Development Of The Zebrafish As An Animal Model For Cardiotoxicity Screening And Cardiac Risk Assessment Of Metoprolol

Many drugs developed by the pharmaceutical industry fail in drugs research anddevelopment stage because of unanticipated toxic side effects. Toxicity may be caused by acompound interacting with a molecular target that is distinct from target selected fortherapeutic benefits, defined as an off-target effect. Toxicity continues to be a major factor inthe attrition of candidate drugs. A recent analysis of nonclinical drug candidate attritionsuggests areas of particular concern include cardiovascular toxicity (26%). With thedevelopment of science and technology, computer-aid drug design and other newtechnology,the number of new chemical substances increase sharply. An important strategyfor lowering drug development costs involves termination of compounds as early in theprocess as possible, before substantial resources have been invested in clinical trials.Drugs are usually tested for in vivo toxicity in mammalian models, such as mouse, rat anddog. Traditional toxicity testing is a time-consuming and expensive part of the modern drugdiscovery process, making them poorly suited for early stage toxicology screening. Newer invitro molecular models that leverage transcriptional profiling, proteomics, and other 'omics'technologies for developing predictive toxicity signatures are being evaluated. Such in vitroassays are amenable to high-throughput screening, but have been hampered by theunexpected difficulty of correlating observed transcriptional profiles with known mechanismsof toxicity as well as a lack of consensus around approaches and study designs. Cell-basedassays are also limited in their ability to mirror the metabolism of a whole animal and toaccurately model multi-cellular processes. Due to the limitations of in vivo and in vitroapproaches, a paradigm shift in the way new and safe compounds are discovered is thuswarranted, and the diminutive zebrafish may lead the way. Because they are small, easy tocare for, inexpensive to maintain, and produce large numbers of transparent embryos thatdevelop outside of the mother, zebrafish have been used to study almost every aspect ofvertebrate biology, including the development and function of the cardiovascular system. Theability to culture large numbers of zebrafish embryos and larvae in small volumes of mediafacilitates rapid testing of compounds for toxicity, while using a minimal amount ofcompound. Compounds in the media are absorbed by the zebrafish through the skin and gillsat embryonic stages and through the digestive system during later larval stages. Embryonic,larval and adult zebrafish have all been used for many years for environmental toxicitytesting. The zebrafish embryo is considered by ECVAM as an alternative to animal experiments.Metoprolol is a selective β1-adrenergic receptor agonist widely used in clinical treatmentseveral diseases of the cardiovascular system, especially hypertension, arrhythmias, heartfailure. Serious side effects, especially with higher dosages, include the following: dizziness,drowsiness, fatigue, diarrhea, unusual dreams, ataxia, and trouble sleeping, depression, andvision problems. It may also reduce blood flow to the hands and feet, causing them to feelnumb and cold.In the present study, based on the physiological and structural characteristics, zebrafish wasused as a model for cardiovascular toxicity screening and safety assessment of exogenouscompounds. And we explore the reliability and security of zebrafish model in cardiovasculartoxicity. With the use of zebrafish as models, we performed experiments to study thecardiovascular toxicity mechanisms of metoprolol.In our experiment,6hpf zebrafish embryos were selected as experimental model andexposed to cardiotoxicity positive drugs (doxorubicin,5-fluorouracil, ibuprofen, andlidocaine). In addition to determining the gross morphology of zebrafish cardiovascularsystem, we also examined the effects on both heart development and function by measuringthe sinus venosus (SV)-bulbus arteriosus (BA) distance and measuring heart rate in thisspecies. The results demonstrate that varying degrees of cardiovascular malformations,including pericardial edema, yolk sac edema, hemorrhage, and other deformities wereinduced after48h of exposure with positive drugs. This SV-BA distance provides an index ofthe change in cardiac looping. There were significant differences in this length betweentreated groups and control groups in every treatment. Heart and vasculature development andfunction are severely impaired, and the heart rate reduced gradually.To investigate the mechanism of metoprolol cardiovascular toxicity, we used the zebrafihas model organism to detect the effects of metoprolol exposure on the heart of developingzebrafish embryos. The results showed that zebrafish embryos exposed to metoprolol developpericardial edema, yolk sac edema and hemorrhage, and so on at72hpf. Hearts frommetoprolol-exposed embryos were elongated and string-like. In control embryos, the normallooping process places the ventricle and atrium side by side, so that the two chambers largelyoverlap each other in lateral view. In contrast, the hearts were stretched out in themetoprolol-treated embryos such that the ventricle was positioned anterior to the atrium. Inthe treated animals the atria were thin and elongated and the ventricles appeared smaller andmore compact than normal. In order to quantify the effect of metoprolol on zebrafish hearts,the distance between the junction of the heart with the inflow tract at the sinus venosus (SV)and the junction with the outflow tract at the region of the bulbus arteriosus (BA) wasdetermined. The resulting numbers provide an index of the change in heart morphology dueto the metoprolol treatment, and reflect the change in cardiac looping. Metoprolol producedsignificant increases in the SV-BA distance. We hypothesized that metoprolol decreases thesize of the developing hearts by reducing the heart tissue. To test this hypothesis we used serial longitudinal section of paraffin-embedded zebrafish at72hpf. The results show thatmetoprolol reduced myocardial cells. The effects of metoprolol exposure on the developingzebrafish heart were not limited to the alterations in heart morphology. In addition, we foundheart function to be profoundly affected. Heart rate was significantly decreased in themetoprolol-exposed embryos.Induction of apoptosis through the β1ARs subtype and its inhibition by β2ARs incardiomyocytes was reported recently. The present study was also undertaken to determinethe significance of metoprolol on apoptotic cell death in zebrafish. The vital dye acridineorange and TUNEL were performed to identify apoptotic cells. The experimental results ofthe present study demonstrate that metoprolol effectively induced apoptosis in zebrafish. Forfurther assessment apoptosis events, total RNA was isolated from zebrafish exposed tometoprolol and subjected to semi-quantitation RT-PCR for zfBax-a, zfBcl-2, zfcaspase-3, andzfcaspase-9. BCL-2family of genes plays an important role in the regulation of apoptosis.Zebrafish genome contains homologs of most mammalian Bcl-2family members and caspasefamily members. The results showed that metoprolol increased the intensity of the signalcorresponding to zfBax-a and concomitantly decreased the signal corresponding to zfBcl-2compared with control. Consequently, the zfBcl-2to zfBax-a ratio was decreased comparedwith control group. Metoprolol treatment increased the intensity of the signal correspondingto zfcaspase-3and zfcaspase-9.In summary, zebrafish is successfully established as vertebrate model with uniqueadvantages amenable for high-throughput in vivo cardiovascular toxicity screening and safetyassessment of drugs in the present study. We explored the cardiovascular toxicological effectsof metoprolol on zebrafish cardiovascular system. These results suggest that metoprololinduced zebrafish cardiomyocyte apoptosis is medicated by regulate the expression of zfBcl-2,zfBax-a, zfcaspase-3and zfcaspase-9. Therefore, we postulate that metoprolol inducesapoptosis via the β2-adrenergic receptor possibly. The experimental results of the presentstudy provide a theoretical basis for further study cardiovascular toxicity mechanism ofβ-adrenergic receptor agonist and drug development.