In the present study, it was observed that the use and/or therapy with omeprazole (OME) in a continuous and prolonged manner, without dose specifications (20, 30 and 40 mg / Kg) can cause cytogenetic risks in cells of the stomach epithelium, byaneugenic and/or clastogenic effects, by inducing micronuclei in patients without gastritis but use the drug, and with gastritis and in therapy, when compared to patients that neither use the medicine and even not in therapy with other drugs. The effects of OME on the formation of micronuclei in human cells are still unwell described. However, there are in silico studiessuggesting that OME can lead to chromosomal changes, as well as contribute to the formation of micronuclei (Rosenkranz and Kjopman, 1991), also promoting covalent connection with DNA, characterizing its genotoxicity (Phillips et al., 1992).
Investigations in gastric epithelial cells of patients with gastritis and with positive results for H. pylori, indicates a risk of genotoxicity, with greater significance in relation to negative patients (Marie and Altahir, 2011), as well as was observed in the study in relation to the formation of micronuclei, in epithelial cells of the stomach. H. pylori infections can cause chronic gastritis, peptic and duodenal ulcers, adenocarcinoma and gastric lymphoma (Trindade et al., 2017). There are reports that OME can induce DNA damage, after its metabolism, by the formation of N-nitrosamines that can generate several nuclear alterations such as MN, pycnosis and cariorrexis (Thongon and Krishnamra, 2011; Novotna et al., 2014). MN can also be provokedby chromosome breaks (Rim and Kim, 2015), or by internal chromosomes that have been separated from the nucleus (Balmus et al., 2015), due to breaks in the double-stranded DNA or as a result of mitotic spindle dysfunction (Fenech, 2007; Fenech et al., 2011). It is taken in consideration that DNA breaks can happen due to oxidative stress (Fang et al., 2015) inducing apoptosis (Pittaluga et al., 2015).
DNA damage, like cytogenetics, can be induced by OME after its metabolism by the production of sulfone, sulfite and hydroxy-omeprazole (Rosenkranz and Kjopman, 1991; Downes and Foster, 2015), as also for its electrophilic potential (Powers et al., 1995) due to covalent bonds with DNA (Burlinson et al., 1990; Furihata et al., 1991). It is observed that OME can increase nuclear cell proliferation antigens (PCNA) (Liu et al., 2016; Zheng et al., 2016), by modulating via lysosomal transport with mechanisms of expression of the LC3 gene associated to autophagy (Udelnow et al., 2011).
In addition to the formation of micronuclei, other cytogenetic risks were observed in the cells of the stomach epithelium of patients without gastritis and with gastritis (without and with H. pylori infection, in and/or OME therapy, such as nucleoplasmicbuds and bridges,the buds are the result of DNA amplification or repair (Fenech et al, 2011; Luzhna, 2013) and the bridges are originated from failures in chromosomal rearrangements or are result from the fusion of chromosomal ends, telomeres that allow the formation of filaments chromatin molecules that link two distinct nuclei (Fenech et al., 2011). Corroborating findings observed in this study, there are previous reports that OME can induce changes in chromosomes and micronucleus formation (Burlinson et al., 1990; Rosenkranz and Kjopman, 1991; Furihata et al., 1991).
The cytotoxicity of OME has been reported in normal human cells (HEK293 and NIH3T3) (Shankar et al., 2019). In cells of the stomach epithelium of patients, that use or are in therapy with OME, cytogenetic risks are indicative of cytotoxicity, due to the induction of picnoses and binucleated cells, especially in patients with H. pylori infection. H. pyloriliberatesa cytotoxin that can provoke apoptosis by alterations in the release of cytochrome C in mitochondria (Gajewski et al., 2016), as well as can destroy the cellular junctions in the gastric epithelium (Alzahrani et al., 2014), causing transient enlargement acid secretion leading to hypochlorhydria and intestinal metaplasia (Trindade et al., 2017). These events are linked to a risk of gastric adenocarcinomas development (Keilberg and Ottemann, 2016; Wessler et al., 2017). It´s necessary takes in considerarion that picnoses occur due to chromatin condensation and dissolution, and binucleated cells result from cytokinesis failures at the end of cell division (Sabharwal et al., 2015).
However, it is needed emphasize that OME has hepatotoxic effects (Cesário et al., 2015) as a result of apoptosis mechanisms by inducement of tumor necrosis factor alpha (TNF-α) (Fontana et al., 2014), as well as by alterations of liver enzymes AST and ALT (Thomas et al., 2016). There is also informationabout organ toxicity (hepatotoxicity), toxicological parameters (Banerjee et al., 2018), hepatotoxicity in pregnant women, observed by the reduction of the enzymes AST and ALT (Thomas et al., 2016) and hepatotoxic and nephrotoxic effects as thrombocytopenia, acute interstitial nephritis, anaphylactic reactions and gynecomastia (Cesário et al., 2015). The by-products OMP-1, OMP-6, OMP-7, OMP-8, OMP-13 and OMP-15 can also combine with the aryl hydrocarbon receptor (AhR) and induce toxic effects to the immune system (Shankar et al., 2019).
In this study, OME induced apoptosis in stomach epithelial cells due to cytogenetic risks of nuclear fragmentation (cariorrexis) and nuclear dissolution (karyolysis), as seen in patients without gastritis and with gastritis in use and/or therapy with OME, with significance also in patients with H. pylori infection, as previously observed in other nuclear alterations. It is necessary mentionate that the induction of apoptosis is one of the mechanisms linked to of acute gastric injury (Lou et al., 2013), however, OME has apoptotic effects in human gastric cancer cells (HGC-27) (Zhazg et al., 2013), colorectal tumor cells (Muerkoster et al., 2008; Kim et al., 2010) and normal human nuclear polymorphic leukocytes (Capodicasa et al., 2018).
According to other investigations, drugs that induce oxidative damage may increase the levels of endogenous enzymes associatedto antioxidant defenses such as catalase and superoxide dismutase (Almenara et al., 2015; Herbet et al., 2016; Poprac et al., 2017). Catalase (CAT) is one of the antioxidant enzymes that have participation in degradation of H2O2 through dismutation reactions, current mainly in the peroxisomes of cells and helps to protect against damage caused by hydrogen peroxide, been considered as an important oxidative biomarker (Pey et al., 2017; Gupta et al., 2012). Superoxide dismutase (SOD) converts the oxygen produced during oxidative stress to H2O2. In this way, to act effectively in maintaining cellular integrity and function, SOD depends on the balance between SOD, GPx and CAT (Pey et al., 2017).
In this study it was possible to detect an increase in antioxidant defenses for these enzymes, especially in patients with H. pylori infection. H. pylori infection can also increase the production of reactive oxygen and nitrogen species in the stomach (Golbabapour et al., 2013). However, gastric lesions can induce oxidative stress, with amplification by OME therapy (Kohler et al., 2010) independently of co-infection with H. pylori, and may also induce an increase in antioxidant enzymes such as SOD and CAT, and glutathione reductase (GSH) (Baldissera and Cruzat, 2014; Glorieux and Calderon, 2017). Drugs contribute to increase oxidative stress levels (Herbet et al., 2016; Almenara et al., 2015; Porto et al., 2015), due to an imbalance between antioxidant defenses and oxidative stress levels (Gunasekarana et al., 2015) and regulation of lipid peroxidation (Ward et al., 2015).
OME is one of the drugs that can induce oxidative stress (Kohler et al., 2010), which culminates in cell apoptosis (Woűniak et al., 2017; Pey et al., 2017; Sies et al., 2017). Free radicals induce gastric lesions (Sofidiya et al., 2012), and that this process can contribute to carcinogenesis (Tsuchiya et al., 2018). Gastric lesions can produce free radicals, which are minimized by enzymes SOD and GPx, which lead to tissue recovery and gastroprotection (Cheng et al., 2014; Chen et al., 2014). OME can also induce lipid peroxidation, with responses to the increase in catalase and superoxide dismutase, and can also be considered as a marker of oxidative stress (Chen et al., 2014).
During its metabolism, OME can generate sulfone, sulfite and hydroxy-omeprazole, compounds that can generate more oxidative damage to genetic material (Brambilla et al., 2010; Brambilla and Martelli, 2009; Downes and Foster, 2015). OME are able to induce an increase of enzyme heme-oxigenease by means independent of the aryl hydrocarbon receptor (AhR), which consequently increases the levels of H2O2 (Patel et al., 2012). Oxidative damage can be one of the OME mechanisms for inducing changes in genetic material in gastric epithelium cells, as it can produce H2O2 when it binds to protein C283, which contains CACT, and C136 generating beta oxidation of fatty acids (Tonazzi et al., 2013). OME can induce oxidative damage in S. cerevisiae, in addition to cytogenetic damage in Sarcoma 180 cells (Paz et al., 2019).
Among these mechanisms, the study points out that the cytogenetic risks that can be induced by oxidative effects that lead to the formation of micronuclei and other nuclear alterations indicative of cytotoxicities and apoptosis. Corroborating these analyzes, positive and negative statistical correlations were observed between micronuclei and the measurements of CAT and SOD, and between pyknosis, respectively. Proton pump inhibiting drugs (PPIs), such as OME, may have genotoxic and/or carcinogenic effects (Downes and Foster, 2015), through several mechanisms including oxidative stress. When the substances that induce oxidative stress are in excess and the antioxidant system is unable to neutralize the oxidative process (Laskoski et al., 2016), several cellular mechanisms induce cell regulation and activation of signaling cascades for cell death (apoptosis or necrosis) (Woźniak et al., 2017), for cell proliferation, metastasis, resistance to apoptosis, angiogenesis and as a consequence of genetic instability (Moloney and Cotter, 2017). A possible mechanism of OME has been shown in Figure 7.