Method of preventing toxic lead effect on experiment animals with chronic poisoning

FIELD: medicine.

SUBSTANCE: invention concerns experimental medicine, particularly experimental toxicology, and can be applied in prevention of toxic lead effect on experiment animals. Method involves daily subcutaneous injection of acyzol in amount of 30 mg/kg simultaneously with lead acetate introduction to stomach through gastric tube in amount of 40 mg/kg.

EFFECT: efficient prevention of toxic lead effect during chronic poisoning due to dosage-dependent effect control.

1 ex, 4 tbl, 2 dwg

 

The invention relates to ecology, medicine, toxicology, experimental biology and can be used in the study of methods of prevention of chronic toxic effects of heavy metals, particularly lead, on the functional state of the kidneys.

One of the urgent problems of modern medicine and Nephrology is the study of the influence of heavy metals in the organs of the urogenital system of the population. In the last years highlights the increase in the frequency of renal pathology in the population living in ecologically unfavorable regions. (Ermachenko A.B., Sawtooth V.I. 1985; Ansary R.A. et al., 1991). As the major organ of excretion of many metals from the body, the kidneys have an increased toxic load, and therefore serve as a target of toxicity. Lead is especially inherent ability to hit the tubular epithelium of the kidneys, so the kidneys are one of the critical organs of intoxication in General (Kireeva, H.E., 2007).

Due to environmental degradation and progressive increase in the concentration of lead in the environment, the search for effective means of preventing its toxic action on the human body is one of the urgent problems of medicine.

There are plenty of detoxicants able to prevent or reduce toxic is some effect of heavy metals on the body. These include thiol compounds - unithiol, sodium thiosulfate, succimer, various chelating agents - pentatsin, the disodium salt of ethylenediaminetetraacetic acid, deferoxamine, and others (Mashkovsky PPM Medicines: in 2 volumes. Vol.2. - Kharkov "Torching", 1998. S-220). But along with therapeutic effect they can cause various side effects: gastrointestinal disorders, allergic reactions, leukopenia, thrombocytopenia, etc. Also they are able to excrete minerals and essential ions like iron, calcium, etc. and cause a violation of metabolic processes, which makes them long and prophylactic use.

As a detoxifier preference was given to azizoglu. Aziza is a comprehensive tsinkorganicheskih connection. The chemical structure of bis (1-vinylimidazole) cindicates. Azizol was developed as an antihypoxic agent in carbon monoxide poisoning and is the only drug for the prevention and life-saving at the specified defeat. Azizol unique product of its pharmacological properties and breadth of use that do not have a toxic action on the organism of man and animal, has no side effects. Azizol fills the concentration of zinc in enzyme systems, helps to optimize the promotion of tissue respiration, improves oxygen-dependent properties of the blood, due antihypoxant and antioxidant action azizol has lipoic property.

Previously azizol was used as hepatoprotector, means for treatment of psoriasis, antidote for poisoning by carbon monoxide, adaptogene, coronary-active antiischemic and antiarrhythmic agent.

Closest to the claimed method is prevention of toxic effects of lead with the use of sodium Selenite, taken as a prototype (Galacian S.M., however F.K. New technologies in reducing the toxic effects of lead with the help of sodium Selenite //Vladikavkaz biomedical journal. - 2003. - T4. - Vol.5-6. - P.64-67), namely, that animals received lead acetate daily with food dose of 30 mg/kg weight of the animal. Simultaneously with this, the animals received twice a week with food sodium Selenite at a dose of 0.4 mg/kg weight of the animal. The duration of the experiment 6 months.

The disadvantage of the prototype is that the lead acetate and sodium Selenite are used as nutrition in the diet, which reduces the accuracy of the control dose-dependent effect.

The claimed invention is directed to the solution of this problem involves the development of a way to prevent the toxic effects of lead in experimental animals etc is chronic poisoning.

The solution to this problem provides a significant reduction in the toxic effects of lead in chronic poisoning in experimental animals by optimizing tissue respiration, improve oxygen-dependent properties of blood. One of the main pathophysiological mechanisms of toxic action of lead on the body as a whole is its inherent ability to negatively affect the processes of tissue respiration. Respiration is a combination of redox processes in cells, tissues and organs, proceeding with the participation of molecular oxygen. Respiration is an important part of metabolism and energy in the body. The intensity of tissue respiration is highest in tissues of the kidneys, perhaps that is why the kidneys are highly sensitive to the toxic effects of heavy metal. Arizol in turn helps to optimize tissue respiration, improving oxygen-dependent properties of the blood, thereby reducing the disturbance of processes of tissue respiration, contributing to stabilization of the pathological process.

To achieve this, the technical result of the claimed invention, a method of preventing the toxic effects of lead in experimental animals with chronic poisoning, including simultaneous introduction of antidote and acetate of lead, as de is oxycanta apply azizol, administered to the animals daily subcutaneous dose of 30 mg/kg, along with a daily injection of lead acetate through a tube into the stomach at a dose of 40 mg/kg

The proposed method differs from the prototype in that, first, is dosed introduction of lead acetate and alzola that facilitates precise control of the dose-dependent effect, and secondly, for the first time applied pharmacological drug - azizol prevention of chronic toxic nephropathy.

Between the features of the proposed method and the result is the following causality, azizol, contributing to stabilization of the processes of tissue respiration, reducing the toxic effects of lead on the body, has a preventive effect.

Prophylactic administration of alzola, subcutaneously at a dosage of 30 mg/kg, every day, at the same time with intragastric introduction of a solution of lead acetate at a dose of 40 mg/kg for 16 days reduces the manifestations of toxic nephropathy, recovery of kidney function. 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".

On mn is 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 the toxic effects of lead acetate in experimental animals with chronic poisoning, including subcutaneous administration of alzola every day, along with daily injection of solution of acetate of lead through noninvasive probe into the stomach for 16 days, 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 effects of lead, by reducing the pathological effects of heavy metal, that allows to make a conclusion about conformity of the invention, the criterion of "industrial applicability".

This method is as follows.

To obtain toxic substances lead acetate dissolved in sterile distilled water so that per unit of solution, 0.2 ml, have 8 mg of lead (in terms of metal). For every 100 g weight rats injected with 0.1 ml toxic solution that is not extremely durable, the volumetric water load on the organism of experimental animals. The solution alzola prepared in such a way that each solution of 1 ml for 30 mg alzola. The solution alzola for each injection is prepared daily before the introduction. The solution of lead acetate is injected through noninvasive probe into the stomach at a dose of 40 mg/kg, daily 1 time per day for 16 days, two groups of animals (No. 1 and No. 2). At the same time, from the first day of the introduction of lead acetate, the group # 2 every day subcutaneously injected arizol in the dosage of 30 mg/kg using an insulin syringe for single use only.

After the time of the experiment (16 days) investigated the functional state of the kidneys, which included the determination of urine output (ml/HR/100 g), glomerular filtration rate (ml/HR/100 g), tubular reabsorption of water (%), osmolarity urine excretion of sodium, potassium, and protein.

Rats were scored with the use of thiopental. The results obtained in group 2 compared with the control group (gr. No. 1) and background indicators intact group.

For histological examination of tissue samples (kidney) 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 magnification 80·200·400.

The method you is by morphologically, which figure 1 in the experimental group identified expressed alterative, necrobiotic and necrotic changes in the tissues of the kidney were noted: hemorrhage in the glomerulus (a); the extension of the lumen of tubules (b), necrosis of individual groups of tubules (b), necrobiosis all tubules (g) on the background of their solid hydropic dystrophy (d). In the glomeruli revealed a pronounced swelling, degenerative changes, there are small hemorrhages in the stroma. Figure 2 histological examination of the kidneys in group No. 2 revealed less pronounced dystrophic and necrobiotic changes in the tissues of the kidney: the regeneration of tubules (a), a moderate expansion of the ducts tubules (b), lymphohistiocytic infiltration of stroma (C), which is an indicator of the regeneration process.

Example. Two groups of male rats Wistar rats weighing 200-300 g were injected through noninvasive probe into the stomach of a solution of lead acetate at a dose of 40 mg/kg every day for 16 days. At the same time, from the first day of the introduction of lead acetate group No. 2 every day subcutaneously administered arizol in the dosage of 30 mg/kg Over 16 days examined renal function in conditions 6-hour spontaneous diuresis, conducted definition: volume of diuresis, glomerular filtration rate by endogenous creatinine, tubular reabsorption of water excretion with urine sodium ions, potassium and calcium excretion in the tree with urine, the osmolarity of the urine.

Sodium, potassium in plasma and urine was determined by the method of breeding photometry, using tribal analyzer liquids PAGE-1, the concentration of calcium and creatinine was determined spectrophotometrically (SF-26) using sets of "Calcium-Arsenazo-agate", "Creatinine-agate", "company" Agat-Honey", Moscow, Russia). The protein concentration was determined spectrophotometrically (SF-26) by the method of Lowry. The osmolarity of the urine was determined using Osmomat-300. The results of all series of experiments processed statistically with the use of the criterion of "t" student test on a PC Pentium-3 using Prizma 2.2.

The results showed (table 1 and 2)that in rats with chronic intoxication with lead acetate (group 1) was significant increase in 6-hour spontaneous diuresis (p<0,05), despite a significant reduction in glomerular filtration rate (GFR) (p<0,05). Politicheskaya reaction was caused by the reduction of reabsorption of water (p<0,05), while there was an increase in Na excretion (p<0,05) (p<0.05), and Sa (p<0.05), the content of protein in the urine (p<0,05), there was a decrease of urine osmolarity (p<0,05), indicating a violation of concentrating kidney function. On the background of the simultaneous introduction of alzola in group No. 2 was significant reduction in 6-hour spontaneous diuresis (p<0,5) compared with group # 1 with isolated introduction of lead acetate, due to the significant increase in tubular reabsorption of water (p<0,05) despite the increase in GFR (p<0,05). Compared with group # 1 were observed significantly lower values of Na excretion (p<0,05) (p<0.05), and Sa (p<0,05). At the same time was increased urine osmolarity (p<0.05), and therefore the use of alzola helps prevent changes concentrating kidney function. The use of alzola contributed to the reduction in the degree of proteinuria (p<0,05) in rats with chronic toxicity of lead acetate.

Toxic effects of lead acetate after intragastric administration caused changes in the electrolyte composition of blood plasma (table 3). The lower sodium levels (p<0,05) associated with a high loss of sodium in the urine due to an earlier defeat tubular apparatus of the kidneys and decrease tubular reabsorption of cations (p<0,05), (table 4). The use of arizola on the background of the toxicity of lead acetate significantly reduced the severity of changes in the electrolyte composition of the blood (p<0,05).

p
Table 1.
The influence of arizola on the basic processes of urine formation and excretion of protein in urine in rats in conditions 6-hour spontaneous diuresis, on the background of intragastric administration of lead acetate 40 mg/kg (M±m).
Conditions of experienceStat. show.The processes of urine formationProtein excretion mg/hour/100 g
Diuresis, ml/HR/100 gGFR, ml/HR/ 100 gEOC, %
BackgroundM±M0,0765±0,0010617,84±0,110399,57±0,00481,213±0,024
p----
16 days lead acetate (40 mg/kg) + azizal (30 mg/kg)M±M0,11±50,001416,49±0,09299,31±0,00721,906±0,028
p*)*)*)+)
16 days lead acetate (40 mg/kg)M±M0,136±0,002514,72±0,1199,07±0,022,795±0,042
+)*)+)+)
Note: (*) - significant (p<0.05) change compared with the background.

List of abbreviations:

• GFR - glomerular filtration rate;

• EOC - the tubular reabsorption of water.

Table 2.
The influence of arizola on the excretion of electrolytes and osmolarity of urine in rats in conditions 6-hour spontaneous diuresis on the background intragastric administration of lead acetate at a dose of 40 mg/kg (M±m).
Conditions of experienceStat. show.Excretion of electrolytes µmol/hour/100 gThe urine osmolarity (OSM/l)
ECENaEna
BackgroundM±m6,36±of) 0.1579,053±0,13030,173±0,00332,125±0,0695
16 days lead Acetate (40 mg/kg) + azizal(30 mg/kg)M±m 7,695±0,15411,76±0,3270,185±0,00421,416±0,0573
p*)*)+)*)
16 days lead Acetate (40 mg/kg)M±m8,54±0,19413,53±0,530,205±0,00810,9254±0,031
p*)*)+)*)
Note: (*) - significant (p<0.05) change compared with the background.

Table 3.
The influence of arizola on the concentration of electrolytes in the blood plasma background intragastric administration of lead acetate at a dose of 40 mg/kg of body weight (M±m).
Conditions of experienceStat. show.The concentration of electrolytes in the blood plasma (mmol/l).
CANaTo
IIIIIIVV
BackgroundM±m2,139±0,022143,48±0,284,259±0,045
16 days lead acetate (40 mg/kg) + azizal (30 mg/kg)M±m2,240±0,0209140,6±0,5035,017±0,056
P*)*)*)
16 days lead acetate (40 mg/kg)M±m2,054±0,0221138,0±0,4785,59±0,114
P*)*)*)
Note: (*) -significant (p<0.05) change compared with the background.

Table 4.
The influence of arizola on the reabsorption of calcium, sodium under conditions of 6-hour spontaneous diuresis on the background intragastric administration of lead acetate at a dose of 40 mg/kg (M±m)
Conditions of experienceStat. show.The reabsorption (%)
CANa
IIIVIVII
BackgroundM±m99,30±0,015399,63±0,0052
16 days lead acetate (40 mg/kg) + azizal (30 mg/kg)M±m99,23±0,019499,47±0,0148
p*)*)
16 days lead acetate (40 mg/kg)M±m98,96±0,040799,30±0,0273
p*)it)
Note: (*) - significant (p<0.05) change compared with the background.

From the foregoing it follows that the use of alzola is an effective way to prevent the toxic effects of lead in chronic poisoning.

The way to prevent the toxic effects of lead in the experiment is lnyh animals in chronic poisoning, involving the simultaneous introduction of antidote and acetate of lead, characterized in that as a detoxifier used azizol, administered to the animals daily subcutaneously at a dose of 30 mg/kg, along with a daily injection of lead acetate through a tube into the stomach at a dose of 40 mg/kg



 

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