1. Caffeine affects early development and mortality in Xenopus tadpoles
Marissa Haeny
Department of Biology, Gustavus Adolphus College Discussion
Increased caffeine concentration retards early growth
The absence of significant differences in body lengths 120
hours after treatment partially supports the original hypothesis.
Caffeine concentration affects mortality, anatomy, and
physiology
The 0-200ppm and 300ppm groups had survival rates of 94%
and 73.07%, respectively. These significant differences among
concentrations for mortality and deformation suggest that caffeine
has negative impacts on Xenopus tadpole development,
supporting the hypothesis.
At 300ppm, only 46.15% of tadpoles developed normally.
Developmental anomalies included kinked spinal cords, enlarged
intestinal regions, and abnormally short body lengths. The drill-like
swimming motion at 300ppm was likely caused by deformed
spinal cords. In rainbow trout, this is known as whirling disease
and is proposed to result from spinal cord constriction3. The
increased caffeine concentration could be causing a similar
process to occur, leading to the same behavior.
Low doses of caffeine can decrease heart rates, explaining the
lower observed rates in the 50ppm and 100ppm groups4. Doses
of caffeine higher than 150ppm can increase heart rates,
explaining the rates seen in the 200ppm and 300ppm groups5.
Acknowledgements
I would like to thank Margaret Bloch-Qazi for her assistance and guidance
throughout this experiment and Maureen Carlson and the Department of Biology at
Gustavus Adolphus College for supplying materials and funding necessary for my
research. This experiment was approved by the Animal Care Review Board.
Methods
Xenopus tadpoles in stages 41-44 developed in varying
caffeine concentrations. The control group developed in water
with 0ppm of caffeine, and the four treatment groups developed in
water with 50ppm, 100ppm, 200ppm, and 300ppm of caffeine,
repectively, at room temperature with ambient light. ~25 tadpoles
were in each group. 0ppm mimics clean drinking water, 50ppm
mimics decaffeinated coffee, 100ppm mimics green tea, 200ppm
mimics black tea, and 300ppm mimics a latte2.
Tadpoles were imaged every 48 hours using a dissecting
microscope and Motic Imaging software, starting 24 hours after
treatment and ending 120 hours after treatment. Measurements
were made using ImageJ software. Body length was defined as
the distance between the snout and the end of the tail. Qualitative
observations were whether or not an tadpole was dead or
deformed. The independent variables were the hours after
administering treatments and the caffeine concentrations.
Dependent variables included body length and the proportion of
tadpoles that died or were deformed.
Differences in body length in the different concentrations at
each of three time points were tested using ANOVAs, and the
proportion of dead or deformed tadpoles among concentrations
were tested using Chi-squared tests.
Introduction
A teratogen is an agent that disrupts normal development.
Caffeine is a common teratogen and has been studied in a wide
variety of model systems, including Xenopus.
Xenopus is a frog species that has been a model system for a
wide variety of developmental and genetic studies. They fertilize
externally, making their development easy to observe, and share
many genetic similarities with humans.
This experiment investigated the effects of caffeine on early
Xenopus tadpole development. Many people consume caffeine
daily, including pregnant women. Because caffeine is readily
absorbed from the digestive tract and freely crosses the placenta,
fetuses can be exposed to caffeine at slightly lower
concentrations than the maternal ingestion concentration1.
Understanding caffeine’s developmental effects in frogs can
inform our understanding of it’s impacts human fetal development.
This experiment’s first hypothesis is that caffeine concentration
affects body length, with tadpoles in higher concentrations having
shorter average body lengths. The second hypothesis is that
caffeine concentration affects normal development, with a positive
relationship between concentration and abnormal tadpole
development existing.
Literature Cited
1. Mihaly GW, Morgan DJ. 1983. Pharmac. Therapeutics. 23(2):253-266.
2. Fenster L, Eskenazi B, Windham GC, Swan SH. 1991. Am. J. Public Health. 81:458-461.
3. Rose JD, Marrs GS, Lewis C, Schisler G. 2000. J. Aquat. Animal Health. 12(2):107-118.
4. McClaran SR, Wetter TJ. 2007. J. Intern. Soc. Sports Nutrit. 4(11).
5. Daniels JW, Molé PA, Shaffrath JD, Stebbins CL. 1998. J. Appl. Physio. 85(1):154-159.
Broader Implications
The 300ppm group was exposed to the same caffeine
concentration as an 8oz cup of coffee. Similar impacts could be
seen on human development if a fetus is exposed to 300ppm of
caffeine continuously. This equates to constant maternal ingestion
of a latte for the duration of the pregnancy1. While this is highly
unlikely, studies have shown that high rates of maternal caffeine
consumption can increase the risk of low birth weight and
intrauterine growth retardation2. Maternal ingestion of over
150ppm of caffeine could also negatively affect fetal heart beats
and development. If caffeine concentration in beverages
increases, the risk of detrimental effects to human fetal
development increases.
Caffeine concentration affects mortality, anatomy, and physiology
There were significant differences between 0-200ppm and 300ppm of caffeine for mortality and deformation (χ2 test,
xmortality=9.78, pmortality<0.005, xdeformation=23.335, pdeformation<0.001, dfall=1) (Figs. 3, 4). Deformations included improperly formed spinal
cords, enlarged intestinal regions, and abnormally short body length (Fig. 4). At 72 and 120 hours post-treatment administration
in the 300ppm treatment group, tadpoles’ tails were whipping in circular motions, propelling them in circles. Elevated heart rates
were observed in the 200ppm and 300ppm groups. At all time points, blood was circulating faster and hearts were beating faster.
Figure 1: Images of normally
developing Xenopus tadpoles
magnified 16x, taken 24 hours
after applying the caffeine
concentrations. The distance
between black lines in each
image is 1mm.
A) 0ppm B) 50ppm
C) 100ppm D) 200ppm
E) 300ppm
*
*
Figure 3: The proportion of dead or deformed Xenopus tadpoles as a
function of caffeine concentration. The 300ppm group had a higher
proportion of dead and deformed tadpoles than the 0-200ppm group.
Statistically significant differences are denoted with an asterisk (*).
Figure 2: The relationship between time post-treatment and average body length
(mm, +/- SE) of Xenopus tadpoles. Significant differences between all
concentrations existed at 24 and 72 hours after treatment, but not at 120 hours.
Statistically significant differences among treatments within a time period are
denoted with an asterisk (*).
Time post-treatment administration (hrs)
Increased caffeine concentration retards early growth
There were overall significant differences in body length among concentrations at 24 and 72 hours after treatment (One Way
ANOVA, F24 hours=247.544, F72 hours=111.133, p24 & 72 hours<0.001) (Figs. 1, 2). At 120 hours after treatment, there were no significant
differences in body length between the caffeine concentrations (One Way ANOVA, F120 hours=1.091, p120 hours=0.365) (Fig. 2).
Results
Figure 4: Images of Xenopus tadpoles, magnified 16x, with
developmental anomalies. The distance between black lines in
each image is 1mm. A) Left side view of a tadpole showing an
enlarged intestinal area 120 hours post-treatment administration.
B) Right side view of a tadpole exhibiting an enlarged intestinal
area and minimal tail growth 24 hours after treatment. C) Dorsal
view of a tadpole with a kinked spinal cord 72 hours after
treatment administration. D) Dorsal view of a tadpole with a
kinked spinal cord 120 hours post-treatment administration.
A) 100ppm B) 300ppm
D) 300ppmC) 300ppm
Enlarged Intestinal
Region
Enlarged Intestinal
Region
Short Tail Length
Spinal Cord Kink Spinal Cord Kinks