The Effects of Zebrafish Development Caused by Caffeine Exposure

Transcription

1 The Effects of Zebrafish Development Caused by Caffeine Exposure By Haley Gill Waukesha North High School Abstract In this study. 40 zebrafish embryos were raised in different amounts of a solution in their environment. (1.0 mg/ml, 0.05 mg/ ml, 0.25 mg/ml,0 mg/ml). The zebrafish are model organisms for humans because 70% of protein coding is found in both humans and zebrafish. This shows that zebrafish are similar to humans because of their response to stimuli. Caffeine is a drug found in many drinks and medicine. It can interfere with your body in many ways, such as heart defects, dehydration, lower birth weight, long term memory, and more. These can happen by consuming too much caffeine. It's important to know the effects on developing embryos so we can relate it to the effects it has on developing human. The purpose of this experiment is to show how caffeine affects zebrafish embryos embryos by recording deformities in the zebrafish with caffeine in it. The zebrafish in the higher amount of caffeine in show more defects in them and were most likely to die. Zebrafish are similar to humans, therefore they were used as a model organism instead of mice. The experiment we did relates to the health of humans because it can show us how it affects the zebrafish and we can assume that it can have the same effect on us. Over time the zebrafish had deformities and their body curved so we and make a connection and assume that if human embryos are introduced to caffeine while in the womb, the baby would come out of the womb deformed.

2 Introduction Caffeine is a drug found in coffee, tea, soda, chocolate, some energy drinks, and medicine. Caffeine is a stimulate which helps you stay awake and caffeine has many effects on the human body. For an example, an article quotes, At the cellular level, caffeine blocks the action of a chemical called phosphodiesterase (PDE), (scientificamerican.com). It also stimulates your central nervous system, which can make you feel awake and a boost of energy. It is also a diuretic, meaning that it helps your body get rid of extra salt and water by urinating more. It may interfere with the absorption of calcium in your body and it also increases the release of acid in your stomach, sometimes resulting in an upset stomach or heartburn. Large amounts of caffeine have been associated with heart defects in a very small number of babies. Large amounts of caffeine have also been related to lower birth weight and an elevated risk of spontaneous abortion, (psychiatry.emory.edu). For most people it is not harmful to consume 400 mg of caffeine a day, but if you consume too much caffeine it can result to restless or shakiness, rapid or abnormal heart rhythm, dehydration, insomnia and many more things. Michael Yassa, an assistant professor of psychological and brain sciences at Johns Hopkins, and his team of scientist found that caffeine has a positive effect on our long term memory. Their research shows that caffeine enhance certain memories at least up to 24 hours after it is consumed. Scientists at Johns Hopkins conducted a double-blind trial in which the participants who did not usually consume caffeine ate or drank caffeinated products, received a 200 milligram caffeine tablet 5 minutes after studying pictures. Salivary samples were also taken to determine their caffeine levels. They did the samples before 1, 3, and 24 hours after consuming the tablet. The next day groups were tested on their ability to recognize those images from the

3 day before. The pictures were different, similar, the same, or new. More people on the caffeine side were able to correctly identify the pictures. The quote, The brain's ability to recognize the difference between two similar but not identical items, called pattern separation, reflects a deeper level of memory retention, the researchers said. from explains that the caffeine is actually good for your long term memory and like it said it goes deeper in the level of memory retention. Which concludes to which if caffeine is consumed then you could retain more information while studying and the information would be kept in your long term memory. The study above can give insights as how humans respond to caffeine. The objective of the experiment is to see the relationship between zebrafish and the human body development. The experiment will also give us a deeper knowledge on how caffeine can affect an embryo which we could connect to how caffeine would affect a baby fetus in which the mother consumes caffeine. It can be hypothesized from this experiment that if the zebrafish embryos were exposed to more caffeine or higher concentrations of it it can cause deformities or deaths. Materials & Method The materials that were used in this project were 40 zebrafish, stock solutions for caffeine (0.0,0.05, 0.25, 1.0 mg/ml caffeine), a small beaker for dead embryos and liquid disposal, a sharpie, tape, instant ocean/embryo media solution (1 bottle), disposable pipette, 1 ml, minimum bore, 1.5 mm for transferring eggs to observation container and manipulating them in the container (1 used each day), a plate with six wells, a 28.5 C incubator, a dissecting

4 microscope, a phone for taking pictures, and a work/data sheet for each person in the group to record data on. First, the well plate was labeled with every group member s name and class hour with a sharpie on a piece of tape. Each well on the plate was also labeled with the stock solution number of caffeine (0.0, 0.05, 0.25, 1.0 mg/ml). Next, a disposable pipette was used to place ten zebrafish eggs into the four wells in the plate with caffeine concentrations labeled on them. The correct concentrations of caffeine were placed into wells using a disposable pipette. Gloves were worn to protect the skin while placing the caffeine solutions in the wells. After, the embryos were observed under a microscope. All the observations were recorded down on the data sheet. Observations included zebrafish characteristics like the color and shape. For an example, a cloudy embryo (discoloration) usually meant it was dead. Quantified data was also recorded, such as number of zebrafish alive, dead, or hatched. Any movement and deformities were also written down. This data was also recorded by taking pictures of the zebrafish under the dissecting microscope. After Day 1, the plate was placed into a 28.5 C incubator overnight. After 24 hours, the plate was taken out of the incubator to record more data and make more adjustments. How many eggs has hatched and how many are dead or alive were recorded. More pictures were also taken under the dissecting scope. After observations were made, another pipette to take out any dead embryos or fish along with the solution and put it in the waste beaker. Then, fresh solutions of caffeine were replaced with the alive zebrafish in the wells of the plate. To protect themselves from the concentrations, gloves were worn again. Then, the plate would be placed back into the incubator for 24 hours. Lastly and most importantly, any mess would be cleaned up and hands were washed. This process would continually repeat for the rest

5 of days so any changes would be recorded. But, Day 5 was a different procedure because it was the last day of recording data. Instead of replacing the previous days solutions of caffeine with fresh ones, all the zebrafish were put back into the nursery (the class tank) for the fish to grow, using a pipette. A chi square analysis was performed to check for statistical significance of data. Results This experiment was conducted to figure out how the exposure of caffeine to zebrafish would affect them and see how it also could affect the human body development in the same way. It was hypothesized that the zebrafish with the highest concentration of caffeine would be most affected. For this experiment, it was set up in a way where you could figure out the differences in the zebrafish contained in the different concentrations. Zebrafish were separated into their own wells and in each well was a different concentration of caffeine making the independent variable the amount of caffeine. The dependent variables would be the number of fish that hatched, lived, or died. The control that the other concentrations were compared against was the 0.0 mg/ml. Controlled variables for this experiment was the well plates, temperature, and the 10 embryos in each well. These all relate to each other because when the concentration was higher in each well, the number of death of zebrafish increased. Zebrafish started to hatch after the 48 hours of post fertilization. All of them lived after the 48 hours but some died off after 72 hours of post fertilization. After these 72 hours, it was noticed that there were some deformities. For an example, the shape of the body was starting to curve inward. This happened in the 0.25 mg/ml concentration the control solution had no

6 deformities. It was also noticed the embryos in the 1.0 mg/ml solution had shorter tails, another observation made was that the yolk sacs of the zebrafish also seemed smaller than it was when they were first hatched. The size of the yolk sacs were different between the solutions 0.25 mg/ml and 1.0 mg/ml. Tabel 1: Hatched and live zebrafish throughout 72 hours Treatments Well # # Of Starting Fish 24 Hour Post Fertilization 24 Hour Post Fertilization 48 Hours Post Fertilization 48 Hour Post Fertilization 72 Hours Post Fertilization 72 Hour Post Fertilization # Hatched # Live # Hatched #Live # Hatched # Live Control (0.0) mg/ml mg/ml mg/ml On this table it shows the well numbers with what concentration was in that one. Also shows how many fish are alive and hatched in 24, 48, and 72 hours, with a starting number of 10 fish. The chi square statistical analysis chart was used to find out if deformities or deaths in the zebrafish was caused by the caffeine. All the probability of the chi squares were greater than the critical value of The probability for the alive zebrafish is 19.2 and the probability for the dead zebrafish is This means that the caffeine did have an affect and that the deaths and deformities were caused by caffeine. This also supports our hypothesis on how it can affect zebrafish and other experiments done. Figure 1;

7 As shown in figure 1, after 48 hours with the concentration of 0.0 mg/m/l, 0.05 mg/ml, and 0.25 mg/ml all the fish were alive but in the 1.0 mg/ml concentration two fish died and eight were alive. This means that the caffeine is starting to affect the fish but not all of them. Figure 2;

8 As shown in figure 2, after 72 hours, in the concentration 0.0 mg/ml all ten fish are still alive. But in 0.05mg/mL, 0.25mg/mL, and 1.0 mg/ml two died in each well and eight stayed alive in each one. This means that the caffeine affected three of the four wells. Figure 3;

9 As shown in figure 3, after 94 hours all 10 fish in 0.0 mg/ml were still all alive. But in 0.05 mg/ml, three died and seven were alive. In 0.25mg/mL, three also died and seven were alive. And in 1.0mg/mL, two died and eight are still alive. This summarizes that caffeine does have an effect on the zebrafish. Figure 4;

10 Figure 4 is the snapshot from the the first day. None of the zebrafish are hatched but they are all living, you can tell they are living because the eggs are not white they are clear. Figure 5;

11 Figure 5 is a snapshot of a hatched, living zebrafish up close. This was taken on day 2. This fish was just starting to hatch, you can see how it is coming out the the egg. Figure 6;

12 Figure 6 is a snapshot of a hatched, living, zebrafish that is deformed. This was taken on day 3. It shows how the caffeine is affecting this fish because it is starting to curve and become deformed. Figure 7;

13 Figure 7 was taken on day 4. In this you notice that multiple zebrafish are deformed because of the caffeine. Discussion

14 The study on caffeine provided results that concluded to the effects on the zebrafish physically. The hypothesis was, that if the Zebrafish embryos were exposed to more caffeine or higher concentrations, it can cause deformities. Previous experiments have found that caffeine can affect pregnant women and its fetus. It can also affect pregnant mice which resulted to the pups having brain changes and lasting memory defects. The zebrafish that was put into the 0.25 mg/ml had three dead zebrafish at the end of day 4. But no zebrafish died in the control treatment after day four. This could relate to the experiment with pregnant mice because it was found that the mice who were given caffeine had lasting effects on their memory and there was also an increased risk of seizures. So we could imply from the results of the experiment to how it could affect a baby fetus that gets exposed in the womb. It was found the larger doses of caffeine have been associated with heart defects in a very small number of babies. It also has been related to lower birth weight and an elevated risk of spontaneous abortion. Caffeine can have a major effect. This experiment could have be more accurate if there was more time given. Longer experiment time could have shown more defects and deformities in the zebrafish which would give more research to prove the hypothesis. Also it could've been more accurate if it was runned more times and with higher solutions of caffeine and more zebrafish. This would have provided more accurate probabilities. These ways to improve the experiment could also help to predict what will happen in the future. There was also some notes and errors. There were two types of wells that were used, both needed different amounts of solution in each well and that could affect how deformed or normal the zebrafish turned out concluding to different results. (5 mg/ml or 3 mg/ml) When recording data under the dissecting scope, the number of dead, hatched, or alive,

15 had to be accurate. If it wasn't accurate then it would be hard to prove your hypothesis or research question. Also, if you messed up with what solution to put in what well, then that could have messed up the whole experiment because it wouldn't be credible. Though there were limitations in the project, there are still some questions that haven't been answer. Such as, relating more the the research about the pregnant mice. It was stated that the mice had brain deformities but with zebrafish you wouldn't be able to do research on their brain or anything internally. To conclude, zebrafish were more likely to have more deformities in deaths in higher concentrations of caffeine. This was proven by the experiment that was performed in class and in other studies with pregnant mice. There were limitations and ways this experiment could have been improved. Zebrafish is a good entity to show humans the discouraging truth about the chemical that is consumed by many everyday, caffeine. Another experiment that could be done is on teen and adult Zebrafish to understand how caffeine affects not only embryos, but also developed Zebrafish. Cited Sources Create a graph. (n.d.). Retrieved December 19, 2017, from

16 Howell, Karen Kuehn (January 2005 ) Ten most frequently asked questions about consuming caffeine during pregnancy. Retrieved December 18, 2017, from Latarsha Gatlin (2014, January 12). Caffeine has positive effect on memory, Johns Hopkins researchers say. Retrieved December 18, 2017, from Smatresk, Neal J. How does caffeine affect the body? (n.d.). Retrieved December 18, 2017, from