Appendix H: Radioactive tracers


The use of radioactive tracers in cell research is an effective and safe means of monitoring molecular interactions. There is simply no other technique which allows the precision and specificity of radioactive tracers.

Radiation is to be taken seriously. At a minimum, its misuse can lead to increased environmental pollution, and at worst can lead to serious long term injury. It can be handled safely, however.

Radioactivity is caused by the spontaneous release of either particulate and/or electromagnetic energy from the nucleus of an atom. Atoms are composed of a positively charge nucleus, surrounded by the negatively charged electrons. In an uncharged atom, the number of orbital electrons equals the number of positively charged protons in the nucleus. In addition, the nucleus contains uncharged neutrons. A proton has a mass of 1.0076 amu (Atomic Mass Units), while a neutron has a mass of 1.0089 amu.

If the mass of a helium nucleus is examined, there is a difference between the expected mass based on its proton and neutron composition, and the actual measured mass. Helium contains two protons and two neutrons in its nucleus, and should have a corresponding mass of 4.0330 amu. It has an actual mass, however, of 5.0028 amu. The difference (0.0302 amu) is the equivalent energy of 28.2 Mev and is known as the binding energy . It would require 28.2 Mev to fuse two protons and two neutrons into a helium nucleus, and the fission of the helium nucleus would yield the same energy.

In addition, the electrons orbit the nucleus with precise energy levels. When the electrons are in their stable orbits, they are said to be in their ground state. If the electrons absorb energy (e.g. from photons), they jump to different, yet characteristic orbits and enter the excited state. The energy difference between a ground state and an excited state can take the form of an electromagnetic radiation.

The number of protons in the nucleus of an atom is called the atomic number, while the number of protons plus neutrons is the mass number. The mass number is approximately equal to the atomic weight. In the representation of an atom used in the periodic table of elements, the atomic number is a subscript written to the left of the letter(s) designating the element, while the mass number is written as a superscript to the left.

The chemical identity of an element is determined by the number of protons in the nucleus of the atom. The number of neutrons may vary, however. Elements sharing the same number of protons, but having different numbers of neutrons are known as isotopes. Hydrogen, for example, has one proton. All nuclei containing one proton are hydrogen nuclei. It may have one, two, or three neutrons. The isotopes of hydrogen would be written as ^1H_1 ^2H_1 ^3H_1 (in all further references, the atomic number subscript 1 is left off for clarity). ^1H is the most stable form of hydrogen and is therefore the most abundant (99.985% of all forms). ^2H is also a stable form of hydrogen, but less stable than ^1H, and constitutes about 0.015% of the total hydrogen found. It is known as deuterium.

^3H is unstable and constitues a very small fraction of the amount of hydrogen available. Termed tritium , this element readily reorganizes its nucleus, and is said to decay. The emission of its sub-atomic particles and energy is therefore known as radioactive decay, or simply radioactivity. Deuterium is a stable, but heavy isotope of hydrogen, tritium is a radioactive isotope of hydrogen.

Note that each of the three will chemically react as hydrogen. This is important for tracer work in cell biology. The substitution of either deuterium or tritium for hydrogen in a molecule will not effect any chemical or physiological changes in the activity of the molecule. Tritium will, however, tag the molecule by making it radioactive.

Radiation emissions have several forms. When an atom reorganizes its sub-atomic structure to a more stable form, it may emit neutrons, protons, electrons, and/or electromagnetic waves (energy). An alpha particle is 2 protons plus 2 neutrons (essentially a helium nucleus). A beta particle is an electron. Gamma rays are electromagnetic energy waves similar to x-rays. The release of sub-atomic particles and energy, resulting in the change of one element to another is known as radioactivity.

Radioactive elements thus, by their very nature, self destruct. The loss of their sub-atomic particles is a spontaneous process, and once it has occurred, the element is no longer radioactive. With time a percentage of all radioactive elements in a solution will decay. Statistically, it is nearly impossible to predict which individual element will radioactively decay, but we can make a prediction about large numbers of the elements. That is, we can say that if we wait 14,000 years, half of the radioactivity in a sample of ^1^4C (a radioactive isotope of ^1^2 C) will be lost (1/2 remains). We then say that ^1^4C has a half- life of 14,000 years. After a second 14,000 years, half of the remaining half would have been lost, or 3/4 of the original amount. Based on this information, could you predict how long it would take for all radioactivity to have disappeared from the sample?

With a half-life of 14,000 years, radioactive carbon will be around for a very long time. This is why it is used for dating rocks and fossils. If one makes some assumptions about the activity of the carbon when the fossil was formed, and measures the current level, the age of the fossil may be determined.

The amount of radioactive material is measured by how many nuclei decay each second, and this value is known as the activity. It is measured in curies Each radioisotope has three important properties; the type of particles emitted, the particle energy, and the half-life. The energy and kind of decay particle will determine the penetration of the radiation, and therefore determine the degree of shielding necessary to protect the user. The half-life determines both the remaining activity after storage or use, and the time that the isotope must be stored before disposal.

In cell biology, only a few of the many radioactive elements are used routinely. The primary elements used are ^3H (Tritium), ^1^5C (Carbon-14), ^3^2P 20(Phosphorus-32), ^1^2^5I 20(Iodine-125) and ^1^3^1 I 20(Iodine-137). The characteristics of each of these are given within the following table:

Not available at this time
TABLE H.1 Radioactive Sources and Emission Types

MEASUREMENT OF DOSE

When alpha or beta particles, or gamma radiation pass through matter, they form ions. They accomplish this by knocking electrons from the orbits of the molecules they pass through. We can monitor the ionization effect by allowing the radiation to pass through dry air and measuring the numbers of ions formed. This is most often done by designing a chamber with an electrical charge capacitance, allowing the radiation to pass through the chamber and monitoring the amount of capacitance discharge caused by the formation of ions. The device is a Geiger-Mueller Counter and has many variations.

The ionizing ability is measured in roentgens, and a roentgen is the number of ionizations necessary to form one electrostatic unit (e.s.u.) in 1 cc of dry air. Since the roentgen is a large unit, dosage for cell research use are normally divided into milliroentgens (mR).

Curies measure the amount of radioactive decay, roentgens measure the amount of radiation transmitted through matter, over distance. Neither unit is useful in determining biological effect, since biological effect implies that the radiation is absorbed by the tissues that are irradiated.

The rad (radiation absorbed dose) is a unit of absorbed dose and equals 100 ergs absorbed in one gram of matter. The roentgen is the amount of radiation exposure in air, while the rad represents the amount of radiation exposure in tissue. The two are usually very close in magnitude, however, since for most biological tissues, 1 roentgen produces 0.96 rad.

Not all radioactive emissions have the same penetrating power, however. If radiation safety (monitoring of dose) is considered, then the rad is insufficient. A linear-energy- transfer dependent factor must be defined for each type of emission. An alpha particle, for example, would not travel very far through tissue, but it is 10 times more likely to be absorbed than a gamma wave of the same energy dose. This factor is known as the Quality Factor (QF) or Relative Biological Effectiveness (RBE). The RBE is limited to work in radiobiology, the QF is used in other exposure monitor schemes. The use of the QF results in a new parameter, the rem. The rem is a unit of dose equivalent and is equal to the product of the QF x rad.

DETECTION OF RADIOACTIVITY

IONIZATION CHAMBERS The most common method of measuring radiation exposure is the use of an ionization chamber. Among the more common forms of ionization chambers are the Geiger counter and the pocket dosimeter.

The chambers are systems that comprise two electrical plates, with a potential established between them by a battery or other electrical source. In effect, they function as capacitors. The plates are separated by an inert gas, which will prevent any current flow between the plates. When an ionizing radiation enters the chamber, it induces the formation of an ion, which in turn is drawn to one of the electrical plates. The negative ions are drawn the the anode (+ plate) while the positive ions are drawn to the cathode (- plate). As the ions reach the plates, they induce an electric current to flow through the system attached to the plates. This is then expressed as a calibrated output, either through the use of a digital or analog meter, or as a series of clicks , by conversion of the current through a speaker.

The sensitivity of the system depends on the voltage applied between the electric plates. Since alpha particles are significantly easier to detect than beta particles, it requires lower voltage to detect the high energy alpha particles. In addition, alpha particles will penetrate through the metal casing of the counter tube, whereas beta particles can only pass through a quartz window on the tube. Consequently, ionization chambers are most useful for measuring alpha emmissions. High energy beta emissions are able to be measured if the tube is equipped with a thin quartz window and if the distance between the source of emission and the tube is minimal.

A modification of the basic ionization chamber can be made by engineering the tube such that it is miniaturized and such that the tube can be charged to hold a voltage without constantly rebuilding the voltage via a battery. This gives rise to the pocket dosimeter . This device is a capacitor, which is charged by a base unit and which can then be carried as a portable unit. They are often the size and shape of a pen and can be thus carried in the pocket of a lab coat. When exposed to an ionizing radiation source, the capacitor discharges slightly. Over a period of time, the charge remaining on the dosimeter can be monitored and used as a measure of radiation exposure. The dosimeters are usually inserted into a reading device which is calibrated to convert the average exposure the dosimeter has had directly into roentgens or rems. 1 Since the instrument works by discharging the built up charge, and the charge is upon a thin wire in the center of the dosimeter, it can be completely discharged by the flexing of that wire, as it touches the outer shell upon impact. When later read for exposure, the investigor will be informed that they have been exposed to dangerously high levels of radiation as there will be no charge left in the dosimeter. Besides causing great consternation with the Radiation Safety Officer, and a good deal of paper work, it also causes some unrest with the investigator. The dosimeters should be worn in a location where they can not impact any other objects.> Since the dosimeters normally lack the fragile and vulnerable quartz windows of a Geiger tube, and carry lower voltage potentials, they are used for the measurement of x- ray and high energy gamma radiation, and will not detect beta emmissions.

PHOTOGRAPHIC FILM Low energy emissions are detected more conveniently through the use of a film badge . This is simply a piece of photographic film sandwhiched between cardboard and made into a badge which can be pinned or clipped onto the outer clothing of the investigator. They can be worn routinely and collected on a regular basis for analysis.

When the film is exposed to radiation, it causes the conversion of the silver halide salts to reduced silver (exactly as exposure of the film to light). When the film is developed, the amount of reduced silver (black) can be measured and calibrated for average exposure to radiation. This is normally done by a lab specializing in the monitoring. Because of the simplicity of the system, its relative low cost and its sensitivity to nearly all forms of radiation, it is the primary means of radiation exposure monitoring of personnel.

SCINTILLATION COUNTERS For accurate quantitative measurement of low energy beta emissions and for rapid measurement of gamma emissions, nothing surpasses the use of scintillation counters. Since they can range from low to high energy detection, they are also useful for the alpha emissions.

Scintillation counters are based on the use of light emitting substances, either in solution, or within a crystal. When a scintillant is placed in solution with a radioactive source (liquid scintillation counter), the radiation strikes the scintillant molecule, which will then fluoresce as it re-emits the energy. Thus the scintillant gives a flash of light for each radiation particle it encounters. The counter than converts light energy (either as counts of flashes, or as an integrated light intensity) to an electrical measure calibrated as either direct counts or counts per minute (CPM). If the efficiency of the system is known (the % of actual radiatioactive decays that result in a collision with a scintillant), then disintegrations per minute (DPM) can readily be calculated. DPM is an absolute value, whereas CPM is a function of the specific instrument used.

Low energy beta emissions can be detected with efficiencies of 40% or better with the inclusion of the scintillant directly into a cocktail solution. Alpha emissions can be detected with efficiencies in excess of 90%. Thus, with a liquid scintillation counter, very low doses of radiation can be detected. This makes it ideal for both sensitivity of detection and for safety.

If the system is modified such that the scintillant is a crystal placed outside of the sample chamber (vial) then the instrument becomes a gamma counter. Gamma emissions are capable of exiting the sample vial and entering into a fluorescent crystal. The light emitted from the crystal is then measured. Gamma counters are usually smaller than liquid scintillation counters, but are limited to use with gamma emittors. Modern scintillation counters usually combine the functional capabilities of both liquid scintillation and direct gamma counting.

Since all use of radioactive materials, and particularly the expensive counting devices are subject to local radiation safety regulations, the specific details for use must be left to the institutional discretion. Under no circumstances should radioactive materials be used without the express supervision of the radiation safety officer of the institution, following all specific institutional guidelines and manufacturer directions for the instrument used.

AUTORADIOGRAPHY

The process of localizing radioactive materials onto a cell is known as autoradiography. ^3H (tritium) is used in cell analysis because it is a relatively weak Beta emitter (thus making it safer to handle) and more significantly, can be localized within cell organelles. ^1^4C and ^3^2P are also used, but are more radioactive, require significantly more precautions in handling and are inherently less capable of resolving intracellular details. They are used at the tissue or organ level of analysis, however.

Radioactive isotopes can be incorporated into cellular molecules. After the cell is labeled with radioactive molecules, it can be placed in contact with photographic film. Ionizing radiations are emitted during radioactive decay and silver ions in the photographic emulsion become reduced to metallic silver grains. The silver grains not only serve as a means of detecting radioactivity but, because of their number and distribution, provide information regarding the amount and cellular distribution of radioactive label.

The process of producing this picture is therefore called autoradiography and the picture is called an autoradiogram.

The number of silver grains produced depends on the type of photographic emulsion and the kind of ionizing particles emitted from the cell. Alpha particles produce straight, dense tracks a few micrometers in length. Gamma rays produce long random tracks of grains and are useless for autoradiograms. Beta particles or electrons produce single grains or tracks of grains. High energy Beta particles (such as those produced by 32P) may travel more than a millimeter before producing a grain. Low energy Beta particles ( ^3H and 14 ° C) produce silver grains within a few micrometers of the radioactive disintegration site and so provide very satisfactory resolution for autoradiography.

The site of synthesis of cellular molecules may be detected by feeding cells a radioactive precursor for a short period and then fixing the cells. During this pulse labeling, radioactivity is incorporated at the site of synthesis but does not have time to move from this site. The site of utilization of a particular molecule may be detected be chase labeling. Cells are exposed to a radioactive precursor, radioactivity is then washed or diluted away and the cells allowed to grow for a period of time. In this case, radioactivity is incorporated at the site of synthesis but then has time to move to a site of utilization in the cell.

^3H-thymidine can be used to locate sites of synthesis and utilization of DNA. Thymidine, the deoxyribose nucleoside of thymine, can be purchased with the tritium label attached to the methyl group of thymine. Thymidine is specifically incorporated into DNA in Tetrahymena. Some organisms can remove the methyl group from thymine, and incorporate the uracil product into RNA. Even in this case RNA would not be labeled because the tritium label would be removed with the methyl group. Methyl labeled thymidine, therefore, serves as a very specific label for DNA.

This is known as pulse labeling, after which the cells are washed free of the radioactive media. All remaining radioactivity would be due to the incorporation of the thymidine into the macromolecular structure of DNA. The cells will be fixed, covered with a photographic emulsion and allowed to develop .

During this time, the activity emanating from the ^3H will expose the photographic emulsion, causing the presence of reduced silver grains immediately above the location of the radioactive source (DNA). Thus, it will be possible to localize the newly synthesized DNA, or that which was in the S phase of mitosis during the time period of the pulse labeling.

RULES FOR SAFE HANDLING OF RADIOACTIVE ISOTOPES


  1. All work with radioactive material must be done in a tray lined with absorbant paper.

  2. All glassware and equipment contacting radioactive material must be appropriately labeled and kept inside the tray. The only exception is that microscope slides of labeled cells may be removed from the tray after the drop of labeled cells has been applied to the slide and allowed to dry.

  3. Plastic gloves should be worn when handling radioactive material.

  4. All waste solutions containing radioisotopes, all contaminated gloves, paper, etc., must be placed in appropriate liquid or dry radioactive waste containers.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu