Preparation of RBC and treatment with uric acid and HgCl2
Young (20–29 year-old), non-smoking and healthy volunteers were used as donors and informed consent was taken from all of them. Venous blood was taken in heparin coated glass tubes and RBC prepared as previously described (Qasim and Mahmood, 2015). Briefly, blood was spun at 1,200 rpm for 10-12 min at 4 0C, supernatant removed, packed RBC washed thrice with phosphate buffered saline (PBS; 0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) and suspended in PBS to give a 10% (v/v) cell suspension or hematocrit.
UA and HgCl2 were purchased from Sigma Aldrich, USA. The 10% hematocrit was first incubated with UA (0.3, 0.4 and 0.6 mM) for 30 min at 37 °C, then 0.05 mM HgCl2 was added and cell suspensions kept for another 60 min at 37 °C. Control RBC were not incubated with UA and HgCl2 and kept at 37 °C for 90 min. RBC incubated with 0.05 mM HgCl2 for 60 min at 37°C served as positive control while those treated with 0.6 mM UA for 90 min at 37 °C showed UA alone effects. After the 37 oC incubations, cell suspensions were spun for 10-12 min at 2600 rpm and the pellets carefully washed thrice with PBS. RBC pellets were resuspended in 10x volume of 5 mM sodium phosphate buffer, pH 7.2, and kept for 2 h at 4 °C to lyse the cells. The samples were spun at 3000 rpm for 12 min, and the hemolysates (supernatants) were either used at once or aliquoted and kept at -20 0C for later use.
Hemoglobin and methemoglobin (MetHb) levels and MetHb reductase
The cyanomethemoglobin method was employed to determine hemoglobin concentration in hemolysates (Drabkin and Austin 1935). Hemolysate absorbance at 630, 576 and 540 nm was taken and MetHb level determined from the equation given below (Benesch et al. 1973):
[MetHb] = [2.985 A630 + 0.194 A576 – 4.023 A540] x 10-4 moles per liter
MetHb reductase activity was determined by adding 0.05 ml hemolysate to 0.1 mM of 2,6-dichlorophenolindophenol and 0.1 mM reduced nicotinamide adenine dinucleotide (NADH) and monitoring the enhancement in absorbance of solution at 600 nm (Kuma et al., 1972).
Heme degradation and free iron release
Degradation of heme was assayed in 50x diluted hemolysates by monitoring fluorescence emission at 480 nm, after excitation at 321 nm (Nagababu et al. 2008). The release of iron (Fe2+) was quantified in hemolysates using ferrozine that chelates free Fe2+ ions to form an adduct that absorbs at 550 nm (Panter, 1994).
Reactive oxygen species (ROS)
Intracellular generation of ROS was monitored by dichlorodihydrofluorescein diacetate (DCFH-DA) while DHE assay was used to detect superoxide radicals (Keller et al., 2004; Wojtala et al. 2014). The 10% RBC suspension was mixed with 10 µM DCFH-DA or DHE, kept in 37 °C water bath for 60 min and then centrifuged. Supernatants were discarded, cell pellets rinsed thrice with PBS and RBC suspended in PBS to again give 10% hematocrit. Cell suspensions were incubated with UA (0.3, 0.4, 0.6 mM) for 30 min, then 0.05 mM HgCl2 was added and RBC left for 15 min at 37 °C. Fluorescence of samples was recorded, setting the excitation and emission wavelengths at 485 and 530 nm (DCFH-DA) or 508 and 605 nm (DHE), respectively.
Reactive nitrogen species (RNS)
Peroxynitrite formation was monitored by adding 15 μM folic acid (final concentration) to hemolysates and after 5 min precipitating proteins with NaOH-ZnSO4. The samples were microfuged at top speed, the supernatants removed and their fluorescence noted at 460 nm after excitation at 380 nm (Huang et al., 2007). Nitric oxide (NO) was quantified from the total nitrite and nitrate content. Proteins were first precipitated by ZnSO4-NaOH and removed by centrifugation. Vanadium(III) chloride was added to supernatants to convert nitrate to nitrite. This was followed by addition of Greiss reagent which reacts with nitrite to give chromophoric azo-derivative that absorbs light at 540 nm (Miranda et al., 2001). A calibration curve was simultaneously constructed using sodium nitrite.
Oxidative stress markers
Oxidative stress parameters were measured in hemolysates. Carbonyl groups, introduced during protein oxidation, give hydrazone adducts with 2,4-dinitrophenylhydrazine that absorb at 360 nm (Levine et al.1990). Advanced oxidation protein products (AOPP) were quantified by adding 0.2 M citric acid and 1.16 M potassium iodide to hemolysates and recording absorbance at 340 nm. A calibration curve was constructed using different concentrations of Chloramine T (Hanasand et al. 2012). Malondialdehyde, a marker and end product of lipid peroxidation (LPO), was quantified from its reaction with thiobarbituric acid. The absorbance of pink chromophore formed was recorded at 531 nm (Buege and Aust 1978). Sulfhydryl groups react with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and form yellow thionitrobenzoate anion (TNB) that absorbs at 410 nm (Sedlak and Lindsay, 1968). Glutathione (GSH) concentration was measured by reaction of its sulfhydryl group with DTNB. Hemolysate proteins were first precipitated by metaphosphoric acid containing reagent and removed by centrifugation. DTNB was added to supernatants and the absorbance of TNB anion was noted at 410 nm (Beutler, 1984).
Total antioxidant capacity
Methods based on metal ion reduction and free radical quenching by sample AOs were employed. Hemolysates were used in the assays.
a) Metal reduction
In the ferric reducing ability of plasma (FRAP) method, sample AOs convert Fe3+ to Fe2+ and the Fe2+ ions give a colored complex with 2,4,6-tris(2-pyridyl)-s-triazine (Benzie and Strain 1996). In the cupric reducing antioxidant capacity (CUPRAC) assay, Cu2+ ions are reduced to Cu+; neocuproine then forms a colored complex with Cu+ (Çekiç et al., 2012). In the phosphomolybdenum method, sample AOs reduce Mo6+ to Mo5+ and absorbance of the green phosphate/Mo5+ product formed was recorded at 695 nm (Prieto et al., 1999).
b) Free radical quenching
In the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, the purple DPP• radical is converted to light yellow non-radical form by H atom donated by sample AOs. The absorbance of solutions was noted at 517 nm (Mishra et al. 2012). In the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method, potassium persulfate converts ABTS to its colored radical form. Sample AOs donate electron to change ABTS.+ to the non-radical colorless ABTS which was monitored from the decrease in absorbance of solution at 734 nm (Re et al., 1999). Trolox was used as standard and results expressed as Trolox equivalents.
The specific activities of RBC AO enzymes were determined in hemolysates. Cu–Zn superoxide dismutase (SOD) inhibits the auto-oxidation of pyrogallol which decreases the formation of colored product (Marklund and Marklund, 1974). The enzyme activity was determined by following the change in absorbance of pyrogallol solution at 420 nm in presence and absence of the enzyme. Catalase was assayed using H2O2 as substrate. The enzyme catalyzes breakdown of H2O2 to water and molecular oxygen which decreases the absorbance of solution at 240 nm (Aebi, 1984). Glutathione reductase cleaves the disulfide bond of oxidized glutathione to give GSH, using NADPH as the reductant. The resulting decrease in absorbance at 340 nm was followed for 3 min (Mannervik and Carlberg, 1985). Thioredoxin reductase activity was monitored from the increase in absorbance at 410 nm upon reduction of disulfide bond of DTNB, in presence of NADPH, to give TNB (Tamura and Stadtman, 1996). Glutathione peroxidase converts peroxides to water or alcohol using NADPH as reductant. The resulting decrease in 340 nm absorbance of solution was monitored for 3 min (Flohé and Günzler, 1984). Glutathione-S-transferase (GST) was assayed as described earlier by Habig et al. (1974); GSH and 1-chloro-2,4-dinitrobenzene were used as substrates.
All enzymes were assayed in hemolysates. The activity of hexokinase was measured by a two enzyme reaction using glucose 6-phosphate dehydrogenase (G6PD) (Bergmayer et al. 1983). Pyruvate kinase was also assayed by a coupled enzymatic reaction involving lactate dehydrogenase. The reaction converts NADH to NAD+ which leads to decrease in the absorbance of solution at 340 nm; this was monitored spectrophotometrically for 5 min (Bergmeyer, 1974). Lactate dehydrogenase, in presence of sodium pyruvate, converts NADH to NAD+ decreasing absorbance at 340 nm (Khundmiri et al., 2004). Activity of G6PD was determined from the enzymatic conversion of NADP+ to NADPH, enhancing the absorbance at 340 nm (Shonk and Boxer 1964). AMP-deaminase liberates ammonia from AMP. The ammonia reacts with phenol-sodium nitroprusside and alkaline hypochlorite to produce a blue indophenol (Pederson and Berry, 1977). The 5’-nucleotidase activity was assayed by quantifying inorganic phosphate released from AMP using arsenomolybdic acid reagent (Heppel and Hilmore, 1951). Assay of glyoxalase-I involves mixing methylglyoxal and glutathione to form hemithioacetal. When enzyme (hemolysate) is added, it converts hemithioacetal to S-lactoyl glutathione which absorbs at 240 nm (Arai et al. 2014).
Membrane bound enzymes
Acetylcholinesterase was assayed in hemolysates using S-acetylthiocholine iodide and DTNB as substrates and recording absorbance of TNB anion formed at 410 nm (Ellman et al. 1961). Na,K-ATPase was assayed by quantifying the inorganic phosphate, formed upon ATP hydrolysis, in absence and presence of 1 mM ouabain (Bonting et al, 1961). The difference between the two gives Na,K-ATPase activity and inorganic phosphate concentration in absence of ouabain yields total ATPase activity.
PMRS and AFR reductase
In the plasma membrane redox system (PMRS) assay, 1 volume of packed RBC was suspended in 9 volumes of PBS containing 5 mM glucose and 1 mM potassium ferricyanide. The cell suspensions were left for 30 min at 37 °C (Rizvi and Srivastava, 2010) and then spun for 10 min at 2000 rpm. The supernatants were analyzed for sodium ferrocyanide content by adding 1,10-phenanthroline and reading sample absorbance at 510 nm (Avron and Shavit, 1963). The method of May et al. (2004) was used to assay ascorbate free radical (AFR) reductase in hemolysates.
Scanning electron microscopy
Pellets of control and treated RBC were carefully rinsed thrice with PBS. Then 0.05 ml cells were suspended in 2.5% glutaraldehyde (fixing agent) and left at room temperature for 1 h. After another wash with PBS, the cell suspensions in PBS were carefully layered on glass slides, air dried and treated with increasing concentrations of ethanol (50%-70%-90%-100%). The dehydrated cells were dried, covered with a 10 nm thick gold-palladium layer and RBC visualized under a scanning electron microscope at 1500 fold magnification (Wang et al., 2009).
Lymphocyte isolation and comet assay
Blood (3 ml) was diluted with 3 ml of 0.9% NaCl (saline), carefully layered over 2 ml Histopaque 1077 and spun for 20 min at 4 °C and 2600 rpm. Lymphocytes present in buffy white layer in the middle were carefully removed and recentrifuged at 2600 rpm for 10 min. The lymphocyte pellets were washed and a 10% suspension prepared in saline. This was then incubated with HgCl2 and UA at 37 °C as mentioned above for RBC.
The comet assay of Singh et al. (1988) was used to detect DNA strand scission but with slight alterations. Briefly, 0.5% low melting point agarose and lymphocyte suspensions were mixed and spread on 1% agarose coated frosted glass slides. After a coat of low melting point agarose, the slides were left on ice for 10 min. Cells were lysed by adding solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 1% Triton-X-100, pH 10.0, and left on ice for 3 h. Slides were immersed for 20 min in ice cold electrophoresis solution (1.5 mM EDTA, 0.42 M NaOH, 0.9% NaCl); the high pH of this solution allows scission of alkali-labile sites and DNA uncoiling. Gel electrophoresis was performed for 20 min at 4 °C (300 mA, 25 V at 0.8 V/cm). Later, slides were dipped in 0.4 M Tris-HCl, pH 7.5, the DNA stained by ethidium bromide and visualized under a fluorescence microscope (CX41, Olympus, Japan) at 100x magnification. Images of 50 cells from every slide were scored. An automated image analysis system (Komet 5.5, Kinetic Imaging, Liverpool, UK) was used to determine comet tail lengths.
Lysosomal membrane destabilization and mitochondrial membrane potential (MMP) in lymphocytes
Equal volumes of 10% lymphocyte suspension and 5 µM acridine orange were mixed and samples left for 10 min at 37 0C. After centrifugation, the cell pellets were rinsed with saline and fluorescence of diffused acridine orange dye, upon lysosomal membrane damage, was recorded at 540 nm after excitation at 470 nm (Pourahmad et al., 2011).
The MMP was also determined fluorometrically in lymphocytes. Control, HgCl2 alone, UA alone and UA + HgCl2 treated lymphocytes (0.2 ml) were mixed with 0.1 ml of 5 µM Rhodamine 123 and left at room temperature for 15 min in the dark. Dye fluorescence was monitored at 525 nm after excitation at 505 nm (Pourahmad et al., 2009).