Determination of cytotoxicity of germicides and disinfectants on the culture of kidney cells of a calf

Kryvoshyya P., Rud O., Lysytsya A.

Research Station of Epizootology

Annotation

Goal. To study the cytotoxicity of certain antibacterial preparations used in the treatment of infectious diseases of the urinary system, as well as some disinfectants for calf kidney cell culture. Methods. Cytological, toxic effects test on primary calf kidney cell culture. Determination of the maximum permissible concentrations of antibiotics, antiseptics, and disinfectants that do not yet lead to degeneration of the cell monolayer of calf kidney cells. Results. Tests of some drugs that are often used in veterinary medicine show that the least toxic to calf kidney cells of all tested antibacterial agents are Oxymicol 20 (active substance -- oxytetracycline base), Norsulfazole sodium, and Farmazin 50 (active substance -- tylosin). Conclusions. These drugs or similar with the same active substances should be used in the treatment of infectious diseases of the kidney and urinary system of cattle and other animals. The possibility of using the method of cultivation and use as test objects of primary cultures of eukaryotic cells in the selection of the optimal composition of therapeutic and prophylactic drugs or disinfectants for veterinary medicine. The obtained results of cytotoxicity can help veterinarians in choosing antimicrobials and disinfectants in terms of their wide range on the market.

Key words: veterinary medicine, chemotherapeutic drugs, cell culture, pyelonephritis, antibiotics, polyhexamethylene guanidine.

The main text

An important issue for veterinary medicine is the choice of optimal and at the same time low-toxic drugs for the treatment of various bacterial infections. At the present stage of development of agro-industrial production in the technological process in the cultivation of animals and poultry uses a huge variety of substances for the treatment and prevention of various diseases. The constant search for new drugs and dosage forms continues. One of the areas of modern pharmacology and toxicology is the development of effective methods of biotesting, including methods for determining the cytotoxic effect of drugs on cell cultures [1]. Every year, pharmaceutical companies register and market a large number of new therapeutic and prophylactic drugs. These processes are regulated by codes of quality laboratory (GLP), clinical (GCP) and manufacturing (GMP) practices [2]. GLP or good laboratory practice regulates the study of pharmacological drugs - candidates for drugs in experimental animals to avoid unexpected adverse effects of their use. GCP or good clinical practice regulates a thorough study of pharmacological agents in humans with a guarantee of reliability and reliability of the data obtained with the protection of human rights. Today, the principles of GCP apply to clinical practice in general. GMP or good manufacturing practice ensures the production of medicines in accordance with standards approved by government agencies.

There have been discussions for a long time about the use of laboratory animals for scientific purposes, including in the development and testing of drugs. It should be noted that animal studies, which are conducted to predict the possible negative consequences of the use of drugs, are often criticized. This applies to both ethical standards and the scientific weight of the results [3, 4]. It is believed that with the current development of science, animal experiments can be replaced by computer simulations. Indeed, certain experiments can now be performed in silico that is, using calculations on supercomputers. But such research requires tens of thousands of dollars to pay for the time required by these calculations. It takes months to study the interaction of only two or three molecules in a vacuum or minimal ionic environment. And this is provided that at some stage no error occurs, then this process can take years. It should be born in mind that a living organism consists of many billions of cells and trillions of molecules that coexist in complex relationships, and we don't know exactly about the structure and functions of many of them. Therefore, it is still best to test on living objects.

Animal cell cultures are increasingly used in vitro as biological systems in determining the toxicity of various compounds due to the possibility of control and greater reproducibility compared to test systems in the living organisms. The 3rd International Congress on Alternative Methods in the Use of Animals in the Life Sciences initiated the Good Cell Culture Practice (GCCP), a project developed by the European Centre for the Validation of Alternative Methods [1].

In determining the cytotoxicity of certain chemical compounds can be guided by Council Regulation (EC) № 440/2008, which establishes test methods in accordance with Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the registration, evaluation, authorization and restriction of chemicals and Drugs (REACH) of 30 May 2008 [5].

As for cell cultures, of course, it is easier to experiment on them, because they are relatively easy to cultivate, they have a controlled balanced environment and in general the set of influencing factors for them is not so great. Therefore, it is possible to study the action of certain types of molecules, the cytotoxicity of a drug substance or certain drugs, their effect on the processes of mitosis, cell death and so on.

One of the urgent issues of veterinary medicine is the treatment of infectious diseases of the urinary tract, including kidneys (pyelonephritis). Pyelonephritis can be caused by various types of pathogenic flora (bacterial, viral, fungal) of exogenous and endogenous origin. The most common pathogens of pyelonephritis are Escherichia coli, Staphylococcus, Proteus, Enterococcus, Pseudomonas aeruginosa, Streptococcus and others. The main method of treatment is antibacterial therapy (antibiotics, sulfonamides, nitrofurans) [6]. At the same time, in recent decades there has been an increase in cases of resistance of many pathogenic microorganisms, including and Escherichia coli to many broad-spectrum antibiotics [7]. In this situation, it becomes important to select the optimal treatment regimens using the least toxic drugs.

We selected a primary calf kidney cell culture (CKC) as the main test object. Another important argument when choosing this object is that when any drug enters the blood of an animal, it will somehow interact with the kidney cells. In addition, in our opinion, the primary cell culture is a more adequate object of testing than transplanted (transformed). And the lack of costs associated with maintaining transplanted cell cultures reduces the cost of testing.

The purpose of the work. Investigate the cytotoxicity of certain antibacterial drugs used in the treatment of urinary tract infections, as well as some disinfectants, on the culture of calf kidney cells.

Materials and methods of research. Antibacterial drugs (antibiotics, antiseptics, disinfectants): Pharmazin 50 (active substance tylosin, antibiotic of the macrolide group, BIOVET, AD, Bulgaria), Tylosomicol 20 (tylosin tartrate, DEVIE, Ukraine), Ceftiolofifur ), Ceftiodev 5% suspension for injection (ceftiofur hydrochloride, "DEVIE", Ukraine), Amoxicillin-Astrapharm (Я-lactam antibiotic amoxicillin, LLC "Astrapharm", Ukraine), Oxymicol 20% solution for injections (oxytetracycline basis, "DEVIE", Ukraine), Azithromycin Grindeks (antibiotic azalide, groups of macrolide antibiotics), Norsulfazole sodium (LLC "PLUTARh-M", Ukraine), "Polyhexamethylene hydrogens", Ukraine), Glutaraldehyde (Runa Inter LLC, Ukraine), 96% ethyl alcohol (Medisept, Ukraine), Formalin (37% formaldehyde solution), Brovadez-20 (alkyldimethylbenzylammonium chloride 20%, "Brovapharma", Ukraine), Sanidez, tablets (trichloroisocyanuric acid - 55%; isocyanuric acid - 10%; excipients: sodium bicarbonate - 30%; sodium carbonate - 5%, "Medisept", Ukraine), Thiomersal (merthiolate, organomercury compound, 98%, Pharm grade, USP, BP).

Determination of cytotoxic effects was performed on primary CKC. Drugs were tested for sterility before biotesting. The studies were performed sequentially in stages: preparation of nutrient media, sowing of cells on microplates and incubation until complete monolayer formation, dilution of test samples and their introduction directly into cell culture in at least 5-10 times, incubation for 3-4 days and accounting results. Primary cell cultures were prepared according to conventional methods [8]. Inoculation of trypsinized cells from embryonic organs was performed at a concentration of 4-6 x 10⁵ cells per 1 cm³. Determination of cell concentration in the nutrient medium was performed according to our own method using a photoelectrocalorimeter KFK-3 [9]. Cell culture was performed in a mixture of nutrient media 199 and Ihla (1:1) with the addition of 10% serum of cattle, preheated at 56°C for one hour. A mixture of 0.02% Versen solution and 0.25% trypsin solution in a ratio of (3:1) was used to obtain a cell suspension for further reseeding. Subcultures were obtained by cell transplantation after monolayer formation after 3-8 days. Cell viability was determined by staining with 0.2% Trypan blue solution. All cell cultures were tested for the absence of contamination by microorganisms.

After determining the cell concentration and obtaining a seed dose of 400-500 thousand cells/cm³ by dilution or concentration, the cell suspension was transferred into 96-well polystyrene plates in a volume of 0.1 cm³. The plates were placed in a special device for cell culture [10]. Kept in a thermostat at t = +37°C for 2-3 days, after the formation of the monolayer in the test wells were added drugs diluted on nutrient medium in titers 10^-1 - 10^-2 - 10³ - 10^-4 - 10^-5 in a volume of 0.1 cm³. The number of wells of the die for each dilution of the drug was 5-10. Observations were made by microscopy and evaluation of experimental and control cultures for 3-4 days in order to establish the manifestation or absence of cytotoxic effects. The degree of cytotoxicity was determined by changes in cell morphology (rounding, shrinkage of cells, separation from the surface of the wells of cells that have undergone degenerative changes). For the maximum concentration of the drug was taken the largest amount, which has not caused yet the degeneration of monolayer cells. To obtain reliable results, the following requirements were met: cell culture was without changes in the external structure of cells and the monolayer as a whole, during and at the beginning of the study did not change the components of nutrient media, studies were performed on cell culture obtained from the same organ animals.

Research results and their discussion. Microscopy and evaluation of experimental and control cultures to determine the manifestation or absence of cytotoxic effects of the studied antibacterial drugs allowed to determine their maximum allowable concentrations (MAC) for primary CKC. The table 1 shows the MACs that did not cause degeneration of the cell monolayer and minimum bactericidal concentrations (MBC), number of replicates are n = 5-10.

1. Cytotoxicity act of antibacterial chemotherapeutics to the primary culture of CKC