Intracellular free calcium concentration and oxidative damage in erythrocytes from workers occupationally exposed to organophosphate pesticides by Martha-Angelica Quintanar-Escorza in Journal of Clinical Case Reports MedicaI Images and Health Sciences

 Intracellular free calcium concentration and oxidative damage in erythrocytes from workers occupationally exposed to organophosphate pesticides by Martha-Angelica Quintanar-Escorza in Journal of Clinical Case Reports MedicaI Images and Health Sciences 

Abstract

Background: Organophosphate (OP) pesticides inhibit the activity of acetylcholinesterase (AChE) in the central nervous system, causing changes in the oxidative state that could be considered as another mechanism of toxicity in erythrocytes related to the concentration of free intracellular calcium ([Ca2 +] i).

Aims: In the present work, the parameters of the oxidative state and [Ca2+]i were evaluated in workers occupationally exposed to OP.

Methods: A comparative and cross-sectional study was conducted in exposed and not exposed to organophosphate pesticides in their workplace and working day (31 subjects per group). In the case of exposure, selected workers with an exposure ≥5 years and with an acetylcholinesterase activity ≤7.9U/ml.  We took a venous blood sample to determine ACgE activity and hematocrit, cholesterol, triglycerides, glucose, total antioxidant capacity, lipoperoxidation, osmotic fragility, and free intracellular calcium.

Results: We found high levels of free intracellular calcium [Ca2+]i, as well as a relationship between free intracellular calcium [Ca2+]i and AChE activity (p = 0.001), higher osmotic fragility, and lower percentage hematocrit. Regarding the oxidative state,  found no differences between groups; however, a relationship between oxidative parameters (total antioxidant capacity and lipoperoxidation) stands out.

Conclusion: The results suggest that chronic intoxication induces changes in [Ca2+]i levels, osmotic fragility, and hematocrit in workers exposed to OP because the antioxidant defense mechanisms compensate part of the damage through a different antioxidant pathway.

KEYWORDS: Organophosphate pesticides, oxidative damage, Intracellular free calcium, Red blood cells, eryptosis.

INTRODUCTION

The Food and Agriculture Organization of the United Nations defines a pesticide as a substance or mixture of substances whose purpose is to prevent, destroy or control any pest, including vectors of human or animal diseases, capable of causing harm (1,2). Widespread use of organophosphate pesticides (OP) in agriculture has increased exposure as an occupational hazard, with higher doses and more extended periods of exposure (3). Due to their fat-soluble capacity, OP can be absorbed through any of the routes: oral, dermal, and respiratory (4), quickly passing through biological barriers. OP toxicity has acute, delayed, and chronic effects (5,6)The decrease in acetylcholinesterase (AChE) activity in the blood is indicative of OP poisoning. The erythrocyte cholinesterase is used as a biomarker to assess chronic exposure to OP due to the difficulty in determining chronic exposure at low doses. The measurement of plasma cholinesterase is used only to diagnose acute poisoning (7). Acetylcholine is an excitatory neurotransmitter; AChE breaks down acetylcholine into choline and acetic acid in the synaptic cleft (8,9). The interaction between organophosphates and AChE in nerves, muscles and postsynaptic muscles, and exocrine glands are irreversibly (4,10); causing overstimulation of the muscarinic receptors and desensitization of nicotinic receptors due to the accumulation of acetylcholine in the postsynaptic cleft, which can alter the functioning of the central nervous system (11,12). Diagnosis of pesticide toxicity is based on medical history, exposure history, symptoms, and laboratory tests (13,14). Moreover, toxicity to OP is still under study, as some authors argue that AChE inhibition does not explain all symptoms related to pesticide toxicity (15,16).

Some OP can alter oxygen metabolism and continuously generate oxidative free radicals in the body, causing the lipid peroxidation phenomenon, which indicates oxidative stress in cells and tissues (17,18). The induction of oxidative stress due to increased lipid peroxidation and a decreased antioxidant capacity, together with the inhibition of AChE, has been evidenced in workers occupationally exposed (WOE) to OP (19). Furthermore, these findings have been replicated in exposed populations with a higher oxidative damage prevalence than unexposed individuals (20). Additionally, oxidative damage has been associated with high levels of eryptosis; therefore, erythrocytes are an excellent biological model to evaluate the oxidative stress caused by the exposition to pesticides OP  (21,22).

Eryptosis is a physiological phenomenon in which old or damaged red blood cells are removed from the circulation before completing the last stages of the death program, preventing hemolysis in the bloodstream (23,24). This process is initiated by complex signaling that includes an increased concentration of [Ca2+]i, ceramide, prostaglandin-E2, activation of caspases, kinases, ion channels, and phosphatidylserine translocation at the external interface of the erythrocyte membrane, which is a signal of "absorbing" macrophages (25-27).

Conformational changes (reduction of cell volume, increased membrane fragility, alteration in structure and organization of the cytoskeleton) in erythrocytes, leading to an increase in [Ca2+]i activating its proteases  (25,28-30). There is a correlation between the increase in the concentration of [Ca2+]i and the changes as mentioned above. Likewise, in the aged erythrocytes, there are increased concentrations that are the mechanism that allows their removal from circulation, carrying out the process of eryptosis (31).

Eryptosis can be induced by osmotic shock, oxidative stress, energy depletion, or mechanical damage to red blood cells (32). Increases in eryptosis have been associated with metabolic diseases, genetic disorders, bacterial and viral infections, and incubation with medications and toxic agents (33).

Exposure to organophosphate pesticides has been reported to provoke oxidative stress on the erythrocyte membrane as well as morphological alterations (34). Additionally, biochemical changes and genotoxicity were observed in the in vitro evaluation of the toxic effects of OP exposure in erythrocytes (35).

This work aimed to study the relation between [Ca2+]i and oxidative damage in human erythrocytes of workers occupationally exposed to organophosphate pesticides. Our findings showed that alterations in the oxidative status and an increase in [Ca2+]i contribute to hematological changes. Furthermore, these data add to the knowledge for populations exposed to low doses of pesticides during long periods, as they are the most susceptible to adverse health effects.

METHODS

A comparative and cross-sectional study with 62 Mexican men from Durango, Dgo, Mexico, was conducted. The participants were divided into exposed and not exposed to organophosphate pesticides in their workplace and working day (31 subjects per group). In the case of exposure, selected workers with an exposure ≥5 years and with an acetylcholinesterase activity ≤7.9U/ml.

The body mass index (BMI) was determined by the bioimpedance method using the body composition monitor scale with sensor D brand Omron Model Hbf-514c by a nutritionist; This measurement was performed on an empty stomach on the same day took the sample a minimum fast of 8 hours. By venipuncture, a total of 5 milliliters of blood was extracted, which was deposited in a tube with heparin as an anticoagulant. The blood was divided into Whole blood: Acetylcholinesterase activity and hematocrit; Plasma: Cholesterol, triglycerides, glucose, and total antioxidant capacity and Erythrocytes: Lipoperoxidation, osmotic fragility, and free intracellular calcium.

The samples were maintained at 4°C until use. An aliquot of the blood was used for obtaining erythrocytes and plasma; each sample of blood was centrifuged at 700xg for 10 min at 4°C, the plasma was preserved by freezing until its use and the white cells were discarded.

Acetylcholinesterase activity

To determine the cholinesterase level in human erythrocytes, previously isolated erythrocytes were used by centrifugation at 3500 rpm for 10 minutes; Enzyme activity was measured using the Sigma-Aldrich MAK119 Acetylcholinesterase Activity Assay Kit. This assay is an optimized version of Ellman's method, which detects the appearance of Thiocholine after hydrolysis of the substrate acetylthiocholine (ATCh) by cholinesterase. To produce a yellow compound, Thiocholine reacts with the chromophore, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) 5,5'-dithiobis-2-nitrobenzoic acid, which can be measured spectrophotometrically at a wavelength of 412 nm; this signal is proportional to the activity of AChE. Present in the samples; One unit of AChE is the amount of enzyme that catalyzes the production of 1.0 μmol of Thiocholine per minute at room temperature at pH 7.5. The analytical method measures the activity of the enzyme in the erythrocyte according to an enzymatic chemical reaction whose action is determined in units U / ml; 8.6 U / ml were taken as average values ​​with a range of 7.9 - 12 U / ml. (20)

Lipid peroxidation.

Lipid peroxidation has been used as a biological marker for oxidative cell damage. The lipid peroxidation of erythrocytes was estimated as reported elsewhere (36). Lipid peroxidation in erythrocytes was measured using thiobarbituric acid reactive substances (TBARS) at absorbance 532 nm using a UV / 125 VIS 730 spectrophotometer (Beckman). TBARS are expressed as nmol MDA / ml erythrocyte equivalents, based on a malondialdehyde calibration curve. (25,37).

Total antioxidant capacity

The chromophore reactive ABTS (2,2'-azino-bis (3-ethyl-6-sulfonic acid)) was used. The reaction catalyzed by peroxidase generates a stable radical; monitored the loss of absorbance at a wavelength of 405 nm spectrophotometrically until the generation of a soluble and green-colored final product. The results were plotted and adjusted to a linear correlation to calculate the Trolox (6-hydroxy-2,5,7,8-tetramethylchrome-2-carboxylic acid) equivalents (nM) which are used as an antioxidant (being an analog of vitamin E). The absorbance is inversely proportional to the concentration of antioxidants. The total antioxidant capacity in plasma to prevent oxidation of ABTS is compared to that of Trolox; the quantification was expressed as Trolox equivalents (mM)/ml of plasma. Calibrations were performed as previously described (38).

Evaluation of [Ca2+]i

Erythrocytes were suspended in saline buffer HEPES, containing NaCl 144 mM, KCl 5 mM, HEPES 10 mM, glucose 5 mM, MgCl2 1.8 m, and CaCl2 1.5 mM. The erythrocytes were incubated with 1 lM Fluo-3 AM in the dark at 37°C for an hour after that, cells were centrifuged at 5009 g, and the final concentration of packed erythrocytes was 1% in an isotonic buffer. For fluorescence measurements, 100 l of cell suspension were added to 2.5 ml of isotonic buffer at 37 ° C with constant magnetic stirring using a Spectro fluorophotometer (RF- Shimadzu 5301PC). The [Ca2+]i was measured with the excitation/emission pair at 500/515 nm. Calibrations were performed as previously described (25, 36).

Osmotic fragility

The evaluation of the osmotic fragility of the erythrocytes was determined with the osmotic resistance technique, using previously isolated erythrocytes; 25 µl of washed erythrocytes were taken and packed in tubes for Falcon type centrifuge 5 ml of each solution in concentrations of: 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.9 g / L of NaCl (pH 7.4), homogenized by inversion and incubated for 1 hour in refrigeration (5-8 ° C). They were mixed by inversion and centrifuged at 1200 xg for 5 min. Carefully transfer the supernatant to an ELISA plate where the absorbance of each hypotonic solution was measured colorimetrically using a UV / VIS spectrophotometer model DU 650 (Beckman) at a wavelength of 540 nm. Determined the percentage of hemolysis by constructing the osmotic profile of each sample analyzed as a basis for determining Os50. (OS50) is the osmolarity that produces 50% of hemolysis and was calculated as previously described (39). The hemolysis curves are drawn in a standard format, plotting the percentage of hemolysis as a function of relative tonicity(40).

Hematocrit percentage

Hematocrit is the fraction of red blood cells in whole blood expressed as a percentage. Determined the rate through the capillarity method (41). This determination is based on the higher density of the erythrocytes concerning the other components in the blood. The capillary tube was filled with whole blood, centrifuging for 5 minutes at 12000 rpm. The length of the column made up of sedimented erythrocytes was measured in the capillary tube; this value was used to determine the hematocrit usin g the following formula: Hematocrit (%) = Erythrocyte length ÷ Total length of the blood-filled × 100.

Statistical analysis

The independent samples t-tests to compare these variables for the two groups and the Pearson correlation analysis were carried out using the SPSS statistical package (α = 0.05) (IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp. Released 2019).

RESULTS

62 male patients with a mean age of 36 ± 10 years (maximum 59, minimum 19) were studied. With higher education 58.6%, upper secondary level 25.81%, and the rest primary education 12.90%, coming mainly from the rural environment (73.3%).

The subjects of the study population were classified according to exposure to POF into two groups: Non-exposed Group (NE); 31 workers not occupationally exposed to POF (50% of the total population) and the exposed group (EX); 31 workers occupationally exposed to POF (50% of the total population) in a 1: 1 ratio.

The subjects included in the exposed group had to meet the inclusion criteria such as exposure ≥ 5 years, have an AchE enzyme activity ≤ 7.9 U / ml. Determined the mean years of exposure 19 ± 14 years. In the evaluation of exposure to OP, it produced intoxication characterized by 38.5 % less AChE activity (U/ml) in erythrocytes of WOE to OP (5.4±2.5 U/ml AChE) compared to those who are not exposed (14.0±7.8 U/ml of AChE), confirming that they have been chronically exposed to lead OP.

To determine the relationship of variables that have been associated with decreased AChE activity in populations occupationally exposed to OP, a statistical analysis was performed; and finding that the variables: Age (p=0.002), workday (p= 0.004), training in pesticide management (p=0.001), and the use of special equipment for application (p=0.001) were associated with the decrease in AChE activity. Nevertheless, this decrease was not related to the time of exposure (p= 0.22) and the presence of other diseases (p=0.24).

Regarding the variable years of exposure to OP pesticides, the mean is 19 ± 14 years, with a minimum of 5 years and a maximum of 46 years.

The mean age, the body mass index (BMI), and the biochemical parameters evaluated by the group are shown in table A.1.

 the relationship between occupational exposure to organophosphate pesticides with increased oxidative damage and increased intracellular calcium concentration in erythrocytes; Oxidative damage variables were determined (Lipoperoxidation and total antioxidant capacity), free intracellular calcium, and hematological variables (Osmotic fragility in erythrocytes and hematocrit levels) both in occupationally exposed subjects and in subjects not exposed to these pesticides.

DISCUSSION

In the search for the evaluation of the chronic effects on workers exposed to OP, multiple and diverse epidemiological studies have been carried out to characterize these effects (42,43). Although the primary mechanism of organophosphate toxicity is through the inhibition of acetylcholinesterase (AChE), some chronic adverse health effects indicate the participation of other molecular mechanisms as additional pathways of damage; In this regard, it has been proposed to evaluate workers at high risk of exposure to OP, evaluating the activity of AChE to estimate the degree of intoxication, suggesting that some signs and symptoms presented by the exposed population are not related to their enzymatic decrease (20, 44, 45). Several studies indicate that certain pesticides, including organophosphates, can alter oxygen metabolism, oxidative stress is one of the most studied mechanisms; Vanova et al., 2018 (16) have described that the overstimulation of the cholinergic nervous system followed by the intensified generation of oxygen from reactive species (ROS) increased oxidative damage in many tissues, in addition to this it has recently been described that the mechanisms of toxicity of some OP include, in addition to the inhibition of the enzyme acetylcholinesterase, the change of the oxidant/antioxidant balance, DNA damage and the facilitation of apoptotic cell damage and the membrane stability of human erythrocytes (29, 46). In animals (in vivo), oxidative stress parameters have been evaluated in different tissues, mainly in rodents' kidneys, liver, and brain, finding an increase in MDA levels in acute poisoning in those treated with high doses of OP. In addition, significant decreases were observed in the tissue levels of the non-enzymatic antioxidant (GS), as well as the GPx, SOD, and CAT enzymes, reflecting the depletion of the cellular antioxidant defense, mechanisms that are activated to counteract oxidative stress induced by the malathion  (34, 47). Our study found the oxidative state of both groups of workers (exposed and not exposed to OP) was compared. Found no differences between groups, it was determined that there is no relationship with AChE activity; Regarding the evaluation of the total antioxidant capacity (CAT), there were no differences between groups, nor was it determined that there is no relationship with AChE levels. The difference in the results could be because the oxidative damage reported in the literature is shown after acute exposure to OP, observed in animals and in in vitro studies. In addition, it could involve individual tolerance due to the activation of compensation mechanisms. This suggests that workers chronically exposed to OP have developed an antioxidant response to prolonged exposure to oxidative damage by increasing CAT; capable of neutralizing the damage caused by prolonged exposure since it was determined that there is an inverse relationship between oxidative damage and CAT; suggesting that adaptive mechanisms are gradually activated overtime under chronic exposure conditions, other authors have made similar observations in fish studies that observed a tissue-specific adaptive response to neutralize oxidative stress after exposure to organophosphates, concluding that this response could be due to the presence of different levels of antioxidants in the tissues ( 8, 48). A previous study carried out in the same study population showed a negative influence of occupational exposure to the OP on oxidative damage and acetylcholinesterase activity in the population of exposed workers (20), which would indicate that the oxidative damage shown in this study has been neutralized by stimulation of the antioxidant system by the exposure to OP itself, which makes a more efficient response system to oxidative damage.

An investigation carried out in the plasma of rats intoxicated with taboo evaluated the activity of the antioxidant enzyme superoxide dismutase (SOD) and the substances reactive to thiobarbituric acid (TBARS), where the group where the tabun was administered showed an activity of SOD notably increased. From 30 minutes to 6 hours after exposure, but the enzymatic activity remained relatively unchanged in a control group, finding no differences in the levels of lipid peroxidation products between these two groups. In conclusion, tabun seems to be inducers of weak oxidative stress; however, slightly different response pathways to oxidative stress are activated (49). These findings suggest that the molecular mechanisms triggered through oxidative damage by exposure to organophosphate pesticides have additional implications to those already described recently in studies that have shown various structural alterations in erythrocytes and associated biochemical alterations induced by OP after in vivo exposures. and in vitro to OP (42,50).

Our results showed that there is a relationship between occupational exposure to OP and increased levels of [Ca2+]i in erythrocytes, showing significant differences between the exposure groups, however, there was no relationship with oxidative state; The contradictions suggest that there could be other mechanisms that are involved in the increase of [Ca2+]i in erythrocytes that is not directly related to oxidative damage, this could be explained by referring to the fact that eryptosis is triggered by osmotic shock, energy depletion mechanisms referred to in the literature as additional pathways to oxidative stress (33) that could result in hematological changes as referred to in a study carried out in agricultural workers where hematological parameters were measured by finding the corpuscular volume of red blood cell media and hematocrit values ​​are significantly lower in the exposed group compared to the reference, concluding that OP exposure over time affects hematobiochemical responses (51). Hematological parameters showed a significant decrease in white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, and platelet count between the study and control groups (52). The detectable serum levels of many pesticides were associated with lower white blood cell counts; the results may suggest that pesticides could cause hematological abnormalities among agricultural workers (53). There is a clear difference in hematological variables in the study groups, as reported in the studies mentioned earlier, supporting our results that show a relationship between decreased AChE activity and increased erythrocyte osmotic fragility and decreased hematocrit percentage. In in vitro evaluations of the toxic effects of exposure to the pesticide OF “Quinalphos” on fish red blood cells, morphological abnormalities of red blood cells were observed in peripheral red blood cells sampled at post-treatment intervals of 0 and 30 days. (35) likewise, Manyilizu et al., 2016 (54), concluded that morphological deformations of fish erythrocytes in vivo have the potential to affect AChE signaling by inducing changes in the size and volume of erythrocytes.

The results found in the present study; suggest that the intensity of the toxic effects of chronic exposure to OP evaluated in occupationally exposed workers is associated with exposure factors such as: work seniority, working hours, age of beginning of work activities, as well as the exposure time expressed with a further decrease in the activity of the enzyme AChE; This enzymatic decrease of the erythrocyte membrane could affect the morphological characteristics of the erythrocyte, causing a greater permeability to calcium, increasing the levels of [Ca2+]i; initiating Ca2+ dependent cell signaling causing greater osmotic fragility in the erythrocyte, accelerating cell death processes such as: hemolysis and/or eryptosis, therefore, a decrease in the percentage of erythrocytes evaluated through the rate of hematocrit in blood total; These changes in the hematological parameters are not related to oxidative processes, reaching a state of eustress due to the physiological compensation of the antioxidant defense systems in direct response to the oxidative aggression caused by chronic exposure to OP. The manifestation of the oxidative effect may be conditioned by intrinsic factors such as the variability of some enzymes that participate in the damage compensation and metabolism processes of some pesticides. In conclusion, the use of chronic intoxication markers provides relevant information as a tool that allows predicting the risk associated with diseases that have not been directly related to exposure to OP, considering that the initial event is the alteration of hematological parameters. One of them is anemia, a common condition that could result from poor formation/accelerated loss of circulating erythrocytes.

In conclusion, the relationship found between exposure to organophosphate pesticides and the increase in [Ca2+]i without oxidative damage are findings, reported for the first time,  should be further investigated to explain other molecular mechanisms involved. The use of the evaluation of the activity of AChE and the oxidative state is insufficient to determine the effects of organophosphate pesticides on the health of the exposed population since the interpretations of the results are very varied and individualized.

Ethics declarations

The Ethics Committee approved the protocol of the General Hospital 450 belonging to the Health Services of the Durango State, Mexico, with Folio number: 124. The protocol was conducted following the Declaration of Helsinki and the second title, Chapter 1, Article 17, paragraph 2 of the General Health Law in research issue. All the subjects signed informed consent, and their participation was voluntary.

Conflict of interest

The authors declare no competing interests.
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