Method of prevention of carcinogenic action of diethylnitrosamine in experimental animals

FIELD: medicine; veterinary science.

SUBSTANCE: prevention of carcinogenic action of diethyl nitrosamine in experimental animals is ensured by daily introduction of anticarcinogen combined with feed. Anticarcinogen is presented with powdered phytoadaptogen "Chagovit" dosed 50 mg/kg within 9 months. On the 5th day, this therapy is added with introduction of diethyl nitrosamine combined with drink dosed 100 mg/l within the first 4 months.

EFFECT: lower rate of neoplastic changes in liver and oesophagus tissues, higher lifetime and survival rate of animals ensured by lower level of lipid peroxidation and improved activity of antioxidant enzymes.

2 ex, 2 tbl, 8 dwg

 

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 [Spom M.B. Suh N. Chemoprevention of cancer // Carcinogenesis. - 2000. No. .3. - P.525-530]. The cause of malignant growth can be carcinogenic nitroso compounds. Nitroso compounds are widely used in industry as components of industrial fuel, antioxidants, are intermediates for the synthesis of dyes, pharmaceuticals, etc. are Extremely important feature of nitrosamines is the possibility of their formation from chemical precursors in the environment, in food and even in the body. It is established that N-nitrosotoluene can be formed in the gastric juice of animals and humans as a result of interaction of nitrite and secondary amines or amides, including some medicinal compounds (amidopirina, piperazine, tetracycline and others) [bogovski P.A., 195; Bellander B.T.D, Osterdahl B.G., Hadmar L., 1984.; Mirvisch S.S., 1975]. These agents have a broad spectrum of biological effects, however, the main and, obviously, the most dangerous of their property, is the ability to cause tumors [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. - Lyon: IARC, IARC Sci. Publ. No. 90. - 1988. - 500 PP].

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 carcinogen-damaged cells and further development blastomogenic process. In this regard, there is an urgent need ways to prevent carcinogenic action of nitrosoguanidine on the human body.

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.51-5] chemoprophylaxis of cancer in the experiment, preventing the development of tumors and suppression of hepatic carcinogenesis induced by 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 ‰/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 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.

For dostigaemogo technical result of the claimed invention is a method of preventing carcinogenic action of diethylnitrosamine in experimental animals has the following essential features: animals injected daily with food anticarcinogen powdered fileadapter "Cahouet" in a dose of 50 mg/kg for 9 months, then with an interval of 5 days carry out simultaneous giving of diethylnitrosamine drinking at a dose of 100 mg/l during the first 4 months.

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

Due to increased morbidity and mortality in the population is increasing production of synthetic drugs with a wide range of side effects. Therefore, an increasing demand for non-toxic natural drugs from traditional medicine. Long-term observations (up to 5-7 years) patients with stage IV cancer has shown that taking chaga helps to improve the quality of life of all patients (except, extremely emaciated), and with long - term use increases life expectancy by 2-3 times compared with patients not taking chaga. Chaga has a number of important body properties: antioxidant, antitoxic, radioprotective, anti-inflammatory, ADAP is ogenyi etc. It increases energy metabolism of the Central nervous system, regulates neuro-reflex reaction, normalizes body defence system [Yakimov P.A., Stupak F Chaga and its therapeutic use in cancer stage IV. Medgiz, 1959.].

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 daily administration with food anticancerogenic, then with an interval of 5 days carry out simultaneous giving of diethylnitrosamine drinking at a dose of 100 mg/l during the first 4 months, characterized in that as anticancerogenic apply powder fileadapter "Cahouet" in a dose of 50 mg/kg for 9 months, which allows to conclude that the criterion of "inventive level.

The set of essential features that characterize the essence of what bretania, in principle, it may be repeatedly used in medicine with the result, consisting in an effective and easily reproducible method of prevention of toxic effects of diethylnitrosamine, by taking the drug "Cahouet", which 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, animals in the experimental group began to enter with food preparation "Cahouet" in a dose of 50 mg/kg of 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 antioxi is based protein plasma - ceruloplasmin (CP), which indicates the manifestation of adaptive responses.

For histological examination of tissue samples (liver, kidney, heart) were fixed in 10% neutral formalin, and 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 b is the left main coronary artery 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., Mayorova I.G. et al. Method for the determination of catalase activity // Laboratoi case. No. 1. P.16-19]. The contents of the CPU was set a simple kilometricas 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 are macro - and microphotography), and also in the form of charts, which reflect the processes of GENDER, the activity of catalase and ceruloplasmin in groups in the intact control animals, animals treated with DAN and DAN in combination with the drug "Cahouet".

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

Figure 2. Hepatocellular cancer immature trabecular. The hematoxylin-eosin. h.

Figure 3. Hepatocarcinoma. The hematoxylin-eosin. h.

Figure 4. The influence of the drug "Cahouet" on the occurrence of liver tumors and esophageal induced in rats by diethylnitrosamine (DAN).

Figure 5. Multiple papillomas of the esophagus.

Phi is .6. Changes in the activity of lipid peroxidation (MDA level, nmol/l) in the liver of rats with the introduction of animals of DAN and DAN in combination with photoadaptation "Cahouet".

7. Changes in the activity of catalase (mkat/l) in serum of rats with the introduction of animals of DAN and DAN in combination with photoadaptation "Cahouet".

Fig. Changes in the activity of ceruloplasmin (mg/l) in blood of rats with the introduction of animals of DAN and DAN in combination with photoadaptation "Cahouet".

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 influence of the drug "Cahouet" 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-diethylnitrosamine (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 the 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 "Cahouet" in a dose of 50 mg/kg daily 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, the average latent 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 (see Figure 2, 3) showed the nalitch the e prostatic ducts and eosinophilic foci and basefilename hyperplasia of hepatocytes. 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 "Cahouet", neoplastic changes in liver tissue was detected in 72.7% of the effective number of animals (22 rats). Microscopic analysis of histological material showed that in this group, 11 cases occurred hepatocellular cancer, 3 - hepatocarcinoma, 2 - cavernous hemangioendothelioma, 1 cholangiocarcinoma, 1 - cholangioma. (Figure 4.)

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 tumor on cu is su. 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 cm (see Figure 5.).

Histologically, the 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 "Cahouet", neoplasms of the esophagus has developed in 63.6% of rats from the effective number of animals (22 rats) when the index of the plurality of 3.4±0,69, i.e. the index of the plurality decreased 1.7 times the percentage inhibition amounted to 35.8 per cent.

Table No. 2.
The influence of the drug "Cahouet" on the occurrence of tumors of the esophagus induced in rats by diethylnitrosamine (DAN).
groupImpactThe number of recorded animalsThe number of animals with tumors (%)The average number of tumors per 1 animal (M±M)Inhibition of tumours in %p
ControlDAN2924 (82,8%) 5,3±0,55
ExperienceDAN + "Cahouet"2214 (63.6 per cent)3,4±0,69-35,8%0,05

In the conditions of the experiment, the drug "Cahouet" resulted 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 to control.

Example 2. The second series of experiments to study the influence of the drug "Cahouet" 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 "Cahouet with feed at a dose of 50 mg/kg throughout the experiment. The experiment lasted 16 weeks. Animals were killed by decapite the Finance in time after 2 weeks, to 4 weeks., 8 weeks, 12 weeks, 16 weeks.

The results obtained (Fig.6-8.) 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.

Thus, on the basis of obtained data we can conclude that the use of the drug "Cahouet 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 /Oncology Issues. 1991. No. 4. S-393; Sporn MV Suh N. Chemoprevention of cancer. Carcinogenesis. 2000; 3: 525-530]. In the conditions of the experiment, the application of the above preparation is tov led to restoration of balance in the system of GENDER-AOD, prospectively, to possibly reduce the risk of primary liver cancer in high-risk groups.

Chaga is one of the most interesting sources of biologically active substances, although poorly understood and controversial. She has specific antitumor and cytotoxic activity, but at the same time, long-term use, even in cancer patients, stage IV, regardless of tumor localization, improved General condition, contributed to the normalization of physiological functions of the body, broken pathological process, except for extremely malnourished patients. Pharmacological action of the fungus is, presumably, the presence of polyphenolic chromogenic humanophobia complex melanin. Obviously, biologically active substances fungus modulate cellular metabolism at the level of the Central and peripheral nervous system, and through them cause the correction processes of Bioregulation protective systems and body reserves [Darkseid YU. Chaga and its therapeutic use in cancer stage IV. Medgiz, 1959, s-325].

The way to prevent carcinogenic action of diethylnitrosamine in experimental animals, including daily administration with food anticancerogenic, then with an interval of 5 days carry out simultaneous giving of diethylnitrosamine drinking at a dose of 100 mg/l for PE is o 4 months characterized in that as anticancerogenic apply powder fileadapter "cahouet" in a dose of 50 mg/kg for 9 months.



 

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