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Sodium nitrite-induced oxidative stress causes membrane damage, protein oxidation, lipid peroxidation and alters major metabolic pathways in human erythrocytes
Accepted Manuscript Sodium nitrite-induced oxidative stress causes membrane damage, protein oxidation, lipid peroxidation and alters major metabolic pathways in human erythrocytes Fariheen Aisha Ansari, Shaikh Nisar Ali, Riaz Mahmood PII: DOI: Reference:
S0887-2333(15)00183-6 http://dx.doi.org/10.1016/j.tiv.2015.07.022 TIV 3595
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Toxicology in Vitro
Received Date: Revised Date: Accepted Date:
10 March 2015 2 July 2015 27 July 2015
Please cite this article as: Ansari, F.A., Ali, S.N., Mahmood, R., Sodium nitrite-induced oxidative stress causes membrane damage, protein oxidation, lipid peroxidation and alters major metabolic pathways in human erythrocytes, Toxicology in Vitro (2015), doi: http://dx.doi.org/10.1016/j.tiv.2015.07.022
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Sodium nitrite-induced oxidative stress causes membrane damage, protein oxidation, lipid peroxidation and alters major metabolic pathways in human erythrocytes
Fariheen Aisha Ansari, Shaikh Nisar Ali, Riaz Mahmood*
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, U.P., India
*Corresponding author
Telephone: 91-571-2700741; +91-9639901935
E. mail: [email protected]
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ABSTRACT Nitrite salts are present as contaminants in drinking water and in the food and feed chain. In this work, the effect of sodium nitrite (NaNO2) on human erythrocytes was studied under in vitro conditions. Incubation of erythrocytes with 0.1-10.0 mM NaNO2 at 37 °C for 30 min resulted in dose dependent decrease in the levels of reduced glutathione, total sulfhydryl and amino groups. It was accompanied by increase in hemoglobin oxidation and aggregation, lipid peroxidation, protein oxidation and hydrogen peroxide levels suggesting the induction of oxidative stress. Activities of all major erythrocyte antioxidant defence enzymes were decreased in NaNO2-treated erythrocytes. The activities of enzymes of glycolytic and pentose phosphate pathways were also compromised. However, there was a significant increase in acid phosphatase and also AMP deaminase, a marker of erythrocyte oxidative stress. Thus, the major metabolic pathways of cell were altered. Erythrocyte membrane damage was suggested by lowered activities of membrane bound enzymes and confirmed by electron microscopic images. These results show that NaNO2-induced oxidative stress causes hemoglobin denaturation and aggregation, weakens the cellular antioxidant defense mechanism, damages the cell membrane and also perturbs normal energy metabolism in erythrocytes. This nitrite-induced damage can reduce erythrocyte life span in the blood. Key words: Nitrite; erythrocytes; methemoglobin; oxidative stress; metabolic pathways; protein oxidation
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Abbreviations: AMP, adenosine 5’-monophosphate; ATP, adeosine 5’-triphosphate; ATPase, adeosine triphosphatase; DTNB, 5,5’-dithiobisnitrobenzoic acid; G6PD, glucose 6phosphate dehydrogenase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; Hb, hemoglobin; H2O2, hydrogen peroxide; LDH, lactate dehydrogenase; MetHb, methemoglobin; NAD+ and NADH, reduced and oxidized nicotinamide adenine dinucleotide; NADP+ and NADPH, reduced and oxidized nicotinamide adenine dinucleotide phosphate; Na,K-ATPase, sodium potassium ATPase; NaNO2, sodium nitrite; NO, nitric oxide; ROS, reactive oxygen species; SEM, scanning electron microscopy; SH, sulfhydryl; SOD, Cu,Zn superoxide dismutase; TR, thioredoxin reductase.
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1. Introduction Nitrite is naturally present in humans and in most green vegetables. In humans it acts directly or indirectly (by reducing to nitric oxide) in maintaining vascular homeostasis. It also plays key physiological roles in signalling, cellular respiration and mediation of innate immunity (Lundberg et al., 2008). Inside the cell, nitrite can be reversibly converted to nitrate and nitric oxide. Sodium nitrite (NaNO2) is widely used as a food preservative and colour fixative in fish and meat. It is also used in the manufacture of dyes, nitroso compounds and rubber chemicals. Medicinally, it is used as a vasodilator, bronchodilator and as an antidote for cyanide poisoning. Human exposure to nitrite has increased sharply in recent years because of extensive use of nitrogenous fertilizers in agriculture, improper disposal of human and industrial wastes and atmospheric nitrogen pollution (Galloway et al., 2003). The nitratenitrite content in food and water in many areas has increased alarmingly beyond the permissible limits (WHO, 2011). In some areas it has greatly exceeded the levels of 1 ppm (nitrite) and 10 ppm (nitrate) in drinking water set by the U.S. Environmental Protection Agency (EPA). Although nitrite has important functions in the cell at physiological concentrations, it can be toxic in high amounts to animals and humans. The immediate and major health implication of nitrite intoxication is methemoglobin (MetHb) formation (Chui et al., 2005) and cyanosis, which can be fatal. Infants are particularly susceptible to nitrite intoxication, causing death in most cases (WHO, 2011). Continuous exposure to non-fatal doses of nitrite can cause physiological disturbances including permanent growth inhibition, neurological disorders, respiratory failure and paralysis (Mensinga et al., 2003). Pregnant women, anaemic and glucose 6-phosphate dehydrogenase (G6PD) deficient individuals are also prone to nitrite toxicity (Huber et al., 2013; WHO, 2011). Nitrite easily transforms into carcinogenic 4
nitrosating compounds in the acidic environment of gut (Brambilla and Martelli, 2007). A relationship between nitrite/nitrate levels and higher incidence of various cancers has been reported in humans (Dellavalle et al., 2013; Coss et al., 2004). NaNO2 administered rats showed increased risk of forestomach neoplasm and squamous papilloma (NTP, 2001). Degenerative changes were observed in various tissues of NaNO2 administered mice (Ozen et al., 2014). Nitrite has thus been classified as cytotoxic, mutagenic, teratogenic and embryotoxic (NTP, 2001). Human exposure to nitrite results in its uptake and subsequent transfer to blood with rapid binding to erythrocytes (Dejam et al., 2007). Blood acts as a biological nitrite buffer to maintain its concentration throughout the body, and supply it as and when required. About 75% of nitrite enters erythrocytes by diffusion, as nitrous acid or other species, while the remaining 25% uptake occurs by the sodium-dependent phosphate transporter (May et al., 2000). Erythrocytes have been used as a simple model to study the cellular effects of various compounds (Takebayashi et al., 2010), especially those that generate reactive oxygen species (ROS). Erythrocytes are particularly susceptible to oxidative insult due to their role as oxygen transporters and high content of polyunsaturated fatty acids, transition metals and redox active hemoglobin (Hb) molecules. We have examined the effect of NaNO2 on human erythrocytes under in vitro conditions using concentrations to which humans could be potentially exposed, especially in areas with nitrate-nitrite contaminated drinking water (WHO, 2011). Our results show that NaNO2 causes significant oxidative damage to erythrocytes which was also visible in electron microscopic images. 2. Materials and Methods 2.1. Materials
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NaNO2, 1-chloro-2,4-dinitrobenzene, glutathione reductase (GR), metaphosphoric acid, 2,4,6-trinitrobenzene sulfonate, N-(1-naphthyl)ethylenediamine dihydrochloride and ouabain were purchased from Sigma–Aldrich, USA. Reduced (GSH) and oxidized (GSSG) glutathione, reduced and oxidized nicotinamide adenine dinucleotide phosphate (NADPH and NADP+), reduced nicotinamide adenine dinucleotide (NADH), N-ethylmaleimide, ophthalaldehyde, pyrogallol, 2,6-dichlorophenolindophenol, 2,4-dinitrophenylhydrazine, 5,5'dithiobisnitrobenzoic acid (DTNB), xylenol orange, sulphanilamide, adenosine 5’monophosphate (AMP), glucose 6-phosphate, sodium nitroprusside, methyl violet and sorbitol were from Sisco Research Laboratory (Mumbai, India). Thiobarbituric acid, tris(hydroxymethyl)aminomethane, trichloroacetic acid and S-acetylthiocholine iodide were purchased from Himedia Laboratories (Mumbai, India). All other chemicals were of analytical grade. 2.2. Isolation of erythrocytes and treatment with NaNO2 This study was approved by the institutional ethics committee which monitors research involving human subjects. Human blood was taken from young (22–30 years), healthy, nonsmoking volunteers after getting their informed consent and used immediately. Blood was collected in heparinized tubes, centrifuged at 1,500 rpm for 10 min at 4 °C in a clinical centrifuge and the plasma and buffy coat were removed. The erythrocyte pellet was washed three times with phosphate-buffered saline (PBS) (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) and resuspended in PBS to give a 10% (v/v) cell suspension (hematocrit). Stock solutions of NaNO2 were prepared in PBS. Erythrocytes were incubated with different concentrations of NaNO2 (0.1–10 mM, corresponding to 0.0069 - 0.69 mg/ml) for 30 min at 37 °C. NaNO2-untreated cells were similarly incubated at 37 °C and served as control. The samples were centrifuged at 2500 rpm for 10 min at 4 °C. Cell pellets were washed three times with PBS and erythrocytes were lysed with ten volumes of 5 mM sodium phosphate 6
buffer, pH 7.4, at 4 °C for 2 h. Samples were centrifuged at 3,000 rpm for 10 min at 4 °C and the supernatants (hemolysates) were quickly frozen in aliquots to be used later for the analysis of several biochemical parameters. 2.3. Osmotic fragility Control and NaNO2 treated erythrocytes were incubated at 37 °C and centrifuged as above. The cell pellet was washed and resuspended in PBS to give 10% hematocrit. Then, 0.05 ml of this cell suspension was added to different tubes containing 5 ml of 0.2 to 0.7% NaCl. After 30 min at 37 °C, the samples were centrifuged at 2500 rpm for 10 min in a clinical centrifuge and the absorbance of supernatants was recorded at 540 nm. The absorbance of untreated erythrocytes, lysed with 5 mM sodium phosphate buffer, pH 7.2, for 2 hr at 4 °C, served as a reference and represents 100% lysis. 2.4. Hemoglobin and methemoglobin levels and methemoglobin reductase Hb concentration in hemolysates was determined by the cyanomethaemoglobin method using a commercially available kit (Hemocor-D Kit, Coral Clinical Systems, Goa, India). The levels of MetHb were determined from the absorbance of diluted hemolysates at 540, 576 and 630 nm (Benesch et al., 1973) and expressed as percent of total Hb. NADHdependent MetHb reductase activity was determined from the increase in absorbance at 600 nm after incubation of hemolysates with NADH and 2,6-dichlorophenolindophenol (Kuma et al., 1972). Heinz bodies were detected by staining control and NaNO2 treated erythrocytes with 0.5% methyl violet (in 0.9% NaCl) for 45 min. Cells were then fixed on glass slides and observed under a light microscope at 100x magnification. 2.5. Reduced (GSH) and oxidized (GSSG) glutathione, amino groups and total sulfhydryl levels 7
GSH and GSSG levels in hemolysates were determined fluorometrically using ophthalaldehyde and N-ethylmaleimide (Hissin and Hilf, 1976). Total sulfhydryl (SH) groups were determined from the yellow colour produced after their reaction with DTNB (Sedlak and Lindsay, 1968). Free amino groups were determined using 2,4,6-trinitrobenzenesulfonate (Snyder and Sobocinski, 1975). 2.6. Carbonyl content, thiobarbituric acid reactive substances, hydrogen peroxide and nitrite levels Protein carbonyl content in hemolysates was determined after reaction with 2,4dinitrophenylhydrazine (Levine et al., 1990). Hemolysates, deproteinized using zinc sulphateNaOH, were used for the analysis of lipid peroxidation, nitrite and H2O2 levels. Malondialdehyde, a product of lipid peroxidation, was measured as thiobarbituric acid reactive substances (Buege and Aust, 1978). Nitrite concentration was determined using Greiss reagent (1% sulfonilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride) as described by Miranda et al., (2001). H2O2 concentrations were determined using ferrous ammonium sulphate-xylenol orange (FOX reagent) as colour reagent in the presence of 100 mM sorbitol (Gay and Gebicki, 2000). 2.7. Antioxidant enzymes All enzymes were assayed in hemolysates. Catalase activity was determined from the conversion of H2O2 to water (Aebi, 1984) and Cu,Zn superoxide dismutase (SOD) from the inhibition of auto-oxidation of pyrogallol (Marklund and Marklund, 1974). Glutathione reductase (GR) was assayed from the conversion of NADPH to NADP + during the reduction of GSSG to GSH (Carlberg and Mannervik, 1985). Glutathione peroxidase (GPx) was assayed at 340 nm from the oxidation of NADPH to NADP+ in the presence of GSSG and GR; azide was added to inhibit catalase activity (Flohe and Gunzler, 1984). Thioredoxin
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reductase (TR) activity was determined from the absorbance at 410 nm due to reduction of DTNB to the yellow coloured thionitrobenzoate anion (Tamura and Stadtman, 1996). 2.8. Metabolic enzymes The activity of AMP deaminase was determined in hemolysates from the ammonia released from AMP (Pederson and Berry, 1977). G6PD was assayed by following the conversion of NADP+ to NADPH at 340 nm in the presence of glucose 6-phosphate (Shonk and Boxer, 1964). Hexokinase activity was determined by the method of Crane and Sols (1953), which measures the depletion in glucose concentration with time. Lactate dehydrogenase (LDH) was assayed from the oxidation of NADH to NAD + at 340 nm using sodium pyruvate as substrate (Khundmiri et al., 2004). Acid phosphatase activity was measured from the yellow coloured p-nitrophenol formed at pH 4.5 upon hydrolysis of p-nitrophenyl phosphate (Mohrenweiser and Novotny, 1982). 2.9. Membrane bound enzymes Acetylcholinesterase was assayed in hemolysates using DTNB to measure thiocholine released from S-acetylthiocholine iodide (Ellman et al., 1961). Na,K-ATPase and total ATPases were assayed from the inorganic phosphate released upon hydrolysis of ATP in the presence and absence of 1 mM ouabain (Bonting et al., 1961). 2.10. Scanning electron microscopy Control and NaNO2 treated erythrocytes were washed three times with PBS and fixed with 2.5% glutaraldehyde for 1 hr at room temperature. After washing again with PBS, the samples were mounted on glass slides and dehydrated with graded ethanol (50%-70%-90%100%). The samples were then coated with gold-palladium layer and examined in a scanning electron microscope (SEM) (Wang et al., 2009). 2.11. Statistical Analysis 9
All data are expressed as mean ± standard error. Statistical evaluation was conducted by oneway ANOVA using the program Origin 6.1 Software (USA). A probability level of P<0.05 was selected as indicating statistical significance. All experiments were done with 6-10 different blood samples to document reproducibility; results of six samples are shown here. 3. Results Human erythrocytes were isolated from fresh blood and treated with different concentrations of NaNO2 for 30 min at 37 °C. The cells were then pelleted by centrifugation, washed with PBS to remove excess nitrite and then lysed with hypotonic buffer. Several biochemical parameters were then determined in the hemolysates. Incubation of erythrocytes with NaNO2 rendered them more osmotically fragile and they lysed at relatively higher salt concentrations compared to control cells (Fig. 1). The activities of membrane bound enzymes, acetylcholinesterase and Na,K-ATPase, were significantly decreased while total erythrocyte ATPase was also lowered compared to control (Table 1). Rapid formation of MetHb was observed within few minutes of NaNO2 treatment. MetHb levels were two times the control value even at 0.05 mM and reached 78% of the total Hb at 10 mM (Fig. 2A). However, the activity of MetHb reductase was enhanced with increase in nitrite concentration (Fig. 2B). Extensive oxidation of Hb causes it to denature, aggregate and form insoluble Heinz bodies which cross-link with membrane proteins. These Heinz bodies were visible in NaNO2 treated erythrocytes, as dark stained spots within and near the periphery of the cell (Fig. 3). Several parameters of induction of oxidative stress in the cell were determined in hemolysates. The carbonyl content was used as a measure of protein oxidation and it increased considerably in NaNO2-treated erythrocytes in a concentration dependent manner (Table 2). A similar increase in lipid peroxidation was seen as determined from the levels of
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malondialdehyde, an end product of lipid peroxidation (Table 2). At higher concentrations of NaNO2, both protein oxidation and lipid peroxidation were more than three times the corresponding control values. Total SH and amino groups were lowered in hemolysates from NaNO2 treated erythrocytes (Table 2). However, a dramatic increase in H2O2 levels was seen, even at low nitrite concentrations (Fig. 4). As expected, nitrite content of the cell increased in a concentration dependent manner (Fig. 5). The effect of NaNO2 on enzymatic and non-enzymatic antioxidant defence system of erythrocytes was determined next. A slight initial increase in GSH levels was followed by a sharp decline at higher NaNO2 concentrations; at 10 mM NaNO2 the GSH content was only 4% of the control value. This was accompanied by a parallel increase in GSSG, which was seen at higher concentrations of NaNO2 (Fig. 6). The activities of all the major (catalase, SOD, GPx) and secondary (TR, GR) antioxidant defence enzymes were decreased in nitrite treated erythrocytes (Table 3). TR was the enzyme most affected and at 10 mM NaNO2 its activity was 10% of control. Nitrite treatment of erythrocytes led to a decrease in the activity of hexokinase, the first enzyme of glycolysis. The activity of G6PD, the major source of NADPH in erythrocytes, declined in a dose dependent manner and was 36% of control at the 10 mM NaNO2. However, there was a ten folds increase (at 10 mM NaNO2) in the activity of AMP deaminase, an enzyme of purine metabolism. The activities of acid phosphatase and LDH also increased upon NaNO2 treatment (Table 4). SEM analysis showed normal biconcave appearance and discocyte shape for control erythrocytes (Fig. 7). Changes in erythrocyte shape and morphology were observed in NaNO2 treated samples. Loss of discocytic morphology and deformation was clearly evident.
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4. Discussion Human exposure to nitrite results in its rapid entry into bloodstream where it can interact with erythrocytes. We have investigated the effects of NaNO2 on human erythrocytes, especially on the cell membrane, Hb oxidation, antioxidant defence and metabolic pathways. Treatment of erythrocytes with NaNO2 did not cause significant hemolysis (<1% at 10 mM NaNO2) but the cells became more osmotically fragile. Alterations in membrane lipid and protein structure cause decrease in membrane integrity. The ability of cells to stretch and deform is reduced, increasing their rigidity and making them more susceptible to lysis. Using fluorescence anisotropy, Zavodik et al. (1999) have also reported that nitrite induces membrane hyperpolarisation and increases its rigidity. NaNO2 treatment led to massive increase in level of H2O2 which can react with transition metals like iron to give the damaging hydroxyl radical. It also resulted in significant increase in lipid peroxidation probably due to increase in superoxide radical and H2O2 levels since the activities of SOD and catalase, enzymes that use them as substrates, were decreased. A three folds increase in protein carbonyl content took place since ROS introduce carbonyl groups in amino acid residues (Halliwell and Gutteridge, 1999). High protein carbonyl and 3nitrotyrosine levels have been reported in NaNO2 treated meat products (Vossen et al., 2015). Amino groups were also oxidized leading to a decrease in their content. The steep decline in GSH, which maintains a reducing environment within the cell, was accompanied by a parallel increase in GSSG levels at higher NaNO2 concentrations. This decrease in GSH might be due to (i) lower activity of GR, which converts GSSG to GSH (ii) direct oxidation of the thiol group of GSH by ROS or (iii) its reaction with NO to form nitrosothiols (Radi, 2004). A concomitant reduction in total SH groups shows that the entire cellular thiol status was affected. This increase in H2O2, protein oxidation and lipid peroxidation along with decrease in SH content, strongly suggests that NaNO2 induces oxidative stress in erythrocytes. 12
Vatassery et al. (2004) have also reported higher oxidative stress in the brain of nitrite treated rats. Nitrite also exacerbates oxidative toxicity when associated with extracellular Hb and potentiates acute renal toxicity in guinea pigs (Baek et al., 2015). Activities of all major antioxidant enzymes were decreased in NaNO2 treated erythrocytes. SOD, catalase and GPx, are known to be inactivated by free radicals and ROS (Escobar et al., 1996; Pigeolot et al., 1990; Yang and Wang, 1991). SOD and catalase contain bound copper and iron, respectively, and the direct reaction of these metals with NO might also be a cause of reduced activity. The decrease in G6PD activity also affects several antioxidant enzymes that require NADPH. TR uses NADPH to keep thioredoxin in its reduced state which, in turn, maintains other proteins in their reduced form by cysteine thiol-disulphide exchange. GR uses NADPH to regenerate GSH from GSSG. Low NADPH level due to nitrite-induced inhibition of G6PD will reduce the activities of both GR and TR. It will also inhibit catalase, which contains four NADPH bound per tetrameric molecule that are needed for the biological activity of this enzyme (Kirkman et al., 1999). Hemoglobin is a major cellular target of ROS in erythrocytes. Oxidation of ferrous iron of Hb to the ferric form gives MetHb, which is inactive as an oxygen transporter. This process releases an electron that is taken up by molecular oxygen to give superoxide radical which can then react with NO to give peroxynitrite (Pryor and Squadrito, 1995) or is converted to H2O2. A high percentage of total Hb was converted to MetHb in nitrite treated erythrocytes, as also reported previously (Chui et al., 2005). MetHb reductase converts MetHb back to Hb, by reducing the ferric iron to ferrous form, using either NADH or NADPH as the electron donor. Surprisingly, the activity of NADH dependent MetHb reductase (major form of the enzyme) was significantly increased in NaNO2 treated erythrocytes. This depicts the adaptive nature of the cell in response to increased MetHb formation due to NaNO2 assault. Another reason for increase in MetHb reductase activity could be the release of inactive membrane 13
bound form into the cytosol (Choury et al., 1981) thereby increasing its activity in cell lysates. However, MetHb levels were too high to be reversed by the enhanced activity of the reductase. Extensive oxidation and cross-linking of some Hb also took place giving Heinz bodies that were attached to the cell membrane. Erythrocyte energy metabolism was also profoundly altered in NaNO 2 treated erythrocytes. Erythrocyte acid phosphatase can dephosphorylate proteins including band 3, a transmembrane protein whose cytoplasmic tail binds several glycolytic enzymes (Campanella et al., 2008). NaNO2-induced increase in acid phosphatase activity can result in dephosphorylation of band 3 protein which will enhance the binding and thus inhibition of glycolytic enzymes, thereby lowering ATP levels. NaNO2-induced decrease in hexokinase activity will also contribute to decrease in cellular energy (ATP) level. AMP deaminase maintains the levels of purine nucleotides and is also a potent marker of oxidative stress in erythrocytes (Tavazzi et al., 2001). A dramatic ten folds increase in the activity of AMP deaminase will cause corresponding increase in inosine monophosphate levels, rather than AMP, parallel to ATP depletion. The erythrocyte membrane contains large amounts of acetylcholinesterase of unknown function. The significant decrease in acetylcholinesterase activity upon NaNO2 treatment is indicative of oxidative stress and membrane damage (O’Malley et al., 1966; Zhou et al., 2003). Erythrocyte ATPases cause asymmetric distribution of ions across the membrane, maintain cell integrity and normal function. Na,K-ATPase (and also Ca2+ ATPase) is known to have an SH group at its active site (Boldyrev et al., 1997) whose oxidation will inhibit the enzyme. Another reason could be increased peroxidation of membrane lipids thereby disturbing the protein environment and leading to enzyme inhibition. LDH is attached to the inner side of plasma membrane and increase in its activity could be due to membrane disruption and consequent release of the bound enzyme into the cytosol. However, there was 14
no release of LDH in the supernatant upon treatment of erythrocytes with nitrite. All these results suggest derangement of membrane function and integrity. SEM images of NaNO2treated cells showed multiple bleb formation on the surface and change in the shape of cells to echinocytes (spiculated erythrocytes). Membrane lipid peroxidation and damage to cytoskeleton proteins cause bleb formation (Singh and Rajini, 2008) which is often accompanied by loss of membrane asymmetry. The SEM analysis greatly supports and confirms the results of our biochemical studies that NaNO2 induces extensive damage in human erythrocytes. 5. Conclusions NaNO2 treatment of human erythrocytes results in oxidation, denaturation and aggregation of Hb and also enhances lipid and protein oxidation. It lowers the enzymatic and non-enzymatic antioxidant defence and alters the activities of enzymes of major metabolic pathways and erythrocyte membrane. The biochemistry of these effects may be attributed to the induction of oxidative stress and consequent increase in ROS and, perhaps partly, due to interaction of NO (generated from nitrite) with thiol groups and transition metal ions, thereby affecting enzymes having either of these species at their active site. The results of nitrite treatment on erythrocytes are shown schematically in Figure 8. In order to prevent nitrite induced toxicity, steps should be taken to reduce its content in feed and food chain and to ensure an antioxidant rich diet in populations living in nitrite contaminated areas. Conflict of interest The authors declare that there is no conflict of interest.
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Pryor WA, Squadrito GL. 1995. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am J Physiol 268(5), L699-7L22. Radi R. 2004. Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci USA 101, 4003-4008. Sedlak J, Lindsay RH. 1968. Estimation of total, protein-bound and non-protein sulphydryl groups in tissue with Ellman’s reagent. Anal Biochem 25, 192–205. Shonk CC, Boxer GE. 1964. Enzyme patterns in human tissues. I. Methods for the determination of glycolytic enzymes. Cancer Res 24, 709–721. Singh N, Rajini PS. 2008. Antioxidant mediated protective effect of potato peel extract in erythrocytes against oxidative damage. Chem Biol Interact 173, 97-104. Snyder SL, Sobocinski PZ. 1975. An improved 2,4,6- trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem 64, 284-288. Takebayashi J, Chen J, Tai A. 2010. A method for evaluation of antioxidant activity based on inhibition of free radical-induced erythrocyte hemolysis. Methods Mol Biol 594, 287-296. Tamura T, Stadtman TC. 1996. A new selenoprotein from human lung adenocarcinoma cells: Purification, properties and thioredoxin reductase activity. Proc Natl Acad Sci USA 93, 1006–1011. Tavazzi B, Amorini AM, Fazzina G, Pierro DD, Tuttobene M, Giardina B, Lazzarino G. 2001. Oxidative stress induces impairment in energy metabolism through the oxygen radicalmediated direct activation of AMP deaminase. J Biol Chem 276, 48083-48092. Vatassery GT, SantaCruz KS, DeMaster EG, Quach HT, Smith WE. 2004. Oxidative stress and inhibition of oxidative phosphorylation induced by peroxynitrite and nitrite in rat brain subcellular fractions. Neurochem Int 45, 963-970.
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Vossen E, De Smet S. 2015. Protein oxidation and protein nitration influenced by sodium nitrite in two different meat model systems. J Agric Food Chem 63(9), 2550-2556. Wang Y, Yang L, Cheng W, Liu M, Chen X, Zhang K, Chen HM, Liao Z. 2009. Scanning electron microscopic observation of erythrocytes and endothelial cells of electrified death rabbits. Leg Med 11, S244-S247. WHO (World Health Organization). 2011. Nitrate and nitrite in drinking-water. Background document for development of WHO Guidelines for Drinking-Water Quality. WHO/SDE/WSH/07.01/16/Rev/1 Yang Q, Wang F. 1991. Effect of sodium nitrite on myocardial glutathione peroxidase and protective action of vitamin E and selenium. Biomed Environ Sci 4, 373-375. Zavodnik IB, Lapshina EA, Rekawiecka K, Zavodnik LB, Bartosz G, Bryszewska M. 1999. Membrane effects of nitrite-induced oxidation of human red blood cells. Biochim Biophys Acta 1421, 306-316. Zhou JF, Zhou YH, Zhang L, Chen HH, Cai D. 2003. 3,4-methylenedioxymethamphetamine (MDMA) abuse markedly inhibits acetylcholinesterase activity and induces severe oxidative damage and liperoxidative damage. Biomed Environ Sci 16(1), 53-61.
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TABLES
Table 1. Effect of NaNO2 on membrane bound enzymes. Control
0.1 mM
0.5 mM
1.0 mM
2.5 mM
5.0 mM
10.0 mM
AChE
57.0 ± 3.8
53.6 ± 3.0
50 ± 4.2*
47.8 ± 3.9*
44.2 ± 3.7*
41.8 ± 3.3*
35.7 ± 2.8*
Total ATPase
0.85 ± 0.06
0.77 ± 0.05
0.68 ± 0.04*
0.61 ± 0.03*
0.56 ± 0.04*
0.52 ± 0.03*
0.44 ± 0.03*
Na,K-ATPase
0.51 ± 0.03
0.44 ± 0.03*
0.41 ± 0.04*
0.39 ± 0.02*
0.35 ± 0.02*
0.34 ± 0.03*
0.31 ± 0.03*
Enzyme activities were assayed in hemolysates from control and NaNO2 treated erythrocytes. Specific activity of acetylcholinestersae (AChE) is in nmoles/min/mg Hb and ATPases are in µmoles/hr/mg Hb. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
Table 2. Effect of NaNO2 on some parameters of oxidative stress Control
0.1 mM
0.5 mM
1.0 mM
2.5 mM
5.0 mM
10.0 mM
Carbonyl content
4.9 ± 0.43
6.3 ± 0.51*
8.3 ± 0.54*
9.2 ± 0.72*
12.2 ± 0.77*
14.1 ± 0.98*
16.3 ± 1.42*
TBARS
0.92 ± 0.06
1.1 ± 0.09
1.3 ± 0.09*
1.6 ± 0.11*
2.1 ± 0.17*
2.5 ± 0.20*
2.8 ± 0.21*
Total SH
0.94 ± 0.06
0.79 ± 0.04
0.7 ± 0.04*
0.59 ± 0.03*
0.52 ± 0.04*
0.47 ± 0.04*
0.41 ± 0.03*
Amino groups
1.2 ± 0.08
1.03 ± 0.07
0.9 ± 0.05*
0.87 ± 0.05*
0.84 ± 0.04*
0.71 ± 0.05*
0.52 ± 0.03*
Thiobarbituric acid reactive substances (TBARS) and carbonyl content are in nmoles/mg Hb while total sulfhydryl (SH) and amino groups are in µmoles/mg Hb. These parameters were determined in hemolysates from control and NaNO2 treated erythrocytes. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Table 3. Effect of NaNO2 on some antioxidant enzymes. Control
0.1 mM
0.5 mM
1.0 mM
2.5 mM
5.0 mM
10.0 mM
CAT
52.6 ± 3.9
48.2 ± 3.1
44.9 ± 2.8*
42.7 ± 3.6*
39 ± 2.7*
36.4 ± 2.1*
34.7 ± 2.3*
SOD
18.7 ± 1.35
17.0 ± 0.98
16.3 ± 1.21*
13.8 ± 0.98*
12.4 ± 0.86*
10.0 ± 0.75*
8.5 ± 0.49*
GPx
18.4 ± 1.14
16.5 ± 1.45*
13 ± 0.92*
11 ± 0.74*
9.6 ± 0.58*
7.4 ± 0.43*
6.6 ± 0.38*
GR
8.5 ± 0.70
7.5 ± 0.54
5.3 ± 0.32*
4.5 ± 0.37*
3.63 ± 0.24*
3.4 ± 0.26*
3.1 ± 0.25*
TR
38.0 ± 2.39
33.3 ± 2.77*
28.5 ± 2.11*
19.5 ± 1.41*
14.1 ± 0.91*
8.0 ± 0.52*
4.0 ± 0.30*
Enzyme activities were assayed in hemolysates from control and NaNO2 treated erythrocytes. Specific activities of GR, GPx and TR are in nmoles/min/mg Hb and CAT is in µmoles/min/mg Hb. Specific activity of SOD is in units/mg Hb (one unit is the amount which causes 50% inhibition of pyrogallol auto-oxidation in a reaction volume of 3 ml). CAT: catalase; SOD: Cu,Zn superoxide dismutase; GR: glutathione reductase; GPx: glutathione peroxidase; TR: thioredoxin reductase Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
Table 4. Effect of NaNO2 on some metabolic enzymes. Control
0.1 mM
0.5 mM
1.0 mM
2.5 mM
5.0 mM
10.0 mM
G6PD
9.4 ± 0.55
7.4 ± 0.47
6.5 ± 0.51*
5.1 ± 0.41*
4.6 ± 0.34*
4.1 ± 0.25*
3.4 ± 0.21*
LDH
34 ± 1.8
38.7 ± 2.1
43.3 ± 3.2*
45.7 ± 3.4*
48.4 ± 3.8*
52 ± 4.2*
58 ± 4.5*
ACP
2.3 ± 0.15
2.6 ± 0.12
3.1 ± 0.17*
3.7 ± 0.15*
4.4 ± 0.15*
5.0 ± 0.26*
5.3 ± 0.32*
HK
0.68 ± 0.06
0.65 ± 0.05
0.6 ± 0.06*
0.58 ± 0.06*
0.55 ± 0.03*
0.52 ± 0.04*
0.46 ± 0.04*
AMP deaminase
0.23 ± 0.03
0.41 ± 0.03*
0.95 ± 0.05*
1.24 ± 0.06*
1.6 ± 0.11*
1.83 ± 0.13*
2.81 ± 0.28*
Enzyme activities were assayed in hemolysates from control and NaNO2 treated erythrocytes. Specific activities of G6PD, LDH and ACP are in nmoles/min/mg Hb, hexokinase in µmoles/hr/mg Hb and AMP deaminase is in units/gm Hb. G6PD: glucose 6-phosphate dehydrogenase; LDH: lactate dehydrogenase; ACP: acid phosphatase; AMP: adenosine 5’-monophosphate. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05. 23
FIGURES
Fig. 1. Effect of NaNO2 on osmotic fragility of erythrocytes. Osmotic fragility of control and NaNO2 treated erythrocytes was determined by adding them to solutions containing different concentrations of NaCl and determining cell lysis (as described in Materials and Methods section). Results of a typical experiment are shown above.
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Fig. 2. Effect of NaNO2 on (A) methemoglobin levels and (B) methemoglobin reductase activity. MetHb and MetHb reductase were determined in hemolysates from control and NaNO2 treated erythrocytes. MetHb levels are expressed as percentage of total hemoglobin in lysates. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Fig. 3. Formation of Heinz bodies. Control and NaNO2 treated erythrocytes were stained with methyl violet and visualized under a microscope at 100 x magnification. (a) Control; cells treated with (b) 2.5 mM and (c) 10 mM NaNO2. Heinz bodies are indicated with arrows.
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Fig. 4. Effect of NaNO2 on H2O2 levels in erythrocytes. H2O2 was determined in hemolysates from control and NaNO2 treated erythrocytes. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Fig. 5. Intracellular nitrite levels in erythrocytes. Nitrite was determined in hemolysates from control and NaNO2 treated erythrocytes using Greiss reagent. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Figure 6. Effect of NaNO2 on reduced (GSH) and oxidized (GSSG) glutathione levels in erythrocytes. GSH and GSSG were determined in hemolysates from control and NaNO2 treated erythrocytes. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Fig. 7. SEM analysis of control and NaNO2 treated erythrocytes. (a) Control; erythrocytes treated with (b) 2.5 mM and (c) 10 mM NaNO2. Magnification is 2000 fold.
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Fig. 8. Schematic representation of nitrite toxicity in human erythrocytes. Nitrite enters the cell where it increases the generation of ROS and RNS. This converts hemoglobin to methemoglobin and decreases the oxygen carrying capacity of erythrocytes. Lowered GSH levels and reduced AO enzyme activities result in compromised AO defence leading to oxidative stress condition. This causes lipid peroxidation, protein oxidation and lowers the cellular energy levels due to derangement of normal metabolism. All these factors contribute to cell damage which can reduce the lifespan of cells in blood (red cell senescence) since damaged erythrocytes are removed from circulation by the spleen. (AO, antioxidant; GSH, glutathione; MetHb, methemoglobin; ROS, reactive oxygen species; RNS, reactive nitrogen species; NADPH, nicotinamide adenine dinucleotide phosphate reduced).
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Graphical abstract
32
Highlights
Sodium nitrite increases hemoglobin oxidation in human erythrocytes. Protein and lipid oxidation are increased while antioxidant power is decreased. ROS generation increases suggesting induction of oxidative stress in erythrocytes. Activities of membrane bound, metabolic and antioxidant enzymes were altered. Gross morphological changes were seen under EM in nitrite treated erythrocytes.
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S0887-2333(15)00183-6 http://dx.doi.org/10.1016/j.tiv.2015.07.022 TIV 3595
To appear in:
Toxicology in Vitro
Received Date: Revised Date: Accepted Date:
10 March 2015 2 July 2015 27 July 2015
Please cite this article as: Ansari, F.A., Ali, S.N., Mahmood, R., Sodium nitrite-induced oxidative stress causes membrane damage, protein oxidation, lipid peroxidation and alters major metabolic pathways in human erythrocytes, Toxicology in Vitro (2015), doi: http://dx.doi.org/10.1016/j.tiv.2015.07.022
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Sodium nitrite-induced oxidative stress causes membrane damage, protein oxidation, lipid peroxidation and alters major metabolic pathways in human erythrocytes
Fariheen Aisha Ansari, Shaikh Nisar Ali, Riaz Mahmood*
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, U.P., India
*Corresponding author
Telephone: 91-571-2700741; +91-9639901935
E. mail: [email protected]
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ABSTRACT Nitrite salts are present as contaminants in drinking water and in the food and feed chain. In this work, the effect of sodium nitrite (NaNO2) on human erythrocytes was studied under in vitro conditions. Incubation of erythrocytes with 0.1-10.0 mM NaNO2 at 37 °C for 30 min resulted in dose dependent decrease in the levels of reduced glutathione, total sulfhydryl and amino groups. It was accompanied by increase in hemoglobin oxidation and aggregation, lipid peroxidation, protein oxidation and hydrogen peroxide levels suggesting the induction of oxidative stress. Activities of all major erythrocyte antioxidant defence enzymes were decreased in NaNO2-treated erythrocytes. The activities of enzymes of glycolytic and pentose phosphate pathways were also compromised. However, there was a significant increase in acid phosphatase and also AMP deaminase, a marker of erythrocyte oxidative stress. Thus, the major metabolic pathways of cell were altered. Erythrocyte membrane damage was suggested by lowered activities of membrane bound enzymes and confirmed by electron microscopic images. These results show that NaNO2-induced oxidative stress causes hemoglobin denaturation and aggregation, weakens the cellular antioxidant defense mechanism, damages the cell membrane and also perturbs normal energy metabolism in erythrocytes. This nitrite-induced damage can reduce erythrocyte life span in the blood. Key words: Nitrite; erythrocytes; methemoglobin; oxidative stress; metabolic pathways; protein oxidation
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Abbreviations: AMP, adenosine 5’-monophosphate; ATP, adeosine 5’-triphosphate; ATPase, adeosine triphosphatase; DTNB, 5,5’-dithiobisnitrobenzoic acid; G6PD, glucose 6phosphate dehydrogenase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; Hb, hemoglobin; H2O2, hydrogen peroxide; LDH, lactate dehydrogenase; MetHb, methemoglobin; NAD+ and NADH, reduced and oxidized nicotinamide adenine dinucleotide; NADP+ and NADPH, reduced and oxidized nicotinamide adenine dinucleotide phosphate; Na,K-ATPase, sodium potassium ATPase; NaNO2, sodium nitrite; NO, nitric oxide; ROS, reactive oxygen species; SEM, scanning electron microscopy; SH, sulfhydryl; SOD, Cu,Zn superoxide dismutase; TR, thioredoxin reductase.
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1. Introduction Nitrite is naturally present in humans and in most green vegetables. In humans it acts directly or indirectly (by reducing to nitric oxide) in maintaining vascular homeostasis. It also plays key physiological roles in signalling, cellular respiration and mediation of innate immunity (Lundberg et al., 2008). Inside the cell, nitrite can be reversibly converted to nitrate and nitric oxide. Sodium nitrite (NaNO2) is widely used as a food preservative and colour fixative in fish and meat. It is also used in the manufacture of dyes, nitroso compounds and rubber chemicals. Medicinally, it is used as a vasodilator, bronchodilator and as an antidote for cyanide poisoning. Human exposure to nitrite has increased sharply in recent years because of extensive use of nitrogenous fertilizers in agriculture, improper disposal of human and industrial wastes and atmospheric nitrogen pollution (Galloway et al., 2003). The nitratenitrite content in food and water in many areas has increased alarmingly beyond the permissible limits (WHO, 2011). In some areas it has greatly exceeded the levels of 1 ppm (nitrite) and 10 ppm (nitrate) in drinking water set by the U.S. Environmental Protection Agency (EPA). Although nitrite has important functions in the cell at physiological concentrations, it can be toxic in high amounts to animals and humans. The immediate and major health implication of nitrite intoxication is methemoglobin (MetHb) formation (Chui et al., 2005) and cyanosis, which can be fatal. Infants are particularly susceptible to nitrite intoxication, causing death in most cases (WHO, 2011). Continuous exposure to non-fatal doses of nitrite can cause physiological disturbances including permanent growth inhibition, neurological disorders, respiratory failure and paralysis (Mensinga et al., 2003). Pregnant women, anaemic and glucose 6-phosphate dehydrogenase (G6PD) deficient individuals are also prone to nitrite toxicity (Huber et al., 2013; WHO, 2011). Nitrite easily transforms into carcinogenic 4
nitrosating compounds in the acidic environment of gut (Brambilla and Martelli, 2007). A relationship between nitrite/nitrate levels and higher incidence of various cancers has been reported in humans (Dellavalle et al., 2013; Coss et al., 2004). NaNO2 administered rats showed increased risk of forestomach neoplasm and squamous papilloma (NTP, 2001). Degenerative changes were observed in various tissues of NaNO2 administered mice (Ozen et al., 2014). Nitrite has thus been classified as cytotoxic, mutagenic, teratogenic and embryotoxic (NTP, 2001). Human exposure to nitrite results in its uptake and subsequent transfer to blood with rapid binding to erythrocytes (Dejam et al., 2007). Blood acts as a biological nitrite buffer to maintain its concentration throughout the body, and supply it as and when required. About 75% of nitrite enters erythrocytes by diffusion, as nitrous acid or other species, while the remaining 25% uptake occurs by the sodium-dependent phosphate transporter (May et al., 2000). Erythrocytes have been used as a simple model to study the cellular effects of various compounds (Takebayashi et al., 2010), especially those that generate reactive oxygen species (ROS). Erythrocytes are particularly susceptible to oxidative insult due to their role as oxygen transporters and high content of polyunsaturated fatty acids, transition metals and redox active hemoglobin (Hb) molecules. We have examined the effect of NaNO2 on human erythrocytes under in vitro conditions using concentrations to which humans could be potentially exposed, especially in areas with nitrate-nitrite contaminated drinking water (WHO, 2011). Our results show that NaNO2 causes significant oxidative damage to erythrocytes which was also visible in electron microscopic images. 2. Materials and Methods 2.1. Materials
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NaNO2, 1-chloro-2,4-dinitrobenzene, glutathione reductase (GR), metaphosphoric acid, 2,4,6-trinitrobenzene sulfonate, N-(1-naphthyl)ethylenediamine dihydrochloride and ouabain were purchased from Sigma–Aldrich, USA. Reduced (GSH) and oxidized (GSSG) glutathione, reduced and oxidized nicotinamide adenine dinucleotide phosphate (NADPH and NADP+), reduced nicotinamide adenine dinucleotide (NADH), N-ethylmaleimide, ophthalaldehyde, pyrogallol, 2,6-dichlorophenolindophenol, 2,4-dinitrophenylhydrazine, 5,5'dithiobisnitrobenzoic acid (DTNB), xylenol orange, sulphanilamide, adenosine 5’monophosphate (AMP), glucose 6-phosphate, sodium nitroprusside, methyl violet and sorbitol were from Sisco Research Laboratory (Mumbai, India). Thiobarbituric acid, tris(hydroxymethyl)aminomethane, trichloroacetic acid and S-acetylthiocholine iodide were purchased from Himedia Laboratories (Mumbai, India). All other chemicals were of analytical grade. 2.2. Isolation of erythrocytes and treatment with NaNO2 This study was approved by the institutional ethics committee which monitors research involving human subjects. Human blood was taken from young (22–30 years), healthy, nonsmoking volunteers after getting their informed consent and used immediately. Blood was collected in heparinized tubes, centrifuged at 1,500 rpm for 10 min at 4 °C in a clinical centrifuge and the plasma and buffy coat were removed. The erythrocyte pellet was washed three times with phosphate-buffered saline (PBS) (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) and resuspended in PBS to give a 10% (v/v) cell suspension (hematocrit). Stock solutions of NaNO2 were prepared in PBS. Erythrocytes were incubated with different concentrations of NaNO2 (0.1–10 mM, corresponding to 0.0069 - 0.69 mg/ml) for 30 min at 37 °C. NaNO2-untreated cells were similarly incubated at 37 °C and served as control. The samples were centrifuged at 2500 rpm for 10 min at 4 °C. Cell pellets were washed three times with PBS and erythrocytes were lysed with ten volumes of 5 mM sodium phosphate 6
buffer, pH 7.4, at 4 °C for 2 h. Samples were centrifuged at 3,000 rpm for 10 min at 4 °C and the supernatants (hemolysates) were quickly frozen in aliquots to be used later for the analysis of several biochemical parameters. 2.3. Osmotic fragility Control and NaNO2 treated erythrocytes were incubated at 37 °C and centrifuged as above. The cell pellet was washed and resuspended in PBS to give 10% hematocrit. Then, 0.05 ml of this cell suspension was added to different tubes containing 5 ml of 0.2 to 0.7% NaCl. After 30 min at 37 °C, the samples were centrifuged at 2500 rpm for 10 min in a clinical centrifuge and the absorbance of supernatants was recorded at 540 nm. The absorbance of untreated erythrocytes, lysed with 5 mM sodium phosphate buffer, pH 7.2, for 2 hr at 4 °C, served as a reference and represents 100% lysis. 2.4. Hemoglobin and methemoglobin levels and methemoglobin reductase Hb concentration in hemolysates was determined by the cyanomethaemoglobin method using a commercially available kit (Hemocor-D Kit, Coral Clinical Systems, Goa, India). The levels of MetHb were determined from the absorbance of diluted hemolysates at 540, 576 and 630 nm (Benesch et al., 1973) and expressed as percent of total Hb. NADHdependent MetHb reductase activity was determined from the increase in absorbance at 600 nm after incubation of hemolysates with NADH and 2,6-dichlorophenolindophenol (Kuma et al., 1972). Heinz bodies were detected by staining control and NaNO2 treated erythrocytes with 0.5% methyl violet (in 0.9% NaCl) for 45 min. Cells were then fixed on glass slides and observed under a light microscope at 100x magnification. 2.5. Reduced (GSH) and oxidized (GSSG) glutathione, amino groups and total sulfhydryl levels 7
GSH and GSSG levels in hemolysates were determined fluorometrically using ophthalaldehyde and N-ethylmaleimide (Hissin and Hilf, 1976). Total sulfhydryl (SH) groups were determined from the yellow colour produced after their reaction with DTNB (Sedlak and Lindsay, 1968). Free amino groups were determined using 2,4,6-trinitrobenzenesulfonate (Snyder and Sobocinski, 1975). 2.6. Carbonyl content, thiobarbituric acid reactive substances, hydrogen peroxide and nitrite levels Protein carbonyl content in hemolysates was determined after reaction with 2,4dinitrophenylhydrazine (Levine et al., 1990). Hemolysates, deproteinized using zinc sulphateNaOH, were used for the analysis of lipid peroxidation, nitrite and H2O2 levels. Malondialdehyde, a product of lipid peroxidation, was measured as thiobarbituric acid reactive substances (Buege and Aust, 1978). Nitrite concentration was determined using Greiss reagent (1% sulfonilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride) as described by Miranda et al., (2001). H2O2 concentrations were determined using ferrous ammonium sulphate-xylenol orange (FOX reagent) as colour reagent in the presence of 100 mM sorbitol (Gay and Gebicki, 2000). 2.7. Antioxidant enzymes All enzymes were assayed in hemolysates. Catalase activity was determined from the conversion of H2O2 to water (Aebi, 1984) and Cu,Zn superoxide dismutase (SOD) from the inhibition of auto-oxidation of pyrogallol (Marklund and Marklund, 1974). Glutathione reductase (GR) was assayed from the conversion of NADPH to NADP + during the reduction of GSSG to GSH (Carlberg and Mannervik, 1985). Glutathione peroxidase (GPx) was assayed at 340 nm from the oxidation of NADPH to NADP+ in the presence of GSSG and GR; azide was added to inhibit catalase activity (Flohe and Gunzler, 1984). Thioredoxin
8
reductase (TR) activity was determined from the absorbance at 410 nm due to reduction of DTNB to the yellow coloured thionitrobenzoate anion (Tamura and Stadtman, 1996). 2.8. Metabolic enzymes The activity of AMP deaminase was determined in hemolysates from the ammonia released from AMP (Pederson and Berry, 1977). G6PD was assayed by following the conversion of NADP+ to NADPH at 340 nm in the presence of glucose 6-phosphate (Shonk and Boxer, 1964). Hexokinase activity was determined by the method of Crane and Sols (1953), which measures the depletion in glucose concentration with time. Lactate dehydrogenase (LDH) was assayed from the oxidation of NADH to NAD + at 340 nm using sodium pyruvate as substrate (Khundmiri et al., 2004). Acid phosphatase activity was measured from the yellow coloured p-nitrophenol formed at pH 4.5 upon hydrolysis of p-nitrophenyl phosphate (Mohrenweiser and Novotny, 1982). 2.9. Membrane bound enzymes Acetylcholinesterase was assayed in hemolysates using DTNB to measure thiocholine released from S-acetylthiocholine iodide (Ellman et al., 1961). Na,K-ATPase and total ATPases were assayed from the inorganic phosphate released upon hydrolysis of ATP in the presence and absence of 1 mM ouabain (Bonting et al., 1961). 2.10. Scanning electron microscopy Control and NaNO2 treated erythrocytes were washed three times with PBS and fixed with 2.5% glutaraldehyde for 1 hr at room temperature. After washing again with PBS, the samples were mounted on glass slides and dehydrated with graded ethanol (50%-70%-90%100%). The samples were then coated with gold-palladium layer and examined in a scanning electron microscope (SEM) (Wang et al., 2009). 2.11. Statistical Analysis 9
All data are expressed as mean ± standard error. Statistical evaluation was conducted by oneway ANOVA using the program Origin 6.1 Software (USA). A probability level of P<0.05 was selected as indicating statistical significance. All experiments were done with 6-10 different blood samples to document reproducibility; results of six samples are shown here. 3. Results Human erythrocytes were isolated from fresh blood and treated with different concentrations of NaNO2 for 30 min at 37 °C. The cells were then pelleted by centrifugation, washed with PBS to remove excess nitrite and then lysed with hypotonic buffer. Several biochemical parameters were then determined in the hemolysates. Incubation of erythrocytes with NaNO2 rendered them more osmotically fragile and they lysed at relatively higher salt concentrations compared to control cells (Fig. 1). The activities of membrane bound enzymes, acetylcholinesterase and Na,K-ATPase, were significantly decreased while total erythrocyte ATPase was also lowered compared to control (Table 1). Rapid formation of MetHb was observed within few minutes of NaNO2 treatment. MetHb levels were two times the control value even at 0.05 mM and reached 78% of the total Hb at 10 mM (Fig. 2A). However, the activity of MetHb reductase was enhanced with increase in nitrite concentration (Fig. 2B). Extensive oxidation of Hb causes it to denature, aggregate and form insoluble Heinz bodies which cross-link with membrane proteins. These Heinz bodies were visible in NaNO2 treated erythrocytes, as dark stained spots within and near the periphery of the cell (Fig. 3). Several parameters of induction of oxidative stress in the cell were determined in hemolysates. The carbonyl content was used as a measure of protein oxidation and it increased considerably in NaNO2-treated erythrocytes in a concentration dependent manner (Table 2). A similar increase in lipid peroxidation was seen as determined from the levels of
10
malondialdehyde, an end product of lipid peroxidation (Table 2). At higher concentrations of NaNO2, both protein oxidation and lipid peroxidation were more than three times the corresponding control values. Total SH and amino groups were lowered in hemolysates from NaNO2 treated erythrocytes (Table 2). However, a dramatic increase in H2O2 levels was seen, even at low nitrite concentrations (Fig. 4). As expected, nitrite content of the cell increased in a concentration dependent manner (Fig. 5). The effect of NaNO2 on enzymatic and non-enzymatic antioxidant defence system of erythrocytes was determined next. A slight initial increase in GSH levels was followed by a sharp decline at higher NaNO2 concentrations; at 10 mM NaNO2 the GSH content was only 4% of the control value. This was accompanied by a parallel increase in GSSG, which was seen at higher concentrations of NaNO2 (Fig. 6). The activities of all the major (catalase, SOD, GPx) and secondary (TR, GR) antioxidant defence enzymes were decreased in nitrite treated erythrocytes (Table 3). TR was the enzyme most affected and at 10 mM NaNO2 its activity was 10% of control. Nitrite treatment of erythrocytes led to a decrease in the activity of hexokinase, the first enzyme of glycolysis. The activity of G6PD, the major source of NADPH in erythrocytes, declined in a dose dependent manner and was 36% of control at the 10 mM NaNO2. However, there was a ten folds increase (at 10 mM NaNO2) in the activity of AMP deaminase, an enzyme of purine metabolism. The activities of acid phosphatase and LDH also increased upon NaNO2 treatment (Table 4). SEM analysis showed normal biconcave appearance and discocyte shape for control erythrocytes (Fig. 7). Changes in erythrocyte shape and morphology were observed in NaNO2 treated samples. Loss of discocytic morphology and deformation was clearly evident.
11
4. Discussion Human exposure to nitrite results in its rapid entry into bloodstream where it can interact with erythrocytes. We have investigated the effects of NaNO2 on human erythrocytes, especially on the cell membrane, Hb oxidation, antioxidant defence and metabolic pathways. Treatment of erythrocytes with NaNO2 did not cause significant hemolysis (<1% at 10 mM NaNO2) but the cells became more osmotically fragile. Alterations in membrane lipid and protein structure cause decrease in membrane integrity. The ability of cells to stretch and deform is reduced, increasing their rigidity and making them more susceptible to lysis. Using fluorescence anisotropy, Zavodik et al. (1999) have also reported that nitrite induces membrane hyperpolarisation and increases its rigidity. NaNO2 treatment led to massive increase in level of H2O2 which can react with transition metals like iron to give the damaging hydroxyl radical. It also resulted in significant increase in lipid peroxidation probably due to increase in superoxide radical and H2O2 levels since the activities of SOD and catalase, enzymes that use them as substrates, were decreased. A three folds increase in protein carbonyl content took place since ROS introduce carbonyl groups in amino acid residues (Halliwell and Gutteridge, 1999). High protein carbonyl and 3nitrotyrosine levels have been reported in NaNO2 treated meat products (Vossen et al., 2015). Amino groups were also oxidized leading to a decrease in their content. The steep decline in GSH, which maintains a reducing environment within the cell, was accompanied by a parallel increase in GSSG levels at higher NaNO2 concentrations. This decrease in GSH might be due to (i) lower activity of GR, which converts GSSG to GSH (ii) direct oxidation of the thiol group of GSH by ROS or (iii) its reaction with NO to form nitrosothiols (Radi, 2004). A concomitant reduction in total SH groups shows that the entire cellular thiol status was affected. This increase in H2O2, protein oxidation and lipid peroxidation along with decrease in SH content, strongly suggests that NaNO2 induces oxidative stress in erythrocytes. 12
Vatassery et al. (2004) have also reported higher oxidative stress in the brain of nitrite treated rats. Nitrite also exacerbates oxidative toxicity when associated with extracellular Hb and potentiates acute renal toxicity in guinea pigs (Baek et al., 2015). Activities of all major antioxidant enzymes were decreased in NaNO2 treated erythrocytes. SOD, catalase and GPx, are known to be inactivated by free radicals and ROS (Escobar et al., 1996; Pigeolot et al., 1990; Yang and Wang, 1991). SOD and catalase contain bound copper and iron, respectively, and the direct reaction of these metals with NO might also be a cause of reduced activity. The decrease in G6PD activity also affects several antioxidant enzymes that require NADPH. TR uses NADPH to keep thioredoxin in its reduced state which, in turn, maintains other proteins in their reduced form by cysteine thiol-disulphide exchange. GR uses NADPH to regenerate GSH from GSSG. Low NADPH level due to nitrite-induced inhibition of G6PD will reduce the activities of both GR and TR. It will also inhibit catalase, which contains four NADPH bound per tetrameric molecule that are needed for the biological activity of this enzyme (Kirkman et al., 1999). Hemoglobin is a major cellular target of ROS in erythrocytes. Oxidation of ferrous iron of Hb to the ferric form gives MetHb, which is inactive as an oxygen transporter. This process releases an electron that is taken up by molecular oxygen to give superoxide radical which can then react with NO to give peroxynitrite (Pryor and Squadrito, 1995) or is converted to H2O2. A high percentage of total Hb was converted to MetHb in nitrite treated erythrocytes, as also reported previously (Chui et al., 2005). MetHb reductase converts MetHb back to Hb, by reducing the ferric iron to ferrous form, using either NADH or NADPH as the electron donor. Surprisingly, the activity of NADH dependent MetHb reductase (major form of the enzyme) was significantly increased in NaNO2 treated erythrocytes. This depicts the adaptive nature of the cell in response to increased MetHb formation due to NaNO2 assault. Another reason for increase in MetHb reductase activity could be the release of inactive membrane 13
bound form into the cytosol (Choury et al., 1981) thereby increasing its activity in cell lysates. However, MetHb levels were too high to be reversed by the enhanced activity of the reductase. Extensive oxidation and cross-linking of some Hb also took place giving Heinz bodies that were attached to the cell membrane. Erythrocyte energy metabolism was also profoundly altered in NaNO 2 treated erythrocytes. Erythrocyte acid phosphatase can dephosphorylate proteins including band 3, a transmembrane protein whose cytoplasmic tail binds several glycolytic enzymes (Campanella et al., 2008). NaNO2-induced increase in acid phosphatase activity can result in dephosphorylation of band 3 protein which will enhance the binding and thus inhibition of glycolytic enzymes, thereby lowering ATP levels. NaNO2-induced decrease in hexokinase activity will also contribute to decrease in cellular energy (ATP) level. AMP deaminase maintains the levels of purine nucleotides and is also a potent marker of oxidative stress in erythrocytes (Tavazzi et al., 2001). A dramatic ten folds increase in the activity of AMP deaminase will cause corresponding increase in inosine monophosphate levels, rather than AMP, parallel to ATP depletion. The erythrocyte membrane contains large amounts of acetylcholinesterase of unknown function. The significant decrease in acetylcholinesterase activity upon NaNO2 treatment is indicative of oxidative stress and membrane damage (O’Malley et al., 1966; Zhou et al., 2003). Erythrocyte ATPases cause asymmetric distribution of ions across the membrane, maintain cell integrity and normal function. Na,K-ATPase (and also Ca2+ ATPase) is known to have an SH group at its active site (Boldyrev et al., 1997) whose oxidation will inhibit the enzyme. Another reason could be increased peroxidation of membrane lipids thereby disturbing the protein environment and leading to enzyme inhibition. LDH is attached to the inner side of plasma membrane and increase in its activity could be due to membrane disruption and consequent release of the bound enzyme into the cytosol. However, there was 14
no release of LDH in the supernatant upon treatment of erythrocytes with nitrite. All these results suggest derangement of membrane function and integrity. SEM images of NaNO2treated cells showed multiple bleb formation on the surface and change in the shape of cells to echinocytes (spiculated erythrocytes). Membrane lipid peroxidation and damage to cytoskeleton proteins cause bleb formation (Singh and Rajini, 2008) which is often accompanied by loss of membrane asymmetry. The SEM analysis greatly supports and confirms the results of our biochemical studies that NaNO2 induces extensive damage in human erythrocytes. 5. Conclusions NaNO2 treatment of human erythrocytes results in oxidation, denaturation and aggregation of Hb and also enhances lipid and protein oxidation. It lowers the enzymatic and non-enzymatic antioxidant defence and alters the activities of enzymes of major metabolic pathways and erythrocyte membrane. The biochemistry of these effects may be attributed to the induction of oxidative stress and consequent increase in ROS and, perhaps partly, due to interaction of NO (generated from nitrite) with thiol groups and transition metal ions, thereby affecting enzymes having either of these species at their active site. The results of nitrite treatment on erythrocytes are shown schematically in Figure 8. In order to prevent nitrite induced toxicity, steps should be taken to reduce its content in feed and food chain and to ensure an antioxidant rich diet in populations living in nitrite contaminated areas. Conflict of interest The authors declare that there is no conflict of interest.
15
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TABLES
Table 1. Effect of NaNO2 on membrane bound enzymes. Control
0.1 mM
0.5 mM
1.0 mM
2.5 mM
5.0 mM
10.0 mM
AChE
57.0 ± 3.8
53.6 ± 3.0
50 ± 4.2*
47.8 ± 3.9*
44.2 ± 3.7*
41.8 ± 3.3*
35.7 ± 2.8*
Total ATPase
0.85 ± 0.06
0.77 ± 0.05
0.68 ± 0.04*
0.61 ± 0.03*
0.56 ± 0.04*
0.52 ± 0.03*
0.44 ± 0.03*
Na,K-ATPase
0.51 ± 0.03
0.44 ± 0.03*
0.41 ± 0.04*
0.39 ± 0.02*
0.35 ± 0.02*
0.34 ± 0.03*
0.31 ± 0.03*
Enzyme activities were assayed in hemolysates from control and NaNO2 treated erythrocytes. Specific activity of acetylcholinestersae (AChE) is in nmoles/min/mg Hb and ATPases are in µmoles/hr/mg Hb. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
Table 2. Effect of NaNO2 on some parameters of oxidative stress Control
0.1 mM
0.5 mM
1.0 mM
2.5 mM
5.0 mM
10.0 mM
Carbonyl content
4.9 ± 0.43
6.3 ± 0.51*
8.3 ± 0.54*
9.2 ± 0.72*
12.2 ± 0.77*
14.1 ± 0.98*
16.3 ± 1.42*
TBARS
0.92 ± 0.06
1.1 ± 0.09
1.3 ± 0.09*
1.6 ± 0.11*
2.1 ± 0.17*
2.5 ± 0.20*
2.8 ± 0.21*
Total SH
0.94 ± 0.06
0.79 ± 0.04
0.7 ± 0.04*
0.59 ± 0.03*
0.52 ± 0.04*
0.47 ± 0.04*
0.41 ± 0.03*
Amino groups
1.2 ± 0.08
1.03 ± 0.07
0.9 ± 0.05*
0.87 ± 0.05*
0.84 ± 0.04*
0.71 ± 0.05*
0.52 ± 0.03*
Thiobarbituric acid reactive substances (TBARS) and carbonyl content are in nmoles/mg Hb while total sulfhydryl (SH) and amino groups are in µmoles/mg Hb. These parameters were determined in hemolysates from control and NaNO2 treated erythrocytes. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Table 3. Effect of NaNO2 on some antioxidant enzymes. Control
0.1 mM
0.5 mM
1.0 mM
2.5 mM
5.0 mM
10.0 mM
CAT
52.6 ± 3.9
48.2 ± 3.1
44.9 ± 2.8*
42.7 ± 3.6*
39 ± 2.7*
36.4 ± 2.1*
34.7 ± 2.3*
SOD
18.7 ± 1.35
17.0 ± 0.98
16.3 ± 1.21*
13.8 ± 0.98*
12.4 ± 0.86*
10.0 ± 0.75*
8.5 ± 0.49*
GPx
18.4 ± 1.14
16.5 ± 1.45*
13 ± 0.92*
11 ± 0.74*
9.6 ± 0.58*
7.4 ± 0.43*
6.6 ± 0.38*
GR
8.5 ± 0.70
7.5 ± 0.54
5.3 ± 0.32*
4.5 ± 0.37*
3.63 ± 0.24*
3.4 ± 0.26*
3.1 ± 0.25*
TR
38.0 ± 2.39
33.3 ± 2.77*
28.5 ± 2.11*
19.5 ± 1.41*
14.1 ± 0.91*
8.0 ± 0.52*
4.0 ± 0.30*
Enzyme activities were assayed in hemolysates from control and NaNO2 treated erythrocytes. Specific activities of GR, GPx and TR are in nmoles/min/mg Hb and CAT is in µmoles/min/mg Hb. Specific activity of SOD is in units/mg Hb (one unit is the amount which causes 50% inhibition of pyrogallol auto-oxidation in a reaction volume of 3 ml). CAT: catalase; SOD: Cu,Zn superoxide dismutase; GR: glutathione reductase; GPx: glutathione peroxidase; TR: thioredoxin reductase Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
Table 4. Effect of NaNO2 on some metabolic enzymes. Control
0.1 mM
0.5 mM
1.0 mM
2.5 mM
5.0 mM
10.0 mM
G6PD
9.4 ± 0.55
7.4 ± 0.47
6.5 ± 0.51*
5.1 ± 0.41*
4.6 ± 0.34*
4.1 ± 0.25*
3.4 ± 0.21*
LDH
34 ± 1.8
38.7 ± 2.1
43.3 ± 3.2*
45.7 ± 3.4*
48.4 ± 3.8*
52 ± 4.2*
58 ± 4.5*
ACP
2.3 ± 0.15
2.6 ± 0.12
3.1 ± 0.17*
3.7 ± 0.15*
4.4 ± 0.15*
5.0 ± 0.26*
5.3 ± 0.32*
HK
0.68 ± 0.06
0.65 ± 0.05
0.6 ± 0.06*
0.58 ± 0.06*
0.55 ± 0.03*
0.52 ± 0.04*
0.46 ± 0.04*
AMP deaminase
0.23 ± 0.03
0.41 ± 0.03*
0.95 ± 0.05*
1.24 ± 0.06*
1.6 ± 0.11*
1.83 ± 0.13*
2.81 ± 0.28*
Enzyme activities were assayed in hemolysates from control and NaNO2 treated erythrocytes. Specific activities of G6PD, LDH and ACP are in nmoles/min/mg Hb, hexokinase in µmoles/hr/mg Hb and AMP deaminase is in units/gm Hb. G6PD: glucose 6-phosphate dehydrogenase; LDH: lactate dehydrogenase; ACP: acid phosphatase; AMP: adenosine 5’-monophosphate. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05. 23
FIGURES
Fig. 1. Effect of NaNO2 on osmotic fragility of erythrocytes. Osmotic fragility of control and NaNO2 treated erythrocytes was determined by adding them to solutions containing different concentrations of NaCl and determining cell lysis (as described in Materials and Methods section). Results of a typical experiment are shown above.
24
Fig. 2. Effect of NaNO2 on (A) methemoglobin levels and (B) methemoglobin reductase activity. MetHb and MetHb reductase were determined in hemolysates from control and NaNO2 treated erythrocytes. MetHb levels are expressed as percentage of total hemoglobin in lysates. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Fig. 3. Formation of Heinz bodies. Control and NaNO2 treated erythrocytes were stained with methyl violet and visualized under a microscope at 100 x magnification. (a) Control; cells treated with (b) 2.5 mM and (c) 10 mM NaNO2. Heinz bodies are indicated with arrows.
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Fig. 4. Effect of NaNO2 on H2O2 levels in erythrocytes. H2O2 was determined in hemolysates from control and NaNO2 treated erythrocytes. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Fig. 5. Intracellular nitrite levels in erythrocytes. Nitrite was determined in hemolysates from control and NaNO2 treated erythrocytes using Greiss reagent. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Figure 6. Effect of NaNO2 on reduced (GSH) and oxidized (GSSG) glutathione levels in erythrocytes. GSH and GSSG were determined in hemolysates from control and NaNO2 treated erythrocytes. Results are mean ± standard error of six different samples. *Significantly different from control at P<0.05.
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Fig. 7. SEM analysis of control and NaNO2 treated erythrocytes. (a) Control; erythrocytes treated with (b) 2.5 mM and (c) 10 mM NaNO2. Magnification is 2000 fold.
30
Fig. 8. Schematic representation of nitrite toxicity in human erythrocytes. Nitrite enters the cell where it increases the generation of ROS and RNS. This converts hemoglobin to methemoglobin and decreases the oxygen carrying capacity of erythrocytes. Lowered GSH levels and reduced AO enzyme activities result in compromised AO defence leading to oxidative stress condition. This causes lipid peroxidation, protein oxidation and lowers the cellular energy levels due to derangement of normal metabolism. All these factors contribute to cell damage which can reduce the lifespan of cells in blood (red cell senescence) since damaged erythrocytes are removed from circulation by the spleen. (AO, antioxidant; GSH, glutathione; MetHb, methemoglobin; ROS, reactive oxygen species; RNS, reactive nitrogen species; NADPH, nicotinamide adenine dinucleotide phosphate reduced).
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Graphical abstract
32
Highlights
Sodium nitrite increases hemoglobin oxidation in human erythrocytes. Protein and lipid oxidation are increased while antioxidant power is decreased. ROS generation increases suggesting induction of oxidative stress in erythrocytes. Activities of membrane bound, metabolic and antioxidant enzymes were altered. Gross morphological changes were seen under EM in nitrite treated erythrocytes.
33