Reflections on the origins and evolution of genetic toxicology and the Environmental Mutagen Society.

This article traces the development of the field of mutagenesis and its metamorphosis into the research area we now call genetic toxicology. In 1969, this transitional event led to the founding of the Environmental Mutagen Society (EMS). The charter of this new Society was to "encourage interest in and study of mutagens in the human environment, particularly as these may be of concern to public health." As the mutagenesis field unfolded and expanded, new wording appeared to better describe this evolving area of research. The term "genetic toxicology" was coined and became an important subspecialty of the broad area of toxicology. Genetic toxicology is now set for a thorough reappraisal of its methods, goals, and priorities to meet the challenges of the 21st Century. To better understand these challenges, we have revisited the primary goal that the EMS founders had in mind for the Society's main mission and objective, namely, the quantitative assessment of genetic (hereditary) risks to human populations exposed to environmental agents. We also have reflected upon some of the seminal events over the last 40 years that have influenced the advancement of the genetic toxicology discipline and the extent to which the Society's major goal and allied objectives have been achieved. Additionally, we have provided suggestions on how EMS can further advance the science of genetic toxicology in the postgenome era. Any oversight or failure to make proper acknowledgment of individuals, events, or the citation of relevant references in this article is unintentional.

In vivo mutation analysis using the ΦX174 transgenic mouse and comparisons with other transgenes and endogenous genes.

The ΦX174 transgenic mouse was first developed as an in vivo Ames test, detecting base pair substitution (bps) at a single bp in a reversion assay. A forward mutational assay was also developed, which is a gain of function assay that also detects bps exclusively. Later work with both assays focused on establishing that a mutation was fixed in vivo using single-burst analysis: determining the number of mutant progeny virus from an electroporated cell by dividing the culture into aliquots before scoring mutants. We review results obtained from single-burst analysis, including testing the hypothesis that high mutant frequencies (MFs) of G:C to A:T mutation recovered by transgenic targets include significant numbers of unrepaired G:T mismatches. Comparison between the ΦX174 and lacI transgenes in mouse spleen indicates that the spontaneous bps mutation frequency per nucleotide (mf(n)) is not significantly lower for ΦX174 than for lacI; the response to ENU is also comparable. For the lacI transgene, the spontaneous bps mf(n) is highly age-dependent up to 12 weeks of age and the linear trend extrapolates at conception to a frequency close to the human bps mf(n) per generation of 1.7 × 10(-8). Unexpectedly, we found that the lacI somatic (spleen) bps mf(n) per cell division at early ages was estimated to be the same as for the human germ-line. The bps mf(n) in bone marrow for the gpt transgene is comparable to spleen for the lacI and ΦX174 transgenes. We conclude that the G:C to A:T transition is characteristic of spontaneous in vivo mutation and that the MFs measured in these transgenes at early ages reflect the expected accumulation of in vivo mutation typical of endogenous mammalian mutation rates. However, spontaneous and induced mf(n)s per nucleotide for the cII gene in spleen are 5-10 times higher than for these other transgenes.

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

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

History of the science of mutagenesis from a personal perspective.

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

Incorporation of mammalian metabolism into mutagenicity testing.

In the 1950's and 1960's it became obvious that many chemicals in daily use were mutagenic or carcinogenic, but there seemed to be little relation between the two activities. As scientists were debating the cause of this discrepancy, it was hypothesized that mammalian metabolism could form highly reactive intermediates from rather innocuous chemicals and that these intermediates could react with DNA and were mutagenic. This commentary presents the historical development of metabolic activation in mutagenicity tests, beginning with Udenfriend's hydroxylation system, which mimics aspects of mammalian metabolism in a purely chemical mixture, and extending through procedures that moved closer and closer to incorporating actual mammalian metabolism into the test systems. The stages include microsomal activation systems, host-mediated assays, incorporation of human P450 genes into the target cells or organisms, and detecting mutations in single cells in vivo. A recent development in this progression is the insertion of recoverable vectors containing mutational targets into the mammalian genome. Since the target genes of transgenic assays are in the genome, they are not only exposed to active metabolites, but they also undergo the same repair processes as endogenous genes of the mammalian genome.

Three origins of phiX174 am3 revertants in transgenic cell culture.

Transgenic systems for measuring mammalian mutagenesis often use recoverable viral vectors. We hypothesize that mutations in these transgenic systems can arise from three different origins of DNA damage and replication errors and that these three origins of mutations (in vivo, ex vivo, and in vitro) can be differentiated in the PhiX174 am3, cs70 single burst assay (SBA) on the basis of burst size (BS). In vivo mutations are fixed in the animal, ex vivo mutations are fixed in bacterial cells during recovery of the phage, and in vitro revertants arise during the first replications of nonmutant phages under selective conditions. PX-2 cells, derived from a homozygous embryo of a PhiX174 transgenic mouse, were treated with vehicle or N-ethyl-N-nitrosourea (ENU). An algorithm was developed to estimate the BS that resulted in the highest induced revertant frequency; the estimate was 56. In vivo revertants were defined as having BS >55, ex vivo revertants as having a BS of 13-56, and in vitro revertants as having a BS of <14. The frequencies of in vivo revertants at 0, 100, and 200 mg/kg ENU were 0.06, 0.36, and 4.10 x 10(-6) (dose response, P = 0.004); ex vivo revertants were 0.36, 0.46, and 0.41 x 10(-6) (P = 0.37), and in vitro revertants were 0.39, 0.46, and 0.41 x 10(-6) (P = 0.55), respectively. These results show that only in vivo revertants reflect mutagen treatment. They also provide a basis for identifying PhiX174 am3 revertants induced in vivo and may increase the sensitivity of the assay for in vivo mutation.

The in vivo but not the in vitro am3 revertant frequencies increase linearly with increased ethylnitrosourea doses in spleen of mice transgenic for phiX174 am3, cs70 using the single burst assay.

The am3 revertant frequencies (RF) in spleens from male mice transgenic for phiX174 am3, cs70 were analyzed 14 weeks after ethylnitrosourea (ENU) treatment, both by the single burst assay (SBA) and the mixed burst assay (MBA). The mean in vivo (burst size >30/assay plate) revertant frequency (MRF) for the vehicle control was 2.6x10(-7). The ENU induced in vivo RF were linear over the dose range 0-150mg/kg, (r(2)=0.999). The concomitant in (burst size G transitions. Sequence analysis of in vivo revertants from ENU treated animals revealed revertants that were 17% A-->G transitions and 83% A-->T transversions, the latter being consistent with the reported A:T base pair alterations induced by ENU. No A-->C transitions were seen. This suggests the occurrence of an ENU-induced O(2) ET-dT lesion leading to a dT base mismatch. The observations in this report both confirm and validate the use of the SBA for distinguishing between in vivo mutations that are fixed in the animal and in vitro mutations that arise from other sources. The ability of the SBA to distinguish the in vivo from the in vitro origin of mutations has increased the specificity, sensitivity and utility of the phiX transgenic system.

Response of the phiX174 am3, cs70 transgene to acute and chronic ENU exposure: implications for protocol design.

Studies of other transgenic assays have shown that time after treatment is a very important variable in the analysis of mutation frequencies but that eventually a plateau frequency is reached, indicating that the mutations are neutral. This neutrality is very important for the design of both experiments and testing protocols. Here we show that the phiX174 am3, cs70 transgene gives qualitatively similar results to the other transgenes studied after exposure of the mice to N-ethyl-N-nitrosourea. In the small intestine, the mutant frequency induced by an acute dose did not change significantly from 10 to 70 days post-treatment, indicating that the mutations induced are, indeed, neutral. Likewise, the mutant frequency increased linearly with duration of exposure to ENU at a constant rate. Mutant frequencies obtained were 10 times higher from the chronic exposure than produced by a nearly lethal acute dose. As in previous comparisons of a transgene and the endogenous Dlb-1 locus in the small intestine, the chronic exposure was much more effective at increasing the sensitivity of the transgene than of the endogenous gene. The Dlb-1 locus shows more complex kinetics in this strain, as in others, with mutations initially accumulating at a slower rate, indicating a differential repair of genetic damage.

Characterization of mutant spectra generated by a forward mutational assay for gene A of Phi X174 from ENU-treated transgenic mouse embryonic cell line PX-2.

The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing. A forward mutational assay exists for Phi X174 that requires an altered, functional Phi X174 protein and therefore should have fewer targets (sense, base-pair substitutions) than forward assays that inactivate a protein. We investigated this assay to determine the number of targets and their suitability for detecting a known mutagen, N-ethyl-N-nitrosourea (ENU). We identified 25 target sites and 33 different mutations in Phi X174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for Phi X174 am3, cs70 (line 54). All six types of base-pair substitution were represented among both the spontaneous and ENU-treated mutant spectra. The mutant spectra from cells treated with 200 and 400 microg/ml ENU were both highly different from the spontaneous spectrum (P < 0.000001) but not from each other. The dose trend was significant (P < 0.0001) for a linear regression of mutant frequencies (R(2) = 0.79), with a ninefold increase in mutant frequency at the 400 microg/ml dose. The spontaneous mutant frequency was 1.9 x 10(-5) and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations. Thirteen mutations at 12 targets were identified only from ENU-treated cells. Seven mutations had highly significant increases with ENU treatment (P < 0.0001) and 15 showed significant increases. The results suggest that the Phi X174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.