1. What bacteria were used in the Ames test?
2. List 3 properties of these bacteria that are important for the test.
3. What is S-9 and what is its role in the test?
4. What is meant by an "activated" carcinogen?
5. What attributes of the chemicals tested in Table 1 make them good carcinogens (the answer is not that they are mutagenic, which they are).
-Proc. Nat. Acad. Sci. USA Vol. 70, No. 8, pp. 2281-2285, August 1973 Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection (frameshift mutagens/aflatoxin/benzo(a)pyrene/acetylaminofluorene) BRUCE N. AMES, WILLIAM E. DURSTON, EDITH YAMASAKI, AND FRANK D. LEE Biochemistry Department, University of California, Berkeley, Calif. 94720 Contributed by Bruce N. Ames, May 14, 1973 ABSTRACT 18 Carcinogens, including aflatoxin Bi, benzo(a)pyrene, acetylaminofluorene, benzidine, and di- methylamino-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 muta- genesis. We propose that these carcinogens, and many others that are mutagens, cause cancer by somatic muta- tion. A simple, inexpensive, and extremely sensitive test for detection of carcinogens as mutagens is described. It con- sists of the use of a rat or human liver homogenate for carcinogen activation (thus supplying mammalian metab- olism) and a set ofSalmonella histidine mutants for muta- gen detection. The homogenate, bacteria, and a TPNH- generating 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 hydrocar- bons, 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. Mam- malian-liver homogenates have been used by Garner et al., (6) to activate aflatoxin B1 to a compound lethal to our bac- terial 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 mu- tagens simply and with great sensitivity by incubation of the carcinogen, a rat or human liver homogenate, and our bac- terial tester strain together on a petri plate. MATERIALS AND METHODS Compounds. Glucose-6-phosphate, TPN, TPNH, and 2- naphthylamine were obtained from Sigma. Benzo(a)pyrene, 2-acetylaminofluorene, and benzidine were from Aldrich. Di- Abbreviation: Me2SO, dimethylsulfoxide. methylsulfoxide (Me2SO), spectrophotometric grade, was ob- tained from Schwarz/Mann, sodium phenobarbital from Mallinckrodt, aflatoxin B1 from Calbiochem, and 3-methyl- cholanthrene 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). Source of Liver. Male rats (Sprague-Dawley/Bio-1 strain, Horton Animal Laboratories) were maintained on Purina laboratory chow. A week before they were killed, their drinking water was made 0.1% in sodium phenobarbital (14). The rats (250-500 g) were killed by a blow to the head and cervical dislocation; the liver was removed and placed in a sterile, ice-cold beaker. A portion of human liver was obtained from an autopsy of a 77-year-old man who had died 7 hr earlier of heart failure. Preparation of Liver Homogenate Fraction "S-9". We have used the procedure of Garner et al. (6). All steps were per- formed at 0-4o with cold and sterile solutions and glassware. The liver (rat livers were 10-25 g each) was washed in an equal volume of 0.15 M KCl, minced with sterile scissors in three volumes of 0.15 M KCl (3 ml/g of wet liver), and homogenized with a Potter-Elvehjem apparatus with a Teflon pestle. The homogenate was centrifuged (Sorvall RC2-B) for 10 min at 9000 X g, and the supernatant, which we call the S-9 fraction, was decanted and saved. 1 ml of S-9 fraction contained microsomes from 250 mg of wet liver; the protein concentrations were fairly constant from preparation to preparation except for the human S-9 fraction which was about half, perhaps due to difficulties in homogenization be- cause of its fibrous nature. The fresh S-9 fractions (rat and human) were distributed in 2-ml portions in small plastic tubes (2-ml liquid nitrogen storage tubes/4-Shore-USA, La Jolla, Calif.), quickly frozen in dry ice, and stored at -80° in a Revco freezer. As required, sufficient S-9 fraction was thawed (at room temperature) and kept in ice; the unused portion was discarded at the end of the day. Mutagenesis Test wvith the S-9 Fraction. The method without the liver activation system has been described in detail (2). The only modification is the addition of S-9 Mix to the top agar. The S-9 Mix contains per ml: 0.3 ml of S-9 fraction, 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM TPN, and 100 mM sodium phosphate (pH 7.4). To 2 ml of 2281 Fatma Alqadi Fatma Alqadi Fatma Alqadi Fatma Alqadi Fatma Alqadi Fatma Alqadi Fatma Alqadi Fatma Alqadi Fatma Alqadi Fatma Alqadi 2282 Genetics: Ames et al. Proc. Nat. Acad. Sci. USA 70 (1973) Carcinogen NH2 2-minoant NH2 2-ainoflu4 ro LY ITNHCOCs 2-acetylm fluorene H 2N H~~~N"2 benzidine &.&NH2 4-aminobip ~ r 4-amino-tr C-C.. stilbene T ~4-dimethyl@ Ad ~~~~trans-St ,0NH2 p-(phenyls (.,NmN aniline 4- (o-tolyl @[NsN CH3 toluidir XaXN(CH3) N,N-dimet} ft NdN p- (r- toI aniline "H2 2-naphthyl JH2 ro[Ino l-aminopyx NH2 6-aminochi benzo(a)p 3 3-methyl-cholantl 7.12-dimebenz (a). ,11"In, aflatoxin OCH Q1 steriguab OCH3 118 S-9 thracene 20 20 - 0 + orene 10 10 - 0 + mino- 50 so 0 + 50 50 - O + phenyl 100 100 _ 0 + rone- 10 + 10 0 lamino- 10 tilbene 10 0 + azo)- 100 + 100 - 0O ,lazo)-o- 10 + ,no 10 0+ Ayl- 100 + lylazo)- 100 I 0 + lamine 100 + 100 0 + rrene 10 + 10 - 0. + TABLE 1. Activation of carcinogens to mutagens rnj~zai~aewrwi per pzaHisi~fne Revertnte per powt TA1S35 TA1536 TA1537 TA1S38 318 11 180 11200 T 27 16 3 12 46 77 2 121 11300 21 0 iT 39 77 0 12 29 117 2 92 13600 86 0 11 21 118 3 12 46 34 1 29 265 38 0 10 38 0 22 36 143 4 108 980 92 2 a 119 3 12 46 23 842 7 17 10 42 57 3 25 896 68 0 6 28 97 1 12 S3 31 3 8 94 15 0 12 13 45 2 18 31 71 1 20 305 94 1 8 T7 97 1 12 29 330 16 41 66 42 2 34 0 8 2 18 1 136 0 23 1 9 rysene 1 + 18 6 127 1 - 30 0 3 0 + 14 2 18 )yrene 5 +' 46 4 148 - 77 1 16 0 + 48 1 28 50 + 22 S 88 threne so - 13 0 2 0 + 11 0 9 ethyl- 50 + 25 1 225 )anthracene 50 - 35 0 is 0 + 29 1 11 BI 1 +t 35 0 36 1 - 23 3 5 0 + 42 0 6 totystin* 0.1 .t 13 3 32 0.1 - 30 1 18 0 + 5 0 13 147 31 81 21 398 59 29 638 30 46 505 44 110 21 27 88 20 36 266 26 26 121 8 32 Revertant colonies (his +) on each plate were scored after 2 days. Numbers underlined are judged to be significantly different from the controls. Rat-liver homogenate (8-9 Mix) was added to the plates, where indicated. Human S-9 fraction gave qualitatively similar results when 2-aminoanthracene, the fluorene derivatives, 4-aminobiphenyl, 6-aminochrysene, and aflatoxin B1 were assayed with TA1538. The amount chosen for each carcinogen was the one that gave maximum mutagenesis of several amounts tried (see Fig. 1). All of the compounds were added from solutions in Me2SO usually 1 mg/ml; the control, lacking compound, had an equiva- lent addition of Me2SO. * The 8-9 preparation was from a 200-g rat that was injected intraperitoneally, 24 hr before it was killed, with 16 mg of 3-meth- ylcholanthrene dissolved in corn oil. t One-third the normal amount of 8-9 fraction was used in the 9-9 Mix. t We are indebted to Dennis Hsieh for a sample and for suggest- ing we test sterigmatocystin. molten top agar at 450 are added 0.1 ml of the bacterial tester strain culture (2 to 3 X 109/ml), up to 0.1 ml of a solution (Me2SO or water) of the compound to be tested, and 0.5 ml of 8-9 Mix; then the tube is rotated quickly and the contents are poured on the agar plate. The additions and pouring should take less than a minute. The colonies on the plates (his+ revertants) are counted after a 2-day incubation at 37°. RESULTS Combined System for Carcinogen Activation and Bacterial Mutagenesi8. We use the TPNH-dependent microsomal-en- zyme systems in liver homogenate to activate carcinogens. The activated carcinogens are detected as mutagens. The test assays, on a petri plate, the number of revertant colonies induced by mutagens in the set of histidine-requiring mutants. Two technical improvements greatly simplify the use of the combined bacterial and liver system. The TPNH-generating system and liver homogenate traction (S-9 Mix) can be in- cubated directly on the petri plate along with the compound to be tested and the bacterial tester strain. Both rat and human liver preparations can be frozen for many months without loss of activity. Carcinogens Detected as Mutagens. Table 1 shows that rat- liver homogenates can activate 18 different aromatic type carcinogens to mutagens. The compounds were chosen for testing because they were known to be carcinogenic in hu- mans or in animals (7, 15). Control values are presented both for the number of revertant colonies on plates with compound and no S-9 Mix, and for the number of colonies on plates A B ~15-N *10 /~~~~~~~~~~~ C0 100 200 0 20 40 Liver Homogenate Per Assay (pil of S-9) Ad g of Compound Fig. 1. Effect of 8-9 fraction and carcinogen concentration on mutagenesis of TA1538. (A) The procedure was as described in Methods except that the amount of S-9 fraction in the Mix was var- ied. As the human 8-9 had half the protein of the rat S-9, the re- sults with the human material are plotted at half the 8-9 amount actually used. 4-Aminobiphenyl (100 Isg per assay) had about 0.1 the activity of