microbiol. 26 (1973) 1004–1005. R-Enfield (R45) Yes Yes Yes Tennant, Evaluation of four in vitro genetic toxicity tests for Y. Acad. Sci. 182 (1971) 141. [46] W.T. Drabble, B.A.D. Stocker, R (transmissible drug-resistance) R-Bradford (R48) Yes Yes Yes predicting rodent carcinogenicity: confirmation of earlier results [24] M. Syvanen, C.I. Kado (Eds.), Horizontal Gene Transfer, second factors in Salmonella typhimurium: pattern of transduction by R-Utrecht (R205)a Yes Yes Yes with 41 additional chemicals, Environ. Mol. Mutagen. 16 (Suppl. ed., Academic Press, London, UK, 2002. phage P22 and ultraviolet-protection effect, J. Gen. Microbiol. R-Peru No No No 18) (1990) 1–14. [25] P. Fredericq, Colicins, Ann. Rev. Microbiol. 11 (1957) 7–22. 53 (1968) 109–123. R-South Africa No No No [5] D. Maron, B.N. Ames, Revised methods for the Salmonella [26] T. Watanabe, H. Nishida, C. Ogata, T. Arai, S. Sato, Episome- [47] N. Datta, Epidemiology and classification of plasmids, in: D. R-Singapore No No No mutagenicity test, Mutat. Res. 113 (1983) 173–215. mediated transfer of drug resistance to Enterobacteriacea. VII. Schlessinger (Ed.), Microbiology, American Society for Micro- R-Munich (R6) Yes Yes Yes [6] D.E. Levin, E. Yamasaki, B.N. Ames, A new Salmonella tester Two types of naturally occurring R factors, J. Bacteriol. 88 biology, Washington, DC, 1974, pp. 9–15. R6-Tc No Yes Yes strain for the detection of frameshift mutagens: a run of cytosines (1964) 716–726. [48] B.N. Ames, The detection of chemical mutagens with enteric N3 Yes Yes Yes as a mutational hot-spot, Mutat. Res. 94 (1982) 315–330. [27] E. Meynell, N. Datta, The relation of resistance transfer factors bacteria, in: A. Hollaender (Ed.), Chemical Mutagens, Princi- R447a Yes Yes Yes [7] D.E. Levin, M.C. Hollstein, M.F. Christman, E.A. Schwiers, to the F-factor (sex-factor) of Escherichia coli K-12, Genet. Res. ples and Methods for their Detection, vol. 1, Plenum, New York, R205a Yes Yes Yes B.N. Ames, A new Salmonella tester strain (TA102) with A:T 7 (1966) 134–140. 1971 , pp. 267–282. R269 No Yes Yes base pairs at the site of mutation detects oxidative mutagens, [28] P. Kontomichalou, M. Mitani, R.C. Clowes, Circular R-factor [49] K. Mortelmans, Effect of R plasmids on Salmonella typhimur- R390 No Yes Yes Proc. Natl. Acad. Sci. U.S.A. 79 (1982) 7445–7449. molecules controlling penicillinase synthesis, replicating in ium: UV-killing, UV-mutagenesis and spontaneous mutability, R3916CPH Yes Yes Yes [8] B.A.D. Stocker, N.D. Zinder, J. Lederberg, Transduction of Escherichia coli under either relaxed or stringent control, J. Ph.D. Thesis, Stanford University, 1975. R9095CPH Yes Yes Yes flagella characteristics in Salmonella, J. Gen. Microbiol. 9 Bacteriol. 104 (1970) 34–44. [50] K. Mortelmans, B.A.D. Stocker, Segregation of the mutator R3729CPH No No No (1953) 410–433. [29] R. Rownds, Replication of a bacterial episome under relaxed property of plasmid R46 from its ultraviolet protection property, R11576CPH No No No [9] B.A.D. Stocker, Abortive transduction of motility in Salmonella; control, J. Mol. Biol. 44 (1969) 387–402. Mol. Gen. Genet. 167 (1979) 317–327. R7842CPH Yes Yes Not tested a non-replicated gene transmitted through many generations to a [30] R.P. Novick, D. Bouanchaud, Extrachromosomal nature of drug [51] D.R. McCalla, D. Voutsinos, On the mutagenicity of nitrofurans, R11687CPH No No Not tested single descendant, J. Gen. Microbiol. 15 (1956) 575–598. resistance in Staphylococcus aureus, Ann. N. Y. Acad. Sci. 182 Mutat. Res. 26 (1974) 3–16.

a [10] W. Hayes, The Genetics of Bacteria and their Viruses, second (1971) 279–294. [52] S.N. Cohen, A.C.Y. Chang, H.W. Boyer, R.B. Helling, Con- Plasmid R205 was available from two different sources [30,31]. ed., John Wiley and Sons Inc., New York, 1968, pp. 96–99, 641– [31] N.S. Willet, The genetics of transmissible plasmids, Ann. Rev. struction of biologically functional bacterial plasmids in vitro, 646. Genet. 6 (1972) 257–268. Proc. Natl. Acad. Sci. U.S.A. 70 (1973) 3240–3244. the three properties evaluated, although R3729CPH was [11] R.J. Roantree, T. Kuo, D.G. MacPhee, B.A.D. Stocker, The [32] D.T. Kingsbury, D.R. Helinski, DNA polymerase as a requirement [53] P.J. Langer, W.G. Shanabruch, G.C. Walker, Functional not tested for its ability to enhance UV-mutagenesis. effect of various rough lesions in Salmonella typhimurium upon for the maintenance of the bacterial plasmid colicinogenic factor organization of plasmid pKM101, J. Bacteriol. 145 (1981) Other plasmids (R6-Tc, R269 and R390) enhanced UV- sensitivity to penicillins, Clin. Res. 17 (1969) 157. E1, Biochem. Biophys. Res. Commun. 41 (1971) 1538–1544. 1310–1316. survival and UV-induced mutagenesis, but did not exhibit [12] R.J. Roantree, T. Kuo, D.G. MacPhee, The effect of defined [33] T. Watanabe, Infective heredity of multiple drug resistance in [54] T. Kato, Y.Shinoura, Isolation and characterization of mutants of lipopolysaccharide core defects upon antibiotic resistances of bacteria, Bacteriol. Rev. 27 (1963) 87–115. Escherichia coli deficient in induction of mutations by ultravio- the spontaneous mutator effect. This result suggests that Salmonella typhimurium, J. Gen. Microbiol. 103 (1977) 223–234. [34] S. Mitsuhashi, Transmissible drug-resistance factor R, Gunma J. let light, Mol. Gen. Genet. 156 (1977) 121–131. at least in these plasmids the mutator effect is determined [13] R.J. Roantree, T. Kuo, D.G. MacPhee, B.A.D. Stocker, Effect of Med. Sci. 13 (1965) 169. [55] K.L. Perry, G.C. Walker, Identification of plasmid (pKM101)- by a gene or genes other than those responsible for various rough lesions in Salmonella typhimurium upon sensitiv- [35] T. Akiba, K. Kayama, Y.Ishiki, S. Kemura, T. Fukushima, On the coded proteins involved in mutagenesis and UV resistance, enhanced UV-survival and UV-induced mutagenesis. ity to antibiotics, Bacteriol. Proc. (1969) 79. mechanism of the development of multiple drug-resistant clones Nature 300 (1982) 278–281. [14] B.N. Ames, F.D. Lee, W.E. Durston, An improved bacterial test of Shigella, Jpn. J. Microbiol. 4 (1960) 219–227. [56] G.C. Walker, P.P. Dobson, Mutagenesis and repair deficiencies of system for the detection and classification of mutagens and [36] C. Ha¨nni, J. Meyer, S. Iida, W. Arber, Occurrence and properties Escherichia coli umuC mutants are suppressed by the plasmid 13. Concluding remarks carcinogens, Proc. Natl. Acad. Sci. U.S.A. 70 (1973) 782–786. of composite transposon Tn2672: evolution of multiple drug pKM101, Mol. Gen. Genet. 172 (1979) 17–24. [15] S.K. Hoiseth, B.A.D. Stocker, Aromatic-dependent Salmonella resistance transposons, J. Bacteriol. 150 (1982) 1266–1273. [57] G.C. Walker, Mutagenesis and inducible responses to deoxyr- The emergence of the bacterial mutagenesis-enhan- typhimurium are non-virulent and effective as live vaccine, [37] R.M. Hall, C.M. Collins, Antibiotic resistance in Gram-negative ibonmucleic acid damage in Escherichia coli, Microbiol. Rev. 48 cing plasmid pKM101 from the Stocker laboratory is a Nature 291 (1981) 238–239. bacteria: the role of gene cassettes and integrons, Drug Resist. (1984) 60–93. [16] B.P. Smith, G.W. Dilling, L. Da Roden, B.A.D. Stocker, Vacci- Updat. 1 (1998) 109–119. [58] A. Gabb, C.J. Kenyon, G.C. Walker, Inducibility of a gene tribute to Bruce’s encyclopedic knowledge of and insight nation of calves with orally administered aromatic-dependent [38] M.G. Kidwell, D.R. Lisch, Transposable elements as sources of product required for UV and chemical mutagenesis, Proc. Natl. into the genetics of S. typhimurium and plasmid behavior. Salmonella dublin, Am. J. Vet. Res. 54 (1993) 1249–1255. genomic variation, in: L. Nancy, R.C. Craig, M. Gellert, A.M. Acad. Sci. U.S.A. 78 (1981) 5749–5753. [17] B.A.D. Stocker, Aromatic-dependent Salmonella as anti-bacter- Lambowitz (Eds.), Mobile DNA II, ASM Press, Washington, [59] W.G. Shanabruch, G.C. Walter, Localization of the plasmid + + Acknowledgments ial vaccines and as presenters of heterologous antigens or of DC, 2002, pp. 59–92. (pKM101) gene(s) involved in recA lexA -dependent mutagen- DNA encoding them, J. Biotechnol. 83 (2000) 45–50. [39] G. Lebek, Uber die Entstehung mehrfach resistenter Salmonel- esis, Mol. Gen. Genet. 179 (1980) 289–297. [18] K. Mortelmans, M. Cox, Evidence that inhibitor(s) are formed len. Ein experimenteller Beitrag, Zentbl. Bakt. Parasit Kde (Abt I [60] S.J. Elledge, G.C. Walker, The muc genes of pKM101 I would like to thank Lynn Johannesen for editing the which may interfere with the growth of revertant colonies in the Orig) 188 (1963) 494. are induced byDNA damage, J. Bacteriol. 155 (1983) manuscript and Scott Bramwell for producing Fig. 2.I Ames Salmonella and the E. coli tryptophan reverse mutation [40] E.S. Anderson, N. Datta, Resistance to penicillins and its transfer 1306–1315. am very grateful to the reviewers for their diligent assays when strictly anaerobic conditions are used, Environ. in Enterobacteriaceae, Lancet 20 (1965) 407–409. [61] J. Kronish, G.C. Walker, The effects of the ultraviolet protecting review of the paper and for their most helpful Mol. Mutagen. 20 (1992) 44–52. [41] C.E. Davis, J. Arrandan, The evolution of R factors. 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[64] G.C. Walker, Plasmid (pKM101) mediated enhancement of [73] D.F. Fix, P.A. Burns, B.W. Glickman, DNA sequence analysis of repair and mutagenesis: dependence on chromosomal genes in spontaneous mutations in a polA1 strain of Echerichia coli Mutation Research 635 (2007) 79–80 Escherichia coli, Mol. Gen. Genet. 152 (1977) 93–103. indicates sequence specific effects, Mol. Gen. Genet. 207 [65] G.C. Walker, Isolation and characterization of mutants of the (1987) 267–272. plasmid pKM101 deficient in their ability to enhance mutagen- [74] D.M. DeMarini, M.L. Shelton, A. Abu-Shakra, A. Szakmary, ☆ esis and repair, J. Bacteriol. 133 (1978) 1203–1211. J.G. Levine, Spectra of spontaneous frameshift mutations at the Mutation Research: The origin [66] P.A. Todd, B.W. Glickman, UV protection and mutagenesis in hisD3052 allele of Salmonella typhimurium in four DNA repair a b uvrD, uvrE, and recL strains of Escherichia coli carrying the backgrounds, Genetics 149 (1998) 17–36. A.T. Natarajan * Mutation Research 635 (2007) 79–80 pKM101 plasmid, Mutat. Res. 62 (1979) 451–457. [75] R.G. Fowler, http://www2.sjsu.edu/depts/Biology/RUMBA/ www.elsevier.com/locate/reviewsmr Community address: www.elsevier.com/locate/mutres [67] K. Mortelmans, L. Dousman, Mutagenesis and plasmids, in: F. FowlerRUMBA.htm. a University of Tuscia, Viterbo, Italy deSerres (Ed.), Chemical Mutagens, Principles and Methods for [76] D.R. Byrd, J.K. Sampson, H.M. Ragonese, S.W. Matson, Struc- Reflections in Mutation Research their detection, Plenum Publishing Company, vol. 10, NY, 1986, ture-function analysis of Escherichia coli DNA helicase I reveals b Leiden University Medical Centre,Mutation Leiden, The Research Netherlands: The origin§ pp. 469–507. non-overlapping transesterase and helicase domains, J. Biol. Accepted 28 December 2006 [68] R.G. Fowler, L.D. McGinty, K.E. Mortelmans, Spontaneous Chem. 277 (2002) 42645–42653. a,b, mutational specificity of drug resistance plasmid pKM101 in [77] R.F. Pohlman, H.D. Gentii, S.C. Winans, Common ancestry Available online 25 March 2007 A.T. Natarajan * Escherichia coli, J. Bacteriol. 140 (1979) 929–937. between IncN conjugal transfer genes and macromolecular a University of Tuscia, Viterbo, Italy [69] R.G. Fowler, L.D. McGinty, K.E. Mortelmans, Mutational spe- export systems of plant ananimal pathogens, Mol. Microbiol. b Leiden University Medical Centre, Leiden, The Netherlands cificity of ultraviolet light in Escherichia coli with and without 14 (1994) 655–668. the R-plasmid pKM101, Genetics 99 (1981) 25–40. [78] C. Baron, M. Llosa, S. Zhou, P.C. Zambryski, VirB1 a compo- Accepted 28 December 2006 [70] D.L. Spanggord, The effects of R plasmids R269, R390 and Rsa nent of the t-complex transfer machinery of Agrobacterium Available online 25 March 2007 on UV-sensitivity, spontaneous and UV-induced mutagenesis in tumefaciens, is processed to a C-terminal secreted protein, Escherichia coli, M.A. Thesis, San Jose State University, San Virb1*, J. Bacteriol. 179 (1997) 1203–1210. Jose, CA, 1982. [79] C. Ho¨ppner, Z. Liu, N. Domke, A.N. Binns, C. Baron, VirB1 Abstract [71] E. Eisenstadt, J.K. Miller, L.-S. Kahng, W.M. Barnes, Influence orthologs from Brucella suis and pKM101 complement defects of uvrB and pKM101 on the spectrum of spontaneous, UV- and of the lytic transglycosylase required for efficient type IV This brief commentary reflects on the founding of the journal Mutation Research. It includes a photograph of the participants in g-ray-induced base substitutions that revert hisG46 in Salmo- secretion from Agrobacterium tumefaciens, J. Bacteriol. 186 the scientific conference held in 1962 at the University of Leiden, The Netherlands, at which Professor F.H. Sobels proposed the nella typhimurium, Mutat. Res. 210 (1989) 113–125. (2004) 1415–1422. establishment of the Journal. [72] A.J. Gordon, C. Bernelot-Moens, B.W. Glickman, Spontaneous [80] K. Mortelmans, B.A.D. Stocker, Ultraviolet light protection, # 2007 Elsevier B.V. All rights reserved. mutagenesis in Escherichia coli harbouring plasmid pKM101: enhancement of ultraviolet light mutagenesis and mutator effect DNA sequence analysis of forward lacI mutations, Mutagenesis of plasmid R46 in Salmonella typhimurium, J. Bacteriol. 128 Keywords: Mutation Research; F.H. Sobels; History of science; DNA repair; University of Leiden 8 (1993) 133–139. (1976) 271–282.

It is 42 years since the journal Mutation Research as well as three great ladies of mutation research— made its first appearance on the international stage, and Charlotte Auerbach, Evelyn Witkin and Tikvah Alper. It it is worthwhile to reflect upon the origin of this was during this meeting that Frits Sobels first proposed important journal. Following the Second International starting a new journal to review and publish new work in Congress of Radiation Research, which was held at any and all fields of mutation research, including both Harrogate, Yorkshire, in the UK, Prof. Frederik H. pure and applied aspects; Professor Sobels’ proposal (Frits) Sobels organized a symposium, the first ever on was unanimously endorsed. DNA repair, entitled ‘‘Repair of genetic radiation Thus was the journal Mutation Research born. The damage and differential radiosensitivity in germ cells,’’ first-ever issue appeared in January of 1964, under the at the University of Leiden, The Netherlands, in August guidance of an Editorial Board that included several of 1962. As a young scientist from India, I was greatly the distinguished scientists who had taken part in the privileged to be able to attend this symposium. 1962 Leiden symposium. A photograph taken at that Numbered among the participants were several of the symposium accompanies this article; in it one can most eminent pioneers in the field, including, for recognize, amongst others, Hermann Muller, Lee example, the 1946 Nobel Prize winner Hermann Muller, Russell, Bentley Glass, Yataro Tazima, Charlotte Auerbach, Tikvah Alper and, of course, Frits Sobels himself. Charlotte Auerbach, whose work included the § This article is part of the Reflections in Mutation Research series. first unequivocal demonstration of chemical mutagen- To suggest topics and authors for Reflections, readers should contact esis, is fourth from the right in the first row of those the series editors, G.R. Hoffmann ([email protected]) or standing. Beside her is Tikvah Alper, fifth from the D.G. MacPhee ([email protected]). right. Frits Sobels is standing immediately behind and * Correspondence address: Burgemeester Warnaarkade 47, 2349 AZ between them. I am one of the young scientists who had Hazerswoude Dorp, The Netherlands. Tel.: +31 172 586757; fax: +31 172 586762. the pleasure of attending this exceptional conference E-mail address: [email protected]. (standing on the far right in the third row).

1383-5742/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2006.12.001

234 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 235 80 A.T. Natarajan / Mutation Research 635 (2007) 79–80 Almost everyone who has ever worked in any field other aspect of what was soon to become the very Mutation Research 635 (2007) 1–16 of mutation research will readily acknowledge that it large and multifaceted field of scientific endeavor that was Prof. Sobels’ 1962 initiative that led to Mutation we all recognize today. Currently there are three Research becoming the first international journal to sections in regular production: Fundamental and specialize in the various fields that were considered of Molecular Mechanisms of Mutagenesis (green cov- Personal reflections on the life and legacy of most relevance at the time; at first the Editorial Board ers), Genetic Toxicology and Environmental Muta- Mutation Research☆ 635 (2007) 1–16 produced the very familiar single-section green multi- genesis (peach and brown covers), and Reviews in Alexander Hollaender www.elsevier.com/locate/reviewsmr paper issue on a monthly basis, but this later Mutation Research (purple covers), as well as the a 1 Community1 address: www.elsevier.com/locate/mutres increased to a point where up to six separate sections now-independent journal DNA Repair (red covers) Mary Esther Gaulden , JohnReflections Jagger *, in Virginia Mutation P. Research White were being printed, each with its own schedule. These that had its origins as part of Mutation Research. § six sections were intended to specialize in one or Long may they continue. aPersonal Radiology Department, reflections University on of the Texas life Southwestern and legacy Medical Center of Alexander at Dallas, 5323 Harry Hollaender Hines Blvd., Dallas, TX 75390-9071,Mary United Esther States Gaulden a, John Jagger 1,*, Virginia P. White 1 Accepted 6 October 2006 a Radiology Department, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Available online 29 November 2006 Dallas, TX 75390-9071, United States Accepted 6 October 2006 Available online 29 November 2006

Abstract Mary Esther Gaulden presents a personal summary of the activities of Alexander Hollaender, from his days at the National Institutes of Health to his becoming Director of the Biology Division of the Oak Ridge National Laboratory in 1947. This appealing story deals with many of her reactions to his personality and organizational style. It reflects the atmosphere of science in those days, and her enthusiasm in this vibrant milieu. Next is a brief account by John Jagger of his first meeting with Dr. Hollaender, arrival in Oak Ridge in April 1956, and wedding to Mary Esther six months later at the house of the Hollaenders in Oak Ridge. The third section is an account by Virginia P. White of how she came to Oak Ridge in 1955 and became Dr. Hollaender’s Laboratory Administrator. She gives a personal account of the many facets of his managerial style, as well as of the personality of his wife, Henrietta. She also describes one of Hollaender’s many avocations, the collection of fossils on Sunday morning hikes in the Cumberland Mountains, accompanied by lab and visiting personnel, and finally comments on the annual research conferences in Gatlinburg TN, for which Hollaender and the lab became very well known, with some closing vignettes on his leadership style. # 2006 Elsevier B.V. All rights reserved.

Keywords: Alexander Hollaender; Mutagenesis; History of science; Radiation biology; Environmental Mutagen Society; Oak Ridge National Laboratory

1. Reflections of Mary Esther Gaulden 1986 in Washington, DC, 13 days shy of his 88th Conference participants, University of Leiden, The Netherlands, August 1962. birthday. The dates that he told me he considered 1.1. Introduction important milestones in his life were: 1921, when he came from Germany to the United States, entering Alexander Hollaender was born on 19 December through the port of Mobile, Alabama; 1925, when he 1898, in Samter, Germany, and died on 6 December married Henrietta Wahlert of St. Louis, Missouri; 1929, when he obtained an A.B. degree in Chemistry at the University of Wisconsin, followed in 1931 by a Ph.D. in § This article is part of the Reflections in Mutation Research series. physical chemistry, including ultraviolet radiation To suggest topics and authors for Reflections, readers should contact effects, under Farrington Daniels; and last but not the series editors, G.R. Hoffmann ([email protected]) or D.G. MacPhee ([email protected]). least, 1946 when he established and was named Director * Corresponding author at: 110 Barnett Blvd. #105, Highland of the Biology Division of the Oak Ridge National Village, TX 75077, United States. Tel.: +1 972 317 0853. Laboratory, in what one dismayed, visiting foreign E-mail address: [email protected] (J. Jagger). 1 scientist called ‘‘the God-forsaken, red clay hills of rural Editors’ note: When Mary Esther Gaulden’s frail health became America.’’ such that she was unable to complete her manuscript in the way she had planned, John Jagger and Virginia White graciously agreed to add Several obituaries and remembrances published some of their own reflections on later events in the Hollaender story. following Dr. Hollaender’s death [1–5] provide some

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236 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 237 2 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 3 information about his activities between 1921 and 1925, on war-rationed gas) to attend a meeting of the Genetics the margins, was impressive. His interpretation was that moment’s hesitation. He said that he had spent the which he was always reluctant to discuss (‘‘not Club of the Washington, DC area. Robert Cook, long- chiasmata result from, rather than cause, crossing-over. previous summer in Hollaender’s lab at NIH, working important’’), and therefore are not generally known. time editor of the Journal of Heredity, led the group, I was therefore thrilled to be in Husted’s graduate on the effects of various wavelengths of ultraviolet light This is not surprising because Alex rarely looked back; calling a meeting whenever a visiting geneticist came class and was all fired up about genes and chromo- on cell division [7,8], and that Hollaender had asked he was always looking to the future with what seemed to near or to town. I should point out that this affair was somes, an enthusiasm that probably caught Alex’s him to return for another summer. I said I had met be binocular, if not microscopic, vision. These held in the basement of the Cosmos Club, because at attention. I asked Alex if he were aware of the papers of Hollaender once but did not know him. He called publications also provide some opinions on the ways that time, unbeknownst to me, women were not only Hermann J. Muller and Lewis Stadler on radiation- Hollaender who readily agreed to get a salaried position in which he organized and administered the Biology ineligible for membership in the Cosmos Club but were induced gene mutations, as well as previous work on for me. I never learned whether he had remembered me Division, as well as some amusing ‘‘Hollaender not allowed in the main rooms of the building! This was radiation-induced chromosome aberrations. His answer from the Cosmos Club ‘‘interview’’ or if he was just anecdotes’’ that reveal aspects of his interactions with one factor that later drove me to help form the National was ‘‘Yes.’’ I had never heard of Hollaender, so I did not accepting a ‘‘pig in a poke’’ in order to get Carlson back people, and which are still exchanged with delight when Organization for Women (NOW) to obtain equal rights know I was talking to a pioneer of radiation biology! to NIH. two or more Biology Division ‘‘Alumni’’ get together. for women. On the way back to Charlottesville that night, the So, in late May 1946, we headed for NIH in The assignment for this article was to focus on my At the dinner served prior to the lecture, I found other students confessed that they had seen to it that I sat Bethesda. By that time I had gotten to know Gordon and personal views of Alexander Hollaender, acquired over myself seated next to an impressive-looking gentleman next to Hollaender, because they had had trouble at his family quite well (lovely wife and two small, a period of 44 years. He was a unique, goals-driven, who started the conversation with ‘‘Vair yo fom?’’ previous meetings making conversation with him. They delightful daughters). They invited me to stay with them prolific scientist (his C.V. in the 1986 American Men Being fresh from a small college (Winthrop) in a small wanted to know how I got him to talk and to look so in a grand, big old house that Henrietta, Hollaender’s and Women of Science included authorship of ‘‘over 300 South Carolina town (Rock Hill), I was not yet dry interested. At the time, I literally did not have a clue. I wife, had found for them, which was about a mile from publications,’’ for a number of which he served as behind the ears. On top of that, I had previously known was a bit piqued at their maneuver, but later I was very NIH and could be rented for the 3 months we expected editor). He was endowed with transcendent determina- only one foreigner, whose English had only a trace of grateful for the introduction-cum-interview. to be there. tion, which was one factor that enabled him to attain accent. After struggling for a bit, I said, ‘‘I’m sorry, but By fall 1945, I had earned an M.S. degree (required prominence by establishing, literally from scratch, a I’m having difficulty understanding your foreign for entry to the Genetics Ph.D. Program) and had 1.3. Alexander Hollaender at NIH large, world-renowned laboratory in a relatively new accent’’—in other words, ‘‘please slow down.’’ Quick completed all requirements for a Ph.D. except a discipline called ‘‘Radiation Biology.’’ In essence, he as lightning, he shot back, ‘‘Und I kand unerstan yo dissertation. Research in the UVA Genetics Program On arriving at Hollaender’s lab, we learned that he took advantage of the increased interest in radiation- suddun akcen eider.’’ With that pronouncement, he took was exclusively oriented to plants, but plant cells were had also arranged for Max Zelle (microbial geneticist) induced biologic, especially genetic, effects that was over the conversation with questions: what was my not then easy to grow in culture. I wanted to work with and Carl Swanson (cytologist) to come to his lab for the generated by the two atomic bombs dropped on Japan in background, why was I in graduate school, why did I living animal cells, whose chromosomes and mitosis summer. They both were at nearby Fort Detrick, August 1945. In July of that year Vannevar Bush had choose genetics as a major, what classes was I taking, could be viewed with high-power microscopy. In Maryland, where they had been involved in ‘‘weapons published a book, Science—the Endless Frontier, what were my goals? He seemed to have no difficulty discussing this with Dr. White, he said he knew a research’’ during World War II, and were now searching calling for increased support for research in the basic understanding my answers. The only information he scientist who was doing that kind of work, Professor J. for academic positions. In addition, Victor Hamburger sciences. This gave a boost to the postwar surge in funds volunteered about himself was: ‘‘I’m a radiation Gordon Carlson at the University of Alabama in (developmental biologist) and his Chinese student- for scientific research, one element being the establish- geneticist.’’ But the ice had been broken and, from Tuscaloosa. He said I could do my research and write assistant, from Washington University in St. Louis, soon ment of the National Science Foundation (NSF) by that evening on, we never had any trouble understanding the dissertation with Carlson. If I successfully defended joined the group. Together with Alex’s lab assistant, the Congress in 1950. In 1944 Congress had passed the each other. There were discussions about differences of it before a dissertation committee, UVAwould grant me group consisted of eight people. Also, 2 days a week, an Public Health Service Act, thereby authorizing NIH to opinion but no more encounters! the Ph.D. degree. Dr. White contacted Carlson and ophthalmology professor at Johns Hopkins School of award grants to university investigators doing medical One item we discussed that evening was a graduate learned that he had a Research Assistant position open Medicine came over to do some ultraviolet experiments research. The Atomic Energy Commission (AEC), course I was taking called Cytology, a term then used to and agreed to supervise my dissertation research, the with Alex. formally established in 1947, became the primary cover chromosomes and cell biology. The Professor, subject to be decided later. So, in November 1945, I Being in close quarters, everybody quickly resorted source of funds for the Biology Division and for Ladley Husted, had recently joined the UVAfaculty after boarded a train for Alabama. Little did I know that this to using first names, and called him ‘‘Alex.’’ I soon extramural radiation biology research. completing three consecutive 1-year postdoctorals with move would lead me back to Hollaender at NIH in found myself addressing him that way. With character- prominent geneticists-cytologists: Lewis J. Stadler at the Washington! istic disregard for pronunciation, he called me 1.2. Early acquaintance with Alexander University of Missouri, Karl Sax at Harvard, and C.D. The project for which Gordon Carlson wanted an ‘‘MayEsta’’ from then on. Later in Oak Ridge he Hollaender Darlington at the John Innes Horticultural Institution in assistant dealt with determining the progression of seemed delighted to have everybody call him ‘‘Alex,’’ London. I had corresponded with Dr. Husted when I was a effects of varying concentrations of colchicine on the but some, especially the support staff, could never bring To begin at the beginning: I first encountered sophomore in an undergraduate genetics class, at the attachment of chromosomes to the mitotic spindle in themselves to stop using ‘‘Dr. Hollaender.’’ Alexander Hollaender in December 1942, at the recommendation of my Winthrop College professor, Dr. situ in living grasshopper neuroblasts [6]. I was so At NIH I had my first exposure to one dominant Cosmos Club, an exclusive private club near the Margaret Hess. She said she was sure Dr. Husted could intrigued by what one could see in these large living aspect of Alex’s lifelong philosophy: THINK BIG. Nine National Academy of Sciences in Washington, DC. I answer my questions about the origin of meiotic cells, 30 mm in diameter with very large chromosomes, researchers were squeezed into three rooms: a typical was a first-year graduate student in the Genetics chromosome chiasmata and their role in crossing-over. that I happily worked 7-day weeks. Within a month, lab with benches, designed to accommodate at most Program of the University of Virginia (UVA), Charlot- Not having access to the current genetics literature, I was Carlson evidently decided that I was suited to the job four people; the second room (about 16 25 ft.) tesville. The Program Head, Dr. Orland E. White (a not aware of the fact that this was a hotly debated topic at and asked if I would like to go to NIH in Bethesda MD contained a monochromator, a small culture room, graduate of Edward M. East’s genetics section at that time. Dr. Husted’s five-page lucid explanation, (‘‘right next to Washington, DC’’) for the next summer. and Alex’s large desk. The third room (about 15 ft.2), Harvard), had wedged 5 students into his car (running accompanied by small instructive diagrams penciled in Always ready for another adventure, I agreed without a across the hall, was allotted to Gordon and me. It

238 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 239 4 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 5 contained a desk for him, and a big table, which we used there. He had the reputation of being contentious and any concern that I would repeat it to others. On and the stem wobbled a bit, unpredictably. Fortunately I for experiments. Gordon requested a desk for me, but very concerned about priority of discovery. I always occasion, I felt a bit like a sounding board, but I had experienced the same pedal when in 1938 I had the only available space, for the small one that was found him most gentlemanly, and he insisted on recognized that he was either ‘‘blowing off steam’’ or driven a Ford of even earlier vintage, which belonged to delivered, was right next to Alex’s desk. addressing me as ‘‘Miss Gaulden.’’ I especially asking for my opinion, which I gave honestly without a friend. Once one conquered that accelerator, driving With all of those people around, my desk was remember one incident at a Genetics Society meeting offense. It was for me a piecemeal course in the politics the car was fun. Alex never complained about it, but he obviously not going to be a place for quiet thinking! Not in New England. I was looking over the program for the of science, which has stood me in good stead. rarely drove it for long distances. The car was on the only were conversations going on around me all the day, while walking along a path, when all of a sudden I Listening to him was indeed something of an movers’ truck when they came to Oak Ridge in 1947. It time, but also in the early afternoon Alex spent a lot of heard someone running toward me. He was waving at education in itself. I learned some of his characteristics should be noted that he kept it until he retired in 1966. I time on the phone. He seemed to assume that everyone me with both arms in the air and yelling ‘‘Ms. Gaulden, I that have served me well, e.g., don’t take ‘‘no’’ for an think that as far as he was concerned it was what I call an he called by long-distance was almost stone deaf, and was delighted with your paper on the nucleolus in the answer. He never used the word unless someone was ideal car: it started, ran, and was paid for. He did not also that no one in the room could hear what he said. He Proceedings of the National Academy of Sciences, disagreeing with him or wanted to change his mind. waste money on cars; he spent it on paintings and thought nothing of having long, loud conversations with which showed that destruction of the nucleolus with a ‘‘Discouragement’’ was not a word he acknowledged. I sculpture. people all over the country, as well as in foreign microbeam of ultraviolet radiation (UV) stopped cell think his brain instantly translated it into ‘‘challenge,’’ The Hollaenders’ ‘‘house in the country’’ was, for countries. Having come of age during the Great division [10]. He said, ‘‘I first pointed out the function which he immediately accepted. For instance, on one me and friend Peg Keister, a delightful vacation away Depression, I thought long distance was used only of the nucleolus in ___’’ I have forgotten the date, but occasion, when he had been calling several people over from the single rooms we rented near NIH. It had been when there was a death in the family, so I was amazed when I got back to Oak Ridge, I looked it up. He was a period of days, trying unsuccessfully to negotiate for an old, small, two-story five-room farmhouse, which that anyone had enough money to run up what would right: he had speculated on its function! Alex had a the use of a building, he finally yelled, ‘‘Vell, fi kannd yo Henrietta had had remodeled, with hardwood floors surely be an enormous phone bill. When I gently similar habit: it seemed that, no matter what we had bill ah fence roun us?’’ That made it a done deal! He got replacing broad pine planks throughout. All rooms were broached the subject with him, he smiled and said, ‘‘Oh, found that we thought was interesting, he would say, that building, and the adjacent one, both surrounded by small, but were somehow made to look bigger with duh govmen pay foit.’’ Obviously, I was not yet fully ‘‘Ve did dot tventy years ago!’’ It often turned out that a security fence! By adding this to snippets from lovely modern paintings, obviously originals, on all of aware of his clever management style, but was sharp Alex had noted the effect but had not studied it nor previous phone conversations, it was obvious that the the walls, even in the bathrooms. There were lots of enough to figure out that he was making BIG plans for a speculated upon its possible importance. A notable buildings were guarded. That puzzled me, but as it was books all over the house, which I thought was as close to BIG lab, and that he did not care whether I overheard his example of this was his finding that bacteria grew none of my business, I never gave it further thought. In heaven as one could get. Among them were a large conversations or not. haphazardly in a petri dish after UV irradiation if some writing of this incident, I realized that by June of 1946 number about Abraham Lincoln. When I later men- This situation made me aware of what turned out to of the plates were left in the light—so he was careful to Alex had already begun mentally to make his ‘‘big lab’’ tioned this to Alex, he said Lincoln was his pick of be his enduring practice of discussing something he leave them in the dark during incubation! He had seen in Oak Ridge a reality. In other words, I was unwittingly American men of stature. wanted to do with a number of people, including a good photoreactivation, but had not discovered it, as Albert privy to the birth of the Biology Division of the Oak The house was set back from the road on probably an sprinkling of prominent ones, before taking action. It Kelner did in 1949, to wide acclaim. Ridge National Laboratory (ORNL). acre of land—fortunately, since there was a golf course led a few of them to puff up, thinking that they had been To get around the noise problem in Alex’s lab at NIH, A car introduced me to Henrietta (Alex’s wife, whom on the other side of the road. Alex enjoyed, and some very important in a successful venture. In fact, Alex I adopted the routine of coming to the lab a little late in he called ‘‘Hen’’) and also revealed Alex’s frugality in times complained about, the many golf balls he gathered usually knew exactly what he wanted to do before he the morning and leaving later in the evening. This gave his personal life that definitely did not apply to his on his property. There was no way he was going to throw called anybody. He just wanted confirmation, and more me the opportunity at the lab to read, write, and plan the administrative life. One afternoon in June 1947, he them back across the two-lane highway—he gave them to importantly, he wanted to use the prominent ones’ next day’s work in a quieter haven. I would catch a bus interrupted my work to ask if I could drive. On getting golfing friends! At that time, his house and the golf course names to support his arguments, especially when he was for the 2-mile ride to the center of Bethesda for dinner at an affirmative answer, he said Hen was joining him for a were the only non-farm properties in that area. When the having trouble getting the powers-that-be to cough up a Howard Johnson restaurant, where a cohort of young 2-week trip to Europe in July, and they needed someone Hollaenders moved to Oak Ridge, they retained own- money for a project. Most of the time that ploy seemed NIH investigators usually gathered to exchange to ‘‘sit with’’ their house. It was several miles north of ership of the land and house, renting it to NIH personnel. to work. accounts of their lab adventures of the day, encounters NIH ‘‘in the country.’’ There was no bus service north of With time, NIH and the Naval Hospital grew rapidly as Alex had a long list of ‘‘prominent’’ scientist-friends. with their bosses, and plans for the next weekend. None NIH and the Naval Medical Center across the road, so did the area to the north, which eventually became a large Those of us who were in his lab at NIH in the summer of had worked for Alex, but they early informed me that he he said I could use his car to get to and from the lab. community now known as Rockville. Needless to say, the 1946 will always remember when he learned that one was reputed to be the most difficult boss on the campus. They had a ride to the airport on leaving, but I was to Hollaenders were delighted to sell their property, making friend had reached ultimate prominence! It was a quiet I quickly challenged that opinion. pick them up at the Bethesda trolley station on their a considerable profit. This was followed by a notable mid-afternoon with everybody absorbed in work, when, Sitting next to Alex, I got to know him much better. return. I remember that when they got off the trolley, increase in their painting collection, as well as the on answering the phone, Alex immediately jumped up In the late afternoon he would begin to relax, and Alex looked over the car thoroughly. I never asked acquisition of some significant sculptures. out of his chair yelling for us all to ‘‘come here.’’ He had sometimes talk to me. These conversations revolved whether he was checking for any damage I may have learned that his longtime friend, Hermann Muller, had around a variety of subjects from modern art to caused (there was none), or whether he had misgivings 1.4. Founding of the Biology Division at ORNL just been told that he would receive a Nobel Prize for his something that was bothering him, e.g., NIH was not about the car’s surviving a driver who was a novice to its demonstration of radiation-induced gene mutation in giving him enough money for his projects. I never peculiarities. By August of 1946, Alex seemed to have obtained Drosophila [9]. I never again saw Alex move so quickly mentioned to anyone anything that I may have This car (of 1938 vintage) was the one that later in some assurance of, but was not talking about, being the and be so exhilarated. Needless to say, no more work overheard in his phone conversations, but he could Oak Ridge was called either the ‘‘Famous Ford’’ or the Director of the new Biology Division in Oak Ridge. I was accomplished in his lab that day! not help but know that I had heard some of them. With ‘‘Green Ford.’’ Everybody in town knew that car. It was recently learned that Eugene Wigner recruited Hol- Muller was a guest in the Biology Division of Oak time it became obvious that he felt he could discuss a challenge to drive, because the accelerator pedal was laender in 1946 to ‘‘form and head a Biology Division’’ Ridge National Laboratory several times when I was what might be considered sensitive information without about the size of a 50-cent piece, slick as greased glass, at ORNL. Wigner, a Nobelist, was a prominent

240 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 241 6 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 7 chemical engineer and physicist—and a friend of accommodate the first recruits, among whom were Third Class in the evening, because that was where the Americans greatly admired the courage and fortitude of Alex—who came to Oak Ridge during the war to help William (Bill) Arnold, William (Bill) Baker, Waldo action was, in spite of the fact that the deck was not the British during thewar, she pulled a huge chocolate bar develop the atomic bomb and design nuclear reactors Cohn, Alan Conger, Gus Doermann, Norman Giles, enclosed. The North Atlantic was predictably gray and from her pocketbook and offered it to me. I knew it was for making weapon materials. After the war, he stayed Richard (Dick) Kimball, and Seymour Pomper. These misty, if not raining, and always cold; the crew would still rationed, because I—a chocoholic—had been unable in Oak Ridge to help reorganize research facilities for were quickly followed by others, including William put up canvas shields when the sea got very rough, but to buy one. I thanked her but handed it back, explaining peacetime operation, before returning to Princeton. (Bill) Russell and his wife Liane (Lee) Russell, who set the deck was not a pleasant place to sit unless one had that I was going home the next day and could easily get a Alex persuaded Gordon Carlson to resign his position at up their large mouse genetics group in the smaller of the woolen clothes. Henrietta Hollaender was in Second bar in the States, if not on the ship. She insisted that I take the University of Alabama and continue working at NIH two buildings, soon dubbed ‘‘the mouse house.’’ By the Class, and she came down every night of the 10 days it it because she ‘‘so wanted to give an American something in his lab, effective 1 September 1946. He got me some end of that year the Division had about 70 scientists and took to reach Sweden. The bar on our deck resembled to show appreciation for all of the aid’’ we had given them kind of appointment, the title of which I don’t technicians. one in an old, primitive Russian movie, so Henrietta during the war, including ‘‘the sharing by US soldiers of remember, but he described it as a ’’predoctoral.‘‘ I Through the next two decades the Biology Division immediately dubbed it the ‘‘Anna Christie Bar’’ and that their rations with the Brits.’’ I was so moved by her had never before heard the term, but it was fine with me, grew to national and international prominence under name stuck. There was a small, upright piano that the motive that I accepted the candy bar while fighting hard to because I was learning a lot, having fun with interesting Alexander Hollaender’s leadership, growing to a size of stewards rolled out onto the deck every night. Larry hold back tears. work, and loving Washington—my first big city. 600 employees, some 150 of whom were scientists. The Snyder (University of Oklahoma) would play the piano To say that I was ‘‘going home’’ was an under- By early spring of 1947, it was public knowledge that Division established a reputation for pioneering work, as long as we kept a glass of beer on top of it. We statement. The next day I was actually boarding a ship Alex had the Directorship of the Biology Division at not only in radiation biology, but also in such other areas ‘‘younguns’’ (as Henrietta called us) would dance on a to return to Go¨teborg, where I would spend the night ORNL. He had already ‘‘dived ankle deep/head first’’ of biological science as biochemistry, genetics, and rocking deck until the wee hours of morning. before boarding the Gripsholm the following day to join into looking for other opportunities in the Oak Ridge environmental biology. During the day, the Third Class deck was, despite the the geneticists for the return trip to New York, whence I area that would enhance and expand the vision he had weather, an ideal place to get to know people and talk would fly to Tennessee. (The only port in Europe served for the Biology Division. The first inkling I had of this 1.5. International Congress of Genetics (1948) genetics. We students took advantage of it, learned a lot, by the Gripsholm was Go¨teborg.) was when he proposed that Gordon join the faculty of and made some lifetime friends among the older, as well Crossing the North Sea for the fourth time provided the nearby University of Tennessee (UT) in Knoxville Alex wanted me to move to Oak Ridge and set up as fellow-student, geneticists. It was essentially a me with confirmation of that sea’s reputation: we sailed by becoming chairman of the Zoology Department. I Carlson’s group, but I insisted that I had to get my delightful course on many aspects of genetics. It also through a rough storm in the afternoon. Tables, chairs, think Gordon was a bit taken aback by Alex’s astuteness degree first. I quickly became acquainted with his made for a hospitable meeting in Stockholm, in that the pianos, etc., in the cloud-darkened social rooms began in having already ascertained that the current chairman characteristic persistence: every time I saw him, he established geneticists we got to know on the ship to roll around, and sailors rushed in to tie down was retiring on 1 September! Alex had also recom- reminded me that I ‘‘was coming out to work in Oak introduced us to a number of those who had not been on everything that could move. Passengers were told to go mended to the Dean that Gordon be offered the Ridge.’’ Another reason for my reluctance to accept his the ship, and to foreign geneticists from many countries. immediately to an inner room that had no windows and chairmanship. After an interview in Knoxville, Gordon job offer was that I had learned that the first post-war The Congress was a great opportunity for Alex to where nothing was moveable, and stay there until got the job. This maneuver assured Alex that he would meeting of the International Congress of Genetics was flex his ‘‘recruitment’’ muscles, which he did very further notice. It was a bit like being in an old black- have a personal connection with UT. His plan was to to be held in Stockholm, Sweden, in the summer of effectively, lining up not only new researchers, but also and-white movie drama. At dinner that night, the promote academic interest in radiation biology by 1948. The American Genetics Society was offering visitors to give lectures, or to stay on for a couple of Captain announced that we had ‘‘passed through a offering graduate courses at UT, while giving students grants to graduate students for travel to Sweden. weeks of consultation. relatively mild storm, just a choppy sea, with no harm.’’ the option of doing experimental work in Oak Ridge. Carlson said that he would write a recommendation for After the Congress, I took a train to Lund and Malmo¨ That seemed a gross understatement to many of us! If it This was the first step in setting up what became the me if I wanted to present a paper based on my for a few days to visit labs doing cytogenetic work with were true, we surely did not want to be in a real North ‘‘University of Tennessee-Oak Ridge Graduate School dissertation. That sounded like a fun adventure not to be plants, and then another train to board a ship at Go¨teborg Sea storm! We were happy to get to the Atlantic Ocean. of Biomedical Sciences.’’ Also, he wanted Gordon to missed, and I did not want to be tied to a job for the for a 2-week visit to labs in England. I had met some The trip back on the Gripsholm was like an extension establish a group in the Biology Division that would summer! I immediately applied for and got a grant, English geneticists at the Congress who invited me to of the Genetics Congress: lots of talk about the papers encompass the area of insect genetics, thus enabling successfully completed the inquisition by the UVA visit their labs, so I went to Edinburgh, and to Oxford, we had heard and the conversations we had had with Gordon to have a lab in Oak Ridge on a part-time basis graduate committee in the spring, and picked up my Cambridge, and the University of London. I enjoyed foreign geneticists. Also, we shared information about during the school year, and fulltime during the diploma at the June graduation exercises, before delightful conversations in their labs and homes. The the various ‘‘adventures’’ we had had between the summers. heading to New York to board the Swedish liner British had not yet completely recovered from WWII, Congress and boarding the ship for home. When By the fall of 1947 Alex was in Oak Ridge, and Gripsholm, bound for Go¨teborg. I was off to see the and it was obvious in many ways, especially in their Henrietta got back to Oak Ridge, she evidently told Gordon and I were about 45 min away at the University world, at least part of it! clothes. For example, the cuffs of men’s and women’s everyone she saw about my ‘‘going alone all over of Tennessee in the Zoology Department. I was working The Gripsholm was a famous old ship, supposedly shirts and jackets were often noticeably frayed. Fresh Sweden and England.’’ I was amazed that she thought it on my dissertation, aiming for a Ph.D., which I received ‘‘refurbished’’ after having brought to America some of meat was still being rationed. At a small party in one so unusual. It revealed a timidity of hers that no one from the University of Virginia in June 1948. the last refugees from Europe at the beginning of WWII. home, an English lady sitting next to me leaned over to would have suspected from the forceful way in which Alex had begun recruiting for his big lab before he The Genetics Society had struck a bargain with the whisper that ‘‘the hostess has obviously been saving she conversed with people. left NIH, but he went full-speed ahead on arriving in company to give reduced rates to all of the geneticists coupons for a month in order to buy that joint (roast)’’she Oak Ridge. While the buildings at the wartime code- who wanted to go by sea to the Congress. Most of us was serving. One day, when I was in London, I stopped at 1.6. The Oak Ridge years named Y-12 area (near the town) were being remodeled students and some older geneticists chose the cheapest a small restaurant for lunch. It was crowded, so the for the Biology Division, Alex obtained some tempor- quarters, i.e., Third Class deck and quarters. A few were hostess seated me with a woman at a tiny table. After we On returning to the University of Tennessee (UT), I ary labs in the X-10 area (about 10 miles from Y-12) to in First and Second Class, but they often joined us in had made small talk for a bit, and I had mentioned that we dived into finishing several projects with Gordon

242 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 243 8 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 9

Carlson, so that I could explore getting a job. By interview. Even years later, I could not bring myself to waited for a response. After some minutes, Alex said, Alex suffered from gout, which often caused him to summer of 1949 I could realistically look to the future, tell him he could have gotten me with his first offer. I ‘‘Vell, try tow spen less.’’ I left the office thinking, walk slowly and with a soft shuffle, so he could be heard so one day in June I took a bus over to Oak Ridge to talk was afraid he would have a heart attack! I have to admit ‘‘What a waste of time! He must be desperate to conduct approaching on the cement floors in the Lab. Kim with Alex about ‘‘the job.’’ He reiterated that he wanted this was the only time in my entire life that I have ever such a dumb exercise!’’ I never figured it out, but never Atwood was a gifted speech mimic, and thought it great me to be the Group Leader for the Insect Genetics been without words! again did any Group Leader, to my knowledge, hear the fun to creep up behind someone who was bent over a Section, which would comprise three investigators: ‘‘Group Leader’’ was in most ways just a titular word ‘‘budget.’’ I certainly didn’t. That was my only microscope and say, ‘‘Vot yo doin?’’ Some claimed it Carlson and myself (grasshopper neuroblasts) and Bill position. Alex used it for the person he held responsible down-and-out argument with Alex. made them jump a foot! Kim never fooled me, because Baker (Drosophila). Both of them were professors at UT not only for the research performed, but also for handling For the whole day of ‘‘budget hearings,’’ Alex had Alex did not intimidate me, but also because he failed to during the school year, so they would be full-time at the all personnel and other problems that arose in a Group. In one Group Leader, Charles (Shep) Shepherd, sit with mimic Alex’s warning shuffle. Biology Division only in the summer. Alex assured me addition, he also looked to the Group Leaders for help in him. Shep was a physicist, expert on all types of Alex was himself a bit intimidated by people who that, with time and available money, the group would recruiting scientists and lab assistants. He did not, radiation, and indispensable for keeping the X-ray found it difficult to converse, much less argue, with him. expand with more investigators. It did, eventually however, give them any responsibility with respect to the machines and the isotopes lab in top running order for He would say, ‘‘He/she won’t talk to me. How am I including Kimball (Kim) Atwood, Alan Conger, Thad budget; in fact, we had no idea how much we could spend. researchers. He was a very bright guy, self-trained in going to get to know them if they don’t talk?’’ This Pittenger, and Jack von Borstel. The Drosophila people ‘‘Budget’’ seemed to be his main task once he got the physiology, and working on theories pertaining to blood would sometimes cause such individuals to falsely think branched off into their own group, headed by Dan Division running at full force. He was a whiz at getting flow. However, in a building full of biologists, he that Alex ‘‘did not like them’’ or ‘‘did not appreciate Lindsley. money for whatever he wanted to do, and he was seemed to feel a bit inferior. He was always giving Alex their work.’’ Such attitudes were sometimes alleviated With that established, I told him there was one thing constantly searching for more to finance a meeting in Oak ‘‘advice’’ about various matters in the running of the at the annual Biology Christmas party, initiated soon that I was not going to be, namely, Dean of Women. Ridge or elsewhere, to publish a book he wanted to edit, Lab, so Alex finally gave him some title (such as ‘‘Vice after the Lab was organized. It included everyone on the There had been several occasions while I was at UT or to fund foreign scientists who wanted to visit the lab Director’’) primarily, I think, to pump up his ego. This is payroll, which made it a big annual event, held in the when he had telephoned, wanting me to catch the next and stay for a time. why Shep was at the ‘‘Budget Inquisition.’’ It was one town’s social hall, and lasting past midnight. AEC bus from Knoxville to Oak Ridge to help him with a I know of only one occasion when Alex acted maneuver that Alex used when he was having trouble money could not be used to purchase alcoholic ‘‘crisis’’ relative to some untoward action taken by some alarmed about ‘‘how much people were spending’’ in keeping a ‘‘valuable investigator’’ happy. beverages, but Alex quickly found funds somewhere young woman lab assistant. For example, one was the Lab. It also illustrates another characteristic: he The day after this little farce, Shep came to my lab to for liquor, dinner, and music for dancing. Those having an affair with her boss, whose wife was could easily, and often unknowingly, intimidate people. tell me, in dismay, that I was the only Group Leader who ingredients loosened tongues and provided him with threatening to leave him. Alex didn’t want such a A notice was sent to all of the Group Leaders, giving the had challenged Alex. Several of them, who were the chance to talk with some people who otherwise were situation in Biology, but, more importantly, he didn’t exact half-hour that each one’s ‘‘expenditures would be definitely not timid types, later told me that they were so tongue-tied in his presence. I’m sure he knew what he want to lose the scientist. In most cases, the problems discussed in detail in Dr. Hollaender’s office.’’ I was the surprised and shocked by his accusations that they would accomplish by having such a party! were of little consequence. All I had to do was to talk last one on the list, in the late afternoon. During the day, decided the better part of valor was not to cross him. I I had gotten the impression in Bethesda that the with these young women and/or get them to talk to me, I had run into several Group Leaders who said that they was surprised at their reactions. Hollaenders did not do much home entertainment, but which usually solved the problem or let it go under- had, from their point of view, barely survived the When Alex was not tied to his office, he would tour that changed when they got to Oak Ridge. The house ground. The only serious incident involved an ‘‘ordeal of the inquisition.’’ I was puzzled by this, but the labs to talk to people about their work. This had been was bigger than the one in Bethesda, and Henrietta ‘‘unhappy lab assistant.’’ I quickly decided she was did not give it much thought, because I did not know a common practice by directors of German scientific made it into a most pleasant and welcoming home- psychotic, and convinced Alex’s secretary to do the what had caused such reactions. When I appeared laboratories, although Alex should not have had any museum. Every time a visiting scientist of note came to ‘‘unthinkable,’’ i.e., pull him out of a budget meeting. I ‘‘before the bench’’ (Alex’s desk), he began with ‘‘Yo direct experience with such practices, since he the Lab, there would be a reception or dinner party at advised him to call in several of the scientists who were Group spending more dan yo budget.’’ presumably had not attended college in Germany. At their home. As a single woman, I was often invited, physicians to help him get psychiatric help for her It took only two seconds for my blood pressure to go any rate, it gave him an opportunity to discuss most probably to even out the number of table guests, immediately, which he did. Sadly, in spite of all efforts into high drive, plus one more to launch into a nonstop experiments and data at the lab bench, as well as and to make conversation. Sometimes the guest, being to help her, she eventually succeeded in committing rebuttal: ‘‘Good grief, Alex, you have never even providing him with first-hand knowledge that he could often a bit shy, would act as though he were suicide—in the lab. mentioned budget to me, much less given me a spending use in justifying demands for further funds. It also overwhelmed by all of the beautiful art and tableware. After Alex saw that I was determined to do nothing limit, so how could I know I was ‘over budget’? When I enabled him to decide who would make good I say ‘‘he’’ because in the early days the vast majority of but science, he moved on to salary, saying ‘‘How about took this job, you directed me to your Office Manager impressions on visiting scientists. This practice guest scientists were men. It was not until much later $..., beginning September 1st? I forget the exact figure, for the procedures used to buy supplies and equipment unnerved some people, especially new employees, that more visitors and resident scientists were women. but the amount so dumbfounded me that I was actually for our lab. He said there were none. I was directed to a who mistook his unannounced appearances, especially speechless. Having struggled to make ends meet for 7 huge room, which looked like a grossly overstocked on Saturdays, as ‘‘checking up’’ on them. He was 2. Reflections of John Jagger years on fellowship stipends, I had not expected what warehouse, containing all sorts of lab supplies. I just had interested in the amount and quality of data, not the time seemed to me a big salary. Seeing my hesitation, he to fill out a form listing what was needed and how many. spent in a lab. He also liked to get to know the I first met Alexander Hollaender in the fall of 1955, immediately increased the amount by another thousand! Period. To get large equipment, one had only to submit investigators on a personal level. Birth of a baby would in the office of Raymond Latarjet, at the Radium Embarrassed, I found enough voice to say, ‘‘OK.’’ After the name of the object (e.g., a microscope), its often bring Alex and Henrietta to a home bearing a Institute (Curie Institute) in Paris. I was a post doc in moving to Oak Ridge and arguing with him about specifications, and name and address of the vendor. present. Illness in a family would evoke concern. The Latarjet’s lab, and approaching the end of my stay. Alex salaries for the people I wanted to bring into the Group, I Cost was never an issue. It was like a dream come true.’’ Hollaenders had no children, but in a sense looked upon offered me a position at Oak Ridge. The salary he saw how stingy he was about starting salaries. I realized At that point, I stopped to catch my breath—one the lab people as their ‘‘family,’’ an attitude that was not offered was half of what I had been offered by Memorial that he had misinterpreted my silence during our could have heard a pin drop on a rug in that room! I just always understood by some researchers. Hospital in New York, so I later asked Latarjet what he

244 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 245 10 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 11 thought. He said, ‘‘Go with Hollaender.’’ So I did. Like sounded good to me, except that she wanted to get Mary Esther, when offered a job at Oak Ridge, any married the next day, a Saturday. I asked her to give me a salary seemed big after living hand-to-mouth as a grad couple of hours to think about that! Not being able to student and post doc. Also I had heard great things about come up with any good reason not to, I agreed. I should the research at Oak Ridge, and thought it the better job. note that we were both in our early thirties and, as she When I left Oak Ridge nine years later, I was finally would say, ‘‘quite dry behind the ears,’’ so we were making the same salary I had been originally offered at aware of what we were doing. Memorial Hospital, but I never regretted my decision. Early the next morning, she called Henrietta Shortly after arrival at Oak Ridge in April 1956, I Hollaender, and told her that we wanted to get married posted a sign on the bulletin board that I had for sale in her house that afternoon (19 October 1956)! Without Lea’s Actions of Radiations on Living Cells. One day, a hesitation, and indeed with enthusiasm, Henrietta agreed. little person popped into my tiny office and declared, Then we shot off to Knoxville, where we got a marriage ‘‘Hi! I’m Mary Esther Gaulden. I’d like to buy your license, and had a blood test. While the test was being book!’’ I told her it was $5. She paid me and scored, we went to Rich’s department store and bought a immediately left, saying, ‘‘Thanks very much!’’ I said ring. Mary Esther said she wanted ‘‘the cheapest ring you ‘‘Thank you,’’ and she was gone. It was my first have’’—the clerk raised his eyebrows and said that would encounter with a Southern tornado. be five dollars. She said we would take it, and I put the Over the next several months, I saw Mary Esther little box into my outside jacket pocket. Back in Oak occasionally, at social events and at seminars. Other- Ridge, at about three 3 PM (we were scheduled to be at wise, we saw little of each other, since she worked on a the Hollaenders’ at four), she called me and asked if I had different floor. But with time, I became more and more the ring. I said no, that I thought she had it. In short, fascinated by her vivacity and intelligence (to say someone must have seen us through the large plate-glass nothing of her good looks and nice legs). window of Rich’s, where the jewelry counter was right at In January 1956, Mary Esther had been sent on a the front, and later picked my pocket. We had no end of Rockefeller Foundation mission to assess genetics pleasure in imagining the look on the face of the thief research in South America. She came down with two when he tried to pawn the ring! types of malaria (vivax and falciparum), contracted in the Henrietta had produced a delicious turkey dinner in no Brazilian jungle. Shewas treated with chloroquine, then a time at all, and we thoroughly enjoyed ourselves in their fairly new drug and, remarkably, never had any relapses. house, decorated with flowers and of course their Fig. 1. The wedding of Mary Esther Gaulden and John Jagger at the Hollaenders’ home in Oak Ridge, 19 October 1956. Left to right: Henrietta She and I really began to connect on a hiking trip in excellent modern paintings, with our guests: Charles Hollaender, John Jagger, Mary Esther Gaulden, and Alexander Hollaender. the Great Smoky Mountains National Park with Max (‘‘Shep’’) and Jane Shepherd, Virginia White and her son Zelle and his wife and several others. Halfway through Charles, Gene and Mary Margaret Joyce, and Gordon and In October 2006, we celebrated our 50th wedding Charles Johnson, a famous sociologist who had been the hike, Mary Esther became too tired to go on, as she Elizabeth Carlson. The wedding cost me a total of $15— anniversary. Neither of us has ever had a doubt about Franklin Roosevelt’s special envoy to several African was still recovering from her malaria. So she sat down five for the ring, and ten for the Unitarian minister—by our quick decision. I had married a most remarkable countries, notably Liberia, where he was sent to on a log and urged the rest of us to go on. I demurred and far the best bargain I ever made! The Hollaenders kept woman, who has been a pleasure to live with, talk with, investigate rumors of slavery. Fisk was a private said that I would stay with her, telling her she couldn’t our wedding picture, showing them and us, on their travel with, and joke with (she laughed at my jokes, university established by the Congregational Church of be left all alone on the trail. So the others went on. We mantel for years afterward, where many other scientists which helped). And even to be alone with. Never a dull New England after the Civil War to give black students talked nonstop for about an hour and a half. We saw it before they had even met us (Fig. 1)! moment. the opportunity to get a college education. My discovered that we had a great deal in common, and Being freethinkers, neither Mary Esther nor I wanted responsibilities were varied and a major one was to loved to read and to travel. She was very frank and a wedding ceremony in which we swore to ‘‘honor and establish and maintain liaison with the white, tradi- straightforward—quite unlike other girls I had known. obey, ‘til death do us part’’ and all that (although it 3. Reflections of Virginia P. White tionally Southern, community of Nashville. Segregation After that, things moved fast. I often drove to her turned out that we did follow such an ethic). Instead, she was still rigidly observed there, and when the university apartment, and helped her paint her hall, among other chose a selection from The Prophet, by Kahlil Gibran, 3.1. Alexander Hollaender held conferences or had visitors requiring hotel things. No one seemed to notice these visits, even on marriage, which includes the following lines: accommodations, the arrangements for such accom- though some of the lab scientists lived in adjacent Alexander Hollaender made up his mind quickly modations had to be negotiated, to put it in the nicest apartment buildings, where they could easily have Love one another, but make not a bond of love: about people. He may have made a few mistakes, but his possible way, very delicately. When we invited elected spotted my blue 1950 Oldsmobile (with a green hood) Let it rather be a moving sea between the shores of your souls. judgment was often on the mark. Going to work in the city or state officials to university events, they were that looked like an army tank, whether coming or going. Fill each other’s cup but drink not from one cup. Biology Division was for me one of those out-of-the- always concerned about the Public Relations coverage, We decided to get married, perhaps in the following Give one another of your bread but eat not from the same loaf. blue, unexpected things that can only be described as the pictures that might appear in the local papers and Sing and dance together and be joyous, but let each one of you be amazing good luck or even a miracle. such. In 1954, that watershed year of the Brown versus spring. But Mary Esther said, ‘‘Why wait? We can get alone, married now, and have a beautiful honeymoon, with all Even as the strings of a lute are alone though they quiver with the same In 1954 I was at Fisk University in Nashville, Board of Education Supreme Court Decision, I was very the fall colors in the Smokies and the Blue Ridge.’’ That music. Tennessee, where I was the Assistant to President fortunate to be at Fisk. We hosted many civil rights

246 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 247 12 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 13 leaders that year including Thurgood Marshall, who were located. Those signs were repugnant to me and I first woman to be elected president of the Biophysical (British, B. America, 1880–1959), which they pur- argued the NAACP case before the Supreme Court and soon discovered that Dr. Hollaender did not like them Society. chased in London from the artist in 1952 and 1953. won a unanimous decision. Marshall was the first black any more than I did. But it was the rule observed Although I did not know it at the time, I became Besides the 20th century works, there are some Supreme Court Justice; he was nominated by President throughout the three AEC installations in Oak Ridge, another Hollaender ‘‘breakthrough’’ in gender equality. Japanese and Chinese hanging scrolls; Russian icons; Lyndon B. Johnson and seated in 1967. and I was a little apprehensive when, after we discussed Every ORNL Division Director had an administrator to and sculptures from the ancient Near East, Africa, In one of those unexpected, rare life-changing events it, he announced that we would remove the signs. manage the non-scientific functions—personnel, bud- Spain, India, and Thailand. From the Americas, there in 1955, a friend of mine who was visiting the Oak We agreed to make no announcement. All we did geting, purchasing, editorial and publications. Those are two pre-Columbian and one Aztec piece, and one Ridge National Laboratory Biology Division and had was reassign change rooms on the basis of their positions were held by men until my appointment in the Native American Eskimo sculpture. met Dr. Mary Esther Gaulden, mentioned to Mary nearness to the employees’ work places. This was the Biology Division. I do not know what happened after I The Hollaenders’ Oak Ridge home was one of the Esther that she had a friend at Fisk University in basis on which assignments were made for white left, but Biology was the only division where a woman larger houses built during World War II designed by Nashville, and told her what I was doing. As a result of employees, whereas the smaller number of black held that position as long as I was there. Skidmore, Owings and Merrill, and situated among the that conversation, Mary Esther wrote me a letter employees, and thus fewer rooms labeled ‘‘Colored,’’ forests of Black Oak Ridge to preserve as many trees as inviting me to visit the Oak Ridge Biology Division. meant that many blacks had to walk long distances to 3.2. Henrietta Hollaender possible. It was assumed that these structures built of That visit turned out to be a job interview with Dr. their work places after changing in the morning and wartime materials would not last long, but now (65 Hollaender. His urbane manner, intelligence, sophisti- back to the change room at the end of the day. We sent a Mrs. Hollaender was an art historian and taught at the years later) most of them sturdily survive. A great many cation, and lack of any pretension put me at ease memo to each employee affected by the change, saying Sidwell Friends School in Washington DC when they have undergone renovations and modernization, adapt- immediately. I told him the things one usually talks that as of a certain date their locker assignment would lived in Bethesda, but she devoted her life to being a ing them to the taste of the owners. The Hollaenders’ about in job interviews, and he told me a bit about be changed and giving the number of their new change helpmeet for her husband. The Hollaenders had no house, with relatively minor alterations, was adapted to himself, and a lot about the work going on in the room. It was evident that the new assignments had been children and she was as dominant in their home as he accommodate art; thus it had a museum-like quality that Biology Division. At the end of the interview he did made on the basis of work place location; it was also was in the laboratory. And she was as unique a delighted the eye of old and young who were privileged something that nobody who interviewed me for a job evident that the signs ‘‘White’’ and ‘‘Colored’’ were personality in her way. She was capable of ordering him to visit there. There was no art gallery in Oak Ridge, and had ever done. He said, ‘‘We have a scientist from gone. around in a stentorian voice, but I never heard her nag some intrepid schoolteachers requested permission to Sweden visiting the Division, and this evening my wife We did not know what to expect when the ‘‘change him, and I never saw him resent her orders. If he reacted bring their students to visit. Mrs. Hollaender not only and I are taking him out to dinner. I would like to have day’’ arrived. It was greeted with silence. It received no at all, it was with an indulgent smile. She never learned welcomed as many as she could but became an you join us.’’ I was overwhelmed and accepted more attention than the reassignment of a telephone to drive a car, and she had a sure-fire way of getting him articulate and well informed ‘‘tour guide.’’ immediately. I instantly felt approved of in a way I number. home from the lab when she thought he had been there The Hollaenders supported all the cultural activities had never felt at the end of a job interview. I knew he About 1 year later, ORNL’s Personnel Director too long, especially on Saturday afternoon. She would in Oak Ridge, and from its earliest days, Biology had talked with Mary Esther after she and I spent some telephoned me and said he wanted to prepare me for a telephone and say, ‘‘If you want any dinner tonight, Division scientists and others were leaders and time together, and that his cordiality was in large part memorandum he was sending because it concerned a you’d better come home and take me to the grocery supporters of music, theatre, art, and related activities due to her, and I have always been very grateful to her. I policy change that some might find objectionable, so it store!’’ If there was anybody around to see him leaving, and institutions that were established in the city. The expressed this gratitude by dedicating to her my book, would have to be handled with great delicacy. Union he would explain, ‘‘I’ve got to take Hen to the grocery first symphony orchestra in Oak Ridge was assembled Handbook of Research Laboratory Management (1988; Carbide had adopted a non-segregation policy, and we store!’’ in 1944, when the city was only 2 years old, by Waldo Philadelphia, ISI Press). must integrate our change rooms and remove the Very modern in her tastes and philosophy, she Cohn, a biochemist in Biology. He played the cello in That was a very pleasant evening; the Swedish ‘‘White’’ and ‘‘Colored’’ signs. I laughed and said, ‘‘Oh, dressed to please herself and presumably Alex, although the orchestra and led the orchestra until the Oak Ridge scientist was intelligent and good company, but I had no we did that a year ago!’’ Then he laughed, relieved that he never seemed to notice what she wore. She owned a Civic Music Association could hire a professional way of knowing that it was my first social meeting with Biology was one Division he did not have to worry few classically beautiful pieces of jewelry, which often conductor. two people who would be very important in my life for about. lent a note of elegance to her costumes. The only Dr. Hollaender was determined to find funds to the next 35 years, Alexander and Henrietta Hollaender. Dr. Hollaender not only believed in racial equality, notable aspect of Henrietta’s wardrobe is that she was support the construction of an Art Center building in The Oak Ridge National Laboratory was operated by he deplored all types of discrimination. When I went never conspicuous. I can never remember seeing her in a Oak Ridge. It was one of the few things he tried and the Union Carbide Corporation for the Atomic Energy there in 1955, two of the group leaders were women, brightly colored or distinctly styled dress, and the only failed at, but the city did eventually have an Art Center. Commission under a cost-plus-fixed-fee contract that Mary Esther Gaulden in Insect Cytology and Genetics, clothing item I ever heard her mention was shoes. She In his search for foundation support, he aroused enough was awarded through an open bidding procedure. Union and Liane B. Russell in Mammalian Cytogenetics and cared only that they be comfortable. Alex was always interest at the Rockefeller Foundation that they sent one Carbide, an international enterprise with branches in Development. Later, other female senior investigators appropriately dressed—he was particularly fond of of their executives to Oak Ridge for a site visit. It several countries, adhered to the advice of St. Ambrose joined the Division, among them Joan Wright Good- Italian silk ties. became my duty to tour the visitor around Oak Ridge all in the fourth century AD, who wrote, ‘‘If you are in man; Rhoda F. Grell; Jane K. Setlow; Dorothy Skinner; They both loved art and amassed an impressive day, and take him to the Hollaenders for cocktails and Rome, live in the Roman style.’’ Therefore, in the and Audrey L. Stevens. All of them received recogni- collection of paintings, sculptures, drawings and prints dinner in the evening. This gave me a notable exhibition segregated state of Tennessee, the laboratory ‘‘change tion for their scientific achievements. Audrey Stevens that can now be seen in the Hollaender Collection at the of one of Henrietta’s most endearing talents: taking the rooms’’ had signs that read ‘‘White’’ and ‘‘Colored.’’ and Liane Russell were elected to the National Elvehjem Museum of Art, University of Wisconsin. It starch out of stuffed shirts. Change rooms were where employees changed from Academy of Sciences, joining Dr. Hollaender and the consists predominantly of 20th century works of The Rockefeller man looked and acted just like street clothes to laboratory garments when they arrived other male members who were brought into the Division modern art. Numbering 256 works in all, there are 89 someone a casting director might send to play the role of a in the morning, and after work changed back into their by Dr. Hollaender: William A. Arnold; Dan L. Lindsley; paintings; 56 sculptures; 32 drawings; 79 prints. The foundation executive: tall, handsome, elegantly dressed, street clothes. That was also where toilets and showers Oscar L. Miller; Richard B. Setlow. Jane Setlow was the sculptures include three works of Sir Jacob Epstein polite, reserved, formal. He was not wearing a top hat, but

248 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 249 14 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 15 my imagination put one on him. It was a trying day for of scientific and cultural information. During the best hope for a peaceful and rational social order must the disfavor of a Senate investigating committee. He me. Nothing I said or did made him relax and act like an thirteen years I was at the Biology Division, a total include free dissemination of scientific knowledge. therefore pleaded with Hollaender not to invite Pauling ordinary person. It was a relief when time came for us to of 160 foreign scientists from 34 countries worked Consistent with that belief, one of the first things he to Oak Ridge. But Hollaender and Doherty held firm. arrive at the Hollaenders’ house. I may have pushed it a there, including postdoctoral students as well as senior did as director of the Biology Division was to establish Strauss resolved the dilemma by offering funds to move bit. Alex had not returned from the lab and Henrietta was investigators. They came from every continent and forums for information exchange among scientists. He the meeting to another location, anywhere but Oak still in the kitchen cooking dinner. After barely some island nations, like The Philippines. Some came inspired, and was the catalyst for, the beginning of very Ridge, and thus the Gatlinburg annual research acknowledging the introduction, she said, ‘‘I’m in the from countries whose cultures were not well known or productive meetings among biologists in South Amer- conferences were born. Ironically, it turned out that kitchen cooking dinner. Come in and we can talk while I understood by Americans, such as Vietnam, Egypt, ica. He was the driving force of a series of conferences, Pauling was unable to attend the conference for other finish.’’ Almost instantly I observed, with admiration Indonesia, Iraq, South Korea, and Turkey. They also beginning in 1957, that led to establishment of the reasons! bordering on disbelief, as she completely disarmed this came from more familiar nations, including Great International Society for Experimental Hematology. Gatlinburg, Tennessee, was then a small village stuffy man. They were soon comparing her utensils and Britain, France, Germany, Sweden, Japan, and Italy. The Biology Division was then creating large programs known as the Gateway to the Great Smoky Mountains appliances with those in his home. I realized he had The Sunday morning hikes thus became a small, to study genetic and somatic effects of radiation. At the National Park, but it had made few concessions to the pegged her as a standard ‘‘housewife’’ type and was informal forum where subjects of international interest same time, chemical protection or neutralization of modern tourist trade. There was only one hotel, the tailoring his conversation to that level. ‘‘Mrs. Hollaen- dominated, and people spoke freely on any scientific, radiation effects inspired medical treatment research, Mountain View, large enough to accommodate a der,’’ he said, ‘‘What do you think is the most important cultural, political, or philosophical matter that inter- such as bone marrow transplantation, which was heavily conference of 50–100 people. It had sparsely furnished thing in a kitchen?’’ Instantly she replied, ‘‘Oh, ested them. funded. It was considered highly experimental. Rou- rooms with ceilings of bare rafters, and no television. somebody besides me to do the cooking!’’ After cocktails There still exists at the Oak Ridge National tinely used today, that treatment was pioneered 50 years There was a long verandah lined with rocking chairs, in their large living room where the walls were covered Laboratory a ‘‘souvenir’’ of those Sunday hikes, in the ago by the Biology Division’s ‘‘Mammalian Recovery’’ and a spacious lobby, one end of which was entirely floor-to-ceiling with art, and sculptures stood on tables or form of a fossilized ‘‘scale tree’’ that lived approximately group headed by Charles C. Congdon. But the most taken up by an enormous stone fireplace where tree-size on pedestals tucked into corners, and after a dinner better 300 million years ago. This was identified as a ‘‘lycopod’’ famous and enduring forum Hollaender created was the logs blazed cheerfully on cool days and chilly evenings. than he might have had at any number of New York’s top or ‘‘wolf’s foot,’’ so called from the form of its Biology Division’s annual research conference. The registration desk was set up in that lobby. On the restaurants, served at their gleaming early American aboveground roots. Lycopods belong to a group of Those conferences were originally held in Oak opening day of conferences, Dr. Hollaender stood dining table, with beautiful linen, china, crystal and plants that includes the club mosses and were the first Ridge. But the town and lab had, for several years nearby and greeted every registrant with the warmth and heavy antique silver, he no longer aimed his conversation plants in the fossil record with true leaves and roots. It following World War II, been designated by the federal ebullience of a genial host at a large house party. He at the ‘‘housewife’’ level. was brought down the mountain in around 1958–1960 by government as a ‘‘security-sensitive’’ area. Although viewed these meetings as a week in the country, and saw two young scientists who regularly joined the Hollaender the Biology Division was located ‘‘outside the fence,’’ to it that the program designated hours for socializing, 3.3. Sunday Morning Hikes in the Cumberland hikes, Heinrich Kroeger from Germany, and Benedetto everyone who worked there had to undergo an FBI including a free afternoon for hiking in the mountains or Mountains Nicoletti from Italy. It is approximately two feet high and investigation and be given a ‘‘Q clearance’’ (the highest otherwise exploring the beautiful countryside. a foot-and-a half in diameter, and resembles a tree trunk. level) to receive classified information. Visitors to the The Mountain View Hotel operated on the American Every Sunday morning that he was in town, Dr. Mounted on a strong base, it stood at the entrance to the Division had to be approved by the Atomic Energy Plan, under which hotel rates included both room and Hollaender led a group of Biology Division personnel, Biology Division when the Division was located in the Y- Commission, which was responsible for the Oak Ridge meals. The outrageously large meals were served family visitors, and/or friends into the strip-mining areas of the 12 area. When Biology was moved to the X-10 area and installation. During the well-known ‘‘McCarthy Era’’ in style, and were sufficiently varied to suit everyone’s Cumberland Mountains, winter and summer, rain, merged with other divisions to form the Life Sciences the early 1950s, Senator Joseph McCarthy and his taste. Thus, people could meet casually in the dining shine, or even snow. Division, the ‘‘tree’’ was at the last minute loaded onto a colleagues could not or would not distinguish between room, join others who had arrived earlier, have a meal That strip-mined area, denuded of vegetation, was truck with scientific equipment by Ken Isham. It was later exchange of scientific information and treason, and to together and leave, without calculating what each littered with items brought to the surface by the installed in the William L. and Liane B. Russell be on the safe side decided that they were one and the person owed. It was all included in the bill. undiscriminating blades of the mining machines, and Genomics Research Laboratory, which was formerly same. Any gathering that included foreigners was There was one drawback to socializing in Gatlin- included an abundance of ancient fossils of plant life. part of the Biology Division in Y-12, and was commonly viewed with suspicion, and such invitees had to be burg. Tennessee law provided for something called The mining companies were interested only in the referred to as ‘‘The Mouse House.’’ The ground-breaking thoroughly vetted. Like all government agencies, the ‘‘county option,’’ meaning that every county decided for cheap, poor quality coal this method brought to the research done through the years in that laboratory AEC was dependent on Congress for funding, so itself whether to permit sale of alcoholic beverages, and surface, which they could market at a profit because it continues in its new independent building in X-10, named invitations to visit Oak Ridge for any purpose were Gatlinburg was in a dry county. Oak Ridgers, who also was produced at minimum cost. We who went on these for the Russells, who came to Oak Ridge in the late 1940s carefully monitored. lived in a dry county, understood this and knew how to outings returned home with as many fossils as we could to join Dr. Hollaender in the earliest days of the Biology The topic for the 1955 conference was ‘‘Structure of deal with it. They arrived well supplied, and assumed carry. We valued them for their history and displayed Division. William Russell died in 2004; his wife Liane Enzymes and Proteins,’’ and the Program Chairman, the obligation to see that everyone had a cocktail before them proudly. We often used them for doorstops, continues to direct the work of the laboratory that bears David Doherty, wanted to invite Linus Pauling. Pauling dinner. Thus, as the dinner hour approached, the hotel paperweights, and other household purposes. I often their names. was awarded the Nobel Prize in Chemistry in 1954 and was a-buzz with cocktail parties. When supplies ran out, wonder about Tennessee fossils that may now be seen in had received many other high awards. Pauling had also it was a short drive to a neighboring ‘‘wet county’’ countries around the world, in the homes of foreign 3.4. Gatlinburg made himself conspicuous by his outspoken political where there was a convenient liquor store just across the scientists who visited the Biology Division and joined views, particularly as a crusader for peace, and he was county line. the hikes and fossil gathering. Alexander Hollaender’s faith in the value of free awarded the 1962 Nobel Peace Prize. Admiral Lewis The Gatlinburg story illustrates two of Hollaender’s The harvest of those hikes was not confined to exchange of scientific information was as strong as the Strauss, then chairman of the AEC, was well aware of characteristics that served him so well in his leadership fossils. They provided an ideal setting for the exchange religious faith of some people. He thought the world’s the vulnerability of a government agency that attracted roles: his unassailable optimism and his cool pragma-

250 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 251 16 M.E. Gaulden et al. / Mutation Research 635 (2007) 1–16 tism. His optimism gave him confidence that anything The subject of the Symposium was Transposable Mutation Research 658 (2008) 1–27 he wanted to happen, that he believed essential and Elements in Mutagenesis and Regulation of Gene Available online at www.sciencedirect.com desirable, he assumed to already exist, and he proceeded Expression. The meeting opened with a plenary session to act on that premise. When something is seen as a devoted to the dedication ceremonies. The major speech reality, objections can be brushed aside as mere trifling was given by Dr. Alvin Weinberg, the eminent physicist Reflections on the impact of advances in the assessment annoyances. He was also exceedingly pragmatic, but and director of the Oak Ridge National Laboratory Mutation Research 658 (2008) 1–27 www.elsevier.com/locate/reviewsmr not a graceful loser. He managed, as in the Pauling story, during most of the years Dr. Hollaender was there. I was of genetic risks of exposure to ionizingCommunity radiation address: www.elsevier.com/locate/mutres on to turn the loss to advantage. Or, in some cases, he asked to give a talk and spoke about the history of the Reflections in Mutation Research resolved it by putting his own interpretation on it. Gatlinburg meetings, and Alex Hollaender’s role. My internationalReflections on radiation the impact protection of advances in recommendations the assessment of genetic One example illustrates his optimism: Alex was trying talk closed as follows: to recruit a well-known biologist to join the Oak Ridge risks of exposure to ionizing radiation on international☆, ☆ ☆ radiation ‘‘Dr. Hollaender really thought of the Biology between the mid-1950s and the present Laboratory. He spoke with the man several times, and Division, especially the scientists, as his children. protection recommendations between the mid-1950s finally wrote him a letter offering him a position and K. Sankaranarayanan a *, J.S. Wassom b The optimism evidenced by the fact that this meeting §,§§ specifying the details, salary, laboratory space and and the present is being held at a time when the future of the Division equipment, and describing the Oak Ridge community as looks very gloomy, is something he would applaud. a Department of Toxicogenetics, Leiden University Medicala, *Centre, Einthovenwegb 20, Building 2, a wonderful place for families, with good schools and K. Sankaranarayanan , J.S. Wassom If he were here today, he would be proud of his Post Zone S-4-P,a Post Office Box 9600, 2300 RC Leiden, The Netherlands ample recreational and cultural facilities. For a long time Department of Toxicogenetics, Leiden University Medical Centre, Einthovenweg 20, Building 2, Post Zone S-4-P, scientific offspring, and encourage them to move Post Office Box 9600, 2300 RC Leiden, The Netherlands he got no reply. Then one day he walked into my office, b Oak Ridge National Laboratory, bOak Ridge, TN 27830, USA1 1 ahead aggressively, in spite of the vicissitudes they Oak Ridge National Laboratory, Oak Ridge, TN 27830, USA waving a letter and the following conversation ensued: face. Perhaps he is here. I can easily imagine him Accepted 31 October 2007, Available online 17 NovemberAccepted 31 October 2007 2007 there in the front row. He always sat in the front row. Available online 17 November 2007 AH: ‘‘I got an answer from Dr. X!’’ And after the presentation of the first paper, some of VW: ‘‘What does he say?’’ us will be half-expecting him to ask the first question, AH: ‘‘He’s interested.’’ He handed me the letter. It Abstract as he often did. The dedication of this meeting to was a cool, not overly polite, letter. Alexander Hollaender is most fitting. We revere him, Efforts at protecting people against the harmful effects of radiation had their beginnings in the early 1900s with the intent of protecting VW: ‘‘Alex, he is telling you to go to Hell!’’ individuals in medicine and associated professions. Such efforts remain vital for all of us more than 100 years later as part of our ‘learning to live and we miss him.’’ AH: (Waving his hand in a dismissive gesture) ‘‘If he with ionizing radiation.’ The field of radiation protection has evolved slowly over time with advances in knowledge on hereditary (i.e., genetic) and vasn’t interested, he vouldn’t have answered!’’ carcinogenic effects of radiation continually improving our ability to make informed judgments about how best to balance risks against benefits of radiation exposure. This paper examines just one aspect of these efforts, namely, how advances in knowledge of genetic effects of radiation have References impacted on the recommendations of the International Commission on Radiological Protection (ICRP). The focus is on the period from the mid- Another example illustrates his pragmatism: Hol- 1950s (when genetic risk estimates were first made) to 2007. This article offers a detailed historical analysis and personal perspective, and laender sometimes discomfited his subordinates (or even [1] M. Bloom (Ed.), Remembering Alexander Hollaender EMS concludes with a synopsis of key developments in radiation protection. his peers) by expressing his displeasure with a stern look Newslett. (1987) 8–15. # 2007 Elsevier B.V. All rights reserved. and a raised voice, but I can recall only once when I [2] R. Latarjet, Alexander Hollaender (1898–1986), in: E. Riklis (Ed.), Photobiology, Plenum Press, New York, 1991, pp. 15–16. Keywords: Ionizing radiation; Genetic risk assessment; Radiation protection; ICRP; UNSCEAR; History of science thought he was really angry. It was one of the rare times I [3] R.B. Setlow, Alexander Hollaender: the man and his work, heard him use profanity. Someone had let him down or Genetics 116 (1987) 1–3 (also printed in Photobiology—see betrayed him in some way—I forget the details. He ended Latarjet, above). 1. Historical background and introduction that a variety of acute and long-term somatic effects of radiation a brief tirade, then added in a bitter voice, ‘‘Dot son-of-a- [4] R.C. von Borstel, C.M. Steinberg, Alexander Hollaender: Myth were discovered. By the end of 1896, 23 cases of severe X-ray- bitch! I’ll never speak to him again!’’ I was stunned and and Mensch, Genetics 143 (1996) 1051–1056. The discovery of X-rays and radioactivity towards the closing induced dermatitis had been reported; three scientific reviews [5] C. Wilson, F.J. de Serres, Alexander Hollaender, 1898–1986, years of the 19th century [1–4] led to the immediate realization published between 1911 and 1914 had collectively identified 198 sat speechless. After a moment, that characteristic Mutat. Res. 179 (1987) 1–3. twinkle came into his eyes; he smiled slightly, leaned and exploitation of their immense diagnostic and therapeutic cases of radiation-induced cancers resulting in 54 deaths [5–7]. [6] M.E. Gaulden, J.G. Carlson, Cytological effects of colchicine on potential. The primary institutional setting for both practice and By 1934, more than 200 radiologists were thought to have died forward a bit, and said, ‘‘Unless I need him.’’ And we both the grasshopper neuroblast in vitro with special reference to the research in medical radiology was the clinic and thus it was there from radiation-induced malignancies [8]. The need to develop origin of the spindle, Exp. Cell Res. 2 (1951) 416–433. laughed. He would never let false pride stand in the way some guidelines to protect both the patient and the practitioner of getting something he wanted. [7] J.G. Carlson, A. Hollaender, Intensity effects of ultraviolet radiation of wavelength 2537 A on mitosis in the grasshopper from the harmful effects of radiation was keenly felt, paving the § This article is part of the Reflections in Mutation Research series. To Oscar Wilde defined a cynic as ‘‘someone who neuroblast, J. Cell Comp. Physiol. 26 (1945) 165–173. way for the emergence of several national and international knows the price of everything and the value of nothing.’’ suggest topics and authors for Reflections, readers should contact the series institutions concerned with radiation protection during the first [8] J.G. Carlson, A. Hollaender, Mitotic effects of ultraviolet radia- editors, G.R. Hoffmann ([email protected]) or D.G. MacPhee Hollaender was not a cynic. He knew the value of tion of the 2250 A region, with special reference to the spindle ([email protected]). four decades of the 20th century. Many have continued their work everything, but insisted on negotiating the price. and cleavage, J. Cell Comp. Physiol. 31 (1948) 149–174. §§ This article is also paper XV in a series entitled ‘Ionizing radiation and up to the present. In April 1998, on the 40th Anniversary of the first [9] H.J. Muller, Artificial transmutation of the gene, Science 66 genetic risks’ authored by K. Sankaranarayanan and colleagues between 1991 The medical uses of X-rays increased during the First World (1927) 84–87. and 2007 and published in Mutation Research. Gatlinburg conference, the Biology Division dedicated War, paralleled by an increase in the incidence of over-exposure [10] M.E. Gaulden, R.P. Perry, Influence of the nucleolus on mitosis * Corresponding author at: Lange Voort 235, 2343 CE Oegstgeest, The to radiation. With major developments in experimental physics the meeting to Alexander Hollaender, who died in Netherlands. Tel.: +31 71 5172507. as revealed by ultraviolet microbeam irradiation, Proc. Natl. (e.g., advances in knowledge on the characteristics and December 1986. Acad. Sci. U.S.A. 44 (1958) 553–559. E-mail address: [email protected] (K. Sankaranarayanan). 1 Oak Ridge National Laboratory is managed by UT-Battelle for the U.S. properties of radioactive substances, the development of new Department of Energy under contract no. DE-AC05-00OR22725. sources of radiation such as man-made radioactivity, etc.), the

1383-5742/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2007.10.004

252 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 253 2 K. Sankaranarayanan, J.S. Wassom / Mutation Research 658 (2008) 1–27 4 K. Sankaranarayanan, J.S. Wassom / Mutation Research 658 (2008) 1–27 hazards became extended to those undertaking work with framework of radiation protection. The main landmarks in Table 1 radioactive substances. In the early years, the emphasis was on radiation protection prior to the mid-1950s are also briefly Explanation of acronyms, technical terms and dose units used in this paper the protection of the radiation worker; the protection of the considered in Section 3 for reasons of historical continuity and Acronym or technical term or dose unit Explanation general public had not been an issue in part because the number to illustrate how intuition and collective wisdom have been and BEAR, BEIR The BEAR Committee is an advisory body of the U.S. National Academy of Sciences on the of facilities that could cause exposure to members to public was continue to be used to supplement scientific knowledge. That is, Biological Effects of Atomic Radiation. From 1972 onwards, the Committee came to be relatively few and fairly isolated. With increasing use of value judgments about the relative importance of different called the BEIR (for Biological Effects of Ionizing Radiation) Committee radiation and radioactive materials, the scope of radiation kinds of risk and the balancing of risks and benefits have always DDREF Dose- and dose-rate effectiveness factor. A judged factor that generalizes the usually lower protection had to be considerably expanded to take into account been a part of the radiation protection decision-making process biological effectiveness (per unit of dose) of radiation exposures at low doses and dose-rates the evolving new realities, including exposures to fallout from [20]. as compared with exposures at high doses and high dose rates Deterministic effects Deterministic effects are those where the severity of the effect increases with the size of the nuclear weapon testing (reviewed in [9–14]). Although ICRPs role in radiation protection encompasses a dose and above a certain threshold dose, the clinical effect is almost certain to appear. These are In the early years, the emphasis was on non-transmissible broad spectrum of activities, the emphasis in this paper is on tissue reactions (injury in populations of cells) which in some cases are modifiable by consequences of radiation exposure. Not until Muller’s 1927 two ‘planned exposure’ situations, namely, occupational post-radiation procedures including biological response modifiers discovery of the mutagenic effects of X-rays in Drosophila exposures and exposures of members of the public. Radiation Dose equivalent (H) Dose equivalent H is the product of D and Q at a point in tissue, where D is the absorbed dose germ cells [15] and its subsequent extension to other kinds of exposure of patients (i.e., medical exposure) also falls under the and Q is the quality factor (related to the linear energy transfer or LET) for the specific radiation at this point, thus, H = DQ. In Publication 9 [39] ICRP defined H = DQN where N is the product ionizing radiation and other biological systems in the years that category of ‘planned exposures’, but is not considered here as of all other modifying factors specified by the Commission and was assigned a value of 1. The followed was there a new dimension to the concern over dose limits that are applicable to occupational and public earlier unit of dose equivalent was rem; now it is the Sievert (Sv). Compare with equivalent radiation effects: one of transmissible genetic risks and genetic exposures cannot be used for medical exposures. This is dose, HT, which is the weighted absorbed dose (weighted for radiation quality), in a tissue health protection. However, in spite of sustained campaigns by because the latter are conducted intentionally, for the direct or organ (also expressed in Sv) Muller on the adverse genetic consequences of carelessly and benefit of the patients and they vary depending on the Equivalent dose (HT) Radiation-weighted dose in a tissue or organ. See radiation weighting factor Effective dose (E) Tissue-weighted sum of the equivalent doses. See tissue-weighting factor avoidably exposing the human gonads to radiation, not much circumstance. Therefore, even though the same principles Genetically significant dose (GSD) Dose which if received by every member of the population would be dose expected to produce attention was paid by either the medical profession or the apply (i.e., justification, optimization and the use of individual the same total genetic injury to the population as do the actual doses received by various individuals general public early on [16,17]. dose limits), different sets of protection guidelines are used for [25]. The following formula taken from the 1972 BEIR report [47] summarizes the concept: GSD DiNiPi= NPi, where D = average gonadal dose to persons age i who received the The genetic effects of radiation exposure did not receive medical exposures. ¼ i radiation dose; N = number of persons in population of age i who received the radiation much attention until the detonation of the atomic bombs over We focus our attention on the activities of two international P i P dose; Pi = expected future number of children for persons of age i and N = number of persons Hiroshima and Nagasaki in World War II in 1945, when the scientific organizations, namely, the UNSCEAR (http://www.un- in the population of age i. (see [26] for a more detailed formula for calculating GSD) resultant radioactive fallout sparked widespread concern over scear.org) and ICRP (http://www.icrp.org), although those of ICRP International Commission on Radiological Protection (ICRP) was established in 1928 following a adverse health effects of exposures of large numbers of people some others such as the NCRP (http://www.ncrponline.org) and decision by the Second International Congress of Radiology, with the name ‘‘International X-ray and to low levels of radiation, some two decades after Muller’s the BEAR/BEIR Committees of the U.S. National Academy of Radium Protection Committee’’. In 1950, it was restructured and given its present name. Over the discovery. At that time, as narrated by Taylor [9], Sciences are also considered when necessary. There are two years, it has greatly broadened its interests to take into account the increasing uses of practices

K. Sankaranarayanan, J.S. Wassom / Mutation Research 658 (2008) 1–27 3

documents (which contain annexes on genetic effects, among concern about public exposures and the risk of stochastic others) published from the mid-1950s onwards which have effects (believed to be mainly hereditary effects at that time) provided the ‘scientific context’ for the general recommenda- looming on the horizon, in 1955, ICRP provisionally tions issued by ICRP in 1959, 1966, 1977, 1991 and 2007 recommended that individuals outside the controlled areas discussed herein. Also listed in Table 2 are the BEAR/BEIR should not receive more than 1/10th of the occupational dose- reports published during the same time frame although only limit (i.e., about 0.03 R/week or 1.5 R/year) [37]. some of these are discussed here. 4. Developments from the mid- to the late-1950s 3. Some landmarks in the history of radiation protection up to the mid-1950s In the mid-1950s, when genetic effects were assumed to be the principal effects of radiation at low doses, the scientific Table 3 summarizes the significant developments in committees involved in radiation protection introduced the radiation protection from the early 1900s up to the mid- concept of regulation of the overall average dose to the 1950s. Arguably, in the early years, deterministic effects were population and advanced the view that genetic hazards should of primary concern, and the protection guidelines, primarily be the main determinant for recommending limits of radiation aimed at the radiation worker, grew out of the radiologists’ exposure of people [38,45,53]. Although it was also well endeavors to establish a maximum ‘dose’ that could be established at that time that irradiation could result in cancer that involve the generation andtolerated,use more or less continuously by the human body. These induction and prenatal developmental effects, these were of radiation and radioactive materials. The existing system of were necessarily qualitative because of the absence of a suitable believed to occur only after large doses (>100–200 rad).2 No unit to define radiation quantitatively. These so-called doubt Muller’s genuine concern about genetic risks associated ‘tolerance doses’ were fractions of the ‘dose’ known to cause with the exposure of a large number of people to small radiation visible effects such as erythema (reddening and blistering of the doses in medical practice coupled with his unquestioned skin) [51] (item 5, Table 3). At that time, low doses of radiation authority and that he was voicing his concerns in the aftermath were deemed beneficial, largely because the uses of radiation of A-bomb explosions played a pivotal role in the genesis and were for medical purposes and there was an abundance of acceptance of these ideas. radioactive consumer products in the market [13]. As discussed below, the most significant developments in By the mid-1930s, adoption of the Roentgen (the R unit) as radiation protection during this time period included: the origin the quantitative unit of radiation exposure made it possible to of a quantitative method for estimating genetic risks; recommend quantitative tolerance doses for occupational incorporation of the concept of genetically significant dose workers in terms of R units. Taylor [9] notes that the values (GSD) in formulating recommendations on gonadal dose limits of 0.2 R/day initially recommended by the ICRP and of 0.1 R/ to workers and the population; and continual re-evaluation of day recommended by the NCRP (items 11 and 12, Table 3) in protection recommendations. the 1930s were obtained by ‘‘a crude summation and evaluation of the available results and were in fact based on an absence of 4.1. State of knowledge on genetic effects of radiation and any kind of observable effect and were probably on the in human genetics in the mid-1950s conservative side, as far as protection purposes were concerned.’’ There were four basic radiation principles guiding the In its 1949 meeting (the proceedings of which were radiation protection committees in the mid-1950s, all of which published in 1954 as NCRP Report no. 17 [52]), the NCRP emerged from extensive work with Drosophila on the induction substituted the concept of tolerance dose by ‘maximum of mutations primarily in mature spermatozoa: (1) Mutations, permissible dose’ (MPD) for workers and lowered these spontaneous or induced, are usually harmful. (2) Any dose of doses from 0.1 R/day to 0.05 R/day (=0.3 R/working week radiation that reaches the reproductive cells entails some or 15 R/year). It also considered the need to specify MPDs genetic risk. (3) The number of mutations produced is for individual tissues such as the bone marrow (the most proportional to the dose such that linear extrapolation from critical tissue) and skin (a critical organ) (see item 16 in high dose data provides a valid estimate of low dose effects. (4) Table 3). When, following a series of meetings held in the early 1950s, ICRP published a comprehensive report in 1955 [37] (item 17 2 The acceptance of the concept that cancer induction could occur at low in Table 3), most of the recommendations set forth in this report doses (and the need for caution with regard to exposures of the public at large) was catalyzed by Lewis [54]. In this paper, Lewis analysed leukemia data from were similar to those of NCRP. This was, in part, due to the fact a variety of sources (i.e., excess leukemia in radiologists, children irradiated as that prior to the ICRP deliberations, several NCRP reports had infants for thymic enlargement, patients treated by radiation for ankylosing been published and drafts of several others were made available spondylitis and A-bomb survivors in Hiroshima) and suggested that the effect at to ICRP and in part due to the considerable overlap in low doses might be linear with no threshold. While Lewis’s linearity was not membership between NCRP and ICRP [9]. In the 1955 ICRP without criticism, the major significance of the paper lies in the emphasis of the risk of cancers as the main stochastic effects of radiation at low doses and in the report, the recommended weekly dose limit of 0.3 R to the endorsement of a model for predicting risks at low doses (for which data are not radiation worker was believed to give sufficient protection available) by linear extrapolation from higher doses where human data are reasons for the focus on UNSCEAR and ICRP. First, UNSCEAR against deterministic effects. However, with widespread available (see also [55]). ‘‘...During 1944–1945, Muller’s concern about the effects radiation protection derives from the recommendations set forth in 1991 in ICRP Publication 60 [43]. publishes critical, extensive and authoritative reviews at regular of x-rays on the genetic material resulted in his undertaking The new set of recommendations [44] is expected to be published in the second half of 2007 intervals on the levels and biological effects of ionizing radiation. NCRP The present National Council on Radiation Protection and Measurements (NCRP) was originally a series of lectures under the auspices of the Society of the ICRP relies on these reviews as its primary scientific foundation established in 1929 with the name ‘‘The Advisory Committee on X-ray and Radium Protection’’ Sigma Xi to inform the public about the harmful effects of when developing recommendations on radiation protection. under the aegis of the U.S. National Bureau of Standards and was intimately related to the founding radiation. While it was probably the possible effects of of the International X-ray and Radium Protection Committee. It was renamed as the National These recommendations, in turn, form the basis for more detailed nuclear radiations that started his concern in this area, it was, Committee on Radiation Protection and Measurements in 1946 and renamed again as NCRP and codes and regulations issued by other international organizations in fact, the effects of medical exposures to radiations that chartered as a non-profit organization by the US Congress in 1964 ‘‘to collect, analyse, develop and by regional and national authorities [20]. and disseminate in the public interest information and recommendations about (a) protection drew his particular attention. This is perhaps the first The second reason for the focus on the activities of against radiation and (b) radiation measurements, quantities and units, particularly those instance where the public had been informed on a broad UNSCEAR and ICRP is a personal one. One of us (KS) had concerned with radiation protection’’ scale about the possible hazards of small amounts of Nominal risk or probability coefficient Sex- and age-at-exposure averaged lifetime risk estimates for a representative population. ICRPs the unique honor and privilege of serving UNSCEAR as radiation...’’ risk estimates are considered ‘nominal’ because they relate to the exposure of a nominal population consultant in genetics from 1970 to 2001 (with a brief of males and females with a typical age distribution and are computed by averaging over age The concern about radiation risks continued to grow with the interruption in the early 1990s) with the responsibility of writing groups and both sexes. The dosimetric quantity, effective dose, recommended for radiological extensive nuclear weapon testing in the mid-1950s: the test over the chapters [called annexes] on genetic effects of radiation for protection, is also computed by age- and sex-averaging. There are many uncertainties inherent in Bikini in Marshall Islands in the South Pacific (code-named the Committee’s reports to the United Nations General the definition of nominal factors to assess the effective dose. As with all estimates derived from epidemiology, the nominal risk coefficients do not apply to specific individuals. If one accepts BRAVO) caused substantial fallout over the atolls Rongelap Assembly. Likewise, KS served ICRP as a member of its these assumptions, then the estimates of fatality and detriment coefficients are adequate both for and Rongerik and over the Japanese fishing vessel, the Fukuryu Committee 1 on Radiation Effects from the mid-1970s to 2003 planning purposes and for general prediction of the consequences of exposure of a nominal population Maru (Lucky Dragon) [18,19]. Finally, in the mid-1950s, steps (with a roughly similar responsibility). Additionally, he was a R, rad and Gy The R (for Ro¨ntgen) was the old unit of radiation exposure based on the quantity of electrical charge 4 1 produced in air by X- or g-irradiation. A radiation field with an intensity of 1 R causes 2.58 10� C kg� ) began to be taken to seriously consider genetic effects in member of the ICRP Task Group of Committee 1 on ‘‘Risks � radiation protection recommendations. These efforts have associated with ionizing radiations’’ [21], ‘‘Genetic suscept- in dry air (C = coulomb, unit of electrical charge). Since the exposure was solely defined for irradiation in air, this quantity is no longer used. Exposure of soft tissue or similar material to 1 R would result in the 1 continued to the present day. ibility to cancer’’ [22], Chairman of the Task Group on ‘‘Risk absorption of about 100 erg of energy per gram or 0.01 joule kg� . This quantity originally called the rad estimation for multifactorial diseases’’[23] and a member of the (for radiation absorbed dose) was used as the special unit for absorbed dose for many years until it was 1 2. Aim and scope of this paper Task Group on the ‘‘Foundation document’’ [24] which provided replaced by Gray (Gy) in 1975. Gy is the special unit of absorbed dose and equals 1 joule kg� . Thus the basis for ICRPs 2007 Recommendations. In all these 1 rad = 0.01 Gy and is approximately equal to 1 R The principal aim of this paper is to capture the essence of capacities KS was intimately involved in the work of both these developments in genetic risk estimation from the mid-1950s international institutions. (i.e., when the first estimates were made) to 2007 and examine Table 1 explains the various acronyms, technical terms and how genetic risks have been incorporated into the evolving radiation units used in this paper. Table 2 lists the UNSCEAR

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Table 1 (Continued ) Table 2 Acronym or technical term or dose unit Explanation Reports published by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR), International Commission on Radiological Protection (ICRP) and the BEAR/BEIR Committees of the U.S. National Academy of Sciences during the period from the early 1900s to the year 2007 that are Radiation weighting factor (wR) Since in radiation protection, it is the absorbed dose averaged over the tissue or organ (rather than a relevant in the context of this paper and equivalent dose (HT) point) and weighted for radiation quality that is of interest, a weighting factor called radiation weighting Time period UNSCEAR ICRP BEAR/BEIR factor, wR, was introduced in Publication 60. The wR was selected for the type and energy of the radiation incident on the body, or in the case of sources within the body, emitted by the source. This weighted Early 1900s to mid-1950s –a 1928, 1934 [35,36] –a absorbed dose is called equivalent dose in a tissue or organ (symbolized by HT) and is given by the expression: HT wRDT R, where wR is the radiation weighting factor, D is the absorbed dose Mid-to-late 1950s 1958 [25] 1955 [37] 1956 [45] ¼ R ; T,R averaged over the tissue organ, T, due to radiation R. The unit of equivalent dose is the Sievert 1959 (Publication 1) [38] P (1 Sv = 100 rem in the old terminology) Early 1960s to late 1970s 1962 [26] 1966 (Publication 9) [39] 1960 [46] Rem and Sievert The rem (roentgen-equivalent-man) unit used in radiation protection grew out of the realization that 1966 [27] 1977 (Publication 26) [40] 1972 [47] equal absorbed doses of different radiations or irradiation conditions may not always give rise to equal 1972 [28] 1977 (Publication 27) [41] risks of a given biological effect. It was defined as the absorbed dose of any radiation which has the same 1977 [29] biological effect as 1 rad of 250 kVp X-rays and is equal to the absorbed dose in rad multiplied by the appropriate radiation weighting factor (wR) and other factors as may be deemed necessary. In 1978, the Early 1980s to early 1990s 1982 [30] 1985 (Publication 45) [42] 1980 [48] rem was replaced by the current unit Sievert (Sv) and 1 Sv = 100 rem. For low LET irradiations, 1 Sv = 1 Gy 1986 [31] 1991 (Publication 60) [43] 1990 [49] Planned exposure Situations where radiological protection can be planned in advance, before exposures occur, and where the 1988 [32] magnitude and extent of the exposures can be reasonably predicted. The term encompasses sources and 1993 [33] situations that have been appropriately managed within the ICRPs previous recommendations for Mid-1990s to 2007 2001 [34] 2007 [24] 2005 [50] ‘practices’. These aspects include as appropriate, design, construction, operation, decommissioning, 2007 [44] radioactive waste management and rehabilitation of the previously occupied land. All categories of a exposure can occur in planned exposure situations, i.e., occupational exposure, public exposure The Committees did not exist in the time period mentioned. and medical exposure of patients Principles of radiation protection A set of principles that apply equally to all controllable exposure situations; includes justification, It is worth stressing here that the LNT assumption remains a while unpublished at that time, were made available to optimization of protection and application of limits on maximum doses in planned situations. Justification refers to the process of determining whether either (a) a planned activity involving radiation is, overall contested issue in radiation protection. While this assumption UNSCEAR [25]. On the radiation side, while there was an beneficial, i.e., whether the benefits to individuals and to society from introducing or continuing the has been used by ICRP over the years and also by the BEIR VII extensive genetic study program involving A-bomb survivors in activity outweigh the harm (including radiation detriment) resulting from the activity; or (b) a proposed Committee [50], the French National Academies of Science Japan, when it was conceived in the late 1940s, the aim was to remedial action in an emergency or existing situation is likely, overall, to be beneficial, i.e., whether the and of Medicine [57] have raised questions about its validity. directly ascertain whether any adverse genetic effects could be benefits to individuals and to society (including the reduction of radiation detriment) from introducing For example, ICRP first rationalized the relevance of LNT in demonstrated in children of the survivors using indicators of or continuing the remedial action outweigh its cost and any harm or damage it causes. Optimization is the process of determining what level of protection and safety makes exposures, and the probability Publication 9 [38] in 1966 (see Section 5.4.2) and has used it genetic damage that were practicable at that time [63]. The goal and magnitude of potential exposures as low as reasonably achievable, economic and societal factors since then. In its most recent document, ICRP [44] reiterated its was never to detect increases in the frequencies of what would being taken into account. The dose limit is the value of the effective dose or the equivalent dose to view that the LNT model is the best practical approach in the formally be called ‘‘genetic diseases.’’ Early results from these individuals from planned exposure situations that shall not be exceeded. Dose limits are determined by protection context by noting that the ‘‘...LNT model, studies were primarily on untoward pregnancy outcomes regulatory authorities and apply to workers and to members of the public in planned exposure situations, combined with a dose- and dose-rate effectiveness factor (which included stillbirths, neonatal deaths and congenital but do not apply to medical exposure of patients, or to public exposures in emergency exposure situations, or to public exposures in existing exposure situations (DDREF) for extrapolation from higher doses and dose-rates, malformations in live births) and did not show any significant Stochastic effects Effects resulting from damage in a single cell for which the probability of the effect, but not the remains a prudent basis for radiological protection at low doses genetic effects attributable to parental radiation exposures [64]. severity, is proportional to the size of the dose. Genetic effects and cancers are the principal stochastic and dose rates.’’ The BEIR VII Committee [50] expressed a effects of radiation. For radiation protection purposes, it is assumed that there is no threshold dose similar view when it concluded that ‘‘...the current scientific 4.2. Goal of genetic risk estimation: estimating increase in for stochastic effects evidence is consistent with the hypothesis that there is a linear the frequency of genetic diseases Tissue-weighting factor (wT ) Since the relationship between the probability of stochastic effects and equivalent dose was and effective dose (E) found also to depend on the organ or tissue irradiated, a further quantity derived from the no-threshold dose–response relationship between exposure to equivalent dose was introduced, to indicate the combination of different doses ionizing radiation and the development of cancer in humans.’’ Not until the early to mid-1950s, after the A-bomb survivor to several different tissues in a way that is likely to correlate well with the total of the stochastic The French National Academies of Science and of Medicine genetic effects studies were underway, did the scientific effect. The factor by which the equivalent dose in tissue or organ T is [57] in contrast, stated that ‘‘...the LNT assumption may committees involved in estimating genetic risks accept the weighted is called the tissue-weighting factor, wT , which represents the relative contribution of greatly overestimate the carcinogenic effects of low doses assumption that exposure to ionizing radiation would cause an that organ or tissue to the total detriment due to these effects resulting from uniform irradiation of the whole body. The weighted sum of the equivalent doses in all (<100 mSv) and even more that of very low doses (< 10 mSv), increase in the frequencies of genetic diseases in the population. specified tissues and organs of the body is called the effective dose, such as those delivered during X-ray examinations’’ [58]. The assumption relied on two well-established facts, namely, E and is given by the expression: E wT HT , where wT is the tissue-weighting factor A detailed discussion of the different arguments advanced that spontaneously arising mutations in single genes have been ¼ T and H is the equivalent dose in a tissue or organ, T T P by these scientific institutions falls outside the intended scope known to cause genetic diseases and radiation was a proven UNSCEAR United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) was of this article. It suffices to note here that we believe that the mutagen. It was therefore considered logical to assume that established by the United Nations General Assembly in 1955 and currently has 21 member states participating in its activities. Starting with the first report [25] issued in 1958, UNSCEAR has approach used by ICRP and the BEIRVII Committee is a sound radiation exposure of germ cells would induce mutations that published 15 major reports on levels and effects of ionizing radiation of which the one on one for radiation protection purposes at this point in time. would cause an increase in the load of genetic diseases in the ‘‘Hereditary effects of radiation’’ published in 2001 [34] is the most recent Readers interested in recent discussion of the LNT model may population. The acceptance of the assumption helped to define consult the series of four papers [58–61] published in 2006. what remains the central goal of genetic risk estimation: the We return now to consider the state of knowledge in human detection and quantification of increases in the frequency of The effect is independent of the rate at which the radiation is linear, no-threshold (LNT) model continues to be the preferred genetics and on radiation effects in humans in the mid-1950s. genetic diseases in populations exposed to ionizing radiation. delivered and of spacing between the exposures ([56]; see Fig. model for developing radiation risk coefficients and for The available human genetic data were limited to baseline While this goal remains central to risk estimation even now, as 1.1. in [11]). predicting radiation effects at low doses by ICRP and other frequencies of several naturally occurring hereditary defects discussed later (in Section 8.1), it has recently been scrutinized The concept of linear relationship between dose and effect organizations. Additionally, this model is used either implicitly and estimates of spontaneous mutation rates for some single in light of advances in molecular understanding of the which first emerged from the above studies has had a profound or explicitly to assess low dose mutagenic and carcinogenic clinical entities. Some of these data, such as those of Stevenson relationships between radiation-induced mutations and their impact on radiation protection between then and now. The effects of chemical agents as well. on genetic defects in the population of Northern Ireland [62] phenotypic effects.

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Table 3 ‘‘equilibrium population’’ is exposed to radiation, additional 4.4. Recommendations for radiation protection in the mid- Some important dates and developments in radiation protection standards from the early 1900s to the late-1950s (based on [9,10,12–14]) mutant genes introduced into its gene pool are subsequently to late-1950s Item Year Development subject to the action of natural selection, and, eventually, the population attains a ‘‘new equilibrium’’ between mutation and Three committees made recommendations for radiation 1 1913 German Ro¨ntgen Society issued radiological protection advice 2 1915 British Roentgen Society recognized the hazards of X-rays in a warning statement selection. The time (in generations) it takes to attain the new protection in the period from the mid- to late-1950s, namely, 3 1921 British adopt qualitative radiation protection proposals equilibrium and the rate of approach to this are dependent on the BEAR Committee, the Committee of the British Medical 4 1922 American Roentgen Society adopts radiation protection rules the duration of radiation exposure (i.e., one generation only or Research Council and the ICRP. Each is discussed in turn below. 5 1925 Concept of ‘tolerance dose’ advanced by Mutscheller [51]; he defined it as the ‘dose which the operator can, for every generation), the type of genetic disease, induced mutation a prolonged period of time, tolerate, without ultimately suffering injury’. He found that members of staff of several rate and the intensity of selection. good radiation installations received what he described as about 1/100 of an ‘erythema dose’ 4.4.1. The BEAR Committee (also known as the skin unit dose) and stated that ‘‘...it is entirely safe if an operator does not receive every 30 days The DD is the amount of radiation required to produce as The BEAR Committee’s [45] recommendation for protec- a dose exceeding 1/100 of an erythema dose.’’ many mutations in a population as those that arise sponta- tion of the general population from man-made sources of 6 1928 International Committee on X-ray and Radium Protection (the present ICRP) established neously in a generation. Ideally, it is estimated as a ratio of the radiation was dictated by a key consideration, namely, that of 7 1928 First international recommendations on radiation protection adopted by second International Congress of Radiology average spontaneous mutation rate of a set of genes to the staying at or below the natural background level. As mentioned 8 1929 U.S. Advisory Committee on X-ray and Radium Protection (the present NCRP) established average-induced mutation rate in the same set of genes. A large 9 1931 Adoption of the Roentgen (R) as a quantitative unit of radiation exposure; consolidation of information leading to the in Section 4.3, the Committee recommended that ‘‘for the conversion of dose measurements from skin erythema dose to R units (a skin erythema unit dose as employed at that DD signifies low relative mutation risk, and a small DD a high present it be accepted as a uniform national standard that X-ray time was about 600 R and consequently 6 R/month was the tolerance dose in entry 5 above) relative mutation risk. The reciprocal of DD (i.e., 1/DD) is the installations (medical and non-medical), power installations, 10 1931 U.S. Advisory Committee recommends a ‘tolerance dose limit’ of 0.2 R/day for radiation workers relative mutation risk (RMR) per unit dose. disposal of radioactive wastes, experimental installations, 11 1934 International Committee on X-ray and Radium Protection establishes a similar limit for radiation workers With very limited data on mutation rates and on the testing of weapons, and all other human controllable sources 12 1936 U.S. Advisory Committee lowers the limit to 0.1 R/day for radiation workers; while this limit initially was intended for incidence of diseases with simple genetic origin, the BEAR X-ray exposure, it was also applied to g-rays from radium, and served as a protection standard for workers on the of radiation be so restricted that members of our general Manhattan Project during World War II Committee assumed that, firstly, about 2% of all live born population shall not receive from such sources an average of 13 1946 U.S. Advisory Committee reorganized to become the National Committee on Radiation Protection (NCRP) children are, or will be, seriously affected by defects with ‘a more than 10 R in addition to background, of ionizing radiation 14 1950 International Commission on Radiological Protection (ICRP) and International Commission on Radiological Units simple genetic origin’ and secondly, the doubling dose was as a total of accumulated dose to the reproductive cells from (ICRU) reorganized from pre-war committees; In its 1950 recommendations, ICRP stated that ‘‘the figure of 0.2 R/week ‘almost surely between 5 and 150 R and probably between 30 R conception to 30 years.’’ Since this limit included medical for (radiation workers) seems very close to the probable threshold for adverse effects’’; it provided a list of health effects and 80 R’. Under the further assumption that for the above to be kept under review which includes the induction of malignant tumors, cataracts (and other less likely effects) exposures as well (estimated as about one half of this), this and genetic effects fraction of human genetic defects, the incidence is proportional meant a limit of 5 R/generation (=30-year period) for all man- 15 1953 Adoption of rad as the unit of absorbed dose by ICRU to mutation rate, and assuming 40 R as a reasonable value for made sources, but excluding medical exposures and natural 16 1954 NCRP report no. 17 [52]: (a) substitution of the concept of ‘maximum permissible dose’ (MPD) for tolerance dose; MPD the DD, the effect at the new equilibrium after a continuing background radiation. Of note is that the NCRP and ICRP defined as ‘the dose of ionizing radiation that, in the light of present knowledge, is not expected to cause appreciable bodily exposure to 10 R/generation3 continued indefinitely was cooperatively worked with the BEAR Committee in the 1950s injury to a person anytime during his life time’; (b) lowering of the MPD for radiation workers from 0.1 R/day to 0.05 R/day computed as a product of disease incidence (P), 1/DD and and expressing it on a weekly basis (i.e., 0.3 R/working week = 15 R/year); (c) concept of critical tissue/organs; blood-forming and so it is not surprising that most of the conclusions reached organs as the most critical tissue (MPD of 0.3 R/week); skin as critical organ (MPD of 0.6 R/week); discussion of radiation dose as follows: by these committees were similar [14]. the general concept of acceptable risk and recommendation that radiation exposure be kept at level ‘as low as practicable’. For radiation workers, the principal recommendation of the Concept of risk in relation to cost or 1 1 BEAR Committee was that no worker should receive more than risk P dose 2% 10 benefit introduced for the first time DD 40 a total cumulative dose of 50 R to the reproductive cells up to 17 1955 The 1955 ICRP recommendations [37]; most were similar to those in NCRP report no. 17; MPD of 0.3 rem/week (=15 rem/year) ¼ � � ¼ � �   age 30, and not more than an additional 50 R up to age 40. At for occupational workers; first recommendations for protection standards applicable to the general public: no member of the public 0:5% or 5000 cases per 106 live births (1) (i.e., any one other than the radiation worker) should be exposed to more than one-tenth of the MPD for occupational worker ¼ those ages, over half or over 9/10ths of their children, respectively, would have been born. For practical purposes, this The value (i.e., 5000 cases per million live births) under the meant limiting the occupational exposure of individual stated radiation conditions represented the predicted total radiation workers to 5 rem/year.4 4.3. The doubling dose method and the estimation of tions regarding the doubling dose (DD), natural incidence of number of ‘new cases’ of tangible inherited defects. It was genetic risks genetic diseases in the population and the fraction of natural assumed that about 1/10th of this number (or 500 new cases per 4.4.2. MRC incidence proportional to mutation rate. Not surprisingly, million births) would be manifest in the first post-radiation The conclusions of the Committee of the British Medical The BEAR Committee was one of the first charged with the Muller was the person who laid the conceptual foundations for generation. UNSCEAR used the same general approach to the Research Council (MRC), set up with objectives similar to task of quantifiably estimating genetic risk [45]. With no actual this method during the 1950s [65–67]. The DD method has problem of risk estimation in its first report published in 1958 those of the BEAR Committee, were essentially the same data on radiation-induced adverse genetic effects in humans, evolved over the years and is still in use [68]. Since, in this [25]. although not expressed in such quantitative terms [53]. let alone data on induced genetic diseases, the Committee paper, we use the risk estimates obtained using the DD method Rather than providing a precise predictive value, the above faced a difficult task. Noting that, firstly, exposure to ionizing to facilitate comparisons, the principles of the method are illustrative arithmetic exercise and calculation of the 5000 cases 4.4.3. ICRP radiation, even in small doses, could cause adverse genetic discussed below in some detail. per million live births value was undertaken mostly to reassure As mentioned in Section 3, the ICRP recommended a effects that could be serious in individual cases and potentially The genetic theory underlying the DD method is known as the scientific community and the public at large that with the weekly limit of 0.3 R to the radiation worker in its 1955 report harmful over a lifetime for the entire population and, secondly, the ‘‘equilibrium theory,’’ a theory that population geneticists recommended dose limit to the population (i.e., 10 R/ [37]. The Commission was cognizant of the fact that if the germ cell mutations resulting from radiation exposure would commonly use to explain the dynamics of mutant genes in generation), the predicted genetic risks would indeed be small. worker were to be occupationally exposed at this rate, 50 weeks cause increased risk for future generations, the Committee populations. It is based on the concept that the stability of sought a method that would provide a risk estimate covering mutant gene frequencies (and thus of disease frequencies) in a 4 both first generation progeny (i.e., following irradiation) and population is a reflection of the existence of a balance between Before making this final recommendation, the BEAR Committee had 3 The 10 R/generation was the exposure limit to the population (in addition to inquired the Atomic Energy Commission (AEC) on the range of practical total genetic risks (i.e., over all future generations, assuming two counteracting forces: spontaneous mutations, which arise the background and including medical exposures) that the BEAR Committee radiation levels that were being reached in normal operation. It turned out that that the population is exposed to radiation in every generation). and enter the gene pool at a finite rate every generation; and recommended in 1956 [45]. Importantly, the recommendation had nothing to do because of the normally cautious procedures of the AEC, it was a rare The Committee devised an indirect method known as the natural selection, which eliminates these same mutations with the numerical estimates of genetic risk but was based on different occurrence for any one to exceed more than about 1/10th of the permissible ‘‘doubling dose method,’’ based on genetic theory, assump- through failure of survival or reproduction. When such an considerations (as discussed in Section 4.4.1). occupational exposure allowed at that time (approximately 15 rem/year).

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a year, for 50 years, the permissible whole body dose would For example, in estimating GSD from medical diagnostic Table 4 amount to 750 R, a large lifetime dose indeed! After significant radiation, the gonadal dose at each age and sex is weighted by Comparison of the estimates of the baseline frequencies of genetic diseases (expressed as number of cases per 100 live born) made in 1959 with those made in public and international debate on this issue, in 1956, the the expected number of future children for a person of that age subsequent years until 2001 Commission adopted changes that corresponded to a reduction and sex. For natural radiation, the GSD is assumed to be the from 0.3 R to 0.1 R/week (=5 R/year) for the radiation worker same as the gonad dose since exposure is uniformly distributed (and 1/10th of this for members of the public). The Commission over all ages. It is somewhat less than the whole body radiation also recommended inclusion of dose limits to a number of because of the shielding of the gonad by other tissues. The ‘critical’ organs, including the gonads, lens of the eye and the occupational exposure is obtained by considering the total blood-forming organs (5 rem/year and a quarterly limit of radiation received by those occupationally exposed, and 3 rem in any 13 consecutive weeks) [14,69,70]. Additionally, treating this total as if it were uniformly distributed over the the total permissible accumulated doses to workers at various whole population. ages were specified: 50 rem up to age 30 years; 100 rem to age Based on exposure data from several countries, in its 1958 40 years and 200 rem lifetime dose to 60 years. report [25], UNSCEAR noted that the highest GSDs were With respect to the general population, in its 1959 caused by diagnostic X-ray exposures, which at that time, were recommendations, the ICRP [38] suggested that ‘‘the genetic frequently carried out with fluoroscopy rather than with dose to the whole population from all sources additional to the radiography. Diagnostic procedures were classified into 23 natural background should not exceed 5 rem plus the lowest types, and the exposure data for these were presented for a few practicable contribution from medical exposure.’’ This meant countries. More than 80% of GSD was found to be contributed a b c d e 5 rem distributed over a 30-year period or 170 mrem/year. As by only six or seven procedures (i.e., those in the region of the Including both trivial and serious anomalies. Including X-linked disorders. Including chromosomal diseases. No information. Including Down syndrome (0.15), will be evident, ICRPs recommendations were similar to those lower abdomen and pelvis) which together made up only about other autosomal trisomies (0.05), Klinefelter syndrome (0.17), Turner syndrome (0.03) and Cri du chat syndrome (0.02) but excluding XXX females (0.12) and individuals with translocations (0.50). fAll but 3% are due to Down syndrome. gFrom chromosomal surveys of newborns (see Table 11 in 1977 UNSCEAR report [29]. of the BEAR Committee. 10% of all procedures. The total GSD from X-ray procedures hFrequency of unknown diseases is not included in the total. ranged from 17 to 150 mrem/year in the various national 4.4.4. The introduction of the concept of age proration estimates. The data indicated that it might be possible to reduce While there was general agreement on the recommended the doses considerably, simply by careful attention to Table 4. By the time of the 1972 BEIR report [47], the led UNSCEAR, BEAR/BEIR and ICRP Committees to revise lowering of the basic occupational exposure from a level of 15 techniques and equipment and the avoidance of all unnecessary consensus view was that about 6% of live births are affected by their genetic risk estimates and protection recommendations to 5 rem/year, NCRP [70] was concerned that with a such a examinations. Recommendations in this regard were provided one or another kind of genetic diseases, the breakdown being: in several ways. The revisions of each are discussed in turn relatively low value, the chance of occasional overrun would by the Joint Study Group report of ICRP and ICRU [71]. In the autosomal dominants and X-linked diseases, 1%; chromosomal below. increase. Therefore efforts were made to find a procedure for same report, the contribution of occupational exposure to the and recessive diseases, 1%, and diseases with complex patterns setting up some kind of reasonable averaging mechanism such annual GSD was estimated at about 0.2–0.5 mrem. of inheritance (including congenital anomalies, constitutional 5.3.1. UNSCEAR that, if there was a small overrun in one period, it might Efforts at estimating the GSD from medical practice in and degenerative diseases), 4.0%. Based on mouse data on reduced effectiveness of chronic somehow be averaged out over the following periods. These different countries continued for many years and have been irradiation in inducing specific locus mutations, UNSCEAR efforts led to the development of a concept of ‘age proration’ reviewed in the 1966, 1972, 1977, 1982, and 1988 UNSCEAR 5.2. Discovery of dose-rate and dose-fractionation effects increased their estimate of the doubling dose (recall that a whereby the worker’s total exposure could be related to his age reports [27–30,32]. These data have been used to devise/ for mutation induction in mice greater DD signifies a lower relative mutation risk) and revised in terms of the relationship: recommend ways of reducing patient doses. However, they are their genetic risk estimates accordingly. As stated in the 1962 of only marginal relevance in the context of the present paper D 5 N 18 rem (2) The discovery, in the late 1950s and early 1960s, of dose-rate report [26], ‘‘... for chronic irradiation of males, new ¼ ð � Þ and are therefore not considered further. effects for mutation induction in germ cell stages of relevance information suggests that the DD is about 4 times the 1958 where D is the maximum permissible cumulative exposure, N is (stem cell spermatogonia in males and oocytes in females) value (for acute irradiation) of 30 rad... for chronic low the age in years. For a person who is occupationally exposed at 5. Advances in human genetics and radiation genetics represents a major advance in mouse radiation genetics. In intensity irradiation of females, mutation rates seem to be lower a constant rate of 5 rem/year from age 18, the formula implies a from the early 1960s to the early 1970s, revision of spermatogonial cells, the yield of mutations at low dose-rates than in males...a permanent doubling of mutation rate would maximum permissible cumulative exposure of 0.1 rem/week. genetic risk estimates and their impact on was only one-third of that obtained at high dose rates [73,74]. In ultimately double the prevalence of the serious defects under In the NCRP guidance, a worker was permitted to receive up to recommendations for radiation protection the early 1970s, Lyon et al. [75] demonstrated that when a large consideration. These are now estimated to have a prevalence at 3 rem/quarter (=12 rem/year) provided the cumulative limit of dose such as 6 Gy was administered in 60 daily fractions of about 1%.’’ Of note here is that UNSCEAR assumed a 1% 5(N 18) rem was not exceeded. In 1959, the ICRP [38] Advances in genetic risk estimation and radiation protection 0.1 Gy to stem cell spermatogonia, the mutation rate was figure for autosomal dominant diseases and tended towards a � adopted the 5 (N 18) rem limit on cumulative dose to the recommendations during this period related to (a) the reduced to one-third of that after a single unfractionated dose of DD value of 100 rad for these disorders (for chronic low linear � worker. These protection recommendations remained in place continuing re-appraisal of Stevenson’s 1959 data [62] on the 6 Gy. The dose-rate effect was even more pronounced in energy transfer [LET] irradiation conditions) but was not for the next 18 years. baseline frequencies of genetic diseases in the population of females: the mutation rate after chronic irradiation was only explicit about the value at that time. Northern Ireland by UNSCEAR and the BEIR Committee (i.e., about 1/20 of that after acute irradiation (sampling of mature In the 1972 UNSCEAR Report, the starting assumption was 4.4.5. Genetically significant dose the same data on which initial genetic risk estimates were and maturing oocytes) [76]. It was thus clear that the early that 30,000 per million live born progeny would be affected by The concept of genetically significant dose (GSD) intro- based; see Section 4.1) and (b) the discovery of dose-rate and BEAR and MRC reports (which had assumed no dose-rate and deleterious traits maintained by mutation. Under the further duced in the late-1950s represents an attempt to estimate the dose-fractionation effects for mutation induction in mouse no dose-fractionation effects for induced mutations) had assumptions that firstly, the above estimate reflects the accumulated gonad dose from various sources (natural back- germ cells and the realization that early estimates of genetic overestimated the genetic risk. situation in an equilibrium population and secondly, the DD ground, medical, occupational exposures, etc.,) that is relevant risk had been overestimated. is 100 rad (as suggested by the analysis of Lu¨ning and Searle for the production of heritable effects. Formally defined, GSD 5.3. Revised genetic risk estimates and protection [77] for five different end-points in male mice), it was ‘‘...is the dose which if received by every member of the 5.1. Advances in human genetics recommendations estimated that 1 rad of irradiation to males of the parental population would be expected to produce the same total genetic generation would add a total of 300 cases of affected children injury to the population as do the actual doses received by Reappraisals of the baseline frequencies of genetic diseases These advances in mouse and human genetics and the per million of which about 6–15 cases would be manifested in various individuals’’ [25]. (by UNSCEAR and BEIR Committee) are summarized in growing awareness of dose-rate and dose-fractionation effects the first generation.

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Table 5 additional information is available or to modify its former Table 6 Estimated genetic effects of 5 rem/generation on a population of one million live births in the 1972 BEIR 1 report [47] recommendation to allow for a probable influence of dose-rate Dose limits for individuals recommended by ICRP in 1966 in Publication 9 [39] Disease classification Current incidence per million (P) Effect of 5 rem/generation on the hereditary effects of radiation in man.’’ Organ or tissue MPD for adults Dose limits The focus of Publication 9, however, was not on genetic exposed in the for members First generation Equilibrium a effects, but on something else, namely, cancers. This was because course of of the public a b their work Dominant + X-linked diseases 10,000 50–500 250–2500 of the fact that the years following the publication of the 1959 b c,d Chromosomal and recessive diseases 10,000 Relatively slight Very slow increase [38] recommendations witnessed a shift in emphasis from Gonads, red bone-marrow 5 rem in a year 0.5 rem in a year Multifactorial diseases Congenital anomalies 40,000 5–500c 50–5000d c e deterministic to stochastic effects, with cancer induction Skin, bone, thyroid 30 rem in a year 3.0 rem in a year (15,000), anomalies expressed later (10,000) Hands and forearms; 75 rem in a yearc 7.5 rem in a year and constitutional and degenerative diseases (15,000) emerging as the principal stochastic effect of radiation. feet and ankles c Total 60,000 60–1000 300–7500 Publication 9 marked a substantial change in radiation protection Other single organs 15 rem in a year 1.5 rem in a year philosophy, with a new focus on stochastic, not deterministic, a Note that the dose limits for members of the public are 1/10th of those for Assumed doubling dose: 20–200 rem. Note: P = baseline frequency; RMR = relative mutation risk (i.e., 1/DD); D = dose = 5 rem; f = fraction of the predicted effects; and it started to lay the groundwork for integrating both equilibrium increase expressed in the first generation (assumed to be 20% for dominant diseases and 10% for multifactorial diseases) and MC = mutation component. occupational workers. b a Risk to the first generation = P RMR D f (assumed to be = 0.2). With a 20 rem DD, the risk = 10000 0.05 5 0.2 = 500 and with a 200 rem DD, cancers and genetic effects into the evolving framework of The gonads and the red bone-marrow are considered the critical organs       RMR is 0.005 and the risk is 1/10th of 500, i.e., 50 cases. radiation protection thus ushering it into what Clarke and when the whole body is exposed uniformly; therefore, throughout these b Risk at equilibrium = P RMR D; with a 20 rem DD, the risk = 10000 0.05 5 = 2500 cases; with a 200 rem DD, it is 1/10th of 2500, i.e., 250 cases. Valentin [13] call the ‘modern era’ in radiation protection. recommendations, dose limits given for these organs also apply to all cases c     Risk to the first generation = P RMR D MC f (assumed to be = 0.1%). The MC has been assumed to be in the range from 5 to 50%. With a 20 rem DD of uniform irradiation of the whole body.     With respect to acceptability of risk, Publication 9 concluded c and MC of 5%, risk = 40000 0.05 5 0.05 0.10 = 50 cases. With MC = 50%, it is 10 times the above value, i.e., 500 cases. Subject to the limitations that in any 1 year, the MPDs should not be d     (in paragraph 52): ‘‘...as any exposure may involve some degree Risk at equilibrium = P RMR D MC. With a 20 rem DD and mutation component of 5%, risk = 40000 0.05 5 0.05 = 500; if the MC is 50%, the exceeded, but in a period of a quarter of a year, up to one-half of the annual estimate will become = 5000 cases. When  the DD is 200 rem (RMR = 0.005) and MC = 0.05, the estimate will be = 50 cases and finally when MC = 0.5, it will be of risk, the Commission recommends that any unnecessary MPD, or for internal exposure, a dose commitment resulting from an intake of 500. Of note here is that in subsequent modeling studies Denniston et al. [88] found that MC will be = 1 at equilibrium also for chronic multifactorial diseases. exposure be avoided, and that all doses be kept as low as is readily radionuclide equivalent in amount to the intake for one-half year at the achievable, economic and social considerations being taken into maximum permissible concentration may be accumulated in conformity with account ’’ 1. (Note that the italicized words above replaced the the considerations of additivity and multiple organ irradiation. 5.3.2. BEIR/BEAR committees exceed 10 R of man-made radiation, and should be kept as far ... d Special recommendations apply for women of reproductive capacity (see The report of the BEIR I Committee published in 1972 [47] below this as is practicable.’’ words ‘lowest possible’ used in Publication 1 [38].) By this time Publication 9 for details). remains a significant one for at least three reasons: for the first The 1972 BEIR I report [47], on the other hand, issued in the history of radiation protection considerations, as Clarke e 1.5 rem to the thyroid of children up to 16 years of age. time in the history of risk estimation, the report (1) rationalized revised recommendations based on its review of the sources and and Valentin stated [13], ‘‘...the problem had become one of the use of human data on spontaneous mutation rates and mouse magnitudes of GSD from natural and man-made sources in the limiting the probability of harm and much of what had infrequent situations) and the expression of MPD for the gonads data on induced rates for estimating DDs; (2) introduced the U.S. together with new data on radiation genetic effects in mice subsequently developed related to the estimation of the and the blood-forming organs as an annual dose of 5 rem (i.e., concept of mutation component (MC) to deal with risk (i.e., dose-rate effects for induced mutations). The GSD from probability of harm and the decision on what level of implied 50 mSv/year). The term ‘‘dose limit’’ was introduced for the estimation for multifactorial diseases for which the relationship natural sources was assessed at about 90 mrem/year and that risk is acceptable, or more importantly, unacceptable’’. annual limit of 0.5 rem recommended for members of the public between mutation and disease is not straightforward, and (3) from man-made sources at about 30–60 mrem/year. The Additionally, Publication 9 endorsed the need to use the (i.e., 1/10th of the MPD for occupational exposures). presented estimates of risk for all classes of genetic diseases, Committee made the following conclusions and recommenda- linearity assumption between dose and effect for cancers. On the basis of data on medical and other exposures but emphasized the tenuous nature of the assumptions that were tions: ‘‘...It seems clear that the genetically significant Paragraph 7 reads: ‘‘...The Commission sees no practical contributing to GSD (as reviewed by UNSCEAR in 1962 [26]), used, especially for multifactorial diseases. exposure from fallout, from nuclear power developments and alternative, for the purposes of radiological protection, to Publication 9 noted that ‘‘...the total genetic dose from all The BEIR I Committee estimated the risks of exposure of the from occupational exposure (treated as part of the overall assuming a linear relationship between dose and effect, and that man-made sources actually being received by the world population to 5 rem/generation (i.e., the exposure limit for the population average) is now very small relative to that from doses act cumulatively. The Commission is aware that the population ...appears considerably lower than 5 rem, and it population recommended in the 1956 BEAR report [45]; see natural radiation. There is no reason to think that the dose assumptions of no threshold and of complete additivity of all seems unlikely that this figure will be approached within the Section 4.4.1) based on DD range of 20–200 rem5 and baseline commitment for the development of nuclear power in the next doses may be incorrect, but is satisfied that they are unlikely to foreseeable future.’’ frequencies of 1% for autosomal dominants, 1.0% for few decades should be more than 1 mrem annually. The 1956 lead to the underestimation of risks.’’ The use of this linearity Table 6 provides a summary of dose limits for occupational chromosomal and recessive diseases, and 4.0% for congenital report and the guides that grew out of it were the result of an assumption has continued to the present day. workers and for members of the public recommended by ICRP anomalies and other multifactorial diseases. These estimates effort to balance genetic risks against the needs of the society. It Also in Publication 9, the ICRP defined a new radiation in Publication 9. are summarized in Table 5 and show that, under the stated now appears that these needs can be met with very much less quantity called the dose equivalent for use in radiation radiation regimen, the total risks (i.e., at the new equilibrium) than the 170 mrem/year of the current Radiation Protection protection. The premise behind the new measure was that 6. Genetic risk estimates in the 1977 UNSCEAR report are of the order of 300–7500 affected cases per million progeny Guides. Accordingly, the 170 mrem seem to provide an equal absorbed doses may not always give rise to equal risks and ICRP recommendations in 1977 (Publication 26) and 60–1000 cases per million progeny in the first post- unnecessarily large cushion. Likewise, we believe that the (since the biological effectiveness may be affected by radiation generation. currently much higher level of radiation from medical sources differences in type of radiation or in irradiation conditions); Major developments during this period included further With regards to protection recommendations based on these (mainly diagnostic) should be examined in view of the same the dose equivalent, symbolized by H (and expressed in rem), revisions of genetic risk estimates based on new baseline estimates, while the 1960 BEAR report [46] had taken note of concept. If it can be reduced without impairing essential was obtained by multiplying the absorbed dose at a point by one frequencies of genetic disease; incorporation of the concept of the data on dose-rate effects for the induction of mutations in medical service, then the present level is unnecessarily high.’’ or more weighting factors as shown below: harm into risk estimates; and development of a new risk-based system of protection. mouse germ cells, that Committee did not consider the need for H DQN (3) any revision in recommendations. It simply reiterated its earlier 5.3.3. ICRPs 1966 recommendations (Publication 9): ¼ conclusion that ‘‘... for the general population, the average paving the way for a focus on cancer and a new system of where D is the absorbed dose in rad, Q the quality factor and N 6.1. Risk estimates in the 1977 UNSCEAR report gonadal dose accumulated during the first 30 years should not protection is the product of all other modifying factors specified by the As was the case with the BEAR 1960 document [46], ICRPs Commission. The quality factor is related to the linear energy When preparing its 1977 report [29], UNSCEAR once again Publication 9 [39] did not consider it necessary to alter the transfer or LETof the radiation and N was assigned a value of 1. re-visited the issue of baseline frequencies of genetic diseases in protection recommendations in the light of dose-rate effects In later years, with the absorbed dose expressed in Gy, the rem light of a paper recently published on the incidence of genetic 5 Based on the assumption that the average spontaneous mutation rate of 6 5 mentioned above and had the following cautious statement was replaced by Sievert (1 Sv = 100 rem). diseases in the population living in the Canadian province of human genes is likely to be in the range between 0.5 10À and 0.5 10À per   gene and that the average induced rate for both sexes, as observed in mice for about this: ‘‘...for the time being...the Commission does not Other notable revisions included the abandonment of the age- British Columbia (BC) [72]. Importantly, the BC study estimates 7 chronic radiation, is 0.25 10À /gene rem. consider it advisable to draw too general conclusions before prorated formula for occupational exposures (except for some for autosomal dominant diseases were not only much lower than Â

262 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 263 K. Sankaranarayanan, J.S. Wassom / Mutation Research 658 (2008) 1–27 13 14 K. Sankaranarayanan, J.S. Wassom / Mutation Research 658 (2008) 1–27 those previously used by UNSCEAR, but that they were also 6.2. The 1977 ICRP recommendations (Publication 26) The ICRP therefore assumed that the risk to radiation workers Table 8 lower than those derived from several ad hoc studies (which used would be acceptable if the average annual cancer mortality risk Estimates of risk factors and tissue-weighting factors (wT ) presented by ICRP in 4 1977 in Publication 26 [40] all available means of ascertainment of cases) on the prevalence For the first time, in its Publication 26, ICRP [40] introduced did not exceed 10À . 4 Tissue at risk Risk factor/Sv ( 10 ) wT of individual dominant conditions. On the other hand, the BC a new risk-based system of protection intended to prevent the The implementation of the new risk-based system of  estimates for multifactorial diseases were higher than those occurrence of deterministic effects and to limit the occurrence protection required several key conceptual changes in addition 1. Gonads 40 0.25a previously used by UNSCEAR. Additionally, extensive data on of stochastic effects to an acceptable level. The three core to those introduced in Publication 9 [39] (see Section 5.4.1): 2. Breast 25 0.15 chromosomal anomalies in newborns from surveys conducted in principles underlying this new system were the following: (1) First, the concept that genetic risk should be the principal 3. Red bone marrow 20 0.12 different countries had become available (see Table 11 in [29]). no practice shall be adopted unless its introduction produces a concern in setting dose limits (which had dominated the field 4. Lung 20 0.12 5. Thyroid 5 0.03 Professor Cedric Carter, an eminent British clinical geneticist positive net benefit (‘‘justification of a practice’’); (2) exposures since the mid-1950s) and that of ‘‘critical’’ organs or tissues 6. Bone surfaces 5 0.03 with considerable knowledge and practical experience in the field shall be kept as low as reasonably achievable, economic and (which had been used in earlier recommendations) were 7. Remainderb 50 0.30 was an adviser to the UK delegation to UNSCEAR; he helped the social factors being taken into account (‘‘optimization of abandoned. These were replaced by the concept that Total 165 1.00 Committee in the re-appraisals (see also [78,79]). The estimates protection’’) and (3) the dose to individuals shall not exceed the emphasized total stochastic risk—that is, the risk of cancers from the BC study and those which UNSCEAR used in its 1977 limits recommended for the appropriate circumstances by the in specified organs/tissues that are susceptible to cancer For all tissues other than gonads, the risk factors pertain to the risk of fatal cancers. For gonads, the risk factor pertains to serious hereditary disease in the report [29] are given in Table 4. Commission (‘‘individual dose limits’’). induction plus genetic effects and their impact. progeny of the first two post-radiation generations under a scenario of the Also in its 1977 report [29], UNSCEAR re-affirmed its view Using this new system, ICRP assessed the levels of risk Second, in order to be able to incorporate the concept of population exposed to radiation generation after generation. expressed in 1972 [28] that, for radiation conditions applicable associated with the dose-equivalent limits for both occupational harm, a new concept called detriment was introduced. a Note that genetic effects have been assessed to contribute only 25% to the to risk estimation (i.e., low dose, low dose rate and low LET exposures and for members of the public that had been in effect Detriment was defined as the mathematical expectation of total detriment. b irradiation), a DD of 100 rad was appropriate. In conjunction for over 20 years (see Table 6) and found that its previously the ‘harm’ incurred from an exposure to radiation which took Includes tissues (e.g., stomach, large intestine and possibly liver) for which no estimates of risk factors could be made because there were no data. with the revised estimates of baseline frequencies of genetic recommended system of dose limitation remained robust (i.e., it into account not only the probability of each type of deleterious diseases, this DD estimate was used to predict risks at the new had not failed to provide an adequate level of safety). In the end, effect, but also a judgment of the severity of the effect. The equilibrium and in the first generation. the Committee reaffirmed the use of a dose equivalent limit of detriment concept permitted the summation of adverse health of each of the tissues/organs to total detriment (i.e., tissue- For the latter, the UNSCEAR accepted the judgment of the 5 rem/year for radiation workers and abolished the genetic dose effects according to the relative sensitivities of the irradiated weighting factors (wT )) normalized to unity. The risk factors BEIR I Committee, namely, that the first generation risk was limit of 5 rem/generation for population exposures. tissues. However, the concept did not really become a formal and wT estimates are presented in Table 8. It is noteworthy that likely to be 20% of that at equilibrium for autosomal dominant With respect to acceptability of risk (an issue first considered part of radiation protection philosophy until it was more genetic effects account for only 25% of the total detriment. and X-linked diseases and 10% of that at equilibrium for in ICRPs 1966 Publication 9), ICRP stated in Publication 26 formally addressed in Publication 27 [41]. Entitled ‘‘Problems Regarding differences in estimating risk for radiation multifactorial diseases. However, it assumed that the MC [40]: ‘‘...for the foreseeable future, a valid method for judging involved in developing an index of harm,’’ this later report was workers versus members of the public, Publication 26 [40] (mutation component) for the latter class of diseases is of the the acceptability of risks in radiation work is by comparing this prepared by Sir Edward Pochin at the request of ICRP. It set the states in paragraph 60: ‘‘...For the purposes of radiation order of 5% (in contrast to the range of 5–50% assumed by the risk with that for other occupations recognized as having high stage for the development of the detriment concept by protection involving individuals, the Commission concludes BEIR I Committee). The newly revised risk estimates obtained standards of safety which are generally considered to be those considering both the limitations and utility of certain indicators that the mortality risk factor for radiation-induced cancers is 2 1 using these values are presented in Table 7. They show that in which the average annual mortality due to occupational of detriment, such as fatality, mean loss of life, various about 10À SvÀ , as an average for both sexes and all ages. The 4 following irradiation at a rate of 1 rad/generation, the risk at hazards does not exceed 10À ...’’ (Paragraph 96). It was occupational injuries and their severity (expressed, for instance, average risk factor for hereditary effects, as expressed in the equilibrium is about 185 affected cases per million progeny suggested that at levels imposed by adherence to the as the total number of working days lost), and it compared these first two generations,7 would be substantially lower than this, (0.17% of baseline frequency) one-third of which (63 cases or recommended dose limits, radiation exposure would be indicators with similar consequences of somatic and genetic when account is taken of the proportion of exposures that is 0.06% of the baseline frequency) is expressed in the first expected to cause very few injuries or illnesses in exposed effects of radiation exposures. Although these different ways of likely to be genetically significant and can be taken as about 3 1 generation. workers other than any malignant disease that may be induced. considering harm were not yet ready for inclusion in 4 10À SvÀ . Both for somatic and for the hereditary risk Publication 26, an initial effort was made to incorporate the factor, the estimates will differ somewhat for workers and for Table 7 concept of harm in risk estimates, as noted below. members of the general public, because of the differences in age The 1977 UNSCEAR [29] estimates of genetic effects of 1 rad/generation of low dose, low dose-rate, low LET radiation on a population of one million live births In the new system of dose limitation, the total health structure of the two populations. These differences in total risk, Disease classificationa Current incidence per millionb Effect of 1 rad/generation detriment was assessed by calculating risk factors for both however, are not sufficiently large to warrant the use of separate cancers and genetic effects and then summing the factors. For values for protection purposes in the two cases.’’ First generation Equilibrium cancers, ICRP chose mortality as the index of harm and for That ICRP emphasized total stochastic effects merits Dominant + X-linked diseases 10,000 20c 100 genetic effects, it was the ‘risk of serious hereditary ill-health’ comment, as it was the basis for proposing that a single Recessive diseases 1,100 Relatively slight Very slow increase dose-equivalent limit based on the total risk of all tissues d e to the progeny of the first two post-radiation generations. The Chromosomal diseases 4,000 38 40 cancer mortality data reviewed in the 1977 UNSCEAR report irradiated replace the previous system of annual dose limits to Multifactorial diseases (Congenital anomalies, 90,100f 5g 45h anomalies expressed later & constitutional provided the basis for the cancer risk calculations. The genetic individual tissues and organs irradiated singly. The concept of and degenerative diseases) risk factor appears to be an average estimate based on figures total risk derives from the fact that almost every exposure of the provided in the 1977 UNSCEAR report and in a report of an body involves irradiation of more than one tissue. Accordingly, Total 105,200 63 185 6 % of current incidence 0.06 0.17 ICRP Task group set up for this purpose [80]. The estimates of the new system was designed to ensure that the total risk from risk factors, in turn, were used to assess the relative contribution Assumed doubling dose: 100 rad. a Follows that given in the 1972 BEIR report [47]. b Based on the results of Trimble and Doughty [72] in the British Columbia survey with certain modifications (see text for details). 7 Although the ICRP [40] noted that in the assessment of the total population c Assumed to be one-fifth of that at equilibrium. 6 The UNSCEAR [29] estimate was: 63 cases per 106 progeny/rad (see detriment, the total risk of hereditary damage that may be expressed in all d 3 1 From newborn surveys; includes those due to numerical and structural abnormalities (see Ref. [29] for details). Table 5), which equals 6.3/10 SvÀ for the reproductive population. Corrected subsequent generations should be taken into account (and that this equilibrium e The first generation incidence is assumed to include all the numerical anomalies and three-fifths of the unbalanced translocations (the remaining two-fifths being for the whole population (i.e., multiplied by 0.4), this figure becomes 2.5/ risk, per unit dose, may be about twice that which is expressed in the first two 3 1 6 1 derived from a balanced translocation in one parent). 10 SvÀ . ICRP’s Task group figure was 125 cases/10 progeny radÀ (=12.5/ generations only), it did not use the equilibrium estimates for calculating the f 3 1 Includes an unknown proportion of numerical (other than Down syndrome) and structural chromosomal anomalies. 10 SvÀ ) for the reproductive population. Corrected for the whole population, risk factor for genetic effects. This is an important point since in its 1991 report g 3 1 3 1 One-tenth of that at equilibrium. it becomes 5/10 SvÀ ). ICRP used 4/10 SvÀ . See Section 7.2 for clarification [43], ICRP did use the equilibrium estimates although it reverted back to the h Based on the assumption that the mutation component is 5%. on the risk to ‘‘total’’ versus ‘‘reproductive’’ populations. estimates for the first two generations in the 2007 [44] recommendations.

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non-uniform irradiation of parts of the body would not exceed population exposures was abolished. For non-stochastic effects, Table 9 that from uniform irradiation of the whole body. This condition the ICRP noted that such effects would be prevented by Estimates of genetic risks presented in the 1988 UNSCEAR report [32] (top half) and those for multifactorial diseases used by ICRP (in addition to those for would be met if: applying a dose equivalent limit of 50 rem (0.5 Sv) in a year to Mendelian diseases) in its 1991 recommendations [43] (bottom half) all tissues except the lens of the eye for which it recommended a Disease classification Current incidence per Effects of 0.01 Gy/generation on a population of 106 children 6 wT HT Hwb;L (4) 10 live births limit of 30 rem (0.3 rem) in a year. 1st generation 2nd generation Equilibrium T  X where w is a weighting factor representing the proportion of 7. Genetic risk estimates from the early 1980s to the UNSCEAR (1988) [32] T Mendelian stochastic risk resulting from tissue (T) to the total risk when the early 1990s and their relevance to the 1990 ICRP Autosomal dominant and X-linked diseases 10,000 15 13 100 a whole body is irradiated uniformly, HT is the annual dose recommendations (Publication 60) Autosomal recessive diseases 2,500 No or negligible increase 15 equivalent in tissue (T) and Hwb,L is the recommended annual Chromosomal diseases due to structural anomalies 400 2.4 1 4 dose-equivalent limit for uniform irradiation of the whole body, 7.1. Risk estimates Sub-total (rounded) 13,000 18 14 120 namely 5 rem (50 mSv), limit [The implied values of such Chromosomal diseases due to numerical anomalies 3,400 Not estimated limits to individual tissues/organs could be obtained, if From the early 1980s through the early 1990s, UNSCEAR Multifactorial required, by dividing the dose-equivalent limit H continued reviewing emerging scientific information and re- Early-acting dominants Unknown Not estimated wb,L Congenital anomalies 60,000b Not estimated (50 mSv in a year) by the relevant wT values (Table 8)]. assessing genetic risks when necessary. The advances, however, Other multifactorials 650,000c Not estimated Support for the view that the annual dose equivalent limit of were incremental and risk estimates made in 1982, 1986, 1988 Heritable tumors Unknown Not estimated 5 rem (recommended in Publication 1 in 1959 [38]) for workers and 1993 [30–33] were essentially similar to those in 1977 [29], remained valid, came from the analysis of empirical data on the at least with respect to Mendelian diseases; for chromosomal ICRP (1991) [43] annual dose equivalents in large occupational groups. The diseases, the estimates varied somewhat depending on the Disease classification Current incidence per 106 live births First two generations Equilibrium analysis showed that the distribution of these values very assumptions used. Mendelian and chromosomal diseases 13,000d 30 120 commonly fitted a log-normal function, with an arithmetic With multifactorial diseases, the situation was different. Multifactorial diseases (congenital anomalies and chronic diseases) 710,000e 67.5f 355f mean of about 5 mSv, and with very few values approaching the First, their baseline frequencies were revised upwards in 1988.  Assumed doubling dose was 1 Gy for chronic low LET irradiation. limit. The application of the risk factors to the above mean dose For congenital anomalies, the revision was from 4.3% (in 1977 a Includes those which are maintained by recurrent mutation as well as those maintained due to heterozygote advantage; in 1977 only those maintained by mutation indicated that the average risk in radiation occupations is [29]) to 6.0% (in 1988 [32]) and for chronic multifactorial were included. comparable with the average risk in other safe industries. diseases, from 4.7% to 65%; these revised estimates have b New estimate based on [82]. c 6 On the genetic dose limit of 5 rem/generation to the remained in place since then (see Table 4). Second, while in New estimate based on [83]; this figure replaces the 600,000/10 cited in the 1988 UNSCEAR report [32] which was based on information from studies in population which had been used thus far, Publication 26 stated: both 1977 [29] and 1982 [30], the UNSCEAR presented progress. d Estimate of baseline frequency and of risks for Mendelian and chromosomal diseases are the same as those in UNSCEAR 1988 report. ‘‘...It has become increasingly clear that the previously estimates of risk for multifactorial diseases (when the total e Estimate of baseline frequency for multifactorials is the same as that in UNSCEAR 1988 report, but risk estimates are those of ICRP[43]; see text for details. suggested level is not likely to be reached, and it is very baseline frequency estimate was around 9%), with the new very f The estimates assume that the MC is 5% and that 10% of the total (i.e., equilibrium) risk will be expressed in each of first two generations. The equilibrium risk is: improbable that responsible authorities would permit the much higher baseline frequencies (now totaling 71%), it grew 710,000 per 106 1/1 0.05 0.01 = 355 cases per 106 progeny. For the first two generations, the estimate is 67.5 cases per 106 progeny. Â Â Â average dose equivalent in a population to reach values that are doubtful of the validity of the assumptions used earlier for this more than small fractions of the former genetic dose limit of class of diseases and therefore refrained from providing risk 5 rem in 30 years. Therefore, continuance of the former genetic estimates in1988 [32] and 1993 [33]. ICRP, however, did 7.2.2. Distinction between genetic risk to the reproductive (30/75) or 40% of the total dose (the 40% figure is also the dose limit could be regarded as suggesting the acceptability of a provide estimates of risk for multifactorial diseases for use in population as opposed to the total population approximate ratio of the reproductive population to the total higher population exposure than is either necessary or probable, the protection context [43,81]. The 1988 UNSCEAR estimates Although the distinction between genetic risks to the population). Algebraically, this is equivalent to assuming that and a higher risk than is justified by any present or easily (for Mendelian and chromosomal diseases and those of ICRP ‘reproductive population’ as opposed to the ‘total population’ the risk to the total population is only 40% of that for the envisaged future development. Furthermore, knowledge gained (for Mendelian, chromosomal and multifactorial diseases) are was implicit in Publication 26, ICRP formalized this distinction reproductive population. over the past two decades indicates that genetic effects while presented in Table 9. in Publication 60 [43]. Estimates of genetic risks presented in important, are unlikely to be of overriding importance, and the UNSCEAR and BEIR reports discussed thus far in this 7.2.3. Risk coefficients for the reproductive and total would need to be related to the sum of all other effects...’’ 7.2. The 1990 ICRP recommendations (Publication 60) paper are risks to the reproductive population since the implicit populations ‘‘ ...In these recommendations, therefore, the Commission assumption here is that all exposed individuals sustain For its 1990 recommendations, the ICRP’s starting point for 7.2.1. Introduction of some additional radiation dosimetric does not propose dose limits for populations. Instead, it genetically significant doses (GSDs) and contribute to the estimating genetic risk coefficients for the reproductive quantities wishes to emphasize that each man-made contribution to next generation. population was the 1988 UNSCEAR risk estimates for Since, in radiation protection, it is the absorbed dose population exposure has to be justified by its benefits, and By contrast, the risk to a ‘total population’ takes into account Mendelian and chromosomal diseases and its own assessment averaged over the tissue or organ (rather than a point) and that limits for individual members of the public refer to the the fact that, while in any given population there are individuals of risk of multifactorial diseases (the latter shown in the bottom weighted for radiation quality that is of interest, in Publication total dose-equivalent from all sources... the limit for of all ages who are exposed to radiation over a lifetime, not all half of Table 9). For Mendelian and chromosomal diseases, the 60, a weighting factor called radiation weighting factor, w , 6 irradiation of a whole population is thus clearly seen as the R individuals transmit the radiation-induced damage to the next rates for equilibrium conditions are: 120 cases per 10 live was introduced. This weighted absorbed dose is called total reached by a summation of minimum necessary generation (i.e., damage sustained by germ cells of individuals births per 0.01 Gy and for multifactorial diseases, 355 cases per equivalent dose, in a tissue or organ and is symbolized by 6 contributions, and not as a total apparently available for who are beyond the reproductive period of their lives or who are 10 live births per 0.01 Gy. The comparable figures for the first H . Further, since the relationship between the probability of 6 apportionment...’’ T not procreating for any reason pose no genetic risks). In other two generations are about 30 cases per 10 live births per stochastic effects and equivalent dose was found also to depend words, in the context of the total population, the genetically 0.01 Gy and 67.5 cases per 106 live births per 0.01 Gy, In summary, Publication 26 launched a new risk-based on the organ or tissue irradiated, a further quantity, called the significant dose will be markedly lower than the total dose respectively. In ICRP’s way of presenting risk figures, the 2 1 system of radiation protection, in which the risk of cancers tissue-weighting factor, wT was introduced which represents received over a lifetime. For the purpose of calculating genetic estimates are, respectively, 1.2 10À GyÀ and 3.55 2 1 Â 2 1 Â assumed a much greater significance than that of genetic effects the relative contribution of that organ or tissue to the total risks to the total population, in Publication 60, the ICRP 10À GyÀ (equilibrium) and: 0.3 10À GyÀ and 0.675 2 1 Â Â in defining dose equivalent limits. The continued use of a dose detriment due to these effects resulting from uniform irradiation assumes that the mean age at reproduction is 30 years and the 10À GyÀ (first two generations). equivalent limit of 5 rem in a year for radiation workers was re- of the whole body. The weighted equivalent dose (a doubly average life expectancy at birth is of the order of 75 years. The As may be recalled, in 1977, ICRP introduced the concept of affirmed and the genetic dose limit of 5 rem/generation for weighted absorbed dose) was called the effective dose, E. implication is that the dose received by 30 years of age is about detriment to correct the rates of induction to derive risk factors

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for cancers and hereditary effects. For cancers, mortality was Table 11 summarizes the estimates of risk factors, Table 11 Estimates of risk factors, aggregated detriment and tissue-weighting factors (w ) presented by ICRP in Publication 60 [43] the criterion for detriment and for genetic effects, it was the risk aggregated detriment and tissue-weighting factors (wT ). T 4 a 4 b c of serious hereditary harm [40] as was explained in Section For comparison, the estimates from 1977 are shown in Tissue at risk Risk factor/Sv ( 10 ) Aggregated detriment/Sv ( 10 ) Relative contribution Assigned wT 5.4.2. In Publication 60 [43], in order to estimate detriment, parenthesis although it should be borne in mind that rigorous   Whole population ICRP used arbitrary severity correction factors of 1 for the rate comparisons are not possible since the procedures used to 1. Gonadsd 100 (40) 133.3 0.183 0.20 (0.25) of induction of Mendelian and chromosomal diseases and 1/3 arrive at these values have been different. There are three 2. Bone marrow (red) 50 (20) 104.0 0.143 0.12 (0.12) for the rate of induction of multifactorial diseases. notable differences between the 1977 and 1991 estimates: (1) 3. Colon 85 102.7 0.141 0.12 Applied to the rates of induction at equilibrium, the The detriment-adjusted probability coefficients for cancer 4. Lung 85 (20) 80.3 0.111 0.12 (0.12) 2 1 5. Stomach 110 100.0 0.139 0.12 risk coefficients become: (1 1.2 10À GyÀ ) = 1.2 mortality and serious genetic effects are higher in 1991 than 2 1    6. Bladder 30 29.4 0.040 0.05 10À GyÀ (for Mendelian and chromosomal diseases) and in 1977; for the cancers, re-evaluations of the earlier risk 7. Breast 20 (25) 36.4 0.050 0.05 (0.15) 2 1 2 1 ([1/3] 3.55 10À GyÀ ) = 1.2 10À GyÀ (for multifac- estimates derived from the survivors of Hiroshima and 8. Liver 15 15.8 0.022 0.05 torial diseases).  The figure for all genetic effects at equilibrium Nagasaki and revisions in the dosimetry showed that the 9. Oesophagus 30 24.2 0.034 0.05 2 1 10. Ovary 10 14.6 0.020 – is therefore 2.4 10À GyÀ . The corresponding figure for the estimates of risk needed to be revised upwards; further, more  2 1 11. Thyroid 8 (5) 15.2 0.021 0.05 (0.03) first two generations are: 0.3 10À GyÀ (Mendelian and tissues/organs could now be included; for genetic effects, the 2 1 12. Skin 2 4.0 0.006 0.01 chromosomal) and 0.23 10À GyÀ (multifactorial), and the equilibrium estimates of risk were used in 1991 (instead of 13. Bone surface 5 (5) 6.5 0.009 0.01 (0.03) 2 1 sum, 0.53 10À GyÀ . Table 10 summarizes the above risks applicable to the first two post-radiation generations as 14. Remaindere 50 (50) 58.9 0.081 0.05 (0.30)  estimates. Also shown in the table are the figures for the total was the case in 1977). (2) The 1991 estimates of aggregated Total 500 (165) 725.3 1.000 1.00 (1.00) population (which are 40% of the above, namely, detriment encompass a greater range of components than the 2 1 2 1 Working age population (18–64 years) 1.0 10À GyÀ and 0.19 10À GyÀ ). For a working 1977 detriment estimates did. (3) The genetic effects now   Gonads 60 80 14.4 population, because of the different age distribution, the risk contribute 20% to the total detriment whereas in 1977, the All other tissues 400 474 85.6 coefficient would be slightly smaller than that for the general figure was 25%. population; the ICRP assumed that it is about 60% of that for Note that the wT values are now based on estimates of aggregated detriment (normalized to unity) and not on the probability of fatal cancers alone. The values shown 2 1 in parenthesis are from Publication 26 [40]; (see Table 8, this paper). the total population, namely, 0.6 10À GyÀ . 7.2.5. Recommendations on dose limits a  For all tissues other than gonads, the risk factors pertain to fatal cancers (i.e., cancer mortality). For gonads, the risk factor pertains to serious ill health to the The dose limits recommended in Publication 60 to progeny estimated for conditions of new equilibrium (i.e.,) over all generations under conditions of irradiation generation after generation. b 7.2.4. Relative contribution of organs to the total detriment occupational workers and the public are shown in Table 12. These values formed the basis of ICRPs wT estimates. c Assigned by ICRP. In considering the relative contributions and recognizing the large uncertainties in deriving them, the ICRP decided that the values could be and wT estimates As with earlier recommendations, the dose limits are aimed at rounded and grouped into a simple system of weights of adequate accuracy for calculations of effective dose. It selected a system which would use no more than four Again, it is instructive to recall here that in Publication 26 ensuring that no individual is exposed to radiation risks that are groups of weights and require no more than about a factor of two rounding between the relative contributions shown in the table. The assigned weighting factors were: [40], ICRP applied the criteria of mortality (for cancers) and judged to be unacceptable. The recommended dose limit for 0.01 each (for bone surface and skin), 0.05 each (for bladder, breast, liver, oesophagus, thyroid and remainder), 0.12 each (bone marrow, colon, lung and stomach) and serious hereditary harm (for genetic effects) for converting the occupational exposures is now revised downwards to 20 mSv/ 0.20 for gonads; these weighting factors were used for both a working population and the general population. rates of induction into detriment-adjusted ‘‘nominal probability year, from the earlier 50 mSv/year limit (more specifically, d Including cancer in ovary. e coefficients.’’ The latter, in turn, provided the basis for 20 mSv/year averaged over 5 years, 100 mSv in 5 years with no For purposes of calculation, the remainder is composed of the following additional tissues and organs: adrenals, brain, upper large intestine, small intestine, kidney, muscle, pancreas, spleen, thymus and uterus. The list includes organs which are likely to be selectively irradiated. Some organs in the list are known to be calculating tissue-weighting factors (wT values). Recall also more than 50 mSv in a single year). susceptible to cancer induction. If other tissues and organs subsequently become identified as having a significant risk of induced cancer, they will then be included that Publication 27 [41] more formally set the stage for the use In order to arrive at the value of 20 mSv/year, ICRP [43] with a specific wT or in this additional list constituting the remainder. The latter may also include other tissues or organs selectively irradiated. of the concept of detriment (see Section 5.4.2). Later, the 1985 made a detriment-based analysis using annual effective dose Publication 45, titled ‘‘Quantitative bases for developing a ‘‘test values’’ of: 10 mSv, 20 mSv, 30 mSv and 50 mSv unified index of harm,’’ which was also written by Sir Edward (corresponding to approximate lifetime doses of, respectively, For public exposures, ICRP concluded that the lowering of constraints, only a small proportion of radiation-induced Pochin, extended the scope of Publication 27 to include a 0.5 Sv, 1.0 Sv, 1.4 Sv and 2.4 Sv). Based on this analysis, the the annual effective dose limit from the earlier 5 mSv/year to mutations in human germ cells is expected to be recovered consideration of radiation-induced non-fatal cancers, non- Commission judged that ‘‘...the dose limit should be set in 1 mSv would be appropriate but a 5-year averaging was in live births. In other words, the rate of induced mutations in stochastic effects and hereditary detriment. Publication 60 [43] such a way and at such a level that the total effective dose allowed in exceptional circumstances (see paragraphs 190–193 human disease-causing genes that are compatible with modified these same calculations by adopting an aggregated received in a full working life would be prevented from in Publication 60 for details). offspring viability will be smaller than the rates estimated in measure of detriment that included the following components: exceeding about 1 Sv received moderately uniformly year by mouse experiments. The latter involve mostly marker genes the probability of attributable fatal cancers, the weighted year and that the application of its system of radiological 8. The 2001 UNSCEAR genetic risk estimates and the which are either known to be not essential for viability or are probability of attributable non-fatal cancer and, for both protection should be such that this figure would only be rarely 2007 ICRP recommendations located in genomic regions not essential for viability. cancers and genetic effects, the relative length of life lost. approached.’’ (paragraph 162). The second related concept addresses the question of the 8.1. Advances in risk estimation recorded in the 2001 phenotypes associated with induced DNA deletions that are Table 10 UNSCEAR report compatible with offspring viability. It predicts that most ICRP’s 1991 estimates of risk coefficients for serious genetic effects of ionizing radiation. All values are expressed in %/Sv (Publication 60 [43]) induced deletions are likely to be associated with multi-system The 2001 UNSCEAR report [34] introduced two new and Time span Disease category Genetic risk coefficient (in % per Sv) for: developmental abnormalities (similar to naturally occurring major concepts, both stemming from advances in molecular congenital anomalies) but which show autosomal dominant Reproductive population Total population Working population biology. The first concept, that of potential recoverability patterns of inheritance [85]. This concept challenges the basic All generations Mendelian and chromosomal 1.2 0.5 correction factor (PRCF), enables one to bridge the gap assumption that has dominated the field thus far, namely, that Multifactorial 1.2 0.5 between rates of radiation-induced mutations empirically radiation-induced mutations cause genetic diseases that are a Total 2.4 1.0 0.6 determined from mouse studies and the risk of inducible similar to those occurring naturally as a result of spontaneous Up to two generations Mendelian and chromosomal 0.3 0.1 genetic diseases in humans [84]. It relies on the premise that mutations in single genes. Multifactorial 0.23 0.09 most radiation-induced mutations are DNA deletions, often The 2001 UNSCEAR report incorporated several other Total 0.53 0.19 encompassing more than one gene and can occur anywhere in advances as well. Firstly, the baseline frequencies of Mendelian a The value used as a basis for estimating the relative contribution of genetic effects to the total detriment. the genome. However, because of structural and functional diseases were revised upward from 1.25% (in 1993) to 2.4% (in

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Table 12 risk estimation, it became possible to obtain risk estimates for Table 14 with genetic effects. This is an important point to stress here, a ICRPs 1991 recommended dose limits in Publication 60 [43] all classes of genetic diseases. The ICRPs 2007 estimates [24,44] of genetic risk coefficients for the repro- since in 1991, the equilibrium estimates of risk were used for ductive and the total population (all values expressed in percent per Sv) and are Application Dose limit up to two generations when the population sustains radiation exposure gen- this purpose. The justification for the use of the estimates of risk Occupational Public 8.2. The risk estimates eration after generation up to generation 2 for detriment calculations relies on, in addition to UNSCEARs arguments (discussed in the preceding c Disease class Reproductive population Total Effective dose 20 mSv per year, 1 mSv in a year Table 13 summarizes the 2001 [34] UNSCEAR risk paragraph), ICRP’s judgement that, for the purpose of radiation averaged over defined population a b periods of 5 yearsb estimates. Note that the risks are expressed as the predicted Range Average Average protection, this approach will not lead to any substantial number of additional cases (i.e., above the baseline number) of underestimation of heritable effects of radiation. Annual equivalent dose in (a) Mendelian diseases 0.13–0.25 0.19 0.08 The lens of the eye 150 mSv 15 mSv different classes of genetic disease per million progeny per Gy (b) Chronic diseases 0.03–0.12 0.08 0.03 The 2007 ICRP estimates of risk coefficients for the The skin 500 mSvd 50 mSv for a population exposed to low LET, low-dose or chronic (c) Congenital abnormalities: 0.24–0.30 0.27 0.11 reproductive and total population are shown in Table 14. The The hands and feet 500 mSv – irradiation generation after generation. Estimates are given for Total for all classes 0.54 0.22 procedure used to calculate these estimates is outlined in a The limits apply to the sum of the relevant doses from external exposure in the first and up to the second post-radiation generation. As will Section 7.2. Briefly, the upper and lower limits of each of a Average of the limits of the indicated ranges. the specified period and the 50-year committed dose (to age 70 years for be evident, the total risk to the first generation is on the order of the estimated ranges are first used to obtain average b Forty percent of that for the reproductive population. children) from intakes during the same period. about 3000–4700 cases per million progeny per Gy which estimates and the latter are then combined. For example, for b 1 With the further provision that the effective dose should not exceed 50 mSv represent about 0.4–0.6% of the baseline risk. The risk up to the Mendelian diseases, the 1300–2500 cases/million SvÀ become in any single year. 2 2 1 c second generation is slightly higher, namely, 3930–6700 cases estimation of risk at equilibrium involve the very unrealistic 0.13 10À to 0.25 10À SvÀ with an average of In special circumstances, a higher value of effective dose could be allowed  2 1  per million per Gy, or about 0.5–0.9% of the baseline risk. assumptions that: (a) the estimates of selection coefficients, 0.19 10À SvÀ . A similar procedure is applied to the other in a single year, provided the average over 5 years does not exceed 1 mSv/year.  d The limitation on the effective dose provides sufficient protection for the With the exception of congenital anomalies, the risk mutation components and other quantities used in the risk classes. The total risk (i.e., for all classes combined) thus 2 1 skin against stochastic effects. An additional limit is needed for localized estimates for other classes of genetic disease have been equation will remain valid for tens or hundreds of human becomes 0.54 10À SvÀ . The above estimates are for a exposures in order to prevent deterministic effects. obtained with the doubling dose method using the following generations and (b) population structures, demography and reproductive population. For the total population, with the same equation: health care facilities will remain constant over many hundreds assumptions as those discussed in Section 7.2, the risk 2 1 2001) [86] (see Table 4). Secondly, human data on spontaneous of years. These assumptions can no longer be sustained. coefficient becomes 40% of the above or 0.22 10À SvÀ . mutation rates and mouse data on induced mutation rates were 1 Table 15 shows ICRP’s most recent and current estimates of risk per unit dose P MC PRCF (5) used to estimate doubling doses [68]. This is an important ¼ � DD � � 8.3. ICRP’s 2007 estimates of detriment associated with nominal and detriment-adjusted risk coefficients for cancers   conceptual change and is based on more extensive data than genetic effects and tissue-weighting factors (wT ) and genetic effects, their relative contributions to the total were available earlier when, in 1993, mouse data were used in where P is the baseline frequency of the disease class under detriment and ICRP’s judgement on tissue-weighting factors, the estimation of both spontaneous and induced mutation rates consideration, DD is the doubling dose, MC is the disease-class Table 13 risk estimates up to generation 2 for the along with similar estimates in Publication 60 (in parenthesis). [33,68]. However, the doubling dose of 1 Gy for chronic low and post-radiation-generation-specific mutation component and ‘reproductive population’ (see Section 6.2) provided the For cancers, the nominal risk coefficients were based on LET radiation conditions used previously, remains somewhat PRCF is the disease-class-specific potential recoverability cor- starting point for ICRP’s assessment of detriment associated lifetime incidence estimates corrected for effects at low doses coincidentally unchanged. Thirdly, mathematical models and rection factor. The risk of congenital anomalies has been esti- methods were developed and used to estimate mutation mated using mouse data on developmental abnormalities (e.g., Table 15 components (MC: the relative increase in disease frequency congenital anomalies ascertained in utero, skeletal abnormalities The ICRPs 2007 estimates [24,44] of nominal risk, detriment-adjusted risk, relative contribution of different tissues to the total detriment-adjusted risk and tissue- per unit relative increase in mutation rate) for both Mendelian and cataracts) without recourse to the doubling dose method. weighting factors for the whole and the worker populations 4 4 and chronic diseases. The methods permit one to estimate MC It is instructive to recall here that in the 1988 UNSCEAR Tissue at risk Nominal risk/Sv ( 10 ) Detriment-adjusted risk/Sv ( 10 ) Relative contribution Assigned wT   for any post-radiation generation of interest under conditions of report [32] risk estimates for the equilibrium situation were also Whole population radiation exposure in every generation or in one generation only included. The reasons for emphasizing the risk for the first two 1. Gonads 20 (100)a 25.4 (133.3)a 0.044 (0.183)a 0.08b (0.20) [87,88]. As a result, for the first time in the history of genetic post-radiation generations in the 2001 report [34] are that the 2. Oesophagus 15 (30) 13.1 (24.2) 0.023 (0.034) 0.04 (0.05) 3. Stomach 79 (110) 67.7 (100.0) 0.118 (0.139) 0.12 (0.12) 4. Colon 65 (85) 47.9 (102.7) 0.083 (0.141) 0.12 (0.12) Table 13 5. Liver 30 (15) 26.6 (15.8) 0.046 (0.022) 0.04 (0.05) The 2001 UNSCEAR [34] estimates of genetic risks from continuing exposure to low LET, low-dose or chronic irradiation 6. Lung 114 (85) 90.3 (80.3) 0.157 (0.111) 0.12 (0.12) 7. Bone 7 (5) 5.1 (6.5) 0.009 (0.009) 0.01 (0.01) Disease class Baseline frequency Risk per Gy per million progeny 8. Skin 1000 (2) 4.0 (4.0) 0.007 (0.006) 0.01 (0.01) (per million live births) 9. Breast 112 (20) 79.8 (36.4) 0.139 (0.050) 0.12 (0.05) 1st generation Up to 2nd generation 10. Ovary 11 (10) 9.9 (14.6) 0.017 (0.020) – (–) Mendelian 11. Bladder 43 (30) 16.7 (29.4) 0.029 (0.040) 0.04 (0.05) Autsomal dominant and X-linked 16,500 750–1500a 1300–2500 12. Thyroid 33 (8) 12.7 (15.2) 0.022 (0.021) 0.04 (0.05) � � Autosomal recessive 7,500 0 0 13. Bone marrow 42 (50) 61.5 (104.0) 0.107 (0.143) 0.12 (0.12) Chromosomal 4,000 bb 14. Other solid cancers 144 (50) 113.5 (58.9) 0.198 (0.081) 0.12 (0.05) 15. Brain 0.01 Multifactorial 16. Salivary glands 0.01 Chronic 650,000c 250–1200 250–1200 � d � e Total 1715 (500) 574.2 (725.3) 1.000 (1.000) 1.00 (1.00) Congenital abnormalities 60,000 2000 2400–3000 � � Total 738,000 3000–4700 3930–6700 Working age population (18–64 years) Total per Gy expressed as percent of baseline �0.41–0.64 �0.53–0.91 Gonads 12 15.3 0.036 � � Other tissues 1167 406.7 0.964 Assumed doubling dose = 1 Gy. a The ranges reflect biological and not statistical uncertainties. For cancers, the nominal risk coefficients are based on incidence estimates (lifetime cancer risk projections). In estimating detriment, these rates were adjusted for b Assumed to be subsumed in part under autosomal dominant and X-linked diseases and in part under congenital abnormalities. fatality, quality of life (i.e., morbidity and suffering associated with non-fatal cancers) and for years of life lost. For genetic effects, the nominal risk coefficient is c Frequency in the population. based on rate of induction of serious hereditary effects and the detriment estimate adjusts for years of life lost. d Estimated from mouse data without recourse to the DD method. a Figures within parenthesis in all columns and rows are the estimates in Publication 60 [43]. e b On the assumption that between 20% and 50% of the abnormal progeny in the first generation may transmit the damage to the second generation. The detriment for heritable effects and cancer following gonadal irradiation were aggregated to give a wT of 0.08.

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Table 16A Table 17 relative importance of genetic risks to the total stochastic risk is a A comparison of the nominal and detriment-adjusted risk coefficients and the relative contribution of genetic effects to total in ICRP Publications 26 [40] and 60 [43] The ICRPs 2007 recommended dose limits [44] in planned exposure situations underestimated. These issues are now addressed in turn. and in 2007 [24,44] Application Dose limit Cancers Genetic effects Sum derived Relative contribution of Assigned wT for a Occupational Public 8.5.1. Arguments suggesting that 2001 UNSCEAR genetic rates (2+4) genetic effects [4/(2 + 4)] genetic effects Basic Derived Basic Derived risk estimates may be over-estimates Effective dose 20 mSv per year, 1 mSv in a yearc (1) (2) (3) (4) The arguments have been discussed in detail in the above averaged over defined Whole population periods of 5 yearsb report [34,89]. We draw attention to three of them. Firstly, there ICRP [40] (Publication 26) – 125b – 40c 165 0.242 0.25 must be substantial overlap between the risk of ‘autosomal Annual equivalent dose in ICRP [43] (Publication 60) 400d 592e 100f 133.3g 725.3 0.183 0.20 The lens of the eye 150 mSv 15 mSv dominant diseases’ (estimated using the DD method) and of ICRP [24,44] 1695h 548.8i 20j 25.4k 574.2 0.044 0.08 The skin 500 mSvd 50 mSv effects grouped under ‘congenital abnormalities’ (which are Working age population (18–64 years) The hands and feet 500 mSv – also predicted to be autosomal dominant but estimated ICRPl [40] – 125 – 40 165 0.242 Note that these recommendations are the same as those in Publication 60 in independently without recourse to the DD method) (see ICRP [43] 400 474 60 80 554 0.144 Table 13). Second, the risk estimate for chronic diseases, with ICRP [24,44] 1167 406.7 12 15.3 422 0.036 1991 (see Table 11). a The limits apply to the sum of the relevant doses from external exposure in the model employed for this purpose, uses the assumption that The ‘basic’ risk coefficients are those without adjustment for detriment and the ‘derived’ ones are those adjusted for detriment as discussed in the text. Rates in the specified period and the 50-year committed dose (to age 70 years for mutations in just two genes may underlie these effects which is columns labeled (1)–(4) and (2 + 4) are per 104 per Sv. children) from intakes during the same period. a b certainly not true in light of what is currently known about the The wT values assigned by ICRP. With the further provision that the effective dose should not exceed 50 mSv b Risk of fatal cancers. in any single year. genetic basis of chronic diseases. Even if one more gene is c Risk of serious hereditary effects up to two post-radiation generations under conditions of radiation in every generation. c In special circumstances, a higher value of effective dose could be allowed involved, then the estimate of risk will drop precipitously [84]. d Risk of fatal cancers unadjusted for detriment. in a single year, provided the average over 5 years does not exceed 1 mSv/year. Third, the value of mutation component (MC) used in e Risk of fatal cancers adjusted for detriment. d The limitation on the effective dose provides sufficient protection for the f calculations of risk for chronic diseases is 0.02 although in Risk of serious hereditary effects at the new equilibrium that already incorporates an arbitrary severity correction factor of 1 for Mendelian and chromosomal skin against stochastic effects. An additional limit is needed for localized several runs of our computer simulations, most of the MC diseases and 1/3 for multifactorial diseases, but unadjusted for detriment. exposures in order to prevent deterministic effects. g Risk of serious hereditary effects at the new equilibrium adjusted for detriment. values were close to 0.01. Had the value of 0.01 been chosen, h Risk of cancers based on predicted life time incidence, unadjusted for detriment. then the risk would be one half of the estimate given [88]. i 4 1 2 1 Risk of cancers adjusted for detriment. (592 10À SvÀ , rounded to 6.0 10À SvÀ ). Given the j Risk of serious hereditary effects in the first two post-radiation generations, unadjusted for detriment.   k different starting points and uncertainties in the procedures used 8.5.2. Arguments suggesting that the 2001 UNSCEAR Risk of serious hereditary effects, adjusted for detriment. to estimate nominal and detriment-adjusted risk coefficients, the genetic risk estimates may underestimate the relative l The ICRP was cognizant of the fact that for both somatic and hereditary risk factors, the estimates would differ somewhat for workers, compared to the whole population because of the differences in age structure of the two populations. It considered, however, that the differences in total risk, were not sufficiently large to small reduction in the estimate of nominal risk since Publication importance of genetic effects in the context of radiation warrant the use of separate values for protection purposes in the two cases. 60 is considered to have no practical significance. protection The main argument here rests on the equilibrium theory using a dose- and dose-rate reduction factor of 2 (unlike and 16B, for the purposes of this paper, is that the 2007 8.4. Recommendations on dose limits in 2007 which predicts that a population sustaining radiation exposure Publication 60 which used mortality data similarly corrected) assessment of relative contribution of genetic effects to the total in every generation will continue to accumulate mutant genes for radiation-associated cancers for 14 organs/tissues averaged detriment is much lower than was implied by either the 1991 or The currently recommended dose limits are shown in over time until an equilibrium is reached between mutation and across seven Western and Asian populations. These nominal 1977 estimates. While this discrepancy partly reflects which Table 17. In light of the discussion in the preceding section, selection. The prediction, which is applicable to all classes of risk coefficients were adjusted for lethality, quality of life and post-radiation generation effects are included in which ICRP concluded in its 2007 report that the dose limits genetic disease, is that if there is an x% increase in mutation rate years of life lost to yield detriment-adjusted risk coefficients. calculations, they also reflect an improved molecular under- recommended in Publication 60 [43] continue to provide an (over the spontaneous rate), there will be an x% increase in For genetic effects, the nominal risk coefficient is based on the standing of the types of genetic effects expected in progeny and appropriate level of protection. The Commission therefore re- disease frequency at equilibrium. Obviously, the equilibrium predicted risk of serious hereditary disease adjusted for years of the development of suitable models for predicting these effects affirmed the continued use of these same limits, noting that risk will be far higher than that in the first post-radiation life lost. and changes over time in the estimated magnitude of the ‘‘...the dose limits apply only in planned exposure situations, generations. Three key differences between the 1991 (Publication 60) detriment-adjusted total risk. but not to medical exposures of patients...the nominal Our studies of these issues have underscored the fact that the estimates and the more recent figures presented in Table 15 For cancers, in ICRP’s judgement, the present detriment- detriment coefficients for both a workforce and the general rate of approach to equilibrium varies markedly between 4 1 merit attention. First, for the whole population, the nominal risk adjusted nominal risk coefficients (548.8 10À SvÀ , rounded public are consistent with, although numerically somewhat dominant and recessive mutations and thus have an impact on 2 1  coefficient for all cancers and genetic effects combined is much to 5.5 10À SvÀ ; again, see Tables 16A and 16B) are lower than those given in 1990. These slight differences are of the magnitude of increase in disease frequencies over time 4 1 4 1  higher (1715 10À SvÀ ) than the 500 10À SvÀ estimate wholly compatible with those presented in Publication 60 no practical significance.’’ Note that Table 17 is the same as [87,88]. The predicted increase in disease frequency is highest reported in Publication 60. This is because the 2007 estimate Table 12 but with a different caption. for autosomal dominants followed by X-linked, much less for for cancers is based on incidence data, not mortality data as Table 16B autosomal recessives and very much lower for chronic diseases. were used previously. Second, the detriment-adjusted estimates A comparison of ICRPs 2007 [24,44] detriment-adjusted nominal risk coeffi- 8.5. Strengths and limitations of the genetic risk estimates In fact, for autosomal recessives, no detectable increase is 4 1 a are somewhat lower in 2007 (574 10À SvÀ ) than in 1991 cients (in percent per Sv) after exposure to radiation at low dose rate for cancer and their use by ICRP predicted before generation 10 or so and for chronic diseases, it 4 1  and genetic effects 725.3 10À SvÀ ). Third, the relative contribution of genetic is much less than for recessive diseases.  effects is lower now (0.044) than was estimated in 1991 (0.183). Exposed population Cancers Genetic Total The rationale for limiting attention to genetic risk estimates Empirical data which support the prediction for recessive The smaller relative contribution of genetic effects is also effects for the first two post-radiation generations (and not projecting mutations come from the work carried out in the late 1960s and evident in Tables 16A and 16B, both of which provide 2007 1991 2007 1991 2007 1991 them to the new equilibrium) was discussed in Sections 8.2 and 1970s by Lu¨ning and colleagues (summarized in [28]). They comparisons of the estimates of nominal risk coefficients over Whole 5.5 6.0 0.2 1.3 5.7 7.3 8.3. While the conclusion that the estimates as presented reflect conducted studies to answer the question of whether radiation- time. Table 16A summarizes estimates both with and without Adult 4.1 4.8 0.1 0.8 4.4 5.6 the current state of knowledge is fully valid, it is instructive to induced recessive lethal mutations would accumulate in mouse adjustment for detriment, as well as tissue-weighting factors, examine the assumptions that have been used, the consequent populations subject to radiation exposure in every generation. a Note that the most significant change from 1991 is the six to eightfold from ICRP’s 1977, 1991 and 2007 reports [40,43,44]. In reduction in the risk coefficient for genetic effects. This reduction comes about uncertainties, and more specifically, whether (a) the estimates They went up to 15 generations of radiation but could not Table 16B, the comparisons are limited to the 1991 and 2007 mainly because the ICRP has chosen to express such risks up to the second are likely to be over- or under-estimates of the true risk and (b) demonstrate the accumulation of recessive lethal mutations documents. The principal take-home message of Tables 16A generation rather than at a theoretical equilibrium. by not considering equilibrium risk for protection purposes, the over time, a result which is unlike that known in Drosophila

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4 1 studies. Several other population genetic studies by others 165 10À SvÀ of which genetic effects constituted about time and DD remained at 1 Gy). In the late 1990s, the field of 11. Some personal perspectives (KS)  4 1 focused on fitness in irradiated mouse populations, but the 25% (i.e., 40 10À SvÀ ). Publication 26 re-affirmed the risk estimation witnessed some notable advances which results were, again, negative and therefore abandoned dose limits of 5 rem/year for radiation workers and of 0.5 rem included, among others, the development of mathematical It has been a privilege and a wonderful experience – (reviewed in [90]). in a year to individual members of the public; at these models to estimate the risk of Mendelian as well of chronic intellectually challenging and emotionally fulfilling – to have In part, the negative results in mouse studies on recessive recommended dose limits, the implied risks were deemed diseases and the emergence of the molecular biology-based been able to serve both the UNSCEAR and ICRP for as long as I lethals could be explained by the sample sizes and techniques acceptable. Importantly, dose limits for exposure of populations concept that the principal adverse effects of radiation exposure have served. The important dividends of the work for that could be used in the mouse for screening for recessive were no longer considered necessary. of germ cells are more likely to be manifest as multi-system UNSCEAR have been the opportunities to develop a broader lethals which were not as efficient as in Drosophila. Perhaps, The years following the appearance of Publication 26 developmental abnormalities rather than as single gene diseases perspective of the whole field of radiation research and get to one can now speculate that this difference in part may also be witnessed the accumulation of substantial amounts of new data in the progeny. This latter concept challenged a basic know the scientists both within and outside the UNSCEAR related to differences in genomic architecture between on radiation-induced cancers, revisions of genetic risk assumption that had dominated the field prior to the emergence framework. I still have vivid memories of my UNSCEAR- Drosophila and mouse and their response to radiation. estimates and further developments in characterizing detriment. of more molecularly based thinking. sponsored periodical visits, in the early years, to several In the 1990 ICRP Recommendations (Publication 60 issued in The estimates of genetic risk presented in the 2001 laboratories in Europe and the USA to discuss science with the 9. Conclusions 1991), the detriment-adjusted nominal risk coefficient for both UNSCEAR report [34] (reproduced in Table 13) show that ‘movers and shakers’ who had made phenomenal contributions 4 1 types of effects was increased to 725 10À SvÀ (see for the low LET low dose and dose-rate conditions used, the to radiation genetics and risk estimation and get directly As pointed out in Sections 8.2 and 8.3, estimation of genetic Table 11), of which genetic effects accounted for about 18% risks are small, compared to the baseline frequency of genetic apprised of the emerging new data. These giants included Bill 4 1 risks at the new equilibrium is fraught with considerable (i.e., 133 10À SvÀ ). The dose limit for workers was diseases and are smaller than what Muller feared they might be and Lee Russell in Oak Ridge, Jim Neel in Ann Arbor, K.G. uncertainties and therefore has not been attempted here. The lowered to 20 mSv/year averaged over 5 years, 100 mSv in 5 in the 1950s; they are also compatible with the lack of evidence Lu¨ning in Stockholm, Mary Lyon, Tony Searle, Charles Ford estimated risk to the first two post-radiation generations is more years with no more than 50 mSv in a single year. For members for any adverse effects in the progeny of A-bomb survivors in and John Evans in the UK, Udo Ehling in Neuherberg, likely to be an overestimation and the relative importance of of the public, the limit was lowered to 1 mSv/year and a 5-year Japan [64,94–97] and a similar lack of evidence in other human Germany, Howard Newcombe and Ben Trimble in Chalk River, genetic risks to the total stochastic risk is therefore not averaging was allowed in exceptional circumstances. studies (e.g. [98–100]). Canada and several others. The UNSCEAR Scientific underestimated in the context of radiation protection. More recently, in ICRP’s 2007 Recommendations, detriment Within the framework of radiation protection, the relative Secretaries with whom I worked with great pleasure during calculations are based on cancer incidence (i.e., not mortality as importance of genetic effects has progressively diminished over the nearly three decades of my association with the Committee 10. Synopsis of major developments in radiation had been the case thus far) and, for the first time in the history of the years. This is not unexpected because in the risk-based system included: Francesco Sella, Dan Beninson, Giovanni Silini, protection the field, genetic risk estimates incorporate advances in of protection, cancer risks (which are spread among multiple Burton Bennett and Norman Gentner. molecular biology. The newly estimated detriment-adjusted organs/tissues) contribute far more to the total stochastic risks In the mid-1970s, when I was elected as a member of The field of radiation protection has been evolving for more nominal risk coefficient for both types of effects combined has than genetic risks (which are limited to the gonads); moreover, Committee 1 (the Radiobiology Committee) of ICRP, I had the 4 1 than 100 years now. The earliest recommendations (in the first been lowered slightly to 574 10À SvÀ (see Table 15), of cancer risks have had to be progressively revised upwards over unique opportunity not only to expand my horizons further, but two to three decades of the 20th century) were concerned with which genetic effects account for only 4% (i.e., the years as new data have become available. The changes in the also look at my science from a different perspective and make 4 1 avoiding threshold (deterministic) effects, initially in a 25 10À SvÀ ). In spite of changes in the cancer risk data assessed relative contribution of genetic effects to the total new friends in the fields of epidemiology, radiobiology, qualitative manner. With the definition of the R unit for used in the calculations, the nominal risk coefficient, albeit detriment-adjusted risk ( 25% in 1977, 18% in 1991 and 4% radiation dosimetry and radiation protection from all over quantifying radiation exposure in the early 1930s, it became somewhat lower than the one in Publication 60, is nonetheless in 2007) reflect, firstly, the changes in the magnitude of the total the world. Among these were: Laurie Taylor, Warren Sinclair, possible to express dose limits to radiation workers in considered compatible with the latter. Consequently, con- (for cancers + genetic effects) detriment-adjusted risk and Art Upton, Michael Fry, Seymour Abrahamson, Jack Schull, quantitative terms. Other developments in radiation dosimetry fidence in the dose limits recommended in Publication 60 is re- changes in how post-radiation generation genetic effects were Jack Little, Julian Preston and Charles Land (from the US), Bill followed. In the mid-1950s, the concept of regulation of the affirmed. included in the calculations (first two in 1977 and 2007 and Pochin, Roger Clarke and Roger Cox (UK), Albrecht Kellerer overall average radiation dose to the population and the view Over the period under review, genetic risks (expressed in equilibrium figures in 1991). It should be borne in mind, however, and Chris Streffer (Germany), Bo Lindell, Lars-Erik Holm and that the risk of genetic effects should be used as the yardstick units of radiation-inducible genetic diseases) have been that cancer risks pertain to the exposed, individuals themselves Jack Valentin (Sweden), Per Oftedal (Norway), Hiromichi for setting exposure dose limits gained acceptance. The dose estimated indirectly on the basis of mouse data on induced and genetic risks, to the descendants of those exposed, and Matsudaira, Masao Sasaki and Ohtsura Niwa (Japan), De limits to radiation workers (5 rem in a year), to individual mutation rates. One of the main breakthroughs in this field therefore these types of risks are not strictly comparable with Chang Wu (China) and several others (H.J. Muller whom I did members of the public (0.5 rem/year) and to the population at occurred early on (the demonstration of dose-rate and dose- each other. Further, in situations where individuals or populations not have the privilege to meet, was associated with ICRP before large (5 rem over a 30-year period or 170 mrem/year) fractionation effects for induced mutations in the late 1950s sustain unexpected or unwanted radiation exposures and are my time, as a member of the main Commission from 1959 to recommended in ICRP Publication 1 (published in 1959) through the early 1970s). From then until the early 1990s, concerned about the risks to themselves or to their progeny, the 1965). although not based on actual observations of radiation-induced progress in conventional mouse mutation studies was risk estimates in the UNSCEAR reports (and not the detriment- The memories of one EU-funded, ICRP-sponsored visit to genetic effects in humans, reflected this point of view. essentially incremental although some of the cytogenetic and adjusted ones in the ICRP documents) are the ones to be used to Madison, WI, to meet with Jim Crow and Carter Denniston, I By the early 1960s, however, the radiation protection molecular studies conducted during this period began to access the needed information. think in the mid-1980s, still remains fresh in my mind. These community recognized that cancer risks are much more shed light on the nature of radiation-induced mutations (e.g. As mentioned in Section 2, in this review the focus has been authors had published a paper in Science on mutation important quantitatively than genetic risks. Over a period of [85,91–93]) enabling comparisons to be made with mutations on genetic effects and their relative importance in radiation component [101] and I wanted to learn about this in the about 15 years, this shift in perspective led to the development underlying naturally occurring genetic diseases in humans. protection recommendations of ICRP with respect to two context of risk estimation for congenital anomalies and chronic and adoption of a risk-based protection system in a ICRP’s This, in turn, set the stage for further developments in risk ‘‘planned exposure situations’’. It should be stressed, however, diseases, a problem in which both ICRP and UNSCEAR were 1977 Publication 26. The three key principles underscored in estimation in subsequent years. that ICRPs role in radiation protection encompasses a much interested. I took Andrew Czeizel with me for discussions that report - Justification of a Practice, Optimization of Estimates of genetic risk have varied over the last 50 years. broader spectrum of activities with its recommendations [Andrew is a Hungarian teratologist/epidemiologist who has Protection and Individual Dose Limits—have remained valid Changes emerging between the mid-1950s and the early 1990s applicable to all sources and to individuals exposed to radiation made major contributions to the study of congenital anomalies since that time. In the new system, rate estimates for both have been driven more by advances in knowledge of baseline (planned exposure situations, emergency exposure situations, in humans and with whom I did collaborative research for over cancer induction and genetic effects in the progeny were taken a frequencies of human genetic diseases than by any real existing exposure situations) and all categories of exposures a decade]. Jim assured me that they were making good progress step further by incorporating a new measure known as breakthroughs in our understanding of the genetic sensitivity of (occupational exposure, public exposure and medical exposure on the issue and that, with luck, he would provide me with a detriment, using mortality for cancers and severity for genetic the human species (see Table 4 and recall that in the risk of patients). Over the years, ICRP has provided guidance on all draft of the paper within about a month. I was ecstatic and was effects as indicators. The estimated detriment-adjusted nominal equation, risk = P 1/DD MC PRCF, the baseline fre- these issues in specific documents. For details of these, the looking forward to the manuscript. [Alas, the luck did not smile risk coefficient for both types of effects combined was quencies of the disease classes, P, were revised from time to reader is referred to the 2007 Recommendations. on us then, but as mentioned later, the problem was handled in

274 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 275 K. Sankaranarayanan, J.S. Wassom / Mutation Research 658 (2008) 1–27 25 26 K. Sankaranarayanan, J.S. Wassom / Mutation Research 658 (2008) 1–27 the mid-1990s]. One unforgettable thing happened during the revised draft versions of the 2007 ICRP Recommendations and [22] ICRP, Genetic Susceptibility to Cancer, ICRP Publication 79, Annals of [43] ICRP, 1990 Recommendations of the International Commission on Wisconsin visit: Jim took me and Andrew to visit with Sewall of Annex A (both currently in press) and Prof. O. Niwa, Kyoto the ICRP, vol. 28, no. 1/2, Pergamon/Elsevier, 1998. Radiological Protection, ICRP Publication 60, Annals of the ICRP, Wright, who, at that time was in his late 80s and staying in a University, Kyoto and a member of Committee 1 of ICRP, for [23] ICRP, Risk Estimation for Multifactorial Diseases, ICRP Publication 83, vol. 21, nos. 1–3, 1991. Annals of the ICRP, 29, Nos. 3/4, Pergamon/Elsevier, 1999. [44] ICRP, 2007 Recommendations of the International Commission on senior citizens’ home. It was an unforgettable visit. I felt so actually providing us with electronic copies of the above. We [24] ICRP, Committee 1 Task Group Report: C1 Foundation Document Radiological Protection, Draft Version, 14 March 2007, Annals of the proud and thrilled to have been able to meet one of the founders acknowledge with gratitude the superb editorial assistance of (Annex A of Main Recommendations), Draft Version, 23 April 2007, ICRP, in press. of mathematical population genetics. Dr. L. Pray (Holyoke, MA, USA). The research for and the Annals of the ICRP, in press. [45] NAS/NRC, National Academy of Sciences/National Research Council. In the late 1980s, when ICRP was working on Publication 60 writing of this paper would not have been possible without the [25] UNSCEAR, Report of the United Nations Scientific Committee on the The Biological Effects of Atomic Radiation (BEAR) Report, NAS/NRC, (which appeared in 1991), the genetic risk estimates I provided kind encouragement and sustained support of Prof. L.H.F. Effects of Atomic Radiation, General Assembly Official Records, 13th Washington, DC, 1956. session, Suppl. 17, United Nations, NY, 1958. [46] National Academy of Sciences/National Research Council (NAS/NRC), in the 1988 UNSCEAR report were used as a starting point for Mullenders (Department of Toxicogenetics, Leiden University [26] UNSCEAR, Report of the United Nations Scientific Committee on the The Biological Effects of Atomic Radiation (BEAR), NAS/NRC, estimating detriment for genetic effects. As noted in Section 7.1 Medical Centre) who generously permitted KS to have access Effects of Atomic Radiation, General Assembly Official Records, 17th Washington, DC, 1960. (this paper), at that time, I was hesitant to provide a risk to the facilities and enjoy the intellectual atmosphere of the session, Suppl. 16, United Nations, NY, 1962. [47] NAS/NRC, The Effects on Populations of Exposure to Low Levels of estimate for multifactorial diseases since the baseline Department. [27] UNSCEAR, Report of the United Nations Scientific Committee on the Ionizing Radiation, Report of the Advisory Committee on the Biological frequencies of these were quite high (71%) and the use of Effects of Atomic Radiation, General Assembly Official Records, 21st Effects of Ionizing Radiations (BEIR I report), NAS/NRC, Washington, session, Suppl. 14, United Nations, NY, 1966. DC, 1972. the 5–50% mutation component (used in the 1977 UNSCEAR [28] UNSCEAR, Report of the United Nations Scientific Committee on the [48] NAS/NRC, The Effects on Populations of Exposure to Low Levels of report) to obtain a risk estimate did not seem right. But the References Effects of Atomic Radiation to the General Assembly, with Annexes, vol. Ionizing Radiation, (BEIR III Report), NAS, Washington, DC, 1980. ICRP did need an estimate for detriment calculations. My II, Effects UN Publication Sales No. E.72.IX.18, United Nations, NY, [49] NAS/NRC, Health Effects of Exposure to Low Levels of Ionizing Radia- argument that there was not much science to give a reliable risk [1] W. Ro¨ntgen, Uber eine neue Art van Strahlen (vorlaufige Mitteilung) 1972. tion (BEIR V Report), National Academy Press, Washington, DC, 1990. estimate for multifactorial diseases at that time was of no avail Sitzungs Berichte der Physikalische-Medicinischen Gesellschaft zu [29] UNSCEAR, Sources and Effects of Ionizing Radiation, Report of the [50] NAS/NRC, Health Risks from Exposure to Low Levels of Ionizing Wurzburg, 9 (1895) 132. United Nations Scientific Committee on the Effects of Atomic Radiation Radiation (BEIR VII, Phase 2 report), National Academy Press, and I ended up providing one that I viewed as ‘reasonable’ [81]. [2] H. Becquerel, Emission de radiations nouvelles par l’uranium me´talli- to the General Assembly, with Annexes, UN Publication Sales No. Washington, DC, 2005. Fortunately, in 1993, the ICRP approved a Task Group on ‘Risk que, C. R. Acad. Sci., Paris 122 (1896) 1086. E77.IX.I, United Nations, NY, 1977. [51] A. Mutscheller, Physical standards of protection against Roentgen ray estimation for multifactorial diseases’ to study the problem in [3] M. Curie, Rayon semis par les composes d’uranium et du thorium, C. R. [30] UNSCEAR, Ionizing radiation: sources and Biological Effects. Report of dangers, Am. J. Roentgenol. 13 (1925) 65. detail. The members (besides myself) were: Carter Denniston, Acad. Sci., Paris 126 (1898) 1101. the United Nations Scientific Committee on the Effects of Atomic [52] NCRP, National Council on Radiation Protection and Measurements, Nori Yasuda, Ranajit Chakraborty, Eric Boerwinkle and [4] M. Curie, Traite´ de Radioactivite´, Gauthier-Villars, Paris, 1910. Radiation to the General Assembly, with Annexes, UN Publication Sales Permissible Dose from External Sources of Ionizing Radiation, Maximum [5] H. Rolleston, Harmful effects of irradiation. A critical review, Ouart. J. No. E82.IX.8, United Nations, NY, 1982. Permissible Exposure to Man, NCRP Report 17 (1954) US Govt Printing Andrew Czeizel. We had the privilege of having Jim Crow Med. 4 (1930) 101. [31] UNSCEAR, Genetic and Somatic Effects of Ionizing Radiation, Report Office, Nat. Bureau of Standards, Handbook 59) Washington, DC. and Jack Schull as corresponding members on this task group. [6] R.S. Stone, Fifty years of radiology: from Roentgen to the era of atomic of the United Nations Scientific Committee on the Effects of Atomic [53] Medical Research Council (MRC), The Hazards to Man of Nuclear and Most of the members were mathematical population geneticists power, Nebraska State Med. J. 31 (1946) 56–66. Radiation to the General Assembly, with Annexes, UN Publication Sales Allied Radiations. Cmd 9780, HMS Office, London, 1956. and very busy people; for me as Chairman, the assignment was [7] R.S. Stone, Maximum Permissible Standards: Protection in Diagnostic No. E86.IX.9, United Nations, NY, 1986. [54] E.B. Lewis, Leukemia and ionizing radiation, Science 125 (1957) 965– as challenging as it was daunting! We did come up with a Radiology, Rutgers University Press, New Brunswick, NJ, 1959. [32] UNSCEAR, Sources, Effects and Risks of Ionizing Radiation, United 972. [8] H. Colwell, S. Russ, X-ray and Radium Injuries: Prevention and Nations Scientific Committee on the Effects of Atomic Radiation, Report [55] J.F. Crow, W. Bender, Perspectives. Anecdotal, historical and critical mathematical model (ICRP Publication 83 [23]), the first one of Treatment, Oxford University Press, London, 1934. to the General Assembly, with Annexes, UN Publication Sales No. commentaries on Genetics. E.B. Lewis, 1918–2004, Genetics 168 (2004) its kind, for use in estimating the radiation risk of chronic [9] L.S. Taylor, Radiation Protection Standards, CRC Press, 1971. E88.IX.7, United Nations, NY, 1988. 1773–1783. diseases [34,88]. [10] D.P. Server, The Rise of Radiation Protection: Science, Medicine and [33] UNSCEAR, Sources and Effects of Ionizing Radiation, United Nations [56] N.V. Timofeeff-Ressovsky, K.G. Zimmer and M. Delbruck, Uber die What I admire most about the committees that I had been Technology in Society, 1896–1935. Brookhaven National Laboratory, Scientific Committee on the Effects of Atomic Radiation, Report to the natur der genmutation und der genstructuur. Vierter teil: Theorie der privileged to be part of, is that the major conclusions/decisions N.Y., BNL 22279, Informal Report, 1976. General Assembly with Scientific Annexes, UN Publication Sales No. genmutation und der genstructuur, Nachr Ges Wiss Gottingen Math [11] K.L. Mossman, A brief history of radiation bioeffects, in: W.R. Hendee, E94.IX.2, United Nations, NY, 1993. Physik KI Fachgruppe VI, I (1935) 156–241. they made over the years regarding both science and policy, by F. Edwards (Eds.), Health Effects of Exposure to Low-Level Ionizing [34] UNSCEAR, Hereditary Effects of Radiation, United Nations Scientific [57] M. Tubiana, A. Aurengo, D. Averbeck, A. Bonnin, B. Le Guen, R. Masse, and large, turned out to be correct in retrospect. I have no real Radiation, IOP Publishing, Bristol, Philadelphia, 1996, pp. 1–26 (Chapter Committee on the Effects of Atomic Radiation, Report to the General R. Monier, A.J. Valleron, F. de Vathaire, Joint Report No. 2, Acade´mie explanations for this observation. My perception is that the 1). Assembly with Scientific Annex, UN Publication Sales No. E01.IX.2, Nationale de Me´decine, Institut de France-Acade´mie des Sciences, Dose collective wisdom transcending the individuals somehow [12] W.R. Hendee, Concepts, history and present status of radiation protec- United Nations, NY, 2001. effect relationships and the estimation of the carcinogenic effects of low provided the right answers. Embedded somewhere there are tion, in: W.R. Hendee, F. Edwards (Eds.), Health Effects of Exposure to [35] International X-ray and Radium Protection Committee (ICRP), Recom- doses of ionizing radiation (http://www.academiemedicine.fr/actualities/ Low-Level Ionizing Radiation, IOP Publishing, Bristol, Philadelphia, mendations of the Second International Congress of Radiology, Br. J. rapports.asp), Edition Nucleon, Paris, 2005. the clues to the strength and longevity of these institutions. This 1996 , pp. 495–525 (Chapter 15). Radiol. 1 (1928) 358. [58] M. Tubiana, A. Aurengo, D. Averbeck, R. Masse, Recent reports on the does not necessarily mean that members did not have different [13] R. Clarke, J. Valentin, A history of the International Commission on [36] International X-ray and Radium Protection Committee (ICRP), 1934 effects of low doses of ionizing radiation and its dose effect relationship, views or arguments; they did (sometimes diametrically Radiological Protection, Health Phys. 88 (2005) 717–732. Recommendations for X-ray and Radium Protection, Radiology 23 Radiat. Environ. Biophys. 44 (2006) 245–251. opposite ones, especially in the UNSCEAR) but with patience, [14] C.G. Jones, A review of the history of U.S. radiation protection regula- (1934) 682–685. [59] A.A. Friedl, W. Ru˝hm, LNT: a never ending story, Radiat. Environ. tact, and tenacity on the part of many, they got resolved over tions, recommendations and standards, Health Phys. 88 (2005) 697–716. [37] International Commission on Radiological Protection (ICRP), Br. J. Biophys. 44 (2006) 241–244. [15] H.J. Muller, Artificial transmutation of the gene, Science 66 (1927) Radiol. Suppl. 6, Brit. Institute of Radiology, London, 1955 (revised [60] D.J. Brenner, R.K. Sachs, Estimating radiation-induced cancer risks at time. Members have come and gone, but the institutions endure. 84–87. December 1, 1954). very low doses: rationale for using a linear no-threshold approach, [16] C. Auerbach, Mutation Research, Problems, Results and Perspectives, [38] ICRP, Recommendations of the International Commission on Radiolo- Radiat. Environ. Biophys. 44 (2006) 253–256. Acknowledgements Chapman and Hall, London, 1976. gical Protection, (adopted September 9, 1958) ICRP Publication 1, [61] J. Breckow, Linear-no-threshold is a radiation protection standard rather [17] E.A. Carlson, Genes, Radiation and Society: The Life and work of H.J. Pergamon Press, Oxford, 1959. than a mechanistic effect model, Radiat. Environ. Biophys. 44 (2006) We thank Professors G.R. Hoffmann and D.G. MacPhee, the Muller, Cornell University Press, Ithaca, NY, 1981. [39] ICRP, Recommendations of the International Commission on Radiolo- 257–260. [18] E.P. Cronkite, V.P. Bond, R.A. Conrad, N.R. Shulman, R.S. Farr, S.H. gical Protection, (adopted September 17, 1965) ICRP Publication 9, [62] A.C. Stevenson, The load of hereditary defects in human populations, Reflections Editors for inviting one of us (KS) to contribute this Kohn, C.L. Dunham, Response of human beings accidentally exposed to Pergamon Press, Oxford, 1966. Radiat. Res. Suppl. 1 (1959) 306–325. article and their very prompt and constructive comments on the significant fallout radiation, JAMA 150 (1955) 430–434. [40] ICRP, Recommendations of the International Commission on Radiolo- [63] Genetics Conference, Committee on Atomic Casualties, NRC, Genetic manuscript. We are indebted to Professors J.F. Crow (Madison, [19] T. Takahashi, K.R. Trott, K. Fujimori, N. Nakashima, H. Ohtomo, M.J. gical Protection, (adopted January 17, 1977) ICRP Publication 26, effects of the atomic bombs in Hiroshima and Nagasaki, Science, 106 WI, USA), R.H. Clarke (Emeritus Chairman of ICRP, Chilton, Schoenmaker, S.L. Simon, Thyroid Diseases in The Marshall Islands. Annals of the ICRP 1-3, Pergamon Press, Oxford, 1977. (1947) 331–333. UK) and R. Cox (formerly Chairman of Committee 1 of ICRP Findings from 10 Years of Study, Tohoku University Press, Sendai, 2001. [41] ICRP, Problems Involved in Developing an Index of Harm, ICRP [64] J.V. Neel, W.J. Schull, The Effect of Exposure to the Atomic Bombs on [20] International Commission on Radiological Protection (ICRP), History, Publication 27, Annals of the ICRP, vol. 1, no. 4, Pergamon Press, Pregnancy Termination in Hiroshima and Nagasaki, Publ. 461, NAS- and currently Vice Chairman of ICRP, and, Director, CRCE, Policies and Procedures, Elsevier Science, Ltd., 1999. Oxford, 1977. NRC, Washington, DC, 1956. Health Protection Agency, Chilton, UK) for their perceptive [21] ICRP, Risks associated with ionizing radiations, Five papers prepared by [42] ICRP, Quantitative bases for developing a unified index of harm, Pub- [65] H.J. Muller, Radiation damage to the genetic material, in: G.A. Baitsell comments on an earlier draft of this paper. We thank Dr. J. a Task Group of Committee 1 of the ICRP, Annals of the ICRP, vol. 22, lication 45, Annals of the ICRP, vol. 5, no. 3, Pergamon Press, Oxford, (Ed.), Science in Progress, 7th series, Yale University Press, New Haven, Valentin, Secretary of ICRP for kindly permitting us to use the no. 1, Pergamon Press, Oxford, 1991. 1985. CT, 1951, pp. 93–177.

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[66] H.J. Muller, The manner of dependence on the permissible dose of [85] K. Sankaranarayanan, Ionizing radiation and genetic risks. X. The Mutation Research 682 (2009) 1–6 radiation on the amount of genetic damage, Acta Radiol. 41 (1954) 5–20. potential ‘‘disease phenotypes’’ of radiation-induced genetic damage Mutation Research 682 (2009) 1–6 [67] H.J. Muller, Advances in radiation mutagenesis through studies on in humans: perspectives from human molecular biology and radiation Drosophila, in: J.C. Burger (Ed.), Progress in Nuclear Energy Series genetics, Mutat. Res. 429 (1999) 45–83. Contents lists available at ScienceDirect VI, Pergamon Press, New York, 1959, pp. 146–160. [86] K. Sankaranarayanan, Ionizing radiation and genetic risks. IX. Estimates [68] K. Sankaranarayanan, R. Chakraborty, Ionizing radiation and genetic of the frequencies of Mendelian diseases and spontaneous mutation rates Electric light causes cancer? risks. XI. The doubling dose estimates from the mid-1950s to the present in human populations: a 1998 perspective, Mutat. Res. 411 (1998) Mutation Research/Reviews in Mutation Research and the conceptual change to the use of human data on spontaneous 129–178. ☆ mutation rates and mouse data on induced mutation rates, Mutat. Res. [87] R. Chakraborty, N. Yasuda, C. Denniston, K. Sankaranarayanan, Ionizing Surely you’rejournal joking, homepage: www.elsMr.evier.com/locate/reviewsmr Stevens 453 (2000) 107–122. radiation and genetic risks. VII. The concept of mutation component and Community address: www.elsevier.com/locate/mutres [69] ICRP, Report on 1956 amendments to the recommendations of the its use in risk estimation for Mendelian diseases, Mutat. Res. 400 (1998) Richard G. Stevens * International Commission on Radiological Protection (ICRP), Radiology 541–552. 70 (1958) 261–262. [88] C. Denniston, R. Chakraborty, K. Sankaranarayanan, Ionizing radiation Reflections in Mutation Research [70] NCRP, Maximum permissible radiation exposure to man—a preliminary and genetic risks. VIII. The concept of mutation component and its use in Department of Community Medicine and Health Care, University of Connecticut Health Center, statement of the National Committee on radiation protection and mea- risk estimation for multifactorial diseases, Mutat. Res. 405 (1998) 57–79. Electric light causes cancer? surements, Radiology 68 (1957) 260. [89] K. Sankaranarayanan, R. Chakraborty, Ionizing radiation and genetic School of Medicine, 263 Farmington Avenue, Farmington,§ CT 06030-6325, USA [71] International Commission on Radiological Protection and International risks. XIII. Summary and synthesis of papers VI to XII and estimates of Surely you’re joking, Mr. Stevens Commission on Radiological Units and Measurements (ICRP and genetic risks in the year 2000, Mutat. Res. 453 (2000) 183–197. ICRU), Joint study group report on exposure of man to ionizing [90] E.L. Green, Genetic effects of radiation on mammalian populations, Ann. Richard G. Stevens * radiation arising from medical procedures, Phys. Med. Biol. 2 Rev. Genet. 2 (1968) 87–120. Department of Community Medicine and Health Care, University of Connecticut Health Center, School of Medicine, 263 Farmington Avenue, Farmington, CT 06030-6325, USA (1957) 107–151. [91] E.M. Rinchik, L.B. Russell, Germ-line deletion mutations in the mouse: [72] B.K. Trimble, J.H. Doughty, The amount of hereditary disease in human tools for intensive functional and physical mapping of regions of the populations, Ann. Hum. Genet. 38 (1974) 199–223. mammalian genome, in: K.E. Davies, S.M. Tilghman (Eds.), Genome [73] W.L. Russell, The effects of radiation dose-rate and fractionation on Analysis, Genetic and Physical Mapping, vol. 1, Cold Spring Harbor ARTICLE INFO ABSTRACT mutation in mice, in: F.H. Sobels (Ed.), Repair from Genetic Radiation Laboratory Press, New York, 1990, pp. 121–158. Damage, Pergamon Press, Oxford, 1963, pp. 205–217. [92] B.M. Cattanach, M.D. Burtenshaw, C. Raspberry, E.P. Evans, Large Article history: Night is no longer dark in the modern world, and the Milky Way has disappeared. Electric light has [74] W.L. Russell, L.B. Russell, E.M. Kelly, Radiation dose rate and mutation deletions and other gross forms of chromosome imbalance compatible Accepted 7 January 2009 benefits but there are also a few detriments. These are (1) loss of the night sky, (2) wasted energy, (3) Available online 16 January 2009 frequency, Science 128 (1958) 1546–1550. with viability and fertility in the mouse, Nat. Genet. 3 (1993) harm to animal and plant life, (4) and perhaps increases in some severe human maladies such as cancers [75] M.F. Lyon, R.J.S. Phillips, H.J. Bailey, Mutagenic effects of repeated 56–61. of breast and prostate. The science on phototransduction for the circadian system and on clock gene Keywords: small radiation doses to mouse spermatogonia. I. Specific-locus mutation [93] B.M. Cattanach, E.P. Evans, C. Raspberry, M.D. Burtenshaw, Incidence function is evolving rapidly, and it provides a rationale for the idea that circadian disruption from light at Breast cancer rates, Mutat. Res. 15 (1972) 185–190. and distribution of radiation-induced large deletions in the mouse, in: U. night could cause disease. Direct evidence from humans and rodent models has also accumulated to the Light-at-night point where the idea is no longer fanciful. Although it may seem logical now, the journey on the path [76] W.L. Russell, L.B. Russell, M.B. Cupp, Dependence of mutation fre- Hagen, D. Harder, H. Jung, C. Streffer (Eds.), Congress Proceedings, Circadian disruption quency on radiation dose-rate in female mice, Proc. Natl. Acad. Sci. Tenth Int. Cong. Rad. Res., vol. 2, Wu¨rburg, Germany, 1996. from electric light to breast cancer has been a tortuous one, at least for me. ß 2009 Elsevier B.V. All rights reserved. U.S.A. 45 (1959) 18–23. [94] J.V. Neel, W.J. Schull (Eds.), The Children of Atomic Bombs, A Genetic [77] K.G. Lu¨ning, A.G. Searle, Estimates of genetic risks from ionizing Study, National Academy Press, Washington, DC, 1991. radiation, Mutat. Res. 12 (1971) 291–304. [95] J.V. Neel, W.J. Schull, A.A. Awa, C. Satoh, H. Kato, M. Otake, Y. [78] C.O. Carter, Monogenic disorders, J. Med. Genet. 14 (1977) 316–320. Yoshimoto, The children of parents exposed to atomic bombs: estimates [79] C.O. Carter, Genetics of common single malformations, Br. Med. Bull. of the genetic doubling dose of radiation for humans, Am. J. Hum. Genet. ‘‘When you’re thinking about something that you don’t understand, 32 (1976) 21–26. 46 (1990) 1053–1072. in adulthood except for a modest effect of alcohol consumption [5]. you have a terrible, uncomfortable feeling called confusion.’’ – [80] P. Oftedal, A.G. Searle, An overall genetic risk assessment for radi- [96] M. Otake, W.J. Schull, J.V. Neel, Congenital malformations, stillbirths, The latest cohort study [6] and a large intervention trial [7] do both Richard Feynman, 1963. ological protection purposes, J. Med. Genet. 17 (1980) 15–20. and early mortality among the children of atomic bomb survivors: a report a small impact of very high fat consumption of borderline [81] K. Sankaranarayanan, Genetic effects of ionizing radiation in man, Ann. reanalysis, Radiat. Res. 122 (1990) 1–11. statistical significance, yet the large effect of adult fat intake ICRP 22 (1) (1991) 75–94. [97] Y. Yoshimoto, J.V. Neel, W.J. Schull, H. Kato, M. Soda, R. Eto, K. 1. The mystery of breast cancer predicted long ago seems now to have been ruled out. [82] A. Czeizel, K. Sankaranarayanan, The load of genetic and partially Mabuchi, Malignant tumors during the first two decades of life in the I got to the Pacific Northwest Laboratory (PNL) in Richland, genetic diseases in man. I. Congenital anomalies: estimates of detriment offspring of atomic bomb survivors, Am. J. Hum. Genet. 46 (1990) 1041– In the mid-1980s I was among the many who were dismayed by Washington, in 1984 at a time when these first diet studies were in terms of years of life lost and years of impaired life, Mutat. Res. 128 1052. the recent reports that dietary fat consumption was unrelated to hitting the press. (I came from the Institute for Cancer Research in (1984) 73–103. [98] A.E. Czeizel, C. Elek, E. Susansky, The evaluation of the germinal risk of breast cancer in women. We had assumed that the large Philadelphia, but that comes later.) Coincidentally, one of our [83] A. Czeizel, K. Sankaranarayanan, A. Losonci, T. Rudas, M. Keresztes, mutagenic impact of Chernobyl radiological contamination in Hungary, international differences in risk must be mainly a result of the sponsors at the Department of Energy, Bob Goldsmith, had a small The load of genetic and partially genetic diseases in man. II. Some Mutagenesis 6 (1991) 285–288. high-fat Western diet. For example, the historically low risk in amount of funding available for research on power frequency selected common multifactorial diseases: estimates of population pre- [99] J. Little, The Chernobyl accident, congenital anomalies and other Japan has been increasing dramatically in recent decades as the electromagnetic fields (EMF) and health. This was an issue of valence and of detriment in terms of years of lost and impaired life, reproductive outcomes, Pediatr. Perinat. Epidemiol. 7 (1993) 121–151. intake of animal fat has correspondingly increased as well. The acrimonious public argument at the time, but the possibility of Mutat. Res. 196 (1988) 259–292. [100] J. Byrne, S.A. Rasmussen, S.C. Steinhorn, Genetic disease in offspring of rodent model is very strong; if rats are fed a diet high in saturated some adverse effects seemed worth investigating to me. Given my [84] K. Sankaranarayanan, R. Chakraborty, Ionizing radiation and genetic long-term survivors of childhood and adolescent cancer, Am. J. Hum. fat, chemically induced tumor yield increases [1]; if polyunstatu- despair at the lack of a dietary explanation of breast cancer, I risks. XII. The concept of ‘‘potential recoverability correction factor’’ Genet. 62 (1998) 45–52. rated fatty acids (e.g., linoleic acid) are added as well, tumor yield decided to find out whether a study of EMF and breast cancer (PRCF) and its use for predicting the risk of radiation-inducible genetic [101] J.F. Crow, C. Denniston, The mutation component of genetic disease, would make sense. I asked Larry Anderson, a staff scientist and disease in human live births, Mutat. Res. 453 (2000) 129–179. Science 212 (1981) 888–893. increases even more [2]. And on a population level, there is a strong international correlation between estimates of per capita fat friend at PNL, if he knew of any biological effects of these very consumption and breast cancer incidence among countries [3]. Yet weak, power frequency fields. He told me that Bary Wilson had after decades of intense research, including many large prospective published a finding of reduced melatonin in rats after exposure cohort studies, estimates of fat consumption for individual women [8]. I had no idea what that might mean so I asked a guy I knew at and risk to those women are almost unrelated [4]. In fact there are the Fred Hutchinson Cancer Research Center in Seattle, David virtually no consistent associations of risk with any dietary factors Thomas, what he knew about melatonin. He gave me a paper by Cohen et al. that had appeared in the Lancet in 1978 [9]. This paper provided a fascinating entre´ e into a field I have been struggling to § This article is part of the Reflections in Mutation Research series. To suggest understand ever since. Cohen proposed that lower melatonin topics and authors for Reflections, readers should contact the series editors, might lead to elevated estrogen and thereby increase breast G.R. Hoffmann ([email protected]) or D.G. MacPhee cancer risk. The notion that lifetime exposure to estrogen was a ([email protected]). * Tel.: +1 860 679 5475 fax: +1 860 679 5464. major factor in causing breast cancer was just beginning to be E-mail address: [email protected]. widely appreciated [10]. On this basis, Cohen further proposed

1383-5742/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2009.01.003

278 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 279 2 R.G. Stevens / Mutation Research 682 (2009) 1–6 R.G. Stevens / Mutation Research 682 (2009) 1–6 3 that international differences in prevalence of pineal calcification surprisingly, had a profound impact on the organization of our long time. Meanwhile, Scott, Dana Mirick, and I finished a paper [41]. In 2001, Bud published his spectral response [42] and later an might therefore explain the differences among societies in their metabolic, cellular, and organismal processes. Suddenly (in showing a significantly increased risk of breast cancer in women important extension of it to phase resetting in a study led by Steve burden of disease. evolutionary time), the bulk of humanity began to be exposed with a history of night work; we submitted it to the Journal of the Lockley, a very talented chronobiologist in Cziesler’s lab [43]. The Although they made no mention of light-at-night (LAN) or to light during the night after the introduction of electric power, National Cancer Institute (JNCI) on May 3, 2001. A couple weeks melatonin response spectrum required hundreds of arduous electricity, Cohen et al. did allude to early laboratory experiments in and to dim, spectrum-restricted light during the day inside later I received a paper to review from JNCI submitted by the NHS I individual experiments in which volunteers were in the lab over which constant light in the animal rooms affected mammary buildings. Everything changed, and not all for the good. reporting increased risk in nurses with a long history of rotating night and exposed to one of many combinations of monochromatic tumorigenesis in rodents. I was curious about this for no good I wanted to have a meeting about all of this, so I called Les shift work. It had been submitted May 8. It was amazing that after a light of a specific wavelength and a specific photon flux density. It reason but tracked down some of these studies nonetheless. It began Reinlib at the National Institute of Environmental Health Sciences dozen years these two studies were submitted to the same journal took over 5 years to do it right. The peak sensitivity turns out to be with pioneering work by Jo¨chle [11] and by Khaetski ([12]; (NIEHS). Much to my pleasure, Les was very supportive and we got within a week of each other. I recommended to JNCI that the NHS I at about 480 nm, which is the wavelength of that beautiful blue we described in [13]) in the 1960s. The tumor outcomes of experiments a healthy budget for the meeting, which Dave Blask and I co- paper be accepted. (I was mildly perturbed that I was not see in the sky on a clear day at mid-morning. It is probably no from a dozen labs over the years since then have been mixed, but chaired. It was an ambitious endeavor because we wanted top mentioned in the acknowledgments in the paper for suggesting the coincidence from an evolutionary perspective that the system for one study from which I learned much was described in a marvelous researchers for all aspects of the subject. I sent an email to Susan idea, although I did not complain in my anonymous review; telling our inner self whether it is day or not is finely tuned to that paper by Shah et al. [14] in which constant light not only increased Golden who had done elegant work [20] on the clock gene fortunately, this was corrected by Sue Hankinson in the NHS II wavelength. The new photoreceptive cell found in the retina, called chemically-induced tumor yield but also altered the normal mechanism in cyanobacteria (a.k.a. ‘pond scum’). She returned an paper several years later [26]; this study also found elevated risk in the intrinsically photoreceptive retinal ganglion cell (ipRGC), also development and differentiation of the mammary tissue in rats. email asking why in the heck would a basic scientist like her, rotating shift workers). responds maximally to this wavelength [44]. A few years earlier, Lewy and co-workers [15] had made the seminal studying bacteria, be invited to a meeting about breast cancer. I It was a coincidence that the two papers were published in the Meanwhile, Dave Blask at the Bassett Research Institute had for observation that bright light in the middle of the night suppressed told her that we need new directions and perspectives in the same issue of JNCI in 2001 [27,28]; a very nice coincidence because years been conducting serious experiments on the mechanism by melatonin production in humans, the first such observation. I was pursuit of understanding breast cancer, and I told her why clock their publication was the turning point for the LAN/breast cancer which melatonin was oncostatic to breast cancer e.g., [45,46]. enduring the anguish of trying to make sense of a new area of genes in cyanobacteria matter to a breast cancer researcher like topic. Despite the date of publication, October 17, 2001, during the (Dave would strongly insist that this work be attributed not only to science when, some months into this journey of confusion, I found me. She emailed back that she would be there. peak worries about anthrax after 9/11, it got a lot of media him but also to his many co-workers over the years.) Dave had an myself fretting about being awake in the middle of the night in my We also invited, among other notables, David Berson [21] who attention. Actually, Johnni Hansen had scooped us with a strong idea that was inspired; he suggested to Bud that he recruit young apartment. I suddenly realized I could almost read a newspaper by had helped find the new photoreceptive cell for the circadian study from Denmark published earlier in the year [29]. Johnni women in Philadelphia into an experiment in which Bud would the street light shining through the shades on my window. Like a system in the retina (ipRGC) which Science Magazine called one of wrote an excellent editorial for JNCI that accompanied the two take blood under three different conditions: during the day, at light bulb going on, it occurred to me that maybe it was the light the Top 10 breakthroughs for 2002 (it is actually much more than papers [30]. Much other evidence has accumulated, and it resulted night in the dark, at night after light exposure. Bud would send the bulb going on that accounted for some of the breast cancer that), Iggy Provencio [22] who found the photopigment for these in the International Agency for Research on Cancer (IARC) blood to Dave’s lab in Cooperstown, where Bob Dauchy would pandemic. (I now know that midnight awakening is normal and new cells (melanopsin), Chris Bradfield [23] who discovered one of concluding that ‘‘shift-work that involves circadian disruption is infuse the blood into xenografts of a human-derived breast cancer healthy as long as you stay quietly in the dark [16].) the core clock genes in mammals (he called it ‘MOP3’ only to have probably carcinogenic to humans (Group 2A)’’ [31]. (MCF7) growing in nude rats. (Bob invented this remarkable There have been a couple dozen epidemiological studies of EMF it renamed ‘BMAL1’ against his will by the Borg Collective), and Another prediction of the LAN theory is that blind women surgical technique [47].) As predicted, blood taken during the night and breast cancer with mixed results but that on balance do not Mark Rea, Director of the Lighting Research Center and editor of the should be at lower risk. Robert Hahn published this idea and the in the dark stopped the growth of the tumors, whereas blood taken support any obvious association. There were also a couple of Lighting Handbook [24], essential reading for the architectural first data in 1991 [32]. The evidence to date is not as strong as for during the day or at night after a light exposure did not slow the groups that conducted toxicological studies using rats exposed to lighting community worldwide. There were no refusals, even shift work, but a half dozen other studies have also reported lower growing cancers at all. This experiment [48] is as close as ethically power-frequency magnetic fields with conflicting results. One though the workshop was in a crummy hotel in DC; all these risk in blind women. Very recently studies have reported an possible to a direct test of whether LAN influences breast cancer group in Germany headed by Wolfgang Lo¨scher, however, has people had done that too many times in the past on various study inverse association of sleep duration and risk of breast cancer. This growth in women. pursued this with startling results including increased tumor yield, sections, yet for this meeting they came again to DC. The result of was based on the idea that reported sleep duration could be a and a strong suggestion of a genetic component to detection of the the meeting was published [25], and although breast cancer was surrogate for hours of dark [33]. It would be very crude, but that is 5. Mechanisms of carcinogenesis and the Jedi Knight fields. Their results are fascinating scientifically, whether or not the original topic, we describe potential importance of circadian what epidemiology usually must deal with. We published a paper they have application to human disease (e.g., [17]). It is astonishing disruption to certain other cancers, such as prostate, and, believe it in 2005 [34] reporting the predicted inverse association in Finland. The historical paradigm for defining a carcinogen is that it is that the work of Lo¨scher and his co-workers, published in major or not, to other chronic diseases such as obesity and diabetes as The association was quite strong so we felt smug. The Nurses’ either genotoxic or mitogenic. In general under this paradigm, the journals for over 10 years, has not gained the attention of other labs well. Health Study immediately checked their data and found no such larger the dose, the greater the cancer yield. For a long time, and funding agencies. inverse relationship, and even a suggestion of the reverse [35]. thought about causes of cancer was confined to initiation and To this day it remains highly contentious whether EMF has any 3. Predictions of the Light-at-Night (LAN) theory Now we felt considerably less smug than before. Then, with the promotion, in which an agent caused a heritable genetic alteration meaningful physiological effects on humans at all, much less in any publication of two more good prospective studies supporting our predisposing to malignant transformation and/or increased turn- way that could affect cancer risk. For light at night, however, there The Light-at-Night (LAN) theory for breast cancer is easy to inverse finding [36,37], we are again feeling smug, though not over of ‘intermediate’ cells, thus increasing the probability that a is no question that there are many physiological effects, some of state: increasing use of electricity to light the night leads to cocky because one never knows. heterozygous genetic alteration would become homozygous which may well have bearing on cancer risk, including suppression circadian disruption which accounts for part of the breast cancer [49,50]. It turns out that it is a little more complicated than that of melatonin. So, I put one and one together (I’m bright, but I’m no burden in the modern world and rising risk in developing 4. Basic biology and Dave’s experiment [51–53]. In particular, agents or environmental factors that Feynman), and published a paper in 1987 proposing that electric countries. But how to test it? Virtually no-one in industrialized increase cell turnover and differentiation in normal tissue could power (EMF and/or electric lighting at night) might increase risk of societies does not use electricity to illuminate part of the daily dark Two areas of rapidly evolving basic biology have excited many have a large impact on breast cancer risk and be neither initiators breast cancer [18]. period, be it at the end of day into the night, or at the beginning of us, both for their inherent scientific interest and for their impact nor promoters; they could actually have effects pre-initiation [54]. Almost immediately, I became somewhat deflated because I before sunrise. One prediction that occurred to me was that shift on the LAN theory for breast cancer causation. One is the Increased risk of breast cancer for a woman who had an early age at realized that breast cancer risk in Japan, though rising, was still working women should be at higher risk due to an even higher mechanism of phototransduction for the circadian system, and menarche may be an example of that. Conversely, agents or much lower than in the U.S., yet Japan has been heavily industrial exposure to light at night than day-workers. the other is the molecular genetics of circadian rhythm generation. exposures that increase the chance that a fully transformed cell or for a long time. I wondered whether the typical household in Japan So, in 1987, I wrote a letter to Walt Willett at Harvard For the clock gene mechanism there are many implications, one of colony (very small cancer) would survive to ever be diagnosed as used as much electricity as the typical household in the U.S. so I suggesting a question be added to the ongoing Nurses’ Health which would be a connection to cell cycle regulatory genes such as cancer could also have a large impact on cancer risk. The evidence called an expert at Lawrence Berkeley Lab; he sent me a paper [19] Study (NHS I). To my astonishment Walt did add a question to the cyclin D1 [38] which could provide a direct rationale for a breast that the recent reduction in use of hormone replacement therapy which estimated that indeed annual Japanese household elec- 1988 NHS I biennial questionnaire. He also put the same question cancer effect [39]. For phototransduction the story has advanced (HRT) by post-menopausal women in the last few years has already tricity consumption in 1973 was much lower than in the U.S. in the inaugural 1989 questionnaire for a new Nurses’ Cohort II greatly as described below. had a noticeable impact on breast cancer incidence may be an (1.9 MW h vs. 8.2 MW h), but was rising very fast, over 50% by (NHS II) that he was just starting. At about the same time I was Bud Brainard at Jefferson Medical College had been working example of that [55]. 1983. I became inflated again, and this is what I have pursued with working with Scott Davis, an early comrade in the electric power furiously for years to define the precise wavelengths of light that So how can exposure to light fit into a model of carcinogenesis? increasing vigor for the past 25 years. struggle, on a grant application to NIH for a case-control study. maximally suppress melatonin, as a marker of circadian rhythmi- The concept of ‘dose’ probably needs a different definition than Scott gets more done before breakfast than I do all day; among city, in the middle of the night. This spectral response function was that for ionizing radiation or a toxic chemical. 2. Evolution and electric power many other honors, he is the only American scientist I know of who critical to figuring out the phototransduction mechanism for the I learned about mechanisms of carcinogenesis at the Institute was elected to the Russian Academy of Medical Sciences for his circadian system. By the late 1990s accumulating evidence was for Cancer Research (ICR) in Philadelphia. I had come there from We, life on the planet, have evolved for several billion years monumental efforts to study health effects of Chernobyl. As PI, he suggesting that the primary mechanism was not vision per se, graduate school in Seattle in 1977 as the research assistant to with a reliable cycle of bright broad-spectrum light (the Sun) and wrote a beautiful grant that was funded in 1992. I checked every so although the retina appeared to be required; some of this evidence Suresh Moolgavkar who had accepted a position. At the time there dark. This fundamental aspect of the environment has, not often on progress of the NHS studies, but nothing happened for a was from blind persons [40] and from retinally degenerate mice were about 50 faculty members at ICR, five of whom were

280 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 281 4 R.G. Stevens / Mutation Research 682 (2009) 1–6 R.G. Stevens / Mutation Research 682 (2009) 1–6 5 members of the National Academy of Sciences (Bob Perry, Tom 6. Past and future necessary, but not sufficient condition for there to be a large effect [25] R.G. Stevens, D.E. Blask, G.C. Brainard, J. Hansen, S.W. Lockley, I. Provencio, M.S. Anderson, Beatrice Mintz, Barry Blumberg, and Ernie Rose). Barry Rea, L. Reinlib, Meeting report: the role of environmental lighting and circadian of LAN on risk. He too is moving fast on this important topic. disruption in cancer and other diseases, Environ. Health Perspect. 115 (2007) had received a Nobel Prize for his work on Hepatitis B virus, and As often happens, I stumbled into a new area while pursuing Now I work at UConn – not the Yukon in northern Canada where 1357–1362. years later, in 2004, Ernie got his for work he was doing on another: into the circadian rhythm arena while searching for huskies pull sleds but the other UConn in central Connecticut [26] E.S. Schernhammer, C.H. Kroenke, F. Laden, S.E. Hankinson, Night work and risk of ubiquitin while I was at ICR. (As my PhD advisor, Barry taught me a answers to the mystery of why breast cancer is so common in the breast cancer, Epidemiology 17 (2006) 108–111. where Huskies shoot hoops – teaching and researching and staring [27] S. Davis, D.K. Mirick, R.G. Stevens, Night shift work, light at night, and risk of lot about how to formulate a hypothesis, gather reliable data, and modern world. Early on, when I told my mentors and senior out the window. At the moment, I’m having fun. breast cancer, J. Natl. Cancer Inst. 93 (2001) 1557–1562. communicate the results coherently; he also sent me to Taiwan colleagues about electric power, they reacted with sympathy and [28] E.S. Schernhammer, F. Laden, F.E. Speizer, W.C. Willett, D.J. Hunter, I. Kawachi, and to the Solomon Islands to do this [56]) Not much later a sixth compassion for my mental state and warned me off this path. At G.A. Colditz, Rotating night shifts and risk of breast cancer in women participating Conflicts of interest in the nurses’ health study, J. Natl. Cancer Inst. 93 (2001) 1563–1568. ICR scientist, Al Knudson, was elected to NAS. the time, I was a hot young science stud publishing in big journals [29] J. Hansen, Increased breast cancer risk among women who work predominantly Part of the ICR culture was ‘tea time’ every day at 3:30. A with big co-authors. My Ph.D. advisor was a Nobel Prize winner, at night, Epidemiology 12 (2001) 74–77. None. wealthy benefactor included in her considerable endowment to Barry, and I had a real career going. Fortunately, I never heard the [30] J. Hansen, Editorial: Light at night, shiftwork, and breast cancer risk, J. Natl. Cancer Inst. 93 (2001) 1513–1515. ICR a provision for a tea time, and she expressly forbade coffee. So word ‘career’ growing up in the home of two artists, Bob and June, [31] K. Straif, R. Baan, Y. Grosse, B.E. Secretan, F.E. Ghissassi, V. Bouvard, et al., Acknowledgments every day, Suresh and I would go down to tea time and talk with in Berkeley during the 1950s and 1960s. To them all that mattered Carcinogenicity of shift-work, painting, and fire-fighting, Lancet Oncol. 8 this amazing collection of scientists. Tom Anderson, a pioneer of was one’s work. So I stuck with the electric power topic through the (2007) 1065–1066. [32] R.A. Hahn, Profound bilateral blindness and the incidence of breast cancer, electron microscopy, was particularly accessible to the younglings 1980s and 1990s, despite it being an oddity and not advancing This work was supported by National Institute of Environmental Health Sciences grant ES11659 (and by the Department of Energy in Epidemiology 2 (1991) 208–210. like me, and a wonderful guy with a great sense of science and how much, and my ‘career’ looking odd and not advancing much either. [33] R.G. Stevens, Epidemiological studies of light and breast cancer (abs), Neuroen- it is done. In the early 1980s ICR was subsumed into the Fox Chase But in fact, by the mid-1990s I was getting scared. What if this the beginning). I greatly appreciate the gentle but effective editing docrinol. Lett. 23 (suppl) (2002) 94. Cancer Center. The place has grown at least 5-fold in staff and electric power stuff really was junk science as some prominent by George Hoffmann and Mutation Research Reviews. [34] P.K. Verkasalo, K. Lillberg, R.G. Stevens, C. Hublin, M. Partinen, M. Koskenvuo, J. Kaprio, Sleep duration and breast cancer: a prospective cohort study, Cancer Res. funding, although there have been no new members elected to physicists were saying publicly and aggressively? Funding from 65 (2005) 9595–9600. NAS. Sometimes bigger is not necessarily better. DOE was drying up, and I did not have much else going on References [35] S.P. Pinheiro, E.S. Schernhammer, S.S. Tworoger, K.B. Michels, A prospective study My luck in mentors has been great, but Suresh had the most anymore. I had never taught students, and I had no NIH grants, so a on habitual duration of sleep and incidence of breast cancer in a large cohort of women, Cancer Res. 66 (2006) 5521–5525. impact in guiding my progression through the rank of Padawan. He new job at a University seemed unlikely. Then, in late 1995, I [1] W.F. Dunning, M.R. Curtis, A.E. Maun, The effect of dietary fat and carbohydrate on diethylstilbestrol-induced mammary cancer in rats, Cancer Res. 9 (1949) 354–361. [36] A.H. Wu, R. Wang, W.P. Koh, F.Z. Stanczyk, H.P. Lee, M.C. Yu, Sleep duration, treated me like a colleague, though I was excessively insecure and received a letter from Sydney Weinhouse. He wrote that as Cover [2] L.M. Braden, K.K. Carroll, Dietary polyunsaturated fat in relation to mammary melatonin and breast cancer among Chinese women in Singapore, Carcinogenesis had a morbid fear of public speaking. We published many papers Editor for the journal Cancer Research he wanted to put my picture carcinogenesis in rats, Lipids 21 (1986) 285–288. 29 (2008) 1244–1248. [37] M. Kakizaki, S. Kuriyama, T. Sone, K. Ohmori-Matsuda, A. Hozawa, N. Nakaya, together on analysis of vital data, while he was also gaining wide and work on the cover. Dr. Weinhouse was past Editor-in-Chief of [3] R.L. Prentice, F. Kakar, S. Hursting, L. Sheppard, R. Klein, L.H. Kushi, Aspects of the rationale for the Women’s Health Trial, J. Natl. Cancer Inst. 80 (1988) 802–814. et al., Sleep duration and the risk of breast cancer: the Ohsaki Cohort Study, Br. J. respect for his work on casting biological models of carcinogenesis the journal, member of the National Academy of Sciences, and [4] M.D. Holmes, W.C. Willett, Does diet affect breast cancer risk? Breast Cancer Res. 6 Cancer 99 (2008) 1502–1505. into mathematical terms, particularly the two-stage model [57]. renowned as a real gentleman. I was astounded that he would want (2004) 170–178. [38] R.G. Stevens, M.S. Rea, Light in the built environment: potential role of circadian disruption in endocrine disruption and breast cancer, Cancer Causes Control 12 Very generously he included me in some of that work as well [54]. my picture for the cover of that prestigious journal. How in the [5] M.B. Michels, A.P. Mohllajee, E. Roset-Bahmanyar, G.P. Beehler, K.B. Moysich, Diet and breast cancer: a review of the prospective observational studies, Cancer 109 (2001) 279–287. Part of what was new here was incorporating growth kinetics of heck did a person like Sidney Weinhouse notice this work and (12 Suppl.) (2007) 2712–2749. [39] A. Arnold, A. Papanikolaou, Cyclin D1 in breast cancer pathogenesis, J. Clin. Oncol. both normal and ‘intermediate’ cells into the mechanism of actually take it seriously? The cover appeared on the July 15, 1996, [6] S. Sieri, V. Krogh, P. Ferrari, F. Berrino, V. Pala, A.C. Thie´ baut, et al., Dietary fat and 23 (2005) 4215–4224. [40] C.A. Czeisler, T.L. Shanahan, E.B. Klerman, H. Martens, D.J. Brotman, J.S. Emens, transformation. issue and was a psychological lifeline that got me through the rest breast cancer risk in the European prospective investigation into cancer and nutrition, Am. J. Clin. Nutr. 88 (2008) 1304–1312. et al., Suppression of melatonin secretion in some blind patients by exposure to My thinking about how LAN and circadian disruption could of the 1990s, saving me from a life of crime. [7] R.L. Prentice, B. Caan, R.T. Chlebowski, R. Patterson, L.H. Kuller, J.K. Ockene, et al., bright light, N. Engl. J. Med. 332 (1995) 6–11. ‘cause’ cancer, has been greatly influenced by this early experience Then in the early 2000s the dam broke and good evidence began Low-fat dietary pattern and risk of invasive breast cancer: the women’s health [41] R.G. Foster, I. Provencio, D. Hudson, S. Fiske, W. De Grip, M. Menaker, Circadian with Suresh. Initiation and promotion can be interpreted as to be published from many research groups. Reports appeared that initiative randomized controlled dietary modification trial, JAMA 295 (2006) 629– photoreception in the retinally degenerate mouse (rd/rd), J. Comp. Physiol. A 169 642. (1991) 39–50. processes that included mutations that render a cell fully shift working women were at higher risk, blind women at lower [8] B.W. Wilson, L.E. Anderson, D.I. Hilton, R.D. Phillips, Chronic exposure to 60-Hz [42] G.C. Brainard, J.P. Hanifin, J.M. Greeson, B. Byrne, G. Glickman, E. Gerner, M.D. transformed. Further mutation may be required to confer risk, sleep duration inversely related to risk. And startling new electric fields: effects on pineal function in the rat, Bioelectromagnetics 2 (1981) Rollag, Action spectrum for melatonin regulation in humans: evidence for a novel metastatic potential. Dave Blask’s brilliant work addresses what reports appeared showing associations with prostate cancer in 371–380. circadian photoreceptor, J. Neurosci. 21 (2001) 6405–6412. [43] S.W. Lockley, G.C. Brainard, C.A. Czeisler, High sensitivity of the human circadian would be called ‘progression’ – melatonin preventing the growth men which had been predicted many years before [68]. The LAN/ [9] M. Cohen, M. Lippman, B. Chabner, Pineal gland and breast cancer, Lancet 2 (1978) 1381–1382. melatonin rhythm to resetting by short wavelength light, J. Clin. Endocrinol. or survival of a small colony of fully transformed cells, cells that if circadian disruption idea suddenly became legitimate. As also [10] M.C. Pike, M.D. Krailo, B.E. Henderson, J.T. Casagrande, D.G. Hoel, ‘Hormonal’ risk Metab. 88 (2003) 4502–4505. released from melatonin’s oncostatic action could then grow and often happens, once an idea becomes attractive, credit for it gains factors, ‘breast tissue age’ and the age-incidence of breast cancer, Nature 303 [44] D.M. Berson, Phototransduction in ganglion-cell photoreceptors, Pflugers Arch. 454 (2007) 849–855. flourish to become a clinically detectable cancer. But there are value, and so there were some publications which minimized or (1983) 767–770. [11] W. Jo¨chle, Trends in photophysiologic concepts, Ann. N. Y. Acad. Sci. 117 (1964) [45] S.M. Hill, D.E. Blask, Effects of the pineal hormone melatonin on the proliferation many other possibilities. denied my role in the genesis of the theory [69,70], to which I 88–104. and morphological characteristics of human breast cancer cells (MCF-7) in At the other end of the carcinogenic process could be an effect of realized I must respond promptly [71,72]. In the long run, it makes [12] I.K. Khaetski, Effect of hypothalamo-pituitary lesions induced by constant illu- culture, Cancer Res. 48 (1988) 6121–6126. [46] D.E. Blask, S.M. Hill, K.M. Orstead, J.S. Massa, Inhibitory effects of the pineal LAN on normal mammary tissue development. Based on Dimitrios no difference who thought of what, but in the short term, it matters mination on development of induced mammary tumors in rats, Vopr. Exp. Oncol. (Kiev) 1 (1965) 87–93. hormone melatonin and underfeeding during the promotional phase of 7,12- Trichopoulos’s [58] intriguing idea that early life experience, even very much to the principals involved. Accurate attribution of ideas [13] V.N. Anisimov, The light-dark regimen and cancer development, Neuroendocri- dimethylbenzanthracene-(DMBA)-induced mammary tumorigenesis, J. Neural beginning in utero, affects lifetime risk of breast cancer, a matters. Too many universities and research institutions assess a nol. Lett. 23 (Suppl. 2) (2002) 28–36. Transm. 67 (1986) 125–138. [47] D.T. Dauchy, D.E. Blask, L.A. Sauer, Preparation of the inguinal fat pad for perfusion prediction is that LAN during these critical developmental periods scientist’s productivity on numbers of grants and data papers, [14] P.N. Shah, M.C. Mhatre, L.S. Kothari, Effect of melatonin on mammary carcino- genesis in intact and pinealectomized rats in varying photoperiods, Cancer Res. 44 in situ in the rat: a surgical technique that preserves continuous blood flow, could also affect lifetime risk [59]. This possibility could have ignoring or undervaluing ideas. For any younglings who might be (1984) 3403–3407. Contemp. Top. Lab. Anim. Sci. 39 (2000) 29–33. serious implications for light exposure to pregnant women (e.g, reading this, stick with what you love. That is the end of the rant for [15] A.J. Lewy, T.A. Wehr, F.K. Goodwin, D.A. Newsome, S.P. Markey, Light suppresses [48] D.E. Blask, G.C. Brainard, R.T. Dauchy, J.P. Hanifin, L.K. Davidson, J.A. Krause, et al., shift work) and for the lighted environment of children. There has now. melatonin secretion in humans, Science 210 (1980) 1267–1269. Melatonin-depleted blood from premenopausal women exposed to light at night [16] W.A. Brown, Acknowledging Preindustrial Patterns of Sleep May Revolutionize stimulates growth of human breast cancer xenografts in nude rats, Cancer Res. 65 been a very limited and indirect look at this important possibility. Presently I am involved with a couple of excellent younger Approach to Sleep Dysfunction, Appl. Neurol., May 26, 2006, available at: http:// (2005) 11174–11184. Shah et al. [14], in their experiment described above, used light colleagues who have addressed some of the other implications of a www.psychiatrictimes.com/display/article/10168/56881. [49] D.R. Charles, E.M. Luce-Clausen, The kinetics of papilloma formation in benzpyr- exposure beginning in utero. My buddies at PNL and I tried to potential impact of LAN and circadian disruption on breast cancer [17] M. Fedrowitz, W. Lo¨ scher, Exposure of Fischer 344 rats to a weak power frequency ene-treated mice, Cancer Res. 2 (1942) 261–263. [50] I. Berenblum, P. Shubik, A new, quantitative, approach to the study of the stages of replicate this but started constant light exposure at age 26 days (with each passing year, more and more people are younger than magnetic field facilitates mammary tumorigenesis in the DMBA model of breast cancer, Carcinogenesis 29 (2008) 186–193. chemical carcinogenesis in the mouse skin, Br. J. Cancer 1 (1947) 383–391. [60]. Much to our surprise (and dismay) we found that this me including for the first time our President). At the basic biology [18] R.G. Stevens, Electric power use and breast cancer: a hypothesis, Am. J. Epidemiol. [51] R.A. Weinberg, Oncogenes, antioncogenes, and the molecular bases of multistep exposure lowered tumor yield apparently by speeding differentia- level, Yong Zhu at Yale has attacked the logical idea that 125 (1987) 556–561. carcinogenesis, Cancer Res. 49 (1989) 3713–3721. [52] H.C. Pitot, Pathways of progression in hepatocarcinogenesis, Lancet 358 (2001) tion of the breast; Jim Morris, an astute observer, noticed at polymorphisms in clock genes might confer different levels of [19] L. Schipper, A. Ketoff, S. Meyers, D. Hawk, Residential electricity consumption in industrialized countries: changes since 1973, Energy 12 (1987) 1197–1208. 859–860. terminal necropsy that the majority of the exposed rats (constant risk of breast cancer, and he was the first to publish such a study [20] G. Dong, S.S. Golden, How a cyanobacterium tells time, Curr Opin Microbiol 11 [53] H.V. Malling, Reflections in Mutation Research: Incorporation of mammalian light) were lactating despite being virgin whereas he saw this in [73]. His work is moving fast, and he has extended these ideas (2008) 541–546. metabolism into mutagenicity testing, Mutation Res. 566 (2004) 183–189. [54] S.H. Moolgavkar, N.E. Day, R.G. Stevens, Two-stage model for carcinogenesis: none of the control rats (on a 12 h light:12 h dark schedule). This is greatly including epigenetics. At the macro level, Itai Kloog at Haifa [21] D.M. Berson, F.A. Dunn, M. Takao, Phototransduction by retinal ganglion cells that set the circadian clock, Science 295 (2002) 1070–1073. epidemiology of breast cancer in females, J. Natl. Cancer Inst. 65 (1980) 559– an understudied area. University has published the first assessment of whether, and to [22] I. Provencio, G. Jiang, W.J. De Grip, W.P. Hayes, M.D. Rollag, Melanopsin: An opsin 569. Finally, the emerging understanding of the role of clock genes in what extent, LAN and breast cancer incidence co-distribute in the in melanophores, brain, and eye, Proc. Natl. Acad. Sci. U.S.A. 95 (1988) 340–345. [55] M. Kumle, Declining breast cancer incidence and decreased HRT use, Lancet 372 (2008) 608–610. expression of so many other genes [61] has led to ideas of how population at large [74]. As predicted, there was a strong [23] M.K. Bunger, L.D. Wilsbacher, S.M. Moran, C. Clendenin, L.A. Radcliffe, J.B. Hogen- esch, et al., Mop3 is an essential component of the master circadian pacemaker in [56] R.G. Stevens, R.P. Beasley, B.S. Blumberg, Iron-binding proteins and risk of canacer these could affect DNA damage [62–64], apoptosis [65], and cell association among Israeli communities of nighttime light level mammals, Cell 103 (2000) 1009–1017. in Taiwan, J. Natl. Cancer Inst. 76 (1986) 605–610. cycle regulation [66,67], all of which have direct implications for and breast cancer incidence, but not lung cancer incidence which [24] M.S. Rea (Ed.), Lighting Handbook, 9th ed., Illuminating Engineering Society of [57] S.H. Moolgavkar, A.G. Knudson, Mutation and cancer: a model for human carci- cancer causation. had been chosen as a test of the specificity of the method. This is a North America, New York, 2000. nogenesis, J. Natl. Cancer Inst. 66 (1981) 1037–1052.

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[58] D. Trichopoulos, Hypothesis: does breast cancer originate in utero? Lancet 335 [66] T. Hunt, P. Sassone-Corsi, Riding tandem: circadian clocks and the cell cycle, Cell Mutation Research 681 (2009) 111–117 (1990) 939–940. 129 (2007) 461–464. Mutation Research 681 (2009) 111–117 [59] R.G. Stevens, Circadian disruption and breast cancer: from melatonin to clock [67] S. Sahar, P. Sassone-Corsi, Circadian clock and breast cancer: a molecular link, Cell genes, Epidemiology 16 (2005) 254–258. Cycle 6 (2007) 1329–1331. [60] L.E. Anderson, J.E. Morris, L.B. Sasser, R.G. Stevens, Effect of constant light on [68] R.G. Stevens, S. Davis, D.B. Thomas, L.E. Anderson, B.W. Wilson, Electric power, Contents lists available at ScienceDirect DMBA mammary tumorigenesis in rats, Cancer Lett. 148 (2000) 121–126. pineal function, and the risk of breast cancer, FASEB J. 6 (1992) 853–860. [61] K.F. Storch, O. Lipan, I. Leykin, N. Viswanathan, F.C. Davis, W.H. Wong, C.J. Weitz, [69] L. Bernstein, The roles of physical activity and electric blankets in breast cancer, A lifetime passion for micronucleus Extensive and divergent circadian gene expression in liver and heart, Nature 417 Epidemiology 12 (2001) 598–600. Mutation Research/Reviews in Mutation Research (2002) 78–83. [70] E.S. Schernhammer, B. Rosner, W.C. Willett, F. Laden, G.A. Colditz, S.E. Hankinson, [62] L. Fu, H. Pelicano, J. Liu, P. Huang, C.C. Lee, The circadian gene Period2 plays an Epidemiology of urinary melatonin in women and its relation to other hormones important role in tumor suppression and DNA damage response in vivo, Cell 111 and night work, Cancer Epidemiol. Biomark. Prev. 13 (2004) 936–943. cytome assays—journal homepage: www.elsevier.com/locate/reviewsmr (2002) 41–50. [71] R.G. Stevens, Letter to editor, Epidemiology 13 (2002) 116. Community address: www.elsevier.com/locate/mutres [63] M. Oklejewicz, E. Destici, F. Tamanini, R.A. Hut, R. Janssens, G.T. van der Horst, [72] R.G. Stevens, Letter to editor, Cancer Epidemiol. Biomark. Prev. 14 (2005) ☆ Phase resetting of the mammalian circadian clock by DNA damage, Curr Biol. 18 551. Reflections from Down Under (2008) 286–291. [73] Y. Zhu, H.N. Brown, Y. Zhang, R.G. Stevens, T. Zheng, Period3 structural variation: a [64] S. Gery, N. Komatsu, L. Baldjyan, A. Yu, D. Koo, H.P. Koeffler, The circadian gene circadian biomarker associated with breast cancer in young women, Cancer Reflections in Mutation Research per1 plays an important role in cell growth and DNA damage control in human Epidemiol. Biomark. Prev. 14 (2005) 268–270. Michael Fenech * cancer cells, Mol. Cell 22 (2006) 375–382. [74] I. Kloog, A. Haim, R.G. Stevens, M. Barchana, B.A. Portnov, Light at night co- A lifetime passion for micronucleus cytome assays—Reflections from Down [65] H. Hua, Y. Wang, C. Wan, Y. Liu, B. Zhu, C. Yang, et al., Circadian gene mPer2 distributes with incident breast but not lung cancer in the female population of overexpression induces cancer cell apoptosis, Cancer Sci. 97 (2006) 589–596. Israel, Chronobiol. Int. 25 (2008) 65–81. § CSIROUnder Human Nutrition, P.O. Box 10041, Adelaide BC, SA 5000, Australia Michael Fenech *

CSIRO Human Nutrition, P.O. Box 10041, Adelaide BC, SA 5000, Australia

ARTICLE INFO ABSTRACT

Article history: A brief account of an improbable career in the field of genetic toxicology is given, extending from my Accepted 18 November 2008 early years in Malta through a life-changing decision to study in Australia (Down Under). I describe the Available online 3 December 2008 circumstances that led to the discovery of the cytokinesis-block micronucleus (CBMN) assay and its evolution into a cytome assay of chromosome breakage and loss (micronuclei), asymmetrical Keywords: chromosome rearrangements or telomere end fusions (nucleoplasmic bridges), gene amplification Micronucleus assay (nuclear buds), cell death (necrosis, apoptosis) and cytostasis (nuclear division index). This paper also Cytokinesis-block describes the role of my laboratory in the beginning of the HUMN project, its achievements, and the Cytome applications of CBMN cytome assays in the fields of radiation biology, genetic toxicology, epidemiology, Cytochalasin-B Reflections biodosimetry and genome health nutrigenomics, leading to the Genome Health Clinic concept. Along the History of science way I mention my encounters with some of the influential people in the field of mutagenesis who provided me with the motivation and guidance needed to realise these achievements. I hope this account provides some inspiration to the next generation of scientists who may be fortunate to see the realisation of the application of the principles of mutagenesis in health optimisation or disease prevention and eventually in mainstream medicine. ß 2009 Published by Elsevier B.V.

1. The formative years (316 km2, population 402,000), and traffic fumes are a major pollution problem on busy roads such as the one I lived on in In my wildest dreams I would have never imagined that my life Birkirkara. Furthermore it was common when swimming to find in research would turn out as it has. The first 25 years of my life your legs stained with tar residues from oil tankers that criss- were spent in Malta where I was born. That I had an inclination for crossed the Mediterranean sea. It was these experiences that first biological sciences was evident in my very early years when I was stimulated my curiosity about scientific issues relating to the fascinated by the response of ants to environmental stress and effects of environmental pollution and diet on human health. their ability to survive extreme conditions. There had never been I graduated with a Bachelor’s degree in Chemistry and Biology any professional scientists in my family, but on my mother’s side from the University of Malta and during this period developed an two uncles were doctors, and on my father’s side one uncle was a interest in such aspects of marine biology as measuring the impact pharmacist. My father was a health inspector, and my mother of pollution on marine organisms and understanding the ecology of trained as a nurse. Therefore, by and large, I was exposed to issues the rocky shores around the island of Malta. The latter was the underlying the importance of health maintenance, and growing up focus of my Master’s degree, in which I studied the zonation of in the Mediterranean there was always strong awareness of the organisms in the intertidal zone in the south of the island as well as importance of diet, although the prevailing philosophy was the the structure and function of the vermetid sessile mollusc more you ate the better, which explains the high incidence of Serpulorbis arenaria which was present in two polymorphic forms. obesity and diabetes in Malta. Malta is a densely populated island This was my first great experience in basic research under the guidance of Dr. Victor Jaccarini who was in charge of the marine biological station located in one of the great fortifications, Fort St. Lucian, built by the Knights of the Order of St. John. My hope was to § This article is part of the Reflections in Mutation Research series. To suggest topics carry on my career in marine biology and ecology but these hopes and authors for Reflections, readers should contact the series editors, G.R. Hoffmann were dashed when the government changed. The Socialist Party ([email protected]) or D.G. MacPhee ([email protected]). * Tel.: +61 8 8303 8880; fax: +61 8 8303 8899. that took power believed that arts and science faculties at the E-mail address: [email protected]. university were a waste of money and decided amongst other

1383-5742/$ – see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.mrrev.2008.11.003

284 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 285 112 M. Fenech / Mutation Research 681 (2009) 111–117 M. Fenech / Mutation Research 681 (2009) 111–117 113 things that the marine biological station should be closed down recover from the long flight to Adelaide due to both the physical 4. Eureka! The cytokinesis-block micronucleus (CBMN) assay and replaced by a maritime museum, even although this meant and emotional stress. The 1st week in Adelaide was unforget- losing a facility which was heavily used and held in very high table as I was unable to communicate properly because, despite After considerable frustration, the ‘‘eureka’’ moment occurred regard by research groups internationally. the friendly reception and welcome by Alec and his team, my to me very early one summer morning in 1984. It suddenly became During the Master’s degree period I was also teaching at pre- accent and body language when using English language were out obvious to me while half awake, almost in a dream, that the point university level and developed an interest in genetics, and a short of synchrony with the Australian way of speaking. Dr. David at which to identify once-divided cells is the short-lived while later I took up a position as a scientist in the pathology Turner, Kevin Trainor, Jack Dempsey, Alexander Kutlaca, Chris binucleated stage in telophase. That same day I enquired with laboratories at the island’s main hospital, St. Luke’s. Malta has a Matthews, Monica McCarron and Sheila Phillips eventually colleagues whether anybody had any idea how to block and great tradition in medicine because of the influence of the Knights straightened me out, and I slowly settled into the research accumulate cells in this stage. I wondered how one could block of the Order of St. John, which was set up initially as a hospitaller laboratory life which was a completely new experience to me. cytokinesis so that the cell, having completed nuclear division, order to care for pilgrims travelling to Jerusalem and the Holy Land. My attempts to break the ice in communication were eventually would no longer be able to complete cellular division. A quick The Knights transferred their headquarters from the island of rewarded when I decided to use some long-forgotten Australian search of the term ‘‘cytokinesis’’ in the Lehninger ‘‘Principles of Rhodes to Malta, which was offered to them after they were colloquialisms during our weekly research meeting such as Biochemistry’’ undergraduate textbook resulted in my finding a evicted from Rhodes in 1522 by the Ottomans under Suleiman the ‘‘You must have kangaroos in your top paddock!’’ meaning ‘‘You brief sentence mentioning cytochalasins as inhibitors of cytokin- Magnificent. The Knights settled in Malta in the early 1500s and must be crazy to have that thought in your head!’’. Participating in esis. They exerted this effect by inhibiting the polymerisation of built massive fortifications as well as establishing one of the first ‘‘happy hour’’ at the hospital common room during this early actin into the microfilaments required for cytokinesis. The medical universities in Europe. The experience at St. Luke’s period on Friday afternoons was also a necessary and cheerful university library happened to have a textbook on cytochalasins Hospital exposed me to the importance of haematology in health short-cut to get to know the younger and established research that included detailed information on the various forms of these assessment, and it was here that for the first time I came face to staff at Flinders Medical Centre where our research group was molecules produced by a wide range of fungi. It was evident from face with the human lymphocyte and the Howell Jolly body located. this information, as well as from a key paper by Carter [2], that (micronucleus) in erythrocytes. Little did I realise then that my life cytochalasin-B was the form that could inhibit cytokinesis most would become consumed by almost three decades of research 3. The tortuous path to micronucleus assays efficiently in lymphocytes and other mammalian cells. devoted to the measurement of DNA damage in human lympho- It happened that colleagues in the Immunology Department cytes using the micronucleus (MN) assay. My initial 3–6 months in the Morley lab proved fruitless as my had some cytochalasin-B which they were using for enucleation attempts to develop a novel point mutation assay based on the ABO experiments. That same day I added cytochalasin-B at 3 mg/ml to 2. From Malta to Australia blood group system did not meet with success. Nevertheless this ongoing lymphocyte cultures, and the following morning I project had required my temporary transfer to the donor–recipient harvested the cells and prepared slides. I distinctly remember At that time Malta offered relatively limited opportunity for blood cross-matching laboratory where I met my future wife Janet, those first moments looking down the microscope and my individuals with active, unconventional and inquisitive scientific and in this sense, my research failure was very much a success on excitement in seeing that a very large proportion of the minds and it was not possible to pursue a Ph.D. on the island as the social and personal level. Professor Morley then suggested that lymphocytes were blocked as binucleated cells. I also recall the university had inadequate facilities for these purposes in perhaps I should change tack completely and pushed in front of me showing the slide to Alec who also was pleasantly surprised by this science. Consequently, I started to explore possibilities for the paper of Countryman and Heddle [1] which had described for development. Thus, the CBMN assay was born. Fig. 1 shows the Fig. 1. The CBMN assay. (a) Micronucleus (MN) and nucleoplasmic bridge (NPB) further studies overseas in the United Kingdom, but possibly the first time the development of a MN assay in cultured concept of the CBMN assay and examples of binucleated cells formation in cells undergoing nuclear division. MN originate from either lagging also in other English-speaking countries. Scholarships were lymphocytes. My failure with the ABO blood group point mutation containing micronuclei. whole chromosomes or acentric chromosome fragments. NPBs originate from limited but I managed to secure one to study in Australia which system was to launch me into a future of great opportunity with The following year was spent verifying the efficacy of the CBMN dicentric chromosomes that may be caused by mis-repair of double strand DNA was my least preferred choice because of distance from home, the MN assay in lymphocytes. technique in terms of reproducibility and sensitivity for detecting breaks or telomere end fusions. These events can only be observed in cells completing nuclear division which are recognised by their binucleated appearance but it turned out to be an excellent experience and opportunity. I set about using Countryman and Heddle’s conventional MN low dose exposures to ionising radiation (5–40 cGy) and demon- after cytokinesis-blocking with cytochalasin-B. (b) Photomicrograph of binucleated With the limited information available to me I came to the assay in lymphocytes and verified their results for ionising strating that the rate of increase of MN frequency with age was cells with one micronucleus (left) and seven micronuclei (right). conclusion that Adelaide was one of the better centres in radiation. However, it soon became apparent that the results underestimated in the conventional method relative to the CBMN Australia to do research in genetics, which had become my key depended on harvest time, and false negative results could be method. I found that acute doses as low as 0.05 Gy X-rays were area of interest. Through a series of coincidences, my letter of generated under conditions of strong inhibition of mitosis as easily detectable by the new method which required much less analysis of gene mutations. Two of the leading laboratories in enquiry to the University of Adelaide ended up with Professor often occurs with chemical exposure. Furthermore it was evident effort than the more laborious metaphase analysis approach [3,4]. these fields at the time were Tony Carrano’s group at the Lawrence Avon Maxwell Clark at Flinders University who was interested in that a greater proportion of lymphocytes respond to mitogen in Livermore National Laboratory and Bryn Bridges’ and Alan using the bone-marrow MN assay to study the genotoxic effects cultures of lymphocytes from young subjects than in those from 5. Postdoctoral research Lehmann’s group at the Medical Research Council Cell Mutation of alkaloids from drought-tolerant weeds toxic to sheep and elderly individuals. In my second annual assessment seminar I Unit (MRC CMU) in Sussex, UK. I visited both laboratories prior to cattle such as Echium plantagineum and Heliotropium europeum. hypothesised that the MN assay in its then-current form had a After being awarded my Ph.D. I remained in Alec’s laboratory for making my decision. Tony Carrano thought it essential that I Professor Clark was about to retire so he forwarded my enquiry to major flaw due to the fact that observed MN frequency depended a further 2 years during which research performed in collaboration should continue with the MN assay and suggested that I work Alexander Morley who was then Professor of Haematology at the on the proportion of lymphocytes that responded to mitogen as with Dr. Jim Denham at the Royal Adelaide Hospital led to the under Joe Gray’s guidance to develop a flow cytometric method for Flinders University Medical School and was soon to become well as the number of divisions that occurred during the culture utility of the CBMN assay for biological dosimetry of in vivo the lymphocyte assay, whilst Alan Lehmann and Bryn Bridges internationally renowned for his research on the measurement of period prior to harvesting cells. I predicted that the assay would radiation exposure being verified for the first time in cancer were happy to take the risk of letting me loose in the molecular point mutations in human lymphocytes using the HPRT and HLA only be robust if a method to accumulate and identify once- patients undergoing radiotherapy [5]. Using kinetochore anti- biology field which effectively clinched the deal. In hindsight I assays. Professor Morley (Alec) wrote back indicating that he divided cells was developed. My examiners recognised this and bodies, we also demonstrated that the great majority of MNi would have been happy to do both projects if it had been at all would be happy to be my Ph.D. supervisor given my interest in wisely suggested that if I solved this problem my Ph.D. would be induced by ionising radiation were kinetochore negative whilst possible. genetics and experience in haematology. I could not believe my secured. Naturally this predicament focuses the mind beautifully, those induced by spindle poisons were kinetochore positive (as My experience at the MRC CMU proved to be a very positive and luck and reasoned that if I was to see the world I might as well and I set about devising a variety of methods to identify once- one would expect given that radiation causes chromosome unforgettable one. The project involved cloning the rad4 gene by start from one of the most distant places from Malta possible. divided cells based on DNA synthesis labelling or parallel cultures breakage leading to acentric chromosome fragments). This complementation. I used the mutant rad4.116 of the fission yeast New Zealand apart, this was Australia. My parents were blocked in mitosis to correct for the proportion of dividing cells. coincided with the beginnings of the era of molecular cytogenetics Schizosaccharomyces pombe, which is temperature-sensitive for somewhat devastated as I was the first member of the extended Neither of these methods was satisfactory because one could not when the specific nature of chromosome lesions could be analysed growth and sensitive to the killing actions of both ultraviolet light family to migrate, and naturally I was somewhat apprehensive be certain whether the labelled cells had actually completed using molecular (antibody or DNA) probes. At this time I recall and ionising radiation. With the help of Tony Carr’s expertise in given that I had no relatives ‘‘Down Under’’—but I could not resist nuclear division or had, in fact, completed only one division. Alec giving a seminar at Flinders Medical Centre titled ‘‘The Micro- transfection of fission yeast I isolated a couple of clones that were the adventure. jokingly said, perhaps with the intention to spur me on, that ‘‘the nucleus Assay at its Limits’’ as I had honestly thought that we had resistant to elevated temperature, and these were eventually used The journey to Adelaide in late April 1980 was memorable conventional MN assay might be like a jacket that does not fit completely optimised the system and subconsciously I was ready to clone the rad4 gene in S. pombe [6]. The postdoc year in Sussex with a stop-over in Bangkok which at the time was on a high because whichever attempt to fix it generates another problem’’. for a new challenge. gave me important insights into the use of molecular biology in state-of-alert due to political unrest. It was my first experience Feeling somewhat optimistic that the solution would soon I had made up my mind to do some further postdoctoral mutagenesis research and a feel for the international nature of of a tropical climate, and it was a complete contrast to the very become evident, I responded that ‘‘It depends on how good the training to learn molecular biology techniques in the emerging research. It also allowed me to meet for the first time most of my dry and hot summers in Adelaide. It took me about 2 weeks to tailor is’’. fields relating to cloning of DNA repair genes and molecular ‘‘heroes’’ in the field of chromosomal mutagenesis including

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Professor A.T. Natarajan, who had written many seminal papers in 7. Micronucleus assays in the study of nutrition fact show that a mid- or high-tertile level of MN frequency MN assay—the ‘‘cytome’’ approach. Using this method, not the field of radiation-induced chromosome aberrations and had predicted an increased cancer risk [24]. only micronuclei but also other DNA damage biomarkers that examined my Ph.D. thesis, Professor Hans Stich who had pioneered The line of research I chose to pursue at CSIRO Human Nutrition we have observed in binucleated cells (i.e., nucleoplasmic the use of the buccal micronucleus assay, and Professor John was inspired by the seminal work of Bruce Ames and Jim 9. Cytome approach to micronucleus assays bridges and nuclear buds) are scored. Cells undergoing cell Heddle who had first described the conventional MN assay in MacGregor showing that erythrocyte MN frequency in humans death by necrosis and apoptosis are also identified and lymphocytes amongst others. is strongly affected by folate status [9–11]. Between 1992 and 2000 From 2000 onwards our laboratory embarked on research to counted. Fig. 2 shows the endpoints considered in the cytome we extended these observations by showing that the MN create and validate a new way to perform the cytokinesis-block approach. 6. Blossoming of the micronucleus assay frequency index in lymphocytes was also associated with plasma vitamin B12, folate and homocysteine status in healthy adults, and A special event during my year in the UK was the ‘‘The that the MN frequency index in lymphocytes could be reduced by Micronucleus Workshop’’ that David Scott and John Ashby had supplementation with folic acid and vitamin B12 [12–14]. The organised in Macclesfield (UK) to bring together the key experts positive association with homocysteine was particularly important in the MN field. Later in the year I participated in the Automated because it showed that DNA damage was associated with a known Micronucleus Workshop in Milpitas, California, in which the first risk factor for cardiovascular disease (CVD) supporting the DNA successful automation prototypes of the erythrocyte MN and the damage hypothesis of CVD [15]. Evidence for this association was lymphocyte CBMN assay were described and exhibited. An later strengthened by the association of MN frequency in invitation the following year to present my work on the CBMN lymphocytes with CVD mortality [16]. assay at the International Conference on Mutagenesis (ICEM) in These studies as well as in vitro studies showed that the Cleveland (USA) was unforgettable, as I was stunned and concentrations of folate and vitamin B12 then considered to be pleasantly surprised to find such an overwhelming interest in adequate for the prevention of anaemia were lower than those this presentation. I had not yet realised the significance and associated with the minimisation of DNA damage (e.g. 150 pmol/l impact of this work, and I would not have guessed the extent vs. 300 pmol/l, respectively, in the case of B12). This raised the to which the CBMN assay would be adopted worldwide. concern that dietary requirements for the prevention of DNA From thereon my working life was transformed, dictated as it damage are likely to be different from those for the prevention of was by the need to further develop and validate the use of this deficiency diseases. The in vitro studies also demonstrated that assay. small differences within the normal physiological range of folate After this euphoric postdoctoral experience overseas I took up concentration (e.g. 10 nmol/l vs. 60 nmol/l folic acid) caused as a position as a radiation biologist at the Australian Nuclear much DNA damage as 10–20 cGy of X-rays, a dose range which is 5– Science and Technology Organisation in Sydney, which unfortu- 10 times higher than the annual allowed limit of radiation exposure. nately at the time was being restructured to become more This meant that small differences in nutrient concentration can commercially focussed. Regrettably, the importance of scientists cause as much DNA damage as doses of a mutagen and carcinogen relative to business managers had been declining for some years, about which there are serious concerns. At this time I published a and the focus on science was being steadily diluted. At about the series of papers promoting the concept that recommended dietary time I began looking for positions elsewhere it happened that Dr. intakes should factor in the prevention of DNA damage, given that it Ivor Dreosti at the Human Nutrition Division of the Common- was becoming increasingly evident that damage to the genome is wealth Scientific and Industrial Research Organisation of implicated in multiple disorders (infertility, developmental defects, Australia (CSIRO) was seeking a postdoc to develop a new immune dysfunction, cancer, accelerated ageing) and can be research area aimed at investigating the mitigating effects of considered among the most fundamental causes of disease [17–19]. dietary factors on oxidative stress, as well as at determining the Growing recognition of the fact that deficiency in dietary factors genotoxic potential of different diets. I reasoned that it would be can modify the genome is indeed one of the main reasons for the interesting to understand how diet might affect base-line DNA importance and emergence of the new field of nutrigenomics. damage rates, and also to explore whether it had any radiation- Several papers have now been published on the impact of diet on protective effects. During my initial year at CSIRO, I simply DNA damage (e.g. [20,21,30]). However, an even more interesting established the lymphocyte CBMN assay there and completed the twist to this science stems from the potential of differences in first improvement by using it in combination with cytosine susceptibility to DNA damage to depend on inherited polymorph- arabinoside in G1 to convert excision-repairable lesions directly isms in genes involved in the folate/methionine metabolism into MN within one cell cycle [7]. I also established the bone- pathways—i.e., from nutrigenetics. This was the dawn of a new marrow erythrocyte MN assay at CSIRO and demonstrated the phase in research, but exploration of the interactive impact of DNA-damaging effect of meat cooked at high temperatures. micronutrient status and genetic background on DNA damage and Collaboration with Youichi Odagiri revealed the possibility of cellular viability would require new research tools. scoring in vivo-induced DNA damage in normoblasts, myelo- blasts and lymphoblasts in mouse bone-marrow cells using the 8. The HUMN project CBMN assay in short-term ex vivo culture of bone-marrow cells following the addition of cytochalasin-B [8]. An interesting aspect The year 1997 proved to be one of the most important in my of this study was the elevated radiation sensitivity of myelo- scientific career because it was then that Stefano Bonassi and I, blasts, which is consistent with observations on the reduction in after a year of email correspondence, decided to launch the HUMN neutrophil cell counts following radiation exposure. A notable project at the ICEM in Toulouse. We were overwhelmed by interest and enduring experience during this period was the 1992 internationally in the HUMN project which had as its primary aims inaugural International Conference in Environmental Mutagen- the collation of data worldwide to determine the main variables esis in Human Populations in Cairo, which was admirably affecting lymphocyte micronucleus frequency, the establishment Fig. 2. The ‘‘Cytome’’ version of the CBMN assay. (a) The various possible fates of cultured cytokinesis-blocked cells following exposure to cytotoxic/genotoxic agents. Using organised by Wagida Anwar and her colleagues. I had the of scoring criteria for this assay, the performance of an inter- and these biomarkers within the CBMN assay it is possible to measure the frequency of chromosome breakage (MN), chromosome loss (MN), chromosome rearrangement, e.g. privilege of sharing in Frits Sobels’ 70th birthday celebrations on intra-laboratory slide scoring exercise, and a prospective study to dicentric chromosomes (NPB), gene amplification (NBUDs), necrosis and apoptosis. In addition, cytostatic effects are readily estimated from the ratio of mono, bi and the Nile. For me, having the opportunity to meet the founding test the hypothesis that MN frequency in lymphocytes predicts multinucleated cells. (b) Photomicrographs of the cells scored in the CBMN ‘‘cytome’’ (CBMN Cyt) assay. (A) Mononucleated cell; (B) binucleated cell; (C) multinucleated cell; (D) early necrotic cell; (E) late apoptotic cell; (F) binucleated cell containing one or more micronuclei; (G) binucleated containing a nucleoplasmic bridge (and a editor of Mutation Research for the first time and to share this cancer risk. All these objectives were met successfully within the micronucleus); (H) binucleated cell containing nuclear buds. The ratios of mononucleated, binucleated, multinucleated, necrotic and apoptotic cells are used to determine special occasion with him and close friends was a truly following 10 years, and the resulting publications are amongst the mitotic division rate or nuclear division index (a measure of cytostasis) and cell death (cytotoxicity). The frequency of binucleated cells with micronuclei, nucleoplasmic unforgettable experience. most cited in the MN field [22–28]. The prospective study did in bridges or nuclear buds provides a measure of genome damage and/or chromosomal instability.

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The cytome approach was validated by the studies of Keizo 10.1. The future Also acknowledged are the funding bodies, NIH/NIAID, NHMRC [24] S. Bonassi, A. Znaor, M. Ceppi, C. Lando, W.P. Chang, N. Holland, M. Kirsch-Volders, E. Zeiger, S. Ban, R. Barale, M.P. Bigatti, C. Bolognesi, A. Cebulska-Wasilewska, E. Umegaki, Philip Thomas, Jimmy Crott, Shauna Brown, Will and Cancer Council Australia who have supported key projects. Fabianova, A. Fucic, L. Hagmar, G. Joksic, A. Martelli, L. Migliore, E. Mirkova, M.R. Greenrod, Bianca Benassi, Sasja Beetstra, and other scientists While this account is mainly about the past, I would also like to Scarfi, A. Zijno, H. Norppa, M. Fenech, An increased micronucleus frequency in and students in my laboratory using models of oxidative stress comment very briefly about the future. During the last 5 years we References peripheral blood lymphocytes predicts the risk of cancer in humans, Carcinogen- esis 28 (March (3)) (2007) 625–631. and/or folate deficiency (references detailed in [29]). The observed focussed more and more attention on the CBMN cytome approach [1] P.I. Countryman, J.A. Heddle, The production of micronuclei from chromosome [25] M. Fenech, J. Crott, J. Turner, S. Brown, Necrosis, apoptosis, cytostasis and DNA positive correlation between micronuclei, nucleoplasmic bridges as it became more evident that this reflected the genetic instability aberrations in irradiated cultures of human lymphocytes, Mutat. Res. 41 (Decem- damage in human lymphocytes measured simultaneously within the cytokinesis- and nuclear buds indicates that these biomarkers of genomic and regenerative potential of tissues within the body. For example, ber (2–3)) (1976) 321–332. block micronucleus assay: description of the method and results for hydrogen instability are mechanistically related to one another. They could the work of El-Zein et al. suggested that nucleoplasmic bridges and [2] S.B. Carter, Effects of cytochalasins on mammalian cells, Nature 213 (January peroxide, Mutagenesis 14 (November (6)) (1999) 605–612. (5073)) (1967) 261–264. [26] M. Fenech, N. Holland, W.P. Chang, E. Zeiger, S. Bonassi, The HUman MicroNucleus be explained by the breakage-fusion-bridge cycle model when the nuclear buds might have a stronger association with lung cancer [3] M. Fenech, A.A. Morley, Measurement of micronuclei in lymphocytes, Mutat. Res. Project—an international collaborative study on the use of the micronucleus nutritional or exposure conditions generated double strand breaks risk than do micronuclei in lymphocytes [39,40]. Philip Thomas’s 147 (February–April (1–2)) (1985) 29–36. technique for measuring DNA damage in humans, Mutat. Res. 428 (July (1–2)) in DNA and led to the formation of dicentric chromosomes Ph.D. research in my laboratory showed that the MN cytome [4] M. Fenech, A.A. Morley, Cytokinesis-block micronucleus method in human lym- (1999) 271–283 (Review). [27] M. Fenech, W.P. Chang, M. Kirsch-Volders, N. Holland, S. Bonassi, Zeiger E, Human [25,26,31–33]. approach using buccal cells can also be remarkably effective in phocytes: effect of in vivo ageing and low dose X-irradiation, Mutat. Res. 161 (July (2)) (1986) 193–198. MicronNucleus project. HUMN project: detailed description of the scoring criteria The work of Kimura et al. [34] demonstrated the power of using demonstrating the reduced regenerative potential of epithelial [5] M. Fenech, J. Denham, W. Francis, A. Morley, Micronuclei in cytokinesis-blocked for the cytokinesis-block micronucleus assay using isolated human lymphocyte the CBMN Cytome (CBMN Cyt) assay in investigating nutrient– tissues in accelerated ageing syndromes, such as Down syndrome, lymphocytes of cancer patients following fractionated partial-body radiotherapy, cultures, Mutat. Res. 534 (January (1–2)) (2003) 65–75. [28] M. Fenech, S. Bonassi, J. Turner, C. Lando, M. Ceppi, W.P. Chang, N. Holland, M. nutrient and gene–nutrient interactions affecting the impact of and in Alzheimer’s disease. These studies relied on biomarkers Int. J. Radiat. Biol. 57 (February (2)) (1990) 373–383. [6] M. Fenech, A.M. Carr, J. Murray, F.Z. Watts, A.R. Lehmann, Cloning and character- Kirsch-Volders, E. Zeiger, M.P. Bigatti, C. Bolognesi, J. Cao, G. De Luca, M. Di folate and riboflavin concentration in individuals who are other than MN frequency (e.g. basal cell frequency) showing an ization of the rad4 gene of Schizosaccharomyces pombe; a gene showing short Giorgio, L.R. Ferguson, A. Fucic, O.G. Lima, V.V. Hadjidekova, P. Hrelia, A. homozygotes for the C or T alleles of the C677T polymorphism association with these conditions [41–43]. In the HUMN project we regions of sequence similarity to the human XRCC1 gene, Nucleic Acids Res. 19 Jaworska, G. Joksic, A.P. Krishnaja, T.K. Lee, A. Martelli, M.J. McKay, L. Migliore, E. Mirkova, W.U. Mu¨ ller, Y. Odagiri, T. Orsiere, M.R. Scarfı`, M.J. Silva, T. Sofuni, J. in the methylenetetrahydrofolatereductase (MTHFR) gene. The have now launched an international collaboration on the buccal (December (24)) (1991) 6737–6741. [7] M. Fenech, S. Neville, Conversion of excision-repairable DNA lesions to micro- Surralles, G. Trenta, I. Vorobtsova, A. Vral, Zijno A, HUman MicroNucleus project. results from this work showed that increasing riboflavin con- MN cytome assay to achieve the same objectives that were realised nuclei within one cell cycle in human lymphocytes, Environ. Mol. Mutagen. 19 (1) Intra- and inter-laboratory variation in the scoring of micronuclei and nucleo- centration in a low folate background increased DNA damage for the lymphocyte assay [44]. (1992) 27–36. plasmic bridges in binucleated human lymphocytes. Results of an international (nuclear buds and nucleoplasmic bridges). Individuals homozy- [8] Y. Odagiri, K. Takemoto, M. Fenech, Micronucleus induction in cytokinesis- slide-scoring exercise by the HUMN project, Mutat. Res. 534 (January (1–2)) blocked mouse bone marrow cells in vitro following in vivo exposure to X- (2003) 45–64. gous for the T allele (which reduced enzyme activity) had reduced 11. A personal afterword irradiation and cyclophosphamide, Environ. Mol. Mutagen. 24 (1) (1994) 61–67. [29] M. Fenech, Cytokinesis-block micronucleus cytome assay, Nat. Protoc. 2 (5) nuclear buds relative to the C allele homozygotes, indicating [9] J.T. MacGregor, R. Schlegel, C.M. Wehr, P. Alperin, B.N. Ames, Cytogenetic damage (2007) 1084–1104. differential effects depending on nutrient concentration combina- It is important to note that none of the above would have been induced by folate deficiency in mice is enhanced by caffeine, Proc. Natl. Acad. Sci. [30] M. Fenech, Genome health nutrigenomics and nutrigenetics—diagnosis and U.S.A. 87 (December (24)) (1990) 9962–9965. nutritional treatment of genome damage on an individual basis, Food Chem. tions and genotype. The observed sensitivity of the CBMN Cyt assay possible without the mentoring of Professor Alec Morley in my [10] J.T. MacGregor, C.M. Wehr, R.A. Hiatt, B. Peters, J.D. Tucker, R.G. Langlois, R.A. Toxicol. 46 (April (4)) (2008) 1365–1370 (Epub 2007 July 5 Review). to nutritional and genetic factors confirmed its suitability as an formative years and the support of friends, colleagues and family Jacob, R.H. Jensen, J.W. Yager, M.K. Shigenaga, B. Frei, B.P. Eynon, B.N. Ames, [31] K. Umegaki, M. Fenech, Cytokinesis-block micronucleus assay in WIL2-NS cells: important tool in the emerging exciting fields of nutrigenetics and throughout the years. I hope that this account provides some ‘Spontaneous’ genetic damage in man: evaluation of interindividual variability, a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils, Mutagenesis 15 (May (3)) (2000) nutrigenomics. The association of folate/methionine metabolism inspiration and encouragement to the next generation of scientists relationship among markers of damage, and influence of nutritional status, Mutat. Res. 377 (June (1)) (1997) 125–135. 261–269. and DNA repair gene polymorphisms with the MN index in in the fields of mutagenesis, radiation biology and nutritional [11] B.C. Blount, M.M. Mack, C.M. Wehr, J.T. MacGregor, R.A. Hiatt, G. Wang, S.N. [32] J.W. Crott, S.T. Mashiyama, B.N. Ames, M. Fenech, The effect of folic acid lymphocytes is becoming an active area of research in nutrige- genomics. In the past 2 years I have been fortunate to receive the Wickramasinghe, R.B. Everson, B.N. Ames, Folate deficiency causes uracil mis- deficiency and MTHFR C677T polymorphism on chromosome damage in human lymphocytes in vitro, Cancer Epidemiol. Biomark. Prev. 10 (October (10)) (2001) nomics and toxicogenomics [35]. Flinders University Convocation medal and the Environmental incorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage, Proc. Natl. Acad. Sci. U.S.A. 94 (April (7)) (1997) 1089–1096. The possibility of measuring nucleoplasmic bridges in the Mutagen Society Alexander Hollaender Award in recognition of 3290–3295. [33] P. Thomas, K. Umegaki, M. Fenech, Nucleoplasmic bridges are a sensitive measure CBMN Cyt assay is of particular importance given that it is a these achievements which, in effect, reflect the success of efforts of [12] M. Fenech, J. Rinaldi, The relationship between micronuclei in human lympho- of chromosome rearrangement in the cytokinesis-block micronucleus assay, Mutagenesis 18 (March (2)) (2003) 187–194. potentially reliable assay for dicentric chromosome formation many people whom I had the good fortune of knowing. Thus, this is cytes and plasma levels of vitamin C, vitamin E, vitamin B12 and folic acid, Carcinogenesis 15 (July (7)) (1994) 1405–1411. [34] M. Kimura, K. Umegaki, M. Higuchi, P. Thomas, M. Fenech, Methylenetetrahy- and therefore relevant to radiation biodosimetry [33].Italso also a story of valuable and rewarding scientific collaboration. I [13] M.F. Fenech, I.E. Dreosti, J.R. Rinaldi, Folate, vitamin B12, homocysteine status and drofolate reductase C677T polymorphism, folic acid and riboflavin are important allows a possible functional assay for telomere dysfunction when never met Alexander Hollaender but much has been written about chromosome damage rate in lymphocytes of older men, Carcinogenesis 18 (July determinants of genome stability in cultured human lymphocytes, J. Nutr. 134 (January (1)) (2004) 48–56. combined with telomere probes [29], given that telomere end this remarkable man and the spirit that he and such other pioneers (7)) (1997) 1329–1336. [14] M. Fenech, C. Aitken, J. Rinaldi, Folate, vitamin B12, homocysteine status and DNA [35] G. Iarmarcovai, S. Bonassi, A. Botta, R.A. Baan, T. Orsie`re, Genetic polymorphisms fusions lead to dicentric chromosome formation and nucleo- of our field as Frits Sobels have engendered that has galvanised damage in young Australian adults, Carcinogenesis 19 (July (7)) (1998) 1163–1171. and micronucleus formation: a review of the literature, Mutat. Res. 658 (3) (2008) plasmic bridge formation. This has opened a possible new environmental mutagenesis research worldwide [45]. My story [15] S. De Flora, A. Izzotti, Mutagenesis and cardiovascular diseases Molecular 215–233. approach for studying the role of nutrition and other environ- owes much to them also. mechanisms, risk factors, and protective factors, Mutat. Res. 621 (August (1– [36] M. Fenech, The Genome Health Clinic and Genome Health Nutrigenomics con- 2)) (2007) 5–17. cepts: diagnosis and nutritional treatment of genome and epigenome damage on mental factors on telomere dysfunction, which is virtually [16] E. Murgia, V. Maggini, R. Barale, A.M. Rossi, Micronuclei, genetic polymorphisms an individual basis, Mutagenesis 20 (July (4)) (2005) 255–269. unexplored as yet. Conflict of interest and cardiovascular disease mortality in a nested case-control study in Italy, [37] H. Ishikawa, Y. Tian, T. Yamauchi, Influence of gender, age and lifestyle factors on Mutat. Res. 621 (August (1–2)) (2007) 113–118. micronuclei frequency in healthy Japanese populations, J. Occup. Health 45 (May (3)) (2003) 179–181. None. [17] M. Fenech, Recommended dietary allowances (RDAs) for genomic stability, 10. The Genome Health Clinic concept emerges Mutat. Res. 480–481 (September) (2001) 51–54. [38] L.J. Moran, M. Noakes, P.M. Clifton, R.J. Norman, M.F. Fenech, Genome instability is [18] M. Fenech, Micronutrients and genomic stability: a new paradigm for recom- increased in lymphocytes of women with polycystic ovary syndrome and is In 2003 I put together various strands of thought that led to the Acknowledgements mended dietary allowances (RDAs), Food Chem. Toxicol. 40 (August (8)) (2002) correlated with insulin resistance, Mutat. Res. 639 (March (1–2)) (2008) 55–63. [39] R.A. El-Zein, M.B. Schabath, C.J. Etzel, M.S. Lopez, J.D. Franklin, M.R. Spitz, Genome Health Clinic concept [30,36]. This concept is based on the 1113–1117. [19] M. Fenech, Genomic stability: a new paradigm for recommended dietary allow- Cytokinesis-blocked micronucleus assay as a novel biomarker for lung cancer premise that damage to the genome is fundamental to disease and None of the experiences and achievements would have been ances (RDAs), Forum Nutr. 56 (2003) 97–100. risk, Cancer Res. 66 (June (12)) (2006) 6449–6456. can be diagnosed and nutritionally prevented. It was based on possible without the support of my wife Janet and my sons [20] B.N. Ames, P. Wakimoto, Are vitamin and mineral deficiencies a major cancer [40] R.A. El-Zein, M. Fenech, M.S. Lopez, M.R. Spitz, C.J. Etzel, Cytokinesis-blocked micronucleus cytome assay biomarkers identify lung cancer cases amongst several lines of evidence showing that the CBMN cytome Nicholas and Benjamin who often have had to endure my absence risk? Nat. Rev. Cancer 2 (September (9)) (2002) 694–704. [21] B.N. Ames, Low micronutrient intake may accelerate the degenerative diseases of smokers, Cancer Epidemiol. Biomark. Prev. 17 (May (5)) (2008) 1111–1119. biomarkers are sensitive to nutritional deficiencies and excess, so I could pursue my research interests with vigour. I would also aging through allocation of scarce micronutrients by triage, Proc. Natl. Acad. Sci. [41] P. Thomas, J. Hecker, J. Faunt, M. Fenech, Buccal micronucleus cytome biomarkers and that alterations to these biomarkers of DNA damage can be like to acknowledge numerous staff members and colleagues who U.S.A. 103 (November (47)) (2006) 17589–17594. may be associated with Alzheimer’s disease, Mutagenesis 22 (November (6)) minimised by appropriate changes in diet and lifestyle and/or contributed to these experiences and achievements including [22] S. Bonassi, M. Fenech, C. Lando, Y.P. Lin, M. Ceppi, W.P. Chang, N. Holland, M. (2007) 371–379. Kirsch-Volders, E. Zeiger, S. Ban, R. Barale, M.P. Bigatti, C. Bolognesi, C. Jia, M. Di [42] P. Thomas, N.J. O’ Callaghan, M. Fenech, Telomere length in white blood cells, supplementation with micronutrients [12,14,36–38]. In 2005 we Alexander Morley, David Turner, Kevin Trainor, Jack Dempsey, Giorgio, L.R. Ferguson, A. Fucic, O.G. Lima, P. Hrelia, A.P. Krishnaja, T.K. Lee, L. buccal cells and brain tissue and its variation with ageing and Alzheimer’s disease, reported a cross-sectional study showing that at least nine Alexander Kutlaca, Chris Matthews, Barbara Sanderson, Pam Sykes, Migliore, L. Mikhalevich, E. Mirkova, P. Mosesso, W.U. Mu¨ ller, Y. Odagiri, M.R. Mech. Ageing Dev. 129 (April (4)) (2008) 183–190. micronutrients are associated with the micronucleus index [36], Alexander Dobrovic, Monica McCarron, Sheila Phillips, Sonia Scarffi, E. Szabova, I. Vorobtsova, A. Vral, A. Zijno, Human MicroNucleus project: [43] P. Thomas, S. Harvey, T. Gruner, M. Fenech, The buccal cytome and micronucleus frequency is substantially altered in Down’s syndrome and normal ageing com- Neville, Julie Turner, Felicia Bulman, Carolyn Salisbury, Philip international database comparison for results with the cytokinesis-block micro- and studies are currently underway to test whether supplementa- nucleus assay in human lymphocytes. I. Effect of laboratory protocol, scoring pared to young healthy controls, Mutat. Res. 638 (February (1–2)) (2008) 37–47. tion with a selection of these micronutrients can reduce the CBMN Thomas, Maryam Hor, Varinderpal Dhillon, Will Greenrod, Jimmy criteria, and host factors on the frequency of micronuclei, Environ. Mol. Mutagen. [44] N. Holland, C. Bolognesi, M. Kirsch-Volders, S. Bonassi, E. Zeiger, S. Knasmueller, cytome biomarkers in a double-blind placebo-controlled trial. The Crott, Sasja Beetstra, Denise Furness, Bianca Benassi, Caroline Bull, 37 (1) (2001) 31–45. M. Fenech, The micronucleus assay in human buccal cells as a tool for biomo- nitoring DNA damage: the HUMN project perspective on current status and Genome Health Clinic concept attracted much attention in Theodora Teo, Jing Wu and Nathan O’Callaghan. A special mention [23] S. Bonassi, M. Neri, C. Lando, M. Ceppi, Y.P. Lin, W.P. Chang, N. Holland, M. Kirsch- Volders, E. Zeiger, M. Fenech, HUMN collaborative group, Effect of smoking habit knowledge gaps, Mutat. Res. 659 (July–August (1–2)) (2008) 93–108. Australia and led to the ‘‘DNA Doctor’’ story on the national of thanks is due to my HUMN project coordinating group on the frequency of micronuclei in human lymphocytes: results from the Human [45] J.S. Wassom, Origins of genetic toxicology and the Environmental Mutagen science program ‘‘Catalyst’’ (http://www.abc.net.au/catalyst/stor- colleagues Stefano Bonassi, Errol Zeiger, Nina Holland, Wushou MicroNucleus project, Mutat. Res. 543 (March (2)) (2003) 155–166. Society, Environ. Mol. Mutagen. 14 (Suppl. 16) (1989) 1–6. ies/s1381311.htm). The opening of the first genome health clinic in Chang, Claudia Bolognesi, Micheline Kirsch-Volders, Siegfried Adelaide, South Australia, in June 2007 is a pioneering attempt to Knasmueller and all the scientists participating in the HUMN put this concept into practice (http://www.csiro.au/partnerships/ project activities. I am also grateful for the contributions of Reach100.html). numerous other collaborators and the volunteers in our studies.

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Table 1 Radiology at Yale, consoled me by pointing out that recA mutants Mutation Research 705 (2010) 71–76 Mutation Research 705 (2010) 71–76 E. coli genes considered in this article. were not conditionally lethal but were still interesting to study. Gene Gene product Microscopic observation showed that the dam cells were not

Contents lists available at ScienceDirect dam DNA adenine methyltransferase uniform in size confirming that for a given optical density in broth dcm DNA cytosine methyltransferase cultures the viable count was always lower for the dam mutant DNA methylationMutation Research/Reviews and mutator in Mutation genes Research in dnaB Replicative helicase than the wild type. In my previous work with dnaB and dnaG dnaE Catalytic alpha-subunit of DNA polymerase III holoenzyme mutants I had also observed this when the cells were grown at the dnaG DNA primase ☆ non-permissive temperature and this led me to look at the DNA Escherichia colijournal homepage:K-12 www.elsevier.com/locate/reviewsmr dnaQ Epsilon-subunit of DNA polymerase III holoenzyme Community address: www.elsevier.com/locate/mutres fpg Synonym for MutM sedimentation profile in alkaline sucrose gradients. There were hexAB MutSL homologs single-strand breaks in the chromosomal DNA of the dam cells and Martin G. Marinus * lig DNA ligase these were amplified in dam polA (Ts) and dam lig (Ts) strains. mutD Allele of dnaQ resulting in defective proofreading These latter strains were inviable at the non-permissive tempera- Reflections in Mutation Research mutH Mismatch repair endonuclease ture as were dam mutations in combination with recA and recBCD Department of Biochemistry and Molecular Pharmacology, University of Massachusetts§ Medical School, mutL Mismatch repair protein DNA methylation and mutator genes in Escherichia coli K-12 mutM Glycosylase specific for oxidized guanine-cytosine basepairs mutant alleles. It was clear that the dam mutants were defective in 55 Lake Avenue North, Worcester, MA 01655, USA mutS Detects base mispairs to initiate mismatch repair some kind of DNA repair but the best that could be done at the time mutT Prevents incorporation of oxidized guanine into DNA was to exclude nucleotide excision repair since the uvr genes had Martin G. Marinus * mutU uvrD Allele of no effect on dam phenotypes [13]. During the mapping of the dam mutY Glycosylase specific for oxidized guanine-adenine basepairs Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA polA DNA polymerase I gene, I noticed that my control plates for the dam mutants often polC Synonym for dnaE had colonies on them while those of the wild type did not. The recA Promotes synapsis of homologous DNA strands mutator phenotype of the dam mutants was quickly confirmed. recBCD Double-strand end-specific exonuclease ARTICLE INFO ABSTRACT These results were published as my three-year appointment at ruvA With RuvB acts as a Holliday junction translocase ruvB With RuvA acts as a Holliday junction translocase Rutgers Medical School was coming to an end, and I was busy Article history: Mutator strains of Escherichia coli have been used to define mechanisms that account for the high fidelity ruvC Holliday junction resolvase trying to find a new position. My wife was seven months pregnant Accepted 27 April 2010 of chromosome duplication and chromosome stability. Mutant strains defective in post-replicative uvrD Mismatch repair associated helicase when in June 1974 she drove our Volvo and I drove the U-Haul Available online 13 May 2010 mismatch repair display a strong mutator phenotype consistent with a role for correction of mismatches truck to Worcester, Massachusetts, where I was to take up a faculty arising from replication errors. Inactivation of the gene (dam) encoding DNA adenine methyltransferase position in the newly formed University of Massachusetts Medical Keywords: results in a mutator phenotype consistent with a role for DNA methylation in strand discrimination methyl-N -nitro-N-nitrosoguanidine (MNNG) and combined the School. I had expected to be there for only a few years before Alkylating agents during mismatch repair. This review gives a personal perspective on the discovery of dam mutants in E. 0 DNA repair survivors in groups of ten. DNA was isolated from the pool and continuing our nomadic existence but I have remained there ever coli and their relationship to mismatch repair and mutator phenotypes. Escherichia coli ß 2010 Elsevier B.V. All rights reserved. assayed for methyl group transfer from tritiated SAM into DNA. If a since. Mutator positive signal was obtained, the pool was split in two for re- In my assistant professor position at UMass Medical School, I Methylation Recombination testing and finally to individuals. This brute force screen led to the isolated more dam mutants by various means and all had the same isolation of 14 DNA and 10 RNA methylation mutants from about range of phenotypes as those previously isolated. In order to 1500 survivors. The RNA mutants were easily identified by testing confirm that these were associated with the dam mutation and not radioactivity in the alkali-treated nucleic acid supernatant fraction something else, advantage was taken of the inviability of dam recA designed to remove RNA. mutants to isolate true revertants. These had none of the The 14 DNA methylation mutants were grown with tritiated phenotypes associated with the dam strains. In addition to the 1. Introduction their effects, and further details can be found in other reviews [3– methionine and the amount of 6-meA and 5-meC quantified. This true revertants there were also suppressed revertants which had 6]. Genes discussed in this review are listed in Table 1 with a brief led to the identification of three mutants lacking 6-meA and 11 mutations in the mutS or mutL genes (see below). These did not Chromosome replication requires a high degree of fidelity, and explanation of each. lacking 5-meC [9]. Seven tRNA methylation mutants were also have the phenotypes associated with dam except for the mutator studies in Escherichia coli K-12 over the last fifty years or so have recovered and in collaboration with Dieter Soll’s lab at Yale were phenotype which was much stronger [14]. The interesting result identified the major mechanisms by which this is achieved. The 2. DNA methylation mutants shown to be deficient in ribothymine (5 isolates), 7-methylguanine was that suppressor mutations of the dam mutator phenotype experimental approach used to solve the fidelity question has and 2-thio-5-methylaminoethyluracil [10]. At this point it was were in mutator genes which had a stronger mutator phenotype. relied mainly on the isolation and characterization of mutator I was appointed to my first faculty position as an Instructor at necessary to identify the genes involved by mapping the mutations In 1974 the SOS hypothesis had not yet been formulated, and it strains. A mutator phenotype (Mut–) is displayed by mutants that Rutgers Medical School (as it was known then) in Piscataway, NJ, in and this was done first by conjugational crosses and then by was a few years later that we showed the dam mutants to be sub- have an increased spontaneous mutation frequency relative to 1971. I had come to join N. Ronald Morris who had been transductional crosses [11]. I tested recombinant classes using the induced for the SOS response. Of all the SOS genes only expression wild type (Mut+). The underlying assumption is that such bacteria researching DNA methylation in eukaryotes. It seemed worthwhile assay above, which was successful but laborious. The 6-meA and 5- of the recA, ruvA and ruvB genes was necessary for dam cell are impaired in systems that normally correct spontaneous studying the problem in E. coli which, unlike eukaryotes, had both meC mutants were designated dam (DNA adenine methylase) and survival. It was also shown subsequently that double-strand replication errors and, in general, this assumption has been 6-methyladenine (6-meA) and 5-methylcytosine (5-meC) in its dcm (DNA cytosine methylase), respectively, although in recent breaks were present in the DNA of dam bacteria. The evidence correct. It took some time, however, for this assumption to take DNA. The approach would be a standard one – isolate mutants years I have been using methyltransferase instead of methylase. I made it clear that mismatch repair is responsible for the formation hold given that the first E. coli mutator strain was described in 1954 lacking methylated bases and deduce their functions by examining had toyed with mad (methyladenine deficient) as the designation of DNA breaks, that DNA ligase is required for repair of single- [1] and systematic screening for mutator strains did not begin until their properties. for the gene but dam won out. The Dam and Dcm methyltrans- strand interruptions, and that homologous recombination is 1970 [2]. The assay we used to isolate methylation-deficient mutants ferases methylate -GATC- and -CC(A/T)GG- sequences, respective- essential for double-strand break repair. What is still not known In this review I have focused on a group of related mutator was based on two prior observations. First, DNA isolated from E. ly, of which there are 19,120 and 12,045, respectively, in the E. coli is how the double-strand breaks are formed, and Fig. 1 shows two strains (and one in particular) that has been the subject of my coli grown in the presence of ethionine, a methionine analog, was chromosome. Stan Hattman’s lab had also isolated dcm mutants at possibilities. First, a replication fork encountering a gap or nick in research for the past few decades. I decided to present a personal found to be deficient in methylation because it was a substrate for the same time by looking for E. coli mutants that would not protect duplex DNA will collapse (Fig. 1A) but can be repaired by view of the developments in this research area in the hope it offers the transfer of methyl groups from S-adenosyl-methionine (SAM) phage lambda from the restriction system encoded by plasmid N3 homologous recombination. It is not known what fraction of insight into the history of these mutants and will be more to such DNA in crude extracts prepared from wild-type cells grown [12]. For many years this was the only phenotype associated with collapsed forks is mended. Second, the presence of a nick on each entertaining than a formal scientific summary. The latter part of without ethionine [7]. DNA isolated from untreated E. coli was not a dcm mutants. From this point on I will deal only with the dam strand at a GATC sequence is equivalent to a double-strand break the review is a more conventional summary of mutator genes and substrate because the DNA was fully methylated. Second, Herb mutants since these had not been previously isolated, and I had (Fig. 1B) which requires a sister chromosome as a template for Boyer’s lab had located the gene (near his) for cytosine methylation concentrated my efforts on them. recombinational repair. on the E. coli K-12 map by using this assay on recombinants The mutations in a clean genetic background were tested for a obtained from crosses between K-12 and B which does not have § variety of phenotypic traits. I had included in the isolation protocol 3. Mismatch repair This article is part of the Reflections in Mutation Research series. To suggest methylated cytosine in its DNA [8]. These findings suggested a way the possibility that the mutations conferred a temperature- topics and authors for Reflections, readers should contact the series editor, G.R. Hoffmann ([email protected]). to detect mutants deficient in methylation – they would sensitive phenotype but none of the mutants were temperature- The existence of this repair system had been postulated by * Tel.: +1 508 856 3330; fax: +1 508 856 2003/3036. incorporate methyl groups into their DNA while wild-type cells sensitive (Ts) for growth. This was somewhat disappointing but K. Holliday [15] to account for gene conversion in fungi. The E-mail address: [email protected]. would not. Accordingly, I treated my wild-type cells with N- Brooks Low, then a junior faculty member in Therapeutic formation of heteroduplex DNA (one DNA chain from the mutant,

1383-5742/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2010.05.001

292 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 293 M.G. Marinus / Mutation Research 705 (2010) 71–76 73 74 M.G. Marinus / Mutation Research 705 (2010) 71–76

strand (from a clear-plaque mutant) to form a heteroduplex transformation frequencies. They also found that correction molecule [19]. In addition, it was possible to obtain strands that occurred only on the incoming strand and not on the recipient’s were unmethylated (propagated in a dam mutant) or fully DNA chains. They proposed that the strand targeted for mismatch methylated (grown in a Dam overproducer). Heteroduplexes were correction was that which had at least one end. Since the made that were unmethylated on both strands; methylated on the chromosomal DNA had no ends and the incoming DNA had two, wild-type or mutant strand (hemimethylated); and methylated on it was the latter that was targeted [16]. This model can be both strands. Infection of a wild-type E. coli host with the extrapolated to the replication fork; the parental strands do not hemimethylated heteroduplexes yielded progeny strongly biased have an end nearby but the newly synthesized strands have 30- to the configuration of the methylated strand. That is, if the wild- ends and therefore are targeted. This model applies to bacteria type strand was methylated the progeny formed mostly turbid with circular chromosomes and eukaryotes with linear chromo- plaques, while if the mutant strand was methylated the progeny somes. were mostly clear-plaque formers. This biased directionality was A puzzling aspect of this general model is why E. coli and its not present if a mutL host was used, consistent with the idea that relatives have abandoned it in favor of a methylation-based this strain was defective in mismatch repair. Directionality of discrimination model. Furthermore, why does not strand discrim- repair was also reduced with the unmethylated heteroduplex. ination occur in a dam mutant? A surprising finding was that if both strands were methylated, there was no correction in a wild-type host indicating that only 5. Antirecombination, Dam methylation, mismatch repair hemimethylated heteroduplex regions were subject to repair [19]. and MNNG Since hemimethylated DNA occurs only transiently behind the replication fork, it was postulated that the mismatch repair system Genetic crosses between E. coli and Salmonella typhimurium are acted on replication errors at this location. The error occurs in the sterile. However, if the recipient in these crosses is mismatch newly synthesized unmethylated strand which is then targeted for repair-deficient (mutS or mutL inactivation), bona fide recombi- repair using the parental methylated strand as template (Fig. 2A). nants are formed [24]. This result suggests that MutS and MutL Further evidence for this model was that dam mutants have a prevent recombination between homeologous sequences, which mutator phenotype because directionality of mismatch correction are related but not identical. This antirecombination effect may be is lost and the error-containing strand is used as template to important in preventing recombination in the DNA of organisms introduce mutations into the genome. In addition, overproduction that have repeated homeologous DNA sequences as such of Dam methyltransferase in a wild-type E. coli host leads to an recombination could lead to genome instability. An initial step Fig. 1. Formation of double-strand breaks in dam bacteria. (A) The encounter of a increased mutation rate because newly synthesized DNA becomes in homologous recombination is the insertion of the 30-end of a replication fork with a strand discontinuity due to mismatch repair (MMR) results methylated before it can be corrected and thereby leads to single strand into duplex DNA to form a D-loop. This reaction is in fork collapse and the formation of a double-stranded end. This end is a substrate introduction of mutations into the chromosome [20,21]. Results for the RecBCD exonuclease which, upon digestion, can lead to the loading of RecA catalyzed by the RecA protein and is not affected by the presence of and the formation of a D-loop after strand invasion. Resolution of the Holliday showing that mutations in the mutHLS genes suppress the lethality MutS and MutL. If the DNA molecules in this reaction are junction by RuvABC can restore the fork. (B) Mismatch repair nicking of both of 2-aminopurine to dam strains were also consistent with this homeologous, however, then MutS and MutL will block the strands at a GATC sequence results in a double-strand break that can be repaired model [22]. formation of RecA product (no D-loops are formed) [25]. This is using a sister chromosome as template. This type of recombinational repair also Although the mutHLS mutants display a strong mutator shown diagrammatically in Fig. 2A where the D-loop formed by requires RecBCD, RecA, and RuvABC. phenotype, that of the dam mutant is relatively weak. This is RecA action between homeologous substrates results in the unexpected since the anticipation is that the mutation frequency formation of multiple mismatches and the reaction is aborted the other from wild type) leads to the creation of base mismatches should be the same as that of the mut strains. It is possible that a by MutS and MutL. Fig. 2. Functions of the mismatch repair system in E. coli. (A) The two known and, depending on the direction of correction, could explain the certain fraction of the dam population is lost due to the inability to functions of mismatch repair are antirecombination (left) and correction of I was on sabbatical leave in 1980–1981 at the Medical Research excess or deficiency of recombinant classes observed as an excess repair all the single- and double-strand breaks that arise. This loss mismatches generated by replication (right). The initial step in antirecombination is Council Cell Mutation Unit at the University of Sussex where Peter of the phenotype conferred by one allele relative to that of the would be reflected in a lower mutator phenotype. the formation of a D-loop by insertion of a single strand into a duplex molecule by Karran and I collaborated on a project on the sensitivity of dam RecA protein. The invading strand is homeologous and therefore creates other. Studies in Streptococcus pneumoniae with heteroduplexes Fig. 2 illustrates the functions of the mismatch repair system in mutants to methylating agents and to MNNG (N-methyl-N0-nitro- mismatches in the duplex DNA that are recognized by MutS and MutL. These showed that transformation frequencies were, in part, dependent E. coli which are correction of replication errors and antirecombi- proteins block the RecA reaction probably by reversing it. MutS and MutL have no N-nitrosoguanidine) in particular. Peter had come to Sussex from on mismatch correction. Furthermore, a mutant strain (hex) nation. Antirecombination will be discussed in detail in the next effect on RecA catalysis if the two molecules are homologous. A mismatch (M) at the the Lindahl lab, then in Gothenburg in Sweden, and was working appeared deficient for this type of repair and had a mutator section. The right side of Fig. 2A depicts a replication fork showing replication fork (right) in hemimethylated DNA is acted upon by mismatch repair, on the biochemistry of enzymes acting on DNA modified by phenotype [16]. a transient section of hemimethylated DNA with a base pair but a mismatch in fully methylated DNA is refractory. Lack of Dam methylating agents. We showed that although dam mutants were methyltransferase results in correction of either the newly synthesized strand or The mutagen 5-bromouracil (5-BU) is known to form base pairs mismatch that is susceptible to mismatch repair while a mismatch rapidly killed by MNNG, dam mut strains were not, indicating that the parental strand. In the latter case, mutations are introduced into the genome. with either adenine or guanine. Rydberg showed that in wild-type in fully methylated DNA is not. Fig. 2B summarizes the biochemical Overproduction of Dam methyltransferase results in premature methylation of mismatch repair was responsible for the inviability. This work E. coli, 5-BU mutagenesis is suppressed at low concentrations studies from Paul Modrich’s laboratory [23] on the correction of DNA behind the fork and stabilization of mismatches by lack of repair. (B) A produced two important findings. First, the differential suscepti- suggesting that 5-BU mispairs were subject to correction. He base mispairs in hemimethylated DNA at the replication fork. Base mismatch in hemimethylated DNA is recognized by the MutS protein (blue and bility of dam mutants to methylating agents indicated that O6- devised an assay to isolate mutants defective in correction and mismatches are recognized and bound by the MutS protein which white) which recruits MutL (red circles) and MutH (scissors), and the latter incises methylguanine (O6-meG) was the lesion recognized by the the unmethylated strand at a GATC sequence. UvrD helicase (green circle) loads at these mapped to the mutH, mutL, mutS and uvrD (=mutU) genes. recruits MutL and MutH. The latter is a latent endonuclease that, the nick created by MutH and unwinds the DNA strand towards the mismatch. mismatch repair system. It was already known that this base 6 6 These mutants all had spontaneous mutation frequencies at least upon formation of the ternary complex, cleaves 50 to the G at a Exonucleases (yellow) digest the unwound single strand including the mismatched leads to ambiguous coding such that O -meG-T or O -meG-C base one-hundred-fold greater than wild type [17]. It was subsequently nearby GATC sequence on the unmethylated strand. The MutH nucleotide. Different exonucleases are used depending on the orientation of the pairs are formed after replication. The second important point was shown that hexAB mutator strains of S. pneumoniae were in genes protein is released from the complex, and the UvrD helicase is mismatch relative to the GATC site (ExoVII or RecJ in one case, and ExoI, ExoVII, or the proposal that mismatch repair of O6-meG-T would lead to homologous to mutSL. recruited to the nick and unwinds the DNA from the nick towards ExoX in the other). The resultant gap is filled by the action of DNA polymerase III formation of O6-meG-C but that this basepair would also be holoenzyme, and DNA ligase seals the nick. Dam methyltransferase acts at the the mismatch. This unwinding can occur in either the 50 to 30 or 30 hemimethylated GATC sites to fully methylate them. subjected to mismatch repair. Repeated mismatch repair attempts 4. Dam methylation and mismatch repair to 50 direction depending on the relative orientation of the GATC to produce a futile repair cycle and eventually lead to cell death [26]. the mismatch. Specific exonucleases are used for each direction: After my sabbatical leave I returned to Worcester to begin work The connection between Dam methylation and mismatch ExoVII or RecJ in one case, and ExoI, ExoVII, or ExoX in the other. strand recognized for targeting by the mismatch repair system in on mutational specificity, and Peter moved to the Imperial Cancer repair was suggested by R. Wagner and M. Messelson; methylation The resultant gap is filled by the action of DNA polymerase III these organisms? This problem was faced by the streptococcal Research Fund (now Cancer Research UK) Clare Hall laboratories was the way E. coli could discriminate between old and new DNA holoenzyme, and DNA ligase seals the nick. Dam methyltransferase geneticists who were using heteroduplexes containing various when Thomas Lindahl became the director. Peter extended the E. chains [18]. To investigate this possibility, advantage was taken of acts at the hemimethylated GATC sites to fully methylate them and base mismatches in transformation experiments. They found that coli work to mammalian cells that are sensitive to killing by MNNG the ability to separate the strands of bacteriophage lambda using to prevent further repair. the transformants fell into two groups yielding either high or low but not when mismatch repair is inactivated. This research gained cesium chloride density gradient centrifugation, thereby allowing All eukaryotes and most bacteria unrelated to E. coli, do not have frequencies. It was proposed that mismatch repair resulted in low importance when (a) a subset of tumors from relapsed cancer annealing of a wild-type strand with a complementary mutant methylated GATC sequences in DNA. How is the newly synthesized transformation frequencies while lack of repair resulted in high patients treated with MNNG-like drugs were found to be mismatch

294 ELSEVIER REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019 295 M.G. Marinus / Mutation Research 705 (2010) 71–76 75 76 M.G. Marinus / Mutation Research 705 (2010) 71–76 repair-deficient [27], and (b) mismatch repair-deficiency was RecA reaction is slowed and in the presence of both MutS and MutL Cancer Society, the National Science Foundation and the National found to be associated with hereditary and sporadic colon cancer the reaction can be blocked (Fig. 3B). The same concentrations of Institutes of Health (currently GM 63790). [28,29]. MutS and MutL have no effect on the RecA reaction in substrate In both E. coli dam mutants and mammalian cells, mismatch molecules lacking O6-methylguanine. This result suggests that References repair therefore sensitizes the cells to killing after exposure to both double-strand-break formation by mismatch repair and the MNNG. We showed that DNA double-strand breaks accumulate in inhibition of double-strand break recombinational repair by MutS [1] H.P. Treffers, V. Spinelli, N.O. Belser, A factor (or mutator gene) influencing mutation rates in Escherichia coli, Proc. Natl. Acad. Sci. U. S. A. 40 (1954) 1064– MNNG-exposed dam mutants to a much higher level than in and MutL may contribute to the killing of dam mutants by MNNG. 1071. untreated dam cells but were not detectable in either case in dam The possibility that double-strand breaks and/or inhibition of their [2] R.M. Liberfarb, V. Bryson, Isolation, characterization, and genetic analysis of mutants that were mismatch repair-deficient [30]. Unrepaired recombinational repair are involved in mismatch repair-depen- mutator genes in Escherichia coli B and K-12, J. Bacteriol. 104 (1970) 363–375. [3] E.C. Cox, Bacterial mutator genes and the control of spontaneous mutation, Annu. double-strand DNA breaks are known to be lethal to the cell, and dent killing of mammalian cells by MNNG has not yet been tested. Rev. Genet. 10 (1976) 548–555. these could account for the sensitivity to MNNG of the dam [4] K.C. Smith, Spontaneous mutagenesis: experimental, genetic and other factors, mutants. Such double-strand breaks might result from futile 6. Other mutator genes in E. coli Mutat. Res. 277 (1992) 139–162. cycling but this possibility remains to be tested. [5] J.H. Miller, Spontaneous mutators in bacteria: insights into pathways of muta- genesis and repair, Annu. Rev. Microbiol. 50 (1996) 625–643. A second mechanism that may promote mismatch repair- In general, mutator strains are defective in functions that (a) [6] J.P. Horst, T.H. Wu, M.G. Marinus, Escherichia coli mutator genes, Trends Microbiol. mediated cell death of MNNG-exposed dam mutants relates to prevent incorporation of mutagenic bases, (b) correct replication 7 (1999) 29–36. antirecombination. Double-strand breaks in E. coli are repaired by errors, or (c) remove potentially mutagenic bases from DNA. An [7] M. Gold, J. Hurwitz, The enzymatic methylation of ribonucleic acid and deoxyr- ibonucleic acid. VI. Further studies on the properties of the deoxyribonucleic acid homologous recombination involving RecA activity. We found that example of a nucleotide-pool cleansing enzyme is MutT. It converts methylation reaction, J. Biol. Chem. 239 (1964) 3866–3874. MNNG-methylated homologous DNA was recognized as if it were oxidized dGTP to oxidized dGMP, which is not used by DNA [8] L. Mamelak, H.W. Boyer, Genetic control of the secondary modification of deox- homeologous in an in vitro reaction using RecA, MutS and MutL [31]. polymerases. Although this mutator strain was first discovered in yribonucleic acid in Escherichia coli, J. Bacteriol. 104 (1970) 57–62. [9] M.G. Marinus, N.R. Morris, Isolation of deoxyribonucleic acid methylase mutants The RecA reaction (Fig. 3A) transfers a homologous strand from 1954 [1], it was not until 1992 that its function was uncovered [32]. of Escherichia coli K-12, J. Bacteriol. 114 (1973) 1143–1150. a duplex molecule (DS) to a single-stranded circular (SS) molecule The dnaE (=polC) gene encodes the catalytic alpha subunit of [10] M.G. Marinus, N.R. Morris, D. Soll, T.C. Kwong, Isolation and partial characteriza- to form a nicked circular (NC) product. When one of the two DNA polymerase III holoenzyme, and dnaQ (=mutD) encodes the tion of three Escherichia coli mutants with altered transfer ribonucleic acid 6 methylases, J. Bacteriol. 122 (1975) 257–265. molecules (6 kb in length) contains 10–20 O -methylguanines, the proofreading epsilon exonuclease subunit. These two proteins are [11] M.G. Marinus, Location of DNA methylation genes on the Escherichia coli K-12 RecA reaction proceeds at the same rate as if no methylated bases in close contact. Although mutations in the catalytic subunit are genetic map, Mol. Gen. Genet. 127 (1973) 47–55. were present (Fig. 3A). With the addition of MutS, the rate of the expected to increase or decrease the fidelity of base selection and [12] S. Hattman, S. Schlagman, L. Cousens, Isolation of a mutant of Escherichia coli defective in cytosine-specific deoxyribonucleic acid methylase activity and in incorporation and thereby impart a mutator phenotype, no such partial protection of bacteriophage lambda against restriction by cells containing bona fide mutations have been isolated. The only mutator allele of the N-3 drug-resistance factor, J. Bacteriol. 115 (1973) 1103–1107. dnaE so far examined appears to affect the activity of the epsilon [13] M.G. Marinus, N.R. Morris, Biological function for 6-methyladenine residues in the DNA of Escherichia coli K12, J. Mol. Biol. 85 (1974) 309–322. proofreading exonuclease subunit. Mutator alleles affecting this + [14] B.R. McGraw, M.G. Marinus, Isolation and characterization of Dam revertants and subunit include the dominant mutD5 allele, which decreases Fig. 4. Action of MutT, MutM and MutY proteins. Guanine is readily oxidized leading suppressor mutations that modify secondary phenotypes of dam-3 strains of correction of replication errors. However, the large number of to various products including 8-oxoguanine (designated *G). The MutT protein Escherichia coli K-12, Mol. Gen. Genet. 178 (1980) 309–315. errors generated in this strain also overwhelms MutHLS mismatch converts d*GTP to d*GMP thereby preventing its utilization by DNA polymerase. [15] R. Holliday, A mechanism for gene conversion in fungi, Genet. Res. 5 (1964) 282– 304. Incorporation of d*GTP into DNA results in base pairing with either A or C. *G–C base repair correction leading to a mutator phenotype due to [16] J.P. Claverys, S.A. Lacks, Heteroduplex deoxyribonucleic acid base mismatch pairs are substrates for the MutM glycosylase which removes the *G. *G–A base repair in bacteria, Microbiol. Rev. 50 (1986) 133–165. transversions, transitions and frameshifts. pairs are substrates for MutY glycosylase which removes the A residue resulting in a The oxidation product 8-oxoguanine can be formed in DNA either [17] B. Rydberg, Bromouracil mutagenesis and mismatch repair in mutator strains of *G–C base pair which is acted upon by MutM. Replication of a *G–A base pair leads Escherichia coli, Mutat. Res. 52 (1978) 11–24. by incorporation as a deoxyribonucleotide triphosphate precursor to a G to T transversion of the original G–C or an A to C transversion of the original [18] R. Wagner, M. Meselson, Repair tracts in mismatched DNA heteroduplexes, Proc. (d*GTP) or by the oxidation of guanine residues in DNA (Fig. 4). The A–T (figure adapted from [6]). Natl. Acad. Sci. U. S. A. 73 (1976) 4135–4139. d*GTP that escapes the cleansing action of MutT can be incorporated [19] P.J. Pukkila, J. Peterson, G. Herman, P. Modrich, M. Meselson, Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia opposite either cytosine or adenine. The *G–C base pair is a substrate coli, Genetics 104 (1983) 571–582. for the MutM glycosylase which removes the oxidized base and 7. Mutator genes in populations of pathogens and commensals [20] G.E. Herman, P. Modrich, Escherichia coli K-12 clones that overproduce dam leads to its replacement with G. The *G–A base pair is acted upon by methylase are hypermutable, J. Bacteriol. 145 (1981) 644–646. [21] M.G. Marinus, A. Poteete, J.A. Arraj, Correlation of DNA adenine methylase activity MutY, which removes the A residue and replaces it with C. The *G–C The frequency of mutator strains in a population of E. coli K-12 with spontaneous mutability in Escherichia coli K-12, Gene 28 (1984) 123–125. pair is then acted upon by MutM (Fig. 4). If the *G–A base pair is (the laboratory strain) is less than one in 100,000. In contrast, this [22] B.W. Glickman, M. Radman, Escherichia coli mutator mutants deficient in meth- replicated before repair, an A–T will result in one daughter molecule frequency increases to at least one percent in pathogenic or ylation-instructed DNA mismatch correction, Proc. Natl. Acad. Sci. U. S. A. 77 and a *G–C in the other, the latter again a substrate for MutM. This (1980) 1063–1067. commensal E. coli strains [33,34]. The relatively high frequency of [23] P. Modrich, DNA mismatch correction, Annu. Rev. Biochem. 56 (1987) 435–466. pathway explains the observed GC to TA changes observed in mutM mutator strains in these populations has led to speculation that [24] C. Rayssiguier, D.S. Thaler, M. Radman, The barrier to recombination between mutY double mutants and the AT to CG specificity of the mutT strain. they may be important when the population is subjected to stress. Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair Loss of both MutM and MutY results in a strong mutator phenotype mutants, Nature 342 (1989) 396–401. Bacterial populations in nature are probably constantly changing [25] L. Worth, S. Clark, M. Radman, P. Modrich, Mismatch repair proteins MutS and (about the same level as the MutHLS mismatch repair system), and the reservoir of mutator bacteria may help to select variants MutL inhibit RecA-catalyzed strand transfer between diverged DNAs, Proc. Natl. indicating the importance of this system [5,6]. Acad. Sci. U. S. A. 91 (1994) 3238–3241. more suited to the new environment. For example, on leaving the 6 The four systems described above (pool cleansing, exonucleolytic [26] P. Karran, M.G. Marinus, Mismatch correction at O -methylguanine residues in E. alimentary tract of an animal, the E. coli population would need to coli DNA, Nature 296 (1982) 868–869. proofreading, MutHLS correction and MutY/MutM action) consti- adapt quickly to extra-intestinal life and the presence of a pre- [27] G.M. Li, The role of mismatch repair in DNA damage-induced apoptosis, Oncol. tute the major mechanisms to prevent or correct base mismatches in existing mutant population may facilitate this change. If the Res. 11 (1999) 393–400. DNA. Other strains displaying a mild mutator phenotype have also [28] F.S. Leach, N.C. Nicolaides, N. Papadopoulos, B. Liu, J. Jen, R. Parsons, P. Peltomaki, mutator subpopulation serves such a function, the variants would P. Sistonen, L.A. Aaltonen, M. Nystrom-Lahti, Mutations of a mutS homolog in been characterized, as have effects of overproducing gene products, need to rapidly lose their mutator phenotype. The mechanism by hereditary nonpolyposis colorectal cancer, Cell 75 (1993) 1215–1225. but the magnitude of these effects is generally small and these are which this could occur is not known. [29] R. Fishel, M.K. Lescoe, M.R. Rao, N.G. Copeland, N.A. Jenkins, J. Garber, M. Kane, R. described in more detail elsewhere [6]. Kolodner, The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer, Cell 75 (1993) 1027–1038. Our knowledge of mutator strains is still largely derived from Conflicts of interest statement Fig. 3. The RecA strand-transfer reaction. (A) A homologous strand from linear [30] A. Nowosielska, M.G. Marinus, DNA mismatch repair-induced double-strand studies with E. coli. The sequencing of genomes of many other breaks, DNA Repair (Amst.) 7 (2008) 48–56. duplex DNA (DS) is transferred to a single-stranded circular (SS) molecule by RecA [31] M.A. Calmann, J.E. Evans, M.G. Marinus, MutS inhibits RecA-mediated strand to form a double-stranded nicked circular (NC) product which can be easily bacteria and Archaea has largely confirmed the existence of the None. transfer with methylated DNA substrates, Nucleic Acids Res. 33 (2005) 3591–3597. 6 error correction pathways described in E. coli, but other more detected. The linear duplex DNA contains O -meG bases (Me). (B) Rate of formation [32] H. Maki, M. Sekiguchi, MutT protein specifically hydrolyses a potent mutagenic 6 of NC product when 10–20 O -meG bases are present in DS DNA. No addition exotic mutator genes probably await discovery. For example, Acknowledgements substrate for DNA synthesis, Nature 355 (1992) 273–275. (triangles), 25 nM MutS (unfilled squares), 100 nM MutS (filled squares), 25 nM organisms living in high temperature, low pH, or high pressure [33] J.E. LeClerc, B. Li, W.L. Payne, T.A. Cebula, High mutation frequencies among MutS and 50 nM MutL (crosses), 100 nM MutS and 100 nM MutL (unfilled circles). Escherichia coli and Salmonella pathogens, Science 274 (1996) 1208–1211. 6 environments presumably have developed special mechanisms The reaction rate in the absence of O -meG modification is denoted by the filled My research on DNA methylation, mismatch repair and [34] F. Taddei, I. Matic, B. Godelle, M. Radman, To be a mutator, or how pathogenic and circles. MutS and MutL do not affect the RecA reaction rate in the absence of DNA preventing damage to their DNA that might elicit a mutator recombination has been funded over the years by the American commensal bacteria can evolve rapidly, Trends Microbiol. 5 (1997) 427–428. modification (figure adapted from [31]). phenotype if not working correctly.

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