**3.2 Effects of gamma rays on living organisms**

Radiation effects on living organisms are mainly associated with the chemical changes but are also dependent on physical and physiological factors. Dose rate, dose distribution, radiation quality are the physical parameters. The most important physiological and environmental parameters are temperature, moisture content and oxygen concentration. The action of radiation on riving organisms can be divided into direct and indirect effects. Normally, the indirect effects occur as an important part of the total action of radiation on it. The Figure 2 shown that radiolytic products of water are mainly formed by indirect action on water molecules yielding radicals OH• , e aq and H•. The action of the hydroxyl radical (OH•) must be responsible for an important part of the indirect effects. Drying or freezing of living organisms can reduce these indirect effects. If we consider pure water, each 100 eV of energy absorbed will generate: 2.7 radicals OH•, 2.6 e- aq, 0.6 radicals H•, 0.45 H2 molecules and 0.7 molecules H2O2. (Borrely et al, 1998).

Several types of microorganism, mainly bacteria and, less frequently, moulds and yeasts, have been found on many medical devices and pharmaceuticals (Takehisa et al, 1998). Complete eradication of these microorganisms (sterilization) is essential to the safety of medical devices and pharmaceutical products. The sterilization process must be validated to verify that it effectively and reliably kills any microorganisms that may be present on the

causing electron displacement within. These reactions, in turn, generate free radicals, which aid in breaking chemical bonds. Disrupting microbial DNA renders any organisms that

Gamma radiation does have some significant advantages over other methods of producing sterile product. These benefits include: better assurance of product sterility than filtration and aseptic processing; no residue like EtO leaves behind; more penetrating than E-beam;

Process validation may be defined as the documented procedure for obtaining, recording and interpreting the results required to establish that a process will consistently yield product complying with a predetermined specification. For sterilization, process validation is essential, since sterilization is one of those special processes for which efficacy cannot be verified by retrospective inspection and testing of the product. Process validation consists of: i. installation qualification of the facility; ii. operational qualification of the facility and iii.

Radiation sterilization of medical products also is currently regulated by two standards, EN 552 (1994) and ISO 11137 (1995). These standards will be harmonized in the very near future into ISO 11137 (2006) part 1, part 2 and part 3. Currently, all three parts of ISO 11137 (2006) are at the Final Draft International Standard Stage (FDIS). These three documents are now published. All sterilization standards consider 'dose' as a key parameter in order to determine if a product is sterile. However, measurement of dose is not a trivial task and a commercial dosimetry system consists of dosimeters, readout equipment and procedure for its use. Dosimeters may be films, small plastic blocks, fluids or pellets where there is a known and reproducible response to radiation dose. The dosimetry system must be calibrated, and the calibration must be traceable to a national standard. ISO/ASTM standard

Radiation effects on living organisms are mainly associated with the chemical changes but are also dependent on physical and physiological factors. Dose rate, dose distribution, radiation quality are the physical parameters. The most important physiological and environmental parameters are temperature, moisture content and oxygen concentration. The action of radiation on riving organisms can be divided into direct and indirect effects. Normally, the indirect effects occur as an important part of the total action of radiation on it. The Figure 2 shown that radiolytic products of water are mainly formed by indirect action

(OH•) must be responsible for an important part of the indirect effects. Drying or freezing of living organisms can reduce these indirect effects. If we consider pure water, each 100 eV of energy absorbed will generate: 2.7 radicals OH•, 2.6 e- aq, 0.6 radicals H•, 0.45 H2 molecules

Several types of microorganism, mainly bacteria and, less frequently, moulds and yeasts, have been found on many medical devices and pharmaceuticals (Takehisa et al, 1998). Complete eradication of these microorganisms (sterilization) is essential to the safety of medical devices and pharmaceutical products. The sterilization process must be validated to verify that it effectively and reliably kills any microorganisms that may be present on the

aq and H•. The action of the hydroxyl radical

survive the process nonviable or unable.

low-temperature process and simple validation process.

performance qualification of the facility (ISO 14937, 2000)

51261 gives guidelines for calibration procedures.

**3.2 Effects of gamma rays on living organisms** 

on water molecules yielding radicals OH• , e-

and 0.7 molecules H2O2. (Borrely et al, 1998).

pre-sterilized product. Radiation sterilization, as a physical cold process, has been widely used in many developed and developing countries for the sterilization of health care products. Earlier, a minimum dose of 25 kGy was routinely applied for many medical devices, pharmaceutical products and biological tissues. Now, as recommended by the International Organization for Standardization (ISO), the sterilization dose must be set for each type of product depending on its bioburden. Generally, the determination of sterilization dose is the responsibility of the principal manufacturer of the medical product, who must have access to a well qualified microbiology laboratory.

Fig. 2. Effect of gamma rays on water molecules

The lethal effect of ionizing radiation on microorganisms, as measured by the loss by cells of colony-forming ability in nutrient medium, has been the subject of detailed study. Much progress has been made towards identification of the mechanism of inactivation, but there still remains considerable doubt as to the nature of the critical lesions involved, although it seems certain that lethality is primarily the consequence of genetic damage. Many hypotheses have been proposed and tested regarding the mechanism of cell damage by radiation. Some scientists proposed the mechanism thought 'radiotoxins' that are the toxic substances produced in the irradiated cells responsible for lethal effect. Others proposed that radiation was directly damaging the cellular membranes. In addition, radiation effects on enzymes or on energy metabolism were postulated. The effect on the cytoplasmic membrane appears to play an additional role in some circumstances (Greez et al, 1983).

It is now universally accepted that the deoxyribonucleic acid (DNA) in the chromosomes represents the most critical 'target' for ionizing radiation because it is responsible for inhibition of cell division.

A DNA strand is composed of a series of nucleotides containing a purine (adenine, guanine) or a pyrimidine base (cytosine, thymine), a sugar (deoxyribose) bond to the base and a phosphate connected to the sugar. The nucleotides are joined by phosphodiester bonds

Sterilization by Gamma Irradiation 177

ionization or to the indirect action of the radiolysis products of water, or both. However, while the work on basic mechanisms continues, much is already known both qualitatively and quantitatively in relation to the radiation inactivation of microbial populations. Just as with heat resistance, there is considerable variability in radiation resistance between microbial species; in general, viruses are more radiation resistant than bacterial spores, which in turn are more resistant than vegetative organisms, yeasts and moulds. Moreover, the inactivation of microbial populations is considerably influenced by conditions of environment during irradiation-for example, gaseous composition, temperature, and nature

When a suspension of a microorganism is irradiated at incremental doses, the number of surviving cell forming colonies after each incremental dose may be used to construct a dose survival curve, as shown in Figure 5. The radiation resistance of a microorganism is measured by the so-called decimal reduction dose (D10 value), which is defined as the radiation dose (kGy) required to reduce the number of that microorganism by 10-fold (one log cycle) or required to kill 90% of the total number (Whitby & Gelda, 1979). The D10 value

Fig. 5. Typical survival curve for a homogeneous microbial population.

of the suspending medium.

**3.2.1 Decimal reduction dose** 

Fig. 4. Combinational repair of DNA double break

between the sugar and the phosphate. DNA is composed of two complementary antiparallel strands linked by hydrogen bonds between the bases. Thymine is complementary to adenine (two hydrogen bonds between them) whilst guanine is the complementary base to cytosine (linked by three hydrogen bonds). In the most frequent configuration, called B form, the two strands are twisted to form a right-handed double helix. Ionizing radiation can affect DNA either directly, by energy deposition in this critical target, or indirectly, by the interaction of radiation with other atoms or molecules in the cell or surrounding the cell like water. In particular, radiation interacts with water, leading to the formation of free radicals (see Figure 2) that can diffuse far enough to reach and damage DNA. It is worth mentioning that the OH• radical is most important; these radicals formed in the hydration layer around the DNA molecule are responsible for 90% of DNA damage. Consequently, in a living cell, the indirect effect is especially significant. In a general sense, the death of a microorganism is a consequence of the ionizing action of the high energy radiation. It is estimated that the irradiation of a living cell at one gray induces 1000 single strand breaks, 40 double strand breaks, 150 cross-links between DNA and proteins and 250 oxidations of thymine (ABCRI, 1992; Borrely et al, 1998) ).

Both prokaryotes (bacteria) and eukaryotes (moulds and yeasts) are capable of repairing many of the different DNA breaks (fractures). Living organisms have developed different strategies to recover from losses of genetic information caused by DNA damages. Damages to DNA alter its spatial configuration so that they can be detected by the cell. In the case of single strand breaks (Figure 3), the damaged DNA strand is excised and its complementary strand is used to restore it. Efficient and accurate repair of the damages can take place as long as the integrity of the complementary strand is maintained. Radiosensitivity is highly influenced by the capability of the strain to repair single-strand breaks. Strains that lack this ability are far more radiosensitive than the others (Tubiana et al., 1990; WHO, 1999). Double strand breaks are far more hazardous since they can lead to genome rearrangements. Two distinct mechanisms have been described for the repair of double strand breaks: non homologous end joining and recombination repair (Broomfield et al., 2001).

Fig. 3. Single strand breaks in DNA


Apart from difficulties in location of the site of primary damage, there is still controversy as to whether the majority of radiation effects on biological systems are due directly to

between the sugar and the phosphate. DNA is composed of two complementary antiparallel strands linked by hydrogen bonds between the bases. Thymine is complementary to adenine (two hydrogen bonds between them) whilst guanine is the complementary base to cytosine (linked by three hydrogen bonds). In the most frequent configuration, called B form, the two strands are twisted to form a right-handed double helix. Ionizing radiation can affect DNA either directly, by energy deposition in this critical target, or indirectly, by the interaction of radiation with other atoms or molecules in the cell or surrounding the cell like water. In particular, radiation interacts with water, leading to the formation of free radicals (see Figure 2) that can diffuse far enough to reach and damage DNA. It is worth mentioning that the OH• radical is most important; these radicals formed in the hydration layer around the DNA molecule are responsible for 90% of DNA damage. Consequently, in a living cell, the indirect effect is especially significant. In a general sense, the death of a microorganism is a consequence of the ionizing action of the high energy radiation. It is estimated that the irradiation of a living cell at one gray induces 1000 single strand breaks, 40 double strand breaks, 150 cross-links between DNA and proteins and 250 oxidations of

Both prokaryotes (bacteria) and eukaryotes (moulds and yeasts) are capable of repairing many of the different DNA breaks (fractures). Living organisms have developed different strategies to recover from losses of genetic information caused by DNA damages. Damages to DNA alter its spatial configuration so that they can be detected by the cell. In the case of single strand breaks (Figure 3), the damaged DNA strand is excised and its complementary strand is used to restore it. Efficient and accurate repair of the damages can take place as long as the integrity of the complementary strand is maintained. Radiosensitivity is highly influenced by the capability of the strain to repair single-strand breaks. Strains that lack this ability are far more radiosensitive than the others (Tubiana et al., 1990; WHO, 1999). Double strand breaks are far more hazardous since they can lead to genome rearrangements. Two distinct mechanisms have been described for the repair of double strand breaks: non

1. For non homologous end joining, the free ends are joined by simple ligation which may result either to perfect reparation or to genetic mutation if sequences are not

2. Combinational repair (Figure 4) necessitates the presence of another copy of the genetic material within the cell since an identical DNA sequence is used as a template. This last mechanism cannot be achieved by all bacteria since some only possess one copy of

Apart from difficulties in location of the site of primary damage, there is still controversy as to whether the majority of radiation effects on biological systems are due directly to

homologous end joining and recombination repair (Broomfield et al., 2001).

genetic material per cell (Hansen, 1978; Kuzminov, 1999).

thymine (ABCRI, 1992; Borrely et al, 1998) ).

Fig. 3. Single strand breaks in DNA

homologue.

ionization or to the indirect action of the radiolysis products of water, or both. However, while the work on basic mechanisms continues, much is already known both qualitatively and quantitatively in relation to the radiation inactivation of microbial populations. Just as with heat resistance, there is considerable variability in radiation resistance between microbial species; in general, viruses are more radiation resistant than bacterial spores, which in turn are more resistant than vegetative organisms, yeasts and moulds. Moreover, the inactivation of microbial populations is considerably influenced by conditions of environment during irradiation-for example, gaseous composition, temperature, and nature of the suspending medium.

Fig. 4. Combinational repair of DNA double break
