Week 10 (Radiobiology) Flashcards
Atomic number
Number of protons (written in subscript)
Mass number =
protons + neutrons (written in superscript)
Isotopes
Number of protons is fixed
- Number of neutrons can vary (isotopes)
- 1H (hydrogen), 2H (deuterium), 3H (tritium)
- This may lead to unstable nuclei (but stable isotopes are also useful!)
Describe Alpha decay
- Low penetration (won’t go very far due to relative large size), intensely ionising (Emission of 2 protons and 2 neutrons
- Atomic number decreases by 2, mass number decreases by 4)
Describe beta decay
Medium penetration, variable energy (Neutron decays to a proton and electron is shot out; atomic number increases by 1, Mass number stays the same) occurs in lighter elements (e.g. tritium decays into helium)
Positrons
Proton decays to a neutron and positive electron (positron) Atomic number goes down by 1, mass number is constant
What is the main property of gamma rays?
High penetrating power (excess energy removed from the nucleus as electromagnetic radiation)
Can cause a lot of ionisation depending on how it leaves the nucleus
Different ways of measuring radioactivty
- Detection
- Quench and efficiency
- Half life and decay curves
What considerations or concerns are there for radiation and Health?
- Mechanisms of damage
- Effects on tissues
- Measuring risk
- Biological half-life
Radiation damage:
What are the long and short term effects?
- Short-term (radiation sickness) which causes cell death
- Long term (cancer + tumours+ birth defects/abnormalities)
What are some uses of radioactivity?
- Isotopes used in biology
- Radioligand binding
- Autogradiography
- Metabolic studies
- =Radioimmunoassay
- 14C dating
- X-rays for structural determination of proteins and other molecules
Explain RADIOLIGAND BINDING ASSAY:
- Incubate constant amount of receptor prep. (tissue homogenates, cells, membranes, purified receptor) with increasing amounts of labelled ligand.
- Allow reaction to come to equilibrium
- Separate bound from free ligand using centrifugation and measure for bound radioactivity.
Describe Autoradiography
It is possible to use radioactive compounds to label receptors in sections from tissue
What are the alternatives to radioactivity?
Mass spectroscopy to identify compounds labelled with stable isotopes (18O, 13C, 15N)
- Stable isotopes of N and O used in NMR to examine structure of macromolecules
- Labelling with fluorophors in binding assays
- FRET (Foster resonance energy transfer)/BRET (Bioluminescence resonance energy transfer)
What is NanoLuc?
Resonance energy technique (to replace radioligand binding) Label receptor with luciferase derivative (Nanoluc) to generate bioluminescence (light) and add ligand with fluorophore that is excited by the luminescence (excite ligand to produce fluorescence)
what is the main principle of alpha radiation?
- There are 2 protons and 2 neutrons
What is the main principle of beta radiation?
-An electron is emitted
What is the main principle of positron radiation?
A positively charged electron is emitted
what is the main principle of electromagnetic radiation?
Gamma rays (a penetrating electromagnetic radiation arising from the radioactive decay of atomic nuclei. It consists of the shortest wavelength electromagnetic waves and so imparts the highest photon energy)
Describe the process of X-rays
- Take a heavy atom e.g. lead
- Fire electrons at it 3.
- As electrons are scattered they loose energy which is converted to X-rays
What device can be used to detect radioactivity?
Geiger-Muller tube
What does a Geiger-Muller tube consist of?
- A hollow tube filled with an inert gas (e.g. Argon)
- An anode in the middle
- Casing that has a negative charge
- Front has a window made of the mineral mica
How does a Giger-Muller tube work?
- Pass radiation through the window Atoms of argon ionise the atoms electrons move to the anode positive ions move to the cathode
- This is registered by the Giger counter (vison count display)
What is one slight disadvantage of the Giger counter?
The sample has to have enough energy to travel from its source through the window, through the middle of the tube
Give an example of a sample that does not have enough energy to penetrate the window of a Giger counter
Tritium
How do you detect low energy radiation?
Scintillation counting (Count light flashes- photomultiplier tubes is the detector)
What is a scintillant?
A chemical that emits light when radiation (typically a beta particle) interacts with it causing it to change conformation
How many photomultiplier tubes are next to your source?
2 (1 either side) Ensures that you are detecting real light flashes and not background light
Why are 2 photomultiplier tubes used?
Eliminates false positives
Explain the process of scintillation counting?
- Dissolve sample into the scintillant
- Put sample in a vile (coincidence gate)
- Load it into the machine
- count each vile to 1-5 mins depending on the level of radioactivity of the sample
- Counts are displayed at the bottom
- More counts= more radioactive
What kind of light flash would you expect to see for a high intensity sample?
Greater intensity of the light flash
Oder P32, tritium and C14 in order of increasing energy
Tritium- low energy
C14- medium energy
P32- High energy
Can the Scintillation counting be tuned?
Yes It can be tuned to count on separate windows
Why can the scintillation value be lower than the radioactivity of the sample?
- Not every radioactive disintegration results in a light flash the photomultiplier tube might miss a light flash
- The solvent that we dissolve the scintillant in can absorb the light flashes
- If the sample is coloured, it can reduce the efficiency of the scintillant -The efficiency of the scintillant can be altered by pH changes
How is the efficiency of counting be measured?
External standard
- A radioactive source of known activity is built into the scintillation counter, it gets pushed out on the rod just below the sample and this activity hits the sample
- We know how much radioactivity is at the end of the rod so we know the excepted number of light flashes
- we measure what the actual number of light flashes are and that tells us the efficiency of counting
What is the channels ratio method measure the efficiency of counting?
- When a sample undergoes quenching (efficiency of counting is impaired, shape of the energy distribution changed and the high energy of the spectrum is lost
- Measure the total amount of counts in channel one and measure the high energy end in channel two (channel ratio) less counts in channel two=more sample being quenched
what is the shape of radioactive decay curve?
As you move toward a stable form, you have less radioactive nuclei
- The shape follows an exponential curve (never quite reaches 0
- Measure of rate= half life (how long it takes for haft the radioactivity to disappear)
What is the half life of tritium?
12.5 years
Regarding half life, more radioactive the sample=
shorter half life
What is the equation for the amount of radioactivity after a certain amount of time?
- A= A.o exp (-kt)
- Ao= starting activity
- k= rate constant t= time
- A= Ao exp(-0.693t/T1/2)
What is the curie unit?
An arbitrary unit= amount of radioactivity in one gram of radium= 2.2x1012 disintegrations per minute
What is a becquerel?
Current unit of readioactivity
1 disintegration per minute (very very small)
This is the correct SI unit
1 Beequerel= 1dpm (disintegration per minute)
What are free radicals?
- an atom/ species with an unpaired electron
- It is very reactive
- it reacts with other atoms/molecules to create more free radicals
how is the superoxide anion created?
An electron is gained by O2
Which organelle (endogenous source) produces superoxide anions?
- Mitochondria leak
- respiratory burst
- enzyme reactions
- autooxidation reactions
What is the body’s defence against free radicals
It has an enzyme called superoxide dismutase that reacts superoxide with hydrogen ions to form hydrogen peroxide which can be broken down by the enzyme catalyse to give water and oxygen
What is produced when Fe reacts with peroxide?
Hydroxyl ion and the hydroxyl radical (extremely reactive)
What are some environmental sources of free radicals?
- cigarette smoke
- pollutants
- UV light
- Ionising radiation
- Xenobiotics
What are some endogenous sources of free radiacals?
- Mitochondiron leak
- Respiratory burst
- Enzyme reations
- Antioxidation reactions
what damage can the hydroxyl radical cause?
- lipid peroxidation (damages membrane bilayer)
- modified DNA bases (and germline DNA passes on radiation damage)
- Protein damage leads to Tissue damage
Background radiation is an important driving force for:
evolution
Which cells are most sensitive to radiation damage?
the ones that are rapidly dividing:
- Hair follicles
- red/white blood cells (leads to anaemia and being prone to infection)
are high or low energy radioactive particles more dangerous?
High energy
are alpha or beta particles larger?
alpha (tend to cause 20x more ionizing damage)
what is the ability of radiation to release energy in any material that absorbs it meaured in?
Grays (used to be Rem)
is radiation more harmful in thymidine or glucose?
thymidine
What are some biologically important isotopes and their half lives?
Isotope Half-life
32P ►14 days
131I► 8.1 days
35S ►87 days
14C ►5570 years
45Ca ►164 days
3H 12.3 ►years
What is metabolic labelling?
Also called pulse chase monitor the radioactive compound throughout different processes and locations
Was first used to see how carbon dioxide was used in photosynthesis
- Plants were exposed to a brief burst of radiolabelled carbon dioxide (pulse)
- Radioactive carbon dioxide was removed
- Determined where the pulse of radioactivity was localised
- Noticed that it was incorporated into a sugar which was metabolised so the radioactive carbon was able to be spread by different metabolites, storage polymers, cellulose and lipids
What are radioimmunoassay’s used for?
Used to find out how much of a compound is present:
Insulin for example
- radiolabel your target sample (insulin)
- Get an antibody to insulin (the target)
- mix the two so all the insulin is bound to the anibody
- Precipitate the antibody out and the antibody will be very radioactive
- Take a blood sample from the patient, if there is no radioactivity in the blood, all the radioactivity will still bind to the antibody so when you separate the antibody it would be very radioactive
- If the patient has a lot of insulin in their blood, it would bind to the antibody but not make it radioactive (harder for radioactive insulin to bind because there is unlabelled insulin in the way)
- Much less radioactive insulin bound to the antibody
- Depending on how much insulin they have in their body, there is less radioativity bound to the antibody
Thiscan be quantified by putting known amounts of unlabelled insulin for radiolabelled insulin so you can work out for any amount of unlabelled insulin how much radioactivity you would expect to find. You can read off the curve how much insulin is present in the samples
- precipitate the antibody
What does carbon dating involve?
A small portion of the carbon dioxide that we breathe in is radioactive (14C) which has a half life of approx 5,500 years
When an organism dies the 12C slowly decays into non-radioactive 12C and
Comparing the ration of 14C (which has a half life of approx 5,500 years) to 12C allows organic samples to be dated, can work out how long ago the organism died
Can be used for 20,000-40,000 years
Descfibe X-ray crystallography?
- Shine a light (x-ray source) at a crystal
- X rays are diffracted by cruystal lattice
- Can go from diffraction pattern to the structure of the molecule
What is ionisation
- Any process by which electrically neutral atoms or molecules are converted to electrically charged atoms or molecules (ions).
- Ionization is one of the principal ways that radiation, such as charged particles and X rays, transfers its energy to matter
What are X-rays?
X rays are electromagnetic radiation that differentially penetrates structures within the body and creates images of these structures on photographic film or a fluorescent screen
Give an example of metabolic labelling
- A pulse-chase experiment in which rpancreatic beta cells were of rats fed with 3H-leucine for 5 minutes (the pulse) followed by excess unlabeled leucine (the chase).
- The amino acid is largely incorporated into insulin, which is destined for secretion.
- After a 10-minute chase the labeled protein has moved from the rough ER to the Golgi stacks
- (A), where its position is revealed by the black silver grains in the photographic emulsion. After a further 45-minute chase the labeled protein is found in electron-dense secretory granules (B).
