Thiram-induced cytotoxicity and oxidative stress in human erythrocytes: an in vitro study

Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx

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Thiram-induced cytotoxicity and oxidative stress in human erythrocytes: an in vitro study Samreen Salam, Amin Arif, Riaz Mahmood

⁎

Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, U.P., India

A R T I C LE I N FO

A B S T R A C T

Keywords: Thiram Antioxidant power Erythrocytes Glucose metabolism Reactive oxygen species

Tetramethylthiuram disulfide, commonly known as thiram, is an organosulfur compound which is used as a bactericide, fungicide and ectoparasiticide to prevent disease in seeds and crops. Being a fungicide there is a high probability of human occupational exposure to thiram and also via consumption of contaminated food. In this work, the cytotoxicity of thiram was studied under in vitro conditions using human erythrocytes as the cellular model. Erythrocytes were incubated with different concentrations of thiram (25–500 μM) for 4 h at 37 °C. Control cells (thiram untreated) were similarly incubated at 37 °C. Whole cells and hemolysates were analyzed for various biochemical parameters. Treatment of erythrocytes with thiram increased protein and lipid oxidation and hydrogen peroxide level in hemolysates but decreased glutathione and total sulfhydryl group content. This was accompanied by hemoglobin oxidation, heme degradation and release of free iron. Activities of all major antioxidant enzymes were inhibited. The antioxidant power of thiram treated erythrocytes was lowered resulting in decreased metal reducing and free radical quenching ability. These results suggest that thiram enhances the generation of reactive species that cause oxidative modification of cell components. This was confirmed by experiments that showed enhanced generation of reactive oxygen and nitrogen species in thiram treated erythrocytes. Activities of marker enzymes of glucose metabolism and erythrocyte membrane were also inhibited. All effects were seen in a thiram concentration-dependent manner. Electron microscopy further supported the damaging effect of thiram on erythrocytes. Thus thiram induces oxidative stress condition in human erythrocytes and causes oxidative modification of cell components.

1. Introduction Pesticides are widely used to protect crops from diseases, insects and organisms that can harm agriculture. It has resulted in human exposure, either occupational or environmental, which can adversely affect human health. Excessive use of pesticides can also damage the ecosystem by affecting surrounding fauna and flora (Paliwal et al., 2009). Pesticide exposure can augment the risk of several human chronic diseases such as amyotrophic lateral sclerosis, diabetes, neurodegenerative disorders (Abdollahi et al., 2004; De Souza et al., 2011; Mostafalou and Abdollahi, 2012). Dithiocarbamates are a class of chemical compounds used in medicine and more commonly in agriculture as pesticides (Thind and

Hollomon, 2018). Tetramethylthiuram, commonly known as thiram, is a member of the dithiocarbamate family. Thiram is a multifaceted compound used as an insecticide, bactericide and mainly as a fungicide to protect seeds and crops from disease. Thiram is used in rubber industry as a vulcanizing agent to provide rigidity to natural rubber (Cereser et al., 2001a; Mathieu et al., 2015). The extensive use of thiram has raised concern about human health, particularly of agricultural workers who are frequently exposed to high level of pesticides. Thiram treatment disrupts the reproductive cycle in female rats by disturbing the hormonal control of ovulation. It also results in neuronal toxicity and behavioral changes in rats (Lee and Peters, 1976). Renal failure, carcinogenicity and developmental toxicity are some other harmful effects of thiram (Rasaputra et al., 2013). Thiram is also toxic

Abbreviations: ABTS, 2,2′-azinobis(3-ethylenebenzothioazoline-6-sulfonic acid); AMP, ADP and ATP, adenosine 5′- mono-, 5′-di- and 5′-tri- phosphate; AFR, ascorbate free radical; AO, antioxidant; ATPase, adenosine triphosphatase; CUPRAC, cupric reducing antioxidant power; DCFH-DA, 2,7-dichlorodihydrofluorescein diacetate; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTNB, 5,5′-dithibisonitrobenzoic acid; FRAP, ferric reducing antioxidant power; G6PD, glucose 6-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; Hb, hemoglobin; H2O2, hydrogen peroxide; LDH, lactate dehydrogenase; MetHb, methemoglobin; NADH, reduced nicotinamide adenine dinucleotide; NADP+ and NADPH, oxidized and reduced nicotinamide adenine dinucleotide phosphate; PBS, phosphate buffered saline; PMRS, plasma membrane redox system; SOD, Cu, Zn-superoxide dismutase; ROS, reactive oxygen species; RNS, reactive nitrogen species ⁎ Corresponding author. E-mail address: [email protected] (R. Mahmood). https://doi.org/10.1016/j.pestbp.2019.12.003 Received 1 October 2019; Received in revised form 5 December 2019; Accepted 15 December 2019 0048-3575/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Samreen Salam, Amin Arif and Riaz Mahmood, Pesticide Biochemistry and Physiology, https://doi.org/10.1016/j.pestbp.2019.12.003

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to goats, fishes, birds and chicken (Oruc, 2009). The cytotoxic effect of thiram may be attributed either to the oxidation of thiol group of peptides and proteins with the disulfide group of thiram or interaction with vital cellular molecules. Thiram has the potential to form carcinogenic nitrosamines in combination with nitrite. Thiram causes tibial dyschondroplasia, a cartilage malformation disease in poultry and is also responsible for thyroid dysfunction in aquatic organisms (Zhang et al., 2019; Chen et al., 2018). Thiram is metabolized in the body to form carbon disulfide and dimethyldithiocarbamate which are also cytotoxic (Dalvi and Deoras, 1986). It is also associated with membrane lipid peroxidation and mitochondrial dysfunction within the cells (Cereser et al., 2001a; Grosicka et al., 2005; Grosicka-Maciąg et al., 2008). Thiram, like many dithiocarbamates, can affect protein function by forming complexes with metal ions (Viquez et al., 2012; Mathieu et al., 2015). Erythrocytes are non-nucleated and most abundant cells in the human body which are specialized to transport oxygen. The presence of large number of polyunsaturated fatty acids in cell membrane and continuous exposure to reactive oxygen species (ROS) make erythrocytes an easy target of oxidative damage (Abdallah et al., 2011). Erythrocytes are a convenient model to study the toxicity of xenobiotics because of their structural and functional simplicity (Farag and Alagawany, 2018). Previous work has shown that exposure to carbamate pesticides alters the activities of antioxidant (AO) enzymes and induces hemolysis in rat erythrocytes (Mansour et al., 2009; Rai et al., 2009). We have done a detailed in vitro study on the concentration dependent effects of thiram on human erythrocytes. We show for the first time that thiram augments generation of reactive oxygen (ROS) and reactive nitrogen species (RNS) that cause oxidative damage to these specialized cells. The consequences of thiram exposure on blood and erythrocyte functions are discussed.

2.2. Isolation of erythrocytes and treatment with thiram This study was approved by the Institutional Ethics Committee of Aligarh Muslim University (Registration number: 714/GO/Re/S/02/ CPCSEA). Blood was collected from non-smoking healthy individuals, who were not on any medication, after taking their informed consent. About 5 ml blood was collected from each donor in heparinized tubes and centrifuged at 1500 rpm (210 ×g) at 4 °C for 10 min. The supernatant was removed and packed erythrocytes washed three times with phosphate-buffered saline (PBS) (10 mM sodium phosphate buffer, 0.9% NaCl pH 7.4). A 10% (v/v) cell suspension (hematocrit) was prepared in PBS. Stock solution of thiram was prepared in dimethyl sulfoxide. Eythrocytes were treated with different concentrations of thiram (25–500 μM; 6–121 μg/ml) for 4 h at 37 °C with continuous shaking. Control cells (thiram untreated) were similarly incubated for 4 h at 37 °C. The samples were then centrifuged at 2500 rpm for 10 min and the supernatants used to determine hemolysis. Ten volumes of hypotonic buffer (5 mM sodium phosphate buffer, pH 7.4) was added to the erythrocyte pellets and kept for 2 h at 4 °C. The lysed cells were microfuged at 3000 rpm and the hemolysates (supernatants) used immediately or stored at −80 °C in aliquots. Hemolysis was determined from absorbance of supernatants at 540 nm. The absorbance of control erythrocytes lysed with ten volumes of hypotonic buffer represents 100% hemolysis. 2.3. Hemoglobin, methemoglobin, and methemoglobin reductase The cyanomethemoglobin method was used to determine the concentration of hemoglobin (Hb) (Balasubramaniam and Malathi, 1992) using Hemocor-D reagent. Concentration of methemoglobin (MetHb), was determined from the absorbance of hemolysates at 560, 576 and 630 nm (Benesch et al., 1973). MetHb reductase was assayed from the conversion of MetHb to Hb, in presence of NADH. Hemolysates were mixed with NADH and 2,6-dichlorophenolindophenol and the change in absorbance at 600 nm was recorded. (Kuma et al., 1972).

2. Materials and methods 2.1. Chemicals

2.4. Heme degradation and free iron release Thiram (97% purity), butylated hydroxyltoluene, 1-chloro-2,4-dinitrobenzene, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 2,4,6-tris(2-pyridyl)-s-triazine, 2,2-diphenyl-1-picrylhydrazyl (DPPH), meta-phosphoric acid, glutathione reductase, 2,4,6-trinitrobenzene sulphonate, ouabain, N-(1-naphthyl)ethylenediamine, copper chloride, chloramine T, methylglyoxal, 2,2′-azinobis(3-ethylenebenzothioazoline-6-sulfonic acid) (ABTS), vanadium chloride and Trolox were purchased from Sigma-Aldrich, St. Louis, USA. Reduced and oxidized glutathione (GSH and GSSG), oxidized and reduced nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH), reduced nicotinamide adenine dinucleotide (NADH), 5,5′-dithibisonitrobenzoic acid (DTNB), trichloroacetic acid, glucose 6-phosphate, triethanolamine, pnitrophenyl phosphate, adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), adenosine 5′-triphosphate (ATP), pyrogallol, xanthine oxidase, ferric chloride (FeCl3), sorbitol, 2,6-dichlorophenolindophenol, sodium nitroprusside, 2,4-dinitrophenylhydrazine, sulphanilamide, potassium ferrocyanide [K3Fe(CN)6], 1,10phenanthroline, NaCl, urea, serum albumin, ethanol, magnesium chloride, sodium pyruvate, triphenylphosphine, phosphoglyceric acid, ascorbic acid, ascorbate oxidase, ammonium acetate, lactate dehydrogenase (LDH), phosphoenolpyruvate, glucose 6-phosphate dehydrogenase (G6PD) and dimethyl sulfoxide were purchased from Sisco Research Laboratory (Mumbai, India). Folic acid, thiobarbituric acid, trichloroacetic acid, neocuproine and acetylthiocholine iodide were obtained from Himedia Laboratories (Mumbai, India). Ammonium ferrous sulfate and potassium iodide were from Merck (Mumbai, India). Hemocor-D reagent was from Clinical Coral System (Goa, India) and dihydroethidium from Genetix Biotech (New Delhi, India). All other chemicals were of analytical grade.

The degradation of heme was determined fluorometrically as described by Nagababu et al. (2008). Briefly, 20 μl hemolysates were diluted to 1 ml with PBS and fluorescence emission was recorded at 480 nm using 325 nm as the excitation wavelength. The concentration of free iron in hemolysates was determined using ferrozine which forms a colored complex with Fe2+ that absorbs at 562 nm (Panter, 1994). 2.5. ROS and RNS Intracellular ROS generation was monitored by the DCFH-DA method (Keller et al., 2004). The 5% hematocrit was mixed with 10 μM DCFH-DA and left for 1 h in the dark. After centrifugation at 2500 rpm, the cell pellets were washed with PBS to remove excess dye and resuspended in PBS to prepare a 10% hematocrit. Different concentrations (25, 50, 100, 250, 500 μM) of thiram were added and samples kept at 37 °C for 15 min. The fluorescence of 50 fold diluted samples was recorded using 485 nm and 535 nm as the excitation and emission wavelengths. Intracellular generation of superoxide radical was determined using dihydroethidium (Wojtala et al., 2014). In this method, 10 μM dihydroethidium was added to 5% hematocrit and left for 1 h in the dark. Samples were centrifuged and the cell pellets washed three times with PBS. The pellets containing erythrocytes were resuspended in PBS to give 10% cell suspensions which were treated with different concentrations of thiram for 1 h at 37 °C. Then, 50 μl samples were diluted to 1 ml with PBS and fluorescence recorded at 605 nm using 518 nm as the excitation wavelength. Hydrogen peroxide (H2O2) concentration was quantified spectrophotometrically in hemolysates using ferrous ammonium sulphate-xylenol orange as the color reagent; 2

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in absorbance of samples at 517 nm (Mishra et al., 2012). In the ABTS assay, ABTS•+ cation radical was first produced by mixing 2.45 mM potassium persulfate and 7 mM ABTS and leaving the solution for 12–16 h in the dark at room temperature. The solution of ABTS•+ was diluted to give an absorbance of 0.70 at 734 nm. Then, 10 μl of sample or standard (Trolox) was mixed with 1 ml ABTS•+ solution and after 5 min the absorbance at 734 nm was recorded (Re et al., 1999). Trolox, a water soluble vitamin E analog, was used as the standard and results expressed as Trolox equivalent antioxidant capacity (TEAC).

100 mM sorbitol was added as color enhancer (Gay and Gebicki, 2000). The generation of peroxynitrite was determined in hemolysates using folic acid which, reacts with peroxynitrite to yield a highly fluorescent product whose fluorescence was recorded at 470 nm using 380 nm as excitation wavelength (Huang et al., 2007). Nitric oxide level was determined spectrophotometrically from the total concentration of nitrite and nitrate in hemolysates by using Griess reagent (Miranda et al., 2001). 2.6. Oxidative stress markers

2.9. Metabolic enzymes

Free radicals induced damage to membrane lipids was determined using thiobarbituric acid. Thiobarbituric reacts with malondialdehyde, an end product of lipid peroxidation, to form a pink colored adduct which absorbs at 535 nm (Buege and Aust, 1978). Lipid hydroperoxides are stable products formed during peroxidation of unsaturated fatty acids. They were quantified in hemolysates using ferrous ammonium sulphate and xylenol orange, as described by Nourooz-Zadeh et al. (1994). Protein oxidation was determined from the content of carbonyl groups that react with 2,4-dinitrophenylhydrazine to form a hydrazone adduct which absorbs at 360 nm (Levine et al., 1990). GSH concentration was determined in protein free hemolysates by reaction with DTNB (Beutler et al., 1963). Total sulfhydryl group content in hemolysates was determined using DTNB that reacts with sulfhydryl groups to form a yellow colored thionitrobenzoate anion which absorbs at 412 nm (Sedlak and Lindsay, 1968). Citric acid and potassium iodide were used for the estimation of advanced oxidation protein products using chloramine T as standard (Hanasand et al., 2012). Free amino groups were determined from their reaction with 2,4,6-trinitrobenzenesulfonate (Snyder and Sobocinski, 1975).

The activity of hexokinase, the first enzyme of glycolytic pathway, was determined by a coupled enzymatic reaction (Bergmayer et al., 1983). In this method, hexokinase in sample converts glucose to glucose 6-phosphate which is then converted to 6-phosphogluconate by glucose 6-phosphate dehydrogenase (G6PD). This reaction results in the reduction of NADP+ to NADPH and increase in absorbance at 340 nm. The activity of glyceraldehyde 3-phosphate dehydrogenase was also monitored by a coupled enzymatic reaction with concomitant oxidation of NADH. The change in absorbance was recorded at 340 nm (Heinz and Freimoller, 1982). Pyruvate kinase, the last enzyme of glycolytic pathway, was assayed by the method of Bergmeyer et al. (1974). In this method, pyruvate kinase converts phosphoenolpyruvate to pyruvate which is then reduced to lactate by lactate dehydrogenase (LDH); NADH serves as the electron donor in the reaction. LDH activity was determined from the decrease in absorbance of solution at 340 nm due to oxidation of NADH to NAD+ in the presence of sodium pyruvate (Khundmiri et al., 2004). The activity of G6PD was monitored from the reduction of NADP+ to NADPH in presence of glucose 6-phosphate and consequent increase in absorbance of solution at 340 nm (Shonk and Boxer, 1964). 5’-Nucleotidase was assayed from the amount of inorganic phosphate released upon hydrolysis of AMP (Heppel and Hilmore, 1951) and acid phosphatase from the hydrolysis of p-nitrophenyl phosphate to give the yellow colored p-nitrophenol which absorbs at 415 nm (Mohrenweiser and Novotny, 1982). Glyoxylase-1 activity was followed spectrophotometrically at 240 nm from the formation of S-lactoylglutathione (Arai et al., 2014).

2.7. Antioxidant enzymes The activity of catalase was determined from its ability to convert H2O2 into H2O (Aebi, 1984) and Cu, Zn-superoxide dismutase (SOD) from the inhibition of autoxidation of pyrogallol (Marklund and Marklund, 1974). The assay of glutathione reductase was based on the oxidation of NADPH to NADP+ during the enzymatic reduction of the disulfide bond of GSSG to yield GSH (Carlberg and Mannervik, 1985). Thioredoxin reductase was assayed from the reduction of DTNB to thionitrobenzoate anion in presence of NADPH; the yellow color produced was read at 410 nm (Tamura and Stadtman, 1996). Glutathione peroxidase was assayed from the oxidation of NADPH to NADP+ in presence of GSSG and glutathione reductase (Flohe and Gunzler, 1984). The resulting decrease in absorbance was recorded at 340 nm. Glutathione-S-transferase was assayed by using GSH and 1-chloro-2,4-dinitrobenzene as substrates (Habig et al., 1974). All enzyme assays were done in hemolysates.

2.10. Membrane-bound enzymes Acetylcholinesterase hydrolyzes S-acetylthiocholine to give thiocholine and acetate. The thiocholine then cleaves the disulphide bond of DTNB to produce the yellow colored thionitrobenzoate anion (Ellman et al., 1961). Na+/K+ ATPase and total ATPase were assayed from the release of inorganic phosphate upon hydrolysis of ATP in the presence and absence of 1 mM ouabain (Bonting et al., 1961). Ascorbate free radical (AFR) reductase was assayed in the presence of an AFR generating system, using ascorbate oxidase and NADH, and following the decrease in absorbance at 340 nm (May et al., 2004). Plasma membrane redox system (PMRS) was monitored by its ability to reduce extracellular and membrane impermeable ferricyanide to ferrocyanide by the transplasma membrane electron transport activity of erythrocytes (Rizvi and Srivastava, 2010).

2.8. Antioxidant power The AO power of cells was determined from the ability of AOs in hemolysates to quench free radicals or reduce metal ions (Fe3+, Cu2+, Mo6+) to their lower states. Cupric reducing antioxidant capacity (CUPRAC) assay utilizes copper(I)-neocuproine as the chromogenic agent to measure the AO power of hemolysates (Cekic et al., 2012). Ferric reducing antioxidant power (FRAP) assay is based on the reduction of Fe3+ to Fe2+ by AOs in sample; the Fe2+ ions form a blue colored complex with 2,4,6-tris(2-pyridyl)-s-triazine (Benzie and Strain, 1996). K3Fe(CN)6 assay is also based on the reduction of ferric ions to ferrous ions by AOs but at near physiological pH (Yen and Chen, 1995). The phosphomolybdenum method involves the reduction of Mo6+ to Mo5+ by AOs in sample and subsequent formation of a green colored complex (Prieto et al., 1999). DPPH assay involves the quenching of colored DPP• free radical to yield the colorless to pale yellow DPPH. This conversion was followed spectrophotometrically from the decrease

2.11. Osmotic fragility Thiram treated and untreated cells were centrifuged, the pellets washed with PBS and resuspended to give a 10% hematocrit. Then, 50 μl of this hematocrit was added to test tubes containing 5 ml of 0.2–0.7% NaCl. The samples were kept at 37 °C for 30 min and then centrifuged at 2500 rpm for 10 min. The absorbance of supernatants was recorded at 540 nm. Absorbance of untreated cells lysed with 10 volumes of 5 mM sodium phosphate buffer was used as a reference and represents 100% lysis (Veena et al., 2007). 3

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2.12. Scanning electron microscopy Control and thiram treated erythrocytes were washed three times with PBS and fixed with 2.5% glutaraldehyde for 1 h at room temperature. The cells were again washed with PBS and mounted on glass slides. The cells were dried at 37 °C in an incubator and washed with graded ethanol (50% - 70% - 90% - 100%). The samples were air dried, coated with gold‑palladium layer and examined under a scanning electron microscope (Wang et al., 2009). 2.13. Statistical analysis All experiments were performed on blood samples taken from six different individuals and statistical significance was reported using one way ANOVA as the mean ± standard error mean. Results were considered significant when the probablity value was P < .05. 3. Results 3.1. Hemolysis and osmotic fragility Incubation of erythrocytes with thiram for 4 h at 37 °C resulted in hemolysis which was measured from the release of Hb in medium and consequent increase in absorbance at 540 nm (Fig. 1A). Hemolysis increased in a thiram concentration-dependent manner and was 11% at 500 μM thiram, the highest concentration used in this study. Control cells showed < 0.8% hemolysis under these conditions. This suggests

Fig. 2. Effect of thiram on (A) methemoglobin (MetHb) levels and (B) MetHb reductase activity in hemolysates. MetHb is in μmoles/L and MetHb reductase activity is in nmoles/min/mg Hb. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎ P < .05.

that thiram damages the erythrocytes plasma membrane. Treatment of erythrocytes with thiram also made these cells more osmotically fragile due to which they lysed at relatively higher NaCl concentrations when compared to the control cells (Fig. 1B). 3.2. Methemoglobin and methemoglobin reductase MetHb is the oxidized form of Hb which contains heme iron in Fe3+ form instead of Fe2+. A significant increase in MetHb was seen in thiram treated cells; MetHb level at 500 μM thiram was four times the control value (Fig. 2A). MetHb reductase is an enzyme that converts MetHb back to Hb using NADH as the reductant. MetHb reductase activity was greatly decreased and was 42% of control value at 500 μM thiram (Fig. 2B). 3.3. Heme degradation and free iron release Hb is a heme protein and also the most abundant protein of erythrocytes. Heme degradation results in the formation of two major products which can be detected from their characteristic fluorescence (Nagababu et al., 2010). Heme degradation increased gradually with the concentration of fungicide (Fig. 3A) and was 1.5 times the control value at 500 μM thiram. Iron is a component of heme porphyrin ring and damage to heme can result in its release. The free iron (Fe2+) concentration was determined from its reaction with ferrozine which

Fig. 1. Effect of thiram on erythrocyte (A) hemolysis and (B) osmotic fragility. Percent hemolysis and osmotic fragility were determined as described under Materials and Methods. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎ P < .05. 4

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Fig. 3. Effect of thiram on (A) heme degradation and (B) release of free iron. Degradation of heme and free iron were determined in hemolysates. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎P < .05.

gives a colored product. Concentration of free iron also increased and was more than two times the control value at 500 μM thiram (Fig. 3B).

3.4. ROS and RNS Enhanced intracellular production of ROS and/or RNS is a characteristic feature of pro-oxidants. The ROS levels were determined fluorometrically using DCFH-DA and dihydroethidium methods. Thiram exposure significantly increased the DCF and dihydroethidium fluorescence, suggesting enhanced ROS generation and induction of oxidative stress in thiram treated erythrocytes (Fig. 4A and B). A two fold increase in concentration of H2O2, a non-radical ROS, was seen in hemolysates prepared from thiram treated cells (Fig. 4C). RNS were measured from the levels of nitric oxide and peroxynitrite. Nitric oxide was determined spectrophotometrically from the total concentration of nitrate and nitrite, the stable end products of nitric oxide oxidation. There was a dramatic increase in nitric oxide level which was seven fold the control value in 500 μM thiram treated erythrocytes (Fig. 5A). Peroxynitrite (ONOO−) reacts with folic acid to give a highly fluorescent product. There was a gradual increase in peroxynitrite production at lower concentrations of thiram (25, 50 and 100 μM) and a dramatic rise at 250 and 500 μM (Fig. 5B). This increase in the intracellular production of ROS and RNS suggests the induction of oxidative stress condition and reflects the pro-oxidant nature of thiram.

Fig. 4. Effect of thiram on ROS production. The intracellular ROS levels were determined fluorometrically by the (A) DCFH-DA and (B) dihydroethidium methods while (C) H2O2 concentration was determined spectrophotometrically. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎P < .05.

3.5. Oxidative stress markers Malondialdehyde and lipid hydroperoxides are markers of lipid peroxidation. They are formed upon oxidation of unsaturated fatty acids present in the cell membrane. Both were increased in thiram treated cells; malondialdehyde level was two fold while lipid hydroperoxides were eight fold of control values (Table 1). Protein oxidation introduces carbonyl groups in the polypeptide backbone. The carbonyl 5

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and at 500 μM thiram it was only 12.7% while total sulfhydryl content was 57.7% of the corresponding control values (Table 1). 3.6. Antioxidant enzymes and antioxidant power The primary (catalase, SOD, glutathione peroxidase) and secondary (thioredoxin reductase, glutathione reductase, glutathione-S-transferase) AO enzymes showed a thiram concentration-dependent decrease in specific activity (Table 2). Thioredoxin reductase was the most affected enzyme and at 500 μM thiram its activity was only one third of control value. The non-enzymatic AO power of erythrocytes was determined next using hemolysates (Table 3). The AO power of hemolysates was determined by their ability to reduce Fe3+, Cu2+ and Mo6+ ions to their lower oxidation states. In the FRAP assay, AOs present in hemolysates reduce ferric ions to ferrous form at low pH (3.6). The principle of K3Fe (CN)6 assay is similar to FRAP but the reduction of ferric to ferrous ions occurs at near physiological pH. In the CUPRAC assay, non-enzymatic AOs present in hemolysates reduce cupric ions to cuprous form while the phosphomolybdenum method involves the reduction of Mo6+ to Mo5+ by accepting electron(s) from the sample. Incubation of erythrocytes with thiram significantly decreased the metal reducing ability of erythrocytes; maximum decrease was in the ability to convert Cu2+ to Cu+ (Table 3). ABTS and DPPH assays determine the AO power of cells in terms of quenching of free radicals. In these assays, the electrons and H atoms donated by AOs present in sample (hemolysates) quench colored free radicals. This results in decrease in absorbance, which is a measure of the free radical quenching power of erythrocytes. DPPH assay showed that treatment of erythrocytes with thiram lowered their free radical quenching by 15.7% compare to control cells while in ABTS assay the AO power of erythrocytes was less than half of control cells at 500 μM fungicide (Table 3). Thus, all these assays showed that thiram significantly decreases the AO power of erythrocytes thereby lowering their free radical quenching and metal reducing ability.

Fig. 5. Effect of thiram on (A) nitric oxide and (B) peroxynitrite. Peroxynitrite was determined fluorometrically using folic acid while nitric oxide was determined spectrophotometrically using Greiss reagent. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎P < .05.

3.7. Metabolic and membrane bound enzymes Glycolysis and hexose monophosphate shunt are the only pathways of glucose metabolism in the erythrocytes, which lack organelles. They provide energy (ATP) and reducing equivalents (NADPH) to erythrocytes. The activities of most metabolic enzymes such as G6PD, hexokinase, pyruvate kinase, glyceraldehyde 3-phosphate dehydrogenase, acid phosphatase, 5′-nucleotidase and glyoxylase-1 decreased while the activity of LDH showed a thiram concentration-dependent increase (Table 4). The activities of membrane bound enzymes, Na+/K+ ATPase and acetylcholinesterase decreased in a thiram concentration-dependent manner. The activity of Na+/K+ ATPase was 41% and acetylcholinesterase 58% of control values at 500 μM thiram (Table 5). The effect of thiram on PMRS was also analyzed. PMRS is an electron transport system that maintains redox homeostasis of cells by

content was significantly higher even at low concentration of thiram (25 μM) and at 500 μM it was 3.5 times the control value. Among the groups oxidized are free amino groups whose level was decreased and was one third of control in 500 μM thiram treated cells. Besides carbonylation, protein oxidation also results in the formation of advanced oxidation protein products. These are a more recent marker of oxidative stress containing several chromophores which absorb light at 340 nm. The level of advanced oxidation protein products in hemolysates increased in thiram treated erythrocytes and was 1.68 fold the control value. The thiol content of erythrocytes was also reduced as the levels of both GSH and total sulfhydryl groups showed a thiram concentration-dependent decrease. The GSH level was dramatically decreased

Table 1 Effect of thiram on some oxidative stress parameters.

MDA LOOH PO AOPP NH2 groups GSH Total SH

Control

25 μM

50 μM

100 μM

250 μM

500 μM

2.81 ± 0.22 0.51 ± 0.06 5.5 ± 0.45 280.8 ± 37.1 12.3 ± 1.17 12.9 ± 1.38 1.42 ± 0.08

3.37 ± 0.30⁎ 0.84 ± 0.07⁎ 7.5 ± 0.60⁎ 332.3 ± 36.4⁎ 10.8 ± 0.79⁎ 10.2 ± 0.98⁎ 1.22 ± 0.09⁎

4.44 ± 0.45⁎ 1.42 ± 0.13⁎ 11.3 ± 1.21⁎ 379.4 ± 32.9⁎ 8.8 ± 0.75⁎ 8.6 ± 0.74⁎ 1.06 ± 0.11⁎

5.06 ± 0.64⁎ 1.83 ± 0.16⁎ 12.5 ± 0.96⁎ 407.6 ± 38.4⁎ 7.6 ± 0.56⁎ 6.8 ± 0.51⁎ 0.97 ± 0.11⁎

5.97 ± 0.67⁎ 3.26 ± 0.36⁎ 15.8 ± 1.89⁎ 431.3 ± 39.8⁎ 6.1 ± 0.67⁎ 3.4 ± 0.29⁎ 0.88 ± 0.09⁎

6.65 ± 0.55⁎ 4.22 ± 0.37⁎ 19.4 ± 1.69⁎ 472.1 ± 40.7⁎ 4.5 ± 0.35⁎ 1.6 ± 0.12⁎ 0.82 ± 0.07⁎

All parameters were determined in hemolysates. MDA, LOOH, PO, AOPP, GSH are in nmoles/mg Hb. Free NH2 groups total SH groups are in μmoles/mg Hb. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎P < .05. MDA, malondialdehyde; LOOH, lipid hydroperoxides; PO, protein oxidation; AOPP, advanced oxidation protein products; NH2 groups, free amino groups; GSH, glutathione; SH, sulfhydryl. 6

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Table 2 Effect of thiram on the activity of antioxidant enzymes. Control CAT SOD GP GR GST TR

40.1 ± 3.5 45.4 ± 3.3 143.2 ± 17.0 10.15 ± 1.5 57.1 ± 4.2 25.0 ± 3.02

25 μM

50 μM ⁎

100 μM ⁎

28.1 ± 3.4 40.0 ± 3.7⁎ 132.5 ± 14.9⁎ 8.58 ± 1.3⁎ 47.6 ± 3.9⁎ 13.2 ± 1.52⁎

32.5 ± 4.3 43.3 ± 3.6 136.6 ± 14.6 9.13 ± 1.2 51.4 ± 4.8⁎ 19.0 ± 1.79

250 μM ⁎

500 μM ⁎

25.4 ± 3.2 37.4 ± 3.6⁎ 128.0 ± 15.5⁎ 8.08 ± 1.1⁎ 46.1 ± 3.4⁎ 11.5 ± 0.97⁎

23.1 ± 2.9 34.1 ± 3.0⁎ 123.4 ± 14.1⁎ 7.11 ± 0.8⁎ 44.1 ± 3.1⁎ 9.6 ± 0.84⁎

20.9 ± 2.8⁎ 31.6 ± 3.1⁎ 117.6 ± 12.7⁎ 6.05 ± 0.7⁎ 38.8 ± 3.3⁎ 8.0 ± 0.72⁎

All enzyme activities were determined in hemolysates. CAT and SOD are in μmoles/min/mg Hb and U/mg Hb, respectively. GP, GR, GST and TR activities are in nmoles/min/mg Hb. Differences were considered significant when the P-value was ⁎P < .05. CAT, catalase; SOD, Cu, Zn superoxide dismutase; GP, glutathione peroxidase; GR, glutathione reductase; GST, glutathione-S-transferase; TR, thioredoxin reductase. Table 3 Effect of thiram on the antioxidant power of erythrocytes.

FRAP K3Fe(CN)6 CUPRAC PMG DPPH ABTS

Control

25 μM

50 μM

100 μM

250 μM

500 μM

145.2 ± 11.0 80.1 ± 6.4 86.9 ± 6.9 384.5 ± 28.6 76.4 ± 6.3 48.9 ± 4.7

139.9 ± 10.8 73.6 ± 5.5⁎ 77.2 ± 6.0 365.5 ± 29.4⁎ 57.3 ± 4.0⁎ 44.04 ± 4.0

131.2 ± 11.9⁎ 65.7 ± 5.2⁎ 67.4 ± 5.2⁎ 346.6 ± 30.5⁎ 46.4 ± 4.1⁎ 37.8 ± 3.8⁎

118.2 ± 9.9⁎ 57.0 ± 4.4⁎ 59.0 ± 4.4⁎ 303.0 ± 30.1⁎ 34.0 ± 2.9⁎ 31.3 ± 2.3⁎

107.7 ± 11.1⁎ 48.1 ± 3.5⁎ 50.2 ± 4.7⁎ 255.1 ± 27.9⁎ 25.3 ± 2.2⁎ 26.2 ± 2.1⁎

87.1 ± 7.8⁎ 38.9 ± 3.3⁎ 36.3 ± 0.3.8⁎ 205.8 ± 20.9⁎ 12.0 ± 1.1⁎ 21.1 ± 1.6⁎

All assays were done in hemolysates. FRAP, CUPRAC and PMG are in nmoles/mg Hb and K3Fe(CN)6 reduction is in μmoles/mg Hb. DPPH is in percent quenching of DPPH radical and ABTS is in μM Trolox/mg Hb. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎P < .05. FRAP, ferric reducing antioxidant power; K3Fe(CN)6, potassium ferricyanide; CUPRAC, cupric reducing antioxidant capacity; PMG, phophomolybdenum green; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline)6-sulfonic acid; TEAC, Trolox equivalent antioxidant capacity.

and anemia.

transferring electrons from intracellular substrates to extracellular acceptors. PMRS transfer electrons, derived mainly from intracellular reductants like GSH and NADH, to extracellular ascorbate free radical (AFR). Activities of both PMRS and AFR reductase decreased with increase in concentration of thiram; PMRS was 71% and AFR reductase 56% of control values at 500 μM (Fig. 6).

4. Discussion The increase in production and use of pesticides in agriculture has resulted in human environmental and occupational exposure. In this work we have investigated the effect of thiram on human erythrocytes. This was done since any toxicant that enters the body soon reaches the blood and erythrocytes, being the most abundant cells of blood, are quickly exposed to its harmful effects. Also, despite many reports documenting thiram toxicity on various cells and tissues, there are none regarding its effect on erythrocytes. Hemoglobin is the most abundant protein in erythrocytes due to which it is a major target of ROS. MetHb level increased in a concentration-dependent manner in thiram treated cells. MetHb binds very tightly to oxygen and does not release it in tissues where oxygen partial pressure is low. Thus elevated MetHb level will reduce the oxygen transporting capacity of blood. The increase in MetHb can be due to inhibition of MetHb reductase, an enzyme that converts MetHb back to Hb by reducing bound Fe3+ to Fe2+ using NADH as the reductant (Geetha et al., 2007). Treatment of erythrocytes with thiram also led to

3.8. Scanning electron microscopy The effect of thiram on erythrocyte morphology was visualized under a scanning electron microscope. The images clearly showed that thiram disrupted the normal biconcave discoid shape of erythrocytes. The biconcave red cells (discocytes) were transformed into spiked red cells (echinocytes) at lower concentration and to spherocytes at higher concentrations of thiram (Fig. 7). A schematic representation of the results obtained above is shown in Fig. 8. Thiram enters the cells where it decreases GSH level and inhibits AO enzymes. This lowers the AO power of erythrocytes and leads to oxidative modification of various cell components. The erythrocyte membrane is also damaged altering the cell morphology. Since damaged erythrocytes are quickly removed by the reticulo-endothelial system, this will result in red cell senescence Table 4 Effect of thiram on activities of some metabolic enzymes. Control HK GAPDH PK LDH G6PD GLO-1 ACP NT

1.39 ± 0.17 5.4 ± 0.51 10.3 ± 2.29 34.7 ± 1.29 3.94 ± 0.49 120.6 ± 10.3 21.7 ± 2.06 260.4 ± 17.8

25 μM

50 μM ⁎

1.21 ± 0.18 4.8 ± 0.39⁎ 9.5 ± 0.71 38.8 ± 2.79⁎ 3.78 ± 0.45 109.4 ± 14.0 19.6 ± 1.42 193.1 ± 22.4⁎

100 μM ⁎

1.03 ± 0.11 4.1 ± 0.24⁎ 8.3 ± 0.79⁎ 43.2 ± 3.00⁎ 2.63 ± 0.26⁎ 102.5 ± 14.2⁎ 18.3 ± 1.38 171.2 ± 20.1⁎

250 μM ⁎

0.92 ± 0.08 3.2 ± 0.21⁎ 6.5 ± 0.54⁎ 50.0 ± 4.68⁎ 2.47 ± 0.34⁎ 94.3 ± 12.1⁎ 14.6 ± 1.53⁎ 145.5 ± 12.1⁎

500 μM ⁎

0.68 ± 0.07 2.4 ± 0.15⁎ 4.8 ± 0.50⁎ 53.7 ± 4.52⁎ 1.58 ± 0.13⁎ 80.8 ± 10.3⁎ 11.1 ± 1.31⁎ 125.8 ± 10.3⁎

0.49 ± 0.06⁎ 1.0 ± 0.09⁎ 3.3 ± 0.36⁎ 58.9 ± 4.20⁎ 1.14 ± 0.12⁎ 67.8 ± 7.0⁎ 8.7 ± 1.20⁎ 100.3 ± 8.2⁎

All enzymes were assayed in hemolysates. All enzymes activities are in nmoles/min/mg Hb. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎P < .05. HK, hexokinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PK, pyruvate kinase; LDH, lactate dehydrogenase; G6PD, glucose 6-phosphate dehydrogenase; GLO-1, glyoxylase-1; ACP, acid phosphatase; NT, 5′-nucleotidase. 7

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Table 5 Effect of thiram on erythrocyte membrane bound enzymes. Control AChE Na+/K+ ATPase Total ATPase

39.6 ± 2.92 32.5 ± 2.48 242.7 ± 41.3

25 μM 35.4 ± 2.67 29.1 ± 1.55⁎ 217.8 ± 28.9

50 μM

100 μM ⁎

32.0 ± 2.84 24.1 ± 1.81⁎ 185.9 ± 24.4⁎

250 μM ⁎

29.3 ± 2.26 19.7 ± 1.53⁎ 157.9 ± 21.3⁎

500 μM ⁎

27.4 ± 2.38 16.3 ± 1.42⁎ 127.8 ± 18.2⁎

23.0 ± 1.96⁎ 13.1 ± 1.14⁎ 86.5 ± 12.1⁎

Enzyme activities were determined in hemolysates. AChE activity is in nmoles/min/mg Hb while Na+/K+ ATPase and total ATPase are in nmoles/h/mg Hb. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎P < .05. AChE, acetylcholinesterase; ATPase, adenosine triphosphatase.

DCFH-DA method is used to quantify all oxidizing species while dihydroethidium is specific for the superoxide radical only (Rastogi et al., 2010; Peshavariya et al., 2007). The increase in fluorescence intensity in both assays shows that thiram enhances the intracellular generation of ROS in erythrocytes (Rana and Shivanadappa, 2010; Kurpios-Piec et al., 2015a). Thiram has been shown to induce ROS in a murine macrophage cell line (Kurpios-Piec et al., 2015b). The concentration of H2O2, a non-radical ROS, was also increased. The production of nitric oxide and peroxynitrite, the two most abundant RNS, was also increased upon exposure of erythrocytes to thiram. This is the first report to show increased peroxynitrite level in dithiocarbamate treated cells. There can be several reasons for elevated RNS and ROS generation in thiram treated erythrocytes. Reduction of Hb to MetHb is accompanied by the release of electron which can react with molecular oxygen present in erythrocytes to yield superoxide radical. Inside erythrocytes the Hb concentration is quite high (5 mM) so even a small increase in MetHb concentration can lead to the formation of large amounts of superoxide. The superoxide radical, which is very sluggish, can react rapidly with nitric oxide to give peroxynitrite (Pryor and Squadrito, 1995; Bashkatova et al., 2004) or is converted to H2O2 (Wu et al., 2009). A Fenton type reaction between ferrous iron and H2O2 yields the highly reactive and damaging hydroxyl radical. The hydroxyl radical, due to its highly reactive nature, can initiate chain reaction by withdrawing electrons from molecules present in its vicinity, thereby converting them into free radicals. This thiram concentration-dependent enhancement in reactive species will result in oxidative modification of cell components. Damaged erythrocytes not only show impaired delivery of oxygen to tissues but also have a shorter life span (Mohanty et al., 2014). Reactive molecular species generated during oxidative stress condition modify cell components especially proteins, thiols and unsaturated lipids. Oxidation of polyunsaturated fatty acids results in the formation of malondialdehyde and lipid hydroperoxides whose levels were elevated in thiram treated erythrocytes. Similarly, increased protein carbonylation suggests oxidation of polypeptide side chains by reactive species. Advanced oxidation protein products, a more recent marker of protein damage, are comprised of several chromophores like dityrosines, protein carbonyls, cross-linked proteins and pentosidine. The advanced oxidation protein products were also increased in thiram treated cells. Protein oxidation is also accompanied by reduction in free amino groups. These results are consistent with a previous report showing enhanced lipid peroxidation and protein oxidation on exposure to thiram (Grosicka et al., 2005). GSH is a thiol containing tripeptide present in millimolar concentration in erythrocytes. It is the major non-enzymatic AO and protects cell components from ROS-induced damage (Schmitt et al., 2015). Exposure of erythrocytes brought about a thiram concentration-dependent decline in GSH levels. Decrease in GSH level could be due to its conversion to GSSG or by direct oxidation of its thiol group by ROS (Cereser et al., 2001b). Another reason could be the lower activity of glutathione reductase, an enzyme that regenerates GSH from its oxidized form GSSG. A concomitant reduction in total sulfhydryl groups showed that the entire thiol status was affected. GSH levels generally decline under severe oxidative stress due to its conversion to GSSG or consumption by direct quenching of

Fig. 6. Effect of thiram on (A) PMRS and (B) AFR reductase activity. The PMRS is in nmoles/30 min/ml PRBC/and AFR reductase activity is in μmoles NADH oxidized/min/ml PRBC. Results are mean ± standard error of six different samples. Differences were considered significant when the P-value was ⁎ P < .05. PRBC, packed red blood cells; PMRS, plasma membrane redox system; AFR, ascorbate free radical reductase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

heme degradation and the release of iron in free form. Hb is the likely source of the degraded heme and free iron since it comprises > 90% of total erythrocyte proteins. However, the possibility of other heme proteins like catalase cannot be ruled out. Enhanced MetHb formation, heme degradation and release of iron are known to take place under oxidative stress condition (Comporti et al., 2002; Nagababu et al., 2008; Ansari et al., 2015). ROS are produced in the cell as a by-product of various metabolic processes. At low concentration these species are beneficial as they participate in cellular signaling pathways and in various physiological functions. But at high concentration they can damage the biological system by oxidizing biomolecules such as lipids, proteins and DNA. The intracellular generation of ROS was determined fluorometrically using membrane permeable compounds DCFH-DA and dihydroethidium. The 8

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Fig. 7. Effect of thiram on erythrocyte morphology as visualized under scanning electron microscope (A) Control erythrocytes and cells treated with (B) 25 μM (C) 100 μM and (D) 500 μM thiram. Magnification is 1500 X.

ROS (Yamada et al., 2006). AO enzymes represent the first line of defense against oxidative stress condition. The activities of all primary (SOD, catalase, glutathione peroxidase) and secondary (glutathione reductase, thioredoxin reductase, glutathione-S-transferase) AO defense enzymes were inhibited in a concentration dependent manner in thiram exposed erythrocytes. Excess concentrations of free radicals and ROS can inactivate AO enzymes (Nabi et al., 2016; Doyotte et al., 1997). Li et al. (2007) have also reported inhibition of liver SOD and glutathione peroxidase in chicken given thiram in their diet for 4 weeks. A decrease in the activity of G6PD will influence several AO enzymes that directly or indirectly require NADPH to function. Thioredoxin reductase uses NADPH as reductant to keep thioredoxin in reduced state which, in turn, maintains the redox status of cell by cysteine thiol-disulphide exchange. Glutathione reductase convert GSSG to GSH using NADPH as reductant while glutathione peroxidase uses GSH to detoxify peroxides to alcohol or water. Glutathione-S-transferase detoxifies reactive molecular species by conjugating them with the thiol group of GSH (Nebbia et al., 1993). Thus the low activities of glutathione peroxidase and glutathione-S-transferase could also be due to decrease in cellular GSH levels in thiram treated cells. Low concentration of NADPH also affects catalase, which contains four NADPH bound per tetrameric molecule that are needed for the catalytic activity of this enzyme. The AO power of the thiram treated and control erythrocytes was determined by several assays due to the complexities of AOs present inside the cell. These assays use the ability of AOs in sample to act as donors of electrons (electron transfer) or hydrogen atoms (H atom transfer). The donated electrons can reduce metal ions (Fe3+, Cu2+, Mo6+) to their lower oxidation states; the electrons or hydrogen atoms can also quench free radicals. All assays showed that the AO power of thiram exposed erythrocytes was significantly lower than the control cells. This represents the first report about the effect of thiram, or any dithiocarbamate, on the AO power of cells and tissues. The reduced concentration of major non-enzymatic AO (GSH) and inhibition of AO

enzymes are likely responsible for the impaired AO power of thiram treated erythrocytes. The compromised free radical scavenging and metal reducing ability of thiram treated erythrocytes will make them more susceptible to oxidative damage. Exposure to toxicants can alter cellular metabolism. Therefore, the effect of thiram on enzymes of several metabolic pathways was also determined. The activities of several marker enzymes of glycolysis (hexokinase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase) were lowered upon thiram exposure. These enzymes could have been inhibited by ROS (Tripathi and Singh, 2002; Anderson et al., 2018; Suneetha, 2012) or by direct reaction of thiram with essential enzyme thiol groups. Decrease in the activity of metabolic enzymes will affect the energy status of cells as glycolysis is the only pathway that produces ATP in erythrocytes. Low ATP level will make these cells more susceptible to damage by ROS. Agalakova and Gusev (2012) showed that oxidative stress induced by xenobiotics causes ATP depletion due to ROS generation. Inhibition of glycolysis will also lower NADH which is required by MetHb reductase. Surprisingly, LDH activity was elevated and showed a direct co-relation with thiram concentration. LDH is bound to the inner side of plasma membrane and, upon disruption of membrane, can dissociate and enter the cytosol; activity of free enzyme is greater than of bound enzyme. G6PD is an enzyme of hexose shunt pathway and its reaction generates NADPH, the major cellular reductant in erythrocytes. G6PD was also inhibited, probably by excess ROS or due to lower concentration of its substrate glucose 6-phosphate upon inhibition of hexokinase (Senturk et al., 2009). As mentioned earlier, low NADPH levels can reduce the activity of several AO enzymes. Glyoxylase-1 is a GSH dependent enzyme that is used to detoxify carbonyl compounds. The decline in activity of glyoxylase-1, with increasing concentration of thiram, may be due to low GSH concentration. The activities of acid phosphatase and 5′-nucleotidase were also inhibited. Thus, thiram significantly inhibits erythrocyte metabolic pathways, especially those of glucose utilization. 9

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Fig. 8. Schematic representation of thiram-induced oxidative stress and erythrocyte damage. ROS, reactive oxygen species; RNS, reactive nitrogen species; AO, antioxidant; GSH, glutathione; MetHb, methemoglobin; O2, oxygen.

The thiram-induced changes reported above can have serious consequences on the functions of blood and erythrocytes. 1. Increased MetHb formation will lower the oxygen carrying capacity of blood and result in hypoxia. 2. Since damaged erythrocytes are rapidly removed from circulation, mainly by spleen, it will reduce the life span of these cells (red cell senescence) leading to anemia (Maita et al., 1991). 3. Excess ROS/RNS produced in thiram treated erythrocytes can leak out of the damaged cells and affect other cells that come in contact with the circulating blood. 4. Blood serves as a mobile AO, with erythrocytes making a major contribution in it. The free radical quenching ability of blood will decline significantly due to lower AO power of erythrocytes 5. Inhibition of PMRS will lower regeneration of ascorbate, major AO of plasma; this will again lower the AO power of blood. 6. Finally, the morphological changes in thiram treated erythrocytes will alter their rheological properties and lower the ability of these cells to deform and pass through microcapillaries, some of whom have smaller diameter than the erythrocytes. This will result in the coagulation of erythrocytes, occlusion of blood vessels and lead to renal failure.

Thiram caused hemolysis in a concentration-dependent manner, as determined by the release of Hb in medium. Enhanced hemolysis with increase in fungicide concentration might be the result of direct or indirect injury to the cells, especially the plasma membrane. Increased hemolysis can result in anemia, as reported by Maita et al. (1991) in thiram dosed dogs and rats. Kumar et al. (1975) showed similar anemic effects of the fungicides, captan and captafol. The thiram treated cells also became more osmotically fragile and, compared to control cells, lysed at higher salt concentration. Modification of membrane proteins and lipids can alter membrane integrity, making them more rigid and more susceptible to lysis (Pangano and Faggio, 2015). Thiram treatment also disrupted the integrity of cell membrane as the activities of bound enzymes– acetylcholinesterase and Na+/K+ ATPase- were also inhibited. Certain insecticides such as organophosphates and carbamates act mainly through inhibition of acetylcholinesterase. Carbamates have been shown to decrease the activity of acetylcholinesterase (Atamaniuk et al., 2013). PMRS and AFR reductase are components of the redox system of erythrocytes. PMRS quenches extracellular oxidants by transfering electrons from intracellular donors to the outside. PMRS also regenerates extracellular ascorbate, a major AO of plasma. Modification of essential -SH groups of PMRS and AFR reductase by ROS results in their inhibition. Due to inhibition of PMRS the regeneration of extracellular ascorbate will decrease because of which the ROS scavenging activity of plasma will decline. The above biochemical results were confirmed by scanning electron microscopy of control and thiram treated erythrocytes. Gross distortion in erythrocyte morphology was seen in thiram treated cells. Morphological changes have been reported for other pesticides like malathion and dicofol (Sawhney and Johal, 2000; Ahmad and Ahmad, 2017).

5. Conclusions Thiram enhances the generation of reactive species in erythrocytes in a concentration dependent manner. This causes oxidative modification of cellular components, especially hemoglobin. It inhibits pathways of glucose metabolism, damages the plasma membrane and alters erythrocyte morphology. Thiram treatment significantly reduces the activity of AO enzymes and lowers the AO power of cell. This will impair the ability of blood to detoxify harmful compounds and provide protection against free radicals. This work represents a step towards elucidating the molecular mechanism of toxicity of thiram, and 10

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structurally related compounds, so that steps can be devised to reduce its damaging effect.

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