Cardiovascular diseases and depression are very prevalent among the general population and often occur simultaneously in the same patient (1). The association between depression and heart disease is two directional. Depression is regarded as a risk factor for coronary heart disease and acute cardiovascular sequelae, including myocardial infarction and heart failure (2). Conversely, patients with heart disease exhibit increased episodes of depression (3,4). Moreover, morbidity and mortality rates in patients with depression and cardiovascular disease have been found to be significantly higher than those in patients with cardiovascular disease without depression (4). It has been observed that patients who experienced a myocardial infarction and became depressed had a 5-fold increase in mortality rate during the 6 months after the infarction (5). Therefore, the effective treatment of depression in patients with cardiovascular disease is vital and requires the simultaneous use of multiple medications.

Clinical trials have indicated that there is an association between cardiovascular improvement and the degree by which low-density lipoprotein cholesterol (LDL-C) levels are reduced (6). The reduction of high blood cholesterol levels is a crucial purpose of pharmacotherapy. The most effective lipid-lowering drugs are statins, also known as 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors. A drug of the statin class, atorvastatin, reduces the risk of cardiovascular events by reducing the total cholesterol, LDL-C and B apolipoprotein levels in serum (7). Atorvastatin is also effective in treating and preventing atherosclerosis, and affects all stages of atherosclerotic plaque formation (8).

Selective serotonin reuptake inhibitors (SSRIs) are the most frequently prescribed class of antidepressants. SSRIs are generally recommended for use in patients with cardiac disease and may also have a protective effect against myocardial infarction. Several studies have documented the cardiovascular effects of fluoxetine treatment (9–11). The SSRI fluoxetine appears to be safe for use in the treatment of depressed patients with cardiac disease and does not cause orthostatic hypotension or slow conduction (10). Another SSRI, paroxetine, is one of the most well-known and effective SSRIs (12,13). According to Nelson et al, depressed patients with ischemic heart disease can be treated with paroxetine effectively and safely as paroxetine has no consistent significant effects on the pulse or blood pressure, and the authors suggest that paroxetine is as effective as nortriptyline but less likely to produce serious side effects (5).

Atorvastatin undergoes extensive microsomal metabolism by the cytochrome P450, CYP3A4 isoenzyme (14). Co-administration of drugs that interact with the CYP450 system may significantly affect the plasma concentrations and potential toxicity of atorvastatin. Fluoxetine and its circulating metabolite norfluoxetine constitute a complex multiple inhibitor system, causing reversible or time-dependent inhibition of CYP3A4, and also CYP2D6 and CYP2C19 in vitro (15). Paroxetine is extensively metabolized and is a mechanism-based inhibitor of CYP2D6 and CYP3A. This inhibition most likely involves the irreversible binding of a paroxetine-reactive metabolite to the heme complex in the P450 enzyme (16). Long-term polypharmacotherapy can lead to increased side effects of drugs and may cause an oxidation-reduction imbalance followed by the generation of reactive oxygen species (ROS) (17–19). ROS are produced intracellularly through multiple mechanisms, and the sources of ROS in cells include CYP450 CYP and NADPH oxidase, with NADPH oxidase complexes being the major producers of ROS in cells. Biochemical and pharmacokinetic studies have revealed that during the biotransformation of drugs, reactive metabolites capable of directly reducing molecular oxygen to generate ROS are often produced. Cytochrome P450 is a superfamily of heme-thiolate proteins that are, in general, the terminal oxidase enzymes in an electron transfer chain that delivers an electron to an oxygen molecule (17,18,20). During phase I metabolism, atorvastatin is extensively metabolized by the monooxygenase CYP3A4, with the insertion of an oxygen atom into the atorvastatin molecule to form o- and p-hydroxyatorvastatin, while the other oxygen atom of molecular oxygen is reduced to water with the concomitant oxidation of NADPH; however, premature and incomplete reduction may occur, to generate the superoxide radical (O₂^·-) (20,21). SSRIs cause reversible or time-dependent inhibition of CYP3A4 and also CYP2D6 which could affect the metabolism of atorvastatin (22).

Drug metabolism generates ROS and reactive metabolites as by-products; however, cells have a defense mechanism for the removal of free radicals to attenuate intracellular damage and ameliorate the harmful effects of ROS (18,23). Drug interactions may alter cell metabolism causing the cellular antioxidant capacity to be exceeded, possibly resulting in increased oxidative stress. Drug-induced oxidative stress has been implicated as a mechanism of toxicity in numerous tissues (18). Therefore, any potential drug interactions and adverse effects require close monitoring.

To the best of our knowledge, there are no published data concerning the effects of concomitant treatment with atorvastatin and SSRIs on redox imbalance. The present study aimed to assess antioxidant defense parameters in the blood of rats after 28 days treatment with atorvastatin in combination with fluoxetine or paroxetine. The activity of glutathione peroxidase (GPX) was determined in whole blood and the glutathione reductase (GR) activity and total antioxidant status (TAS) were determined in serum.

The formation of chemically reactive metabolites of drugs is associated with numerous adverse drug reactions (18,19). Reactive metabolites may react with cellular proteins, lipids and nucleic acids and thus cause oxidative stress. Atorvastatin is extensively metabolized in the gut and liver by oxidation, lactonization and glucuronidation. Metabolism in the liver produces two active hydroxy metabolites, o-hydroxyatorvastatin and p-hydroxyatorvastatin (21). During the bioactivation of atorvastatin, the reduction of molecular oxygen by reactive metabolites may generate ROS (17,8,20). The risk of ROS being generated increases during simultaneous treatment with several drugs metabolized by cytochrome P450 and this can lead to redox imbalance (17). Oxidative stress causes cell damage through a number of mechanisms including lipid peroxidation, protein oxidation, DNA oxidation and mitochondrial damage. This impairment of cellular function can result in cell death and possible organ failure (27,28). However, cells contain antioxidant enzymes that function to maintain the redox status.

In the present study, the combined administration of atorvastatin with fluoxetine caused an increase of GPX activity and did not influence GR activity. GPX is one of the body's most potent antioxidant defense agents. Its purpose is to balance pro-oxidative and antioxidant actions and to prevent the excessive accumulation of ROS in the body. A change in the activity of this enzyme may reflect the impairment of antioxidant activity in the blood and can be useful in monitoring the effects of treatment (23). In the present study, the increase of GPX activity indicated that endogenous antioxidant levels were increased. This might indicate that the production of free radicals was increased following the concomitant application of atorvastatin with fluoxetine. Changes in GPX activity have been observed in animals exposed to pro-oxidative drug action (18). In our previous study, increases in the activity of both GPX and GR were observed after the combined 14-day administration of rosuvastatin with fluoxetine to rats (24). In the present study, the simultaneous application of atorvastatin with fluoxetine caused an increase in the TAS, although with a high error of the mean. In the body, the total antioxidant system consists of multiple components providing protection against the molecular damage of cell structures. The TAS is defined as the ability of the serum to quench free radical production, and is determined in order to indicate the efficiency of cellular antioxidant mechanisms. A reduction in the TAS suggests an increase in the number of oxygen free radicals and a reduction of the activity of the antioxidant defense system. The TAS also indicates the antioxidant potential of drugs and can indicate whether a new treatment is detrimental to the body's anti-oxidation system. In the present study, an increase of the TAS following the co-administration of atorvastatin with fluoxetine is probably associated with metabolic mobilization and may indicate that the amounts of endogenous antioxidants were increased. Our previous study carried out on rats treated simultaneously with rosuvastatin and fluoxetine for 14 days revealed a reduction in the TAS (24). This difference may result from the shared metabolism of rosuvastatin and fluoxetine; these drugs are biotransformed by cytochrome P450 isoenzyme CYP2C9, while atorvastatin is metabolized primarily by isoenzyme CYP3A4 (15,21,28). It is also important to note that different durations of treatment were applied (14 days in the previous study, and 28 days in the current study). The present study was a preliminary study, and did not include the monitoring of transcription factors, such as Nrf2, that are responsible for activating the antioxidant capacity of cells.

Combined administration of atorvastatin with paroxetine caused an increase in the activity of both GPX and GR (only in comparison with the control group) and did not affect the TAS. GR is closely associated with GPX and plays an important role in protecting cells against oxidative damage by increasing the level of reduced glutathione in the process of aerobic glycolysis (29). The increased activity of GR may be influenced by the reduced form of glutathione, and potentially indicates that there is an increased activity of the enzyme system that prevents oxidation. Similar results were obtained in our previous study, in which rats were treated with rosuvastatin and paroxetine simultaneously for 14 days (25).

The present study revealed no significant changes in the activity of GPX and GR enzymes or in the TAS in rats treated with only atorvastatin. However, a number of studies support the role of atorvastatin in the maintenance of the antioxidant status (30–32). The possible antioxidant mechanisms of atorvastatin include reducing the generation of ROS by the inhibition of vascular NAD(P)H oxidase, modifying redox homeostasis in LDL particles, modulating RNA expression, increasing the synthesis of nitric oxide in the vasculature and binding to the surface phospholipids of lipoproteins (30,33). In the current study, no statistically significant changes concerning the oxidative stress parameters in the groups of rats receiving antidepressants individually were noted. Previous studies suggest that SSRIs have a beneficial effect in maintaining the oxidative-reductive balance (34,35). The potentially favorable antioxidant effect of fluoxetine may be mediated by three mechanisms: Inhibition of lipid peroxidation, increase of glutaminergic transmission and reduction of immune and inflammatory components that favor the generation of ROS (36). Similar results, which did not reveal any crucial changes in oxidative stress parameters in rats receiving fluoxetine for 14 days, were obtained in a previous study (24). Other research indicates that the antidepressive effect of paroxetine is at least partially associated with the attenuation of oxidative stress imbalance (37).

An increase in GPX activity in the rats receiving atorvastatin with fluoxetine may be associated with the defense mechanism against the increased ROS load. An increase in the TAS is probably connected with the metabolic mobilization of the rats treated with atorvastatin plus fluoxetine, and may indicate an increasing amount of endogenous antioxidants. Concurrent increases in the activity of GPX and GR are likely to be due to the augmentation of free oxygen radical production during the simultaneous administration of atorvastatin with paroxetine. The present study was a preliminary study, aiming to assess antioxidant defense parameters in the blood of rats by assaying the activity of antioxidant enzymes and TAS level. In the future, this research may be expanded, for example, to determine the level of glutathione.