PRINCIPLES OF TOXICOLOGY

may not be repaired, resulting in a permanent break; or (3) they may be misrepaired or join with another
chromosome to cause a translocation of genetic material. A second theory is the “chromatid exchange”
hypothesis. If the exchange occurs with a chromatid from another chromosome, an “exchange figure”
results. This theory assumes that the initial lesion is not a break and that the lesion can either be repaired
directly or may interact with another lesion by a process called exchange initiation. Most chromosomal
abnormalities result in cell lethality and, if induced in germ cells, generally produce dominant lethal
effects that cannot be transmitted to the next generation. The traditional method for determining
chromosomal aberrations is the direct visual analysis of chromosomes in cells frozen at the metaphase
of their division cycle. Thus, metaphase-spread analysis evaluates both structural and numerical
chromosome anomalies directly.
Chemicals inducing changes in chromosomal number or structure also may be identified by the
micronucleus test, an assay that assesses genotoxicity by observing micronucleated cells. It is a
relatively simple assay because the number of cells with micronuclei are easily identified microscopically.
At anaphase, in dividing cells that possess chromatid breaks or exchanges, chromatid and
chromosome fragments may lag behind when the chromosome elements move toward the spindle
poles. After telophase, the undamaged chromosomes give rise to regular daughter nuclei. The lagging
elements are also included in the daughter cells, but a considerable proportion are included in secondary
nuclei, which are typically much smaller than the principal nucleus and are therefore called micronuclei.
Increased numbers of micronuclei represent increased chromosome breakage. Similar events can
occur if interference with the spindle apparatus occurs, but the appearance of micronuclei produced
in this manner is different, and they are usually larger than typical micronuclei. Historically, lymphocytes
and epithelial cells have been the most commonly used cell populations.
Many point mutations are detected by the cell and are deleted by various repair mechanisms. Some,
however, remain undetected and are passed to daughter cells. The significance of the mutations varies
with the type of cell, and the location within the DNA. If the cell is of somatic lineage, altered gene
products can result from gene expression. If the cell is a gonadal cell (or germ cell), the change can be
passed on to offspring and may cause problems in future generations. Much of the DNA in organisms
is never expressed. If the mutation occurs in that portion of the DNA that is not expressed, no problem
occurs. However, if the mutation occurs in the active portion of the DNA, the altered gene products
can be expressed. An example of a problematic point mutation is in the gene that causes sickle cell
anemia. A change of one basepair (a transversion from thymine to adenine) results in the amino acid
glutamate being replaced by another amino acid, valine, in one of the molecules that makes up
hemoglobin, the oxygen-carrying molecule in red blood cells. When the blood becomes deoxygenated,
such as under heavy exercise conditions, the valine allows the red blood cells to assume a sickle shape
instead of the normal circular shape. This leads to clumping of blood cells in capillaries, which in turn
may limit blood flow to the tissues. This behavior of the blood cells exacerbates other effects of sickle
cell anemia, which result in oxygen deprivation because the hemoglobin content of the blood in persons
with sickle cell anemia is about half that of other persons.
12.3 NONMAMMALIAN MUTAGENICITY TESTS
12.3 NONMAMMALIAN MUTAGENICITY TESTS 251
Because results from bacterial or prokaryotic assays often establish priorities for other testing
approaches, it is of interest to briefly describe the assays currently used to screen for mutagenic
capacity, particularly those done in industrial settings.
Rapid cell division and the relative ease with which large quantities of data can be generated
(approximately 10 8 bacteria per test plate) have made bacterial tests the most widely utilized routine
means of testing for mutagenicity. These systems are the quickest and most inexpensive procedures.
However, bacteria are evolutionarily far removed from the human model. They lack true nuclei as well
as the enzymatic pathways by which most promutagens are activated to form mutagenic compounds.
Bacterial DNA has a different protein coat than seen in eukaryotes. Nevertheless, bacterial systems
have great utility as a preliminary screen for potential mutagens.