2. PREDECESSORS OF LIPOFUSCIN AGE PIGMENT -
PROBABLE ROLE IN THE PROCESSES OF RADIATION DAMAGE
AND PROTECTION
V. T. Vertushkoff
Dnepropetrovsk,
49128, Ukraine
Polymeric products of unsaturated fatty acids' oxidation are involved both into development of radiation damage and protection of animals by means of chemical radio-protectors.
The polymeric compounds are the main product of oil thermal oxidation at temperature 200°-300° C. The higher the temperature of oil oxidation is, the lesser is the maximum concentration of peroxides, achieved in the course of oxidation. At temperatures above 200°C, peroxide concentration is close to zero, and it doesn't increase under oxidation. Perkins [1], having summarized the results of wide range of investigations on auto- and thermal oxidation of fats, considers that ethyl linoleate oxidation leads to appearance of conjugated hydroperoxides which can cyclize with further polymer formation. Cyclization and polymerization of peroxides is the cause of oil coloring. Maximum polymer molecular mass resulting upon superheating of corn oil (200°C, 24 hours) exceeds 10.000 dalton [2].
It is revealed [3] that addition of a number of radio-protectors (catecholamines, serotonin, histamine, cystamine, cysteamine, mecsamine, amino-ethyl isothiouronium, propyl gallate, hydroxylamine) to oleic acid, which oxidizes at relatively low temperature (75°C in presence of Fe²+ ions and air oxygen) results in quick accumulation of colored polymeric products in oleic acid. Biogenic amines are the most active ones in this respect. For example, in 2-3 minutes after infusing of serotonin (in 75% methyl alcohol) or adrenaline into oleic acid (in a drop of ice-cold acetic acid) in 2,5x10-3 mol/l concentration, the quantity of lipopolymers amounted to 1,3 - 1,5%
of the acid original mass. Fractioning on the column with sephadex LH-20 (1 : 1 eluting chloroform-methanol mixture) calibrated by polyethylene glycol displayed that maximum molecular mass of polymers exceeded 6.000 dalton. The results obtained have demonstrated that addition of above listed radio-protectors to oleic acid is equivalent to rising in temperature of its oxidation up to 200°-400°C (depending on the added radio-protector). Therefore, radio-protectors are the catalysts of oxidative polymerization of unsaturated fatty acids. But for all that, radio-protector impact on fatty acids is distinguished by specific nature. Polymers formed in oleic and linoleic acids under the influence of various radio-protectors are varying between each other by absorption and fluorescence spectra, as well as biologic action [4-7].
When analyzing the structure of the most efficient radio-protecting preparations, Moszhukhin and Rachinsky [8] note that amino-mercapto- and oxy-group may be referred to active groupings in radio-protectors structure. For the preparation to possess strongly pronounced protective properties, it is necessary to combine the above mentioned groups with each other or to associate them with the other groups, such as imidazole, indole or guanide ones. Also, the distance between active groups should not exceed 2-3 carbon atoms. Preparations with such a structure, which come into compound with any chemical group, will form the most stable five- and six-component rings, with the help of both active groups simultaneously. At the same time, preparations featuring separation of the active groups by one or four and more carbon atoms, fail to form stable rings. The authors suppose that the ability to form cyclic compounds is one of the aspects of protective action mechanism displayed by radio-protectors [8]. Evidently, these cyclic compounds are the polymeric products of unsaturated fatty acids' oxidation, which are formed of peroxides under the influence of radio-protectors.
Intraperitoneal injection of white mice with radio-protectors (including reduced glutathione and cysteine) in radio-protective doses also induces the occurrence of polymeric products of unsaturated fatty acids' oxidation in organs and tissues. The lipids were extracted from tissues with 2:1 chloroform-methanol mixture [9]. Presence of colored polymers in the lipids' solution in chloroform was determined by absorption at 400 nm. Lipid concentration in small intestines is 1%, whereas the other tissues contain 0,15% of lipids (Fig. 1, 2) [5, 10]. As shown in the figures, maximum concentration of lipopolymers in a number of tissues falls on the period of maximum protection with these radio-protectors. Thus, after injection of mice with aminoethylisothiouronium (150 mg/ kg of animal weight), maximum absorption in 10 - 15 minutes at 400 nm is found in spleen and bone marrow. Injecting mice with adrenaline (4 mg/kg) causes accumulation of lipopolymers in blood and small intestines within the same periods. The results above have given rise to assumption that protective action of radio-protectors used in the present work was based upon the phenomenon that they formed polymeric products of unsaturated fatty acids' oxidation in the lipid structures of cells. If the radio-protective action of cystamine is maintained during several hours after its introduction [11], lipopolymer concentration in mice tissues remains at high level during three hours of observation (Fig. 3) [5]. Obviously, protective effect of lipopolymers is based on their ability to inhibit the formation of peroxide compounds in lipid phase of the exposed animals' cells (Fig. 4) [10].
To prove this assumption, cystamine was added to oleic acid being oxidized at 75°C. Lipopolymers formed were extracted by means of gel filtration method on column with sephadex LH-20. Analysis according to the method [12] has shown the absence of thio-groups in polymers. Out-bred white mice males of 18-22 g by weight were taken for experiments. Group consisting of 20 animals was chosen for obtaining each test point. Mice were exposed to radiation at X-ray apparatus ђ“Њ-13, dose rate being equal to 34 r/ min. It was single radiation exposure of the whole body, from the back. Lipopolymers extracted from the oleic acid were introduced to animals in 0,02 ml of olive oil using intraperitoneal method, amount being equal to 125 - 175 mg per 1 kg of the animal weight. The control animals were injected with olive oil. Lipopolymers were introduced 90 minutes before exposure to radiation. Efficiency of the protective action was determined according to the number of mice survived on the 30th day.
Experimental results have shown the availability of strongly pronounced protective action of lipopolymers with approximate value of the dose reduction factor - 1, 2 (Fig. 5) [13]. Relatively low value of this dose reduction factor is connected, apparently, with selective absorption of lipopolymers by the animal lymph [14], spleen [4] and, possibly, by the liver.
The results obtained confirm the theory, according to which the biologic action of ionizing radiation is based upon self-accelerated chain reactions of oxidation in lipid structures of cells [15-19]. Number of peroxides formed in the course of methyl oleate and methyl linoleate exposure to radiation, is growing in the ratio, directly proportional to the radiation dose [20]. Lipopolymers appearing in lipid phase of cells under the influence of radio-protectors, reduce the rate of generation of unsaturated fatty acids' peroxides in the process of radiation treatment, thus decreasing the effective radiation dose. As a result, under the same radiation dose the amount of peroxides is less in those lipids, which contain lipopolymers. Also it should be noted, that in the process of oxidative polymerization of unsaturated fatty acids under the influence of radio-protectors, the level of lipid peroxides found in sub-cell structures in normal amounts [21, 22] is virtually reduced to zero.
During five days after total X-ray exposure of mice in dose of 750 r, their spleen, bone marrow and blood are featuring appearance of two sharply defined maximums of lipopolymer concentration, registered by absorption at 400 nm in the solution of tissue lipids in chloroform. The first maximum falls on 6-12 hour period after exposure to radiation, and the second and more significant one occurs on the 3-5th days (Fig. 6) [3]. Occurrence of the first maximum concurs with the progressive damage and destruction of hemopoietic organs' cells. The second rise in lipopolymer concentration correlates with degenerative changes and mass death of cells of the intestinal mucous membrane (intestinal syndrome). Spleen lipids fractioning on the column with sephadex LH-20 has shown that maximum molecular mass of lipopolymers exceeded 4,000 dalton. On the fourth day after radiation treatment, lipopolymers content was equal to 40% of total spleen lipids mass. At the same time, dark-red color of spleen lipids solution in chloroform was observed visually.
Appearance of lipopolymers in tissues of the exposed animals results from the activity of the integrated system of primary response, warning and protection of the organism [23]. The individual elements of this system are: 1) diffusive endocrine system [24], which hormone-producing cells are dissipated in epithelial and connective tissues of all the organs in fact; 2) immune system; 3) sense organs and receptors of epithelial tissues connected with peptidergic neurons of the nervous system.
The immunological reaction is activated when the organism is affected by any stress factor, initiating damage and destruction of cells in tissues [7]. Responding to tissue damage (in particular, to the damage of hemopoietic organs and intestines by ionizing radiation), cells of the local diffusive endocrine system evolve excessive amounts of biogenic amines. Apparently, hypophysis-adrenal system (hormones of adrenal gland cortex) takes part in activation of diffusive endocrine system. Under the organism stress reaction condition, high concentrations of catecholamines, serotonin, histamine act as the catalysts of oxidative polymerization of unsaturated fatty acids included into the structure of phospholipids found in plasma membrane and sub-cell structure membranes. Since lipids in the membranes are associated with proteins, the action of massed biogenic amine doses on the cells results in the formation of colored lipoproteins of polymeric structure. Considerable amount of lipoproteins formed in plasma membrane, comes off the cells and goes to lymph, lymph nodes, spleen, liver. (Lipopolymers generated in cytoplasm are forming, eventually, lipofuscin granules). In spleen polymeric lipoproteins are absorbed by macrophages where lipopolymers are subjected to de-structuring [7]. Macrophages present the released protein antigens to T-lymphocytes thus initiating antigen-specific immune response. Possibly, bone marrow and liver of animals are capable to destruct lipopolymers.
It should be noted that damage of the actively proliferating cells of hemopoietic organs and intestinal mucous membrane is caused by the action of unsaturated fatty acids peroxides which amount increases pro rata to the radiation dose. Subsequent lipopolymer formation in the organism of exposed mice means that lipid peroxides' content in the damaged tissues is close to zero.
There is every reason to believe that the ability of animal spleen after total exposure to radiation to polymer destruction is broken. Barakina [25, 26] was investigating the patterns of radiation damage development in mice spleen. Results of the experiments have displayed the following:
1) When the mouse is totally exposed to radiation dosed 1000 r and its spleen is surgically detached and left in the abdominal cavity, there are no destructive changes observed in tissues of this spleen in 4 hours. Under detachment of a part of spleen, necrotic changes are found in 4 hours in the part connected with the organism only.
2) Application of ligature for the period of 4 hours onto neuro-vascular fascicle of spleen immediately after exposure prevents necrotic changes in spleen cells. Removal of ligature causes development of typical necroses, but they are developing slower that necroses without prior application of the ligature.
3) Under injecting suspension of cells of the spleen exposed to radiation "in vitro" to unexposed mouse spleen, in 4 hours the place of injection is featuring the focuses of necrotic cells. In the control sample (injection of suspension of unexposed spleen cells) no necroses are found.
As based on these facts, Barakina draws the conclusion that "destruction of the hemopoietic organs' cells damaged by radiation occurs as a result of implementing certain specific functions running in the organism".
Presence of considerable lipopolymer amounts in the exposed animal results in the intoxication of the organism. That is, lipopolimers may be considered as radio-toxins. Toxic effect of thermally oxidized fats (200°-300°C) is pro rata correlated with content of polymer products in them [27, 28]. Introducing polymeric fractions extracted from superheated corn oil into ration for rats in the amount of 2,5 % of diet causes the death of animals within seven days [29]. Lipopolymers formed in oleic acid under biogenic amine action suppress the respiration of liver homogenate in pigs, owing to the failure of oxidative phosphorylation [7].
Apparently, diffusive endocrine system is not activated under removal of hypophysis or adrenal glands in animals. As a result, the tissues of animals exposed to radiation display development of chain reaction of unsaturated fatty acids oxidation with formation of highly toxic peroxides, aldehydes, ketones, epoxides. Sensitivity of mice with removed adrenal glands to the lethal action of X-rays is remarkable even under low doses [30]. When the mice with removed adrenal glands are injected with certain dose of the adrenal gland cortex extract every day, their survival rate is equivalent to that of healthy mice [31]. White rats were exposed to various doses of radiation, from 800 r to 15.000 r, within the interval not depending on the dose. No constant time of survival was observed in animals with the adrenal glands removed, and significant dependence of life span on radiation dose was found. In this case the rats life span was inversely proportional to the amount of lipid peroxides formed under treatment with various doses of radiation. Animals receiving corticosterone were displaying all the interval of the 3,5th day effect not depending on the dose, in full [32]. Obviously, corticosterone activates diffusive endocrine system, which causes polymerization of lipid peroxides with biogenic amines.
Absorption, %
Time, min
Fig.1. Absorption dynamics at 400 nm in lipids solution in chloroform after injection of aminoethylisothiouronium to mice in dose of 150 mg/kg. 1 - blood, 2 - spleen, 3 - bone marrow, 4 - small intestines. Here and further the dotted lines mark absorption values in lipids of corresponding tissues of control (unexposed) mice.
Absorption, %
Time, min
Fig.2. Absorption dynamics at 400 nm in lipids solution in chloroform after injection of adrenalin to mice in dose of 4 mg/kg. 1 - blood, 2 - small intestines, 3 - spleen, 4 - bone marrow.
Absorption, %
Time, min
Fig.3. Absorption dynamics at 400 nm in lipids solution in chloroform after injecting of cystamine to mice in dose of 150 mg/kg. 1 - blood, 2 - small intestines, 3 - bone marrow, 4 - spleen.
Peroxides, µmol/g
Time, min
Fig.4. Kinetics of peroxide accumulation in oleic acid oxidized at 75°C with additives: 1 - serotonin; 2 - vitamin E; 3 - lipopolymers extracted from mice spleen in 15 minutes after aminoethylisothiouronium injection; 4 - polymers extracted from oleic acid after its interaction with aminoethylisothiouronium; 5 - oleic acid, control.
Survival rate, %
Dose, r
Fig.5. Influence of polymers formed in oleic acid under the action of cystamine, on the survival of exposed mice on the 30th day. 1 - experiment, injection of polymers (125 mg/kg) 90 min before exposure; 2 - control.
Absorption, %
Time, days
Fig.6. Absorption dynamics at 400 nm in chloroform solution of lipids of tissues in mice exposed to 750 r dose of radiation. 1 - blood, 2 - bone marrow, 3 - spleen.