Page 189 - Veterinary Toxicology, Basic and Clinical Principles, 3rd Edition

156 SECTION | I General

VetBooks.ir Typically, the amount of ATP in cell lysates is deter- two-dimensional PAGE, chromatographic or protein chip
fractionation of proteins from cell/tissue lysates or subcel-
mined by the light released from firefly luciferase-
lular in order to identify changes in specific protein levels
catalyzed oxidation of D-luciferin in the presence of ATP
and oxygen. ATP and its major metabolites (ADP and following toxin exposure. Proteins of interest are then
AMP) in cell culture extracts can also be determined normally digested with trypsin, and the tryptic fragments
using an HPLC method (Yang and Gupta, 2003). are initially identified by “peptide mass fingerprinting” in
a matrix-assisted laser desorption ionization time of
Dehydrogenase Activity flight mass spectrometer, followed by further confirma-
tion using, e.g., tandem mass spectrometry (Steen and
The activity of cellular dehydrogenases can be assayed by
Mann, 2004). Identification is facilitated by the use of
the reduction of the yellow methyl tetrazolium dye to its
freely available Internet-based gene and protein sequence
blue formazan reaction product. The reduced dye forms
databases such as MASCOT, SwissProt, etc.
crystals in metabolically active cells and, after removal of
growth medium, can be solubilized in an organic solvent
such as dimethyl sulfoxide and quantified spectrophoto- Genomic Analysis
metrically in the microtiter plate format, e.g., in assays of
Changes in the levels of specific proteins detected by pro-
in vitro toxicity of OPs (Hargreaves et al., 2006).
teome analysis could reflect changes in the regulation of
Although metabolic assays such as those indicated
protein turnover (i.e., how rapidly it is degraded once syn-
above are not direct measurements of cell viability, a sig-
thesized) or changes in gene expression. Furthermore,
nificant fall in either measurement is normally taken to
depending on the cell or tissue fractionation procedures
indicate a fall in cell viability or cell number.
adopted, changes in low abundance proteins that could be
functionally important may not necessarily be detected by
Calcium Homeostasis proteomic methods. Changes in gene expression following
Many toxins have a direct or indirect effect on mechan- exposure to toxin can be determined by a number of
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isms involved in the control of Ca homeostasis. Such methods including reverse transcriptase polymerase
effects could be related to a breakdown in plasma mem- chain reaction (RT-PCR) and DNA microarray analysis
brane integrity, which would also be detected by other (Koizimo and Yamada, 2003).
methods discussed previously (e.g., leakage of LDH), or RT-PCR is a targeted approach involving the produc-
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it may be the result of a selective effect on Ca channels tion of complementary DNA (cDNA), by treatment of
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or Ca ATPases that regulate Ca movements across cell or tissue RNA with RT. The resultant cDNA is then
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cellular membranes. Changes in Ca flux can be mea- incubated with primers (probes) that recognize specific
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sured using a variety of Ca binding dyes that fluoresce genes or DNA sequences of interest, followed by amplifi-
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when bound to Ca (e.g., Quin 2 and Fura 2). Detection cation of such sequences by the Taq polymerase reaction.
requires spectrofluorimetric analysis that is potentially Changes in the level of amplified target sequence would
applicable to high throughput analysis of average change indicate corresponding changes in the expression of the
for a given cell population. However, analysis of changes target gene to which the primers are matched. This
in Ca 21 flux in individual cells requires a system that has approach is only semiquantitative although accuracy can
an integrated microscope and image analysis software be improved by continuous measurement of amplicon
package. levels using real-time quantitative RT-PCR. Levels of
amplicon are normally compared to changes in household
Cell or Tissue-Specific Markers of Toxicity genes such as glyceraldehyde dehydrogenase or β-actin,
as internal controls.
This approach is useful for targeting key proteins or
A less targeted approach that, nevertheless, could help
enzyme activities involved in the normal function(s) of
to identify novel changes in gene expression is that of
the target cells or tissues. It is particularly useful in suble-
DNA microarray analysis. In this technique, up to several
thal/chronic studies of exposure to toxin, in order to deter-
thousand genes are immobilized on a template and probed
mine whether a potentially reversible functional deficit
with labeled RNA from control and treated cells or tissues.
occurs. The principal approaches used at the protein level
Image analysis software is then used to determine up- or
are those of antibody-based detection and enzyme assays.
down-regulation of genes due to toxin exposure. Controls
usually involve averaged changes for a series of household
Proteomic Analysis genes. The researcher should be aware that a change in
This approach can be useful in mechanistic studies levels of gene expression does not necessarily indicate a
of toxicity as it helps to identify novel protein markers corresponding change in protein levels or vice versa, as
of toxicity. Techniques of this type include the use of there are multiple levels of control. A multidisciplinary