Prevention method of carcinogenic action of diethyl nitrosamine in experimental animals

FIELD: medicine.

SUBSTANCE: prevention of carcinogenic action of diethyl nitrosamine in experimental animals is ensured by introduction of kaskorutol dosed 0.4 g/kg within 9 months as an anticarcinogen, while diethyl nitrosamine is introduced in a dose 100 mg/l 5 days after introduction of kaskorutol within 4 months.

EFFECT: reduced carcinogenic action of diethyl nitrosamine due to inhibition of blastomogenic process, lowered level of lipid peroxidation, improved activity of antioxidant enzymes.

2 tbl, 8 dwg, 1 ex

 

The invention relates to ecology, medicine, toxicology, experimental biology, veterinary medicine and can be used when hepatocarcinogenesis prophylactically in high-risk groups in the incidence of liver cancer.

The steady increase in the incidence of malignant neoplasms makes the problem of prevention, i.e. the problem of development and application of funds, preventing neoplastic cell transformation and further development of tumors [Sporn M.B. N. Suh, 2000]. The cause of malignant growth can be carcinogenic nitroso compounds [N-Nitroso Compounds: Occurrence and Biological Effects and Relevance to Human Cancer. - Eds. I.K. O Nei, R.C. von Borstel, C.T.Miller, J.Long and H.Bartsch, 1988]. In this regard, there is an urgent need ways to prevent carcinogenic action of nitrosoguanidine on the human body.

Known methods of preventing the occurrence of benign and malignant tumors induced carcinogenic nitrosoguanidine, using substances that affect the different stages blastomogenic process: endogenous formation of carcinogens, in particular nitrosoguanidine from non-cancer precursors in the body, the metabolism of chemical carcinogens in the body, the interaction of chemical carcinogens with DNA, tumor transformation by povidoneiodine cells and further development blastomogenic process.

A significant number of works devoted to the study of anticarcinogenic properties of natural antioxidants (vitamins E, C, P, beta-carotene) and synthetic (equivalent and others). It is established that in the process of hepatocarcinogenesis the intensification of the processes of peroxide oxidation occurring in particular in lipids and the formation of free radicals [Cerovecki V.B. have been, Sitkovskiy M.V. et al., 1974]. It is proved that the imbalance in the system of lipid peroxidation - antioxidant protection, leading to the accumulation of free radicals, is one of the reasons run malignant transformation and tumor progression [Jakubowski R.I., 2000]. Based on views about the involvement of free radicals in the mechanism of carcinogenesis, several researchers tried to use antioxidants as inhibitors of blastogenesis [Dzhioev F.K., 1984; Emanuel N.M., 1974; Sporn M.V. N. Suh, 2000; L.W. Wattenberg, 1972].

The closest is the way [Dzhioev REVEALED the Influence of selenium, phenobarbital, teturama and carbon tetrachloride on the carcinogenic effects of diethylnitrosamine and 1,2-dimetilgidrazina. // Materials of III, proc.: "Carcinogenic N-nitroso compounds - action, synthesis, determination". Tallinn. - 1978. - P.51-53] cancer chemoprophylaxis in the experiment, preventing the development of tumors and suppression of hepatic Kanz is regenta, induced diethylnitrosamine, the country which was carried out with water at a dose of 100 mg/l during the first 4 months, prophylactic used se-acidic sodium (sodium Selenite) at a dose of 6 ppm/l of water for 6 months, which was taken as a prototype.

The disadvantage of the prototype is that se-acidic sodium is toxic in high doses, its use impractical in areas with high concentrations of selenium in water, soil and food products, the timing of the introduction has been reduced.

The claimed invention is directed to the solution of this problem involves the development of preventive methods carcinogenic action of diethylnitrosamine in experimental animals.

The solution to this problem provides a significant reduction of the carcinogenic action of diethylnitrosamine, resulting in a significant reduction in the incidence of neoplastic changes in the liver tissue, and multiplicity of tumors of the esophagus, later malignancy in the liver and esophagus in the experimental group compared with the control, the increase in life expectancy and survival of animals, decreasing the level of lipid peroxidation, increased the activity of antioxidant enzymes - catalase and ceruloplasmin.

To achieve this, the technical result of the claimed invention is a method of preventing enterogenic actions of diethylnitrosamine in experimental animals has the following essential features: animal 4 times a week administered with food anticarcinogen - multivitamin preparation "Cascarita" in a dose of 0.4 g/kg for 9 months, and diethylnitrosamine at a dose of 100 mg/kg after 5 days from the beginning of the introduction of Casorate" for 4 months.

Casarural is a multivitamin complex preparation containing 1 beta-carotene pills 2.5 mg, ascorbic acid 25 mg, alpha-tocopherol 12.5 mg, rutin and 12.5 mg. It can be regarded as a kind of vzaimorezerviruemym AOC. In this system, beta-carotene absorbs and neutralizes singlet oxygen, turning it into a triplet state and other free radicals, together with α-tocopherol, which regenerates beta-carotene. Ascorbic acid converts α-tocopherol from the oxidized form in restored, and rutin, in turn, regenerates ascorbic acid. The synergistic action of the components of the complex allows to obtain a high effect when used in physiological doses.

The proposed method differs from the prototype in that as anticancerogenic used multivitamin complex drug "Cascarita", which leads to inhibition blastomogenic process in the liver and esophagus, increasing life expectancy and survival of animals, reducing the level of peroxidation okisleniyu, the increased activity of antioxidant enzymes - catalase and ceruloplasmin.

The inventive method is efficient, cost effective and easily reproducible.

According to the authors the information set of essential features that characterize the essence of the claimed invention, is not known, which allows to make a conclusion about conformity of the invention, the criterion of "novelty".

According to the authors, the essence of the claimed invention should not be for experts explicitly known level of medicine, since it is not detected above the possibility of obtaining a way to prevent carcinogenic action of diethylnitrosamine in experimental animals, including the introduction of diethylnitrosamine drinking and anticancerogenic with food, characterized in that as anticancerogenic enter "Cascarita" in a dose of 0.4 g/kg for 9 months, and diethylnitrosamine at a dose of 100 mg/kg after 5 days from the beginning of the introduction of Casorate" within 4 months, which allows to conclude that the criterion of "inventive step".

The set of essential features that characterize the invention, in principle, can be repeatedly used in medicine with the result, consisting in an effective and easily reproducible method of prevention of toxic action is ethylnitrosourea by taking the drug "Cascarita", that allows to make a conclusion about conformity of the invention, the criterion of "industrial applicability".

This method is as follows. Tumors of the esophagus and liver induced by adding to the drinking water of diethylnitrosamine (DAN) at a concentration of 100 mg/kg during the first 4 months. The solution DAN gave the control and experimental groups. Five days before giving carcinogen to animals in the experimental group began to enter with food 4 times a week drug "Cascarita" in a dose of 0.4 g/kg body weight. After the time of the experiment (9 months) conducted a microscopic examination of the internal organs, histological examination of the tissues of the esophagus and liver. The results obtained in the control and experimental groups were compared. After the second series of the experiment (4 months) estimated the intensity of peroxidation processes in the accumulation of secondary products of lipid peroxidation, the main of which is malonic dialdehyde (MDA), and determined the status of antioxidant defense (AOD) on the level of activity of key antioxidant enzyme catalase and the main antioxidant protein of blood plasma - ceruloplasmin (CP), which indicates the manifestation of adaptive responses.

For histological examination of tissue samples (liver, kidney, heart) were fixed in 10% neutral is th formalin, then was subjected to fill in paraffin and subsequent preparation of slices of a thickness of 7-8 microns. Sections were stained with hematoxylin and eosin. The study sections were performed in transmitted light using a microscope Micmed-1 under the magnification of 80×120×600.

The intensity of peroxidation processes investigated the accumulation of secondary products of lipid peroxidation, the main of which is malonic dialdehyde (MDA) [Gavrilov V.B. have been, Gavrilova, A.R., Mazhul L.M. Analysis methods for the determination of GENDER in the serum test thiobarbiturate acid. // Issues of honey. chemistry. 1981. No. 1. Pp.118-122]. MDA content was determined spectrophotometrically in the test with thiobarbituric acid with the formation of trimethylboron complex with maximum absorption at 532 nm by the method of Hunter E.A. (1963), M.Uchiyama, M.Mihara (1978) modified Leeandrew et al. [Andreeva LI, Kozhemyakina L.A., Kiskun A.A. Modification method determination of lipid peroxides in the test with thiobarbiturate acid. // Laboratory work. 1988. No. 11. P.41-43]. The antioxidant protection (AOD) studied on the level of activity of key antioxidant enzyme catalase and the main antioxidant protein of blood plasma - ceruloplasmin (CP), which indicates the manifestation of adaptive responses. The catalase activity in the blood was determined photometrically by the method according to Bach and Zubkova [Korolyuk M.A., MA is arava I.G. et al. Method for the determination of catalase activity. // Laboratoi case. No. 1. P.16-19]. The contents of the CPU was determined by a simple colorimetric method developed Ravines (1956) and is based on the oxidation of p-phenylenediamine [Kolb V.T., Kamyshnikov B.C. Handbook of clinical chemistry. Minsk: Belarus, 1982. 366 S.].

The essence of the proposed method is confirmed macroscopically and morphologically (for clarity, changes in the organs of macro - and microphotographs see in colour.

Figure 1. The liver tumor induced by diethylnitrosamine (DAN) in rats; 9 months after the start of injection.

Figure 2. Multiple papillomas of the esophagus.

Figure 3. Hepatocellular cancer is poorly differentiated. The hematoxylin-eosin. h.

Figure 4. Hepatocarcinoma. The hematoxylin-eosin. h.

In rats male Wistar rats with initial body weight of 130-140 grams were conducted two series of experiments.

Example 1. The first series of experiments to study the effects of multivitamin preparation "Cascarita" on the occurrence of tumors of the liver and esophagus induced by means of diethylnitrosamine (DAN). The experiments were conducted on rats male Wistar rats with initial body weight of 130-140 grams, obtained from the vivarium of Pyatigorsk pharmaceutical Academy. The animals were kept on the diet of the vivarium. As a carcinogenic agent used substance - N-d is ethylnitrosourea (DAN), synthesized in the Institute of Oncology. New health Ministry. Tumors of the liver and esophagus induced by the described method (FCCIV, 1973) [Dzhioev REVEALED ABOUT the impact of some of adaptogens on experimental carcinogenesis. // Author's abstract on competition of a scientific degree of Cand. the honey. Sciences. - Leningrad, 1966] adding to the drinking water of DAN at a concentration of 100 mg/l of water for 4 months. Animals were divided into 2 groups-control (30 rats) and experimental (23 rats). Animals of the control group received only hepatocarcinogen. Animals in the experimental group, along with a carcinogen, received the multivitamin preparation "Cascarita" in a dose of 0.4 g/kg (single dose of 4 pills/kg) 4 times a week with food throughout the experiment. The experiment lasted 36 weeks. Animals at the end of the experiment were killed pairs of halothane, opened and conducted a microscopic examination of the internal organs.

In the analysis of microscopic changes in the liver and esophagus of rats was based on the classification of tumors in laboratory animals, the International Agency for research on cancer [Pathology of tumors in laboratory animals. Vol.1. Tumors of the rat. Sekond adetion. Edited by V.S.Turusov and U.Mohr.-Lyon: IARC Scientific Publicaitions No. 99. - 1990. - 754 R.]. About the effectiveness of modifying effects on carcinogenesis tried to change the number of rats with neoplastic processes, medium latte is these period of occurrence of neoplasms, index of multiplicity in the experimental group compared to control.

Results: in the control group neoplastic changes in liver tissue was detected in 89,7% of the effective number of animals (29 rats) (see Figure 1). The first rat with precancerous changes in the liver fell on the 12th week of the experiment. Microscopic examination of the liver tissue showed the presence of prostatic ducts and eosinophilic foci and basefilename hyperplasia of hepatocytes (see figure 3, 4). Malignant neoplasms were first identified from three rats of the control group, died on 21 week experience: well-differentiated hepatocellular cancer and cholangiocellular cancer developed on a background of cirrhosis and hyperplasia of the ducts and/or hepatocytes. In total the morphological analysis of neoplastic changes in 16 rats of the control group identified highly, moderately or poorly differentiated hepatocellular carcinoma, 8 rats, hepatocellular adenoma, 3 - cholangiocellular cancer, the 5 - cholangioma, 6 - cavernous hemangioendothelioma. These processes were developed on the background of diffuse and focal hyperplasia of hepatocytes and/or ducts, as well as cirrhotic processes of different severity (Table 1).

In the group of animals treated "Cascarita", neoplastic changes in liver tissue was detected in 77,3% of the effect of the main numbers of the animals (22 rats). Microscopic analysis of histological material showed that in this group, 13 cases of hepatocellular carcinoma, 5 - hepatocarcinoma, 1 cholangiocarcinoma, 2 - cholangioma, 1 - cavernous hemangioendothelioma, 11 animals identified hyperplasia duct, including cystic (Figure 5).

Tumors of the esophagus (table 2) developed in 82.8% of rats in the control group from the effective number of animals (29 rats), the index of multiplicity was 5.3±0,55 tumors in the rat (see Figure 2). In all cases the tumor was located in the mucosa of the esophagus and had the appearance of circular formations with a diameter of 0.2-0.6 see Histologically, tumors classified as papillomas with dysplasia and malignancy basal cell type, well-differentiated and poorly-differentiated squamous cell carcinoma. In the group of animals treated "Cascarita", neoplasms of the esophagus occurred in 50% of rats from the effective number of animals (22 rats) when the index of the plurality of 3.0±0,81, i.e. the index of the plurality decreased 1.8 times, the percentage of inhibition was -43,4%.

In the conditions of experiment, application of comprehensive multivitamin preparation "Cascarita" resulted in a significant reduction in the incidence of neoplastic changes in the tissue is liver, multiple tumors of the esophagus, later malignancy in the liver and esophagus in the experimental group compared to control.

The second series of experiments to study the influence of the drug "Cascarita" activity of processes of lipid peroxidation (LPO) and antioxidant efficiency of protection (AOP) by evaluating changes in the catalase activity of the main antioxidant protein of blood plasma - ceruloplasmin (CPU).

Animals were divided into two groups - control (30 rats) and experimental (23 rats). Animals of the first group received only DAN by the method described above. Animals in the experimental group along with the carcinogen was given the drug "Cascarita with feed at a dose of 0.4 g/kg (single dose of 4 pills/kg) 4 times a week throughout the experiment. The experiment lasted 16 weeks. Animals were killed by decapitalise in time after 2, 4, 8, 12, 16 weeks.

The results indicate significant increase in the activity of antioxidant enzymes in erythrocytes of rats experimental groups in comparison with these figures in animals from group "without impact". Thus, the impact of drugs restores the intensity of the reactions of free radical oxidation to appropriate healthy body (Fig.6 - 8).

Thus, on the basis of the received the data we can conclude that, that the use of the drug "Cascarita" led to the improvement of the General condition of the animals, which is manifested in the reduction of lipid peroxidation and increasing antioxidant activity, reducing the degree of intoxication. These results support expressed by a number of researchers opinion that the imbalance in the system of GENDER-AOD is one of the main mechanisms contributing to the progression of the malignant process. Consequently, it should be recognized pathogenetically justified the use of drugs with antioxidant activity when hepatocarcinogenesis [Alexandrov VA, Bespalov V.G. Principles and prospects for chemoprevention of cancer. // Problems of Oncology. 1991. No. 4. S-393.; 8.Sporn MV Suh N. Chemoprevention of cancer. Carcinogenesis. 2000; 3: 525-530]. In the conditions of the experiment, the use of the above drugs have led to a restoration of balance in the system of GENDER-AOD, prospectively, to possibly reduce the risk of primary liver cancer in high-risk groups.

Anticarcinogenic activity of complex drug "Cascarita" is primarily due to the synergy of the antioxidant properties of its constituent vitamins, protecting cells from the damaging effects of free radicals, and specific immunomodulatory activity component is s drug.

The way to prevent carcinogenic action of diethylnitrosamine in experimental animals, including the introduction of diethylnitrosamine drinking and anticancerogenic with food, characterized in that as anticancerogenic enter casarural at a dose of 0.4 g/kg for 9 months, and diethylnitrosamine administered in a dose of 100 mg/l after 5 days from the beginning of the introduction of Casorate within 4 months.



 

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