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Constant darkness conditions modulate the effects of melatonin and luzindole on the antioxidant enzyme activities and levels of retinol and a-tocopherol in rats
Svetlana Kalinina, Viktor Ilyukha, Evgeniy Khizhkin, Irina Baishnikova, Ekaterina Antonova, and Artem Morozov
Institute of Biology of the Karelian Research Centre of the Russian Academy of Sciences (IB KarRC RAS), Pushkinskaya Str., 11, Petrozavodsk, Karelia, 185610, Russian Federation
Address correspondence and requests for materials to Svetlana Kalinina, cvetnick@yandex.ru
Abstract
Citation: Kalinina, S., Ilyukha, V., Khizhkin, E., Baishnikova, I., Antonova, E., and Morozov, A. 2019. Constant darkness conditions modulate the effects of melatonin and luzindole on the antioxidant enzyme activities and levels of retinol and a-tocopherol in rats. Bio. Comm. 64(3): 211-218. https://doi.org/10.21638/ spbu03.2019.305
Author's information: Svetlana Kalinina, PhD, Head of Laboratory, orcid.org/0000-0003-1906-092X; Viktor Ilyukha, Dr. of Sci. in Biology, Director of Institute, orcid.org/0000-0002-7085-4154; Evgeniy Khizhkin, PhD, Senior Researcher, orcid.org/0000-0001-7831-8025; Irina Baishnikova, PhD, Senior Researcher, orcid.org/0000-0001-5064-3731; Ekaterina Antonova, PhD, Researcher, orcid. org/0000-0002-4740-2141; Artem Morozov, Assistant, orcid.org/0000-0001-7840-939X
Manuscript Editor: Michael Firsov, Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Saint Petersburg, Russia
Received: March 20, 2019;
Revised: June 18, 2019;
Accepted: June 21, 2019;
Copyright: © 2019 Kalinina et al. This is an open-access article distributed under the terms of the License Agreement with Saint Petersburg State University, which permits to the authors unrestricted distribution, and self-archiving free of charge.
Funding: The study was carried out under the state order (project № 0218-2019-0073).
Competing interests: The authors have declared that no competing interests exist.
This study was conducted to evaluate the effects of both exogenous melatonin and melatonin receptor antagonist luzindole on the activities of antioxidant enzymes (AOE) (superoxide dismutase, SOD; catalase, CAT) and the level of low-molecular antioxidant vitamins (retinol, a-tocopherol) in male Wistar rats kept in normal light conditions (LD 12:12) or constant darkness (DD). In LD, while melatonin had no influence on the studied antioxidants, luzindole caused an increase in retinol and a decrease in a-tocopherol contents in the liver compared to the control. In DD, with no influence on AOE activities, both drugs exerted similar effects on the liver retinol and kidney a-tocopherol contents, increasing them in comparison with control. Exposing the animals to DD induced an increase in kidney SOD activity and in liver retinol content. Moreover, DD-mel rats had higher SOD activity in the liver and kidney and a higher retinol level in the liver compared to LD-mel ones; DD-luz rats had a higher liver retinol content compared to LD-luz ones. Liver retinol level seems to be the most sensitive to influence of DD, melatonin and luzindole; the data are probably connected with the involvement of vitamin A in the regulation of circadian rhythms. Keywords: constant darkness, melatonin, luzindole, antioxidant enzymes, reti-nol, a-tocopherol.
List of abbreviations
AOE
ATRA
CAT
DD
GPx
HPLC
LD
LD (DD)-control LD (DD)-mel LD (dd)-Iuz MT1 and MT2 ROS
RZR/RORa and
NR1F2 (RZR/RORß)
SCN
SOD
U
antioxidant enzymes; all-trans-retinoic acid; catalase;
constant darkness, 24 hours dark;
glutathione peroxidase;
high performance liquid chromatography;
normal light conditions, 12 hours light : 12 hours dark;
group of rats received placebo;
group of rats received melatonin;
group of rats received luzindole;
melatonin receptors;
reactive oxygen species;
nuclear orphan receptors; suprachiasmatic nucleus; superoxide dismutase; enzyme activity unit.
Introduction
Cellular antioxidant responses are crucial for both redox signaling and redox damage. Antioxidant molecules are able to react with free radicals and oxidant species which may behave as deleterious and toxic products, involved in the dysfunction of cells and tissues (Espinoza-Diez et al., 2015). The part of the cellular defense against oxidative stress is such antioxidant enzymes (AOE) as superoxide dismutases (SOD, EC 1.15.1.1), which catalyze the dismutation of the extremely toxic superoxide radical into potentially less toxic hydrogen peroxide; catalase (CAT, EC 1.11.1.6), which stops the formation of the hy-droxyl radical by converting hydrogen peroxide to oxygen and water; and glutathione peroxidase (GPx), which metabolizes hydrogen peroxide and lipid hydroperoxides, etc. Among low molecular-weight antioxidants, the lipid-soluble substances such as retinol (one of the major forms of vitamin A) and a-tocopherol (the main and most active component of vitamin E) also act as direct scavengers of reactive oxygen species (ROS). Besides the lipoperoxyl radical-scavenging activity of retinol, it is an essential micronutrient for several biological processes, including vision, embryonic development, cell differentiation, growth and development, and regulation of the immune system (Owusu and Ross, 2016). a-Tocopherol localizes mainly at cell membranes and suppresses the formation of lipid hydroperoxides.
These enzymatic and nonenzymatic antioxidants are necessary for sustaining life by maintaining a delicate intracellular redox balance and minimizing undesirable cellular damage caused by ROS (Espinoza-Diez et al., 2015). Besides the regulation of antioxidants by the redox status of the cell, evidence for their hormonal modulation also exists, and the study of such regulation becomes very important for physiological and therapeutic approaches (Feingold, Longhurst and Colby, 1993; Bolzan, Brown, Goya and Bianchi, 1995; Mayo et al., 2002; Fernández et al., 2005).
It is well known that AOE activities exhibit an endogenous rhythm with a nighttime rise under normal light/dark (LD) conditions that is considered to be correlated with physiological melatonin levels (Benot et al., 1998; Albarrán et al., 2001; Mayo et al., 2002). It remains unclear whether circadian rhythms of retinol and a-tocopherol exist in tissues. In human blood plasma the daily changes of retinol and a-tocopherol are negligible (Nierenberg and Stukel, 1987). However, the synthesis and signaling of all-trans-retinoic acid (ATRA), an active metabolite of retinol, exhibit diurnal changes in the pineal gland (Ashton, Stoney, Ransom and McCaffery, 2018).
Melatonin (N-acetyl-5-methoxytryptamine) is the pineal hormone, which is produced and secreted with a circadian rhythm (low level during the day, or in light,
and peak values at night, or in darkness) (Tapia-Osorio, Salgado-Delgado, Angeles-Castellanos and Escobar, 2013). Chronobiotic effects of melatonin, modulation of sleep processes and influences on bone growth and osteoporosis are mediated through the interaction of mel-atonin with at least two G-protein coupled membrane-bound melatonin receptors MT1 and MT2 or indirectly with nuclear orphan receptors (namely, RZR/RORa and NR1F2 (RZR/RORP)) (Dubocovich et al., 1998; Dubo-covich, Rivera-Bermudez, Gerdin and Masana, 2003).
Besides such well-known functions of melatonin as its involvement in circadian rhythm regulation and in the modulation of a variety of neural and endocrine functions, it also acts as the most powerful antioxidant. Due to the fact that melatonin can readily pass through cell membranes and has access to all sub-cellular organelles, this indoleamine and its metabolic derivates are able to function as free radical scavengers (at high concentrations) by receptor-independent pathway (Tan et al., 2015). Also, melatonin can induce the gene expression and the activities of AOE (at physiological concentrations and under non-oxidative conditions), and this ability is receptor-mediated (Antolín et al., 1996; Kotler et al., 1998; Liu and Ng, 2000; Mayo et al., 2002) through RORa (Fang et al., 2019) and MT1 (Barberino et al., 2017). However, it is not fully elucidated whether melatonin might affect not only the enzymatic antioxidants but also the level of low molecular-weight ones such as retinol and a-tocopherol. Previously (Sergina et al., 2013) we have noted the increase in the level of both these substances in the kidney and heart of raccoon dogs (Nyctereutes procyonoides, Carnivora, Mammalia) treated with a subcutaneous continuous-release melatonin implant.
To study the receptor-mediated actions of melato-nin, the melatonin receptor antagonist luzindole (2-ben-zyl-N-acetyltryptamine) is widely used — it is considered a non-selective ligand, although it has a 15- to 25fold higher affinity for the MT2 melatonin receptor as compared to the MT1 receptor (Dubocovich et al., 1998). It was shown that luzindole blocks melatonin-mediated phase advances of circadian rhythms (Dubocovich et al., 1998), exerts antidepressant-like effects in the C3H/ HeN mouse (Dubocovich, Mogilnicka and Areso, 1990), exhibits antioxidant actions in vitro (Mathes, Wolf and Rensing, 2008) and reduces the level of lipid peroxida-tion in vitro (Requintina and Oxenkrug, 2007). Moreover, luzindole does not block the antioxidant effects of melatonin (Behan, McDonald, Darlington and Stone, 1999). It remains unclear whether luzindole acts as an antioxidant in vivo and whether it influences the level of antioxidants.
Although it is generally known that constant conditions (light or darkness) strongly influence circadian rhythmicity and melatonin secretion (Tapia-Osorio, Salgado-Delgado, Angeles-Castellanos and Escobar,
2013), little information is available concerning changes in antioxidant level of mammals following darkness exposure (Delibas, Tuzmen, Yonden and Altuntas, 2002; Pang et al., 2008). Moreover, it is not known whether the altered lighting conditions — for example, constant darkness (DD) — can affect the potent antioxidant effects of melatonin and luzindole in vivo. Exposure of mammals to continuous light (LL) or DD chronically attenuates internal 24-hr rhythms, leading to detrimental effects on multiple biological processes. In DD the circa-dian rhythm of melatonin persists, but it is determined to be free-running as a result of a steady expansion of its secretion durations, while LL rats show an arryhtmic temporal pattern (Tapia-Osorio, Salgado-Delgado, Angeles-Castellanos and Escobar, 2013). In LD 12:12 conditions the melatonin receptor density in the suprachi-asmatic nucleus (SCN) was shown to be specifically reduced during the night, while in DD it did not show any variation throughout the 24 h subjective day and night (Gauer, Masson-Pevet, Stehle and Pevet, 1994).
The aim of this study was to evaluate the effects of both exogenous melatonin and melatonin receptor antagonist luzindole on the activities of AOE (SOD, CAT) as well as retinol and a-tocopherol levels in rats exposed to LD 12:12 or DD conditions.
Materials and methods
ETHICAL PROCEDURES
The research was carried out using the equipment of the Core Facility of the Karelian Research Centre of the Russian Academy of Sciences and according to EU Directive 2010/63/EU for animal experiments with the special permission of the Local Ethics Committee of the Institute of Biology. All efforts were made to minimize the number of animals and their suffering.
SUBJECTS AND EXPERIMENTAL DESIGN
Seven-month-old male Wistar rats weighing 450-500 g were housed at constant temperature (23 ± 1 °C) with laboratory food and water ad libitum. Animals were synchronized for 14 days to a 12:12 light/dark cycle (LD, lights on at 07:00, light intensity of 200 lx). After that, a portion of the rats was kept under LD conditions, while another portion was exposed to constant darkness (DD) beginning at the time of lights off for 14 days. Then, rats kept under either LD or DD conditions were randomly divided into three groups (n=3-6 in each group): LD (DD)-control (received placebo); LD (DD)-mel (received melatonin), and LD (DD)-luz (received luz-indole). After 14 days the rats were terminated by decapitation at 10.00 after anesthesia with ether. All treatments of the DD rats were performed under a dim red light (640-700 nm, <2 lx) within 3 min.
DRUGS AND DOSES
The drugs used were melatonin (N-acetyl-5-methoxy-tryptamine) (Sigma, USA) and melatonin receptor antagonist luzindole (2-benzyl-N-acetyltryptamine) (Bachem AG, Switzerland). For each drug, 10 mg were dissolved in a minimal amount of 95 % ethanol and further diluted to 100 ml distilled water to yield stock solution, which was then diluted in 1 liter tap water (final ethanol concentration 0.1 %). The dose of both melatonin and luzindole was 0.22 mg/kg BW of rat. Control rats received tap water containing 0.0001 % ethanol. All drugs were given to the rats with drinking water at night for 14 days. The last administration of drugs was given the day before death.
TISSUE COLLECTION
After decapitation, the liver and kidney samples were immediately placed on ice for transportation to the laboratory, where samples were stored at -80 °C awaiting analyses. All samples were analyzed in triplicate.
DETERMINATION OF ANTIOXIDANT ENZYMES ACTIVITIES AND CONTENTS OF RETINOL AND A-TOCOPHEROL
Antioxidant enzyme activities
For determination of AOE activities, tissue samples were homogenized in 0.05 M phosphate buffer, pH 7.0, and centrifuged at 6000 x g for 15 min.
Superoxide dismutase (EC 1.15.1.1)
The total SOD activity was measured by the adreno-chrome method based on the spontaneous autoxidation of epinephrine with the formation of product with an absorbance peak at 480 nm (Misra and Fridovich, 1972). This reaction depends on the presence of superoxide anions and is specifically inhibited by SOD. The amount of enzyme that caused 50 % inhibition of epinephrine au-toxidation is defined as 1 unit (U). SOD activity was expressed as U per mg protein after normalization with estimated total protein in milligrams in the respective tissues.
Catalase (EC 1.11.1.6)
CAT activity was evaluated by measuring the decrease in H2O2 concentration at 240 nm (Bears and Sizes, 1952). One enzyme unit (U) is defined as the amount of CAT capable of transforming 1.0 ^mol of H2O2 for a minute. CAT activity was expressed as U per mg protein after normalization with estimated total protein in milligrams in the respective tissues.
Total protein assay
Results for AOE activities were standardized to total soluble protein content in tissue homogenates. Total tissue
Fig. 1. The activity of SOD in both melatonin- and luzindole-treated rats, exposed to either normal light conditions 12:12 (LD) or constant darkness (DD). Results (in U/ mg protein) are expressed as mean ± SEM. Significant difference with: ◊ LD-control, * LD-mel rats (p<0.05, Kruskall-Wallis test).
Fig. 2. The activity of CAT in both melatonin- and luzindole-treated rats, exposed to either normal light conditions 12:12 (LD) or constant darkness (DD). Results (in U/ mg protein) are expressed as mean ± SEM.* Significant difference with LD-mel rats (p<0.05, Kruskall-Wallis test).
Fig. 3. The retinol content in both melatonin- and luzindole-treated rats, exposed to either normal light conditions 12:12 (LD) or constant darkness (DD). Results (in pg/g wet tissue) are expressed as mean ± SEM. Significant difference with: ◊ LD-control, ♦ DD-control, * LD-mel, « LD-luz rats (p<0.05, Kruskall-Wallis test).
Fig. 4. The a-tocopherol content in both melatonin- and luzindole-treated rats, exposed to either normal light conditions 12:12 (LD) or constant darkness (DD). Results (in pg/g wet tissue) are expressed as mean ± SEM.Significant difference with: ◊ LD-control, ♦ DD-control rats (p<0.05, Kruskall-Wallis test).
protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.
Retinol and a-tocopherol levels
The contents of retinol and a-tocopherol in the tissues were determined by HPLC. Proteins in the samples were precipitated by ethanol. Retinol and a-tocopherol were extracted by n-hexane. Chromatographic separation was carried out by microcolumn chromatography with a UV detector with n-hexane and isopropanol as an elu-ent (98.5:1.5). The eluate was monitored at 292 nm for a-tocopherol and 324 nm for retinol, and the substances were identified by retention time compared with pure standards (MP Biomedicals, USA).
STATISTICAL ANALYSIS
All the collected and calculated numerical data were transformed into SI units and processed statistically as mean ± standard error of the mean. Statistical analysis was done using the Kruskal-Wallis analysis of variance with the Mann-Whitney U post hoc testing as appropriate. A p-value of <0.05 was taken as significant. Relationships between light conditions, drugs and studied values were analyzed by means of the Pearson's and Spearman's correlation coefficients. The statistical analyses were performed using Sigma-Stat 2.03 (SPSS Science Software Ltd., USA), while figures were prepared using Grapher 7.0 (Golden Software Inc., USA).
Results
INFLUENCE OF DD CONDITIONS ON STUDIED PARAMETERS
Results of the study are presented in Figures 1-4. DD-control rats had higher kidney SOD activity (Fig. 1) and higher liver retinol content (Fig. 3) than LD-control ones. No significant differences in liver SOD activity, in both liver and kidney CAT activities, in kidney retinol level and in both liver and kidney a-tocopherol contents were found between LD- and DD-control groups (Figs. 1-4).
INFLUENCE OF BOTH MELATONIN AND LUZINDOLE ON STUDIED PARAMETERS IN RATS EXPOSED TO LD CONDITIONS
No influence of melatonin on studied antioxidants in LD-rats was observed in our study (Figs. 1-4). Treatment of the LD-rats with luzindole had no effect on AOE activities but caused an increase in retinol and a decrease in a-tocopherol contents in the liver in comparison with the control (Figs. 3, 4). Also LD-luz rats had higher kidney AOE activities and higher liver retinol content than LD-mel ones (Figs. 1-3).
INFLUENCE OF BOTH MELATONIN AND LUZINDOLE ON STUDIED PARAMETERS IN RATS EXPOSED TO DD CONDITIONS
In DD-rats, along with having no effect on AOE activities, both melatonin and luzindole induced an increase in the liver retinol content and in the kidney a-tocopherol level compared with DD-control (Figs. 1-4).
INFLUENCE OF DD CONDITIONS ON STUDIED PARAMETERS IN RATS TREATED WITH MELATONIN AND LUZINDOLE
DD-mel rats had higher SOD activities in the liver and kidney and a higher retinol level in the liver compared to LD-mel ones (Figs. 1, 3). DD-luz rats had a higher liver retinol content compared to LD-luz ones (Fig. 3).
CORRELATIONS
Table 1 shows correlation coefficients between light conditions, drugs, and studied parameters. Only significant correlations are presented. The results are mostly similar for the Pearson and Spearman coefficients. Most coefficients of correlation were positive. The highest correlation (positive) occurred between liver retinol content and light conditions (Pearson: r=0.88; Spearman: r=0.84; p=0.000). No correlations were observed between the drugs and AOE activity, nor between either light conditions or drug and CAT activity.
Table 1. Correlations and between light conditions,
significance levels (p) in rats drugs, and studied values
Parameter correlation Pearson Spearman
r p r p
Liver retinol level — light conditions 0.88 0.000 0.84 0.000
Liver retinol level — liver SOD activity 0.62 0.001 0.56 0.007
Liver SOD activity — liver CAT activity 0.53 0.008 0.46 0.027
Liver SOD activity — light conditions 0.62 0.001 0.62 0.003
Kidney SOD activity — light conditions 0.56 0.005 0.52 0.013
Kidney a-tocopherol level — drug 0.60 0.002 0.61 0.003
Liver a-tocopherol level — drug -0.43 0.036 -0.44 0.034
Kidney retinol level — kidney a-tocopherol level 0.49 0.016
Liver retinol level — liver a-tocopherol level -0.43 0.034
Kidney retinol level — light conditions 0.42 0.042
Liver retinol level — kidney a-tocopherol level 0.42 0.047
Discussion
The significance of melatonin and melatonin receptor antagonist luzindole in the regulation of AOE activities and retinol and a-tocopherol levels in Wistar rats was evaluated through studies on the effects of exogenously administered drugs during rat exposure to normal light conditions (LD 12:12) or constant darkness (DD).
In DD conditions the clock gene expression in the SCN does not show a 24-h rhythmicity because the clock is not entrained to the environmental lighting cycle (Okamura et al., 1999). Although a circadian rhythm of melatonin with higher values during the subjective night and lower values during the subjective day persists in DD, a smoothing of the melatonin secretion rhythm per day is observed—the day portion slightly increases and the night portion slightly decreases in comparison with the LD regime, but the total concentration of the hormone per day in DD does not exceed that in LD (Tapia-Osorio, Salgado-Delgado, Angeles-Castellanos and Escobar, 2013).
Little information is available concerning the levels of tissue antioxidants following darkness exposure
(Delibas, Tuzmen, Yonden and Altuntas, 2002; Pang et al., 2008). It is well known that under normal LD conditions the AOE activities exhibit endogenous rhythms with a nighttime rise that is considered to be correlated with physiological melatonin levels (Benot et al., 1998; Albarran et al., 2001; Mayo et al., 2002). In DD the cir-cadian rhythm of melatonin is determined to be free-running, therefore in DD conditions, circadian rhythms of AOE activities could differ from those in a normal LD cycle. We have noted the higher kidney SOD activity in DD-control rats compared to LD-control ones. This is in accordance with earlier observations by Delibas N. et al. (2002), the increase in SOD and GPx activities in the hippocampus of rats exposed to DD in comparison with control ones.
Although the SOD activity was shown to be higher in the kidneys of DD-control rats in comparison with LD-control ones, the activity of another studied enzyme — CAT — did not change. The balance between the activities of SOD and CAT is necessary to provide adequate antioxidant protection. The increase in SOD activity, which dismutates the superoxide anion, causes an increase in the hydrogen peroxide concentration, which if not sufficiently removed by CAT or GPx (not determined in our study) will produce hydroxyl radicals that are highly toxic for the cell (Antolin et al., 1996). However, although the imbalance between the antioxi-dant enzymes may cause cell injury (Toborek, Kopiecz-na-Grzebieniak, Drozdz and Wieczorek, 1995), the protective role of SOD is widely accepted even when other AOEs do not change (Warner et al., 1993). Moreover, SOD seems to be more sensitive to hormonal changes (including changes in melatonin level) than CAT or GPx (Kotler et al., 1998; Barp et al., 2002).
Previous studies have suggested that melatonin could induce the gene expression and the activity of AOEs such as SOD, CAT and GPx, and this effect is considered to be receptor-mediated through MT1 (Barber-ino et al., 2017) and RORa (Fang et al., 2019). However, in our study we did not find a statistically significant effect of exogenous melatonin on AOE activities in LD-rats, but DD-mel rats exhibited higher SOD activities in the liver and kidney compared to LD-mel ones. This illustrates that changes in physiological levels of mela-tonin are adequate to alter this component of the tissue antioxidative defense. Probably it could be due to alteration in circadian rhythms of melatonin and therefore of SOD activity in DD conditions.
We also detected a higher liver retinol content in DD-control rats compared to LD-control ones. The liver is known to play an important role in vitamin A ho-meostasis, because in this organ retinol can follow several metabolic pathways, including oxidation to ATRA (Wolf, 2001). When the plasma concentration of ATRA is increased, there is an accompanying reduction of cir-
culating levels of retinol, suggesting that ATRA may have a regulatory effect on retinol metabolism in vivo (Wang, Krinsky and Russell, 1993). The DD conditions may influence the homeostasis of vitamin A by decreasing circulatory levels of ATRA, as was shown in mice (Pang et al., 2008) and increasing liver retinol content, as was observed in rats in our study.
Although data on the melatonin influence on the levels of retinol and a-tocopherol in tissues are lacking in the literature, the protective effect of melatonin against oxidative stress related to restoration of retinol availability was shown in rats treated with melatonin after heroin administration (Cemek et al., 2011). Besides that, melatonin at various concentrations combined with a-tocopherol decreases lipid peroxidation in human placental mitochondria with the effect being additive (Milczarek et al., 2010). In our study, in contrast to LD conditions, where there was no effect of melatonin on the retinol and a-tocopherol contents, in DD mela-tonin induced an increase in liver retinol content and in the kidney a-tocopherol level compared with the DD-control. Also, we have noted a higher liver retinol content in DD-mel rats compared to LD-mel animals. The increase in kidney vitamin E content after mela-tonin administration observed in our study may be a result of a-tocopherol mobilization from adipose and other tissues as a consequence of antioxidant metabolic adaptation of the organism. Moreover, melatonin may recycle several oxidized antioxidants including vitamin C, a-tocopherol, glutathione, and NADH, and these an-tioxidants reportedly also recycle the melatonin neutral radical (Tan et al., 2015).
Most studies of luzindole action in the organism revolve around it being a melatonin receptor antagonist, but others have indicated that luzindole has a function of its own — for example, protection of rat retinal pho-toreceptors from light damage (Sugawara et al., 1998). Moreover, luzindole's antioxidant effect is considered to exceed that of melatonin (Oxenkrug and Requintina, 2005). Unfortunately, data on the antioxidant actions of luzindole are scarce in the literature and have been revealed only in vitro (Requintina and Oxenkrug, 2007; Mathes, Wolf and Rensing, 2008), therefore it remains unclear whether this substance acts as an antioxidant in vivo. It was shown that luzindole does not suppress the antioxidant effects of melatonin (Behan, McDonald, Darlington and Stone, 1999) and in a model of melatonin plus luzindole co-administration, radical scavenging may be far more intense than with melatonin alone, but in the experiments where luzindole is usually given without melatonin, results may be influenced by its an-tioxidant properties (Mathes, Wolf and Rensing, 2008).
As in the case of melatonin, luzindole administration to LD- and DD-rats had no effect on AOE activities in comparison with corresponding controls. How-
ever, in LD conditions this drug caused an increase in retinol and a decrease in a-tocopherol contents in the liver compared to the control group. According to the antagonism between retinol and a-tocopherol reported previously in the literature (Olivares, Rey, Daza and Lopez-Bote, 2009), this result would be expected. In DD conditions luzindole induced an increase in liver reti-nol content and in the kidney a-tocopherol level compared to the control. Although no significant differences in studied values were found between melatonin- and luzindole-treated rats in DD, in LD conditions the luz-indole-treated rats had higher kidney AOE activities and a higher liver retinol level than the melatonin-treated ones. According to recently published data (Barberino et al., 2017; Fang et al., 2019), it could be hypothesized that MT2 blocking by luzindole can increase the affinity of melatonin to MT1 and potentiate its antioxidant actions.
In conclusion, our results indicate that DD exerts influence only on the kidney SOD activity and the liver retinol level and in several cases significantly modulates the effects of exogenous melatonin and melatonin receptor antagonist luzindole on the studied antioxidants mainly by increasing their activity or content. In the case of melatonin, these parameters are the liver and kidney SOD activities and liver retinol, and in the case of luz-indole it is liver retinol content. Probably due to the important role of ATRA in regulation of circadian rhythms (Pang et al., 2008; Sherman et al., 2012), the liver retinol level seems to be the most sensitive to the influence of DD, melatonin and luzindole. The mechanisms of effects of these factors on studied parameters need to be further investigated.
Acknowledgements
The authors are extremely grateful to our colleagues from the Institute of Biology, Karelian Research Centre of Russian Academy of Sciences (Petrozavodsk, Russia) for their assistance in this study.
References
Albarran, M.T., Lopez-Burillo, S., Pablos, M. I., Reiter, R.J., and Agapito, M.T. 2001. Endogenous rhythms of melatonin, total antioxidant status and superoxide dismutase activity in several tissues of chick and their inhibition by light. Journal of Pineal Research 30:227-233. https://doi. org/10.1034/j.1600-079X.2001.300406.x Antolin, I., Rodriguez, C., Sainz, R.M., Mayo, J.C., Uria, H., Kotler, M.L., Rodriguez-Colunga, M.J., Tolivia, D., and Menendez-Pelaez, A. 1996. Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes. The FASEB Journal 10(8):882-890. https://doi.org/10.1096/fasebj.10.8.8666165 Ashton, A., Stoney, P. N., Ransom, J., and McCaffery, P. 2018. Rhythmic diurnal synthesis and signaling of retinoic acid in the rat pineal gland and its action to rapidly down-regulate ERK phosphorylation. Molecular Neurobiology 55(11):8219-8235. https://doi.org/10.1007/s12035-018-0964-5
Barberino, R. S., Menezes, V. G., Ribeiro, A. E., Palheta Jr, R. C., Jiang, X., Smitz, J.E., and Matos, M.H.T. 2017. Melatonin protects against cisplatin-induced ovarian damage in mice via the MT1 receptor and antioxidant activity. Biology of Reproduction 96(6):1244-1255. https://doi. org/10.1093/biolre/iox053 Barp, J., Araújo, A.S.D.R., Fernandes, T.R.G., Rigatto, K.V., Llesuy, S., Belló-Klein, A., and Singal, P. 2002. Myocardial antioxidant and oxidative stress changes due to sex hormones. Brazilian Journal of Medical and Biological Research 35(9):1075-1081. https://doi.org/10.1590/S0100-879X2002000900008 Bears, R. F. and Sizes, I. N. 1952. A spectral method for measuring the breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry 195(1):133-140. Behan, W.M.H., McDonald, M., Darlington, L.G., and Stone T.W. 1999. Oxidative stress as a mechanism for quinolinic acid-induced hippocampal damage: protection by melatonin and deprenyl. British Journal of Pharmacology 128(8):1754-1760. https://doi.org/10.1038/ sj.bjp.0702940
Benot, S., Molinero, P., Soutto, M., Goberna, R., and Guerrero, J.M. 1998. Circadian variations in the rat serum total antioxidant status: correlation with melatonin levels. Journal of Pineal Research 25:1-4. https://doi. org/10.1111/j.1600-079X.1998.tb00378.x Bolzan, A., Brown, O., Goya, R., and Bianchi, M. 1995. Hormonal modulation of antioxidant enzyme activities in young and old rats. Experimental Gerontology 30:169175. https://doi.org/10.1016/0531-5565(94)00053-0 Cemek, M., Büyükokuroglu, M.E., Hazman, O., Konuk, M., Bulut, S., and Birdane, Y.O. 2011. The roles of melatonin and vitamin E plus selenium in prevention of oxidative stress induced by naloxone-precipitated withdrawal in heroin-addicted rats. Biological Trace Element Research 142(1):55-66. https://doi.org/10.1007/s12011-010-8744-8
Delibas, N., Tuzmen, N., Yonden, Z., and Altuntas, I. 2002. Effect of functional pinealectomy on hippocampal lipid peroxidation, antioxidant enzymes and N-methyl-D-as-partate receptor subunits 2A and 2B in young and old rats. Neuroendocrinology Letters 23(4):345-350. Dubocovich, M. L., Mogilnicka, E., and Areso, P. M. 1990. An-tidepressant-like activity of the melatonin receptor antagonist, luzindole (N-0774), in the mouse behavioral despair test. European Journal of Pharmacology 182(2):313-325. https://doi.org/10.1016/0014-2999(90)90290-M Dubocovich, M. L., Rivera-Bermudez, M.A., Gerdin, M.J., and Masana, M. I. 2003. Molecular pharmacology, regulation and function of mammalian melatonin receptors. Frontiers in Bioscience 8:d1093-108. https://doi. org/10.2741/1089 Dubocovich, M. L., Yun, K., Al-Ghoul, W. M., Benloucif, S., and Masana, M.I. 1998. Selective MT2 melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms. The FASEB Journal 12:1211-1220. https://doi.org/10.1096/fasebj.12.12.1211 Espinosa-Diez, C., Miguel, V., Mennerich, D., Kietzmann, T., Sánchez-Pérez, P., Cadenas, S., and Lamas S. 2015. Antioxidant responses and cellular adjustments to oxidative stress. Redox Biology 6:183-197. https://doi. org/10.1016/j.redox.2015.07.008 Fang, Y., Zhang, J., Li, Y., Guo, X., Li, J., Zhong, R., and Zhang, X. 2019. Melatonin-induced demethylation of antioxidant genes increases antioxidant capacity through RORa in cumulus cells of prepubertal lambs. Free Radical Biology and Medicine 131:173-183. https://doi.org/10.1016Zj.fre-eradbiomed.2018.11.027
Feingold, I.B., Longhurst, P.A., and Colby, H.D. 1993. Regulation of adrenal and hepatic a-tocopherol content by androgens and estrogens. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1176(1-2):192-196. https:// doi.org/10.1016/0167-4889(93)90196-V Fernández, V., Tapia, G., Varela, P., Castillo, I., Mora, C., Moya, F., Orellana, M., and Videla, L.A. 2005. Redox up-regulated expression of rat liver manganese superoxide dismutase and Bcl-2 by thyroid hormone is associated with inhibitor of KB-a phosphorylation and nuclear factor-KB activation. Journal of Endocrinology 186(3):539-547. https://doi.org/10.1677/joe.1.06261 Gauer, F., Masson-Pevet, M., Stehle, J., and Pevet P. 1994. Daily variations in melatonin receptor density of rat pars tuberalis and suprachiasmatic nuclei are distinctly regulated. Brain Research 641(1):92-98. https://doi. org/10.1016/0006-8993(94)91819-8 Kotler, M., Rodríguez, C., Sáinz, R. M., Antolín, I., and Menén-dez-Peláez, A. 1998. Melatonin increases gene expression for antioxidant enzymes in rat brain cortex. Journal of Pineal Research 24(2):83-89. https://doi.org/10.1111/ j.1600-079X.1998.tb00371.x Liu, F. and Ng, T.B. 2000. Effect of pineal indoles on activities of the antioxidant defense enzymes superoxide dismutase, catalase and glutathione reductase, and levels of reduced and oxidized glutathione in rat tissues. Biochemistry and Cell Biology 78:447-453. https://doi. org/10.1139/o00-018 Lowry, O. H., Rosenbrough, N.J., Farr, A. L., and Randan, R.J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193(1):265-275. Mathes, A. M., Wolf, B., and Rensing, H. 2008. Melatonin receptor antagonist luzindole is a powerful radical scavenger in vitro. Journal of Pineal Research 45:337-338. https://doi.org/10.1111/j.1600-079X.2008.00583.x Mayo, J. C., Sainz, R. M., Antolín, I., Herrera, F., Martin, V., and Rodriguez, C. 2002. Melatonin regulation of antioxidant enzyme gene expression. Cellular and Molecular Life Sciences (CMLS) 59(10):1706-1713. https://doi.org/10.1007/ PL00012498
Milczarek, R., Hallmann, A., Sokotowska, E., Kaletha, K., and Klimek, J. 2010. Melatonin enhances antioxidant action of a-tocopherol and ascorbate against NADPH- and iron-dependent lipid peroxidation in human placental mitochondria. Journal of Pineal Research 49(2):149-155. https://doi.org/10.1111/j.1600-079X.2010.00779.x Misra, H. P. and Fridovich, F. 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. Journal of Biological Chemistry 247(10):3170-3175. Nierenberg, D.W., and Stukel, T. 1987. Diurnal variation in plasma levels of retinol, tocopherol, and p-carotene. The American Journal of the Medical Sciences 294(3):187-190. https://doi.org/10.1097/00000441-198709000-00010 Okamura, H., Miyake, S., Sumi, Y., Yamaguchi, S., Ya-sui, A., Muijtjens, M., Hoeijmakers, J.H., and van der Horst, G.T. 1999. Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock. Science 286(5449):2531-2534. https://doi.org/10.1126/sci-ence.286.5449.2531 Olivares, A., Rey, A. I., Daza, A., and Lopez-Bote, C.J. 2009. High dietary vitamin A interferes with tissue a-tocopherol concentrations in fattening pigs: a study that examines administration and withdrawal times. Animal 3(9):1264-1270. https://doi.org/10.1017/S175173110900487X
Owusu, S. A. and Ross, A. C. 2016. Retinoid homeostatic gene expression in liver, lung and kidney: ontogeny and response to vitamin A — retinoic acid (VARA) supplementation from birth to adult age. PLoS ONE 11(1):e0145924. https://doi.org/10.1371/journal.pone.0145924 Oxenkrug, G.F. and Requintina, P.J. 2005. N-acetyldo-pamine inhibits rat brain lipid peroxidation induced by lipopolysaccharide. Annals of the New York Academy of Sciences 1053(1):394-399. https://doi. org/10.1111/j.1749-6632.2005.tb00047.x Pang, W., Li, C., Zhao, Y., Wang, S., Dong, W., Jiang, P., and Zhang, J. 2008. The environmental light influences the circulatory levels of retinoic acid and associates with hepatic lipid metabolism. Endocrinology 149(12):6336-6342. https://doi.org/10.1210/en.2008-0562 Requintina, P.J. and Oxenkrug, G. F. 2007. Effect of luzindole and other melatonin receptor antagonists on iron- and lipopolysaccharide-induced lipid peroxidation in vitro. Annals of the New York Academy of Sciences 1122(1):289-294. https://doi.org/10.1196/annals.1403.021 Sergina, S., Baishnikova, I., Ilyukha, V., Lis, M., Lapinski, S., Niedbala, P., and Barabasz, B. 2013. Comparison of the antioxidant system response to melatonin implant in raccoon dog (Nyctereutes procyonoides) and silver fox (Vulpes vulpes). Turkish Journal of Veterinary and Animal Sciences 37(6):641-646. https://doi.org/10.3906/vet-1302-48 Sherman, H., Gutman, R., Chapnik, N., Meylan, J., le Coutre, J., and Froy, O. 2012. All-trans retinoic acid modifies the expression of clock and disease marker genes. The Journal of Nutritional Biochemistry 23(3):209-217. https://doi. org/10.1016/j.jnutbio.2010.11.017 Sugawara, T., Sieving, P.A., Iuvone, P.M., and Bush, R.A. 1998. The melatonin antagonist luzindole protects retinal photoreceptors from light damage in the rat. Investigative Ophthalmology & Visual Science 39(12):2458-2465. Tan, D.X., Manchester, L. C., Esteban-Zubero, E., Zhou, Z., and Reiter, R.J. 2015. Melatonin as a potent and inducible endogenous antioxidant: synthesis and metabolism. Molecules 20(10):18886-18906. https://doi.org/10.3390/ molecules201018886 Tapia-Osorio, A., Salgado-Delgado, R., Angeles-Castellanos, M., and Escobar, C. 2013. Disruption of circadian rhythms due to chronic constant light leads to depressive and anxiety-like behaviors in the rat. Behavioural Brain Research 252(1):1-9. https://doi.org/10.1016Zj.bbr.2013.05.028 Toborek, M., Kopieczna-Grzebieniak, E., Drózdz, M., and Wieczorek, M. 1995. Increased lipid peroxidation as a mechanism of methionine-induced atherosclerosis in rabbits. Atherosclerosis 1 15(2):217-224. https://doi. org/10.1016/0021-9150(94)05516-L Wang, X. D., Krinsky, N. I., and Russell, R. M. 1993. Retinoic acid regulates retinol metabolism via feedback inhibition of retinol oxidation and stimulation of retinol esteriflcation in ferret liver. The Journal of Nutrition 123(7):1277-1285. https://doi.org/10.1093/jn/123.7J277 Warner, B., Papas, F.T., Heile, K., Spitz, D., and Wispe, J. 1993. Expression of human Mn-SOD in Chinese hamster ovary cells confers protection from oxidant injury. American Journal of Physiology-Lung Cellular and Molecular Physiology 264:L598-L605. https://doi.org/10.1152/ ajplung.1993.264.6.L598 Wolf, G. 2001. Retinoic acid homeostasis: retinoic acid regulates liver retinol esteriflcation as well as its own cata-bolic oxidation in liver. Nutrition Reviews 59(12):391-394. https://doi.org/10.1111/j.1753-4887.2001.tb06968.x
