News Feature
Ames test success paves way for short-term cancer testing Acceptance of Ames' approach has set the stage for development of new and ingenious techniques that, used together, may provide accurate information on the possible hazards of chemicals Jeffrey L. Fox C&EN, Washington "It doesn't mean that we'll ban all mutagens and carcinogens. We can learn to live with these chemicals, but must treat them with respect. But when there is human exposure, the chemical industry will need to come to terms with biology. Those that realize and do it will survive, and those that won't will not survive."—Bruce N. Ames, University of California, Berkeley Seventeenth century English philosopher Thomas Hobbes, in his political treatise Leviathan, reminded his contemporaries that their environment could be hostile. Twentieth century scientists have wrested a share of warning duties from the philosophers, and offer frequent reminders of how the small fish in the environment can come in hostile schools. Low concentrations of many environmental factors, they warn, can cause cancer. One effort to fend off that threat is to seek cancer-causing agents and limit (or eliminate) human exposure to them. But to do that, carcinogens must be fished out from the vast pool of chemicals, most of which are innocuous. Though not yet easy, that enormous task is. beginning to look possible. There are new ways to plumb the murky environs where chemicals are often found. Several dozen tests are being developed, the most conspicuous being that devised by Dr. Bruce N. Ames and his colleagues at the University of California, Berkeley. This test, which uses mutants of the bacterium Salmonella typhimurium, offers a quick way to look for chemicals that cause mutations (changes in genes). Many such chemicals, called mutagens, also cause cancer in animals and are thus carcinogens. In fact, nearly 90% of chemicals that have tested positive in the Ames test are carcinogens. Hence, the Ames test is a good net for a first cast among suspect chemicals. Other short-term tests under development are like nets but with different 34
C&EN Dec. 12, 1977
patterns of meshing. So far, they haven't been checked so thoroughly as has the Ames test, and most are neither so reliable nor so simple to do. But the most promising already are in limited use by industry and under consideration by several federal regulatory agencies. Estimates of the validity of these tests vary widely. Yet some companies are including them in recently launched in-house testing programs, whereas still others want little to do with them. Regardless of any particular company's stance on short-term testing, a fear threads its way through industry. That fear is spinning out of Washington, D.C., where several regulatory agencies already have drafted guidelines specifying potential applications of testing programs—for example, to pesticides. Most companies regard such guidelines as premature, saying in effect that regulations ought not to be written before the tests are better validated. To appreciate the scope of this matter and the potential burden it entails, it's best to look at several of the short-term tests. All the candidates, from the simplest bacterial screening to the sophisticated tests with cultured animal cells, offer enticing benefits. For one, they're fast, giving results within weeks or even days. For another, they're relatively cheap, costing from several hundred to several thousand dollars.
That's in sharp contrast to the standard tests for carcinogens that expose small mammals such as rats and mice to suspect chemicals. This long-term testing, which usually extends over the animal's twoyear life, costs plenty (more than $200,000) and sops up highly trained personnel. However, animal tests are considered more definitive than shortterm tests. An important problem with animal tests is that weak carcinogens can be missed because the tests don't include sufficient numbers of animals. This is particularly true when high doses of the substance under test are acutely toxic, and it can be administered only in low doses. "Most animal cancer studies are models of nonthoroughness," Ames asserts. Thus short-term tests with their lower costs and fewer variables can complement the cumbersome long-term animal tests. Of the several most promising shortterm approaches, the Ames Salmonella test is the best known and most widely used. Ames says that he began developing the test in 1964 as sort of a hobby. Then, his interest was strictly in looking for mutagens. "I was curious what was out there in the modern chemical world that might be mutating people," he says. "It was years before I thought about using the test for picking up carcinogens." Since then, Ames and colleagues have put together a highly sensitive test that
Without adding mutagen, few Salmonella colonies form in Ames test (left). But one microgram of furyIfuiamide boosts the number considerably (right).
forms a γ-carboline derivative 3-aminol,4-dimethyl-5-H-pyrido[4,3-b]indole, called trp-p-1
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Ames: short-term tests are agreeing checks a chemical's ability to change the genetic material of several bacterial strains. This ability correlates well with carcinogenicity for the several hundred known carcinogens that have been tested. For some classes of compounds tested, that correlation is as high as about 90%. "It's never going to be 100%," Ames says. "Some carcinogens simply don't work by causing mutations, such as diethylstilbestrol, or will cause mutations in eucaryotes but not in bacteria. Even if you had an absolutely perfect test, you would be wrong 10% of the time because of the problems of animal cancer tests," he adds. The case of the Japanese food additive, furylfuramide, demonstrates the fallibil ity of animal tests. Tested against rats and mice, the compound seemed safe and was used widely for about a decade starting in 1965. But in 1972 Japanese scientists ex amined the compound for mutagenicity in bacteria and found that it is extremely potent. Its use in food has since been curtailed, and further animal testing verified that it is also carcinogenic. Besides complementing animal cancer tests, the Ames test is a versatile tool for screening complex mixes. Dr. Takashi Sugimura, head of the National Cancer Center Research Institute of Tokyo, in a recent interview with C&EN related a fascinating example of the Ames test as a bioassay. Sugimura and colleagues tracked down mutagenic ingredients in broiled, and particularly charred, meat. They find that the amino acid tryptophan
that is more potent as a mutagen than one CH3 of the most potent carcinogens known, aflatoxin B. By using the Ames test as a bioassay, Sugimura's group also finds that several other amino acid derivatives, which form during heating, are mutagenic. This in formation is especially important in Japan where stomach cancer is a leading killer. Because Japanese who emigrate have a lower incidence of this cancer, diet and food preparation are suspected causes of the disease. Sugimura sees the Ames test as a very useful way of delving into this question. "We need very urgently to deal with many compounds," he says. Short-term tests offer that possibility. None of the other short-term tests is developed so extensively as the Ames test. But several provide advantages, or po tential advantages, that attract the at tention of toxicologists in industry and regulators in government. And several tests used together might make a finer net than just one. Ames notes that a "lot of short-term tests are agreeing with one another so that several positives are really meaningful." This fall, for instance, the Office of Technology Assessment released a study entitled "Cancer Testing Technology and Saccharin." The study tackled two knotty problems simultaneously: Is saccharin a hazard?, and, how well do short-term tests match up with one another and to data from animal tests? To the first question, the report responds that saccharin is an extremely weak carcinogen. The answer to the second question emphasizes both the immaturity and the potential of short-term tests. Dr. Joyce C. McCann, a colleague of Ames at Berkeley, coordinated the short-term testing of saccharin in a dozen of the more promising tests. McCann notes that the list is neither exhaustive nor ideal, but that time constraints kept the project from growing any larger. In the 10 tests so far completed, saccharin tested weakly positive in three—two tests in volving mammalian chromosomes and one involving mammalian mutations. It tested negative in the three microbial tests included in the battery. Among the 12 tests, a few such as un scheduled DNA synthesis, sister chro matid exchange (SCE), mammalian cell mutagenesis, and mammalian cell trans formation are being used more widely and have been tested with more chemicals than have most of the other methods. For example, she refers to unscheduled DNA synthesis currently as "one of the
better tests." It's being developed by Dr. Hans Stich of the University of British Columbia, Vancouver. Human fibro blasts, a kind of skin cell, grown in culture are subjected to chemicals. He examines the amount of DNA damage and subse quent repair that takes place. This is fol lowed by adding radioactive DNA pre cursors that are taken up into the cells' chromosomes after DNA repair enzymes go into action. The amount of radioac tivity in the chromosomes is measured by autoradiography (exposing photographic film to radioactive emission). Saccharin was not active in this test. But the test does pick up many other carcinogens. Though the test is relatively easy to do, the film exposure step slows down an otherwise rapid procedure. Sister chromatid exchange, in which saccharin tested positive, entails looking at changes in chromosomal structure within mammalian cells. The test is quick and seems very sensitive to low doses of carcinogens and mutagens. Dr. Sheldon Wolff of the University of California, San Francisco, says that so far the test corre lates well with other short-term tests. Though the test requires scoring chro mosomes by microscopy—a potential slowdown in mass screening—statistically significant results are obtained when as few as 20 cells are counted, Wolff says. That's a marked improvement over other chromosomal aberration tests, he adds. Sister chromatid exchange might prove versatile enough to screen for chronic exposure to mutagens among humans. For example, human lymphocytes (a cell type) can be removed from blood and cultured in preparation for the special staining technique of this test. The cells then are exposed to a suspected carcino gen. SCE also can be observed in cells from rabbits that have been exposed to particular chemicals. If rabbits show ele-
Sugimura: deal with many compounds Dec. 12, 1977 C&EN
35
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SAC = saccharin TCE = trich loroethy lene MET= metronidazole MMS = methyl methanesulfonate NA = β-naphthylamine SAF = safrole MOCA = 4,4'-methy!ene-bis-2chloroaniline CCI 4 = carbon tetrachloride UR = urethane
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BZD = benzidene TRIS = tris (2,3-dibromopropyl) phosphate PS = propane sultone NP = nitrosopyrrolidine 2AAF=2-acetylaminofluorene DBCP = dibromochloropropane PI = propylenimine DMN = dimethylnitrosamine 3MCA=3-methylcholanthrene STRG = sterigmatocystin AFBI = aflatoxinB1
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a To give 50% animals cancer in lifetime. Note: Preliminary analysis of data extracted from literature shows that chemicals vary over a million-fokJ range in carcinogenic potency. Location of each carcinogen is subject to change as calculations are refined, according to Ames. Source: Office of Technology Assessment report, "Cancer Testing Technology and Saccharin"
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vated SCE's after acute exposures to carcinogens, Wolff reasons, it's plausible that chronically exposed humans will show similar changes. Two other tests in the saccharin battery resemble the Ames test in principle, but take mutation from the realm of bacteria into that of mammalian cells. Only results from the test that uses mouse lymphoma cells are available. This test, which was conducted by Dr. Donald Clive of Bur roughs Wellcome Co., Research Triangle Park, N.C., subjects cells growing in cul ture to a particular DNA inhibitor that can kill them. Adding mutagens can alter the cells' DNA, rendering the cells resis tant to the inhibitor and thus able to grow in its presence. The number of survivors is a measure of the mutagenicity of the chemical. Saccharin is weakly active in this test, and only at doses that are toxic to the cells. This belies a general sensitivity problem with this test. Many substances fail to test positive until doses are raised to toxic or near toxic levels, according to the OTA report. Mammalian cell transformation tests, though more complicated than many of the other short-term tests, also are more attractive on theoretical grounds. Transformation involves a heritable change in appearance and behavior of animal cells that are growing in tissue culture. Ordinarily, such cells grow in an orderly fashion, and cease growing when they contact one another. But, if trans formed, growth is disordered and cells pile on one another. Moreover, when such cells are removed from the culture medium and injected into appropriate animals (usually animals like the one from which 36 C&EN Dec. 12, 1977
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