David M. DeMarini, Ph.D. US Environmental Protection Agency Research Triangle Park, North Carolina Disclaimer

This Presentation Does Not Necessarily Reflect the Policy of the U.S. EPA The Mutagenesis Moonshot: The Propitious Beginnings of the Environmental Society

David M. DeMarini

U.S. Environmental Protection Agency Outline

• Mutagen 1 (X-rays): mutating genes experimentally • Mutagen 2 (UV): genetic material & genetic repair • Mutagen 3 (mustard gas): chemical • Fallout from fallout: mutagenesis at Oak Ridge • Pollution, politics and persuasion: influence of the environment on our germ cells • Emergence of the environmental movement • EMS takes off in 1969 • Early successes • Legacy of action without activism Before the Mutagens Gregor Mendel

In 1866 this Czech priest deduced that particulate units explain hereditary traits in peas. Hugo de Vries (The Netherlands) 1. In 1900 he coined the term “.” 2. In 1901 he noted that the artificial induction of will permit production of better plants and animals. 3. In 1904 he suggested in a lecture to U.S. scientists that X-rays might be able to alter the hereditary particles in the germ cells. Mutagen 1: X-Rays Early Studies in Germany in 1905- 1912 on the Mutagenicity of X-rays

First studies of X-ray-induced mutagenicity were done for morphological changes in Bacillus and Aspergillus Erwin Baur Elizabeth Schiemann X-Rays Are Mutagenic • Hermann Muller at U of Texas (Science 64:84, 1927) reported on the first mutagenicity assay (SLRL in Drosophila) and first mutagen (X-rays), but he did not show any data. He reported the data in PNAS 14:714, 1928.

• Lewis Stadler at U of Missouri showed the mutagenicity of X- rays in (PNAS 14:69, 1928) and (Science 68:186, 1928). X-Rays and Cancer?

• In his 1927 paper, Muller suggested that mutations might cause cancer.

• In a 1934 paper Muller (Proc Int Radiol Congr 4th, 2:100) noted “…producing mutations in somatic cells by radiation … in which mitosis occurs, may result in cancer, leukemia, etc.” Mutagen 2: UV Light UV Light is Mutagenic • Edgar Altenburg at Rice University in Houston, Texas showed UV was mutagenic in Drosophila (Science 78:587, 1933).

• Lewis Stadler at U of Missouri showed UV was mutagenic in maize (PNAS 22:584, 1936). UV Light Suggests Nucleic Acid as the Genetic Material

Frederick Gates at the Rockefeller Institute in New York showed that the maximum bactericidal effect of UV was the absorption spectrum of nucleic acids (265 nm) and suggested that UV damage to nucleic acid caused the cells to die (FL Gates, J Gen Physiol 13:231, 1929). Alexander Hollaender • Published in 1929 on UV bactericidal effects while at the Univ of Wisconsin.

• At NIH, he showed UV induced maximal mutagenesis in fungal spores at 265 nm and suggested that UV acted on nucleic acid to produce mutations. (Emmons & Hollaender, Am J Bot 26:467, 1939; Hollaender, Proc 7th Int Cong Genet 153, 1941; J Bacteriol 46:531, 1943). Hollaender Gave First Suggestion That Cells Can Repair UV Damage

Based on studies showing higher survival of UV-irradiated fungal spores after being held in liquid medium prior to plating, Hollaender suggested “the possibility of recovery of the irradiated bacteria is not entirely excluded.”

Hollaender & Curtis (Proc Soc Exp Biol Med 33:61, 1935), and Hollaender & Claus (J Gen Physiol 19:753, 1936). First UV-Sensitive Mutant in 1946

Evelyn Witkin at Rutgers Univ in New Jersey isolated the first UV-sensitive mutants of E. coli.

(PNAS 32:59, 1946; Genetics 32:221, 1947). DNA Repair Emerges from UV Work

• Repair replication in the gap generated by excision of UV- induced DNA damage was discovered by Pettijohn & Hanawalt at Stanford (J Mol Biol 9:395, 1964) and Setlow & Carrier at Oak Ridge Richard Setlow Phil Hanawalt (PNAS 81:7397, 1964). • XP cells were deficient in repair replication after UV James Cleaver discovered by Cleaver at UC- San Francisco (Nature 218:652, 1968). Mutagen 3: Chemicals

Allyl isothiocyanate Auerbach and Robson • Mustard gas induced SLRL in Drosophila (reported to UK Ministry of Supply 1942).

• Allyl isothiocyanate induced SLRL (Auerbach & Robson, Nature 154:81, 1944).

• Mustard gas data published (Auerbach & Robson, Nature 157:302, 1946). More Chemical Mutagens (1946-1948)

• Iosif Rapoport (USSR) reported that epichlorohydrin, ethylene oxide, and glycidol induced SLRL (Rapoport, C R Dokl Acad Sci USSR 54:65, 1946; 60:469, 1948).

• Milislav Demerec at Cold Spring Harbor, New York confirmed Auerbach’s work and was the first to show a rodent carcinogen, 1,2,5,6-dibenzanthracene, was a germ-cell mutagen in Drosophila (Demerec, Nature 159:604, 1947). Fallout from Fallout: Mutagenesis Comes to Oak Ridge Ionizing Radiation Causes Heritable Chromosomal Mutations in Mice

• Snell (1937) in the U.S. and Hertwig and Brenneke (1937) in Germany had provided the foundation for the establishment of the rodent dominant-lethal and heritable- translocation tests to detect heritable chromosomal effects of ionizing radiation.

• No methods were available to detect heritable gene mutations in mammals. Ionizing Radiation and Concern for Germ-Cell Mutation • The detonation of the atomic bombs in 1945 caused the U.S. government to establish the Biology Divison at the Oak Ridge National Laboratory in Tennessee in 1947 with Alexander Hollaender as the Director.

• Hollaender hired William (Bill) Russell to determine if ionizing radiation caused germ-cell mutations in mice because the government was unwilling to make public policy based on data from an insect. Ionizing Radiation Induces Germ-Cell Gene Mutations in Mice in 1951 • Bill Russell (CSHSQB 16:327, 1951) developed the mouse 7-locus specific locus assay and showed that ionizing radiation was a germ-cell mutagen in mice.

• With Liane Russell, Bill showed that fractionated doses and different dose rates gave first evidence of DNA repair in mammals (Russell et al., Science 128:1546, 1958). Radiation Risk Assessment

• Risk for germ-cell mutations by ionizing radiation in mice was extrapolated to humans in the 1950s (Neel & Lewis, Annu Rev Genet 24:327, 1990) and extended to chemicals and cancer risk assessment in the 1970s (Calabrese, James Neel Chemico-Biol Interact 308:110, 2019).

• Work by Mary Lyon (1966) at Harwell, UK with a 5-locus assay and Udo Ehling (1983) in Berlin added to these assessments. Mary Lyon Oak Ridge Scientists & Founders of EMS Fred de Serres

Fred de Serres designed a forward- mutation assay at the ad-3 locus in Neurospora to mimic Russell’s mouse specific-locus assay.

It was the first microbial assay that detected both gene & chromosomal (multi-locus) mutations and permitted one to infer the nature of Fred de Serres the mutation (base substitution, frameshift, or deletion) (de Serres & Kolmark, Nature 182:1249, 1958). Ernest H.Y. Chu

With Heinrich Malling, Ernie Chu developed one of the first gene-mutation assays in mammalian cells, the assay in V79 cells that detected BrdU- resistant mutants (Chu and Malling, PNAS 61:1306, 1968). Abraham W. Hsie

Abe Hsie developed the quantitative CHO/Hprt assay, which became a standard mammalian cell mutagenesis assay (Hsie et al., Somatic Cell Genet 1:383, 1975) Heinrich V. Malling • Improved the Neurospora assay • Co-invented the V79/BrdU assay. • Invented first tester set (Malling & de Serres (Mutat Res 6:181, 1968). • Invented enzymatic in vitro (S30) metabolic activation (Malling, Mutat Res 13:425, 1971). • Invented biochemical specific-locus assay (Malling & Valcovic,1977). • Invented transgenic mouse assay (Malling & Burkhart, Mutat Res 212:11, 1989). More Chemical Mutagens Discovered, Producing Increasing Concern for Environmental Germ-Cell Mutagens More Chemical Mutagens Discovered • Horowitz (1946) at Stanford found Edward mustard gas mutagenic in Tatum Neurospora. • Tatum (1947) at Yale found derivative of mustard gas was Milislav mutagenic in E. coli. Demerec • Demerec (1951) at Cold Spring Harbor found formaldehyde mutagenic in E. coli. Waclav • Szybalski (1958) at Rutgers found Szybalski 22/431 chemicals mutagenic in E. coli. • Freese (1968) at NIH found base Ernst analogues, alkylating agents, drugs Freese were mutagenic. Scientific Concern About Environmental Germ-Cell Mutagens

• Many untested chemicals being introduced into the environment after World War II. • Muller expressed concern for chemical germ- cell mutagens along with radiation. • Joshua Lederberg at U of Wisconsin wrote to Muller in 1950 to bring this concern to the National Academies of Science (NAS). • Lederberg raised this concern directly to the NAS in 1955. More Concern • Lederberg stressed concern at 1962 genetics conference. Joshua • Matthew Meselson at Lederberg Harvard asked James Crow at U of Wisconsin to organize an NIH genetics conference Matthew in 1966 to address this issue. Messelson • Crow published influential summary of the NIH conference (Crow, Scientist James Citizen, June-July 113, 1968). Crow Even More Concern • K. Luning (1966) in Stockholm screened 6 derivatives of phenobarbital for mutagenicity in Drosophila. • S. Epstein (1968) at Harvard screened 39 chemicals in mouse Sam Epstein dominant-lethal assay (aflatoxin, extracts of air and drinking water, food additives, pesticides and drugs). • M. Legator and S. Epstein (1969) proposed in the Mrak Report that pesticides should be tested for mutagenicity prior to approval. Marvin Legator The Environmental Movement

Oil Spill off coast of Santa Barbara 1969

Cuyahoga River on fire in Cleveland, Ohio 1969 Some Environmental Events of the 1960s • Thalidomide removed from U.S. market due to teratogenic effects in 1961. • London smog kills 750 in 1962. • Berne Accord to Protect Rhine River 1963. • Nuclear Test Ban Treaty (US/USSR) 1963. • Weather inversion in NYC kills 80 in 1964. • U.S. Senate hearings on leaded gasoline in 1966. • France begins nuclear testing in the Pacific 1966. • Oil spill of coast of Santa Barbara 1969. • U.S. Dept of Health Education & Welfare recommends genetic toxicity testing of pesticides in 1969. • EMS is founded, and men land on the moon in 1969. Popular Books and Articles

• A Sand County Almanac by Aldo Leopold (U Wisconsin) 1949 • Silent Spring by Rachel Carson (Maryland) 1962 Aldo Leopold Rachel Carson • The Population Bomb by Paul Ehrlich (Stanford) 1968 • The Tragedy of the Commons by Garrett Hardin (UC-Santa Paul Ehrlich Barbara) 1968 Garrett Hardin EMS Takes Off Pre-flight Planning Time-Line • Summer 1967: Hollaender retired from Oak Ridge and spent that summer touring European labs convincing people of the importance of chemical mutagenesis. • April 1968: Hollaender organized an “Informal Discussion” on mutagenesis at Oak Ridge with his Oak Ridge colleagues. • July 1968: Crow published his summary of 1966 NIH meeting on mutagenesis, which had 4 recommendations, 3 of which Hollaender adopted for EMS. Pre-flight Planning Cont. • September 1968: Hollaender organized a “Roundtable on Mutagenesis” in Gaithersburg, Maryland for 40 mostly U.S. scientists to discuss the 4 recommendations. • This group agreed there should be a society, and mailed a questionnaire to ~100 mostly U.S. scientists to see if they thought so too. • January 8, 1969: Hollaender, de Serres, Malling, Legator, Epstein and Ernst Freese met in NYC, and saw that the responses to the questionnaire were in favor of forming a society, and they all agreed and named it EMS. The Flight Plan • The group elected Hollaender president, Epstein secretary, Legator treasurer, and council was de Serres, Freese, Malling, Crow, and Bruce Ames. • August 4, 1969: First council meeting at NAS; all 5 requests of Hollaender from January had been started: a literature database, a newsletter, a book series on methods, a proposal written for funding a conference, and organizing started for first meeting. Liftoff • October 7, 1969: Scientists in Germany had a symposium on chemical mutagenesis (F. Vogel, I- D Adler), and Legator, Malling, de Serres, Chu, and Epstein also attended. • March 22-25, 1970: First EMS meeting in Washington, DC. 268 attendees, 452 members (64 from Europe). • Symposia were on: testing, mutational mechanisms, population monitoring, and nitroso compounds and cyclamates. • Registration $10, $2 for wife; Jacqueline Verrett (US FDA) was only invited female scientist. H. Bentley Glass Gave Opening Talk

• Glass gave the opening talk at the first EMS meeting. • Student of Muller, VP of State U of New York at Stony Brook. • Leading public intellectual of his day. • FBI followed him, he was a president of the Maryland chapter of the ACLU, and he fought nuclear proliferation, racism, and loyalty oaths. Early Successes • Environmental Mutagen Information Center (EMIC): first John Wassom electronically accessible, curated, database (Malling & John Wassom); predated PubMed by 30 years. John Drake • Committee 17 Report Environmental Mutagenic Hazards (Drake, Science 187:503, 1975) • Helped establish and implement Mike Waters Toxic Substances Control Act (TSCA) in 1976 by Gene-Tox Program at EPA (M. Waters) More Successes • EMS Newsletter (1969) (de Serres) • Chemical Mutagens: Principles and Methods for Their Detection (1971- Bruce Ames 1986) (Hollaender & de Serres). Ames introduced his assay in the first volume. • Environmental Mutagenesis (1979) (S. Abrahamson) Seymore Abrahamson • Hollaender started training courses in Latin America; he died in 1986, and Patricia Ostrosky (Mexico) re- initiated the Hollaender Courses in 1993 Patricia Ostrosky Legacy of Action without Activism • The EMS was established firmly in science and not activism, giving it credibility without bias. • It was one of the first multi-disciplinary societies at the time. • It accomplished all of its initial goals except for one: identifying human germ-cell mutagens. • Advances in genomics caused the Society to rename itself the Environmental Mutagenesis and Genomics Society (EMGS) The Mission is Not Complete

• Genomic methods need to be applied to determine whether there are human germ-cell mutagens. • Epigenetic effects on the germline from environmental exposures need to be determined. • Better test methods need to be developed. • Improved biomarkers are needed to assess genetic effects of environmental exposures. • Improved legislation is needed to protect the environment and populations from environmental exposures to mutagens and epigenetic agents. Our Fragile Blue Marble Home

Earthrise photo taken by Apollo 8 astronaut Bill Anders in December 1968. We owe a big debt of gratitude to these scientists and may others whose work and efforts helped to found the EMS Acknowledgments • Errol Zeiger, Phil Hanawalt, Mike Waters, George Hoffmann, and many others for their comments. • More than 70 EMS/EMGS members, especially John Wassom and Scott Frickel, whose writings on the history of the Society were essential to construct this talk. • My professor, Herman Brockman, who brought me into EMS in 1974. • The members of EMS, who granted to me the privilege of being president of EMS 2001-2002.