PRINCIPLES OF TOXICOLOGY

10.5 TESTS FOR DETECTING IMMUNOTOXICITY IN ANIMAL MODELS 197
normally produces cellular proliferation and increased immunoglobulin synthesis. This response
requires both TH and B cells, and provides an indication of the capability of these two cells to interact
properly and of B cells to produce immunoglobulins. Lipopolysaccharide (LPS) is a mitogen effective
selectively on B cells, while phytohemaglutinin (PHA) and concanavalin A (con A) are selective T-cell
mitogens. Other stimulants to lymphocyte activation can be used, such as tetanus toxoid, diptheria
toxoid, Candida, and PPD, if the subject has been previously exposed to these. The rapid cell division
characteristic of a normal response to these mitogens is typically assessed by measuring incorporation
of 3 H-thymidine into DNA of the cells. Other endpoints of stimulation, such as increased expression
of IL2 receptors on T cells, can also be evaluated. The results of these tests are particularly prone to
variability, and the tests should be repeated on several occasions in order to demonstrate an abnormal
response.
In the mixed-lymphocyte reaction (MLR) test, lymphocytes from the test subject and another
individual are mixed. Normally, contact with the allogenic lymphocytes will cause the test subject’s
lymphocytes to become activated and proliferate. To conduct this assay, the target lymphocytes are
rendered incapable of replication, often by irradiation or by treatment with mitomycin C. Test subject
lymphocytes are then added, and the rate of their replication is evaluated by measuring incorporation
of 3 H-thymidine. The cytotoxic lymphocyte (CTL) assay takes the lymphocyte interactions one step
further to evaluate the ability of cytotoxic T cells (T C ) to destroy target cells. After incubation of the
test subject and target lymphocytes, the subject T C are isolated, washed, and reincubated with target
lymphocytes preloaded with 51 Cr. As the target cells are destroyed, 51 Cr is released into the medium
and can be measured, providing an index of cytotoxic capabilities of the T C lymphocytes.
Fluorescent Antinuclear Antibody Assay (FANA) The indirect immunofluorescence antinuclear
antibody assay (FANA) may be the initial screening test used to show autoimmunity. However, several
FANA patterns are recognized in various connective-tissue diseases and some low-titer staining
patterns have also been reported in sera from persons exposed to environmental agents. The following
staining patterns may be observed:
1. The diffuse (homogenous) staining pattern, which is usually associated with antibody directed
to DNA-histone or histone subfractions. This staining pattern is frequently found in sera from
patients receiving chronic treatment with procainamide, hydralazine, isoniazid, anticonvulsant
drugs, and some environmental chemical agents.
2. A peripheral (rim) pattern, which is attributed to antibody reacting with native DNA and
soluble DNA-histone complexes. This staining pattern is frequently seen in sera from patients
with systemic lupus erythematosus (>95 percent).
3. Speckled FANA staining, which is usually attributed to antibodies reacting with saline-soluble
antigens. These antibodies are directed to nonhistone antigens and include Sm, ribonucleoprotein,
SS-A/Ro, SS-B/La, PM-1, and SCl-70. While these staining patterns frequently occur in
patients with mixed connective tissue diseases, including Sjögren’s syndrome, polymyositis
and progressive systemic sclerosis, they have also been found in sera from persons exposed to
immunotoxic agents.
4. The nucleolar staining pattern, which has been restricted to antibodies reactive with nucleolar
RNA. This pattern is associated with a particular form of systemic sclerosis (progressive
systemic sclerosis).
10.5 TESTS FOR DETECTING IMMUNOTOXICITY IN ANIMAL MODELS
For most chemicals, an assessment of their potential to produce immunotoxicity in humans is based
on testing in animals. Many of the tests used in animal studies are the same as, or at least analogous
to, those available for clinical assessment described above. However, studies in animals offer the