Fishing to Design Inherently Safer Nanoparticles - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 279

20.4 TIER 2: CELLULAR TOXICITY AND
DISTRIBUTION
20.4.1 Cellular Level Responses
Cellular targets can be identified in vivo by using (1) transgenic zebrafish that express
fluorescent protein in specific cell populations and (2) fluorescent dyes that respond to
changes in the cellular oxidation environment.
20.4.2 Transgenic Zebrafish
There are many transgenic zebrafish available, most of which can be found on the
Zebrafish Model Organism Database (ZFIN) web site (www.zfin.org). The morpho-
logical effects observed in tier 1 are used as a guide to the selection of the reporter
transgenic line used to assess target toxicity. Nanoparticle-induced disruption of
specific cell populations can be easily investigated using this approach (Lele and
Krone, 1996; Blechinger et al., 2002). For example, if nervous system end points are
significantly impacted (e.g., brain malformation or abnormal behavior), then specific
neuronal effects will be identified using transgenic fish such as huc -GFP (GFP
produced in most neurons), islet1 -GFP (GFP produced only in secondary motor
neurons), nbt1 -GFP (GFP expressed in primary spinal neurons), and/or neurog1 -GFP
(GFP produced in Rohon-Beard cells, and later in dorsal root ganglion cell bodies
and axons).
After the most appropriate transgenic lines have been selected, 8hpf embryos are
exposed as described in tier 1. After exposure, embryos are rinsed, anesthetized, and
mounted in 1% agarose on a glass slide for microscopic analysis. Images are captured
from live embryos expressing cell-specific fluorescent proteins via digital micros-
copy. There are multiple software programs available to quantify the amount of GFP
positive cells, but image analysis using Image ProPlus 5.1 and tools in Axiovert 4.0
(Zeiss) has been successfully used. If more precise images are necessary, confocal
laser scanning microscopy can be used.
20.4.3 Oxidative Stress
Nanoparticles have been implicated in disrupting the oxidative environment of cells
(Tsuchiya et al., 1996; Hussain, 2001; Gharbi et al., 2004; Nemmar et al., 2004;
Oberdorster, 2004). Excessive quantities of reactive oxygen species can cause
oxidation of lipids, proteins, and DNA, cellular events that have been implicated
in cardiovascular diseases (hypertension) and neurodegenerative diseases
(Parkinson's and Alzheimer's). To directly measure oxidative stress in vivo , whole
mount oxidative stress assays have been developed to identify temporal and spatial
localization. To conduct these assays, we use embryonic exposures identical to that
described for tier 1, except a positive control for oxidative stress (hydrogen peroxide)
is also included.