Toxicity of carbaryl on the cardiovascular system in early developing zebrafish (Danio rerio) embryos
Student thesis: Master's Thesis
Every year, few billion kilograms of pesticides are applied for agricultural and domestic usage. A report from U.S. Environmental Protection Agency indicated that more than five billion pounds of pesticide active ingredients were used in 2001 over the world. Since pesticides are usually used in relatively large amounts and concentrated in particular areas, any leaching or accidental leakage has the potential to greatly affect aquatic organisms living nearby, especially developing embryos. During embryonic development, the cardiovascular system is a very important system as it functions to transport resources and remove wastes. Alteration of this system can eventually cause death. In this study, the toxicities of different pesticides were studied to determine their effects on the cardiovascular system of early developing zebrafish embryos. Chlorpyrifos, an organophosphate insecticide, was studied. Cardiac edema was the only malformation observed in early developing zebrafish embryos at 28 hours post-fertilization (hpf). However, the percentage in all concentrations examined was as low as in control group embryos. Moreover, there was no obvious change in mortality within these concentrations. Hence, dosage response curves could not be plotted. Neither 50%-lethal (LC50) nor 50%-effective concentrations (EC50) could be determined for this pesticide. Embryos exposed to glyphosate, a non-selective and post-emergent organophosphate herbicide, caused several defects, including cardiac edema, malformations in the tail region, debris inside the cavity of the chorion, and membrane-like structures formed between the embryo and chorion. No cardiovascular dysfunctions were observed. The LC50 and EC50 values for glyphosate at 28 hpf were 105 μg/ml and 42.9 μg/ml, respectively. Carbaryl, an acetylcholinesterase inhibitor, is known to be moderately toxic to adult fish, and has been reported to cause heart malformations and irregular heartbeat in medaka. The LC50 and the EC50 values for carbaryl at 28 hpf were 44.7 μg/ml and 7.52μg/ml, respectively. Delayed hatching and cardiac edema were found in carbaryl-exposed embryos as described in some previous reports. Moreover, accumulation of red blood cells (RBCs), impaired RBC flow and abnormal heart rate were also observed. The toxicity of 1-naphthol, the main metabolite of carbaryl, was studied to see whether the effects were induced by the pesticide itself or by its metabolites. Exposure to 1-naphthol resulted in higher toxicity compared with carbaryl. The LC50 and the EC50 values of 1-naphthol at 28 hpf were 10.8 μg/ml and 5.36 μg/ml, respectively. However, we only found that carbaryl, but not 1-naphthol, affects the cardiovascular system. Carbaryl (10.0 μg/ml) was used in subsequent detailed studies on the developing cardiovascular system and the pesticide’s function as an acetylcholinesterase inhibitor was confirmed using an enzymatic method. Exposure to 10μg/ml carbaryl affected cardiovascular function in several ways. Blood vessels developed normally but blood flow was affected. RBCs did not move easily into intersegmental vessels (SEs) and flowed slowly in the system. In some serious cases, only a few or no RBCs were observed to flow in arteries and veins. The window exposure and the phenotype recovery experiments suggested that the effects may occur through physiological responses. On the other hand, heart malformations were observed in zebrafish embryos as described in some previous reports. The distance between the sinus venosus, the point where blood enters the atrium, and the bulbus-arteriosus, the point where blood leaves the ventricle, indicated normal looping of the heart tube. Immunostaining with the ventricle-dominated antibody MF20 and the atrium-specific antibody S46 on myosin heavy chains showed normal development of heart chambers. However, the chronic exposure data demonstrated carbaryl-induced bradycardia, which is a common effect of acetylcholinesterase inhibitors due to the accumulation of acetylcholine, in embryos from 24 hpf to 120 hpf. At the same time, the acute exposure data showed that carbaryl-induced bradycardia was a physiological response. The heart rate dropped significantly after a 10-minute exposure to 100 μg/ml carbaryl, but recovered when carbaryl was removed. Exposure to atropine, a non-selective acetylcholine receptor blocker, suggested that the carbaryl-induced bradycardia observed in 24 and 48 hpf embryos occurred through a mechanism other than acetylcholinesterase inhibition, since acetylcholine receptors in zebrafish are not functional until 3 days post-fertilization (dpf). The preliminary data concerning nitric oxide (NO), which is a vasodilation factor, indicated NO may also be affected by carbaryl. Immunostaining of endothelial nitric oxide synthase was down-regulated in expression while in vivo labelling of NO was also reduced in zebrafish embryos at 28 hpf. In summary, different pesticides were examined in early developing zebrafish to determine their effects on cardiovascular activity. In the study, CPF, glyphosate and carbaryl were examined and only carbaryl was found to impair the cardiovascular system. Our data suggested that carbaryl did not cause malformations in angiogenesis and heart development in early developing zebrafish embryos but induced significant bradycardia from 24 hpf to 96 dpf. However, effects mediated through the heart-specific M2 mAChR, which is known to be functionally mature at 3 dpf, could not explain the bradycardia observed at 24 and 48 hpf. These results suggest that carbaryl, apart from inhibiting AChE, may work via one or more alternate mechanisms to induce bradycardia in early developing zebrafish embryos. Possible alternate mechanisms involved may include inhibition of different ion channels which generate action potentials in cardiomyocytes or impairment of NO production. Originally, digital motion analysis showed that heart rate was significantly increased in zebrafish embryos and the speed of blood flow was decreased. Moreover, the caudal arterial diameter was narrowed significantly, indicating that vasoconstriction may be one reason for the reduced blood flow in SEs. However, the significantly increased heart rate was likely the result of warming up of the embryos during the experiment. Therefore, all data generated by the same method were discarded as the heart rate was found to be decreased in the later experiments.
- Toxicology, Embryos, Carbaryl, Zebra danio, Physiology