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Physiological Effects of Polycyclic Aromatic Hydrocarbons in the Leopard Frog,
Rana pipiens
Anna Sasser and Crystal Schulte
Department of Biology, Bradley University
Faculty Advisor: Dr. Erich K. Stabenau
A cknow ledgm ents
This research w as supported and partially financed by a Bradley U niversity Special Em phasis Student/Faculty C ollaborative R esearch Grant,
Student
U ndergraduate R esearch Funds, and a B radley U niversity Biology D epartm ent Bjorkland G rant.
Introduction
•Recent studies have suggested populations of leopard frogs are declining in the Midwest.
•For decades, pollutants such as heavy metals and organics have been detected in the Illinois River (IEPA,
1998).
•These contaminants are a result of increased industrial, agricultural, and recreational burden on the river.
Specifically, PAHs are formed during the incomplete combustion of coal, oil, gas, and garbage. PAHs are
comprised of two or more benzene rings and have a low solubility in water. As a result of their
hydrophobicity, they tend to readily absorb in the sediment, where they can remain for years (Varanasi,
1989).
•A recent inspection of Illinois River segments (Figure 1) by our lab identified PAHs in sites deemed “clean”
by the IEPA (Table 1).
•These PAHs
may cause mutations resulting in phenotypic deformities (see picture to the right).
•While such a physical deformity would likely pose difficulties for a leopard frog, it is unknown whether
PAHs affect leopard frogs on a cellular basis. It is possible that metabolic processes are altered such that they
are unable to use their muscles.
If so, such PAH exposure may result in reproductive, foraging, and
swimming deficiencies.
Objective of the Research
The objective of the experiment was to measure the contractile force of the gastrocnemius muscle of the
leopard frog,
Rana pipiens
, after electrical stimulation in control and PAH-exposed animals.
Specifically,
the goal of the study was to determine if muscle performance is limited following PAH exposure.
Figure 1.
Illinois R iver sam pling area.
Abstract
Polycyclic arom atic hydrocarbons (PA Hs) are carcinogens that our lab has found in Illinois R iver sedim ent. The consequences of
in
situ
PAH exposure have not been docum ented. Therefore, this study used in-w ater colum n exposures to determ ine the effect of PA Hs
on m uscle tissue contractility in leopard frogs.
The results show that PA Hs cause a significant decrease in m uscle contractile tension,
w hich lim its exercise tolerance.
U nder natural w ater exposures, contam inated m uscles m ay be inefficient in perform ing norm al
physiological functions, possibly com prom ising the ability of the anim al to survive.
Methods
Rana pipiens
weighing 31.7±4.8 (Mean ± SD, n=16) of either sex were used in this experiment.
Animals
were housed and cared for under approved Bradley University Institutional Animal Care and Use Committee
guidelines.
• Frogs were randomly placed into either a control tank with water or an experimental tank with a saturated
solution of pyrene, both of which were maintained at 20-25°C and completely covered in aluminum foil to
prevent photolysis of the pyrene.
The concentrated pyrene solution was replenished during the exposure
period via circulation through a reservoir of solid pyrene.
•After an acute exposure of 7 days (Weber and Janz, 2000), frogs from both treatment tanks were either
exercised until exhaustion and then anesthetized or simply taken from the exposure tank and immediately
anesthetized with 10% MS-222 dissolved in frog Ringers solution
(in mM: 111.1 NaCl, 1.9 KCl, 1.1 CaCl2,
and 2.4 NaHCO3, pH 7.5 at room temperature).
•Exhaustion was defined as the point
when the frogs were no longer able to turn themselves over after being
placed on their backs.
•Following anesthesia, frogs were pithed to avoid cerebral control of muscle contraction. The gastrocnemius
muscle (calf muscle) was subsequently isolated and bathed in frog Ringers.
•The muscle was electrically challenged with a Grass S9 Stimulator at 6 to 48 V, and the contractile force was
measured with a transducer (see photos to the right) connected to a computer data acquisition system.
•Data are expressed as mean ±SD. The data was analyzed using one-way ANOVA to determine overall
treatment effects, and post-hoc Bonferroni tests were used to compare the treatments means with a
significance level of P<0.05.
Results
•As expected, increasing the stimulus to the isolated muscle produced increased contractile force (Figures 2-5).
•At 6 V, there were no significant differences in the control versus PAH-exposed frogs in non-exercised frogs.
Exercise of control
and PAH-exposed frogs resulted in a significant contractile force (Figure 2) .
•At 12 V, contractile force of isolated muscle was not affected by PAH exposure and/or exercise (Figure 3).
•At an increased electrical challenge of 24 V, a significant decrease in contractile force was measured in frogs exercised to
exhaustion prior to electrical stimulation (Figure 4).
This suggests that severe exercise compromised the ability of the frog muscles
to respond to additional stimuli.
•At 48 V, PAH exposure produced significant decreases in contractile force in non-exercised frogs.
Thus, the PAH exposure
compromised the ability of the muscle to perform, much like that observed following severe exercise (Figure 5).
•Figures 6A-D show representative traces of the effects of continuous stimulation on the isolated frog muscle.
Clearly, PAH
exposure reduced the initial contractile force to such a significant degree that further degradation of the signal was not observed.
Similar results were seen in control and PAH-exposed frogs following severe exercise (Figures 6B-D).
•Figure 7 shows a summary of the continuous stimulation experiments for control and PAH-exposed frogs without exercise as a
function of voltage stimulus from 12 to 48 V.
PAH-exposed frogs did not exhibit as much of a change with continual stimulation at
any voltage when compared to control frogs.
Discussion
•To our knowledge, no information is available in the literature on the effects of PAHs on muscle properties in any vertebrate.
However, as shown in the representative traces, it is clear that PAH-exposed frogs did not have the energy to generate the same
contractile force as measured in control frogs and/or that the mechanical properties of the muscle were adversely affected by the in-
water PAH exposure.
•Previous studies by Horton et al. (2003) revealed that PAH accumulated in frog muscles when the whole animal was exposed to an
in-water pyrene environment.
This PAH caused activation of heat shock protein (HSP70), which is commonly activated during
periods of cellular damage.
The PAH exposure may also cause DNA adducts and inhibit proper DNA transcription (Weber and
Janz, 2000).
•Additional studies in our laboratory (Giczewski, 2002) determined that CO
2
excretion from the frog after PAH exposure was
significantly less than measured in control frogs.
These data suggested that cellular metabolism or direct tissue damage occurred as
a result of the PAH exposure.
In this study, it was possible that PAHs produced significant effects on the leopard frog at the
molecular and physiological level.
However, it is unknown whether this occured because of inhibition of cellular metabolism in
R.
pipiens
or because of direct PAH accumulation and damage at the tissues.
•Investigation of muscle metabolic enzymes will be required to elucidate the targeted system affected by PAHs.
Literature Cited
Giczew ski, D . 2002.
The effects of napthalene exposure on
Rana
pipiens
. M .S. Thesis Bradley U niversity.
Peoria, IL.
Horton, J., A . Sasser, and E. Stabenau. Illinois river project:
C ytochrom e P450 and HSP70 induction by polycyclic arom atic
hydrocarbons. 11
th
Annual Bradley U niversity Student Exposition.
2003.
IEPA C lean W ater Act Section 303(d) List-Illinois’ Subm ittal for
1998.
Illinois Environm ental Protection Agency, Springfield, IL.,
1998.
W eber, L., and D . Janz.
2000.
Effect of
beta
-napthaflavone and
dim ethylbenz[alpha]anthracene on apoptosis and HSP70 expression in
juvenile channel catfish ovary.
Aquatic T oxicology
54: 39-50.
V eranasi, U .
M etabolism of Polycyclic A rom atic H ydrocarbons in the
Aquatic Environm ent.
C RC Press, Inc:
Boca R aton, FL. 1989; pp. 10,
311-313.
S u p erim p o s itio n o f F ig u r es 6 A -D .
+P AH, +E X
0
5
1 0
1 5
2 0
2 5
3 0
0
5
1 0
1 5
2 0
2 5
Figure 6D.
Treatment with data acquistion at 200 samples/second.
Contractile force measured at 48V after PAH exposure and exercise.
Time (s)
+P AH, -E X
0
5
1 0
1 5
2 0
2 5
3 0
0
5
1 0
1 5
2 0
2 5
Figure 6C.
Data acquistion at 200 samples/second from isolated frog muscle.
Contractile force measured at 48V after PAH exposure without exercise.
Time (s)
-PAH, +EX
0
5
1 0
1 5
2 0
2 5
3 0
0
5
1 0
1 5
2 0
2 5
Figure 6B.
Data acquistion at 200 samples/second from isolated frog muscle.
Contractile force measured at 48V with exercise but without PAH exposure.
Time (s)
0
3 6
1 2
2 4
4 8
0
3
6
9
1 2
1 5
1 8
+PAH, -ex
-PAH, -ex
F i g u r e 7 .
A n a ly s i s o f th e c h a n g e i n c o n tr a c ti le f o r c e w i th c o n ti n u o u s s ti m u la ti o n
a s a f u n c ti o n o f s ti m u lu s v o lta g e i n n o n - P A H a n d P A H - e x p o s e d f r o g s .
V o lt a g e
1
2
3
4
0
3
6
9
1 2
1 5
1 8
-PAH, -ex
+PAH, -ex
-PAH, +ex
+PAH, +ex
*
*
Figure 2. Contractile force (Mean
±
SD) as a function of four treatments with an
electrical stimulus of
6V. The treatments are animals reared in the absence or
presence of PAH, with no exercise or exercise provided the day of force
measurement.
The asterisks represent signicant dif erence from non-exercised,
control frogs (-PAH, -ex).
Treatment
1
2
3
4
0
3
6
9
1 2
1 5
1 8
-PAH, -ex
+PAH, -ex
-PAH, +ex
+PAH, +ex
*
*
*
Figure 5. Contractile force (Mean
±
SD) as a function of four treatments with an
electrical stimulus of
48V. The treatments are animals reared in the absence or
presence of PAH, with no exercise or exercise provided the day of force
measurement. The asterisks represent signicant dif erence from non-exercised,
control frogs (-PAH, -ex).
Treatment
1
2
3
4
0
3
6
9
1 2
1 5
1 8
-PAH, -ex
+PAH, -ex
-PAH, +ex
+PAH, +ex
Figure 3. Contractile force (Mean
±
SD) as a function of four treatments with an
electrical stimulus of
12V. The treatments are animals reared in the absence or
presence of PAH, with no exercise or exercise provided the day of force
measurement. The asterisks represent signicant dif erence from non-exercised,
control frogs (-PAH, -ex).
Treatment
1
2
3
4
0
3
6
9
1 2
1 5
1 8
*
*
-PAH, -ex
+PAH, -ex
-PAH, +ex
+PAH, +ex
Figure 4. Contractile force (Mean
±
SD) as a function of four treatments with an
electrical stimulus of
24V. The treatments are animals reared in the absence or
presence of PAH, with no exercise or exercise provided the day of force
measurement. The asterisks represent signicant dif erence from non-exercised,
control frogs (-PAH, -ex).
Treatment
0
5
1 0
1 5
2 0
2 5
3 0
0
5
1 0
1 5
2 0
2 5
- P A H , - e x
- P A H , + e x
+ P A H , - e x
+ P A H , + e x
T im e ( s e c )
C
o
n
t
r
a
c
t
i
l
e
F
o
r
c
e
(
g
r
a
m
s
)
C
o
n
t
r
a
c
t
i
l
e
F
o
r
c
e
(
g
r
a
m
s
)
C
o
n
t
r
a
c
t
i
l
e
F
o
r
c
e
(
g
r
a
m
s
)
C
o
n
t
r
a
c
t
i
l
e
F
o
r
c
e
(
g
r
a
m
s
)
C
o
n
t
r
a
c
t
i
l
e
F
o
r
c
e
(
g
r
a
m
s
)
C
o
n
t
r
a
c
t
i
l
e
F
o
r
c
e
(
g
r
a
m
s
)
-P AH, -E X
0
5
1 0
1 5
2 0
2 5
3 0
0
5
1 0
1 5
2 0
2 5
Figure 6A.
Data acquistion at 200 samples/second from isolated frog muscle.
Contractile force measured at 48V without exercise or PAH exposure.
Time (s)