Influence of long-term administration of non-steroidal anti- inflammatory drugs on the level of whole genome dna methylation and its fragmentation in rats

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Zaporizhzhia State Medical University

Influence of long-term administration of non-steroidal anti- inflammatory drugs on the level of whole genome dna methylation and its fragmentation in rats

Burlaka K. A.

In recent years, there has been rapid growth in epigenetic research associated with the development of new molecular and cytological approaches. Recent studies have established that chemical compounds can have an epigenetic effect, influencing the expression activity of specific genes. And most often, it is connected with people with chronic diseases because they are forced to use medicines for a long time to improve their quality of life. It is known that long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) leads to the formation of erosions, ulcers and oncological diseases of the stomach and intestines. It is suggested that side effects result from damage to the mucous membrane under the influence of NSAIDs and the possible effect of NSAIDs on genes that regulate the cell cycle, especially with their long-term use. In the research, 60 male rats were used, which were divided into four groups of 15 animals each, which were administered NSAIDs daily for 3 months: 1st - control (CG), 0.9% sodium chloride solution, 2nd - rats, which were injected with indomethacin, the 3rd group - rats, which were injected with acetyl- salicylic acid, and the 4th group - rats, which were injected with meloxicam. Long-term use of all NSAIDs led to a statistically significant increase in genome-wide DNA methylation and intensification of its fragmentation processes. It is important to note that according to the degree of epigenetic influence, the effect of the studied NSAIDs was of varying severity. Indomethacin had the most pronounced effects (increase of MspI/HpaII by 99%; DNA fragmentation by 90% compared to intact). Taking into account the established effects of NSAIDs, regarding their ability to increase the level of whole-genome DNA methylation and their fragmentation, a promising direction is to study the level of methylation of genes that regulate the cell cycle of the stomach and intestines, as well as the study of their expression/protein synthesis function.

Key words: DNA methylation, epigenetics, ulcerogenic effect, non-steroidal anti-inflammatory drugs.

Connection of the publication with planned research works.

The work was carried out as part of the research work of the Department of Clinical and Laboratory Diagnostics of the Zaporizhzhia State Medical University "Development of new effective ways of diagnosis and endogenous cytoprotection of ischemic damage of the coronary and cerebral blood circulation (clinical and experimental research)", state registration number - 0118U004369.

Introduction.

In human life, the body is subject to the influence of negative external environment factors, leading to epigenetic changes and, accordingly, to a violation of the expression/protein synthesizing function of the body's cells. Researches of the last decade have established that drugs can act as epigenetic factors, especially with their long-term administration, some drugs are considered as epigenetically active xenobiotics, which can chemically interact with the nucleic acids of the cell, leading to histone modification and DNA methylation [1, 2].

It is known that epigenetic modification of DNA affects the expression activity of certain genes at several levels, which leads to a change in the phenotype of the cell. In the body, there are molecular-biochemical systems aimed at inactivating xenobiotics, including drugs, but it is known that drugs change not only molecular reactions, physiological functions but also can model gene expression [3].

It has long been known that gene mutations play an important role in the formation of oncogenesis. However, only recently was it recognized that epigenetic changes significantly contribute to the development of oncological diseases [3, 4, 5].

Based on the above, studying the epigenetic effect of drugs on epigenetic changes in genes is interesting. In recent years, there has been a rapid increase in epigenetic research associated with the development of new molecular and cytological approaches. It has been established that there are basic epigenetic mechanisms, or, as they often say, epigenetic markers. Currently, DNA methylation is the most studied. DNA methylation is catalyzed by a family of DNA methyltransferases (Dnmts), which transfer a methyl group from S-adenylmethionine (SAM) to the fifth carbon of a cytosine residue to form 5mC. Dnmt3a and Dnmt3b can establish a new meth- ylation pattern of unmodified DNA and are therefore known as Dnmt de novo. On the other hand, Dnmtl functions during DNA replication by copying the DNA methylation pattern from the parental DNA strand to the newly synthesized daughter strand [2, 3, 6, 7].

Today, the ability of drugs to epigenetically affect gene expression is considered one of the likely mechanisms of unwanted side reactions.

There are data that long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) leads to the formation of erosions, ulcers and oncological diseases of the stomach and intestines. It is suggested that these side effects result from damage to the mucous membrane under the influence of NSAIDs and the possible effect of NSAIDs on genes that regulate the cell cycle, especially with their long-term use [8].

The aim of the study.

Study of the effect of various NSAIDs on the level of genome-wide DNA methylation and its fragmentation processes.

Object and research methods.

The research used 60 male Wistar rats weighing 200400 grams, obtained from the kennel of the SI "Institute of Pharmacology and Toxicology NAMS of Ukraine" All experimental studies complied with the main provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986). Care, maintenance and feeding of animals were carried out in accordance with the requirements of regulatory documents in standard vivarium conditions. Before starting the research, the Bioethics Commission of the State Medical University reviewed and approved the research protocol (No. 7 dated 11.11.2019) and all procedures related to keeping animals, humane treatment of animals, and their use in experiments. Each group included 15 animals (15 males). The duration of quarantine (acclimatization period) for all animals was 14 days. During the quarantine, each animal was examined daily (behavior and general condition), twice a day the animals were observed in the cells (morbidity and mortality).

Sixty laboratory rats weighing 200-400 g. were divided into four groups of 15 animals as follows: 1st - control (CG), which were injected with a 0.9% sodium chloride solution daily for 3 months, 2nd - rats, which were injected with indomethacin (Ind) daily for 3 months months at a dose of 0.6 mg/kg, the 3rd group - rats that were administered daily for 3 months with acetylsali- cylic acid (Asa) for 3 months at a dose of 0.6 mg/kg and the 4th group - rats that were administered daily with meloxicam (Mxc) in a dose of 0.1 mg/kg - 3 months.

DNA fragmentation and methylation levels were determined using the DNA-comet assay method. MspI and HpaII restriction enzymes (Abcam, USA) were used to study methylation processes. The degree of DNA fragmentation was expressed as a percentage. The level of DNA methylation was expressed as a percentage of MspI and HpaII in the nuclei of blood cells [9].

Statistical evaluation of the research results was performed using the Statistica 10.0 software package (Stat- soft Inc., USA). The obtained results are presented in the form of the median of the interquartile range. Differences between groups were assessed using nonparametric methods using the Mann-Whitney test. Differences at p<0.05 were considered statistically significant.

Research results and their discussion.

Long-term use of all NSAIDs led to a statistically significant increase in genome-wide DNA methylation and intensification of its fragmentation processes (table). It is important to note that according to the degree of epigenetic influence, the effect of the studied NSAIDs was of varying severity. Thus, the most significant effect on the amount of MspI/HpaII was exerted by long-term administration of indometacin (increase by 99% compared to the intact group of animals), then acetylsalicylic acid and meloxicam (by 56% and 41%, respectively). Similar dynamics of NSAIDs was also established when studying the degree of total DNA fragmentation.

We established the ability of NSAIDs to affect DNA methylation processes, to some extent confirming many researchers' assumptions regarding the presence of NSAIDs in the epigenetic mechanism of their unwanted side effects.

Table - Whole-genome DNA methylation (MspI/ HpaII) and its fragmentation in blood lymphocytes of rats against the background of long-term administration of NSAIDs

Note: * - p<0.05 in relation to the intact group of animals.

gene expression lymphocyte

DNA methylation is regulated by a family of DNA methyltransferase enzymes (DNMTs), of which DNMT1 methylates DNA during replication, DNMT3A and DN- MT3B carry out de novo methylation, and DNMT3L, in turn, is responsible for binding the listed methyltrans- ferases to the methyl group donor - S - adenosyl-L-me- thionine. There are 2 main mechanisms of epigenetic regulation of transcription by DNA methylation: direct and indirect. The direct mechanism of action is based on the steric hindrance of methyl groups in the interaction of DNA with transcription factors. In the case of a mediated mechanism, methylated regions interact with methyl binding proteins (MBPs) MeCP-1 and MeCP-2, which bind to DNA. MeCP-1 sterically blocks the binding of DNA to the transcription apparatus, which leads to repression of transcription [6, 8, 10]. MeCP-2, in turn, recruits histone deacetylases (HDACs) together with transcriptional corepressors, leading to compaction of chromatin structure and transcriptional repression, leading to DNA fragmentation.

The molecular processes described above lead to disruption of gene expression/protein synthesis function, which can lead to genomic instability and disruption of cell proliferation and differentiation processes. A number of studies have shown that NSAIDs can cause oncological diseases of the gastrointestinal tract with their long-term use. An epigenetic effect of NSAIDs on genes regulating the cell cycle of the stomach (CDH1, MHL1) and intestines (SEPT9, APC) was established. It is known that the SEPT9 gene encodes the synthesis of septin-9 protein. DNA methylation of this gene stops its active work and "turns off" the cancer growth suppressor protein synthesis. Loss of SEPT9 gene expression is associated with the development of colorectal cancer (CRC) [8, 10]. The presence of an inactive tumor suppressor gene in the blood indicates the processes of tumor development in the intestine, reflecting such events as cell proliferation and angiogenesis in the tumor [11]. In addition, our previous studies established a probable MMP-mediated mechanism of side effects of NSAIDs, due to changes in the expression activity of genes encoding the synthesis of matrix metalloproteinases, an increase in their concentration and their participation in the degradation of the extracellular matrix.

Conclusions

Thus, the experimental studies we conducted established the ability of NSAIDs to increase whole-genome DNA methylation and intensify their fragmentation processes with long-term administration. Indomethacin had the most pronounced effects (increase of MspI/ HpaII by 99%; DNA fragmentation by 90% compared to intact).

Prospects for further research.

Considering the established effects of NSAIDs regarding their ability to increase the level of genome-wide DNA methylation and their fragmentation, a promising direction is to study the methylation level of genes that regulate the cell cycle of the stomach (CDH1, MHL1) and intestines (SEPT9, APC), as well as the study of their expression/protein-synthesizing function.

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