About Our Project
Our goal is to determine if the combination of hypergravity and microgravity (i.e. space travel) affects cellular function.
We chose to analyze the effects of both, because we could not come up with a way to have the cells on board the plane and experience only hypergravity or only microgravity. Furthermore, the combination of both is more relevant to what astronauts experience during space travel.
Image 1: Human Lung Cells Under the Microscope
We used human lung cells as our test subject, because lungs are a vital organ and can be easily exposed to dust particles in the air (especially in zero gravity).
Because we used lung cells, we must keep them at body temperature (37oC, 98oF), which is much higher than room temperature (27oC, ~80oF). To do this, we brought a small oven on board the plane with us. Also, a few of our experiments involved stopping cellular function. One way to do this is to decrease the temperature of the cells' environment; thus, we moved them from the oven into an even smaller cooler. The cold temperature stops cellular function, therefore stopping treatment and bringing the treatment period closer to just the flight. SpaceWorks Engineering, Inc. and Maine Tool and Machine assisted us with developing a "rig" which we could use to secure the oven and cooler to the floor of the plane. We cannot thank them enough for the assistance they provided us.
Image 2: In These Images of the Rig, the Cooler is Secured to the Rig Above the Oven; (A) Side View of the Rig With Electrical Hardware, (B) Front of the Rig, (C) Side View of the Rig, and (D) Rear View of the Rig With the Clipbaord.
We are looking at four cellular processes (more detail below):
- Production of DNA damage - grown in flasks and bags
- Repair of DNA strand damage - grown in chamber slides
- Ion uptake - grown in flasks and bags
- Particle internalization - grown in flasks
Image 3: Materials Used to Grow Cells; (A) Flask, (B) Bag, and (C) Chamber Slide
We are looking at two chemicals:
Processes 1-3 were treated with SODIUM CHROMATE. It dissolves in water. It breaks metaphase chromosomes and DNA strands. Its chromate ion gets into cells by facilitated diffusion. Thus we know what it will do and can see if hyper/microgravity have an effect.
Image 4: Sodium Chromate Dissolved in Water (in Test Tube) and in a Solid (in Tray).
Process 4 was treated with SILVER NANOPARTICLES. We know they get into cells. We will see if hyper/microgravity affects this. We do not know if it breaks DNA. We also did not want to start our experiments with particles for processes 1-3 as it adds a lot more complexity to the work, so we used one dose with a negative control for treatment. If we find an effect this year with sodium chromate, we will try next year with a particle.
Image 5: Silver Nanoparticles Suspsend in Water.
To give you an idea of the complexity of our experiments...they were very time oriented; cells had to be seeded two days before they could be treated. The majority of our experiments were prepared for flight several hours before flight. Thus, if there had been bad weather or some complication with the plane that delayed our flight, we would have lost all our experiments for that flight. We planned on having two sets of experiments each day. One set of experiments included three concentrations of sodium chromate (low, medium, and high) and a negative control (as that is proper toxicology design); and each set had to have a ground trial and a flight trial. We are very pleased to report our experiments were successful; we didn’t have any failed experiments while working at Johnson Space Center.
Metaphase Chromosome Damage:
Image 6 shows the phases of a cell's life cycle - we are studying them in METAPHASE, when the chromosomes are easiest to see. By adding demecolchicine we can see the chromosome condensed. Because metaphase is a phase - repair studies are not possible and must be done separately. Image 7 displays a spread of chromosomes in metaphase. Note the "space" in the arms (indicated by red arrows) - this is a type of break which we are looking for.
Image 6: Phases of a Cell's Life Cycle
Image 7: Spreads of Chromosomes in Metaphase(Red Arrows Point to Damage)
DNA Strand Damage and Repair:
DNA is a double helix and is often visualized as a ladder you. The rungs are the bases of DNA and the outer part is the phosphate backbone. Chromium breaks the outer part. If it breaks both outer parts it is called a "double strand break." These breaks are very dangerous and difficult to repair.
Image 8: An illustration of a double strand break.
In response to such a break, a protein called H2A.X accumulates at the break and starts the repair process. Enough H2A.X protein accumulates so that if you stain the cells with an antibody to H2A.X (an H2A.X antibody is a protein with a green fluorescent tag that ONLY recognizes H2A.X so it binds wherever H2A.X is) - you get bright green spots called "foci". We can then count these foci in the microscope.
The more foci present = more double strand breaks.
Then by washing out treatment we can measure the foci disappearing with time - and that is repair.
Image 9: (A) Shows the nuclear H2A.X foci in green; (B) same cell nucleus in red. So we look for red cells and then switch the light filter to count green dots in that red cell.
Ions enter the cell via facilitated diffusion. During facilitated diffusion, a chemical compound (sodium chromate in this case) breaks apart into its ions, and the ions enter the cell through protein channels. Protein channels act like doorways in the cell membrane. If we see a difference in the damage or repair - a key question will be; is it a difference in effect or in the amount of Cr that got into the cell? Thus, we will measure the amount of Cr that gets into the cell under all of the damage and repair conditions above. We can measure the amount of Cr in a cell and outside the cell by using an ICP machine. By knowing how much Cr is outside the cell and how much is inside the cell, we can determine how much Cr got into the cell.
Image 10: A Diagram of Facilitated Diffusion.
If we see a difference in the amount of particles internalized by phagocytosis, we will return to Johnson Space Center next year to analyze the toxic effects of a particle. Phagocytosis is a process by which cells take particles from the extracellular environment and bring them into food vacuoles (where they break down the particles).
Image 11: A Diagram of Phagocytosis.