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About 300 carcinogens and non-carcinogens of a wide variety of chemical types have been tested for mu- There is considerable evidence that a large proportion of human cancer may be caused by exposure to toxic chemicals in the environment, very few of which have been tested for carcinogenicity or mutagenicity. A program of cancer prevention aimed at identifying and eliminating human exposure to hazardous chemicals requires the development of rapid, inexpensive, screening methods as complements to expensive, long-term animal tests, to pinpoint dangerous chemicals among the thousands to which humans are exposed. The Salmonella/microsome mutagenicity test (1-4) has been sufficiently developed and validated to be seriously considered for widespread use in this way. The considerable evidence (5), much of it obtained using this test (1)(2)(3)(4)(6)(7)(8)(9)(10)(11)(12)(13), that with few exceptions carcinogens are mutagens, supports the desirability of using this type of rapid and economical test system as a screening technique (1-3).Chemicals are tested for mutagenicity on petri plates with several specially constructed mutants of Salmonella typhimurium (2-4). Homogenates of rat (or human) liver, (S-9 Mix), are added directly to the petri plates, thus incorporating an important aspect of mammalian metabolism into the in vitro test (2). In this way, a wide variety of carcinogens requiring metabolic activation can be detected easily as mutagens (2, 4, 5). The system has been recently reviewed (6) and the test method described in detail (7).The present paper presents mutagenicity data on a large number of carcinogens and non-carcinogens of many different classes that have been examined in the system using a standard methodology (7) All non-mutagens, and most mutagens, have been tested, using the recently improved standard methodology (7), on the new R factor tester strains (4) as well as the earlier standard tester strains (3). Non-mutagens have been tested over a wide dose range both with and without the liver microsome activating system.In addition to our previously published studies and new work presented here, results have been contributed to this compilation by a number of laboratories using this test, including a large contribution from Japan. Some of the chemicals tested are also specified in a contract sponsored by the National Cancer Institute (V. Simmon and H. Rosenkranz, to be published). Some of the non-carcinogens we tested were specified in a contract sponsored by the Environmental Protection Agency to B. Commoner.We have not reported on any metal carcinogens, though three or four that have been tested are negative in the standard test. The test system is not suitable for metals entering the bacteria because of the large amount of Mg salts, citrate, and phosphate in the minimal medium. A number of carcinogenic metals have been shown to be mutagens in bacteria by means of a different methodology, e.g. ref.14.In addition to the compounds presented we have tested 46 common biochemicals that are non-ca...

ABSTRACT18 Carcinogens, including aflatoxin Bi, benzo(a)pyrene, acetylaminofluorene, benzidine, and dimethylamino-trans-stilbene, are shown to be activated by liver homogenates to form potent frameshift mutagens. We believe that these carcinogens have in common a ring system sufficiently planar for a stacking interaction with DNA base pairs and a part of the molecule capable of being metabolized to a reactive group: these structural features are discussed in terms of the theory of frameshift mutagenesis. We propose that these carcinogens, and many others that are mutagens, cause cancer by somatic mutation. A simple, inexpensive, and extremely sensitive test for detection of carcinogens as mutagens is described. It consists of the use of a rat or human liver homogenate for carcinogen activation (thus supplying mammalian metabolism) and a set of Salmonella histidine mutants for mutagen detection. The homogenate, bacteria, and a TPNHgenerating system are all incubated together on a petri plate. With the most active compounds, as little as a few nanograms can be detected.We have previously described the use of a set of mutants of Salmonella typhimurium for detecting and classifying chemical mutagens with great simplicity and sensitivity (1, 2). With this test we have also shown that the active forms of a large number of known carcinogens are mutagens (1-5). The active forms of carcinogens such as aflatoxin, polycyclic hydrocarbons, dimethylnitrosamine, and various aromatic amines are formed by mammalian metabolism, in particular by the TPNH-dependent microsomal enzymes of liver (6-11). The principal limitation of any bacterial system for detecting carcinogens as mutagens is that bacteria do not duplicate mammalian metabolism in activating carcinogens. Mammalian-liver homogenates have been used by Garner et al., (6) to activate aflatoxin B1 to a compound lethal to our bacterial tester strain lacking excision repair, by Malling (12) to activate dimethylnitrosamine to a compound that reverts one of our bacterial tester strains, and by Slater et al. (13) to activate dimethylnitrosamine to a compound lethal for bacteria lacking polymerase I. In this study we have extended this work and shown that carcinogens can be detected as mutagens simply and with great sensitivity by incubation of the carcinogen, a rat or human liver homogenate, and our bacterial tester strain together on a petri plate. MATERIALS AND METHODSCompounds. Glucose-6-phosphate, TPN, TPNH, and 2-naphthylamine were obtained from Sigma. Benzo(a)pyrene, 2-acetylaminofluorene, and benzidine were from Aldrich. DiAbbreviation: Me2SO, dimethylsulfoxide. methylsulfoxide (Me2SO), spectrophotometric grade, was obtained from Schwarz/Mann, sodium phenobarbital from Mallinckrodt, aflatoxin B1 from Calbiochem, and 3-methylcholanthrene from Eastman; 7,12-dimethylbenz(a)anthracene was a gift of P. L. Grover. Schuchardt (Munich) was the source for the other carcinogens.Bacterial Strains used are mutants of S. typhimurium LT-2 and have been discussed in detail (2).Sourc...

A method is described for concentrating mutagens/carcinogens from human urine about 200-fold for subsequent assay in the Salmonella/mammalian microsome mutagenicity test. The method is also applicable for other aqueous liquids and for other in vitro tests for mutagens/carcinogens. The urine (up to 500 ml) is put through a column with a 1.5-cm3 bed volume of XAD-2 (styrene-divinylbenzene polymer) and the adsorbed material is then eluted with a few milliliters of acetone. The acetone is taken to dryness and the residue is dissolved in dimethyl sulfoxide. This is the urine concentrate that is assayed for mutagenicity. Various mutagens/carcinogens have been added to human urine and the recoveries have been measured after adsorption on XAD-2, XAI4, and Tenax GC (diphenyl-p-phenylene oxide polymer). We propose that this method be used in monitoring the urine of human populations and of experimental animals in toxicological studies.It is shown with this procedure that cigarette smokers have mutagenic urine while nonsmokers do not.Humans are being exposed to a wide variety of environmental chemicals that are mutagens/carcinogens (1-4). A number of rapid in vitro systems for detecting these chemicals have been developed (1), such as the Salmonella/mammalian microsome mutagenicity test (5). The Salmonella test is about 90% accurate in detecting a wide variety of carcinogens as mutagens (6-9). Chemical mutagens enter people through the diet, or in other ways, and are often excreted unchanged, as conjugates, or as other metabolites in the urine. In order to demonstrate exposure to mutagens/carcinogens it is of particular interest to have a rapid way of examining urine of large numbers of people.Testing urine directly in the Salmonella system has been done (10-14), but only a small amount of urine can be added to the test system because of volume limitations and interfering histidine (10), and therefore only mutagens present in high concentration or those of exceptional potency can be detected. We (10) and others (11) have used solvent extraction techniques for concentrating mutagens from urine, but because of the inconvenience of solvent extraction and methods such as lyophilization and subsequent selective extraction, we have explored resin adsorption techniques. Various nonpolar resins such as XAD-2, XAD-4, and Tenax GC have been used for adsorbing drugs and their metabolites from urine (15-18), and organic compounds from water (19-24) and from sea water (25-27).MATERIALS AND METHODS Materials. XAD-2 and XAD-4 resins were washed by swirling and decanting several times with 10 volumes of acetone followed by absolute methanol and distilled water, and then stored in water at 4°. Glass Econo columns, 0.7 cm (inside diameter) X 10 cm (Bio-Rad, Richmond, CA), were filled with distilled water before addition of sufficient washed resin (0.7 g dry weight) to give a bed height of 4 cm (1.5-cm3 bed volume). Flow was regulated with a 3-way nylon stopcock (American Hospital Supply). Distilled water (about 50 ml) was passed th...

We have previously described a sensitive bacterial test for detecting carcinogens as mutagens. We show here that 89% (150/169) The present work stems from a biochemistry class experiment in which hundreds of commercial products were tested for mutagenicity. Only two products were found to be mutagenic: cigarette smoke tar (8) and an oxidative-type hair dye.Since 20,000,000 people (10), mainly women, dye their hair in the U.S. we have made a detailed study of hair dye mutagenicity. 2423The two main types of hair dyes are the "semi-permanent" or direct color dyes and the "permanent" or oxidative-tN pe dyes in which H202 is used to oxidize aromatic diamines with the production of larger colored molecules which are trapped in the hair shaft. We have concentrated on the oxidativetype dyes since they account for about 75% (11) of the $250,000,000/year hair dye market. The oxidative dye package consists of one bottle containing a mixture of aromatic amines, aromatic nitro derivatives, and phenols in a vehicle liquid (12,13), and a bottle of H202 with which it is mixed immediately before use. RESULTSWe first tested about 30 oxidative-type hair (lye fz cmulations for reversion of our standard tester strains. Almost all of these were mutagenic when tested on strain TA1538, indicating the production of frameshift-type mutations. Strain TA1535 was not reverted, indicating a lack of base-pair substitution mutations; the other frameshift tester strain, TA1537, with a different specificity also was not reverted. We have, therefore, used strain TA1538 to test 169 different oxidative-type dye formulations; these were all the products we could find in two local drug stores. Each hair dye preparation has been tested both before and after mixing with H202.