The toxicity testing procedures will be demonstrated by Dr.Ashok Aspatwar, a senior researcher from my laboratory. Our protocol identifies off-target effects and detects subtle toxic effects of chemicals in the early phase of discovery that may be missed in cell culture or other animal models. The main advantages of our technique are:it is rapid and efficient, requires very small amount of chemical, and the basic facilities in the laboratory.
Toxicity screening will help us in deciding the concentration for studying its efficiency against mycobacterium that causes tuberculosis and for further preclinical calculation. This method uses insights into off-target effects of chemicals, and hence this method can be used in any area of research where the toxicity of chemical is of primary importance. This method is very simple and can be carried out by anybody without prior experience.
Individuals with no experience should plan the experiment carefully and use just one compound. This method provides toxic effects of the chemicals in the form of phenotypic changes in the developing zebrafish embryos and hence safety of the compounds tested. To begin, place two to five adult male zebrafish and three to five adult female zebrafish into mating tanks overnight.
In the morning, turn on the light as breeding is induced by the automatic dark and subsequent light cycle. To avoid handling stress to the animals, allow the animals to rest for two weeks before using the same individuals for breeding. The next day before noon, collect the embryos using a fine mesh strainer, and transfer them onto a petri dish containing E3 embryo medium.
Place the petri dish under a stereo microscope to examine each batch of embryos. Identify the opaque appearance and remove them as unfertilized or dead embryos. Keep the embryos at 28.5 degrees Celsius in an incubator overnight.
The next morning, examine the embryos under a stereo microscope and remove any unhealthy or dead embryos. Carefully use a pasteur pipette to transfer one embryo into each well of a 24 well plate. Make sure each well contains enough E3 medium to cover the embryos.
Take out the vials containing inhibitor compounds stored at 4 degrees Celsius in a refrigerator. Use an analytical balance to weigh appropriate amount of the compound, and prepare 250 microliters of 100 millimolar stock solution for each compound in E3 medium or another appropriate solvent. Then, use E3 medium to make serial dilutions of the stock solutions based on toxicity levels in 15 milliliter centrifuge tubes.
From the wells that each contains one DPF embryo, use a pasteur pipette and 1 milliliter pipette to remove the E3 water one row at a time. Immediately distribute 1 milliliter of each diluent of the stock solutions into the wells of the 24 well plate starting from lower and moving to higher concentration. For the control groups, add E3 water or another relevant solvent.
Label 24 well plates with the name and concentration of the compound and keep the plates at 28.5 degrees Celsius in an incubator. Take care that the dilutants of the chemicals in the wells is not evaporated in the incubator by sealing the sides of 24 well plate. Twenty four hours after exposure of the chemical compounds, use a pasteur pipette to transfer the larvae exposed to each concentration of the compound in a small petri dish containing 3%high molecular weight methyl cellulose.
With a metal probe, lay it sideways. Place the petri dish under a stereo microscope attached to a camera. Take the images, and save the images in a separate folder each day until the end of the experiment.
Enter all the observations in a table each day either in an online table or on a printed sheet. For neurotoxic compound exposure, the four to five DPF larvae may show abnormal swim pattern. In that case, make a record of such changes by capturing a short 30 seconds to one minute video of the larvae.
After five days of exposure to the chemical compounds, note the concentration at which half of the embryos die as the half maximal lethal concentration 50 of each chemical. Construct a curve for mortality of embryos for all the concentrations using a suitable program. In this protocol, the critical part of the evaluation of toxicity is testing different concentrations of one or multiple chemical compounds in a single experiment.
For the compounds that induce any phenotypic defects in the larvae, the defects were recorded every 24 hours over the period of one to five days post exposure to the chemical. The embryos treated with beta CA inhibitors at the concentrations of 250 micromolar and 125 micromolar exhibit various phenotypic defects compared with the control. For example, unhatched embryos even at Day 3, curved body structure, unutilized yolk sac and pericardial edema, and absence of otolith sacs in the larvae at five days after treatment.
In another study, the embryos treated with CA inhibitors showed the absence of otolith sacs and swim bladder. The experiments identified a representative compound carbonic anhydrase 9, which induced minimal or no phenotypic changes during the embryonic development at 500 micromolar. The compound showed a high LC50 concentration.
However, in a comparison with a control, the same compound at 300 micromolar was found to be neurotoxic and induced ataxia in the larvae after five days of exposure. Good quality of embryos are important for toxicity screening of chemicals. For obtaining good quality embryos, use young pair of adult zebrafish for breeding.
The compounds that emerge as safe can be further characterized by performing relevant in vitro and in vivo experiments. In the field of antituberculosis drug development, this technique helped to set up further experiments for in vivo inhibition of mycobacterium marinum in zebrafish embryos. The protocol involves use of chemicals that may be toxic to humans.
Persons involved in the experiments should take proper care when handling the chemicals.
