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microscopes are used to capture images using cells that have been treated with fluo-
rescent tags. Tags are available to monitor such endpoints as oxidant status; sulfhydryl
content; intracellular Ca
2
, H
, Na
, and K
; mitochondrial function; and membrane
potential. Digital electronic imaging and computerized data analysis can enhance the
sensitivity of this technique and provide information on temporal relationships between
multiple responses within a given cell. The disadvantage is poor resolution. However,
this can be overcome through the use of laser-scanning confocal light microscopy,
which can optically limit the image to thin slices within the depth of the monolayer.
The use of various molecular biological methods has greatly increased the pace
of research in the biomedical sciences in general, and toxicology in particular. One
common method is the use of recombinant DNA technology and molecular cloning.
This is a method whereby strands/sequences of DNA that one would not normally
encounter can be created and multiplied using vectors. A vector is simply a DNA mol-
ecule that originates from a plasmid or a virus that has the capacity to self-replicate
when placed into a host cell. In this manner, hundreds of exact copies of the DNA
strand of interest are produced within the cell. These cloned genes can then be used
for a variety of purposes. The gene could be sequenced to determine the amino acid
sequence that would be generated from the DNA or it could be put into a mammalian
expression vector and expressed in mammalian cells.
PROTEOMICS
The proteome can be defined as that portion of the genome that is expressed as pro-
teins in a cell or organism over time. Proteomics is a global analysis to study the struc-
ture and function of the proteome, involving its separation and identification. Finally,
toxicoproteomics is the study of proteomics applied to toxicology in such a way as to
identify critical proteins and pathways affected by toxicant exposure. There are seven
attributes of proteins needed for comprehensive protein expression analysis: identity,
quantity, post-translation modification, structure, protein-protein interactions, cellular-
spatial relationships, and function. Given that proteins such as enzymes and cellular
receptors are the preferred targets for virtually all pesticides, the field of proteomics
promises to contribute significantly to the study of pesticide toxicology. Large-scale,
high-throughput omics technologies are increasingly being used to elucidate highly
complex networks of proteins and protein interactions within a biological system
(
Figure 2.2
). The methods employed in these complex studies include two-dimen-
sional gel electrophoresis, chromatographic separations, stable isotope labeling, mass
spectroscopy, and protein bioinformatics.
In its simplest form, two-dimensional gel electrophoresis (2D-GE) involves run-
ning out proteins on a gel, rotating the gel 90°, and then further separating the pro-
teins by a different property. For instance, proteins may be separated by mass and then
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