Radioprotective Effect of Chlorophyll-Based Agents in the Process of Exposure of Animals to Gamma Radiation

N.P. Lysenko, A.V. Pozdeev, L.A. Romodin, A.V. Novikova, L.V. Rogozhina,
M.V. Shchukin, V.B. Chernetsov.

Federal State Budgetary Educational Institution of Higher Education “K.I. Skyabin Moscow State Academy of Veterinary Medicine and Biotechnology”, Moscow, Russia

Abstract

The issue of searching new ways to protect living organisms against ionizing radiation is still topical. Development of chemical compounds leveling the negative impact of ionizing radiation of living organisms (radioprotectors) is one of the most optimal ways. This paper provides findings of a study of a chlorophyll-based radioprotector we conducted. In order to assess efficacy of the agent we determined concentrations of lipid peroxidation markers (malondialdehyde (MDA) concentration and intensity of NADPH-dependent and ascorbate-dependent lipid peroxidation) induced by γ-rays and hematological parameters of exposed laboratory animals. The total ionizing radiation dose was 5 Gy in all our experiments except for the experiment with assessment of effects of the agent on cortisol production resulting from exposure, the total radiation dose in which was 13.05 Gy.  

The chlorophyll-based radioprotector was administered by intramuscular injection to different study groups of laboratory animals before and after radiation exposure, while animals exposed to γ-rays without administration of the chlorophyll-based agent and animals which were neither treated with the agent nor exposed to ionize radiation were used as control groups. The parameters we estimated in animals which received the radioprotector and were exposed to γ-rays came back to normal during 30 days after exposure to ionized radiation, and this fact suggests that the chlorophyll-based agent under investigation is efficient as a radioprotector.

Keywords

Radiation injury, radioprotectors, chlorophyll, antioxidants, active forms of oxygen, ionizing radiation, gamma radiation

Abbreviations

LPO – lipid peroxidation;
MDA – malondialdehyde;
NADPH – reduced nicotinamide-adenine dinucleotide phosphate;
ATP – adenosine triphosphate;
ELISA – enzyme-linked immunosorbent assay;
ARS – acute radiation syndrome;
DNA – deoxyribonucleic acid;
EDTA – ethylenediamine tetraacetate.

Introduction

The risk of radiation injury our environment poses is still rather high. This risk stems not from nuclear weapon testing or a possibility of its military use only, but also from possible accidents at nuclear power plants and facilities and from an ever growing amount of radioactive waste. Thus, the risk of radiation injury for humans and animals is growing higher year by year. Therefore, development of measures and discovery of ways to eliminate consequences of nuclear pollution of the environment has become one of the major tasks of the world science. In terms of medicine, i.e. wellbeing of individuals, development of new radioprotectors and investigation of mechanisms of their effects on various systems of organs in an organism or evaluation of their safety with regard to toxicity is a topical issue. This paper provides findings of a study of effects of a chlorophyll-based agent on parameters reflecting lipid peroxidation, including blood and liver malondialdehyde (MDA) concentration and intensity of NADPH-dependent and ascorbate-dependent lipid peroxidation (LPO) after radiation exposure, blood cortisol levels, and hematological parameters (red blood cell (RBC) and white blood cell (WBC) percentage).    

Role of Peroxidation Reactions in Radiation Injury

Peroxidation reactions which take place in organisms of humans and animals are more or less harmful for vital functions of the organism. However, there are various antioxidant mechanisms which can reduce peroxidation processes to a minimum level, such as molecules of fat-soluble antioxidants embedded into biological membranes (tocopherols, retinoids, etc.) [10]. Any failure in such antioxidant systems results in activation of peroxidation processes which, in turn, cause various abnormalities. Ionized radiation is one of the factors triggering peroxidation processes [7,8]. Malondialdehyde (MDA) concentration in blood plasma is one of the indicators of activation of peroxidation processes [14].   

Changes in Hematological Parameters after Radiation Exposure

The most frequently phenomenon in an exposed organism reported in literature is leukocyte “left shift”, i.e. emergence of young “immature” forms of neutrophils in blood, which are normally present in bone marrow only. This phenomenon is mainly attributable to transient leucopenia which, in most cases, develops due to low count of neutrophils [5]. Investigators note that the degree of leukocyte “left shift” becomes higher with the increase in the radiation dose received, and there is evidence to the fact that after radiation exposure neutrophils counts remain rather low during the lifetime and that phagocytic and lysosomal activity is reduced [6]. Low ATP level in a cell at a radiation dose of 10 Gy is one of the factors contributing to such decrease in neutrophils (and granulocytes in particular) [4]. Biosher at al. [1] suggested that a radiation dose of 50 Gy results in inhibition of oxidative phosphorylation processes in neutrophils.

Quantitative changes in white blood cells after exposure to lethal and sublethal doses can be divided into three phases:

1^st phase (first minutes and hours) – short-term mild drop in white blood cell (WBC) count;

2^nd phase (at 6 to 8 h) – rising of the baseline level by 10 to 15%;

3^rd phase (by the end of the day) – WBC count drops abruptly to the baseline level and remains stable [6].

It is a well-known fact that quantitative changes in white blood cells are radiation dose-dependent. Reduced WBC count is most prominent in the second or third week after exposure to ionized radiation (in semilethal doses). During this period the WBC count is three and more time less than normal [6].

Red Blood Cells

Mature anucleated red blood cells are more resistant to deleterious effects of ionizing radiation than other cells. This can be explained by absence of mitochondria, genome and metabolic processes inherent to other cells. Radiation exposure in a dose of 200 Gy causes disruption of red blood cell morphology, emergence of binucleated cells, vacuolation of nuclei, granularity of cytoplasm, and impaired permeability of red blood cell (RBC) membranes [2]. It should be noted that ionized radiation plays an important role in hemolysis and disruption of red blood cell membrane structures. Back in 1904 Henry and Meier [3] were the first to describe hemolytic properties of ionizing radiation. A.P. Kozlov et al. showed that radiation exposure in a dose of 600 Gy causes immediate hemolysis [9]. The greatest changes in red cells are observed at semilethal doses. During the first three days after exposure RBC counts and hemoglobin content in 1 mm³ grow by 10 to 15% and then the exposed organism develops anemia reaching its peak on the 15^th to 20^th day and the RBC count and hemoglobin content become 2‒3 times lower.

The purpose of the present study was to investigate effects of a chlorophyll-based agent on blood parameters of laboratory animals, which are markers of deleterious effects of ionizing radiation.

We selected CD-1 male mice (body mass of 25‒30 g) as the subject of our study.

A chlorophyll-based agent manufactured by “Limex-Farma” LLC (Moscow) of plant raw materials was diluted in saline solution in concentration 1:5 and used as the agent under investigation. The preparation was administered by intramuscular injection in a specific volume of 10 ml/kg.

The same volume of saline solution was used as control.

1.      Estimation of malondialdehyde (MDA) in blood and liver tissue of mice exposed to radiation using the chlorophyll-based agent:

We formed 3 groups of 50 mice each according to the time of administration of the agent, and a control group as follows:

• Group 1 (Control): The animals were exposed without administration of the agent;

• Group 2: The agent was administered before exposure;

• Group 3: The agent was administered after exposure; and

• Group 4 (Biological Control): The animals were not exposed to ionized radiation and did not receive the agent.

2.      Estimation of activity of NADPH-dependent LPO in blood and the liver of mice exposed to radiation using the chlorophyll preparation:

We formed 3 groups of 50 mice each according to the time of administration of the preparation, and a control group as follows:

• Group 1: Control;

• Group 2: The agent was administered before exposure; and

• Group 3: The agent was administered after exposure.

 

3.      Estimation of activity of ascorbate-dependent LPO in blood of exposed mice after administration of the chlorophyll preparation (nmol/g):

We formed 3 groups of 50 mice each according to the time of administration of the preparation, and a control group as follows:

• Group 1: Control;

• Group 2: The agent was administered before exposure; and

• Group 3: The agent was administered after exposure.

4.      Estimation of cortisol levels in blood of exposed mice on the background of administration of the chlorophyll preparation:

The mice were exposed with a gamma ray therapy unit “Panorama”. The total single dose was 13.05 Gy which caused development of clinical signs of acute radiation syndrome. Cortisol levels in blood were estimated using the ELISA method. We formed 3 groups of 10 mice:

Group 1 – without exposure;

Group 2 ‒ exposure without administration of the preparation;

Group 3 – administration of the preparation in a dose of 10 ml/1 kg before exposure.

Orbital sinus blood sampling was performed while the mice were alive.

5.      RBC count in blood of exposed mice with administration of the chlorophyll preparation:

We formed 3 groups of 50 mice each according to the time of administration of the preparation, and a control group as follows:

• Group 1: Control;

• Group 2: The agent was administered before exposure; and

• Group 3: The agent was administered after exposure.

6.      WBC count in blood of exposed mice with administration of the chlorophyll preparation:

We formed 3 groups of 50 mice each according to the time of administration of the preparation, and a control group as follows:

• Group 1: Control;

• Group 2: The agent was administered before exposure; and

• Group 3: The agent was administered after exposure.

Conclusion

This study was aimed at investigation of changes in some blood parameters of laboratory animals in response to exposure to ionizing radiation with administration of a chlorophyll preparation and assessment of radioprotective properties of the latter. The findings give evidence to the fact that values of such blood parameters as MDA concentration in blood and the liver, activity of NADPH-dependent LPO, cortisol concentration and counts of blood corpuscles (red blood cells and white blood cells) were close to the physiological norm when the chlorophyll-based agent was administered before and after radiation exposure, while those parameters in the control group which did not receive the preparation deviated from the norm significantly.

On the basis of the aforesaid we suggest that the chlorophyll preparation has a positive effect on animal organisms after radiation exposure, and since this agent is plant-based the risk of development of side effects of the agent is low. However, in order to simplify its administration an oral form of the agent should be developed.

 

References:

1.      Buescher E.S., Holland P.V., Gallin J.I. Radiation induced defective oxygen metabolism in leukocytes prepared for transfusion as assessed by nitroblue tetrazolium (BTT) reduction // Clin Res / 31, 1983.
2.      Davis R.W., Dole N., Izzo M.J., Young L.E. Hemolytic effect of radiation // Clin Med / 35, 1950.
3.      Henri V., Mayer A. Action des radiations du radium sur les colloïdes d'hémoglobin, les ferments, et les globules rouges // Compt Rend /, 1904.
4.      Kotelba-Witkowska B., Dudа W., Witas H. Effect of ionizing radiation on the morphological blood elements // Acta Physiol / Pol. 30 1979. p.:15 - 6.
5.      Ts.I. Abakelia, I.S. Tsomaia, M.G. Odishvili. [On Matters of Presence of Leukopoietically Active Substances in Ionized Radiation-Induced Leukopenias]. Theses of the 6^th All-Russian Scientific Conference “Regenerative and Compensatory Processes in Radiation Injuries”. Leningrad: 1973. P. 60‒1.
6.      M.D. Brilliant, A.I. Vorobyov. [Changes in Some Peripheral Blood Parameters in Total Exposure of Humans]. Problems of Hematology and Blood Transfusion. No.1, 1972. 
7.      A.A. Greshilov. [Products of U-235, U-238 and Ru-239 Prompt Fussion within the Period from 0 to 1 h: Handbook]. Moscow: Atomizdat, 1969. 105 p.
8.      Y.G. Grigoryev. [Nuclear Safety during Spaceflights]. Moscow: Atomizdat, 1975. 253 p.
9.      A.P. Kpzlov, E.A. Krasavin, A.V. Bereyko et al. [Investigation of Red Blood Cell Membrane Damage after Exposure to γ-Rays in a Wide Dose Range]. Letters to “Physics of Elementary Particles and Atomic Nuclei” Jounral. V. 5, No.2, 2008. P. 219‒25.
10.    N.A. Korneev, A.I. Sirotkin, I.M. Rasin, S.A. Geraskin. [Topical Issues of Agricultural Radioecology]. Bulletin of Agricultural Science. No.11, 1987. P. 113‒20.
11.    V.I. Legeza. [Syndrome of Primary Response to Radiation Exposure]. Military-Medical Journal. No.10, 1991. P. 20‒2.
12.    V.I. Legeza. [Means of Early Pathogenetic Therapy of Radiation Injuries: Problems and Prospects]. Radiobiology, Radioecology, and Nuclear Safety: Thesies of the 4^th Congress on Nuclear Research. Moscow. V. 2, 2001. P. 426.
13.    A.V. Pozdeev. [Experimental Study of Cortisol Concentration in Blood after Radiation Exposure]. Bulletin of Kursk State Agricultural Academy, 2013.
14.    G.L. Shreiberg. [Neurohumoral Mechanisms of Regulation of the Hypothalamus‒Pituitary‒Adrenal Cortex System Function]. Hypothalamus Physiology and Pathology. Moscow, 1963. P. 30‒8.