Nicotine and Epinephrine Impair Heart Function and Cause Premature Death in Daphnia magna

Nicotine and Epinephrine Impair Heart Function and Cause Premature Death in Daphnia magna

Ingrid Schoonover
General Biology II
February 15, 2019

Abstract

The Daphnia magna is a widely used model organism for studying the effects of pharmaceutical agents, especially those present in aquatic ecosystems. Here, we show that Daphnia magna is excellent for the use of studying the long-term toxicity of these agents and their effect on heart function. A 10uM epinephrine solution decreased the heartrate beat by 78% over one hour and induced arrhythmia. 5uM had no effect on heart rate (0.7% increase over 48 hours). 40uM nicotine solution decreased heartbeat rate by 227.7% over one hour. 5uM nicotine solution decreased heartbeat rate by 80.2% over 48 hours. Nicotine and epinephrine act on Daphnia to alter heartbeat rate, increase blood pressure, as well as to induce vasoconstriction and arrhythmia. It was concluded that long term exposure to nicotine and epinephrine does not increase the heartbeat rate in Daphnia magna, but eventually results in a stress-induced death as a combined result of high blood pressure and exposure to toxic chemicals.

Introduction

Model organisms such as Daphnia magna are used in laboratories to perform experiments that would be unethical in humans. They are ideal organisms for studying the effects of heavy metals12 and pharmaceuticals10 because of their short lifespan and ability to reproduce quickly. They have a transparent carapace that allows for the monitoring of their organs, which allows for the counting of their heartbeat rate1. Daphnia are very sensitive to environmental stress, this makes them an excellent indicator species, which is an organism that represents the health of the overall environment1. The Daphnia genome has been sequenced6 which allows for human/daphnia homologies to be identified. Through identifying structural and molecular homologies experimental results can be used to make predictions in other species.

Pharmaceuticals and chemicals have become an intricate part of human life, they are used recreationally, medically, industrially, and agriculturally. Much of these chemicals are new and the entirety of their effects are still unknown, which is why it is important to explore their impact on the health of model organisms. Whether chemicals are dumped into the soil or released into the atmosphere most will eventually find their way into bodies of water, over time these chemicals have the potential to accumulate(1).

The most common(9) trash item collected on beaches are cigarette filters, which constitute 19.1%(9) of all items collected in the 1997 International Coastal Cleanup Project. Humans smoke cigarettes for the recreational purpose of a nicotine high. Nicotine (C10H14N2) is a highly addictive central nervous system stimulant(7) that is absorbed(9) through the respiratory tract, buccal mucous membrane, skin, and gastrointestinal tract. Nicotine is an agonist(7), it activates nicotinic cholinergic receptors(7) (a type of ionotropic receptor) found on muscle-cell membranes and neurons within the presynaptic terminals. This results in the opening of ion-conducting(6) channels which allow cations(6) to pass through indiscriminately. In the liver, lungs, gut, and kidney 80-90% of the nicotine is oxidized into 209 metabolites, the remaining product is eliminated in urine. Nicotine is a toxin and has numerous adverse effects on human health(7) including increased heart rate, vasoconstriction, increased blood pressure, spontaneous abortions(7), and birth defects in children. These long term effects can be ethically examined by using Daphnia magna as a model organism.

Epinephrine (C9H13NO3) is a hormone that occurs naturally in many species but it has particular use in the medical field(8) for resurrections after cardiac failure, relaxing smooth muscles, increases the ease of breathing, and increasing heart rate. Epinephrine is an agonist, acting on the sympathetic nervous system by stimulating alpha, beta 1, and beta 2 adrenergic receptors(5) located throughout the cardiovascular system. Similar to nicotine, this opens ion-conducting channels altering the influx and efflux of cations(5). In low doses it appears that epinephrine can have beneficial effects, however, it increases the demand for oxygen(8) which can lead to hypoxia, and overdoses can be lethal resulting in cerebrovascular hemorrhage(8). For these reasons a greater understanding of epinephrine would benefit the medical community, this information can be gathered from studying Daphnia magna.

While the anatomy of the Daphnia differs from humans similarities can be noted by the presence of a brain, digestive system, and heart(1). Additionally, Daphnia magna have a number of receptor proteins, including ionotropic receptors(4), beta-adrenergic receptors, and alpha-adrenergic receptors. When nicotine is present in Daphnia’s aquatic environment(4) the molecules permeate their carapace and activate ionotropic receptors.  Similarly, epinephrine in Daphnia’s aquatic environment permeates their carapace and activates beta and alpha-adrenergic receptors. Then biomolecules involved in ion transport(6) and proteolytic processes(6) metabolize the drugs similar to humans. Daphnia sp. possess degrading enzymes(4) to cleave the agonist from the receptors and membrane transporters(4) to remove the chemicals and their metabolites. Chemicals are excreted(12) by transfer to offspring, in fecal matter, and through molting.

This experiment will be using 24 Daphnia magna to model the short-term and long-term health effects of exposure to 10uM epinephrine, 5uM epinephrine, 40uM nicotine, and 5uM nicotine. Daphnia will be separated into three groups: control, treatment group 1 (epinephrine), and treatment group 2. It is standard to keep daphnia in well(11) plates(3) for toxicological experiments so they will be kept in 12 well plates. Nicotine has been widely accepted as highly toxic to Daphnia in other experiments, especially to neonates and embryos(3). Neonates grown in nicotine solutions experienced an increased number of mutations(3). It has been proposed that the toxicity of nicotine can be attributed to its ability to mimic acetylcholine, consequently, disrupting neurotransmission within the neuromuscular junction(3). Similarly, but very few experiments have been conducted with Daphnia to determine the effects of epinephrine. In one experiment a group of Daphnia were exposed to 100uM epinephrine, it increased their heartbeat rate by 10-15% within 5-15 minutes(2). This study confirmed that pharmacological agents in an aquatic environment interfere with the ion channels and cell signaling within Daphnia. Although these experiments have been conducted with Daphnia, none have involved studying the effects of long-term exposure or drug exposure at these concentrations so there is still much to learn from using them as a model organism.

Hypothesis: Exposure to nicotine and epinephrine increases the heartbeat rate in Daphnia magna and eventually results in a stress-induced death as a combined result of high blood pressure and exposure to toxic chemicals.
Null Hypothesis: Exposure to nicotine and epinephrine does not increase the heartbeat rate in Daphnia magna and eventually results in a stress-induced death as a combined result of high blood pressure and exposure to toxic chemicals.

Methods and Materials

For the first trial, three solutions were prepared: water (control), 10uM epinephrine (treatment 1), 40uM nicotine (treatment 2). A 12 well plate was obtained and 4mL of the appropriate treatment was added. The control was added to row A, treatment 1 was added to row B, and treatment 2 was added to row C. One healthy adult Daphnia magna was added to each of the 12 wells. Once all the Daphnia were placed in their appropriate wells their baseline vitals were recorded as time 0. This was achieved by observing the Daphnia under a microscope and counting their heartbeat rate and movement. The presence of any neonates was also noted in addition to the adult’s sex. These measurements were repeated and recorded at the time intervals of 0.5 hours, 1 hour, 24 hours, and 120 hours.

For the second trial, three solutions were prepared: water (control), 5uM epinephrine (treatment 1), and 5uM nicotine (treatment 2). The wells were prepared in the same manner as in trial 1. Measurements were recorded in the same manner as trial 1 at time intervals of 0 hours and 48 hours.

The data from both trials was collected and organized into tables and graphs for purposes of analysis.
To quantify the changes in heartbeat rate the percent change equation was used:

Results

Table 1: Displaying the raw data for trial 1 of drug treatments. Showing that 40uM nicotine was toxic and killed the Daphnia within 0.5 hours. 10uM epinephrine was toxic, altering the heart contractions of the Daphnia and killing them within 24 hours. The control treatment killed all daphnia by 120 hours.

At concentrations of 40uM, the nicotine solution proved to be highly toxic to the Daphnia, all 4 test subjects had died by the time interval of 0.5 hours. Daphnia exposed to epinephrine at concentrations of 10uM survived as long as the control group but experienced a much slower heartbeat rate. Additionally, it caused visible effects such as lethargic and erratic heart contractions.

Table 2: Displaying the raw data for trial 2 of drug treatments. Showing that the population was unhealthy to begin with. Also that a concentration of 5uM nicotine was toxic and killed the Daphnia within 48 hours. 5uM epinephrine caused Daphnia to move erratically and killed 50% within 48 hours. The control treatment killed 75% of the Daphnia within 48 hours.

The nicotine solution from trial one was obviously too toxic so for trial two the concentration was diluted to 5uM. The epinephrine solution was also diluted to 5uM in hopes to gain more insight. Unfortunately, this batch of Daphnia was in worse health than those in trial one, many showing signs of near-death before the experiments were able to begin. Even at this concentration, the nicotine solution was still extremely toxic to the Daphnia. The effects of epinephrine were less noticeable at this concentration, they behaved very similarly to the control group.

Graph 1: Displaying the average heartbeat rates of Daphnia magna over time, after exposure to the control, epinephrine, or nicotine solution. Showing that nicotine concentrations reduce the heartbeat rate by the greatest amount and that higher concentrations of either drug treatment reduce the heartbeat rate quicker.

The average heartbeat rates at each time interval were calculated by adding the heartbeat rates of all the daphnia within each treatment group for each trial (control, nicotine, and epinephrine) and dividing that value by 4. Viewing the data in this manner ensures that variations between individuals do not discredit the data. In trial one, the average heartbeat rate for the control group at each time interval was 154bpm, 118.5bpm, 107bpm, 34bpm, and 0bpm respectively. The average heartbeat rate for the 10uM epinephrine group at each time interval was 162bpm, 145bpm, 74bpm, 0bpm, and 0bpm respectively. The average heartbeat rate for the 40uM nicotine group at each time interval was 156bpm, 0bpm, 0bpm, 0bpm, and 0bpm respectively. In trial two, the average heartbeat rate for the control group at the time intervals was 90bpm, and 40bpm respectively. The average heartbeat rate for the 5uM epinephrine group at the time intervals was 152bpm, and 67bpm respectively. The average heartbeat rate for the 5uM nicotine group at the time intervals was 86bpm, and 0bpm respectively.

Graph 2: Displaying the fatality rates of Daphnia magna over time after exposure to the control, epinephrine, or nicotine. Showing that nicotine is the most toxic and that control is the least toxic.

The fatality rate at each time interval was calculated from the number of deceased daphnia within each treatment group for each trial (control, nicotine, and epinephrine). In trial one, the average fatality rate for the control group at each time interval was 0%, 0%, 25%, 75%, and 100% respectively. The average fatality rate for the 10uM epinephrine group at each time interval was 0%, 0%, 0%, 100%, and 100% respectively. The average fatality rate for the 40uM nicotine group at each time interval was 0%, 100%, 100%, 100%, and 100% respectively. In trial two, the average fatality rate for the control group at the time intervals was 25%, and 75% respectively. The average fatality rate for the 5uM epinephrine group at the time intervals was 0%, and 50% respectively. The average fatality rate for the 5uM nicotine group at the time intervals was 50%, and 100% respectively.

Graph 3: Displaying the heartbeat rate decreases in Daphnia magna within 1 hour of drug exposure for trial #1 and within 48 hours for trial #2. Showing that in trial #2 the control group experienced a larger heartbeat rate decrease than in trial #1. Calculated from the percent change equation.

To determine how the heartbeat rate of Daphnia changed during exposure to each drug treatment group percent change was calculated. These values demonstrate the change in Daphnia heartbeat rate between a time interval and their initial baseline measurements. In trial one, within one hour, the heartbeat rate of the control group decreased by 30.5%, a 54.3% decrease for the 10uM epinephrine group, and a 100% decrease for the 40uM nicotine group. In trial two, within 48 hours, the heartbeat rate of the control group decreased by 55.5%, a 55.9% decrease for the 5uM epinephrine group, and a 100% decrease for the 5uM nicotine group.

Graph 4: Displaying the heartbeat rate change from the control group in Daphnia magna within 1 hour of drug exposure for trial #1 and within 48 hours for trial #2. Showing that higher concentrations of drug treatments had a greater effect on heartbeat rate than lower concentrations. Calculated from the percent change equation.

To account for external factors contributing to a heartbeat rate decrease in Daphnia during the experiments, the percent change in respect to the control was calculated. These values demonstrate the change in Daphnia heartbeat rate after drug exposure which can be solely attributed to the drug treatment. In trial one, the Daphnia within the 10uM epinephrine group experienced a 78% decrease in heartbeat rate in relation to the control, and the Daphnia within the 40uM nicotine group experienced a 227.7% decrease in heartbeat rate in relation to the control. In trial two, the Daphnia within the 5uM epinephrine group experienced a 0.7% increase in heartbeat rate in relation to the control, and the Daphnia within the 5uM nicotine group experienced an 80.2% decrease in heartbeat rate in relation to the control.

Discussion

In this experiment, Daphnia magna was used as a model organism to study how heartbeat rate is effected over time by two pharmacological agents, nicotine and epinephrine. The Daphnia were exposed to treatment solutions containing a control, 10uM epinephrine, 5uM epinephrine, 40uM nicotine, or 5uM nicotine, and then heart rate was measured over the course of several hours.

The findings show that the control from trial one experienced a 30.5% decrease in heartbeat rate in the first hour of the experiment (Graph 3). This decrease in heartbeat rate can be attributed to stress from handling, decreased water volume, and light exposure during observation. Since this group was not exposed to drug treatment, this value can be compared to the decreased heartbeat rates for exposure to 10uM epinephrine and 40uM nicotine to determine the change from normal. Although some of the Daphnia magna initially increased their movements (Table 1) in response to the 40uM nicotine treatment, their heartbeat rate slowed down by 100% (Graph 3) indicating the initial boost in activity was a shock response. Their consequential death suggests that nicotine is highly toxic to Daphnia magna. Shortly after exposure to the 10uM epinephrine treatment, the daphnia began moving erratically (Table 1). Their heart contractions were irregular (arrhythmia) suggesting that the drug was acting directly on the heart. With time they stopped moving and their heartbeat rate decreased by 54.3% (Graph 3). With respect to the control group, this is a 78% decrease (Graph 4). They also experienced a higher fatality rate than the control (Graph 2). Thus, it can be concluded that a 10uM concentration of epinephrine decreases the heartbeat rate of Daphnia magna.

Additionally, the control group from trial two experienced a 55.5% decrease in heartbeat rate in the first 48 hours of the experiment (Graph 3). This decrease in heartbeat rate can be attributed to stress from handling, decreased water volume, hypoxia, and light exposure during observation. Since this group was not exposed to drug treatment, this value is compared to the decreased heartbeat rates in Daphnia exposed to 5uM epinephrine and 5uM nicotine, to determine the change from normal. Daphnia exposed to the 5uM epinephrine treatment were hypoxic as a result of increased oxygen demand, and their heartbeat decreased by 55.9% (Graph 3) throughout the experiment. Although, with respect to the control group this is actually a 0.7% increase (Graph 4). This group also had a lower fatality rate than the control and no arrhythmia. Thus, it can be concluded that a 5uM concentration of epinephrine does not have any effects on the health or heartbeat rate of the Daphnia magna. The Daphnia exposed to the 5uM nicotine treatment experienced a 100% decrease for their heartbeat rate (Graph 3). Their consequential death suggests that 5uM nicotine is highly toxic to the Daphnia magna. It is important to recall that the trial 2 population was in poor health prior to experimentations. This should not impact the results significantly because the effects of the drug treatments in each trial was analyzed in respect to their control.

In conclusion, the drug treatments of 40uM nicotine and 5uM nicotine proved to be detrimental to the health of the Daphnia magna due to the drug’s effect on heart rate, and increased blood pressure and vasoconstriction, leading to a decreased heartbeat rate and rapid death. The drug treatments of 10uM epinephrine also have negative implications for Daphnia health, decreasing their heartbeat rate and the likelihood of premature death, most likely due to cerebrovascular hemorrhage. Additionally, nicotine and epinephrine interfere with the natural influx and outflux of cations in ion-channels. Surely, long term impairment of these ion-channels induces a stress response reducing the lifespan of the Daphnia. In contrast to these treatments, the drug treatment of 5uM epinephrine had no overall effect on Daphnia health or their heart.

Since none of the groups experienced an increase in heartbeat rate the hypothesis must be rejected. Instead, we accept the null hypothesis: Exposure to nicotine and epinephrine does not increase the heartbeat rate in Daphnia magna and eventually results in a stress-induced death as a combined result of high blood pressure and exposure to toxic chemicals.

The fatality rates observed in this experiment correspond with other findings(2), however, the decreased heartbeat is in conflict with other findings. In humans nicotine and epinephrine increase the heartbeat rate(7). In similar experiments, higher doses of epinephrine caused a slight increase in the heartbeat rate of daphnia2. This experiment showed the opposite, that nicotine and epinephrine decrease the heartbeat rate of Daphnia magna. There are two possible explanations for this observation. The first being that the autonomy and physiology of Daphnia and humans are different, thus the two species respond to drug treatments differently. The second being that these drug treatments only initially increase heartbeat rate and that long term exposure decreases the rate. The latter is the more likely explanation because other studies examining the relationship between drug treatments and heartbeat rate took measurements at time intervals of 15 seconds(2), enabling more precise data analysis. In this experiment, the heartbeat rate was measured in very large time intervals (.5-48 hours) leaving the possibility for a lot of unknowns. To confirm this explanation than further experimentation is necessary with more precise tracking. To determine if Daphnia magna experience an initial increase in heartbeat rate immediately after exposure, then, heartbeat measurements should be recorded every few seconds after drug exposure.

However, if upon replication the same decrease in heartbeat rate is found, then it is possible that long-term use of epinephrine and nicotine could impair long term heart function in humans. Additionally, the effects of other concentrations of nicotine should be examined, particularly in proportion to the concentrations found in cigarettes or vaporizers. In conclusion, due to the inconsistencies of this study, the findings cannot be applied to a human model until further testing is done on a larger population of Daphnia magna.

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