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The eg oes A Bacterial Test System for: Chemical Carcinogenicity LEARNING OBJECTIVES Once you have completed this experiment, you should be able to 1. Screen for potential chemical carcinogens using a bacterial test system. Principle Our exposure to a wide variety of chemical com- pounds has increased markedly over the past decades. Oncological epidemiologists strongly suspect that the intrusion of these chemicals in the form of industrial pollutants, pesticides, food addi- tives, hair dyes, cigarette smoke, and the like may play a significant role in the induction of malignant transformations in humans. From a genetic aspect there is strong evidence linking carcinogenicity to mutagenicity. Research indicates that approxi- mately 90% of the chemicals proved to be carcino- gens are mutagens; they cause cancer by inducing mutations in somatic cells. These mutations are most frequently a result of base substitutions, the substitution of one base for another in the DNA molecule, and frameshift mutations, a shift in the reading frame of a gene because of the addition or deletion of a base. In view of the rapid advent of new products and new industrial processes with their resultant pollutants, it is essential to determine their poten- tial genetic hazards. Despite the fact that mamma- lian cell structure and human enzymatic pathways differ from those in bacteria, the chemical nature of DNA is common to all organisms; this permits the use of bacterial test systems for the rapid detection of possible mutagens and therefore possible carcinogens. The Ames test is a simple and inexpensive procedure that uses a bacterial test organism to screen for mutagens. The test organism is a histidine-negative (his) and biotin-negative (bio) auxotrophic strain of Salmonella typhimurium ow that will not grow on a medium deficient in his- tidine unless a back mutation to his* (histidine- positive) has occurred. It is recognized that the mutagenic effect of a chemical is frequently in- fluenced by the enzymatic pathways of an organ- ism, whereby nonmutagens are transformed into mutagens and vice versa when introduced into human systems. In mammals, this toxification or detoxification frequently occurs in the liver. The Ames test generally requires the addition of a liver homogenate, S-9, which serves as a source of acti- vating enzymes, to make this bacterial system more comparable to a mammalian test system. In the Ames test, by means of the spot method, molten agar containing the test organism, S-9 mix, and a trace of histidine to allow the bacteria to undergo the several cell divisions necessary for mutation to occur is poured on a minimal agar plate. A disc impregnated with the test chemi- cal is then placed in the center of the test plate. Following diffusion of the test compound from the disc, a concentration gradient of the chemical is established. Following incubation, a qualitative indication of the mutagenicity of the test chemical can be determined by noting the number of colo- nies present on the plate. Each colony represents ahis” — his* revertant. A positive result, indicat- ing mutagenicity, is obtained when an obvious increase in the number of colonies is evident com- pared to the number of spontaneous revertants on the negative control plate. In the following procedure, you will perform a modified Ames test; you will not use the S-9 mix to test for the mutagenicity of nitro compounds, which, as in humans, are activated by the bacterial nitroreductases. Four minimal agar plates are in- oculated with the S. typhimurium test organism. One plate, the negative control, is not exposed to atest chemical. Any colonies developing on this plate are representative of spontaneous his > his* mutations. The second plate, the positive control, is exposed to a known nitrocarcinogen, 2-nitrofluorene. The remaining two plates are used to determine the mutagenicity of two commercial hair dyes. 381 > Cs 
