Effects of Varying Levels of Arsenic

Trioxide Bioaccumulation on
Melanophore Index in Channa punctata
Background - Max Berman
Experimental Design - Gehad Abdelaal
Results - Kailyn Broughton
Discussion - Shrey Mittal
Limitations - Tayler Blair
 Research Question
How does arsenic trioxide bioaccumulation in
Channa punctata affect dermal chromatophores,
specifically dermal melanophores, and how can
this information be used as a biomarker for
arsenic pollutants in aquatic environments.
 Background
● The expansion of industry and the advancement of human civilization has brought with it
increases in pollution, namely wastewater runoff filled with pesticides, toxins, and heavy
metal pollutants.
● Heavy Metal pollutant arsenic- Arsenic trioxide is one of the most abundant forms of
arsenic in nature and it is heavily used in both the manufacturing and agriculture
industries (Lin et al., 2014).
○ Levels of soluble arsenic have beens shown to be above permissible levels of 0.010 mg/L,
and this is a global occurrence (Kumari et. al, 2016).
● Fish chromatophores, in particular, were shown to have been impacted by the pollutants
in the water, with changes in dispersal being detected (Kaur and Dua, 2011).
● Channa punctata is a common species that can be used as a model system in various
pollution studies because they are readily available and suitable to evaluate the effects of
the toxicity of pesticides.
○ Channa punctata does not have the ability for robust physiological color change, such
as in other teleosts like Fundulus heteroclitus, they undergo frequent morphological
color change as they develop.
 Background
● Color change is mediated by dermal chromatophores, including dermal melanophores.
There are two main methods for color change in teleosts:
○ Morphological- long-term color change affected by hormones that change chromatophore
density and form (Sugimoto, 2002).
○ Physiological- rapid color change controlled by neurotransmitters utilizing motor proteins
and cytoskeleton intracellular tracks (Ligon and McCartney, 2016)
● Biomarkers refer to an organism or tool that can be used to show the relationship
between changes in cellular responses, and environmental pollutants or toxic effects.
○ Previous research used Betta splendens chromatophores as cytosensors for various
pollutants and toxins (Chaplen et al., 2002).
Research Objectives:
● Establish how Channa punctata dermal chromatophores bioaccumulate arsenic.
● Observe how melanophore index is affected by arsenic trioxide bioaccumulation in
dermal melanophores
● Extend this information to another model organism, Fundulus heteroclitus, in order to
generate another biomarker and examine the impacts of arsenic bioaccumulation in
other ecosystems
 Experimental Design
Hypothesis: Arsenic trioxide bioaccumulation for prolonged exposure leads to structural changes
in the dermal melanophores in Channa punctata making them a model organism to use as a
biomarker for arsenic pollution.
 Experimental Design
Independent Dependent
Variable Variable
Duration of Level of overall
exposure to 1 mg pigment dispersion
1-1 of arsenic according to
trioxide (7, 14, 30, Hogben MI
60, 90 days)

Experimental Controlled
Control Group Group Variables
20 fish not 4 fish per group - Weight of Channa punctata : 35
+/- 5 g
exposed to exposed to arsenic
- Temperature of water in tanks :
arsenic for any for 7, 14, 30, 60, 25 C
time split and 90 days - Number of feedings/day : 1
randomly into 5 Type of food : Kijaro Japan
groups to serve as - Number of fish used
- Fish kept in individual tanks
parallel controls
 Methods
● Before any treatment was given, the fish were kept under laboratory conditions for two
weeks
● In order to estimate the arsenic concentrations in the fish scales, the removed scales
were digested in concentrated nitric acid that was diluted by double distilled water.
○ A 1.0g scale sample was digested in 10 mL of nitric acid at 80℃ for one hour.
After the hour, 2 mL aliquots of the digested scale resultant were tested for As3+.
This was done by using hydride generation at a pH of 6.0, with sodium
borohydride being used as the reducing agent. The resultant hydride was
collected on a column and tested through coupled plasma emission
spectroscopy. The samples were tested at each duration level this way and the
spectroscopy was performed at USIC Indian Institute of Technology.
● Scales from both experimental and control groups were isolated and stained
with borax carmine and examined under a microscope
● The melanophore index was then calculated using the Hogben Method (Hogben
1942).
 Results
Table 1. Accumulation of arsenic (μ g g-1) in scales
of the fresh water fish C. punctatus.
A number of four fish were used per each exposure
group in days. The data is depicted as the mean ± the
standard error of the mean with a p-value set as less
than 0.05. An asterisk (*) denotes significant statistical
difference when compared with the control fish group.
N.D represents arsenic values that were not detected.
Figure 1. Arsenic Trioxide Concentration in the Scales of
Channa punctata
The line graph depicts the data collected and stated in Table 1. The
concentration of arsenic trioxide ( g g-1) in the scales of the Channa
punctata fish in fresh water was found at different amounts of days of
exposure ranging from 0, 7, 14, 30, 60, and 90 days. Each point on the
above graph is the arsenic concentration measured at these 6 different
exposure ranges that were previously mentioned and at day 90 there was
not enough arsenic collected to form an arsenic concentration value. At
each data point there were four fish exposed (n=4). The bars at each data
point represent the calculated standard error of the mean..

Table 2. Effect of AsIII on melanophores of C.
punctatus.
A number of four fish were used per each exposure
group in days. The data is depicted as the mean ±
the standard error of the mean with a p-value set as
less than 0.05. An asterisk (*) denotes significant
statistical difference when compared with the
control fish group.
Results
Figure 2. Melanophore Index Based on Various Arsenic Treatment
Days
The following line graph depicts the data stated in Table 2. The mean
melanophore index (MI) of freshwater Channa punctata was found at
different points in terms of the number of days exposed to arsenic in the
environment; 0, 7, 14, 30, 60, or 90 days. At each data point there were
four fish exposed (n=4). The Channa punctata fish were exposed to
arsenic for varying amounts of days and then the dermal melanophores
examined at each of those days to determine the MI through microscopic
examination. The error bars at each data point represents the standard
error of the mean of the MI calculated from the fish scales examined on
those days.
Results
Figure 3. Chromatophores of Channa
punctata After Each Duration Period; Images
1–6. From left to right starting from the upper
left hand corner, the following six images are
labeled as 1, 2, 3, 4, 5, and 6 and are all
captured at 400x magnification. Image 1
depicts reticulated chromatophores of an
unexposed fish. Image 2 depicts both
reticulated and punctuated chromatophores
after 7 days of exposure to arsenic trioxide.
Image 3 depicts only punctuated
chromatophores that are present after 14 days
of exposure to arsenic trioxide. Image 4 depicts
many reticulated chromatophores after 30 days
of exposure to arsenic trioxide. Image 5 depicts
all reticulated chromatophores after 60 days of
exposure to arsenic trioxide. Lastly, image 6
depicts punctuated chromatophores after 90
days of exposure to arsenic trioxide.
 Conclusions
● The results of this study indicate that there is not a direct correlation between
days of exposure to arsenic trioxide and arsenic concentration in scales.
● The key results from the study support the hypothesis that, the bioaccumulation
of arsenic trioxide over a long period of time in the Channa punctata fish would
lead to structural changes in the dermal melanophores of the fish. These dermal
melanophores would then be valid biomarkers for arsenic.
● These results also indicate that the fish also may have found a way to adapt to the
arsenic in water over time due to the fish scales not accumulating arsenic near the
end of the study.
● The metalloid arsenic has been shown to accumulate in fishes in the aquatic
system through uptake by gills, skin, and food sources in the arsenic
contaminated environment (Kumari, et. al 2016).
 Conclusions
• The overall change in MI was mostly due to a morphological change
• The mechanism of change may have involved alpha-MSH released from the
pituitary of the fish (Melanocyte stimulating hormone) (Sugimoto, 2002)
• MSH mediates the effects of cAMP dependent PKA (protein kinase A) and high
levels of MSH induce dispersion (Sugimoto, 2002)
• Rho family GTP binding proteins affect the organization of the microfilament actin
filaments (Sugimoto, 2002)
• Arsenic has also been shown to cause decrease in ATP production which may go
on to cause decrease in cAMP level (Kumari et. al, 2016)
• Low cAMP would limit PKA activation leading to activation of motor protein dynein
causing aggregation of pigment (Ligon and McCartney, 2016))
 Limitations
● Melanophore Index
○ It was noted that they used the Hogben Melanophore index which runs on
a scale from 1 (punctate) to 5 (reticulate). We know that this is not true
because their results indicate MI values of 22 to 54.
● Concentrations not mentioned
○ The concentration of nitric acid, sodium borohydride, and borax carmine
was not given which creates difficulties for any researcher trying to
replicate the experiment when digesting the scales in solution
● Location of Harvested Scales
○ There is very limited information on where the scales were harvested from
on the fish. We speculated that it was harvested from the dorsal side of the
fish, but it remains unknown, causing difficulty for other researchers to
replicate this experiment as it would have been a controlled variable if
location was specified.
 Future Study
• Extend this information to another model organism, Fundulus heteroclitus, in
order to generate another biomarker and examine the impacts of arsenic
bioaccumulation in other ecosystems.
• Further investigate the mechanism of melanosome dispersion in the dermal
melanophores at 90 days of exposure.
• Due to the similarities between the environments and melanophore index
observation between Channa punctata and Fundulus heteroclitus, we would like
to conduct a similar experiment to see if Fundulus heteroclitus will yield similar
results to Channa punctata.
• Observe the effects of increased amounts of arsenic concentration on
melanophores to understand how high levels of of unchecked pollution may
change the Channa punctata pigmentation.
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