English
中文Abstract
The cladoceran Daphnia is well established as a model for ecotoxicology. Here, we show that Daphnia is also useful for investigating the effects of alcohol on heart rate. The results may then be extrapolated to determine the effect of alcohol on human heart rate and the possible heart diseases that may result from too much alcohol consumption. Daphnia heart was shown to respond to ethanol in the bathing medium. Ethanol decreased the heart rate when in concentration of 0.05% and above. Three measurements of the heart rate for each concentration of ethanol using three different Daphnia for each measurement was taken and the mean determined. Though the sample size was small and the results cannot be conclusive due to such a small sample size, the results show that Daphnia can be used as a novel model system for studying the effects of alcohol on heart rate and it may be used in further studies to determine the possible receptors that an alcohol molecule may bind to bring about an effect on the heart rate.
Introduction
Daphnia are small, mostly planktonic, crustaceans, between 0.2 and 5 mm in length. They belong to the order Cladocera and are also called water fleas. They are extremely hardy organisms as they have the ability to survive in a variety of aquatic environments such as acidic swamps, freshwater lakes, ponds, streams and rivers. The most commonly found species are Daphnia pulex and Daphnia magna. They are excellent organisms for use in bioassays for they are sensitive to small chemical changes of water and rapidly respond to these changes. Furthermore, they are simple and inexpensive to raise even in an aquarium. Their life cycle is short and thus they mature in a very short period of time so it does not take long to grow a culture of test organisms. They have been established as useful model systems in ecotoxicology and for investigating the ecological impact of toxic substances in freshwater (Diamantino et al., 2000, Guilhermino et al., 2000 and De Coen and Janssen, 2003).
Daphnia have also been reported to have a myogenic heart (Bekker and Krijgsman, 1951). The heart is thus able generate a cardiac contraction independent of nervous input. The Daphnia heart responds to a range of agonists and antagonists that affect heart rate and rhythm in humans (Villegas-Navarro et al., 2003).
Because Daphnia are transparent organisms, the use of these organisms give the option to conduct bioassays using endpoints other than death. The heart rate which is a measurement of stress, can be measured under a microscope making the process convenient and efficient.
The aim of the experiment described here was carried out to show whether Daphnia could be used to investigate the effects of alcohol (ethanol) on the heart rate and to establish Daphnia as a model for investigating the role of ethanol in human diseases related to the heart.
Alcohol has always been expected to have a direct effect on the human heart rate. Although a quickening of the pulse is an effect that is generally accepted as a consequence of alcohol use, studies in which the actions of alcohol on heart rate have been assessed have not produced consistent findings. Following alcohol administration, heart rate has been found to increase (Blomqvist et al. 1970), not change (Ahmed et al. 1973), or merely show a transient, inconsistent increase.
In order to test the effect of alcohol on the heart rate of Daphnia, a model system was required that could be used to examine the effects of ethanol on the heart. Thus, a microscope system was established to measure the effect of ethanol on heart rate when ethanol was added to the water in which the Daphnia were swimming.
Here, we report that ethanol, at concentrations above 0.05%, caused a dramatic decrease in Daphnia heart rate. Our results provide further evidence of Daphnia as a unique model system in biology and medicine.
Materials and Methods
Daphnia
Daphnia were obtained from a pond and were kept in 10- or 80-l tanks on the window ledge of a laboratory at approximately 22 °C. The tanks generated enough microalgae to sustain a colony of several hundred for at least 6 months. The walls of the tanks were scraped clean every 2-3 months and topped up alternatively with pond water or distilled water to replenish water lost by evaporation. The Daphnia were observed in a specially constructed cooled chamber, maintained in most experiments between 10 and 11 °C, using a binocular dissecting microscope, magnification 20-40×. The Daphnia were approximately 1-2 mm in length, with a heart 100-200 μm long and 50 μm across. The heart is situated above the brooding chamber, though the animal normally swims what appears at first glance upside down, with the feelers that move food into the mouth facing upwards. The highest magnification (40×) was required in order to see the heart beat adequately for counting. Each Daphnia was maintained within a 50-μl droplet throughout the experiment.
Measurement of heart rate
Daphnia were incubated in 50-μl drops of pond water containing the ethanol at various concentrations (0, 0.01, 0.05, 0.1 and 0.2%). The Daphnia were free to swim around in each droplet. It was necessary to cool the Daphnia since the heart rate was too fast at room temperature to obtain accurate measurements. The heart rate was counted manually for 60 s. Three sets of measurements were taken for each concentration using a different Daphnia in a different droplet and the mean obtained for each concentration of ethanol. This was a blind study where measurement was carried out by a person who was not aware of the concentration of ethanol in each droplet. To further ensure that the study was accurate, the concentration of ethanol in each droplet was labelled differently for each set of measurements ensuring there was no biasness in the measurement. The temperature of the drops was monitored throughout each experiment using a small thermocouple and remained within ±0.5 °C of the initial temperature. This was important in view of the sensitivity of the heart rate to temperature.
Statistical analysis was carried out as paired t-tests using SPSS and the results are expressed as probability (p) of two values being significantly different when p<0.05.
Results and analysis where appropriate
Table 1: Results of the heart rate of Daphnia for three separate sets of measurements for 5 various concentrations of ethanol.
Figure 1. The effect of ethanol on Daphnia heart rate for 3 sets of measurements and the mean of the 3 measurements for 5 different concentrations of ethanol
As can be seen from Figure 1, the mean heart rate of Daphnia is shown to first increase and then decrease when the concentration of ethanol it is exposed to exceeds 0.05%. The results of run 3 however are not in consensus with the other two runs as the heart rate increased when Daphnia was exposed to 0.2% ethanol.
Discussion
Our results show that ethanol can bring about a dramatic effect on the rate of the heart in the cladoceran Daphnia. The overall decrease in heart rate observed when the exposure of the Daphnia to ethanol increases above the concentration of 0.05% may be explained by these possible mechanisms.
As is the case in most animals, crustacean heartbeats are regulated primarily by nerve pulses. These impulses are generated by pacemaker neurons located in a group of nerve cells called the cardiac ganglion. The impulses are transferred to larger follower neurons which carry the signal to the cardiac muscle, causing it to beat.
In general, there are two possible ways that a small molecule like ethanol can get across the cell membrane of Daphnia. There can either be a transport protein on the membrane of Daphnia that mediates passage across the membrane or the ethanol can diffuse across down a concentration gradient. Ethanol can do both. Since ethanol is not a charged molecule and is small, it can diffuse across the membrane. It is dissolved in the water outside the cell, then dissolves in the lipid of the membrane when it contacts the membrane through random motion. Once in the membrane it will diffuse through the membrane by random motion and eventually dissolve itself back into the water. It is random which side of the membrane it exits. It may also be possible that Daphnia have a protein on their membrane that can transport ethanol. These transport proteins will have a binding site for ethanol. Once bound, the ethanol will cause the protein/transporter to change shape so that when the ethanol dissociates from the protein, it ends up inside the cell. A transporter has, usually, more direction than just random diffusion. It will preferentially move the substrate (ethanol) in one direction.
Once ethanol enters the Daphnia, it interferes with the heart rate. The method of action is probably by interfering with the nerves as mentioned above. (If the quality of the heart beat was affected, we might consider the cardiac muscle to be the site of action. In this case, however, only the rate is involved, so it’s most likely the nerves that are affected.) It may be possible that the nerve fibers conducting pulses to the hearts of Daphnia may contain receptors that inappropriately bind to ethanol (or a product of ethanol after it is broken down) causing an inappropriate decrease in nerve activity.
To further confirm these findings and to determine the possible receptors found on the heart that are involved in binding of ethanol, further studies have to be carried out with a larger sample size.
