Ractopamine at the Center of Decades-Long Scientific and Legal Disputes: A Lesson on Benefits, Safety Issues, and Conflicts.

Ractopamine (RAC) is a synthetic phenethanolamine, ß-adrenergic agonist used as a feed additive to develop leanness and increase feed conversion efficiency in different farm animals. While RAC has been authorized as a feed additive for pigs and cattle in a limited number of countries, a great majority of jurisdictions, including the European Union (EU), China, Russia, and Taiwan, have banned its use on safety grounds. RAC has been under long scientific and political discussion as a controversial antibiotic as a feed additive. Here, we will present significant information on RAC regarding its application, detection methods, conflicts, and legal divisions that play a major role in controversial deadlock and why this issue warrants the attention of scientists, agriculturists, environmentalists, and health advocates. In this review, we highlight the potential toxicities of RAC on aquatic animals to emphasize scientific evidence and reports on the potentially harmful effects of RAC on the aquatic environment and human health.

Cross-species pharmacological characterization of the allylglycine seizure model in mice and larval zebrafish.

Treatment-resistant seizures affect about a third of patients suffering from epilepsy. To fulfill the need for new medications targeting treatment-resistant seizures, a number of rodent models offer the opportunity to assess a variety of potential treatment approaches. The use of such models, however, has proven to be time-consuming and labor-intensive. In this study, we performed pharmacological characterization of the allylglycine (AG) seizure model, a simple in vivo model for which we demonstrated a high level of treatment resistance. (d,l)-Allylglycine inhibits glutamic acid decarboxylase (GAD) - the key enzyme in γ-aminobutyric acid (GABA) biosynthesis - leading to GABA depletion, seizures, and neuronal damage. We performed a side-by-side comparison of mouse and zebrafish acute AG treatments including biochemical, electrographic, and behavioral assessments. Interestingly, seizure progression rate and GABA depletion kinetics were comparable in both species. Five mechanistically diverse antiepileptic drugs (AEDs) were used. Three out of the five AEDs (levetiracetam, phenytoin, and topiramate) showed only a limited protective effect (mainly mortality delay) at doses close to the TD50 (dose inducing motor impairment in 50% of animals) in mice. The two remaining AEDs (diazepam and sodium valproate) displayed protective activity against AG-induced seizures. Experiments performed in zebrafish larvae revealed behavioral AED activity profiles highly analogous to those obtained in mice. Having demonstrated cross-species similarities and limited efficacy of tested AEDs, we propose the use of AG in zebrafish as a convenient and high-throughput model of treatment-resistant seizures.

Zebrafish bioassay-guided microfractionation identifies anticonvulsant steroid glycosides from the Philippine medicinal plant Solanum torvum.

Medicinal plants used for the treatment of epilepsy are potentially a valuable source of novel antiepileptic small molecules. To identify anticonvulsant secondary metabolites, we performed an in vivo, zebrafish-based screen of medicinal plants used in Southeast Asia for the treatment of seizures. Solanum torvum Sw. (Solanaceae) was identified as having significant anticonvulsant activity in zebrafish larvae with seizures induced by the GABAA antagonist pentylenetetrazol (PTZ). This finding correlates well with the ethnomedical use of this plant in the Philippines, where a water decoction of S. torvum leaves is used to treat epileptic seizures. HPLC microfractionation of the bioactive crude extract, in combination with the in vivo zebrafish seizure assay, enabled the rapid localization of several bioactive compounds that were partially identified online by UHPLC-TOF-MS as steroid glycosides. Targeted isolation of the active constituents from the methanolic extract enabled the complete de novo structure identification of the six main bioactive compounds that were also present in the traditional preparation. To partially mimic the in vivo metabolism of these triterpene glycosides, their common aglycone was generated by acid hydrolysis. The isolated molecules exhibited significant anticonvulsant activity in zebrafish seizure assays. These results underscore the potential of zebrafish bioassay-guided microfractionation to rapidly identify novel bioactive small molecules of natural origin.

Validation of the zebrafish pentylenetetrazol seizure model: locomotor versus electrographic responses to antiepileptic drugs.

Zebrafish have recently emerged as an attractive in vivo model for epilepsy. Seven-day-old zebrafish larvae exposed to the GABA(A) antagonist pentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-like behavior, and epileptiform electrographic activity. A previous study showed that 12 out of 13 antiepileptic drugs (AEDs) suppressed PTZ-mediated increases in larval movement, indicating the potential utility of zebrafish as a high-throughput in vivo model for AED discovery. However, a question remained as to whether an AED-induced decrease in locomotion is truly indicative of anticonvulsant activity, as some drugs may impair larval movement through other mechanisms such as general toxicity or sedation. We therefore carried out a study in PTZ-treated zebrafish larvae, to directly compare the ability of AEDs to inhibit seizure-like behavioral manifestations with their capacity to suppress epileptiform electrographic activity. We re-tested the 13 AEDs of which 12 were previously reported to inhibit convulsions in the larval movement tracking assay, administering concentrations that did not, on their own, impair locomotion. In parallel, we carried out open-field recordings on larval brains after treatment with each AED. For the majority of AEDs we obtained the same response in both the behavioral and electrographic assays. Overall our data correlate well with those reported in the literature for acute rodent PTZ tests, indicating that the larval zebrafish brain is more discriminatory than previously thought in its response to AEDs with different modes of action. Our results underscore the validity of using the zebrafish larval locomotor assay as a rapid first-pass screening tool in assessing the anticonvulsant and/or proconvulsant activity of compounds, but also highlight the importance of performing adequate validation when using in vivo models.

Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae.

Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2-3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies - e.g. omics analyses -should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish.

Zebrafish bioassay-guided microfractionation for the rapid in vivo identification of pharmacologically active natural products.

The rapid acquisition of structural and bioactivity information on natural products (NPs) at the sub- milligram scale is key for performing efficient bioactivity-guided isolations. Zebrafish offer the possibility of rapid in vivo bioactivity analysis of small molecules at the microgram scale - an attractive feature when combined with high-resolution fractionation technologies and analytical methods such as UHPLC-TOF-MS and microflow NMR. Numerous biomedically relevant assays are now available in zebrafish, encompassing most indication areas. Zebrafish also provide the possibility to screen bioactive compounds for potential hepato-, cardio-, and neurotoxicities at a very early stage in the drug discovery process. Here we describe two strategies using zebrafish bioassays for the high-resolution in vivo bioactivity profiling of medicinal plants, using either a one-step or a two-step procedure for active compound isolation directly into 96-well plates. The analysis of the microfractions by microflow NMR in combination with UHPLC-TOF-MS of the extract enables the rapid dereplication of compounds and an estimation of their microgram quantities for zebrafish bioassays. Both the one-step and the two-step isolation procedures enable a rapid estimation of the bioactive potential of NPs directly from crude extracts. In summary, we present an in vivo , microgram-scale NP discovery platform combining zebrafish bioassays with microscale analytics to identify, isolate and evaluate pharmacologically active NPs.