- Email: [email protected]
Effects of nicotine and vitamin E on glutathione reductase activity in some rat tissues in vivo and in vitro
European Journal of Pharmacology 554 (2007) 92 – 97 www.elsevier.com/locate/ejphar
Effects of nicotine and vitamin E on glutathione reductase activity in some rat tissues in vivo and in vitro Mustafa Erat a , Mehmet Ciftci a,b,⁎, Kenan Gumustekin c , Mustafa Gul c a
b
Biotechnology Application and Research Center, Ataturk University, 25240, Erzurum, Turkey Department of Chemistry, Arts and Science Faculty, Ataturk University, 25240, Erzurum, Turkey c Department of Physiology, Faculty of Medicine, Ataturk University, 25240, Erzurum, Turkey Received 11 May 2006; received in revised form 29 September 2006; accepted 5 October 2006 Available online 17 October 2006
Abstract Effects of nicotine, and nicotine + vitamin E on glutathione reductase (Glutathione: NADP+ oxidoreductase, EC 1.8.1.7) activity in the muscle, heart, lungs, testicles, kidney, stomach, brain and liver tissues were investigated in vivo and also in vitro. The groups were: nicotine [0.5 mg/kg/ day, intraperitoneal (i.p.)]; nicotine + vitamin E [75 mg/kg/day, intragastric (i.g.)]; and control group (receiving only vehicles). There were eight rats per group and supplementation period was 3 weeks. The results showed that nicotine (0.5 mg/kg, i.p.) inhibited glutathione reductase activity significantly in the liver, lungs, heart, stomach, kidney, and testicles by ∼ 61.5%, ∼ 65%, ∼70.5%, ∼ 72.5%, ∼ 64% and ∼ 71.5%, respectively, while it had activated glutathione reductase activity in the brain by ∼ 11.8%, and had no effect on the muscle glutathione reductase activity. Vitamin E supplementation prevented this nicotine-induced decrease in glutathione reductase activity in liver, lungs, heart, stomach, and kidney. However, it did not prevent this nicotine-induced decrease in testicles. In vitro studies were also carried out to elucidate the effects of nicotine and vitamin E on glutathione reductase activity. In vitro results correlated well with in vivo experimental results in liver, lungs, heart, stomach, and testicular tissues. These results show that vitamin E administration generally restores the inactivation of glutathione reductase activity due to nicotine administration in various rat tissues in vivo, and also in vitro. © 2006 Elsevier B.V. All rights reserved. Keywords: Glutathione reductase; Nicotine; Rat tissues; Vitamin E
1. Introduction Glutathione reductase (Glutathione: NADP+ oxidoreductase, E.C.1.8.1.7), a member of the pyridine-nucleotide disulfide oxidoreductase family of flavoenzymes, catalyzes the reduction of glutathione disulfide (GSSG) to reduced form (GSH) in the presence of NADPH. In order to maintain a high ratio of [GSH]/ [GSSG], the enzyme has a crucial role (Kondo et al., 1980). GSH is the major nonprotein sulfhydryl compound in all living organisms and it has been shown to be involved in the regulation of protein synthesis and enzyme organization, in ⁎ Corresponding author. Department of Chemistry, Arts and Science Faculty, Ataturk University 25240, Erzurum, Turkey. Tel.: +90 442 2314436; fax: + 90 442 2360948. E-mail address: [email protected] (M. Ciftci). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.10.008
formation of the deoxyribonucleotide precursors of DNA, in maintaining the sulfhydryl groups of intracellular proteins and in protection of the cells against free radicals and reactive oxygen species such as H2O2, O2· and ·OH (Deneke and Fanburg, 1989). The formation of reactive oxygen species in cells leads to the formation of free radicals in metabolic processes. These harmful species cause damage to many molecules such as lipids, proteins and nucleic acids. These harmful effects are controlled by antioxidant defense system in cells. The most important free radical chain breaking molecule in antioxidant defense system in various tissues of the body is glutathione (Ames et al., 1993; Bondy and Orozco, 1994; DeLeve and Kaplowitz, 1991; Gul et al., 2000; McCord, 1993). Furthermore, the enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase are
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97
necessary to remove these radicals and keep the cells stable. Under normal conditions, the reductive and oxidative capacity of the cell (redox state) is in favor of oxidation (Halliwell and Gutteridge, 1989; Rubin, 1993; Somani, 1996). However, reactive oxygen species produced in oxidative stress are removed by the antioxidant defense system. A number of drugs and chemicals increase the reactive oxygen species production in specific organs of the body. Many researchers have determined that nicotine contributes to reactive oxygen species production (Guemouri et al., 1993; Jenkins and Goldfarb, 1993; Leanderson and Tagesson, 1992; Wetscher et al., 1995). Cigarette smoking is common in many societies. Two third of the American adults are addicted to alcohol and 30% of them are addicted to both cigarettes and alcohol (Mehta et al., 1998; US Department of Health and Human Services, 1990; US Environmental Protection Agency, 1992; Weksler et al., 1990). Nicotine, the major toxic component of cigarette smoke (Del Boccio et al., 1990; Hoffmann et al., 1996; Maser, 1997; McGinnis and Foege, 1993; Pryor and Stone, 1993), is a risk factor for various cardiovascular diseases and cancer (Del Boccio et al., 1990; Hoffmann et al., 1996; Maser, 1997; McGinnis and Foege, 1993; Pryor and Stone, 1993). Kessler et al. (1996) have determined a marked increase in nicotine content in all kinds of cigarettes in the last decade in the United States. Shaw et al. (2000) reported that one cigarette decreases lifespan by 11 min. Nicotine is oxidized to its metabolite cotinine, which has a long half life and may contribute to vascular diseases (Sastry et al., 1995; Sastry and Gujrati, 1994; Shaw et al., 2000). Half lives of nicotine and its metabolite cotinine are 1.3–2.7 and 15–19 h, respectively (Buccafusco and Terry, 2003; Gilman et al., 1980). It is reported that chronic nicotine treatment decreases the cytochrome P450 IIE1, increases free radical formation and leads to oxidative damage in rats (Anandatheerthavarada et al., 1993; Ashakumary and Vijayammal, 1991; Bhagwat et al., 1998; Leanderson and Tagesson, 1992; Pryor and Stone, 1993; Weksler et al., 1990). We used vitamin E whether it counteracts against nicotineinduced adverse effects on glutathione reductase activity. Vitamin E is well accepted as nature's most effective lipidsoluble, chain-breaking antioxidant, protecting cell membranes from peroxidative damage (Brigelius-Flohe and Traber, 1999). Owing to widespread use of cigarettes, we thought that it is important to examine the effect of nicotine on glutathione reductase activity, and for this purpose, we investigated the in vivo effects of nicotine and nicotine + vitamin E on glutathione reductase activities in the rat muscle, heart, lungs, testicle, kidney, stomach, brain and liver in vivo, and we also carried out in vitro enzyme inhibition experiments. Effects of many chemicals and drugs on the red cell glutathione reductase enzyme activity have been investigated, such as streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, cefodizime, ofloxacin, levofloxacin, cefepime, cefazolin, and netilmicin (Erat and Ciftci, 2003; Erat et al., 2003, 2005), but no studies on other tissues were encountered in the previous reports.
93
2. Materials and methods 2.1. Materials NADP+, glucose 6-phosphate, protein assay reagent were purchased from Sigma Chem. Co. Germany. All other chemicals used were of analytical grade and were purchased from either Sigma or Merck. 2.2. Animals Twenty-four rats (Sprague–Dawley strain with a body weight of 225 ± 28 g), fed with standard laboratory chow and water, were used in the study. They were randomly divided into 3 groups (8 rats per group) and placed in separate cages during the study. The groups were as follows: Group I Nicotine (0.5 mg/kg/day, i.p.), Group II Nicotine (0.5 mg/kg/day, i.p.) + Vitamin E (75 mg/kg/day, i.g.) Group III Control group (received only the same amounts of vehicles, 0.9% NaCl solution, i.p., and corn oil, i.g.). Supplementation period was 3 weeks. Previously, it was shown that nicotine (0.6 mg/kg/day) induces oxidative stress in various rat tissues and supplementation of vitamin E (100 mg/kg/day) for 3 weeks counteracts against this nicotine induced oxidative stress (Helen et al., 2000). Animal experimentations were carried out in an ethically proper way by following guidelines as set by the Ethical Committee of the Ataturk University. 2.3. Preparation and administration of nicotine Hydrogen tartrate salt of nicotine (Sigma N-5260) was dissolved in 0.9% NaCl solution to get a 0.15 mg/ml concentration of nicotine. Then, pH of the nicotine solution was adjusted to 7.4 by 0.1N NaOH. Nicotine (0.5 mg/kg/day) was administered by i.p. injection to groups I and II for 3 weeks. 2.4. Preparation and administration of vitamin E Vitamin E (Ephynal 300 mg capsule, Roche, France) was dissolved in corn oil (30 mg/ml) and administered orally by a stomach tube (approximately 75 mg/kg/day) to group II for 3 weeks. 2.5. Sample collection At the end of the experiment, the animals were anesthetized with ketamine–HCl (Ketalar, Eczacibasi, Turkey, 20 mg/kg, i.p.). The animals were killed by exsanguination by cardiac puncture after thoracotomy. Then, each tissue was carefully removed, rinsed in saline and stored at −80 °C until homogenization. 2.6. Preparation of homogenate A piece of each tissue (approximately 300 mg) was homogenized by an OMNI TH International, model TH 220
94
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97
glutathione reductase from tissue homogenates of control animals were investigated by incubating for 5 min. Activities were measured by adding 20, 40, 60, 80, and 100 μl of 5 mM nicotine, and nicotine (5 mM) + vitamin E (10 mM). Control cuvette activity was accepted as 100%. 2.9. Activity determination
Fig. 1. In vivo effects of nicotine and nicotine + vitamin E on glutathione reductase activity in various rat tissues. ⁎: nicotine vs. control, #: nicotine + vit. E vs. control, #: nicotine + vit. E vs. nicotine. The results are means ± SD. One way ANOVA with post-hoc LSD test, P b 0.05 was accepted as statistically significant.
(Warrenton, VA 20187 USA) homogenizer in 20 mM Tris–HCl, pH 7.4 (1/10 weight/volume) on ice for 10 s at the first speed level. Then, the homogenates were centrifuged at 10,000×g for 15 min at 4 °C. The supernatants were stored at − 80 °C in aliquots until biochemical measurements.
The enzymatic activity was measured by Beutler's (1984) method. Protein was determined by Bradford's (1976) method by using bovine serum albumin as standard. The enzymatic activity and protein content were measured in 1 ml of each sample of enzyme. Then, enzyme activity was determined as EU/mg protein. One EU was defined as the oxidation of 1 μmol NADPH per min at 25 °C at optimal pH (pH 8.0). 2.10. Statistical analysis One-way analysis of variance (ANOVA) with post-hoc leastsignificant difference (LSD) test was used to compare the group means and P b 0.05 was considered statistically significant. SPSS for Windows (version 10.0) was used for statistical analyses.
2.7. Ammonium sulphate fractionation and dialysis 3. Results Ammonium sulphate (20–60%) precipitation was made in homogenate. Ammonium sulphate was slowly added for complete dissolution. It was centrifuged at 5000 ×g for 15 min and precipitate was dissolved in 20 mM Tris–HCl (pH 7.4), then dialysed at 4 °C in 20 mM Tris–HCl (pH 7.4) for 2 h with two changes of buffer. Thus, partially purified total glutathione reductase was obtained by ammonium sulphate fractionation and dialysis from tissue homogenates (muscle, heart, lungs, testicle, kidney, stomach, brain and liver). 2.8. In vitro studies In vitro effects of nicotine (5 mM), nicotine (5 mM) + vitamin E (10 mM dissolved in corn oil) on partially purified total
Nicotine (0.5 g/kg, i.p.) inhibited glutathione reductase activity significantly in the liver, lungs, heart, stomach, kidney, and testicles by ∼61.5%, ∼65%, ∼70.5%, ∼72.5%, ∼64% and ∼71.5%, respectively, while it had activated glutathione reductase activity in the brain by ∼11.8%, and it had no effect on the muscle glutathione reductase activity in vivo (Fig. 1). In addition, nicotine inhibited glutathione reductase activity in the liver, lungs, heart, stomach, and testicles by ∼20%, ∼20%, ∼14%, ∼18%, and ∼15% respectively in in vitro studies, except kidney and brain. Nicotine had no effect on muscle, kidney and brain glutathione reductase activities in in vitro studies. Vitamin E supplementation prevented this nicotine-induced decrease in glutathione reductase activity in liver, lungs, heart,
Table 1 In vitro effects of nicotine and nicotine + vitamin E on glutathione reductase activity in various rat tissues after 5 min incubation (n = 8) Tissues
Muscle (activity%)
Liver (activity%)
Lungs (activity%)
Heart (activity%)
Stomach (activity%)
Kidney (activity%)
Testicles (activity%)
Brain (activity%)
Control
100.0 (0.40EU/mg protein)
100.0 (0.40EU/mg protein)
100.0 (0.28EU/mg protein)
100.0 (0.26EU/mg protein)
100.0 (0.41EU/mg protein)
100.0 (0.44EU/mg protein)
100.0 (0.25EU/mg protein)
100.0 (0.40EU/mg protein)
20 40 60 80 100 20 40 60 80 100
100.1 100.4 100.0 104.3 102.3 100.0 100.0 101.0 101.0 101.0
85.2 88.1 80.2 80.4 85.2 100.0 103.0 102.0 102.0 99.5
79.4 79.5 78.5 79.2 78.0 99.1 95.3 97.8 98.4 100.2
85.3 88.1 88.4 90.6 86.9 98.5 98.6 100.4 100.1 101.0
91.1 88.4 87.3 87.3 82.4 97.3 99.8 96.9 99.2 100
96.2 97.8 99.4 100.2 100.4 102.4 100.0 101.0 100.0 100.0
84.6 83.5 82.4 81.2 84.7 94.5 90.4 94.5 94.5 94.5
100.0 100.0 99.3 100.0 99.5 100.0 100.0 100.0 100.0 100.0
Volume (μl)
Nicotine (5 mM)
Nicotine (5 mM) + Vitamin E (10 mM)
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97
stomach, kidney, and testicles in in vivo and in liver, lungs, heart, stomach, and testicles in in vitro studies. However, it did not prevent this nicotine-induced decrease in brain in in vitro studies. The results of the in vitro inhibition studies with nicotine (5 mM) and nicotine (5 mM) + vitamin E (10 mM) are shown in Table 1. 4. Discussion Glutathione reductase is essential for the maintenance of cellular glutathione in its reduced form, which is highly nucleophilic for many reactive electrophils (Carlberg and Mannervik, 1985). GSH is involved either as a substrate in the cytosolic GSH redox cycle, or is able to directly inactivate free radicals and reactive oxygen species, which are known to be effective stress agents (Meister, 1981). Many chemicals and drugs are known to have adverse or beneficial effects on human enzymes and metabolic events and the effects can be dramatic and systemic (Christensen et al., 1982). The inhibition of some important enzymes plays a key role in a metabolic pathway, e.g., some metabolic diseases such as diabetes mellitus are affected by enzyme activity (Gupta et al., 1997). Similarly, acetazolamide has an inhibitory effect on the carbonic anhydrase enzyme leading to diuresis (Warnock, 1989). Additionally, epiandrosterone was found to inhibit red blood cell glucose 6phosphate dehydrogenase uncompetitively and suppress hexose monophosphate shunt activity by more than 95% (Grossman et al., 1995). Some chemicals and drugs, such as nitrofurazone, nitrofurantoin, 5-nitroindol, 5-nitro-2-furoic acid, 2,4,6-trinitrobenzene sulfonate (McCallum and Barrett, 1995) and polyphenolic compounds inhibit glutathione reductase enzyme activity (Zhang et al., 1997). Furthermore, it is reported that human erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) are inhibited by some drugs (Beydemir et al., 2000; Bulbul et al., 2002, 2003); human erythrocyte glucose-6-phosphate dehydrogenase, human skin catalase are inhibited by some drugs (Ciftci et al., 2000; Ozdemir and Ciftci, 2006; Altikat et al., 2006) and chemicals, such as metamizol and magnesium sulfate (Ciftci et al., 2001); and bovine erythrocyte glutathione reductase is inhibited by cefotaxime and cefodizime (Erat and Ciftci, 2003). We have previously reported the inhibition of glucose-6-phosphate dehydrogenase and carbonic anhydrase by nicotine and restoration of these enzyme activities by vitamin E in vivo and in vitro (Ciftci et al., 2005; Gumustekin et al., 2005). Glutathione levels in liver and testicles decrease markedly after chronic nicotine treatment. Nicotine is oxidized to its main metabolite cotinine in liver and causes the formation of free radicals in tissues. The formation of these radicals causes oxidative damage. The decrease in GSH in tissues leads to oxidative tissue damage (Husain et al., 2001). There are many studies about the effects of nicotine on the enzyme activities. For example, inhibition of kidney and testicular superoxide dismutase and activation of liver superoxide dismutase by nicotine treatment in rats were shown. Inhibition of liver catalase and activation of kidney, lungs and testicular catalase by nicotine treatment in rats was reported (US Environmental Protection Agency, 1992).
95
Gumustekin et al. (2003) have reported that nicotine has increased the activity of glutathione peroxidase of the brain while vitamin-E abolished this effect. Nicotine has also inhibited the brain GST activity and this activity was restored by vitamin E, too. In addition, Suleyman et al. (2002) have reported that nicotine has inhibited the activities of glutathione peroxidase and superoxide dismutase of erythrocytes while vitamin-E reversed these effects. The increased mRNA levels of endothelial nitric oxide synthase by nicotine (Zhang et al., 2001), inhibition of typtophan hydroxylase by alcohol and nicotine-treated rats (Jang et al., 2002), while activation of typtophan hydroxylase by nicotine (1 mg/kg) were reported (Lee et al., 2002). Activation of the enzymes in liver metabolism in rats, which were exposed to cigarette smoke (Vanscheeuwijck et al., 2002), and the increased (2.5 fold) expression of enzymes involved in energy metabolism in nicotine-treated rats (Hu et al., 2002) were found. In addition, activation of adenylate cyclase in nicotine (6 mg/kg)-treated rats was also demonstrated (AbreuVillaca et al., 2003). In this study, nicotine inhibited glutathione reductase activity in the liver, lungs, heart, stomach, kidney, and testicular tissues in vivo compared with the control group. Nicotine can cause competitive inhibition by binding the active site of glutathione reductase. It is also possible that it can cause noncompetitive inhibition by binding other sites affecting three dimensional structure of the enzyme (Lehninger et al., 1993). However, it had no effect on the muscle glutathione reductase activity and it activated brain glutathione reductase. Administration of vitamin E with nicotine prevented this inhibition in the liver, lungs, heart, stomach, and kidney. However, nicotine + vitamin E had no effects on the muscle glutathione reductase activity compared with the control group. The reason for the increased glutathione reductase activity in brain tissue may be a compensatory increase to remove the increased reactive oxygen species due to nicotine administration in brain (Gumustekin et al., 2003). Vitamin E might have diffused in the brain to activate the glutathione reductase activity as a lipid soluble antioxidant (Brigelius-Flohe and Traber, 1999). However, the glutathione reductase activity in muscles may not be affected as much as it was affected in the brain tissue from the nicotine administration. As seen in Table 1, nicotine inhibited glutathione reductase activity in vitro, in liver, lungs, heart, stomach and testicular tissues moderately and this inhibition was eliminated by vitamin E. In vitro glutathione reductase inhibition results correlated well with in vivo experimental results in muscle, liver, lungs, heart, stomach and testicles. In the in vivo study, when average volume of blood present in a rat is accepted to be about 15 ml, 0.5 mg/kg dose corresponds to 0.0513 mM nicotine concentration. In the in vitro study, enzyme activities were determined by using 0.04, 0.08, 0.12, 0.16 and 0.20 mM cuvette concentrations. Since similar nicotine concentrations have been used in both in vivo and in vitro experiments, our results obtained by in vitro and in vivo studies were comparable. However, in vitro and in vivo results did not correlate well in kidney tissue. Although it is difficult to envisage how nicotine and vitamin E modify the glutathione reductase activity in various tissues, the
96
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97
inhibition of glutathione reductase by nicotine may result from the binding of the active site of glutathione reductase in liver, lungs, heart, stomach and testicles. On the other hand, facilitating proton transfer may be responsible for the increased glutathione reductase activity, which may be a different isoenzyme of glutathione reductase in kidney tissue (Supuran et al., 2003). The results of this study show that vitamin E administration may generally restore the glutathione reductase activity inactivated due to nicotine administration in various tissues in vivo, and also in vitro. References Abreu-Villaca, Y., Seidler, F.J., Slotkin, T.A., 2003. Impact of adolescent nicotine exposure on adenylyl cyclase-mediated cell signaling: enzyme induction, neurotransmitter-specific effects, regional selectivities, and the role of withdrawal. Brain Res. 988, 164–172. Altikat, S., Coban, T.A.K., Ciftci, M., Ozdemir, H., 2006. In vitro effects of some drugs on catalase purified from human skin. J. Enzyme Inhib. Med. Chem. 21, 231–234. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U. S. A. 90, 7915–7922. Anandatheerthavarada, H.K., Williams, J.F., Wecker, L., 1993. The chronic administration of nicotine induces cytochrome P450 in rat brain. J. Neurochem. 60, 1941–1944. Ashakumary, L., Vijayammal, P.L., 1991. Lipid peroxidation in nicotine treated rats. J. Ecotoxicol. Environ. Monit. 1, 283–290. Beutler, E., 1984. Red Cell Metabolism: A Manual of Biochemical Method. Grune and Stration, New York. Beydemir, S., Ciftci, M., Ozmen, I., Buyukokuroglu, M.E., Ozdemir, H., Kufrevioglu, O.I., 2000. Effects of some medical drugs on enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Pharmacol. Res. 42, 187–191. Bhagwat, S.V., Vijayasarathy, C., Raza, H., Mullick, J., Avadhani, N.G., 1998. Preferential effects of nicotine and 4-(N-methyl-N-nitrosamine)-1-(3pyridyl)-1-butanone on mitochondrial glutathione S-transferase A4-4 induction and increased oxidative stress in the rat brain. Biochem. Pharmacol. 56, 831–839. Bondy, S.C., Orozco, J., 1994. Effects of ethanol treatment upon sources of reactive oxygen species in brain and liver. Alcohol Alcohol. 29, 375–383. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brigelius-Flohe, R., Traber, M.G., 1999. Vitamin E: function and metabolism. FASEB J. 13, 1145–1155. Buccafusco, J.J., Terry Jr., A.V., 2003. The potential role of cotinine in the cognitive and neuroprotective actions of nicotine. Life Sci. 72, 2931–2942. Bulbul, M., Saracoglu, N., Kufrevioglu, O.I., Ciftci, M., 2002. Bile acid derivatives of 5-amino-1,3,4-thiadiazole-2-sulfonamide as new carbonic anhydrase inhibitors: synthesis and investigation of inhibition effects. Bioorg. Med. Chem. 10, 2561–2567. Bulbul, M., Hisar, O., Beydemir, S., Ciftci, M., Kufrevioglu, O.I., 2003. The in vitro and in vivo inhibitory effects of some sulfonamide derivatives on rainbow trout (Oncorhynchus mykiss) erythrocyte carbonic anhydrase activity. J. Enzyme Inhib. Med. Chem. 18, 371–375. Carlberg, I., Mannervik, B., 1985. Glutathione reductase. Methods Enzymol. 113, 484–490. Christensen, G.M., Olson, D., Riedel, B., 1982. Chemical effects on the activity of eight enzymes: a review and a discussion relevant to environmental monitoring. Environ. Res. 29, 247–255. Ciftci, M., Kufrevioglu, O.I., Gundogdu, M., Ozmen, I., 2000. Effects of some antibiotics on enzyme activity of glucose-6-phosphate dehydrogenase from human erythrocytes. Pharmacol. Res. 41, 109–113.
Ciftci, M., Ozmen, I., Buyukokuroglu, M.E., Pence, S., Kufrevioglu, O.I., 2001. Effects of metamizol and magnesium sulfate on enzyme activity of glucose 6-phosphate dehydrogenase from human erythrocyte in vitro and rat erythrocyte in vivo. Clin. Biochem. 34, 297–302. Ciftci, M., Bulbul, M., Gul, M., Gumustekin, K., Dane, S., Suleyman, H., 2005. Effects of nicotine and vitamin E on carbonic anhydrase activity in some rat tissues in vivo and in vitro. J. Enzyme Inhib. Med. Chem. 20, 103–108. Del Boccio, G., Lapenna, D., Porreca, E., Pennelli, A., Savini, F., Feliciani, P., Ricci, G., Cuccurullo, F., 1990. Aortic antioxidant defence mechanisms: time-related changes in cholesterol-fed rabbits. Atherosclerosis 81, 127–135. DeLeve, L.D., Kaplowitz, N., 1991. Glutathione metabolism and its role in hepatotoxicity. Pharmacol. Ther. 52, 287–305. Deneke, S.M., Fanburg, B.L., 1989. Regulation of cellular glutathione. Am. J. Physiol. 257, L163–L173. Erat, M., Ciftci, M., 2003. In vitro effects of some antibiotics on glutathione reductase from sheep liver. J. Enzyme Inhib. Med. Chem. 18, 545–550. Erat, M., Sakiroglu, H., Ciftci, M., 2003. Purification and characterization of glutathione reductase from bovine erythrocytes. Prep. Biochem. Biotechnol. 33, 283–300. Erat, M., Demir, H., Sakiroglu, H., 2005. Purification of glutathione reductase from chicken liver and investigation of kinetic properties. Appl. Biochem. Biotechnol. 125, 127–138. Gilman, A.G., Goodman, L.S., Rall, T.W., Murad, F., 1980. Goodman and Gilman's The Pharmacological Basis of Therapeutics. MacMillan Publishing Co., New York. Grossman, S., Budinsky, R., Jollow, D., 1995. Dapsone-induced hemolytic anemia: role of glucose-6-phosphate dehydrogenase in the hemolytic response of rat erythrocytes to N-hydroxydapsone. J. Pharmacol. Exp. Ther. 273, 870–877. Guemouri, L., Lecomte, E., Herbeth, B., Pirollet, P., Paille, F., Siest, G., Artur, Y., 1993. Blood activities of antioxidant enzymes in alcoholics before and after withdrawal. J. Stud. Alcohol 54, 626–629. Gul, M., Kutay, F.Z., Temocin, S., Hanninen, O., 2000. Cellular and clinical implications of glutathione. Indian J. Exp. Biol. 38, 625–634. Gumustekin, K., Altinkaynak, K., Timur, H., Taysi, S., Oztasan, N., Polat, M.F., Akcay, F., Suleyman, H., Dane, S., Gul, M., 2003. Vitamin E but not Hippophea rhamnoides L. prevented nicotine-induced oxidative stress in rat brain. Human Exp. Toxicol. 22, 425–431. Gumustekin, K., Ciftci, M., Coban, A., Altikat, S., Aktas, O., Gul, M., Timur, H., Dane, S., 2005. Effects of nicotine and vitamin E on glucose 6-phosphate dehydrogenase activity in some rat tissues in vivo and in vitro. J. Enzyme. Inhib. Med. Chem. 20, 497–502. Gupta, B.L., Nehal, M., Baquer, N.Z., 1997. Effect of experimental diabetes on the activities of hexokinase, glucose-6-phosphate dehydrogenase and catecholamines in rat erythrocytes of different ages. Indian J. Exp. Biol. 35, 792–795. Halliwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine. Clarendon Press, Oxford. Helen, A., Krishnakumar, K, Vijayammal, P.L., Augusti, K.T., 2000. Antioxidant effect of onion oil (Allium cepa. Linn) on the damages induced by nicotine in rats as compared to alpha-tocopherol. Toxicol. Lett. 116, 61–68. Hoffmann, D., Rivenson, A., Hecht, S.S., 1996. The biological significance of tobacco-specific N-nitrosamines: smoking and adenocarcinoma of the lung. Crit. Rev. Toxicol. 26, 199–211. Hu, D., Cao, K., Peterson-Wakeman, R., Wang, R., 2002. Altered profile of gene expression in rat hearts induced by chronic nicotine consumption. Biochem. Biophys. Res. Commun. 297, 729–736. Husain, K., Scott, B.R., Reddy, S.K., Somani, S.M., 2001. Chronic ethanol and nicotine interaction on rat tissue antioxidant defense system. Alcohol 25, 89–97. Jang, M.H., Shin, M.C., Lee, T.H., Kim, Y.P., Jung, S.B., Shin, D.H., Kim, H., Kim, S.S., Kim, E.H., Kim, C.J., 2002. Alcohol and nicotine administration inhibits serotonin synthesis and tryptophan hydroxylase expression in dorsal and median raphe of young rats. Neurosci. Lett. 329, 141–144. Jenkins, R.R., Goldfarb, A., 1993. Introduction: oxidant stress, aging, and exercise. Med. Sci. Sports Exerc. 25, 210–212.
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97 Kessler, D.A., Witt, A.M., Barnett, P.S., Zeller, M.R., Natanblut, S.L., Wilkenfeld, J.P., Lorraine, C.C., Thompson, L.J., Schultz, W.B., 1996. The Food and Drug Administration's regulation of tobacco products. N. Engl. J. Med. 335, 988–994. Kondo, T., Dale, G.L., Beutler, E., 1980. Glutathione transport by inside-out vesicles from human erythrocytes. Proc. Natl. Acad. Sci. U. S. A. 77, 6359–6362. Leanderson, P., Tagesson, C., 1992. Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation. Chem. Biol. Interact. 81, 197–208. Lee, T.H., Jang, M.H., Shin, M.C., Lim, B.V., Choi, H.H., Kim, H., Kim, E.H., Kim, C.J., 2002. Nicotine administration increases serotonin synthesis and tryptophan hydroxylase expressions in dorsal raphe of food-deprived rats. Nutr. Res. 22, 1445–1452. Lehninger, A.L., Nelson, D.L., Cox, M.M., 1993. Principles of Biochemistry. Worth Publishers, New York. Maser, E., 1997. Stress, hormonal changes, alcohol, food constituents and drugs: factors that advance the incidence of tobacco smoke-related cancer? Trends Pharmacol. Sci. 18, 270–275. McCallum, M.J., Barrett, J., 1995. The purification and properties of glutathione reductase from the cestode Moniezia expansa. Int. J. Biochem. Cell Biol. 27, 393–401. McCord, J.M., 1993. Human disease, free radicals, and the oxidant/antioxidant balance. Clin. Biochem. 26, 351–357. McGinnis, J.M., Foege, W.H., 1993. Actual causes of death in the United States. JAMA 270, 2207–2212. Mehta, M.C., Jain, A.C., Billie, M., 1998. Combined effects of alcohol and nicotine on cardiovascular performance in a canine model. J. Cardiovasc. Pharmacol. 31, 930–936. Meister, A., 1981. Metabolism and functions of glutathione. Trends Biochem. Sci. 6, 231–234. Ozdemir, H., Ciftci, M., 2006. In vitro effects of some drugs on human red blood cell glucose-6-phosphate dehydrogenase enzyme activity. J. Enzyme Inhib. Med. Chem. 21, 75–80. Pryor, W.A., Stone, K., 1993. Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann. N. Y. Acad. Sci. 686, 12–27 (discussion 27-18). Rubin, E., 1993. The chemical pathogenesis of alcohol-induced tissue injury. Alcohol Health Res. World 17, 272–278. Sastry, B.V., Chance, M.B., Singh, G., Horn, J.L., Janson, V.E., 1995. Distribution and retention of nicotine and its metabolite, cotinine, in the rat as a function of time. Pharmacology 50, 128–136.
97
Sastry, B.V., Gujrati, V.R., 1994. Activation of PAF-acetylhydrolase by nicotine and cotinine and their possible involvement in arterial thrombosis. Ann. N. Y. Acad. Sci. 714, 312–314. Shaw, M., Mitchell, R., Dorling, D., 2000. Time for a smoke? One cigarette reduces your life by 11 minutes. BMJ 320, 53. Somani, S.M., 1996. Exercise, drugs and tissue specific antioxidant system. In: Somani, S.M. (Ed.), Pharmacology in Exercise and Sports. CRC Press, Boca Raton FL, pp. 57–95. Suleyman, H., Gumustekin, K., Taysi, S., Keles, S., Oztasan, N., Aktas, O., Altinkaynak, K., Timur, H., Akcay, F., Akar, S., Dane, S., Gul, M., 2002. Beneficial effects of Hippophae rhamnoides L. on nicotine induced oxidative stress in rat blood compared with vitamin E. Biol. Pharm. Bull. 25, 1133–1136. Supuran, C.T., Scozzafava, A., Casini, A., 2003. Carbonic anhydrase inhibitors. Med. Res. Rev. 23, 146–189. US Department of Health and Human Services, 1990. Seventh Special Report to the US Congress on Alcohol and Health. Washington, DC: National Institute on Alcohol Abuse and Alcoholism (US Department of Health and Human Services Publication ADM 90, 1656). US Environmental Protection Agency, 1992. Respiratory Health Effects of Passive Smoking, Lung Cancer and Other Disorders. Washington, DC: US Environmental Protection Agency (US Environmental Protection Agency Publication 600, 6-90/006F). Vanscheeuwijck, P.M., Teredesai, A., Terpstra, P.M., Verbeeck, J., Kuhl, P., Gerstenberg, B., Gebel, S., Carmines, E.L., 2002. Evaluation of the potential effects of ingredients added to cigarettes. Part 4: Subchronic inhalation toxicity. Food Chem. Toxicol. 40, 113–131. Warnock, D.G., 1989. Diuretic agents. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. Appletion and Lange, London, pp. 183–197. Weksler, B.B., Moore, A., Tepler, J., 1990. Hematology. In: Andreoli, T.E., Carpenter, C.C.J., Plum, F., Smith, L.H.J. (Eds.), Cecil Essentials of Medicine. WB Saunders Co., Philadelphia, pp. 341–363. Wetscher, G.J., Bagchi, M., Bagchi, D., Perdikis, G., Hinder, P.R., Glaser, K., Hinder, R.A., 1995. Free radical production in nicotine treated pancreatic tissue. Free Radic. Biol. Med. 18, 877–882. Zhang, K., Yang, E.B., Tang, W.Y., Wong, K.P., Mack, P., 1997. Inhibition of glutathione reductase by plant polyphenols. Biochem. Pharmacol. 54, 1047–1053. Zhang, S., Day, I., Ye, S., 2001. Nicotine induced changes in gene expression by human coronary artery endothelial cells. Atherosclerosis 154, 277–283.
Effects of nicotine and vitamin E on glutathione reductase activity in some rat tissues in vivo and in vitro Mustafa Erat a , Mehmet Ciftci a,b,⁎, Kenan Gumustekin c , Mustafa Gul c a
b
Biotechnology Application and Research Center, Ataturk University, 25240, Erzurum, Turkey Department of Chemistry, Arts and Science Faculty, Ataturk University, 25240, Erzurum, Turkey c Department of Physiology, Faculty of Medicine, Ataturk University, 25240, Erzurum, Turkey Received 11 May 2006; received in revised form 29 September 2006; accepted 5 October 2006 Available online 17 October 2006
Abstract Effects of nicotine, and nicotine + vitamin E on glutathione reductase (Glutathione: NADP+ oxidoreductase, EC 1.8.1.7) activity in the muscle, heart, lungs, testicles, kidney, stomach, brain and liver tissues were investigated in vivo and also in vitro. The groups were: nicotine [0.5 mg/kg/ day, intraperitoneal (i.p.)]; nicotine + vitamin E [75 mg/kg/day, intragastric (i.g.)]; and control group (receiving only vehicles). There were eight rats per group and supplementation period was 3 weeks. The results showed that nicotine (0.5 mg/kg, i.p.) inhibited glutathione reductase activity significantly in the liver, lungs, heart, stomach, kidney, and testicles by ∼ 61.5%, ∼ 65%, ∼70.5%, ∼ 72.5%, ∼ 64% and ∼ 71.5%, respectively, while it had activated glutathione reductase activity in the brain by ∼ 11.8%, and had no effect on the muscle glutathione reductase activity. Vitamin E supplementation prevented this nicotine-induced decrease in glutathione reductase activity in liver, lungs, heart, stomach, and kidney. However, it did not prevent this nicotine-induced decrease in testicles. In vitro studies were also carried out to elucidate the effects of nicotine and vitamin E on glutathione reductase activity. In vitro results correlated well with in vivo experimental results in liver, lungs, heart, stomach, and testicular tissues. These results show that vitamin E administration generally restores the inactivation of glutathione reductase activity due to nicotine administration in various rat tissues in vivo, and also in vitro. © 2006 Elsevier B.V. All rights reserved. Keywords: Glutathione reductase; Nicotine; Rat tissues; Vitamin E
1. Introduction Glutathione reductase (Glutathione: NADP+ oxidoreductase, E.C.1.8.1.7), a member of the pyridine-nucleotide disulfide oxidoreductase family of flavoenzymes, catalyzes the reduction of glutathione disulfide (GSSG) to reduced form (GSH) in the presence of NADPH. In order to maintain a high ratio of [GSH]/ [GSSG], the enzyme has a crucial role (Kondo et al., 1980). GSH is the major nonprotein sulfhydryl compound in all living organisms and it has been shown to be involved in the regulation of protein synthesis and enzyme organization, in ⁎ Corresponding author. Department of Chemistry, Arts and Science Faculty, Ataturk University 25240, Erzurum, Turkey. Tel.: +90 442 2314436; fax: + 90 442 2360948. E-mail address: [email protected] (M. Ciftci). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.10.008
formation of the deoxyribonucleotide precursors of DNA, in maintaining the sulfhydryl groups of intracellular proteins and in protection of the cells against free radicals and reactive oxygen species such as H2O2, O2· and ·OH (Deneke and Fanburg, 1989). The formation of reactive oxygen species in cells leads to the formation of free radicals in metabolic processes. These harmful species cause damage to many molecules such as lipids, proteins and nucleic acids. These harmful effects are controlled by antioxidant defense system in cells. The most important free radical chain breaking molecule in antioxidant defense system in various tissues of the body is glutathione (Ames et al., 1993; Bondy and Orozco, 1994; DeLeve and Kaplowitz, 1991; Gul et al., 2000; McCord, 1993). Furthermore, the enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase are
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97
necessary to remove these radicals and keep the cells stable. Under normal conditions, the reductive and oxidative capacity of the cell (redox state) is in favor of oxidation (Halliwell and Gutteridge, 1989; Rubin, 1993; Somani, 1996). However, reactive oxygen species produced in oxidative stress are removed by the antioxidant defense system. A number of drugs and chemicals increase the reactive oxygen species production in specific organs of the body. Many researchers have determined that nicotine contributes to reactive oxygen species production (Guemouri et al., 1993; Jenkins and Goldfarb, 1993; Leanderson and Tagesson, 1992; Wetscher et al., 1995). Cigarette smoking is common in many societies. Two third of the American adults are addicted to alcohol and 30% of them are addicted to both cigarettes and alcohol (Mehta et al., 1998; US Department of Health and Human Services, 1990; US Environmental Protection Agency, 1992; Weksler et al., 1990). Nicotine, the major toxic component of cigarette smoke (Del Boccio et al., 1990; Hoffmann et al., 1996; Maser, 1997; McGinnis and Foege, 1993; Pryor and Stone, 1993), is a risk factor for various cardiovascular diseases and cancer (Del Boccio et al., 1990; Hoffmann et al., 1996; Maser, 1997; McGinnis and Foege, 1993; Pryor and Stone, 1993). Kessler et al. (1996) have determined a marked increase in nicotine content in all kinds of cigarettes in the last decade in the United States. Shaw et al. (2000) reported that one cigarette decreases lifespan by 11 min. Nicotine is oxidized to its metabolite cotinine, which has a long half life and may contribute to vascular diseases (Sastry et al., 1995; Sastry and Gujrati, 1994; Shaw et al., 2000). Half lives of nicotine and its metabolite cotinine are 1.3–2.7 and 15–19 h, respectively (Buccafusco and Terry, 2003; Gilman et al., 1980). It is reported that chronic nicotine treatment decreases the cytochrome P450 IIE1, increases free radical formation and leads to oxidative damage in rats (Anandatheerthavarada et al., 1993; Ashakumary and Vijayammal, 1991; Bhagwat et al., 1998; Leanderson and Tagesson, 1992; Pryor and Stone, 1993; Weksler et al., 1990). We used vitamin E whether it counteracts against nicotineinduced adverse effects on glutathione reductase activity. Vitamin E is well accepted as nature's most effective lipidsoluble, chain-breaking antioxidant, protecting cell membranes from peroxidative damage (Brigelius-Flohe and Traber, 1999). Owing to widespread use of cigarettes, we thought that it is important to examine the effect of nicotine on glutathione reductase activity, and for this purpose, we investigated the in vivo effects of nicotine and nicotine + vitamin E on glutathione reductase activities in the rat muscle, heart, lungs, testicle, kidney, stomach, brain and liver in vivo, and we also carried out in vitro enzyme inhibition experiments. Effects of many chemicals and drugs on the red cell glutathione reductase enzyme activity have been investigated, such as streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, cefodizime, ofloxacin, levofloxacin, cefepime, cefazolin, and netilmicin (Erat and Ciftci, 2003; Erat et al., 2003, 2005), but no studies on other tissues were encountered in the previous reports.
93
2. Materials and methods 2.1. Materials NADP+, glucose 6-phosphate, protein assay reagent were purchased from Sigma Chem. Co. Germany. All other chemicals used were of analytical grade and were purchased from either Sigma or Merck. 2.2. Animals Twenty-four rats (Sprague–Dawley strain with a body weight of 225 ± 28 g), fed with standard laboratory chow and water, were used in the study. They were randomly divided into 3 groups (8 rats per group) and placed in separate cages during the study. The groups were as follows: Group I Nicotine (0.5 mg/kg/day, i.p.), Group II Nicotine (0.5 mg/kg/day, i.p.) + Vitamin E (75 mg/kg/day, i.g.) Group III Control group (received only the same amounts of vehicles, 0.9% NaCl solution, i.p., and corn oil, i.g.). Supplementation period was 3 weeks. Previously, it was shown that nicotine (0.6 mg/kg/day) induces oxidative stress in various rat tissues and supplementation of vitamin E (100 mg/kg/day) for 3 weeks counteracts against this nicotine induced oxidative stress (Helen et al., 2000). Animal experimentations were carried out in an ethically proper way by following guidelines as set by the Ethical Committee of the Ataturk University. 2.3. Preparation and administration of nicotine Hydrogen tartrate salt of nicotine (Sigma N-5260) was dissolved in 0.9% NaCl solution to get a 0.15 mg/ml concentration of nicotine. Then, pH of the nicotine solution was adjusted to 7.4 by 0.1N NaOH. Nicotine (0.5 mg/kg/day) was administered by i.p. injection to groups I and II for 3 weeks. 2.4. Preparation and administration of vitamin E Vitamin E (Ephynal 300 mg capsule, Roche, France) was dissolved in corn oil (30 mg/ml) and administered orally by a stomach tube (approximately 75 mg/kg/day) to group II for 3 weeks. 2.5. Sample collection At the end of the experiment, the animals were anesthetized with ketamine–HCl (Ketalar, Eczacibasi, Turkey, 20 mg/kg, i.p.). The animals were killed by exsanguination by cardiac puncture after thoracotomy. Then, each tissue was carefully removed, rinsed in saline and stored at −80 °C until homogenization. 2.6. Preparation of homogenate A piece of each tissue (approximately 300 mg) was homogenized by an OMNI TH International, model TH 220
94
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97
glutathione reductase from tissue homogenates of control animals were investigated by incubating for 5 min. Activities were measured by adding 20, 40, 60, 80, and 100 μl of 5 mM nicotine, and nicotine (5 mM) + vitamin E (10 mM). Control cuvette activity was accepted as 100%. 2.9. Activity determination
Fig. 1. In vivo effects of nicotine and nicotine + vitamin E on glutathione reductase activity in various rat tissues. ⁎: nicotine vs. control, #: nicotine + vit. E vs. control, #: nicotine + vit. E vs. nicotine. The results are means ± SD. One way ANOVA with post-hoc LSD test, P b 0.05 was accepted as statistically significant.
(Warrenton, VA 20187 USA) homogenizer in 20 mM Tris–HCl, pH 7.4 (1/10 weight/volume) on ice for 10 s at the first speed level. Then, the homogenates were centrifuged at 10,000×g for 15 min at 4 °C. The supernatants were stored at − 80 °C in aliquots until biochemical measurements.
The enzymatic activity was measured by Beutler's (1984) method. Protein was determined by Bradford's (1976) method by using bovine serum albumin as standard. The enzymatic activity and protein content were measured in 1 ml of each sample of enzyme. Then, enzyme activity was determined as EU/mg protein. One EU was defined as the oxidation of 1 μmol NADPH per min at 25 °C at optimal pH (pH 8.0). 2.10. Statistical analysis One-way analysis of variance (ANOVA) with post-hoc leastsignificant difference (LSD) test was used to compare the group means and P b 0.05 was considered statistically significant. SPSS for Windows (version 10.0) was used for statistical analyses.
2.7. Ammonium sulphate fractionation and dialysis 3. Results Ammonium sulphate (20–60%) precipitation was made in homogenate. Ammonium sulphate was slowly added for complete dissolution. It was centrifuged at 5000 ×g for 15 min and precipitate was dissolved in 20 mM Tris–HCl (pH 7.4), then dialysed at 4 °C in 20 mM Tris–HCl (pH 7.4) for 2 h with two changes of buffer. Thus, partially purified total glutathione reductase was obtained by ammonium sulphate fractionation and dialysis from tissue homogenates (muscle, heart, lungs, testicle, kidney, stomach, brain and liver). 2.8. In vitro studies In vitro effects of nicotine (5 mM), nicotine (5 mM) + vitamin E (10 mM dissolved in corn oil) on partially purified total
Nicotine (0.5 g/kg, i.p.) inhibited glutathione reductase activity significantly in the liver, lungs, heart, stomach, kidney, and testicles by ∼61.5%, ∼65%, ∼70.5%, ∼72.5%, ∼64% and ∼71.5%, respectively, while it had activated glutathione reductase activity in the brain by ∼11.8%, and it had no effect on the muscle glutathione reductase activity in vivo (Fig. 1). In addition, nicotine inhibited glutathione reductase activity in the liver, lungs, heart, stomach, and testicles by ∼20%, ∼20%, ∼14%, ∼18%, and ∼15% respectively in in vitro studies, except kidney and brain. Nicotine had no effect on muscle, kidney and brain glutathione reductase activities in in vitro studies. Vitamin E supplementation prevented this nicotine-induced decrease in glutathione reductase activity in liver, lungs, heart,
Table 1 In vitro effects of nicotine and nicotine + vitamin E on glutathione reductase activity in various rat tissues after 5 min incubation (n = 8) Tissues
Muscle (activity%)
Liver (activity%)
Lungs (activity%)
Heart (activity%)
Stomach (activity%)
Kidney (activity%)
Testicles (activity%)
Brain (activity%)
Control
100.0 (0.40EU/mg protein)
100.0 (0.40EU/mg protein)
100.0 (0.28EU/mg protein)
100.0 (0.26EU/mg protein)
100.0 (0.41EU/mg protein)
100.0 (0.44EU/mg protein)
100.0 (0.25EU/mg protein)
100.0 (0.40EU/mg protein)
20 40 60 80 100 20 40 60 80 100
100.1 100.4 100.0 104.3 102.3 100.0 100.0 101.0 101.0 101.0
85.2 88.1 80.2 80.4 85.2 100.0 103.0 102.0 102.0 99.5
79.4 79.5 78.5 79.2 78.0 99.1 95.3 97.8 98.4 100.2
85.3 88.1 88.4 90.6 86.9 98.5 98.6 100.4 100.1 101.0
91.1 88.4 87.3 87.3 82.4 97.3 99.8 96.9 99.2 100
96.2 97.8 99.4 100.2 100.4 102.4 100.0 101.0 100.0 100.0
84.6 83.5 82.4 81.2 84.7 94.5 90.4 94.5 94.5 94.5
100.0 100.0 99.3 100.0 99.5 100.0 100.0 100.0 100.0 100.0
Volume (μl)
Nicotine (5 mM)
Nicotine (5 mM) + Vitamin E (10 mM)
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97
stomach, kidney, and testicles in in vivo and in liver, lungs, heart, stomach, and testicles in in vitro studies. However, it did not prevent this nicotine-induced decrease in brain in in vitro studies. The results of the in vitro inhibition studies with nicotine (5 mM) and nicotine (5 mM) + vitamin E (10 mM) are shown in Table 1. 4. Discussion Glutathione reductase is essential for the maintenance of cellular glutathione in its reduced form, which is highly nucleophilic for many reactive electrophils (Carlberg and Mannervik, 1985). GSH is involved either as a substrate in the cytosolic GSH redox cycle, or is able to directly inactivate free radicals and reactive oxygen species, which are known to be effective stress agents (Meister, 1981). Many chemicals and drugs are known to have adverse or beneficial effects on human enzymes and metabolic events and the effects can be dramatic and systemic (Christensen et al., 1982). The inhibition of some important enzymes plays a key role in a metabolic pathway, e.g., some metabolic diseases such as diabetes mellitus are affected by enzyme activity (Gupta et al., 1997). Similarly, acetazolamide has an inhibitory effect on the carbonic anhydrase enzyme leading to diuresis (Warnock, 1989). Additionally, epiandrosterone was found to inhibit red blood cell glucose 6phosphate dehydrogenase uncompetitively and suppress hexose monophosphate shunt activity by more than 95% (Grossman et al., 1995). Some chemicals and drugs, such as nitrofurazone, nitrofurantoin, 5-nitroindol, 5-nitro-2-furoic acid, 2,4,6-trinitrobenzene sulfonate (McCallum and Barrett, 1995) and polyphenolic compounds inhibit glutathione reductase enzyme activity (Zhang et al., 1997). Furthermore, it is reported that human erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) are inhibited by some drugs (Beydemir et al., 2000; Bulbul et al., 2002, 2003); human erythrocyte glucose-6-phosphate dehydrogenase, human skin catalase are inhibited by some drugs (Ciftci et al., 2000; Ozdemir and Ciftci, 2006; Altikat et al., 2006) and chemicals, such as metamizol and magnesium sulfate (Ciftci et al., 2001); and bovine erythrocyte glutathione reductase is inhibited by cefotaxime and cefodizime (Erat and Ciftci, 2003). We have previously reported the inhibition of glucose-6-phosphate dehydrogenase and carbonic anhydrase by nicotine and restoration of these enzyme activities by vitamin E in vivo and in vitro (Ciftci et al., 2005; Gumustekin et al., 2005). Glutathione levels in liver and testicles decrease markedly after chronic nicotine treatment. Nicotine is oxidized to its main metabolite cotinine in liver and causes the formation of free radicals in tissues. The formation of these radicals causes oxidative damage. The decrease in GSH in tissues leads to oxidative tissue damage (Husain et al., 2001). There are many studies about the effects of nicotine on the enzyme activities. For example, inhibition of kidney and testicular superoxide dismutase and activation of liver superoxide dismutase by nicotine treatment in rats were shown. Inhibition of liver catalase and activation of kidney, lungs and testicular catalase by nicotine treatment in rats was reported (US Environmental Protection Agency, 1992).
95
Gumustekin et al. (2003) have reported that nicotine has increased the activity of glutathione peroxidase of the brain while vitamin-E abolished this effect. Nicotine has also inhibited the brain GST activity and this activity was restored by vitamin E, too. In addition, Suleyman et al. (2002) have reported that nicotine has inhibited the activities of glutathione peroxidase and superoxide dismutase of erythrocytes while vitamin-E reversed these effects. The increased mRNA levels of endothelial nitric oxide synthase by nicotine (Zhang et al., 2001), inhibition of typtophan hydroxylase by alcohol and nicotine-treated rats (Jang et al., 2002), while activation of typtophan hydroxylase by nicotine (1 mg/kg) were reported (Lee et al., 2002). Activation of the enzymes in liver metabolism in rats, which were exposed to cigarette smoke (Vanscheeuwijck et al., 2002), and the increased (2.5 fold) expression of enzymes involved in energy metabolism in nicotine-treated rats (Hu et al., 2002) were found. In addition, activation of adenylate cyclase in nicotine (6 mg/kg)-treated rats was also demonstrated (AbreuVillaca et al., 2003). In this study, nicotine inhibited glutathione reductase activity in the liver, lungs, heart, stomach, kidney, and testicular tissues in vivo compared with the control group. Nicotine can cause competitive inhibition by binding the active site of glutathione reductase. It is also possible that it can cause noncompetitive inhibition by binding other sites affecting three dimensional structure of the enzyme (Lehninger et al., 1993). However, it had no effect on the muscle glutathione reductase activity and it activated brain glutathione reductase. Administration of vitamin E with nicotine prevented this inhibition in the liver, lungs, heart, stomach, and kidney. However, nicotine + vitamin E had no effects on the muscle glutathione reductase activity compared with the control group. The reason for the increased glutathione reductase activity in brain tissue may be a compensatory increase to remove the increased reactive oxygen species due to nicotine administration in brain (Gumustekin et al., 2003). Vitamin E might have diffused in the brain to activate the glutathione reductase activity as a lipid soluble antioxidant (Brigelius-Flohe and Traber, 1999). However, the glutathione reductase activity in muscles may not be affected as much as it was affected in the brain tissue from the nicotine administration. As seen in Table 1, nicotine inhibited glutathione reductase activity in vitro, in liver, lungs, heart, stomach and testicular tissues moderately and this inhibition was eliminated by vitamin E. In vitro glutathione reductase inhibition results correlated well with in vivo experimental results in muscle, liver, lungs, heart, stomach and testicles. In the in vivo study, when average volume of blood present in a rat is accepted to be about 15 ml, 0.5 mg/kg dose corresponds to 0.0513 mM nicotine concentration. In the in vitro study, enzyme activities were determined by using 0.04, 0.08, 0.12, 0.16 and 0.20 mM cuvette concentrations. Since similar nicotine concentrations have been used in both in vivo and in vitro experiments, our results obtained by in vitro and in vivo studies were comparable. However, in vitro and in vivo results did not correlate well in kidney tissue. Although it is difficult to envisage how nicotine and vitamin E modify the glutathione reductase activity in various tissues, the
96
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97
inhibition of glutathione reductase by nicotine may result from the binding of the active site of glutathione reductase in liver, lungs, heart, stomach and testicles. On the other hand, facilitating proton transfer may be responsible for the increased glutathione reductase activity, which may be a different isoenzyme of glutathione reductase in kidney tissue (Supuran et al., 2003). The results of this study show that vitamin E administration may generally restore the glutathione reductase activity inactivated due to nicotine administration in various tissues in vivo, and also in vitro. References Abreu-Villaca, Y., Seidler, F.J., Slotkin, T.A., 2003. Impact of adolescent nicotine exposure on adenylyl cyclase-mediated cell signaling: enzyme induction, neurotransmitter-specific effects, regional selectivities, and the role of withdrawal. Brain Res. 988, 164–172. Altikat, S., Coban, T.A.K., Ciftci, M., Ozdemir, H., 2006. In vitro effects of some drugs on catalase purified from human skin. J. Enzyme Inhib. Med. Chem. 21, 231–234. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U. S. A. 90, 7915–7922. Anandatheerthavarada, H.K., Williams, J.F., Wecker, L., 1993. The chronic administration of nicotine induces cytochrome P450 in rat brain. J. Neurochem. 60, 1941–1944. Ashakumary, L., Vijayammal, P.L., 1991. Lipid peroxidation in nicotine treated rats. J. Ecotoxicol. Environ. Monit. 1, 283–290. Beutler, E., 1984. Red Cell Metabolism: A Manual of Biochemical Method. Grune and Stration, New York. Beydemir, S., Ciftci, M., Ozmen, I., Buyukokuroglu, M.E., Ozdemir, H., Kufrevioglu, O.I., 2000. Effects of some medical drugs on enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Pharmacol. Res. 42, 187–191. Bhagwat, S.V., Vijayasarathy, C., Raza, H., Mullick, J., Avadhani, N.G., 1998. Preferential effects of nicotine and 4-(N-methyl-N-nitrosamine)-1-(3pyridyl)-1-butanone on mitochondrial glutathione S-transferase A4-4 induction and increased oxidative stress in the rat brain. Biochem. Pharmacol. 56, 831–839. Bondy, S.C., Orozco, J., 1994. Effects of ethanol treatment upon sources of reactive oxygen species in brain and liver. Alcohol Alcohol. 29, 375–383. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brigelius-Flohe, R., Traber, M.G., 1999. Vitamin E: function and metabolism. FASEB J. 13, 1145–1155. Buccafusco, J.J., Terry Jr., A.V., 2003. The potential role of cotinine in the cognitive and neuroprotective actions of nicotine. Life Sci. 72, 2931–2942. Bulbul, M., Saracoglu, N., Kufrevioglu, O.I., Ciftci, M., 2002. Bile acid derivatives of 5-amino-1,3,4-thiadiazole-2-sulfonamide as new carbonic anhydrase inhibitors: synthesis and investigation of inhibition effects. Bioorg. Med. Chem. 10, 2561–2567. Bulbul, M., Hisar, O., Beydemir, S., Ciftci, M., Kufrevioglu, O.I., 2003. The in vitro and in vivo inhibitory effects of some sulfonamide derivatives on rainbow trout (Oncorhynchus mykiss) erythrocyte carbonic anhydrase activity. J. Enzyme Inhib. Med. Chem. 18, 371–375. Carlberg, I., Mannervik, B., 1985. Glutathione reductase. Methods Enzymol. 113, 484–490. Christensen, G.M., Olson, D., Riedel, B., 1982. Chemical effects on the activity of eight enzymes: a review and a discussion relevant to environmental monitoring. Environ. Res. 29, 247–255. Ciftci, M., Kufrevioglu, O.I., Gundogdu, M., Ozmen, I., 2000. Effects of some antibiotics on enzyme activity of glucose-6-phosphate dehydrogenase from human erythrocytes. Pharmacol. Res. 41, 109–113.
Ciftci, M., Ozmen, I., Buyukokuroglu, M.E., Pence, S., Kufrevioglu, O.I., 2001. Effects of metamizol and magnesium sulfate on enzyme activity of glucose 6-phosphate dehydrogenase from human erythrocyte in vitro and rat erythrocyte in vivo. Clin. Biochem. 34, 297–302. Ciftci, M., Bulbul, M., Gul, M., Gumustekin, K., Dane, S., Suleyman, H., 2005. Effects of nicotine and vitamin E on carbonic anhydrase activity in some rat tissues in vivo and in vitro. J. Enzyme Inhib. Med. Chem. 20, 103–108. Del Boccio, G., Lapenna, D., Porreca, E., Pennelli, A., Savini, F., Feliciani, P., Ricci, G., Cuccurullo, F., 1990. Aortic antioxidant defence mechanisms: time-related changes in cholesterol-fed rabbits. Atherosclerosis 81, 127–135. DeLeve, L.D., Kaplowitz, N., 1991. Glutathione metabolism and its role in hepatotoxicity. Pharmacol. Ther. 52, 287–305. Deneke, S.M., Fanburg, B.L., 1989. Regulation of cellular glutathione. Am. J. Physiol. 257, L163–L173. Erat, M., Ciftci, M., 2003. In vitro effects of some antibiotics on glutathione reductase from sheep liver. J. Enzyme Inhib. Med. Chem. 18, 545–550. Erat, M., Sakiroglu, H., Ciftci, M., 2003. Purification and characterization of glutathione reductase from bovine erythrocytes. Prep. Biochem. Biotechnol. 33, 283–300. Erat, M., Demir, H., Sakiroglu, H., 2005. Purification of glutathione reductase from chicken liver and investigation of kinetic properties. Appl. Biochem. Biotechnol. 125, 127–138. Gilman, A.G., Goodman, L.S., Rall, T.W., Murad, F., 1980. Goodman and Gilman's The Pharmacological Basis of Therapeutics. MacMillan Publishing Co., New York. Grossman, S., Budinsky, R., Jollow, D., 1995. Dapsone-induced hemolytic anemia: role of glucose-6-phosphate dehydrogenase in the hemolytic response of rat erythrocytes to N-hydroxydapsone. J. Pharmacol. Exp. Ther. 273, 870–877. Guemouri, L., Lecomte, E., Herbeth, B., Pirollet, P., Paille, F., Siest, G., Artur, Y., 1993. Blood activities of antioxidant enzymes in alcoholics before and after withdrawal. J. Stud. Alcohol 54, 626–629. Gul, M., Kutay, F.Z., Temocin, S., Hanninen, O., 2000. Cellular and clinical implications of glutathione. Indian J. Exp. Biol. 38, 625–634. Gumustekin, K., Altinkaynak, K., Timur, H., Taysi, S., Oztasan, N., Polat, M.F., Akcay, F., Suleyman, H., Dane, S., Gul, M., 2003. Vitamin E but not Hippophea rhamnoides L. prevented nicotine-induced oxidative stress in rat brain. Human Exp. Toxicol. 22, 425–431. Gumustekin, K., Ciftci, M., Coban, A., Altikat, S., Aktas, O., Gul, M., Timur, H., Dane, S., 2005. Effects of nicotine and vitamin E on glucose 6-phosphate dehydrogenase activity in some rat tissues in vivo and in vitro. J. Enzyme. Inhib. Med. Chem. 20, 497–502. Gupta, B.L., Nehal, M., Baquer, N.Z., 1997. Effect of experimental diabetes on the activities of hexokinase, glucose-6-phosphate dehydrogenase and catecholamines in rat erythrocytes of different ages. Indian J. Exp. Biol. 35, 792–795. Halliwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine. Clarendon Press, Oxford. Helen, A., Krishnakumar, K, Vijayammal, P.L., Augusti, K.T., 2000. Antioxidant effect of onion oil (Allium cepa. Linn) on the damages induced by nicotine in rats as compared to alpha-tocopherol. Toxicol. Lett. 116, 61–68. Hoffmann, D., Rivenson, A., Hecht, S.S., 1996. The biological significance of tobacco-specific N-nitrosamines: smoking and adenocarcinoma of the lung. Crit. Rev. Toxicol. 26, 199–211. Hu, D., Cao, K., Peterson-Wakeman, R., Wang, R., 2002. Altered profile of gene expression in rat hearts induced by chronic nicotine consumption. Biochem. Biophys. Res. Commun. 297, 729–736. Husain, K., Scott, B.R., Reddy, S.K., Somani, S.M., 2001. Chronic ethanol and nicotine interaction on rat tissue antioxidant defense system. Alcohol 25, 89–97. Jang, M.H., Shin, M.C., Lee, T.H., Kim, Y.P., Jung, S.B., Shin, D.H., Kim, H., Kim, S.S., Kim, E.H., Kim, C.J., 2002. Alcohol and nicotine administration inhibits serotonin synthesis and tryptophan hydroxylase expression in dorsal and median raphe of young rats. Neurosci. Lett. 329, 141–144. Jenkins, R.R., Goldfarb, A., 1993. Introduction: oxidant stress, aging, and exercise. Med. Sci. Sports Exerc. 25, 210–212.
M. Erat et al. / European Journal of Pharmacology 554 (2007) 92–97 Kessler, D.A., Witt, A.M., Barnett, P.S., Zeller, M.R., Natanblut, S.L., Wilkenfeld, J.P., Lorraine, C.C., Thompson, L.J., Schultz, W.B., 1996. The Food and Drug Administration's regulation of tobacco products. N. Engl. J. Med. 335, 988–994. Kondo, T., Dale, G.L., Beutler, E., 1980. Glutathione transport by inside-out vesicles from human erythrocytes. Proc. Natl. Acad. Sci. U. S. A. 77, 6359–6362. Leanderson, P., Tagesson, C., 1992. Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation. Chem. Biol. Interact. 81, 197–208. Lee, T.H., Jang, M.H., Shin, M.C., Lim, B.V., Choi, H.H., Kim, H., Kim, E.H., Kim, C.J., 2002. Nicotine administration increases serotonin synthesis and tryptophan hydroxylase expressions in dorsal raphe of food-deprived rats. Nutr. Res. 22, 1445–1452. Lehninger, A.L., Nelson, D.L., Cox, M.M., 1993. Principles of Biochemistry. Worth Publishers, New York. Maser, E., 1997. Stress, hormonal changes, alcohol, food constituents and drugs: factors that advance the incidence of tobacco smoke-related cancer? Trends Pharmacol. Sci. 18, 270–275. McCallum, M.J., Barrett, J., 1995. The purification and properties of glutathione reductase from the cestode Moniezia expansa. Int. J. Biochem. Cell Biol. 27, 393–401. McCord, J.M., 1993. Human disease, free radicals, and the oxidant/antioxidant balance. Clin. Biochem. 26, 351–357. McGinnis, J.M., Foege, W.H., 1993. Actual causes of death in the United States. JAMA 270, 2207–2212. Mehta, M.C., Jain, A.C., Billie, M., 1998. Combined effects of alcohol and nicotine on cardiovascular performance in a canine model. J. Cardiovasc. Pharmacol. 31, 930–936. Meister, A., 1981. Metabolism and functions of glutathione. Trends Biochem. Sci. 6, 231–234. Ozdemir, H., Ciftci, M., 2006. In vitro effects of some drugs on human red blood cell glucose-6-phosphate dehydrogenase enzyme activity. J. Enzyme Inhib. Med. Chem. 21, 75–80. Pryor, W.A., Stone, K., 1993. Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann. N. Y. Acad. Sci. 686, 12–27 (discussion 27-18). Rubin, E., 1993. The chemical pathogenesis of alcohol-induced tissue injury. Alcohol Health Res. World 17, 272–278. Sastry, B.V., Chance, M.B., Singh, G., Horn, J.L., Janson, V.E., 1995. Distribution and retention of nicotine and its metabolite, cotinine, in the rat as a function of time. Pharmacology 50, 128–136.
97
Sastry, B.V., Gujrati, V.R., 1994. Activation of PAF-acetylhydrolase by nicotine and cotinine and their possible involvement in arterial thrombosis. Ann. N. Y. Acad. Sci. 714, 312–314. Shaw, M., Mitchell, R., Dorling, D., 2000. Time for a smoke? One cigarette reduces your life by 11 minutes. BMJ 320, 53. Somani, S.M., 1996. Exercise, drugs and tissue specific antioxidant system. In: Somani, S.M. (Ed.), Pharmacology in Exercise and Sports. CRC Press, Boca Raton FL, pp. 57–95. Suleyman, H., Gumustekin, K., Taysi, S., Keles, S., Oztasan, N., Aktas, O., Altinkaynak, K., Timur, H., Akcay, F., Akar, S., Dane, S., Gul, M., 2002. Beneficial effects of Hippophae rhamnoides L. on nicotine induced oxidative stress in rat blood compared with vitamin E. Biol. Pharm. Bull. 25, 1133–1136. Supuran, C.T., Scozzafava, A., Casini, A., 2003. Carbonic anhydrase inhibitors. Med. Res. Rev. 23, 146–189. US Department of Health and Human Services, 1990. Seventh Special Report to the US Congress on Alcohol and Health. Washington, DC: National Institute on Alcohol Abuse and Alcoholism (US Department of Health and Human Services Publication ADM 90, 1656). US Environmental Protection Agency, 1992. Respiratory Health Effects of Passive Smoking, Lung Cancer and Other Disorders. Washington, DC: US Environmental Protection Agency (US Environmental Protection Agency Publication 600, 6-90/006F). Vanscheeuwijck, P.M., Teredesai, A., Terpstra, P.M., Verbeeck, J., Kuhl, P., Gerstenberg, B., Gebel, S., Carmines, E.L., 2002. Evaluation of the potential effects of ingredients added to cigarettes. Part 4: Subchronic inhalation toxicity. Food Chem. Toxicol. 40, 113–131. Warnock, D.G., 1989. Diuretic agents. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. Appletion and Lange, London, pp. 183–197. Weksler, B.B., Moore, A., Tepler, J., 1990. Hematology. In: Andreoli, T.E., Carpenter, C.C.J., Plum, F., Smith, L.H.J. (Eds.), Cecil Essentials of Medicine. WB Saunders Co., Philadelphia, pp. 341–363. Wetscher, G.J., Bagchi, M., Bagchi, D., Perdikis, G., Hinder, P.R., Glaser, K., Hinder, R.A., 1995. Free radical production in nicotine treated pancreatic tissue. Free Radic. Biol. Med. 18, 877–882. Zhang, K., Yang, E.B., Tang, W.Y., Wong, K.P., Mack, P., 1997. Inhibition of glutathione reductase by plant polyphenols. Biochem. Pharmacol. 54, 1047–1053. Zhang, S., Day, I., Ye, S., 2001. Nicotine induced changes in gene expression by human coronary artery endothelial cells. Atherosclerosis 154, 277–283.