Evaluation of Embryotoxicity Using the Zebrafish Model - Essay Example

Evaluation of Embryotoxi Using the Zebrafish Model of Evaluation of Embryotoxi Using the Zebrafish Model Abstract: The model of the embryonic zebrafish puts forward investigations on whole animal (e.g. complete organism, properly working homeostatic feedback mechanisms and intercellular signaling) with the benefit of cell culture (e.g. economic, reduced framework and less portion of solutions required). The exemplary structure puts behind the present drawbacks by fast to high-throughput testing of medicines and produces an expansive profit to calculate and accommodate consequences of system. Furthermore it is perfect stage to check out proposed studies which are targeted at components of harmful reactions. Displays occur at 96 well plates in order to use less quantity of solutions appropriate for calculations. Various semantic advancement and behavioral extremities can be assessed without meddling because of the clear essence of embryos. 1. Introduction Various biological structures can be incorporated for toxoid assessment. Artificial approaches such as cell culture systems are favored due to their economical and less time consuming traits. These researches are beneficial but unambiguous rendition to complete organisms and human well-being is challenging to interpret. Within living organism investigations give revised forecast of biological solutions in unblemished systems but at times need expansive aptitude and framework. (1) Zebrafish (Danio rerio) provide feasible assets as an exemplary organism that overshadows certain drawbacks, this trait makes these vertebrates susceptible for toxic studies. Zebrafish can be incorporated as a potent in vivo exemplary system to scan biological intercommunications; also they are a remarkable stage to schedule the workings through which substances show definitive results. A significant correlation in cellular structures, signaling techniques, physiology and anatomy are present in zebrafish and other vertebrates especially in initial development (2-6). Present assessments show that 90% of human exposed reading frames are analogous to fish gene (7). Hence tests incorporating this structural system affirm slight reactions that are bound to be preserved trans versing species. Points of zebrafish’s biology are affirmative for familiarizing this model’s working to through evaluations. The female zebrafish have the capability to lay hundreds of eggs on a weekly basis. The samples are abundant and hence samples for studies are achieved easily. The abundant embryos make toxoid testing of large samples achievable in a shortened period. The vertebrate’s fast evolution in comparison to other warm blooded animals makes it optimal for testing (8). For example, neuronal plate construction happens at 10 hours after fertilization (hpf) followed by organ formation at 24 hpf, which in contrast to a rat happens at 9.5 days and 5-6 days respectively. The initial heartbeat happens at 30 hpf for the zebrafish and 10.2 days for rats (9). Zebrafish embryos independently exposed in wells of a multi-well plate so the compulsory volume required for the structure is minute, hence finite supply of materials are required to calculate a complete suite of biological reactions and feedbacks. Initial formation stages of organism are prone to environmental abuse, due to the various changes in cellular discrimination, propagation and migration necessary for various cell types and organs. Because the process of development is definitive requiring cell to cell associations. During this crucial period if embryo comes in contact with a material, reactions and formation is expected to be distorted. Embryos endured in water come across chemicals using an endless process, in which 24 embryos face per concentration in solo wells of a multi-well plate from 8-120 hpf. Disclosure until 120 hpf is the desired period for toxoid formation testing, initially because of the vertebrate’s capability to sustain its nutrients from its yolk sac till 5 days. Flustered formation can come out as structural abnormalities, behavioral abnormalities or cessation of the embryo. Zebrafish form outside and are clear to the naked eye so there is a a chance to resolve solo cells in the organism through the period of disclosure using microscopic procedures and various possessions can be tested by not interfering over the period of formation. 2. Materials Zebrafish Husbandry Fish water: 0.3 gL instant ocean salts in reverse osmosis Incubator temperature 28 degrees centigrade Dechorination Compound microscope for observing embryos 60 mm glass petri dish 50 gm ml pronase. Measure 50 mg pronase in 1.5 mL centrifuge tube and fill with 1 mL water. Place centrifuge tube in freezer box. Timer Exposure Multi-well plates 8 or 12 multichannel pipette 50 mL reagent reservoir Wide bore Pasteur pipette Assessment Anesthesia: 4mg/mL of 3-aminobenzoate ethyl ester methanesulfonate Methyl cellulose 3. Methods Methods involved in this procedure include zebrafish husbandry, dechorination, exposure and assessment. Firstly in zebrafish husbandry the fish are reared in standard laboratory conditions of 28 degrees in a pH of 7 ± 0.2 on a 14 h light/10 h dark photoperiod. Then the zebrafish are housed in 2.0 liter of polycarbonate tanks that recirculate water. Then adult fish are placed in groups so a large numbers of embryos can be obtained increasing the chances of genetic diversity. The fish are to be fed twice daily with crushedTetraMinR Tropical Flake or live Artemia. Spawning is also carried out by which male and female zebrafish are placed in spawning baskets in a tank with polycarbonate, an afternoon before the embryos are required. The fish spawn after light appears. The next morning newly fertilized eggs are obtained and washed a few times in water, then placed in fresh water in 150 mm plastic petri dish. Removal of unfertilized, noxious and embryos that are not at the same maturity level as the majority is necessary before placing petri dishes into the incubator until they are 6 hours post fertilization. Due to different spawning various embryos have different development patterns. Another method can be used, y which male and female fish are put in tanks that have dividers, the embryos that match stages can then be put together and randomly selected. Dechorination is done next. To hinder blockade effects that the chorion possess, all the embryos should be dechorionated 6 hours after fertilization by a specific version of Westerfirld for pronase breakdown. Bleaching is avoided because it modifies the chorion and pronase is ineffective. Then placement of 6 embryos in 60 mm glass petri dish with 25 mL fish water is done, glass petri dishes are preferred because embryos do not stick in to glass while they stick to plastic. Add 50μl of 50 mg/mL pronase to the center of the dish and continuously swirl gently to mix the solution. Timer is set for 7 minutes and embryos are swirled while keeping an eye on the petri dish under the microscope. This is done to observe the embryos that lack chorions, chorion pieces or deflated chorions. After 7 minutes or after the microscopic changes have occurred, pronase solution is extracted by addition of fresh water dilution. Decantation over the edge of the petri dish is done for 1 minute; it is repeated for 10 minutes. Decantation of fresh fish water should be done gently as embryos are fragile; some have not left their chorions. Post cleaning with fresh water embryos should be allowed to recover in petri dishes and not bleached. Chemicals should be mixed in water to confirm activation. If this is not possible the preferred solvent for exposure is dimethyl sulfoxide, studies have proven this chemical causes no developmental deformities. Then test solutions are poured into 50 mL reagent reservoir which accommodates a multichannel pipette. Each exposure tested a multichannel pipette to fill 24 solo wells in a multi plate 100 μl of chemical solution is required. Seven concentrations and 1 control can be tested using two 96 well plates. At 8 hpf transfer viable and formed embryos into solo wells of multi-well plate using a wide bore pipette. It should be kept in mind that the embryos be allowed to reach bottom of wide bore pipette before touching solution in wells. If an embryo distorts when in solution it is a must to replace solution and add a new embryo in well. Incubation is done at 28°C until 24 hpf, then perform tests. Methods for chemical delivery are direct and continuous. If no analytical method is present for biological uptake direct delivery of chemical into the animal is by microinjection. The transparency of the embryos makes it possible to asses’ tissue dose and distribution by fluorescent materials and laser scanning microscopy. Arrange 8 hpf embryos in troughs that are engraved in 1% agarose plate filled with fish water as described by The Zebrafish Book (9, 11). Then inject each embryo with 2.3 nL of the desired chemical concentration or the appropriate vehicle control directly into the yolk sac. After that each embryo is set into solo wells of a 96-well plate, each filled with 100 μl of fish water. When injecting chemical directly any concentration above 0.1% DMSO caused developmental defects is not attributed to the chemical. If any chemical requires a solvent, two sets of serial dilutions should be made. The first serial dilution should be 100 times more potent than the last one. It should be made sure to vortex each micro centrifuge tube before next dilution so solution is homogenous. The second set of dilutions from the 100% DMSO serial dilution, make a 1:10 dilution from the first serial dilution. An appropriate control for each chemical, which includes the correct percentage of solvent, is used in each solution. Lastly, Incubate at 28°C until first assessments at 24 hpf. Lastly, assessments are made at 24 hpf, in which embryos are assessed for viability, formation and movements (earliest behavior in zebrafish). Formation is considered stunted if zebrafish are more than 12 hours late in comparison to control animals. Rapid movements are tested over 2 minute time frame, and is considered perturbed if there is lack of embryonic movement. At 120 hpf, larval morphology (body axis, eye, snout, jaw, otic vesicle, notochord, heart, brain, somite, fin, yolk sac, trunk, circulation, pigment, swim bladder; Fig. 2) is assessed and written down and behavioral endpoints (motility, tactile response) are thoroughly evaluated within the organism. Test for behavioral endpoints and then anesthetize animals for thorough morphological testing. At the end of the assessments, zebrafish are euthanized with tricaine. Assessments are completed ion binary form (present or not present). If 2 grown animals die in a control group the experiment is considered invalid and is not repeated. Test chemicals have certain targets in humans but those targets are not achieved in vertebrates. Structural discriminations among human and vertebrates are false positive or false negative. For example if a drug is designated to target a human specific structure that isn’t present in zebrafish, when exposed the drug would affect the zebrafish. The results observed when this happens are labelled false negative. On the contrary a false positive can occur when drug affects are seen due to drug affecting a certain target present only in zebrafish, keeping in mind this structure is not present in humans. Assessment can also be carried out on the basis of metabolic activity. False negative and positives may also arise when metabolic activity in zebrafish is different from metabolic activity in humans. Exogenous metabolic activation system can be incorporated to decrease number of false positive and negatives. REFERENCES Akimenko MA, Johnson SL, et al. Differential induction of four msx homeobox genes during findevelopment and regeneration in zebrafish. Development. 1995; 121(2):347–57. [PubMed: 7768177] Aparicio S, Chapman J, et al. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science. 2002; 297(5585):1301–10. [PubMed: 12142439] Blechinger SR, Warren JT Jr, et al. 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