Objective

To study the toxicity of compounds using zebrafish embryo.

Theory

Most of toxicity  studies  are conducted to assess the degree to which substance are toxic for humans or animals , to investigate the mechanism  of toxic chemicals, or to develop new or improved tests for specific types of chemically induced effects. The toxicological studies include acute toxicity studies where as the examination of adverse effects that may occur on first exposure to a single dose of a substance, genotoxicity studies that seek to assess the potential of substance to interact with genetic materials and the repeated–dose toxicity studies that are undertaken to find out whether cancer may be develop as a result of exposure to certain chemicals and studies to ensure safety of medicine.

Toxicity has two main components they are the effect caused and the level of dose at which the effect is observed. Some tests are designed specifically to detect a particular effect such as skin sensation and mutagenicity studies. And the other tests such as sub chronic and chronic studies, are designed to detect a wider range of less- specific effects on organs or body systems and the dose range over which the effect develops. The degree to which a toxic substance can harm animals or humans, acute toxicity involves harmful effects in an organism through a single or short –term exposure. Subchronic toxicity is the ability of a toxic substance to cause effects for more than one year but less than the lifetime of the exposed organism. Chronic cytotoxicity is the ability of a substance or a mixture of substance to cause harmful effects over an extended period, usually upon repeated or continuous exposure, sometimes lasting for the entire life of the exposed organism.

Animal models, while more physiologic, have an even lower throughput, restricting their ability to screen systematically at scale for drug- drug interaction which may involve a large proportion of the current formulaory. Zebrafish have long been the genetic model of choice for vertebrate developmental biologists, as it provides several advantages for investigating organ and tissue development not available through other model system. The advanced genetics and convenient developmental features of the zebrafish embryo make it is the best vertebrate model for high- through phenotyping. It perhaps the only vertebrate model compatible with the predictive Toxicology Needs of Drug Discovery to Alleviate the Pipeline Problems. Therefore it makes a logical sense that zebrafish has become a powerful model organism for investigating the molecular and cellular mechanism by which the environmental chemical disrupt normal developmental processes.

Most embryo toxicity tests have been conducted with zebrafish in the laboratories. The zebrafish embryo toxicity test is based on a 48 hours exposure of newly fertilized eggs in a static or semi- static system.

Advantages of Zebrafish

There are several advantages for the use of zebrafish as a toxicological model over other vertebrate. The main benefit of using zebrafish related to their size, husbandry and early morphology. Instead of other fish species, zebrafish adults are only approximately 1-1.5 inches long. Also zebrafish have been utilized as a laboratory species for quite some time so the optimum breeding and maintenance conditions have been well determined. In severalties to larger species, the minute size of the larval and adult zebrafish minimizes quantities of lab ware and chemicals, both for treating and maintaining live fishes and also costs through low quantities of dosing substance and performing various assays.

In addition to these another important feature of the zebrafish embryos are fecundity and transparent embryos. The rapid maturation of zebrafish also allows easy experimentation for transgenerational endpoints required for mutagenesis screening, establishing transgenic lines assessing chemicals for teratogenicity. Their optical clarity allows for easy developmental staging, identification of phenotypic traits during mutagenesis screening and assessment of endpoints of toxicity during toxicity studies. The advantages of the zebrafish both in identifying endpoints of toxicity and in elucidating mechanism of toxicity are highlighted. By using zebrafish to study vertebrate development that mutations are relatively easy to obtain and to screen. Finding mutation is very parents difficulty in the case of the others but in zebrafish mutation can be easily identified and there can be still too bred to keep the mutation in the heterozygotes.

Principle

Zebrafish embryos are individually exposed in microtiter plate to a range of concentration of toxic chemical substance. The test is initiated immediately after fertilization and is continued for 24-48 hours. Lethal effects, as described through the endpoint are determined by comparison with controls to identify the LD50. Lethal dose 50 (LD50) is the measurement used in toxicology studies to determine the potential impact of toxic substance on different types of organism. It provides an objective measure to compare and rank the toxicity of substance. When comparing LD50 values, lower value is regarded as more toxic, as it means the smaller amount of the toxin is required to cause death. The LD50 depend on the route of entry into the body of the organism being tested. The zebrafish embryos may be more or less sensitive to toxins than the other organisms, therefore the lowest value can be considered as the starting point. The method is based on using a minimum of five test concentration as well as appropriate control with individual embryos per exposure concentration. After treatment the embryos are all dead or all alive, reevaluate the range of the test concentration. The percentage of dead vs. concentration is plotted to produce the toxicity curve.