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

https://doi.org/10.1016/j.pestbp.2019.12.003Get rights and content

Highlights

  • Pesticide thiram causes hemoglobin oxidation, heme degradation and free iron release in human erythrocytes.

  • Protein and lipid oxidation was increased while antioxidant power was compromised.

  • Membrane bound and antioxidant enzymes were inhibited.

  • Thiram induced gross alterations in erythrocyte morphology.

  • Thiram increased generation of reactive oxygen and nitrogen species in erythrocytes.

Abstract

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.

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 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.

Section snippets

Chemicals

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.

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 that thiram damages the erythrocytes plasma membrane. Treatment of erythrocytes with thiram also

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

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

Declaration of competing interest

The authors declare that there is no conflict of interest in this work.

Acknowledgements

Financial support to the Department of Biochemistry by the following schemes is gratefully acknowledged: DST-FIST II, DBT-PURSE and UGC-SAP-DRS III. SS and AA are the recipients of Junior Research Fellowship from Aligarh Muslim University.

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