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Changes in Retina Morphology in Vertebrates Following Exposure
to Polychlorinated Biphenyls
violence Endocrine disrupting contaminants (EDCs) are chemical pollutants that interfere with normal
endocrine function. They either mimic hormones, causing a synergistic effect, or they inhibit the binding of
hormones to their receptors, causing an antagonistic effect. While EDCs are most commonly known for
their estrogenic disruption, they are also thought to alter the activity of other hormones including
androgens, progesterone, glucocorticoids, and thyroid hormones (Kelce et al. 1995).
Polychlorinated biphenyls (PCBs) are a group of industrial chemicals that have contributed to a global pollution
crisis. PCBs are lipid-soluble biphenyl rings with varying degrees of chlorination, and their structures are very
similar to those of thyroid hormones. Since cell membranes are made up of a lipid bilayer and PCBs are lipid
soluble, PCBs are capable of penetrating cell membranes, thereby interfering with the cell's machinery. Owing to
their lipid solubility, PCBs aggregate in adipose tissue after they enter the organism's body. Finally,
PCBs bioaccumulate as they ascend through higher trophic levels in the food chain.
Cellular mechanisms of PCBs
Early exposure to PCBs has been implicated in disruption of central nervous system (CNS) development
(Laessig 1999), but the cellular mechanisms of developmental neurotoxicity from PCBs are still unknown. Given
that thyroid hormone receptors are present in the vertebrate CNS, that thyroid hormones play a major role in
CNS development, and that PCBs can bind to these receptors as a result of their structural similarity to
thyroid hormones, one plausible neurotoxic result of PCB exposure is via the thyroid hormone system.
Interference with normal thyroid hormone signaling during development, such as might occur from PCB
exposure, could cause alterations in neuronal proliferation and differentiation, neuron migration, synaptogenesis,
and expression of neurotransmitters, as well as cognitive and behavioral changes and morphological abnormalities
in the CNS.
For this project, I exposed zebrafish (Danio rerio) to Aroclor 1260, a commercial mixture of PCBs, to study the
effects of PCBs on CNS development. These fish were exposed at critical developmental stages to a range of
Aroclor concentrations, including environmentally relevant PCB concentrations. I then compared retinal
morphology between exposed and unexposed fish to quantify the developmental impact of Aroclor exposure.
MATERIALS AND METHODS
Aroclor dissolved in methanol (100 pg A1260 per mL methanol) was obtained for the PCB exposures (ULTRA
Scientific RPCK-3A). Each fish was exposed to either one of four Aroclor concentrations (0.010, 0.10, 1.0, and 10
pg/L A1260), 0.01% methanol (as a control for the Aroclor carrier), or pure water. The effect of each
concentration and the controls were studied at each of four developmental stages (30, 48, 72, and 96 hpf).
Each treatment combination consisted of six fish replicates, for a total of 144 fish. Exposures were conducted
in amber glass jars that were pre-cleaned to meet US EPA specifications for contaminant free sampling (VWR
After exposure, the fish were killed and fixed overnight at 4woC in 4 % paraformaldehyde (EMS Cat.
# 15710). After fixation, the fish were rinsed in several changes of phosphate-buffered saline
(PBS; contains 8 g/L NaCl, 0.2 g/L KCI, 1.15 g/L Na2HP04, and 0.2 g/L KH2PO4), dehydrated for 30
min each in two changes of 70 % ethanol, followed by 30 min dehydrations in two changes of 100
% ethanol. This was followed by 1-hour room temperature incubations in two changes of LR
White plastic pre-polymer (EMS Cat. # 14382). The tubes were placed in an oven at 60ooC for 24 hours
to polymerize the LR White.
Blocks containing the fish were sectioned at 2 mm with glass knives on an ultramicrotome (Du Pont
MT 5000 Sorvall Ultra Microtome). The sections were stained with toluidine blue (0.2 g toluidine in 50
mL water and 2 g borax) for 5 minutes on a 125moC hot plate. The slide was rinsed with distilled
water, destined in ethanol for 6 minutes, dried, and coverslipped with Cytoseal (Stephen Scientific Cat
Our plex layer Inner pledor
Rods arnd Manblage
Pgomen.d Posterior O.tic eww
Figure 1. The major retinal regions of the zebrafish retina (www.zebrafish.org).
Figure 1 illustrates the terminology used to describe different regions of the retina. Sections
were examined under the microscope (Olympus ZX70) and digital pictures (Nikon CoolPix 990)
were taken of control and exposed sections that were comparable in retinal location. The thickness
of retinal layers such as the inner nuclear layer (INL), the outer nuclear layer (ONL) and the ganglion
cell layer (GCL), and the width of the retina were measured using ImageJ software (NIH) and
averages were calculated. The color-coded lines in Figure 2 represent the thickness measurements of
the retinal width and of the cell layers.
Fish at all four developmental stages (30, 48, 72 and 96 hpf) survived each of the six
conditions, indicating that the Aroclor exposures were not lethal. By gross observation,
swimming behavior appeared the same in all the conditions. Retinal morphology could not be
examined in the smallest fish (30 and 48 hpf) because they were too small to be oriented
appropriately during embedding, excluding 72 of the 144 fish from morphological analyses.
Furthermore, even when the fish were oriented accurately in the pre-polymer, the orientation of
some specimens changed during the 24-hour polymerization period, further reducing the number
that could be analyzed. Ultimately, sections from only four fish at the 72 hpf developmental stage
were appropriate for morphological analyses: two methanol control fish and two fish exposed to 10 pg/
Figure 2. The nasal region of the retina of exposed and control fish 72 hpf. Red lines indicate
retinal width, yellow lines INL thickness, black lines ONL thickness and green lines GCL thickness.
Figure 2 illustrates some of the differences between control and exposed fish in the nasal region of
the retina. Qualitatively, the general health of the control retina section looks better than that of
the Aroclor retina. The pigmented epithelium of the Aroclor retina has ripples, while that of the
control retina appears more taut. The cell outlines of the control retina are clearly discernable
in comparison to the retina of the exposed fish. Finally, Aroclor retinas have more space
between individual cells.
The width of the retina and the thickness of the INL, ONL, and GCL are always greater in the
exposed fish, irrespective of whether the mid-nasal or the mid-temporal region is measured (Fig. 3 A,
B, C and D). The most striking difference between the two conditions is observed in the width of the
mid-nasal region (Fig. 3A, left cluster). The width of the exposed fish (269 pm) was 42 % greater
than that of the control (189 pm).
B. Inner Nuclear Layer
C. Outer Nuclear Layer
D. Ganglion Cell Layer
Figure 3. The retinal width, and the thickness of the INL, ONL and GCL of the mid-nasal and mid-
temporal regions of control and exposed fish.
Even more striking is the cell density difference between the retinas of the exposed and control fish
(Fig. 4). The density of the INL cells in the exposed fish (4.8*10-4 cells pm-1) is only one-fourteenth
that of the control fish (6.7*10-3 cells pm-1).
Figure 4. Cell density of the control and the exposed fish in nasal region of the INL of the retina.
Fish retina as a model
One objective of this study was to test the feasibility of using the fish retina as a model to
evaluate cellular effects of PCBs on the developing vertebrate CNS. Unlike other sensory organs,
the vertebrate retina evaginates from the brain during early embryonic development and is a part of
the CNS. The mature vertebrate retina is extremely well characterized physiologically, and
its development is well-studied. Furthermore, when compared with the rest of the CNS, the
retina contains fewer cell types, many of which have recognizable morphologies and consistent
positions, making the investigation of CNS mechanisms in the retina less daunting.
The teleost retina develops in a laminar fashion that may be analogous to mammalian
cortex development. Therefore, understanding how toxins alter retinal development would provide
some direction for studies attempting to determine the impact of these contaminants on
mammalian brain development. The fish retina grows throughout the life of the organism by adding
new cells to its margins (Lyall 1957). Consequently, a cross-section of the retina displays various
stages of development with the most recent at the margins and the oldest at the center of the
retina (Olsen et al. 1999). This permits the examination of different developmental stages in one
animal, rather than sacrificing animals at different ages.
The zebrafish, Danio rerio, was selected for this study primarily because its genome has been
sequenced. The data collected in this study can be used to conduct more advanced molecular
research such as looking for gene expression responses to chemical stress. Since zebrafish
develop rapidly, a large amount of data can be collected within a short period of time. The advantages
of being inexpensive, readily accessible, and easy to maintain make the zebrafish an attractive
subject for studying development.
Significance of results
The data in this study provide insight into the mechanisms through which PCBs may impact
CNS development. Compared with age-matched fish exposed to the methanol control, the thickness
was increased in all three retinal layers (GCL, INL and ONL) in the retinas of fish exposed to Aroclor.
This increased thickness could be due to increased cell division or increased spacing between cells.
I found that the latter of these was the case, since INL cell density of fish treated with Aroclor was
much lower than that of the control fish (Fig. 4). Furthermore, the rippled pigmented epithelium may
also indicate that that fish exposed to Aroclor were not as healthy in comparison to the controls.
When the fish retina is undergoing normal development, cells from the margins proliferate to occupy
the available space. The lower cell densities in fish exposed to Aroclor could be explained by
several possibilities. First, the margins might not be proliferating as actively in the exposed fish as in
the control. Since thyroid hormones actively participate in cell proliferation, it is possible that the
Aroclor bound to thyroid receptors and prevented hormonal regulation of cell proliferation. The
second explanation for the decreased cell density is that Aroclor exposure affected the
intercellular connections necessary to maintain cells within close proximity of each other. Finally,
Aroclor exposure may have caused cell death resulting from apoptosis or necrosis, which would result
in decreased cell density. The morphological changes I observed in the retinas of fish treated
with Aroclor may be representative of changes that occur in other regions of the CNS in
higher vertebrates, and could result in functional deficiencies.
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