PRINCIPLES OF TOXICOLOGY - Biology East Borneo

252 MUTAGENESIS AND GENETIC TOXICOLOGYIn addition to bacteria, fungi have been used in genotoxicity assays. The Saccharomyces andSchizosaccharomyces yeasts, as well as the molds Neurospora and Aspergillus, have been utilized inforward mutation tests, which are similar in design to the salmonella histidine revertant assays thatwill be described in the next section.Typical Bacterial Test SystemsThe most widely utilized bacterial test system for monitoring gene mutations and the most widelyutilized short-term mutagenicity test of any type is the Salmonella typhimurium microsome testdeveloped by Dr. Bruce Ames and co-workers and commonly called the Ames assay. The phenotypicmarker utilized for the detection of gene mutations in all the Ames Salmonella strains is the ability ofthe bacteria to synthesize histidine, an amino acid essential for bacterial division. The tester strains ofbacteria have mutations rendering them unable to synthesize histidine; thus, they must depend onhistidine included in the culture medium in order to be able to multiply. Bacteria are taken directlyfrom a prepared culture and incorporated with a trace of histidine into soft agar overlay on a dishcontaining minimal growth factors. The bacteria undergo several divisions, which are necessary forthe expression of mutagenicity and, after the available histidine has been used up, a fine bacterial lawnis formed. Bacteria that have back-mutated in their histidine operon sites (and thus have reverted tothe ability to synthesize histidine) will keep on dividing to form discrete colonies, while the nonmutatedbacteria will die. A chemical that is a positive mutagen will demonstrate a statistically significantdose-related increase in “revertants” (colonies formed) when compared to the spontaneous revertantsin control plates.Five Ames S. typhimurium tester strains are recommended for routine mutagenicity testing:TA1535, TA1537, TA1538, TA98, and TA100. The TA1535 tester strain detects basepair substitutionmutations. The TA1538 tester strain detects frameshift mutagens that cause basepair deletions. TheTA1537 tester strain detects frameshift mutagens that cause basepair additions. The TA100 (basepairsubstitution) and TA98 (frameshift) strains are sensitive to effects caused by certain compounds, suchas nitrofurans, which were not detectable with the previous three strains.The lack of oxidative metabolism to transform promutagens (those mutagens requiring bioactivationto the active form) is overcome in these bacterial assays by two means. First, a suspension of ratliver homogenate containing appropriate enzymes may be added to the bacterial incubation. The liverpreparation is centrifuged at 9000g for 20 min at 4°C, and the resultant supernatant (S9) is added tothe culture medium. In a slightly more complex procedure, called the host-mediated assay, the bacterialtester strains are injected into the body cavity of a test animal such as the mouse. This host is treatedwith the suspected mutagen and, after a selected period, the bacteria are harvested and assayed formutation (revertants) as described earlier. Other bacterial species used in mutagenicity screens includeEscherichia coli and Bacillus subtilis.Assays that measure DNA repair in bacterial systems have also been developed. These tests arebased on the premise that a strain deficient in DNA repair enzymes will be more susceptible tomutagenic activity than will a similar strain that possesses repair enzymes that can correct themutagenic damage. A “spot” test consists of placing the chemical to be tested in a well or on a paperdisk on top of the agar in a petri dish. The test chemical will diffuse from the central source, causinga declining concentration gradient as the distance from the source increases. A strain deficient in repairenzymes will exhibit a greater diameter of bacterial kill than the repair-sufficient strain tested with amutagen. In a “suspension” test, a given number of bacteria are preincubated with and without thetest compound. The bacteria are then plated and the colonies counted. The repair-deficient strain willdemonstrate a greater percentage kill than will the sister DNA-repair-sufficient strain. A liver S9activation system can also be incorporated in bacterial DNA repair tests. The most widely used bacterialDNA repair test utilizes the polA + and polA – strains of E. coli. The polA – strain is deficient in DNApolymerase I, whereas the polA + strain is sufficient in this enzyme.