Study of antimutagenic effect of hydrophilic cryofraction of bovine spleen

High production loads and deterioration of the environmental situation lead to an increase in the effect of various factors that can cause mutations in the cells of farm animals. The accumulation of mutations leads to the occurrence of diseases, disruption of immune functions, decreased weight gain, partial or complete sterility, loss of valuable breed traits, causes death and also affects the next generations. The use of drugs with an antimutagenic effect is considered to be one of the ways to reduce the accumulation of DNA damage in the body. Pharmacological substances obtained from animal tissues, such as bovine spleen, can become a promising basis for drugs with such properties. In this work, we have established the antimutagenic effect of hydrophilic cryofraction of bovine spleen on bone marrow cells of mice for the first time, as well as the DNA-protective effect on mitochondrial DNA (mtDNA) of mouse liver under conditions of cytogenetic instability induced by the experimental mutagen mitomycin C. We have also shown the effect of hydrophilic cryofraction of bovine spleen on some markers of oxidative stress in mouse liver cells upon administration of mitomycin C to animals. The aim of the work was to determine the effect of hydrophilic cryofraction of bovine spleen on the cytogenetic stability of bone marrow cells and the integrity of mtDNA of mouse liver, as well as to assess its antimutagenic and DNA-protective effects on mice with mitomycin C (MMC)-induced cytogenetic instability. Hydrophilic cryofraction of bovine spleen (HCBS) was obtained at the FSBSI ARVRIPP&T. The positive control was the drug Mitomycin C Kyowa (Kyowa Hakko Kogyo Co., Ltd, Japan), containing mitomycin as the active substance. The experiments were conducted in 2024. Male white outbred mice (Mus albus officinarum) (n = 30) weighing 26.0±2.0 g were used. Five groups of animals ( n = 6) were formed. In group I (negative control), the animals were given a single intramuscular injection of sterile isotonic sodium chloride solution in a volume of 0.1 ml. In group II, the mice were given a single intramuscular injection of HCBS at a dose of 0.5 ml/kg in a volume of 0.1 ml. The animals of group III were administered HCBS at a dose of 0.5 ml/kg in a volume of 0.1 ml and together with the last injection of HCBS intramuscularly three times with an interval of 24 h, a single intraperitoneal injection of MMC at a dose of 10 mg/kg in a volume of 0.5 ml. The animals of group IV were administered HCBS at a dose of 0.5 ml/kg in a volume of 0.1 ml intramuscularly once and a single intraperitoneal injection of MMC similarly to group III. The mice from group V (positive control) were given a single intraperitoneal injection of MMC at a dose of 10 mg/kg in a volume of 0.5 ml. The mice were eliminated from the experiment 24 hours after the last injection by means of an overdose of carbon dioxide in a special chamber. To study the frequency of polychromatophilic erythrocytes (PCE) with micronuclei (micronucleus test), bone marrow cells from femurs were added to inactivated fetal bovine serum (BioloT, Russia) and applied to glass slides, then the preparations were dried and stained according to Papenheim. The frequency of PCE with micronuclei per 1000 PCE was studied, and the ratio of PCE to normochromic erythrocytes (NE) was also accounted. The relative number of damages in mtDNA was estimated by qPCR. For this purpose, total DNA was isolated from 25 mg of homogenized mouse liver using the PROBA-GS kit (DNA Technology, Russia). Calculation of mtDNA damage was performed in the regions encoding the 12S and 16S rRNA (12S-16S) and the ND5 gene (ND5). The total relative content of intracellular reactive oxygen species (ROS) was determined using a cellular probe, 2',7'-dichlorodihydrofluorescein diacetate (Sigma-Aldrich, USA), which, when oxidized inside living cells, forms a fluorescent form (DCF) detected using a RF-5301 spectrofluorimeter (Shimadzu, Japan). The content of intracellular ROS was estimated in a suspension of mouse liver cells (3×106 cells/ml). The concentration of malondialdehyde (MDA) was determined in mouse liver homogenate using a UV-1700 spectrophotometer (Shimadzu, Japan) by staining the solution at l = 535 nm with trimethine complex. As a result of the experiments, we did not find any significant differences from the negative control of all the studied parameters in the mice from group II. Thus, in group II, the frequency of PCE with micronuclei was 0.41±0.080 %, the number of mtDNA damages in the 12S-16S and ND5 fragments was 0.0±0.92 and 2.2±0.45, respectively. The introduction of MMC in group V led to an increase in the frequency of PCE with micronuclei in the bone marrow of mice to 11.20±1.000 %.