Biology tests using embryos and larvae

In the previous STEPS you have seen that  the Biology Laboratory uses cells to see   the effect of chemicals. Although very useful and  handy, these tests are not complete. In this step   we will show you the use of larvae and embryos. Our body is not a mass of independent cells,   but of cells organized into perfect society,  cells move, dialogue and interact. This is SOO   evident when looking at the hormones. So,  to study chemicals that interfere with our   perfect funcioning, we need tests  that consider the whole organism. In the first example we  will talk about the thyroid.   This is an important gland at the base  of the neck, that produces hormones   called T3 and T4.

These hormones have a special  formula – that you see here - and contain Iodine.   This is why the introdution of a low  amount of Iodine in our diet is essential. Thyroid hormones have several actions in  our body, they control body temperature,   they control general metabolims, they control  energy consumption and fat deposition… ….they control our heart rate, and… they  are very important for fetal development. 

The question is : can the chemicals present  in the packaging migrate into food and drinks,   or in the environment, that mimick or  interfere with our Thyroid Hormones ? Here comes useful the tadpole of  the african toad, called Xenopus,   an animal that lives easily in the acquarium.  You see here the tadpoles and the adult frog.

You may remember that the  tadpoles at a certain time   “transform” to became young toads : they  lose their tail and the gills and grow legs.   This is called Metamorphorsis,  and … guess what … it is initiated   and strictly dependent on thyroid hormones.  These are the same hormones that we have.

Since the frog has a thyroid hormone identical  to ours, the test is : young tadpoles   are exposed to a sample of water containing  an extract from packaging, from kitchenware,   from food, from drinks, or the environment.  After sometime we look at their metamorphosis.   If it is ANTICIPATED, it means  POSITIVE INTERFERENCE, if it is DELAYED   it means NEGATIVE INTERFERENCE.  Or perhaps we see NO EFFECT. Today this can be assisted with  molecular and endocrine analysis   on the DNA, RNA and proteins of the  tadpoles, to be even more precise.

Another set of hormones well studied and  very important has to do with reproduction.   To have babies is a natural desire as  well as a necessity for every species.   The is no need to explain that .. thanks to love,  sex and reproduction that we are here today. Sexual maturation and fertility goes by itself,  but for our body it is not a simple task.   There are many hormones that need to  come up and go down at the right time   and the right amount. Just think  about puberty and the female cycle.

The chief hormone, for brevity we call GnRH,  comes from the hypothalamus of the brain,   others come from pituitary,  others from the gonades…   ecc. and these establish a natural  connection between organs and cells. You remember from Week 2 that sexual  maturation and fertility are threatened in   our modern society, with an increased frequency  of deficits or anticipations.

The question is:   may chemicals we are exposed interfere with  our sex hormones ? how do we study this ?

Here comes the Zebrafish : this is a small  fish that lives happily in the acquarium.   Produces lots of eggs and larvae, has a  short life-cycle and is nearly trasparent.

In the DNA of these fishes researchers have  introduces something fantastic : a fluorescent   gene that shows the cells and neurons that we are  interested in. These here are the smell neurons in   the nose.

See how beautify: we just turn off the  light, look in the microscope and take pictures. Here is another example. These are the  famous cells in the brain that produce   the CHIEF Hormone GnRH, in green. See  the green cells and their extensions ?

So the typical experiment will be: 1. breed males  and females 2. obtain the eggs and larvae 3.   separate in group 4. add samples or treatment  5. Place in the incubator for 2 or 3 days   6. Examine at the microscope. Let us show you another example. Do you remember  the DeepWater Horizon oil spill, in the Carribean   sea, April 2010 ? Or the methyl isocyanide  spill in Bophal INDIA in 1984 ? Or the spill   of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)  in Seveso Italy, in 1976 ?

The question is : how   toxic are these chemicals ? For how long they stay  in the environment ? and at what distance we can   be sure there is no effect ? Here you see  zebrafish larvae treated with water samples,   and it is clear that there is a damaging effect. Another method uses quail eggs. The  fertilized egg is carefully opened,   the tiny embryo is treated with a sample, then the  egg is closed again and incubated for few days or   allowed to hatch. Then, the results are observed  with a microscope or with molecular tests.

Finally for specific purposes we can use  mice, to test for example in utero effects.   In this case a sample or a chemical  is injected in the embryonic space,   the baby mice are allowed to be born and then  analysed for DNA, RNA and proteins. These test   is used as the last evidence, first we prefer to  use frog tadpoles, fish larvae, or quail eggs.

The take-home messages are : 1. Chose the right test  depending on the question you ask  2. Minimize the use of animals  and maximize the results  3. Think about doses relevant  for us and consider the mixes  4. Have professionals to conduct the  experiments and to judge on the result