Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
Toxicology
Protective effect of omega-3 fatty acid against mercury chloride intoxication in mice Mahmut Karapehlivan a , Metin Ogun a , Inan Kaya b,∗ , Hasan Ozen c , Haci Ahmet Deveci d , Musa Karaman c a
Department of Biochemistry, Faculty of Veterinary Medicine, Kafkas University, 36100 Kars, Turkey Department of Molecular Biology and Genetic, Faculty of Arts and Science, Kafkas University, 36100 Kars, Turkey c Department of Pathology, Faculty of Veterinary Medicine, Kafkas University, 36100 Kars, Turkey d Atatürk Health Services Vocational School, Kafkas University, 36100 Kars, Turkey b
a r t i c l e
i n f o
Article history: Received 4 June 2013 Received in revised form 3 August 2013 Accepted 30 August 2013 Keywords: Mercury chloride Omega-3 fatty acid Sialic acid Malondialdehyde Nitric oxide
a b s t r a c t The aim of this study was to investigate the protective effect of omega-3 fatty acid in HgCI2 toxicity in mice. Levels of malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO) and total sialic acid (TSA), and histopathological changes in selected organs were evaluated. Twenty-eight mice were equally divided into 4 groups, namely Groups I–IV. Group I animals received intraperitoneal (ip) injection of physiological saline solution; Group II animals received ip injection of 0.4 mg/kg/day HgCI2 ; Group III animals received ip injection of 0.4 mg/kg/day HgCI2 in addition to subcutaneous (sc) injection of 0.5 g/kg/day omega-3 fatty acid; and Group IV animals received sc injection of 0.5 g/kg/day omega-3 fatty acid. All treatments lasted 7 days. The levels of MDA, NO and TSA were significantly higher in Group II and lower in Groups III and IV as compared to the Group I. GSH level was the highest in Group IV. In histopathology, severe degeneration in liver and kidney was observed in Group II animals. These degrading changes were seen to be reduced greatly in Group III animals. The results suggested that omega-3 fatty acid might attenuate HgCI2 -induced toxicity by improving antioxidant status and acute phase response in mice. © 2013 Elsevier GmbH. All rights reserved.
Introduction Mercury is a well-known toxic heavy metal to animals as well as humans. It is widespread and distributed throughout the environment by countless processes such as volcanic activities, water movements and various biological activities. Industrial processes such as mining activities, combustion of fossil fuels, pesticides and medical wastes have also become significant contributors to the environmental distribution of mercury and its compounds [1,2]. Mercury causes various metabolic changes as a result of its toxic effects especially in central nervous system, kidney and liver tissues of animals. It manifests its toxicity by interacting with reactive oxygen species (ROS) and binding to thiol groups in several proteins such as glutathione (GSH) and many of the antioxidant enzymes [3]. High levels of ROS, which indicates the oxidative stress, are known to induce lipid peroxidation that can be further shown by increased
∗ Corresponding author. Tel.: +90 474 225 1150; fax: +90 474 225 1179. E-mail address: inankaya @hotmail.com (I. Kaya). 0946-672X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jtemb.2013.08.004
malondialdehyde (MDA) levels [4,5]. Nitric oxide (NO) is involved in various biological processes such as vasodilatation, neurotransmission and leukocyte mediated killing of pathogens. Moreover, it is involved in the process of peroxynitrite anion (ONOO− ) production reacting with peroxides, which further yields lipid peroxidation [6]. Total sialic acid (TSA) can be used as an indicator of tissue damage. Sialic acids have many important physiological and pathological functions such as cellular transmission, embryogenesis, organ development, immune system regulations, leucodiapedesis, metastasis of neoplastic cells, and carrying out membrane receptor functions [7–10]. The omega-3 fatty acids are polyunsaturated fatty acids and commonly found in marine oils. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) compose of the major parts of omega3 fatty acids. In recent years, it has been reported that consumption of long-chain omega-3 fatty acids may have beneficial effects in many health problems such as cardiovascular, neuronal and gastrointestinal diseases related particularly to electrical signal modulation [11,12]. In the present study, potential protective effect of omega-3 fatty acids in HgCI2 toxicity in mice was investigated by biochemical and histopathological means.
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
95
Materials and methods Experimental design The ethical approval of the study was confirmed by I˙ nönü University Animal Care and Use Committee (Registration Number: 2013/A-41). All procedures were conducted in accordance with the ‘Guide for Care and Use of Laboratory Animals’ published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain. Experiments were carried out on 28 male Swiss albino mice each weighing 29–34 g at 14–16 weeks of age. Animals were divided into 4 equal groups namely Group I through Group IV and housed in a room maintained at 18 ± 1 ◦ C with an alternating 12 h light-dark cycle. Food and water were provided ad libitum. All experimental injections were carried out for 7 days and at the same hours of the light cycles of the study. Treatments were performed as follow; Group I: ip injection of physiological saline solution, Group II: ip injection of 0.4 mg/kg/day HgCI2 . Group III: ip injection of 0.4 mg/kg/day HgCI2 plus sc injection of 0.5 g/kg/day omega-3 fatty acid, and Group IV: sc injection of 0.5 g/kg/day omega-3 fatty acid.
Biochemical analysis At the end of the 7th day, blood samples were collected into EDTA tubes from the hearts via cardiac puncture under ether anesthesia for GSH, MDA and NO measurements. The blood samples were centrifuged (1200 × g, 4 ◦ C) for 10 min, and the plasma were obtained and then kept at −25 ◦ C until the analyzes were carried out. At necropsy liver, kidney and brain tissues were collected to determine tissue GSH, MDA, NO and TSA concentrations. For this purpose, the tissues were rinsed with ice-cold 0.9% NaCl. Then, 1 g of tissue was homogenized in phosphate buffer (pH 7.4) in an ice bath and the homogenates were centrifuged (1200 × g, 4 ◦ C) for 15 min. TSA was measured calorimetrically using a spectrophotometer (UV-1201, Shimadzu, Japan) by the method of Sydow [13] in that all bound sialic acid were separated by acid per-chloride in plasma and tissue homogenates, and then the supernatants were boiled by Erlich reagent, and finally the product was read at 525 nm. GSH concentration was assayed by the method of Beutler et al. [14] based on the spectrophotometric measurement of sulphydril ( SH) groups forming complexes with 5,5 -(2-dithiobis nitrobenzoic acid) which give rise to colored products which were read at 412 nm. Measurement of MDA concentrations was carried out by the method of Yoshoiko et al. [15] based on the reaction between thiobarbituric acid and MDA produced as an end product of lipid peroxidation. The end products were read at 535 nm. NO levels were determined according to the method described by Miranda et al. [16] in that nitrate is reduced to nitrite by VaCl3 , and then in acidic environment nitrite was reacted with sulphanilamide to produce colored diazonium compound, which was read at 540 nm.
Fig. 1. Levels of the liver, kidney and brain TSA in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
Statistical analysis SPSS software (Windows version 20.0) was used for statistical evaluation of data which were expressed as median (X) ± standard deviation (SD). Importance level of difference among the groups was determined by variance analysis (ANOVA) and Duncan multiple comparison test. Results TSA, MDA, GSH and NO levels The results of TSA, MDA, GSH and NO levels in liver, kidney, and brain tissues were shown in Figs. 1–4 respectively. Blood MDA, GSH and NO levels were also shown in Fig. 5. The levels of MDA and NO in liver, kidney, brain and blood tissues were significantly higher in mice received HgCI2 alone compared to that of Group I animals used as control. On the other hand, compared to the control group, the levels of MDA and NO were lower in mice received omega3 fatty acid alone. Comparably, the levels of TSA in liver, kidney and brain tissues were significantly higher in Group II compared to Group I. Contrary to TSA, MDA and NO, GSH levels were significantly lower in liver, brain and blood, while no change was detected in kidney. In liver, omega-3 fatty acid injection in HgCl2 received mice had significant effects by decreasing TSA, GSH and NO levels and increasing GSH level compared to that of mice received HgCl2 alone. Similar results were also observed for brain and blood. In kidney, GSH levels were comparable among the groups, however TSA, MDA
Histopathological investigations Tissue sections of liver, kidney and brain were sliced and fixed in 10% phosphate buffered formalin. After the specimens were dehydrated in serial ethanol and xylene, they were embedded in paraffin. Tissue sections were then cut and processed for hematoxylin and eosin (HE) staining. The sections were examined unbiased under a light microscope.
Fig. 2. Levels of the liver, kidney and brain MDA in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
96
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
Fig. 3. Levels of the liver, kidney and brain GSH in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
trilobuler degeneration characterized by cloudy appearance of hepatocytes especially around the central veins was observed. Occasional necrotic cells were also noted in some sections. Organization of hepatic cords seemed to be slightly or moderately disrupted in degenerated regions. Prominent hyperemia and occasional hemorrhages were also detected in livers of mice given HgCl2 alone (Fig. 6b). In HgCl2 plus omega-3 fatty acid given animals, degenerative changes seen in HgCl2 alone given animals were minimized. In these animals, either no recognizable degenerative changes were seen or minimal degeneration was noted (Fig. 6c). Normal architecture of liver tissue was observed in the control (Fig. 6a) and omega-3 fatty acid alone given (Fig. 6d) groups. In kidneys, severe histopathological changes were noted in HgCl2 alone given mice. Granular degeneration especially in the proximal tubules was prominent. Some tubule lumens were occluded with homogenous pink hyalinous material. Moderate to severe hyperemia and occasional hemorrhages were also seen. Focal monocytic and granulocytic accumulations were recorded in some sections (Fig. 7b). Some degenerative changes together with hyperemia and tubular changes were also noted in kidneys of HgCl2 plus omega-3 fatty acid given animals, though with reduced severity compared to the HgCl2 alone given mice (Fig. 7c). Control (Fig. 7a) and omega-3 fatty acid alone given (Fig. 7d) mice showed normal morphology of kidney tissue. No significant changes were observed in cerebral tissue in terms of neuronal degeneration, hyperemia, or else in HgCl2 given animals (Fig. 8a–d). In cerebellum, no prominent changes were either noted.
Discussion
Fig. 4. Levels of the liver, kidney and brain NO in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
and NO levels were significantly lowered in mice received omega3 fatty acid plus HgCl2 compared to that of mice received HgCl2 alone. Histopathological changes In histopathological examination of liver tissues, severe hepatic degeneration was observed in HgCl2 alone given mice. A cen-
Fig. 5. Levels of the blood MDA, GSH and NO in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
Oxidative damage and imbalance between the oxidant and antioxidant defense mechanisms have been the most important mechanisms behind heavy metal toxicity [17]. Recent studies have shown that heavy metals such as mercury generates reactive radicals which in turn cause cellular damages by depleting important enzyme activities and damaging membrane lipid bilayers and nucleic acids [18]. Though these cellular alterations might affect many organ systems, hepatotoxic, nephrotoxic and neurotoxic alterations are generally seen. Similarly, the toxic effects of mercury in several organs have been observed [18–20]. In spite of the great efforts in the search for development of new drugs that counteract the toxic or metabolic changes induced by mercury, an effective treatment that would suppress its toxic effects have yet found. In the present study, administration of omega-3 fatty acid was tested in HgCI2 toxicity in mice. The results shown that the toxic alterations detected by biochemical and histopathological means were ameliorated by sc injection of omega-3 fatty acid. In biochemical analysis, significant increases at the levels of TSA, MDA and NO were determined in HgCI2 alone treated mice in liver, kidney, brain and blood tissues. In addition, GSH level decreased in liver, brain and blood. Omega-3 fatty acid administration was seen to normalize these values to that of the control animals. Glutathione is known to be an important mediator in detoxifying lipid hydroperoxides. In the process of mercury toxicity, mercury can bind to cysteinyl residues of thiol containing proteins [21]. It was suggested that depletion of GSH might be the mechanism leading to increases in levels of ROS, such as superoxide anion radicals, hydrogen peroxide and hydroxyl radicals and increasing to the mercury induced toxicity [18,22]. Therefore, low levels of GSH with high MDA concentrations could be used as an indicator of oxidative stress [23,24]. In a previous study, mice exposed to HgCI2 showed significant changes at the levels of glutathione reductase (GR), glutathione peroxidase (GPx) and GSH in several organs, though differences were observed among liver, kidney and
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
97
Fig. 6. Microscopic view of liver in a control mouse (a) showing normal architecture of the tissue as compared to a mouse given HgCI2 alone (b) showing centrilobular degeneration and hyperemia. Preservation of normal architecture of the tissue in a mouse given HgCI2 plus omega-3 fatty acid (c) and a mouse given omega-3 fatty acid alone (d). HE.
brain tissues [25]. Higher enzyme activities in liver and kidney than brain were suggested to play role in different tissue answers [26]. As in mice, rats exposed to mercury showed reduced GSH and increased lipid peroxidation with formation of H2 O2 in kidney tissue [27]. Therefore, it is reasonable to assume for the current study that the peroxidative effect of mercury might be related to
thiol depletion and H2 O2 production in mitochondria as suggested previously [28]. Levels of NO in a manner similar to levels of GSH and MDA are widely used to reflect oxidative tissue degeneration. NO has many physiological functions such as alleviating oxidative damage, vasodilatation and neurotransmission. However, excess NO may
Fig. 7. Kidney of a control mouse (a); HgCI2 alone given mouse (b) showing tubular degeneration (black arrows), proteinous substance in the tubular lumens (white arrows), hyperemia (white arrowhead) and monocytic cellular infiltration (black arrowheads); kidney of a mouse given HgCI2 plus omega-3 fatty acid (c) showing only hyperemia (white arrowhead) but no signs of degenerative changes; kidney of a mouse given omega-3 fatty acid alone (d). HE.
98
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
Fig. 8. Representative microscopic views of brain tissues from control (a), HgCI2 alone (b), HgCI2 plus omega-3 fatty acid (c), and omega-3 fatty acid (d) given mice.
involve in the production of peroxynitrite and impair the function of certain enzymes [29]. Large part of pathologic conditions originated from NO depends on the formation of secondary intermediates such as ONOO− and nitrogen dioxide that are usually more reactive and toxic than NO alone. Reactive nitrogen species originated from NO require the presence of oxidants such as superoxide radicals and H2 O2 [6]. HgCI2 has the mechanism leading to increases in levels of ROS [18,22]. It has been shown that sodium nitroprusside as a NO donor causes significant toxic effects, which was evaluated by electrophysiological and morphological means [30]. In search of the physiological effects of fish oil, it has been shown that excretion of NO metabolites increases in volunteers taking fish oil rich in eicosapentaenoic and docosahexaenoic acids; and the observed effect was determined to be due to mainly docosahexaenoic acid [31]. In addition, it has been shown that rats given n-3 essential fatty acids present reduced NO levels in brain tissue [32]. Consistent with the previous study, reduced level of NO was also detected in brain tissue, as well as liver, kidney and blood, with the use of omega-3 fatty acid in the current study. Therefore, in light of the results obtained in this study, it could be assumed that omega3 fatty acid might be effective in regulation of NO metabolism and hence the oxidative status of tissues which was also confirmed by increased GSH levels. Histopathological changes observed in HgCI2 treated rats might therefore, be due to increased level of NO in tissues. However, absence of any pathological changes in brain might be explained by the different status and origin of NO produced in tissues, especially the brain. Although not seen in brain by histopathology, the results of biochemical analysis showed that subcutaneous omega-3 fatty acid injection might present significant corrections at the level of NO both in brain and the other tissues investigated. On the other hand toxic effects of mercury has been previously reported in oligodendrocytes, astrocytes, cerebral cortical and cerebellar granular neurons obtained from embryonic and neonatal rat brains [33]. Altered SA level has been reported in various disease conditions such as cancer, rheumatoid arthritis, bacterial infections and liver dysfunctions as well as heavy metal intoxications, presenting clinical potential as a marker to indicate tissue degeneration [10,34,35].
Although the exact mechanisms of how SA level increases in metabolic disorders is unclear, a plausible explanation might be that tissue sialic acid originates from the terminal oligosaccharide chain of several acute phase proteins, such as fibrinogen, haptoglobin, ceruloplasmin, and transferring, which are known to increase in these conditions [36]. In a previous study, the level of liver ␣1 -acid glycoprotein, which contains sialic acid, was found higher in lead poisoned rats compared to the control [37]. Moreover, it was shown that application of gangliosides, which are rich at SA concentration, to neuronal tissue following experimental trauma increases some important enzyme activities such as Na+ K+ ATPase and presents a better resistance to anoxia and ionic unbalances [38]. Since sialylated glycosphingolipids are found at high amount in the outer leaflet of the lipid bilayer in the plasma membranes of neurons SA level is normally higher in brain tissue than those of other. In the present study, TSA level was also found higher in brain than liver and kidney. It was also determined that HgCI2 treated rats showed increased TSA concentrations in brain, liver and kidney compared to that of control animals, and omega-3 fatty acid use significantly decreases these levels. Under physiological conditions, the balance between the tissue oxidant and antioxidant molecules show the oxidant/antioxidant statue of the tissue. Changes in favor to the oxidant molecules and/or decreased antioxidant molecules might therefore be indication of the tissue oxidative stress and degeneration. GSH levels for antioxidant response and MDA levels for lipid peroxidation may be an indicator in terms of oxidative stress [39]. In the present study, statistically significant changes at all the studied tissue MDA and GSH levels were observed in mice treated with HgCI2 and omega-3 fatty acid. Decreased levels of tissue NO and MDA levels together with increased GSH levels, as a result of omega-3 fatty acid treated, clearly show the protective effect of omega-3 fatty acid treatment against lipid peroxidation and oxidative stress directed by NO. Our findings support the idea of Barbosa et al. [40] in that omega-3 fatty acid supplementation may have free radical scavenger activity. Omega-3 fatty acid may stimulate vitamin E incorporation into membranes to avoid lipid peroxidation and ROS resulting from increased membrane omega-3 fatty acid treatment [41]. ROS,
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
which are also produced physiologically, are essential as regulatory molecules to many normal biological processes. However, ROS can create risk to cells and tissues if their generation is not securely controlled. According to Sarsilmaz et al. [42], EPA as a member of omega-3 essential fatty acid affects phospholipid metabolism in cell membranes. Inhibition of phospholipase A2 enzyme by EPA may cause stabilization of membrane structure and decrease ROS generation and hence lipid peroxidation [42]. Dietary supplementation of polyunsaturated fatty acids has been suggested to be beneficial in reducing the susceptibility to HgCI2 -induced lipid peroxidation [22,43]. The results of the present study also confirms the previous results, and adds that subcutaneous injection of omega-3 fatty acid would be quite effective in ameliorating the toxic effects of HgCl2 toxicity in liver, kidney and brain tissues. Although the results indicate that omega-3 fatty acid performs these effects by improving antioxidant status, acute phase response and reducing lipid peroxidation as well as protecting cellular membranes the underlying mechanisms are warranted further studies. Conflict of interest None declared. References [1] Clarkson TW. The toxicology of mercury: critical reviews. Clin Lab Sci 1997;34:369–403. [2] WHO. Elemental mercury and inorganic mercury compounds: human health aspects. Concise International Chemical Assessment Document 50. Geneva: World Health Organization; 2003. [3] Zhao JQ, Wen YF, Bhadauria M, Nirala SK, Sharma A, Shrivastava S, et al. Protective effects of propolis on inorganic mercury induced oxidative stress in mice. Indian J Exp Biol 2009;47:264–9. [4] Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186:421–31. [5] Kaya I, Karapehlivan M, Yilmaz M, Ersan Y, Koc E. Investigation of effects on plasma nitric oxide, malondialdehyde and total sialic acid levels of glyphosate in Kars creek transcaucasian barb (Capoeta capoeta [Guldenstaedt, 1773]) in Turkey. Fresenius Environ Bull 2012;21:123–6. [6] Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, et al. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem 1994;269:26066–75. [7] Mohan SK, Priyav V. Serum total sialic acid, lipid peroxidation, and glutathione reductase levels in patients with rheumatoid arthritis. Turk J Med Sci 2010;40:537–40. [8] Schauer R. Chemistry, metabolism, and biological functions of sialic acids. Adv Carbohydr Chem Biochem 1982;40:131–234. [9] Schroder PN, Giannis A. From substrate to transition state analogues: the first potent inhibitör of sialyltransferases. Angew Chem Int Ed 1999;38: 1379–80. [10] Yilmaz M, Karapehlivan M, Kaya I. Effects of zinc sulphate on transcaucasian barb, (Capoeta capoeta [Guldenstaedt, 1773]) plasma nitric oxide, malondialdehyde and total sialic acid levels. Kafkas Univ Vet Fak Derg 2012;18:61–4. [11] Yuen AW, Sander JW, Fluegel D, Patsalos PN, Bell GS, Johnson T, et al. Omega3 fatty acid supplementation in patients with chronic epilepsy: a randomized trial. Epilepsy Behav 2005;7:253–8. [12] Mazza M, Pomponi M, Janiri L, Bria P, Mazza S. Omega-3 fatty acids and antioxidants in neurological and psychiatric diseases: an overview. Prog Neuropsychopharmacol Biol Psychiatry 2007;31:12–26. [13] Sydow G. A simplified quick method for determination of sialic acid in serum. Biomed Biochim Acta 1985;44:1721–3. [14] Beutler E, Duran O, Kelley BM. Improved method for determination of blood glutathione. J Lab Clin Med 1963;28:882–8. [15] Yoshioka T, Kawada K, Shimada T, Mori M. Lipid peroxidation in maternal and cord blood and protective mechanism against active-oxygen toxicity in the blood. Am J Obstet Gynecol 1979;135:372–6.
99
[16] Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001;5:62–71. [17] Leonard SS, Harris GK, Shi X. Metal-induced oxidative stress and signal transduction. Free Radic Biol Med 2004;37:1921–42. [18] Stohs SJ, Baqchi D. Oxidative mechanisms in the toxicity of metal-ions. Free Radic Biol Med 1995;18:321–36. [19] Flora SJ, Mittal M, Mehta A. Heavy metal induced oxidative stress and its possible reversal by chelation therapy. Indian J Med Res 2008;128:501–23. [20] Moretto MB, Lermen CL, Morsch VM, Bohrer D, Ineu RP, Silva AC, et al. Effect of subchronic treatment with mercury chloride on NTPDase, 5 -nucleotidase and acetylcholinesterase from cerebral cortex of rats. J Trace Elem Med Biol 2004;17:255–60. [21] Farina M, Brandão R, de Lara FS, Pagliosa LB, Soares FA, Souza DO, et al. Profile of nonprotein thiols, lipid peroxidation and ␦-aminolevulinate dehydratase activity in mouse kidney and liver in response to acute exposure to mercuric chloride and sodium selenite. Toxicology 2002;184:179–87. [22] Farina M, Soares FA, Feoli A, Roehring C, Brusque AM, Rotta L, et al. In vitro effects of selenite and mercuric chloride on liver thiobarbituric acid-reactive substances and non-protein thiols from rats: influences of dietary cholesterol and polyunsaturated and saturated fatty acids. Nutrition 2003;19:531–5. [23] Rao MV, Sharma PS. Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice. Reprod Toxicol 2001;15:705–12. [24] Cigremis Y, Turel H, Adiguzel K, Akgoz M, Kart A, Karaman M, et al. The effects of acute acetaminophen toxicity on hepatic mRNA expression of SOD, CAT, GSH-Px, and levels of peroxynitrite, nitric oxide, reduced glutathione and malondialdehyde in rabbit. Mol Cell Biochem 2009;323:31–8. [25] Mahboob M, Shireen KF, Atkinson A, Khan AT. Lipid peroxidation and antioxidant enzyme activity in different organs of mice exposed to low level of mercury. J Environ Sci Health B 2001;36:687–97. [26] De Freitas AS, Funck VR, Rotta Mdos S, Bohrer D, Mörschbächer V, Puntel RL, et al. Diphenyl diselenide, a simple organoselenium compound, decreases methylmercury-induced cerebral, hepatic and renal oxidative stress and mercury deposition in adult mice. Brain Res Bull 2009;79:77–84. [27] Lund BO, Miller DM, Woods JS. Studies on Hg(II)-induced H2 O2 formation and oxidative stress in vivo and in vitro in rat kidney mitochondria. Biochem Pharmacol 1993;45:2017–24. [28] Lund BO, Miller DM, Woods JS. Mercury-induced H2 O2 production and lipid peroxidation in vitro in rat kidney mitochondria. Biochem Pharmacol 1991;42:181–7. [29] Ruan RS, Leong SK, Yeoh KH. Ototoxicity of sodium nitroprusside. Hear Res 1997;114:169–78. [30] Kong WJ, Ren T, Nuttall AL. Electrophysiological and morphological evaluation of the acute ototoxicity of sodium nitroprusside. Hear Res 1996;99:22–30. [31] Harris WS, Rambjør GS, Windsor SL, Diederich D. n-3 Fatty acids and urinary excretion of nitric oxide metabolites in humans. Am J Clin Nutr 1997;65:459–64. [32] Songur A, Sarsilmaz M, Sogut S, Ozyurt B, Ozyurt H, Zararsiz I, et al. Hypothalamic superoxide dismutase, xanthine oxidase, nitric oxide, and malondialdehyde in rats fed with fish omega-3 fatty acids. Prog Neuropsychopharmacol Biol Psychiatry 2004;28:693–8. [33] Yee S, Choi BH. Oxidative stress in neurotoxic effects of methylmercury poisoning. Neurotoxicology 1996;17:17–26. [34] Sydow G, Wittmann W, Bender E, Starick E. The sialic acid content of the serum of cattle infected with bovine leukosis virus. Arch Exp Vetmed 1988;42:194–7. [35] Sillanaukee P, Pönniö M, Jääskeläinen IP. Occurence of sialic acids in healthy humans and different disorders. Eur J Clin Invest 1999;29:413–25. [36] Kushner I. The acute phase response: an overview. Methods Enzymol 1988;163:373–83. [37] Patel AB, Venkatakrishna Bhatt H. Effect of lead on the blood serum, liver and brain sialoglycoconjugate levels in rats. Hum Exp Toxicol 1992;11:89–92. [38] Gorio A. Ganglioside enhancement of neuronal differentiation, plasticity, and repair. CRC Crit Rev Clin Neurobiol 1986;2:241–96. [39] Kalaiselvi T, Panneersalvam C. Effect of l-carnitine on the status of lipid peroxidation and antioxidants in aging rats. J Nutr Biochem 1998;9:575–81. [40] Barbosa DS, Cecchini R, El Kadri MZ, Rodríguez MA, Burini RC, Dichi I. Decreased oxidative stress in patients with ulcerative colitis supplemented with fish oil omega-3 fatty acids. Nutrition 2003;19:837–42. [41] Chautan M, Calaf R, Léonardi J, Charbonnier M, Andre M, Portugal H, et al. Inverse modifications of heart and liver alpha-tocopherol status by various dietary n-6/n-3 polyunsaturated ratios. J Lipid Res 1990;31:2201–8. [42] Sarsilmaz M, Songur A, Ozyurt H, Kus I, Ozen OA, Ozyurt B, et al. Potential role of dietary omega-3 essential fatty acids on some oxidant/antioxidant parameters in rats’ corpus striatum. Prostaglandins Leukot Essent Fatty Acids 2003;69:253–9. [43] Sprecher H. Interactions between the metabolism of n-3 and n-6 fatty acids. J Intern Med Suppl 1989;731:5–9.
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
Toxicology
Protective effect of omega-3 fatty acid against mercury chloride intoxication in mice Mahmut Karapehlivan a , Metin Ogun a , Inan Kaya b,∗ , Hasan Ozen c , Haci Ahmet Deveci d , Musa Karaman c a
Department of Biochemistry, Faculty of Veterinary Medicine, Kafkas University, 36100 Kars, Turkey Department of Molecular Biology and Genetic, Faculty of Arts and Science, Kafkas University, 36100 Kars, Turkey c Department of Pathology, Faculty of Veterinary Medicine, Kafkas University, 36100 Kars, Turkey d Atatürk Health Services Vocational School, Kafkas University, 36100 Kars, Turkey b
a r t i c l e
i n f o
Article history: Received 4 June 2013 Received in revised form 3 August 2013 Accepted 30 August 2013 Keywords: Mercury chloride Omega-3 fatty acid Sialic acid Malondialdehyde Nitric oxide
a b s t r a c t The aim of this study was to investigate the protective effect of omega-3 fatty acid in HgCI2 toxicity in mice. Levels of malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO) and total sialic acid (TSA), and histopathological changes in selected organs were evaluated. Twenty-eight mice were equally divided into 4 groups, namely Groups I–IV. Group I animals received intraperitoneal (ip) injection of physiological saline solution; Group II animals received ip injection of 0.4 mg/kg/day HgCI2 ; Group III animals received ip injection of 0.4 mg/kg/day HgCI2 in addition to subcutaneous (sc) injection of 0.5 g/kg/day omega-3 fatty acid; and Group IV animals received sc injection of 0.5 g/kg/day omega-3 fatty acid. All treatments lasted 7 days. The levels of MDA, NO and TSA were significantly higher in Group II and lower in Groups III and IV as compared to the Group I. GSH level was the highest in Group IV. In histopathology, severe degeneration in liver and kidney was observed in Group II animals. These degrading changes were seen to be reduced greatly in Group III animals. The results suggested that omega-3 fatty acid might attenuate HgCI2 -induced toxicity by improving antioxidant status and acute phase response in mice. © 2013 Elsevier GmbH. All rights reserved.
Introduction Mercury is a well-known toxic heavy metal to animals as well as humans. It is widespread and distributed throughout the environment by countless processes such as volcanic activities, water movements and various biological activities. Industrial processes such as mining activities, combustion of fossil fuels, pesticides and medical wastes have also become significant contributors to the environmental distribution of mercury and its compounds [1,2]. Mercury causes various metabolic changes as a result of its toxic effects especially in central nervous system, kidney and liver tissues of animals. It manifests its toxicity by interacting with reactive oxygen species (ROS) and binding to thiol groups in several proteins such as glutathione (GSH) and many of the antioxidant enzymes [3]. High levels of ROS, which indicates the oxidative stress, are known to induce lipid peroxidation that can be further shown by increased
∗ Corresponding author. Tel.: +90 474 225 1150; fax: +90 474 225 1179. E-mail address: inankaya @hotmail.com (I. Kaya). 0946-672X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jtemb.2013.08.004
malondialdehyde (MDA) levels [4,5]. Nitric oxide (NO) is involved in various biological processes such as vasodilatation, neurotransmission and leukocyte mediated killing of pathogens. Moreover, it is involved in the process of peroxynitrite anion (ONOO− ) production reacting with peroxides, which further yields lipid peroxidation [6]. Total sialic acid (TSA) can be used as an indicator of tissue damage. Sialic acids have many important physiological and pathological functions such as cellular transmission, embryogenesis, organ development, immune system regulations, leucodiapedesis, metastasis of neoplastic cells, and carrying out membrane receptor functions [7–10]. The omega-3 fatty acids are polyunsaturated fatty acids and commonly found in marine oils. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) compose of the major parts of omega3 fatty acids. In recent years, it has been reported that consumption of long-chain omega-3 fatty acids may have beneficial effects in many health problems such as cardiovascular, neuronal and gastrointestinal diseases related particularly to electrical signal modulation [11,12]. In the present study, potential protective effect of omega-3 fatty acids in HgCI2 toxicity in mice was investigated by biochemical and histopathological means.
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
95
Materials and methods Experimental design The ethical approval of the study was confirmed by I˙ nönü University Animal Care and Use Committee (Registration Number: 2013/A-41). All procedures were conducted in accordance with the ‘Guide for Care and Use of Laboratory Animals’ published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain. Experiments were carried out on 28 male Swiss albino mice each weighing 29–34 g at 14–16 weeks of age. Animals were divided into 4 equal groups namely Group I through Group IV and housed in a room maintained at 18 ± 1 ◦ C with an alternating 12 h light-dark cycle. Food and water were provided ad libitum. All experimental injections were carried out for 7 days and at the same hours of the light cycles of the study. Treatments were performed as follow; Group I: ip injection of physiological saline solution, Group II: ip injection of 0.4 mg/kg/day HgCI2 . Group III: ip injection of 0.4 mg/kg/day HgCI2 plus sc injection of 0.5 g/kg/day omega-3 fatty acid, and Group IV: sc injection of 0.5 g/kg/day omega-3 fatty acid.
Biochemical analysis At the end of the 7th day, blood samples were collected into EDTA tubes from the hearts via cardiac puncture under ether anesthesia for GSH, MDA and NO measurements. The blood samples were centrifuged (1200 × g, 4 ◦ C) for 10 min, and the plasma were obtained and then kept at −25 ◦ C until the analyzes were carried out. At necropsy liver, kidney and brain tissues were collected to determine tissue GSH, MDA, NO and TSA concentrations. For this purpose, the tissues were rinsed with ice-cold 0.9% NaCl. Then, 1 g of tissue was homogenized in phosphate buffer (pH 7.4) in an ice bath and the homogenates were centrifuged (1200 × g, 4 ◦ C) for 15 min. TSA was measured calorimetrically using a spectrophotometer (UV-1201, Shimadzu, Japan) by the method of Sydow [13] in that all bound sialic acid were separated by acid per-chloride in plasma and tissue homogenates, and then the supernatants were boiled by Erlich reagent, and finally the product was read at 525 nm. GSH concentration was assayed by the method of Beutler et al. [14] based on the spectrophotometric measurement of sulphydril ( SH) groups forming complexes with 5,5 -(2-dithiobis nitrobenzoic acid) which give rise to colored products which were read at 412 nm. Measurement of MDA concentrations was carried out by the method of Yoshoiko et al. [15] based on the reaction between thiobarbituric acid and MDA produced as an end product of lipid peroxidation. The end products were read at 535 nm. NO levels were determined according to the method described by Miranda et al. [16] in that nitrate is reduced to nitrite by VaCl3 , and then in acidic environment nitrite was reacted with sulphanilamide to produce colored diazonium compound, which was read at 540 nm.
Fig. 1. Levels of the liver, kidney and brain TSA in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
Statistical analysis SPSS software (Windows version 20.0) was used for statistical evaluation of data which were expressed as median (X) ± standard deviation (SD). Importance level of difference among the groups was determined by variance analysis (ANOVA) and Duncan multiple comparison test. Results TSA, MDA, GSH and NO levels The results of TSA, MDA, GSH and NO levels in liver, kidney, and brain tissues were shown in Figs. 1–4 respectively. Blood MDA, GSH and NO levels were also shown in Fig. 5. The levels of MDA and NO in liver, kidney, brain and blood tissues were significantly higher in mice received HgCI2 alone compared to that of Group I animals used as control. On the other hand, compared to the control group, the levels of MDA and NO were lower in mice received omega3 fatty acid alone. Comparably, the levels of TSA in liver, kidney and brain tissues were significantly higher in Group II compared to Group I. Contrary to TSA, MDA and NO, GSH levels were significantly lower in liver, brain and blood, while no change was detected in kidney. In liver, omega-3 fatty acid injection in HgCl2 received mice had significant effects by decreasing TSA, GSH and NO levels and increasing GSH level compared to that of mice received HgCl2 alone. Similar results were also observed for brain and blood. In kidney, GSH levels were comparable among the groups, however TSA, MDA
Histopathological investigations Tissue sections of liver, kidney and brain were sliced and fixed in 10% phosphate buffered formalin. After the specimens were dehydrated in serial ethanol and xylene, they were embedded in paraffin. Tissue sections were then cut and processed for hematoxylin and eosin (HE) staining. The sections were examined unbiased under a light microscope.
Fig. 2. Levels of the liver, kidney and brain MDA in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
96
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
Fig. 3. Levels of the liver, kidney and brain GSH in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
trilobuler degeneration characterized by cloudy appearance of hepatocytes especially around the central veins was observed. Occasional necrotic cells were also noted in some sections. Organization of hepatic cords seemed to be slightly or moderately disrupted in degenerated regions. Prominent hyperemia and occasional hemorrhages were also detected in livers of mice given HgCl2 alone (Fig. 6b). In HgCl2 plus omega-3 fatty acid given animals, degenerative changes seen in HgCl2 alone given animals were minimized. In these animals, either no recognizable degenerative changes were seen or minimal degeneration was noted (Fig. 6c). Normal architecture of liver tissue was observed in the control (Fig. 6a) and omega-3 fatty acid alone given (Fig. 6d) groups. In kidneys, severe histopathological changes were noted in HgCl2 alone given mice. Granular degeneration especially in the proximal tubules was prominent. Some tubule lumens were occluded with homogenous pink hyalinous material. Moderate to severe hyperemia and occasional hemorrhages were also seen. Focal monocytic and granulocytic accumulations were recorded in some sections (Fig. 7b). Some degenerative changes together with hyperemia and tubular changes were also noted in kidneys of HgCl2 plus omega-3 fatty acid given animals, though with reduced severity compared to the HgCl2 alone given mice (Fig. 7c). Control (Fig. 7a) and omega-3 fatty acid alone given (Fig. 7d) mice showed normal morphology of kidney tissue. No significant changes were observed in cerebral tissue in terms of neuronal degeneration, hyperemia, or else in HgCl2 given animals (Fig. 8a–d). In cerebellum, no prominent changes were either noted.
Discussion
Fig. 4. Levels of the liver, kidney and brain NO in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
and NO levels were significantly lowered in mice received omega3 fatty acid plus HgCl2 compared to that of mice received HgCl2 alone. Histopathological changes In histopathological examination of liver tissues, severe hepatic degeneration was observed in HgCl2 alone given mice. A cen-
Fig. 5. Levels of the blood MDA, GSH and NO in HgCI2 and omega-3 fatty acidstreated and without treated groups. Results with different superscripts within the same row are significantly different (P < 0.001).
Oxidative damage and imbalance between the oxidant and antioxidant defense mechanisms have been the most important mechanisms behind heavy metal toxicity [17]. Recent studies have shown that heavy metals such as mercury generates reactive radicals which in turn cause cellular damages by depleting important enzyme activities and damaging membrane lipid bilayers and nucleic acids [18]. Though these cellular alterations might affect many organ systems, hepatotoxic, nephrotoxic and neurotoxic alterations are generally seen. Similarly, the toxic effects of mercury in several organs have been observed [18–20]. In spite of the great efforts in the search for development of new drugs that counteract the toxic or metabolic changes induced by mercury, an effective treatment that would suppress its toxic effects have yet found. In the present study, administration of omega-3 fatty acid was tested in HgCI2 toxicity in mice. The results shown that the toxic alterations detected by biochemical and histopathological means were ameliorated by sc injection of omega-3 fatty acid. In biochemical analysis, significant increases at the levels of TSA, MDA and NO were determined in HgCI2 alone treated mice in liver, kidney, brain and blood tissues. In addition, GSH level decreased in liver, brain and blood. Omega-3 fatty acid administration was seen to normalize these values to that of the control animals. Glutathione is known to be an important mediator in detoxifying lipid hydroperoxides. In the process of mercury toxicity, mercury can bind to cysteinyl residues of thiol containing proteins [21]. It was suggested that depletion of GSH might be the mechanism leading to increases in levels of ROS, such as superoxide anion radicals, hydrogen peroxide and hydroxyl radicals and increasing to the mercury induced toxicity [18,22]. Therefore, low levels of GSH with high MDA concentrations could be used as an indicator of oxidative stress [23,24]. In a previous study, mice exposed to HgCI2 showed significant changes at the levels of glutathione reductase (GR), glutathione peroxidase (GPx) and GSH in several organs, though differences were observed among liver, kidney and
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
97
Fig. 6. Microscopic view of liver in a control mouse (a) showing normal architecture of the tissue as compared to a mouse given HgCI2 alone (b) showing centrilobular degeneration and hyperemia. Preservation of normal architecture of the tissue in a mouse given HgCI2 plus omega-3 fatty acid (c) and a mouse given omega-3 fatty acid alone (d). HE.
brain tissues [25]. Higher enzyme activities in liver and kidney than brain were suggested to play role in different tissue answers [26]. As in mice, rats exposed to mercury showed reduced GSH and increased lipid peroxidation with formation of H2 O2 in kidney tissue [27]. Therefore, it is reasonable to assume for the current study that the peroxidative effect of mercury might be related to
thiol depletion and H2 O2 production in mitochondria as suggested previously [28]. Levels of NO in a manner similar to levels of GSH and MDA are widely used to reflect oxidative tissue degeneration. NO has many physiological functions such as alleviating oxidative damage, vasodilatation and neurotransmission. However, excess NO may
Fig. 7. Kidney of a control mouse (a); HgCI2 alone given mouse (b) showing tubular degeneration (black arrows), proteinous substance in the tubular lumens (white arrows), hyperemia (white arrowhead) and monocytic cellular infiltration (black arrowheads); kidney of a mouse given HgCI2 plus omega-3 fatty acid (c) showing only hyperemia (white arrowhead) but no signs of degenerative changes; kidney of a mouse given omega-3 fatty acid alone (d). HE.
98
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
Fig. 8. Representative microscopic views of brain tissues from control (a), HgCI2 alone (b), HgCI2 plus omega-3 fatty acid (c), and omega-3 fatty acid (d) given mice.
involve in the production of peroxynitrite and impair the function of certain enzymes [29]. Large part of pathologic conditions originated from NO depends on the formation of secondary intermediates such as ONOO− and nitrogen dioxide that are usually more reactive and toxic than NO alone. Reactive nitrogen species originated from NO require the presence of oxidants such as superoxide radicals and H2 O2 [6]. HgCI2 has the mechanism leading to increases in levels of ROS [18,22]. It has been shown that sodium nitroprusside as a NO donor causes significant toxic effects, which was evaluated by electrophysiological and morphological means [30]. In search of the physiological effects of fish oil, it has been shown that excretion of NO metabolites increases in volunteers taking fish oil rich in eicosapentaenoic and docosahexaenoic acids; and the observed effect was determined to be due to mainly docosahexaenoic acid [31]. In addition, it has been shown that rats given n-3 essential fatty acids present reduced NO levels in brain tissue [32]. Consistent with the previous study, reduced level of NO was also detected in brain tissue, as well as liver, kidney and blood, with the use of omega-3 fatty acid in the current study. Therefore, in light of the results obtained in this study, it could be assumed that omega3 fatty acid might be effective in regulation of NO metabolism and hence the oxidative status of tissues which was also confirmed by increased GSH levels. Histopathological changes observed in HgCI2 treated rats might therefore, be due to increased level of NO in tissues. However, absence of any pathological changes in brain might be explained by the different status and origin of NO produced in tissues, especially the brain. Although not seen in brain by histopathology, the results of biochemical analysis showed that subcutaneous omega-3 fatty acid injection might present significant corrections at the level of NO both in brain and the other tissues investigated. On the other hand toxic effects of mercury has been previously reported in oligodendrocytes, astrocytes, cerebral cortical and cerebellar granular neurons obtained from embryonic and neonatal rat brains [33]. Altered SA level has been reported in various disease conditions such as cancer, rheumatoid arthritis, bacterial infections and liver dysfunctions as well as heavy metal intoxications, presenting clinical potential as a marker to indicate tissue degeneration [10,34,35].
Although the exact mechanisms of how SA level increases in metabolic disorders is unclear, a plausible explanation might be that tissue sialic acid originates from the terminal oligosaccharide chain of several acute phase proteins, such as fibrinogen, haptoglobin, ceruloplasmin, and transferring, which are known to increase in these conditions [36]. In a previous study, the level of liver ␣1 -acid glycoprotein, which contains sialic acid, was found higher in lead poisoned rats compared to the control [37]. Moreover, it was shown that application of gangliosides, which are rich at SA concentration, to neuronal tissue following experimental trauma increases some important enzyme activities such as Na+ K+ ATPase and presents a better resistance to anoxia and ionic unbalances [38]. Since sialylated glycosphingolipids are found at high amount in the outer leaflet of the lipid bilayer in the plasma membranes of neurons SA level is normally higher in brain tissue than those of other. In the present study, TSA level was also found higher in brain than liver and kidney. It was also determined that HgCI2 treated rats showed increased TSA concentrations in brain, liver and kidney compared to that of control animals, and omega-3 fatty acid use significantly decreases these levels. Under physiological conditions, the balance between the tissue oxidant and antioxidant molecules show the oxidant/antioxidant statue of the tissue. Changes in favor to the oxidant molecules and/or decreased antioxidant molecules might therefore be indication of the tissue oxidative stress and degeneration. GSH levels for antioxidant response and MDA levels for lipid peroxidation may be an indicator in terms of oxidative stress [39]. In the present study, statistically significant changes at all the studied tissue MDA and GSH levels were observed in mice treated with HgCI2 and omega-3 fatty acid. Decreased levels of tissue NO and MDA levels together with increased GSH levels, as a result of omega-3 fatty acid treated, clearly show the protective effect of omega-3 fatty acid treatment against lipid peroxidation and oxidative stress directed by NO. Our findings support the idea of Barbosa et al. [40] in that omega-3 fatty acid supplementation may have free radical scavenger activity. Omega-3 fatty acid may stimulate vitamin E incorporation into membranes to avoid lipid peroxidation and ROS resulting from increased membrane omega-3 fatty acid treatment [41]. ROS,
M. Karapehlivan et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 94–99
which are also produced physiologically, are essential as regulatory molecules to many normal biological processes. However, ROS can create risk to cells and tissues if their generation is not securely controlled. According to Sarsilmaz et al. [42], EPA as a member of omega-3 essential fatty acid affects phospholipid metabolism in cell membranes. Inhibition of phospholipase A2 enzyme by EPA may cause stabilization of membrane structure and decrease ROS generation and hence lipid peroxidation [42]. Dietary supplementation of polyunsaturated fatty acids has been suggested to be beneficial in reducing the susceptibility to HgCI2 -induced lipid peroxidation [22,43]. The results of the present study also confirms the previous results, and adds that subcutaneous injection of omega-3 fatty acid would be quite effective in ameliorating the toxic effects of HgCl2 toxicity in liver, kidney and brain tissues. Although the results indicate that omega-3 fatty acid performs these effects by improving antioxidant status, acute phase response and reducing lipid peroxidation as well as protecting cellular membranes the underlying mechanisms are warranted further studies. Conflict of interest None declared. References [1] Clarkson TW. The toxicology of mercury: critical reviews. Clin Lab Sci 1997;34:369–403. [2] WHO. Elemental mercury and inorganic mercury compounds: human health aspects. Concise International Chemical Assessment Document 50. Geneva: World Health Organization; 2003. [3] Zhao JQ, Wen YF, Bhadauria M, Nirala SK, Sharma A, Shrivastava S, et al. Protective effects of propolis on inorganic mercury induced oxidative stress in mice. Indian J Exp Biol 2009;47:264–9. [4] Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186:421–31. [5] Kaya I, Karapehlivan M, Yilmaz M, Ersan Y, Koc E. Investigation of effects on plasma nitric oxide, malondialdehyde and total sialic acid levels of glyphosate in Kars creek transcaucasian barb (Capoeta capoeta [Guldenstaedt, 1773]) in Turkey. Fresenius Environ Bull 2012;21:123–6. [6] Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, et al. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J Biol Chem 1994;269:26066–75. [7] Mohan SK, Priyav V. Serum total sialic acid, lipid peroxidation, and glutathione reductase levels in patients with rheumatoid arthritis. Turk J Med Sci 2010;40:537–40. [8] Schauer R. Chemistry, metabolism, and biological functions of sialic acids. Adv Carbohydr Chem Biochem 1982;40:131–234. [9] Schroder PN, Giannis A. From substrate to transition state analogues: the first potent inhibitör of sialyltransferases. Angew Chem Int Ed 1999;38: 1379–80. [10] Yilmaz M, Karapehlivan M, Kaya I. Effects of zinc sulphate on transcaucasian barb, (Capoeta capoeta [Guldenstaedt, 1773]) plasma nitric oxide, malondialdehyde and total sialic acid levels. Kafkas Univ Vet Fak Derg 2012;18:61–4. [11] Yuen AW, Sander JW, Fluegel D, Patsalos PN, Bell GS, Johnson T, et al. Omega3 fatty acid supplementation in patients with chronic epilepsy: a randomized trial. Epilepsy Behav 2005;7:253–8. [12] Mazza M, Pomponi M, Janiri L, Bria P, Mazza S. Omega-3 fatty acids and antioxidants in neurological and psychiatric diseases: an overview. Prog Neuropsychopharmacol Biol Psychiatry 2007;31:12–26. [13] Sydow G. A simplified quick method for determination of sialic acid in serum. Biomed Biochim Acta 1985;44:1721–3. [14] Beutler E, Duran O, Kelley BM. Improved method for determination of blood glutathione. J Lab Clin Med 1963;28:882–8. [15] Yoshioka T, Kawada K, Shimada T, Mori M. Lipid peroxidation in maternal and cord blood and protective mechanism against active-oxygen toxicity in the blood. Am J Obstet Gynecol 1979;135:372–6.
99
[16] Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001;5:62–71. [17] Leonard SS, Harris GK, Shi X. Metal-induced oxidative stress and signal transduction. Free Radic Biol Med 2004;37:1921–42. [18] Stohs SJ, Baqchi D. Oxidative mechanisms in the toxicity of metal-ions. Free Radic Biol Med 1995;18:321–36. [19] Flora SJ, Mittal M, Mehta A. Heavy metal induced oxidative stress and its possible reversal by chelation therapy. Indian J Med Res 2008;128:501–23. [20] Moretto MB, Lermen CL, Morsch VM, Bohrer D, Ineu RP, Silva AC, et al. Effect of subchronic treatment with mercury chloride on NTPDase, 5 -nucleotidase and acetylcholinesterase from cerebral cortex of rats. J Trace Elem Med Biol 2004;17:255–60. [21] Farina M, Brandão R, de Lara FS, Pagliosa LB, Soares FA, Souza DO, et al. Profile of nonprotein thiols, lipid peroxidation and ␦-aminolevulinate dehydratase activity in mouse kidney and liver in response to acute exposure to mercuric chloride and sodium selenite. Toxicology 2002;184:179–87. [22] Farina M, Soares FA, Feoli A, Roehring C, Brusque AM, Rotta L, et al. In vitro effects of selenite and mercuric chloride on liver thiobarbituric acid-reactive substances and non-protein thiols from rats: influences of dietary cholesterol and polyunsaturated and saturated fatty acids. Nutrition 2003;19:531–5. [23] Rao MV, Sharma PS. Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice. Reprod Toxicol 2001;15:705–12. [24] Cigremis Y, Turel H, Adiguzel K, Akgoz M, Kart A, Karaman M, et al. The effects of acute acetaminophen toxicity on hepatic mRNA expression of SOD, CAT, GSH-Px, and levels of peroxynitrite, nitric oxide, reduced glutathione and malondialdehyde in rabbit. Mol Cell Biochem 2009;323:31–8. [25] Mahboob M, Shireen KF, Atkinson A, Khan AT. Lipid peroxidation and antioxidant enzyme activity in different organs of mice exposed to low level of mercury. J Environ Sci Health B 2001;36:687–97. [26] De Freitas AS, Funck VR, Rotta Mdos S, Bohrer D, Mörschbächer V, Puntel RL, et al. Diphenyl diselenide, a simple organoselenium compound, decreases methylmercury-induced cerebral, hepatic and renal oxidative stress and mercury deposition in adult mice. Brain Res Bull 2009;79:77–84. [27] Lund BO, Miller DM, Woods JS. Studies on Hg(II)-induced H2 O2 formation and oxidative stress in vivo and in vitro in rat kidney mitochondria. Biochem Pharmacol 1993;45:2017–24. [28] Lund BO, Miller DM, Woods JS. Mercury-induced H2 O2 production and lipid peroxidation in vitro in rat kidney mitochondria. Biochem Pharmacol 1991;42:181–7. [29] Ruan RS, Leong SK, Yeoh KH. Ototoxicity of sodium nitroprusside. Hear Res 1997;114:169–78. [30] Kong WJ, Ren T, Nuttall AL. Electrophysiological and morphological evaluation of the acute ototoxicity of sodium nitroprusside. Hear Res 1996;99:22–30. [31] Harris WS, Rambjør GS, Windsor SL, Diederich D. n-3 Fatty acids and urinary excretion of nitric oxide metabolites in humans. Am J Clin Nutr 1997;65:459–64. [32] Songur A, Sarsilmaz M, Sogut S, Ozyurt B, Ozyurt H, Zararsiz I, et al. Hypothalamic superoxide dismutase, xanthine oxidase, nitric oxide, and malondialdehyde in rats fed with fish omega-3 fatty acids. Prog Neuropsychopharmacol Biol Psychiatry 2004;28:693–8. [33] Yee S, Choi BH. Oxidative stress in neurotoxic effects of methylmercury poisoning. Neurotoxicology 1996;17:17–26. [34] Sydow G, Wittmann W, Bender E, Starick E. The sialic acid content of the serum of cattle infected with bovine leukosis virus. Arch Exp Vetmed 1988;42:194–7. [35] Sillanaukee P, Pönniö M, Jääskeläinen IP. Occurence of sialic acids in healthy humans and different disorders. Eur J Clin Invest 1999;29:413–25. [36] Kushner I. The acute phase response: an overview. Methods Enzymol 1988;163:373–83. [37] Patel AB, Venkatakrishna Bhatt H. Effect of lead on the blood serum, liver and brain sialoglycoconjugate levels in rats. Hum Exp Toxicol 1992;11:89–92. [38] Gorio A. Ganglioside enhancement of neuronal differentiation, plasticity, and repair. CRC Crit Rev Clin Neurobiol 1986;2:241–96. [39] Kalaiselvi T, Panneersalvam C. Effect of l-carnitine on the status of lipid peroxidation and antioxidants in aging rats. J Nutr Biochem 1998;9:575–81. [40] Barbosa DS, Cecchini R, El Kadri MZ, Rodríguez MA, Burini RC, Dichi I. Decreased oxidative stress in patients with ulcerative colitis supplemented with fish oil omega-3 fatty acids. Nutrition 2003;19:837–42. [41] Chautan M, Calaf R, Léonardi J, Charbonnier M, Andre M, Portugal H, et al. Inverse modifications of heart and liver alpha-tocopherol status by various dietary n-6/n-3 polyunsaturated ratios. J Lipid Res 1990;31:2201–8. [42] Sarsilmaz M, Songur A, Ozyurt H, Kus I, Ozen OA, Ozyurt B, et al. Potential role of dietary omega-3 essential fatty acids on some oxidant/antioxidant parameters in rats’ corpus striatum. Prostaglandins Leukot Essent Fatty Acids 2003;69:253–9. [43] Sprecher H. Interactions between the metabolism of n-3 and n-6 fatty acids. J Intern Med Suppl 1989;731:5–9.
