PRINCIPLES OF TOXICOLOGY

252 MUTAGENESIS AND GENETIC TOXICOLOGY
In addition to bacteria, fungi have been used in genotoxicity assays. The Saccharomyces and
Schizosaccharomyces yeasts, as well as the molds Neurospora and Aspergillus, have been utilized in
forward mutation tests, which are similar in design to the salmonella histidine revertant assays that
will be described in the next section.
Typical Bacterial Test Systems
The most widely utilized bacterial test system for monitoring gene mutations and the most widely
utilized short-term mutagenicity test of any type is the Salmonella typhimurium microsome test
developed by Dr. Bruce Ames and co-workers and commonly called the Ames assay. The phenotypic
marker utilized for the detection of gene mutations in all the Ames Salmonella strains is the ability of
the bacteria to synthesize histidine, an amino acid essential for bacterial division. The tester strains of
bacteria have mutations rendering them unable to synthesize histidine; thus, they must depend on
histidine included in the culture medium in order to be able to multiply. Bacteria are taken directly
from a prepared culture and incorporated with a trace of histidine into soft agar overlay on a dish
containing minimal growth factors. The bacteria undergo several divisions, which are necessary for
the expression of mutagenicity and, after the available histidine has been used up, a fine bacterial lawn
is formed. Bacteria that have back-mutated in their histidine operon sites (and thus have reverted to
the ability to synthesize histidine) will keep on dividing to form discrete colonies, while the nonmutated
bacteria will die. A chemical that is a positive mutagen will demonstrate a statistically significant
dose-related increase in “revertants” (colonies formed) when compared to the spontaneous revertants
in 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 substitution
mutations. The TA1538 tester strain detects frameshift mutagens that cause basepair deletions. The
TA1537 tester strain detects frameshift mutagens that cause basepair additions. The TA100 (basepair
substitution) and TA98 (frameshift) strains are sensitive to effects caused by certain compounds, such
as nitrofurans, which were not detectable with the previous three strains.
The lack of oxidative metabolism to transform promutagens (those mutagens requiring bioactivation
to the active form) is overcome in these bacterial assays by two means. First, a suspension of rat
liver homogenate containing appropriate enzymes may be added to the bacterial incubation. The liver
preparation is centrifuged at 9000g for 20 min at 4°C, and the resultant supernatant (S9) is added to
the culture medium. In a slightly more complex procedure, called the host-mediated assay, the bacterial
tester strains are injected into the body cavity of a test animal such as the mouse. This host is treated
with the suspected mutagen and, after a selected period, the bacteria are harvested and assayed for
mutation (revertants) as described earlier. Other bacterial species used in mutagenicity screens include
Escherichia coli and Bacillus subtilis.
Assays that measure DNA repair in bacterial systems have also been developed. These tests are
based on the premise that a strain deficient in DNA repair enzymes will be more susceptible to
mutagenic activity than will a similar strain that possesses repair enzymes that can correct the
mutagenic damage. A “spot” test consists of placing the chemical to be tested in a well or on a paper
disk on top of the agar in a petri dish. The test chemical will diffuse from the central source, causing
a declining concentration gradient as the distance from the source increases. A strain deficient in repair
enzymes will exhibit a greater diameter of bacterial kill than the repair-sufficient strain tested with a
mutagen. In a “suspension” test, a given number of bacteria are preincubated with and without the
test compound. The bacteria are then plated and the colonies counted. The repair-deficient strain will
demonstrate a greater percentage kill than will the sister DNA-repair-sufficient strain. A liver S9
activation system can also be incorporated in bacterial DNA repair tests. The most widely used bacterial
DNA repair test utilizes the polA + and polA – strains of E. coli. The polA – strain is deficient in DNA
polymerase I, whereas the polA + strain is sufficient in this enzyme.