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Changes in Retina Morphology in Vertebrates Following Exposure to Polychlorinated Biphenyls

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Changes in Retina Morphology in Vertebrates Following Exposure to Polychlorinated Biphenyls
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Lavakumar, Mallika
Julian, David ( Mentor )
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Gainesville, Fla.
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University of Florida
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English

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Changes in Retina Morphology in Vertebrates Following Exposure
to Polychlorinated Biphenyls

Mallika Lavakumar


INTRODUCTION


EDCs


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 Exposures


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

Cat. #15900-184).


Histology



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

# 8310-4).


Morphological analysis





Tecltm?


Optic chiasm


Bipolare Ganglion



Our plex layer Inner pledor
layer

Rods arnd Manblage

Pgomen.d Posterior O.tic eww
opithellurn


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.



RESULTS


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/


L Aroclor.

















- Mt


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


A. Width

300o

250

200

S150

S100


o


Md-niaIa Mid-ternpolal
Region1


* Control
S*Arocor


Mind-tasal Mdic-ernporal
Region


C. Outer Nuclear Layer

60

50



30



10

0
Mid-nasal Mid-temporal
Region


D. Ganglion Cell Layer

80
70

T 60

240
z30
~20
10
0
Mlid-nasal Mid-temnporal
Region


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).




8


Control


Exposed


Treatment


Figure 4. Cell density of the control and the exposed fish in nasal region of the INL of the retina.


140

120
100

80


j 40
20
0








DISCUSSION


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.






REFERENCES


Barron, M. G., Galbraith, H., and Beltman D. (1995). Comparative Biochemistry and Physiology

C-Pharmacology Toxicology & Endocrinology. 112: 217-227.


Cameron, D. A. (2000). Cellular proliferation and neurogenesis in the injured retina of adult

zebrafish. Visual Neuroscience. 17: 789-797.


Carnevali, M. D. C., Bonasoro F., Patruno M., Thorndyke M. C., and Galassi S. (2001). PCB exposure

and regeneration in crinoids (Echinodermata). Marine Ecology Progress Series. 215:155-167.


Colborn, T., F. S. vom Saal, and A. M. Soto. (1993). Developmental effects of endocrine-

disrupting chemicals in wildlife and humans. Environmental Health Perspectives 101:378-384.


Kelce, W. R., C. R. Stone, S. L. Laws, L. E. Gray, J. A. Kemppaainen, and E. M. Wilson. (1995).

Persistent DDT metabolite p,p'DDE is a potent androgen receptor antagonist. Nature 375:581-585.


Laessig, S. A. (1999). Neurotoxic effects of endocrine disruptors. Current Opinions in Neurology. 12

(6): 745-751.


Lyall, A. H. The growth of the trout retina. Q. J. Micros. Sci. 98: 101-110.


Ng L., Hurley L. B., Dierks B., Srinivas M., Salto C., Vennstrom B., Reh T. A., and Forrest D. (2001).

A thryroid hormone receptor that is required for the development of green cone photoreceptors.

Nature Genetics. 27 (1): 94-98.


Olson AJ, Picones A, Julian D and Korenbrot 31 (1999). A developmental time line in a retinal slice

from rainbow trout. Journal of Neuroscience Methods 93(2): 91-100.


Raloff, J. (1994). The gender benders--are environmental "hormones" emasculating wildlife?

Science News 145:24-27.





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