Page 188 - Veterinary Toxicology, Basic and Clinical Principles, 3rd Edition

Toxicological Testing: In Vivo and In Vitro Models Chapter | 9 155

VetBooks.ir Membrane Leakage on living cells. Furthermore, high throughput assays could
be developed that measure the underlying molecular
Leakage of macromolecules through the plasma mem-
changes determined from follow-up studies.
brane into the culture medium is an effective means of
detecting early or late stages of membrane damage. Cells Morphological changes can take various forms, as
3
can be incubated in the presence of H-thymidine, which indicated below.
is incorporated into DNA during cell proliferation.
3
Subsequent loss of H-labeled DNA would be indicative Cell Volume
of cell lysis. An alternative approach is to label cells in An increase or decrease in cell volume could indicate
the presence of 51 Cr, which binds to many cellular pro- osmotic changes or represent the early stages of cell death
teins. Loss of labeled proteins from treated cells would be by necrosis or apoptosis. Cell death or viability changes
indicative of membrane leakage/cell lysis. In this sense, would then need to be made to confirm the type of cell
the 51 Cr release assay is more sensitive than that for death as indicated earlier.
3
H-labeled DNA as the former will detect signs of
damage to the membrane at a much earlier stage. Cell Shape
An attractive alternative to radioisotopic methods is
Changes in shape may occur following exposure to a
the release of lactate dehydrogenase (LDH) into cell cul-
toxin. These could include rounding up, flattening,
ture medium, which is measured spectrophotometrically.
spreading, or a process outgrowth in cell culture mono-
The assay, which is commercially available in kit form, layers. Such changes would give an initial indication of
shows sensitivity comparable to the 51 Cr release assay
altered cell attachment, migration, proliferation, or differ-
and is amenable to the microtiter plate format, which
entiation, indicating potential targets for follow-up molec-
would facilitate medium to high throughput analysis.
ular studies. For example, OP-induced changes in axon
outgrowth in cultured neurons indicated possible changes
Cell Growth and Proliferation in proteins associated with axon growth and maintenance,
Cell growth or proliferation can be measured in several which were then targeted in molecular studies (Hargreaves
ways. The simplest method is that of cloning efficiency. et al., 2006).
The mitotic index of cell cultures can be determined by
counting the percentage of mitotic figures following stain- Membrane Integrity
ing of DNA with hematoxylin. Another approach is the
Changes in membrane integrity may be indicated by sur-
measurement of cell growth in toxin-exposed cells via the
3 face blebbing, which can occur as a result of cellular
incorporation of H-thymidine into DNA (Flaskos et al.,
stress (e.g., oxidative stress) or during the early stages of
1994). Alternatively, cell proliferation can be measured
apoptosis. These parameters could be measured further by
by the incorporation of 5-bromo, 2-deoxyuridine incorpo-
biochemical methods to confirm the underlying molecular
rated into S-phase cultured cells, and subsequent quantifi-
events associated with these morphological changes (e.g.,
cation is achieved by enzyme-linked immunoabsorbent
free radical generation, lipid peroxidation, caspase activa-
assay (Lanier et al., 1989). Total DNA content can also
tion, etc.).
be determined by detecting fluorescence after incubation
of cells with DNA binding dyes such as Hoechst 33258,
Growth Patterns
using a spectrofluorimetric microplate reader or by FACS
Growth patterns may change as a result of exposure to
analysis (Downs and Wilfinger, 1983).
toxin. Thus, the proportion of cells growing in colonies or
Cell growth can also be measured by total protein con-
singly would indicate changes in cell cell interactions
tent or by protein synthesis. Protein content can be esti-
and potential changes in cell adhesion proteins, which
mated by a number of dye binding assays in microtiter
could be targeted in subsequent molecular studies.
plate format, such as the bicinchoninic acid assay
(Tuszynski and Murphy, 1990).
Metabolic Assay
Cell Morphology ATP Levels
Changes in cell morphology can be measured microscopi- ATP is an essential requirement for many energy-
cally and are very useful in studies of mechanisms of tox- dependent processes and its levels can be affected by a
icity. However, medium to high throughput analysis of variety of toxins. It can be used as a marker for cell via-
toxicity would require the use of image analysis software bility as it is present in all metabolically active cells and
to produce more consistent data. Specialist techniques its levels decline rapidly when cells undergo apoptosis or
such as Allen video-enhanced contrast differential inter- necrosis (Kangas et al., 1984). A number of reagents and
ference contrast microscopy facilitate analysis of effects kits suitable for high throughput screening are available.