Keywords

1 Introduction

The advancement of civilization, including the fast pace of life and profound changes in nutrition, has lead to serious health problems in individuals and the entire population. Lifestyle modifications have led to an increase in the prevalence of obesity and related diseases. Obesity is described as a chronic inflammatory process occurring in the body with the participation of visceral adipose tissue (Popko et al. 2010). It is highly correlated with many diseases such as diabetes mellitus type 2, hypertension and metabolic syndrome (Winklewski et al. 2015; Goodwin et al. 2012; Goodwin and Stambolic 2011). Factors related to lifestyle such as nighttime work, insomnia, poor diet, and a high dietary fat content, are associated with increased risk of malignant transformation. The potential mechanisms responsible for increased production of toxic metabolites and reactive oxygen species (ROS), resulting in neoplasm development, constitute an important component of basic research.

Neoplasms accounted for 7 million deaths in 2000, expected to reach approximately 10 million deaths in 2020. In Poland, there were 130,000 new cases of cancer and 85,000 deaths from cancer in 2000, and 160,000 new cases of cancer and 100,000 deaths attributable to cancer in 2010 (Syczewska-Weber and Rucinski 2008). Cancer is responsible for 25% of all causes of deaths. One of the most common forms of female malignancy in Europe is breast cancer (464,000 cases, 13.5% of all cancer cases) (Ferlay et al. 2013). In Poland, approximately 12,000 new cases of breast cancer are reported each year (Didkowska et al. 2007).

Several studies support the concept that ROS are involved in the etiology and progression of breast cancer. Importantly, high levels of the biomarkers of oxidative stress, including lipid peroxidation products, malondialdehyde or isoprostanes, protein oxidation products (carbonyls and diene conjugates), and DNA modifications are frequently identified in breast cancer patients (Kedzierska et al. 2012). It has recently been shown that altered redox status is directly correlated to estrogen levels. ROS may also control antioxidant gene expression (Sekkin et al. 2015; Carpentieri et al. 2012). Oxidative stress participates in the structural modification of estrogen and progesterone receptors in patients with endocrine-responsive breast cancer (Panis et al. 2012, Sosa et al. 2013). A high level of ROS in cancer cells may lead to a variety of biological responses, such as cell adaptation, the development of DNA mutations and genetic instability, increased proliferation rate, and resistance to some drugs used in anticancer therapy (Jou et al. 2007; Tas et al. 2005).

There are reports demonstrating that cancer patients often have symptoms of metabolic syndrome, a component of which is glucose intolerance and diabetes mellitus type 2. Therefore, an evaluation of oral antidiabetic preparations for anti-tumor properties is particularly important. Oral antidiabetics with pleiotropic properties that show anti-tumor activity have drawn considerable attention, as the incidence of diabetes mellitus type 2 and cancer continue to rise. So far, anti-tumor properties have been reported in two groups of oral antidiabetics: biguanides, particularly metformin (MF) (Erdem et al. 2015; Zhu et al. 2015; Anisimov 2014; Leone et al. 2014; Pollak 2013) and thiazolidinediones (Bojková et al. 2014).

Melatonin (MEL), N-acetyl-5-methoxytryptamine, a hormone produced primarily by the pineal gland as indolamine and secreted in a circadian pattern, protects against cancer development (Viswanathan and Schernhammer 2009), functioning as an antioxidant (Reiter et al. 2003), with properties that reduce oxidative stress (Kurhaluk et al. 2017; Esrefoglu et al. 2012) and inflammation (Ren et al. 2015; Shin et al. 2015). MEL also stabilizes mitochondrial function (Carrasco et al. 2015), protects DNA against apoptosis (Jou et al. 2007), attenuates metabolic disorders in the streptozotocin-induced diabetes model in rats (Sudnikovich et al. 2007), and is involved in the regulation of inflammatory cell infiltration and obesity-induced adipokine alteration (Favero et al. 2015).

We have recently reported a preventive or curative effect of MEL on mammary carcinogenesis, consisting of increased survival time, preferably when MEL is given in combination with other oncostatic substance (Kubatka et al. 2014; Orendáš et al. 2014; Orendáš et al. 2009; Kubatka et al. 2002). The aim of the present study was to evaluate the effect of the oral antidiabetic metformin and the pineal hormone melatonin administered alone and in combination on oxidative stress and antioxidant enzyme activity in the heart tissue in female Sprague-Dawley rats fed a high fat diet and subjected to mammary tumor cell proliferation in vivo induced by N-methyl-N-nitrosourea (NMU) administration.

2 Methods

The experiments were conducted following the guidelines of the European Union Council and the current laws of the Slovak Republic for animal experiments. The study was approved by the Ethics Committee of the Pavol Jozef Šafárik University and by the State Veterinary and Food Administration in Slovakia (no. Ro-2054/13–221). The animals were treated and sacrificed in a humane manner according to the principles provided in Laws No. 289/2003, 489/2003, and 23/2009 of the Slovak Republic on the care and use of laboratory animals.

2.1 Animals and Experimental Design

Mammary Carcinogenesis Model

Female rats of the Sprague-Dawley strain (Velaz; Prague, Czech Republic) aged 30 days were used in the experiment. This strain is most commonly used in in vivo mammary carcinogenesis models, as other strains (e.g., Wistar) are not sensitive to mammary tumor induction by chemocarcinogens. The animals were adapted to standard vivarium conditions with temperature of 23 ± 2 °C, relative humidity of 60–70%, and an artificial light-dark cycle of 12:12 h (lights on from 7 a.m., light intensity 150 lx/cage). During the experiment, the animals (6 per cage) were fed ad libitum with a high fat diet (10% of total fat, 2.5% from lard, 7.5% from palm oil; Biofer, Slovakia) and tap water, or received MEL solution.

Mammary carcinogenesis was induced by N-methyl-N-nitrosourea (NMU, cat. no. N4766; Sigma-Aldrich; Deisenhofen, Germany) administered intraperitoneally in a dose of 50 mg/kg on the 42nd postnatal day. The NMU solution was freshly prepared before administration by dissolving NMU in 0.9% NaCl. A single volume of injectant was 0.5 mL.

Experimental Groups

Chemoprevention with MF and MEL was initiated 12 days prior to carcinogen application and lasted until the termination of the experiment. MF (Actos; Lilly, Alcobendas, Spain) was administered in the diet at a concentration of 2000 ppm. MEL (cat. no. M5250, Sigma-Aldrich Deisenhofen, Germany) was administered in tap water at a concentration of 20 mg/L daily from 3 p.m. to 8 a.m. Only pure water was given at other times.

Animals were assigned randomly to one of five experimental groups (18 rats per group), except the control group (Group 1), having ten rats. Group 2 was NMU model group without chemoprevention, Group 3 – NMU + MF chemoprevention, Group 4 was NMU + MEL chemoprevention, and Group 5 was NMU + MF + MEL chemoprevention combined.

All rats, including control animals, used in this experiment were fed a high fat diet. All rats were weighed weekly during the experiment and palpated to register the presence, number, location, and size of each palpable tumour. NMU rats were given only the carcinogen and the values of NMU rats were compared to the control group to determine the effects of the carcinogen. The groups given the chemopreventive treatment were compared to the NMU group and to each other, to determine the effects of chemoprevention.

Food and water intake over a 24-h period was monitored during the 4th, 9th, and 14th week of the experiment (dated from NMU administration). During the experiment, the average daily intake of MF ranged from 28 to 32 mg/rat/day, which equals to ca 880 mg/m2, considering the body surface of 0.034 m2 in a 245 g rat. For comparison, the recommended daily dose of MF in diabetic patients is 1700 mg, which equals to ca 940 mg/m2, considering the body surface of 1.8 m2.

The average daily intake of MEL ranged from 0.36 to 0.48 mg/rat/day. The MEL dose was supraphysiological, about fourfold higher than the usual dose used for the treatment of sleep disorders and jet lag disorder in humans, according to the body surface area. In the last week of the experiment (week 16), the animals were killed by a quick decapitation and mammary tumors, and selected organs and tissues were removed for further evaluation.

Drugs and Solutions

EDTA, HEPES, KCl, K2CO3, KH2PO4, EDTA, 2-thiobarbituric acid were purchased from Sigma-Aldrich (Deisenhofen, Germany). All drugs were freshly prepared. All reagents used in the study were of analytical grade.

Tissue Isolation

The heart tissues were removed. Briefly, the heart was excised, weighed, washed in ice-cold buffer, and minced. The minced tissue was rinsed with cold isolation buffer to remove blood and was homogenized on ice in a glass Potter-Elvehjem homogenizer (Kennesaw, GA) with a motor-driven Teflon pestle. The isolation buffer consisted of 120 mM KCl, 2 mM K2CO3, 10 mM HEPES, and 1 mM EDTA. The pH was adjusted to 7.2 with KOH.

Heart homogenates were used for the determination of thiobarbituric acid reactive substances (TBARS) and oxidative modified protein (OMP) levels, total antioxidant status (TAS), as well as catalase (CAT) and superoxide dismutase (SOD) (1:1000), glutathione reductase (GR) and glutathione peroxidase (GPx) (1:20) activities. For the quantification of proteins, the Bradford method with bovine serum albumin as a standard was used. Absorbance was recorded at 595 nm.

2.2 Biochemical Assays

Conjugated Dienes

The level of conjugated dienes was determined according to Kamyshnikov’s (2004) method. Conjugated dienes are formed during the lipid peroxidation process as a result of the reconfiguration of double bonds after the detachment of hydrogen from the polyunsatured fatty acid chain. Conjugated dienes were determined by absorption at a wavelength of 233 nm and expressed in nmol per mg protein.

Thiobarbituric Acid Reactive Substances (TBARS)

TBARS also were estimated using according to Kamyshnikov’s (2004) method and were expressed in μmol of malondialdehyde (MDA) per mg protein from heart tissue.

Protein Carbonyl Derivatives

The OMP rate was estimated using the reaction detecting carbonyl derivatives of amino acids with 2,4-dinitrophenyl hydrazine (DNFH) according to Levine et al.’s (1990) method in Dubinina et al.’s (1995) modification. The final solution was centrifuged to remove any insoluble material. The carbonyl content was calculated from the absorbance measurements at 370 nm and 430 nm with an absorption coefficient of 22,000 M−1·cm−1. Carbonyl groups were determined spectrophotometrically at 370 nm (aldehyde derivatives (AD); OMP370) and 430 nm (ketone derivatives (KD); OMP430), and expressed as nmol per mg protein.

Total Antioxidant Status (TAS)

The TAS level in the plasma and liver was estimated by measuring the TBARS level according the oxidation of Tween 80. This level was determined calorimetrically according to Galaktionova et al.’s (1998) method based on the reaction of Fe2+/ascorbate-induced oxidation of Tween80, resulting in a stable level of 2-thiobarbituric acid reactive substances. The maximum absorbance was measured at 532 nm. The absorbance of the blank was defined as 100%. The percentage of TAS in a sample was calculated in reference to the absorbance of the blank.

Superoxide Dismutase Activity

Superoxide dismutase (SOD, E.C. 1.15.1.1) activity in the supernatant was determined according to Kostiuk et al.’s (1990) method. SOD activity was assessed by its ability to produce superoxide dismutase during quercetin auto-oxidation in an alkaline medium (pH 10.0). The absorbance at 406 nm was measured immediately and after 20 min and the activity was expressed in units of SOD per mg protein.

Catalase Activity Assay

Catalase (CAT, E.C. 1.11.1.6) activity was determined by measuring a decrease in H2O2 in the reaction mixture using Koroliuk et al.’s (1988) method. One unit of CAT activity was defined as the amount of enzyme required for decomposition of 1 μmol H2O2 per min per mg protein.

Glutathione Reductase Activity

Glutathione reductase (GR, E.C. 1.6.4.2) activity in the blood and tissues was measured according to Glatzle et al.’s (1974) method. The enzymatic activity was assayed spectrophotometrically by measuring NADPH consumption. A blank without NADPH was used and the GR activity is expressed in nmol NADPH per mg protein.

Glutathione Peroxidase Activity

Glutathione peroxidase (GPx, EC 1.11.1.9) activity was determined by the detection of non-enzymatic utilization of reduced glutathione (GSH) as the reacting substrate at 412 nm after incubation with 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) according to Moin’s (1986) method. GPx activity was expressed in nmol GSH per mg protein.

For protein quantification, Bradford’s (1976) method was used with bovine serum albumin as the standard. The absorbance was recorded at 595 nm. All enzymatic assays were carried out in duplicate at 22 ± 0.5 °C using a Specol 11 spectrophotometer (Carl Zeiss Jena, Germany).

2.3 Statistical Evaluation

Results were expressed as means ±SE. All variables were tested for a normal distribution using Kolmogorov-Smirnov and Lilliefors’ tests (p > 0.05). Homogeneity of variance was checked using Levene’s test. The significance of differences in the level of conjugated dienes, lipid peroxidation, amino acid carbonyl derivatives, and antioxidant enzyme activities between the control and experimental groups were examined using Student’s test and one-way analysis of variance (ANOVA), with post-hoc Bonferonni’s test. Differences were considered significant at p < 0.05. In addition, associations between individual data were evaluated using Pearson’s correlation analysis. All statistical calculations were performed with Statistica v8.0 software (StatSoft Inc., Cracow, Poland).

3 Results

Since the process of lipid peroxidation (LPO) occurs in multiple stages, we decided to analyze the process at its beginning and end. Diene conjugation products, as the first stage of LPO primary product formation, are presented in Fig. 1. The model of mammary carcinogenesis formation induced by NMU in the rats given a high fat diet caused statistically significant changes in the substrate accumulation in the heart during the first stage of free radical production. MF or MEL chemoprevention alone after NMU administration decreased the level of LPO intensity compared to NMU rats (F = 22.51, p = 0.0001). A reduction in LPO, particularly in the content of conjugated dienes, during MEL chemoprevention was greater than that during MF chemoprevention. MF and MEL combined did not cause significant changes in tissue compared to the MF + NMU-treated and MEL + NMU-treated rats.

Fig. 1
figure 1

Conjugated dienes (E233) in the heart tissue of female Sprague-Dawley rats subjected to a high fat diet in the mammary carcinogenesis model induced by N-methyl-N-nitrosourea (NMU). Metformin (MF) or melatonin (MEL) was administered alone and in combination (MF + MEL). Values are means ±SE. Significant differences between groups were designated as follows: a – NMU group vs. control group; aa – MF + NMU and MEL + NMU groups vs. NMU group; bb – MF + MEL + NMU group vs. MF + NMU and MEL + NMU group

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TBARS, estimated by the MDA level, are the end product of the terminal stages of free radical production, and are a biomarker of oxidative stress. The TBARS content increased significantly in the heart tissue after NMU treatment (F = 5.45, p = 0.001) and decreased significantly after MEL chemoprevention (Fig. 2). The intensity of the LPO process also was lower after MF and MEL chemoprevention combined (F = 4.61, p = 0.002) compared to MF + NMU-treated rats.

Fig. 2
figure 2

TBARS products in the heart tissue of female Sprague-Dawley rats subjected to a high fat diet in the mammary carcinogenesis model induced by N-methyl-N-nitrosourea (NMU). Metformin (MF) or melatonin (MEL) was administered alone and in combination (MF + MEL). Values are means ±SE. a – NMU group vs. control group. Significant differences between groups were designated as follows: aa – MF + NMU and MEL + NMU groups vs. NMU group; bb – MF + MEL + NMU group vs. MF + NMU and MEL + NMU group

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We further found significant effects of NMU administration on the level of OMP-AD and OMP-KD derivatives in the heart tissue and then after MF and MEL chemoprevention. MEL chemoprevention substantially limited the ROS processes compared to NMU-treated rats (Table 1).

Table 1 Content of oxidatively modified proteins (OMP) (aldehyde derivatives (AD); ketone derivatives (KD)) in the heart tissue of female Sprague-Dawley rats subjected to a high fat diet in the mammary carcinogenesis model induced by N-methyl-N-nitrosourea (NMU)
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The TAS level was statistically lower in the heart tissue after NMU treatment compared to that found in the control rats (Fig. 3). MF or MEL chemoprevention alone increased TAS compared to the NMU group (F = 21.34, p = 0.0001). Percentagewise, the effect of MEL was greater than that in both MF and NMU groups.

Fig. 3
figure 3

Percentage of TAS changes in the heart tissue of female Sprague-Dawley rats subjected to a high fat diet in the mammary carcinogenesis model induced by N-methyl-N-nitrosourea (NMU). Metformin (MF) or melatonin (MEL) was administered alone and in combination (MF + MEL). Values are means ±SE. a – NMU group vs. control group; aa – MF + NMU and MEL + NMU groups vs. NMU group; bb – MF + MEL + NMU group vs. MF + NMU and MEL + NMU group. Significant differences between groups were designated as follows: a – NMU group vs. Control group; aa – MF + NMU and MEL + NMU group vs. NMU group

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Antioxidant enzyme activity is shown in Table 2. NMU treatment increased SOD and GR activity, while it diminished CAT and GPx activity. MF chemoprevention augmented GR, CAT, and GPx activities compared to NMU-treated rats. MEL chemoprevention elevated GPx, and reversed the effects of NMU on SOD and GR activities. Combined chemoprevention with MF + MEL in the NMU model increased SOD activity compared to the MEL + NMU group. GR activity was lower in MF + MEL rats compared with the MEL + NMU rats.

Table 2 Antioxidant enzyme activity in the heart tissue of female Sprague-Dawley rats subjected to the mammary carcinogenesis model induced by N-methyl-N-nitrosourea (NMU)
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The following associations were present in the heart tissue in the NMU model of carcinogenesis: MDA-TAS (r = 0.89, p = 0.001), AMP AD-TAS (r = −0.91, p = 0.000), and diene conjugates-CAT (r = −0.93, p = 0.0001). The association of oxidative stress biomarkers after NMU with MF chemoprevention included diene conjugates-TAS (r = 0.88, p = 0.004) and TAS-GR (r = 0.89, p = 0.001) and with MEL chemoprevention included TAS-MDA (r = −0.92, p = 0.0001), diene conjugates-OMP AD (r = 0.87, p = 0.001), and OMP AD-TAS (r = −0.89, p = 0.001). No changes in food and water intake were found in comparison with the control (intact) group. Metformin and melatonin had no significant effect on parameters of mammary tumour growth (data not shown).

4 Discussion

The present study was designed to investigate the effects of the oral antidiabetic metformin and the pineal hormone melatonin, administered alone and in combination on oxidative stress and antioxidant enzyme activity in the heart tissue of female Sprague-Dawley rats. The rats were kept on a high fat diet and were subjected to the mammary carcinogenic process induced by N-methyl-N-nitrosourea. The main finding was that metformin and melatonin, in all combinations used, prevented NMU-induced toxicity and oxidative stress. A detailed comparative analysis demonstrates that melatonin was superior to metformin as antioxidant prevention in the heart tissue. Melatonin increased a total antioxidant capacity through a substantial reduction in ROS generation and in the content of oxidatively modified proteins. The antioxidant effects consisted of reductions in lipid peroxidation processes at the initial (diene conjugation) and terminal stages (TBARS estimated from MDA), protein destruction (estimated from OMP AD and OMP KD), and from the enhancement of the total antioxidant status in the heart tissue estimated from the antioxidant enzyme activity (SOD, CAT, GR, and GPx).

Data referring to free radicals and cell homeostasis suggest a coordinated activity between the enzymatic and non-enzymatic systems of ROS generation, and their removal. The literature shows a sequence of cell events in cancer cells, related to ROS generation (Tas et al. 2005). Oxidative stress promotes tumor development, but it can also be useful in the search for new therapeutic strategies for cancer treatment (Reiter et al. 2003). In breast cancer, dysregulated ROS metabolism has been observed, detected by various indicators in plasma or blood cells, including red blood cells and platelets (Sosa et al. 2013; Kedzierska et al. 2012).

The present study demonstrates that NMU-induced mammary carcinogenesis featured an enhanced oxidative stress in the heart tissue of female Sprague-Dawley rats, as estimated from increased TBARS and oxidatively modified proteins. Oxidative stress has been proposed as a key mechanism in the toxic effects of carcinogenesis in many organs of the body, including the liver, heart, and kidney (Sekkin et al. 2015). The role of two main groups of oral antidiabetics consisting of biguanides and thiazolidinediones is debated in the literature. However, due to a risk of inducing lactic acidosis, only metformin has been used in clinical practice. The oncostatic effects of metformin are reported in numerous neoplasms (Zhu et al. 2015; Anisimov 2014), including a mammary carcinogenic model in vivo (Bojková et al. 2009).

In the present study, metformin with NMU exposure and a high fat diet reduced the oxidative stress in the heart tissue of rats, which was accompanied by a decrease in diene conjugates, and an elimination of ROS products as assessed from the TAS level. These results suggest that metformin reversed the effects of NMU on the oxidative stress, notably activating the CAT and GR proteins. Metformin exerts its effects through AMP-activated protein kinase activation and a subsequent mammalian target of rapamycin (mTOR) pathway inhibition. Metformin decreases the protein synthesis and cell proliferation. AMPK activation is also reported in the effects of thiazolidinediones, yet another group of antidiabetics that improve insulin resistance in diabetic patients. Thiazolidinediones are synthetic ligands of peroxisome proliferator-activated receptors. These receptors act as transcription factors and are involved in the immune response modulation, cell proliferation, and lipid transport and accumulation (He et al. 2012). These drugs may inhibit carcinogenesis through the cell cycle arrest, induction of apoptosis, suppression of angiogenesis, and anti-inflammatory activity. They also inhibit malignant growth, including mammary cancer cells, in numerous in vitro and in vivo models. We have previously found a prominent oncostatic effect of pioglitazone in chemically-induced mammary carcinogenesis (Bojková et al. 2010); rosiglitazone exerted only a partial effect in a similar experimental model (Bojková et al. 2014).

The present findings are in line with those of Favero et al. (2015), who have shown that melatonin reduces body weight, adipose tissue depots, adipocyte hyperplasia and hypertrophy, blood glucose, pro-inflammatory factors, and restores the adipokine physiological profile. We found that melatonin suppresses the pathophysiological mechanisms underlying the influence of a high fat diet and toxic effects of NMU. Several studies support the hypothesis that melatonin, a potent antioxidant, may be of benefit in a variety of conditions, acting as an anti-inflammatory, immunomodulatory, anti-proliferative, pro-apoptotic, and anti-angiogenic compound (Carrasco et al. 2015; Esrefoglu et al. 2012; Carpentieri et al. 2012; Jou et al. 2007; Reiter et al. 2003). These properties may underlie melatonin’s oncostatic effect, first reported in the MCF-7 mammary adenocarcinoma cell line. The therapeutic effect of melatonin has been reported in many experimental neoplasms and in human cancers (Borin et al. 2016). The multifaceted effects of melatonin are mediated by increased activity of genes encoding the antioxidant SOD, CAT, GR, and GPx proteins (Okatani et al. 2000). Several authors have demonstrated the preventive effects of melatonin, administered alone or in combination with other agents, in female rat mammary carcinogenesis (Kubatka et al. 2014; Orendáš et al. 2014; Orendáš et al. 2009). The important finding of the present work is that such effects were greater than those exerted by metformin in the heart tissue in the NMU model of carcinogenesis.

Experimental findings support the oncostatic role of melatonin in the hormone-dependent mammary tumors (Borin et al. 2016). The mechanisms of the anti-tumor action of melatonin include interactions with the tumor cell estrogen-dependent pathways, cell cycle regulation, inhibited telomerase activity, modulation of fatty acid transport, and the metabolic and anti-invasive characteristics of a hormone (Viswanathan and Schernhammer 2009). Direct effects of melatonin, associated with the estrogen-dependent pathways of cellular metabolism, have been intensively studied in vitro, mainly in the cultured human breast tumor cells (MCF-7). These cells express receptors for melatonin, estrogen, and progesterone, and their growth is dependent on estrogen (Cos and Sanchez-Barcelo 2000). Estrogen-induced transcription and proliferation in the synchronized MCF-7 cells are inhibited by melatonin (Rato et al. 1999). Plausibly, cAMP and calmodulin constitute a link between the melatonin and estrogen signaling pathways. cAMP and other protein kinase activators stimulate estrogen-mediated transcription, through a mechanism involving the phosphorylation of estrogen receptors. In MCF-7 cells, estrogens activate adenylate cyclase via a non-transcriptional pathway and significantly increase the concentration of intracellular cAMP (Zivadinovic et al. 2005). In contrast, melatonin, acting through membrane receptors, inhibits adenylate cyclase and reduces the level of cAMP (Kiefer et al. 2002).

This study has some limitations related to the experimental methodology. Due to a high number of analyses, we resigned from the investigation of the effects exerted by a high fat diet alone. We believe that such evaluations are sufficiently present in the literature. Also, we believe we eliminated this factor by giving a high fat diet to all the rats investigated. This approach enabled us to obtain the data on the influence of metformin and melatonin on oxidative stress in the heart tissue of female Sprague-Dawley rats remaining on a high fat diet and subjected to the mammary carcinogenic process induced by NMU; the data that have not been hitherto reported. The evidence provided demonstrates that metformin or melatonin alone, and both combined, modulate various metabolic pathways of oxidative stress in the heart tissue. The results suggest that metformin is less effective than melatonin in reversing the effects of NMU and a high fat diet on oxidative stress. Thus, melatonin might be useful as supportive therapy in the conditions associated with elevated oxidative stress caused by a high fat diet and related carcinogenesis.

5 Conclusions

Mammary carcinogenesis induced by N-methyl-N-nitrosourea (NMU) and a high fat diet leads to increased oxidative stress by generating of ROS in the heart tissue of female Sprague-Dawley rats, as estimated from the enhanced content of TBARS, OMP (aldehyde and ketone derivatives), and antioxidant defense enzymes such as SOD, GR, and GPx. Metformin reduced the oxidative stress, which was accompanied by a decrease in diene conjugates and the elimination of ROS as indicated by a stable TAS level and the activation of CAT and GR enzymes. Administration of melatonin to the NMU-exposed rats staying on a high fat diet caused an increase in TAS through a substantial reduction in ROS generation, as estimated by OMP AD and OMP KD levels, and LPO processes in the initial (diene conjugates) and terminal stages (TBARS) and by increases in the antioxidant enzymes CAT and GPx. A combination of metformin and melatonin reversed the effect of NMU toxicity on oxidative stress. Finally, melatonin acted significantly stronger than metformin in oxidative stress reduction in the heart tissue, caused by NMU carcinogenesis and a high fat diet.