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) 300ppm C) 300ppm

Enlarged Intestinal Region

Enlarged Intestinal Region

Short Tail Length

Spinal Cord Kink Spinal Cord Kinks