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...
We previously described a set of four strains of Salmonella typhimurium designed for detecting the various types of mutagens, and showed their utility in detecting a wide variety of carcinogens as mutagens. The lipopolysaccharide that normally coats these bacteria is a barrier to penetration of mutagens to the cell membrane. The set of tester strains has been improved by adding a mutation ( rfa: deep rough) that results in a deficient lipopolysaccharide. The techniques for using these strains for detecting mutagens are presented and the tests are shown to be extremely sensitive and convenient. The specificity of frameshift mutagenesis is clarified. As adjuncts to the test with the four strains, we describe a test that compares mutagenic killing in deep rough strains with and without DNA excision repair, and a test using forward mutagenesis in a deep rough strain lacking excision repair.
We described previously a simple test on petri plates for detecting many carcinogens as mutagens using an especially sensitive set of bacterial strains to detect mutagens and a rat, or human, liver homogenate for carcinogen activation. We now extend the utility of the method for the detection of mutagenic metabolites in urine. The addition of commercial 6-glucuronidase (EC 3.2.1.31) to the petri plates along with the urine, liver homogenate, and bacteria allows detection of metabolites that are excreted in urine as jB-glucuronide conjugates.By this method mutagenic activity is readily demonstrated with urine of rats that were administered as little as 200 jug (1.6 mg/kg) of the carcinogen, 2-acetylaminofluorene.In this case the major urinary metabolite that we detect appears to be a glucuronide conjugate. We propose that the method be used for the screening of human urines in order to detect mutagenic metabolites of drugs and of dietary components. It may also be useful for testing of urinary metabolites of drugs and food additives in experimental animals.We have previously described a very sensitive and simple bacterial test system for detecting chemical mutagens (1-4). The compounds are tested on petri plates with specially constructed mutants of Salmonella typimurium as tester strains. Four tester strains have been selected for sensitivity and specificity in being reverted from a histidine requirement back to prototrophy by a variety of mutagens. One strain can be used to detect mutagens causing base-pair substitutions and three to detect various kinds of frameshift mutagens. In addition to the histidine mutation, each tester strain has two additional mutations that greatly increase their sensitivity to mutagens: one causes loss of the excision repair system and the other loss of the lipopolysaccharide barrier that coats the surface of the bacteria. We have shown that by adding a microsomal activation system of rat (or human) liver to the petri plates a wide variety of carcinogens can be activated to mutagens and thus detected easily (3). Thus, an important aspect of mammalian metabolism can be duplicated in an in vitro test. The present study examines the testing of urine in the combined bacterial/liver system. A wide variety of metabolites of drugs and other ingested compounds appear in the urine. Since many metabolites are excreted in urine as glucuronides, we added fl-glucuronidase (EC 3.2.1.31) to our test plates. The method is demonstrated by administering the carcinogen, 2-acetylaminofluorene (AcAF), to rats and detecting mutagenic metabolites in the urine.AcAF, like many other compounds, gives rise to a large number of metabolites (5-li). The oxidation products Nhydro.xy-2-acetylaminofluorene (N-OH-AcAF), N-hydroxy-2-aminofluorene (N-OH-AF), and 2-nitrosofluorene (NOF), are thought to be proximate or ultimate carcinogens formed from AcAF and 2-aminofluorene (AF) by the microsomal systems of liver (5). They are more carcinogenic than the parent compounds at the site of application in rodents an...
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