Plasma, erythrocyte membrane bound enzymes and tissue histopathology in male Wistar rats exposed to common insecticides

Abstract

Low doses (one-fourth of the LD₅₀) of dichlorvos (DDVP), lambda-cyhalothrin (LMB), cypermethrin (CPM), and imidacloprid (IMP) were administered to adult male Wistar rats for 3 weeks. Erythrocyte antioxidant enzymes, biomarkers of tissue toxicity and histopathology of some visceral organs, were assessed. Glucose-6-phosphate dehydrogenase (G6PD), glutathione-S-transferase (GST), acetylcholine (ACH), and body weight decreased significantly in DDVP- and LMB-treated rats only. However, glutathione (GSH) levels decreased significantly in rats treated with DDVP and IMP. Lipid peroxidation (LPO) increased significantly in plasma of DDVP and erythrocyte of DDVP, CPM, and IMP as compared to the control. Plasma urea and creatinine were insignificant, while aspartate aminotransferase (AST) and alanine amino transferase (ALT) increased significantly in DDVP only; these observations are consistent with the histopathology.

Introduction

Pesticides are used worldwide each year in vast amounts for controlling pests for households, public health, product storage, farming, animal husbandry, and industrial purposes.¹⁾ Organochlorine and organophosphorous were used extensively in the past, but more recently, they have been replaced by synthetic pyrethroid insecticides for both pest control and increased production of agricultural products.²⁾ Organophosphorous and organochlorine have been shown to be absorbed through all routes and are acetylcholinesterase inhibitors, yet they have not been banned and are still in use in some countries such as the United State of America and Nigeria, probably due to cost as compared to synthetic pyrethroid insecticides.³⁾

The most commonly used pesticides in Nigeria are dichlorvos, lambda-cyhalothrin, cypermethrin, and imidacloprid, which are readily available commercially. LMB and CPM are synthetic pyrethroids and have been shown to cause disruption of seminiferous tubules and spermatogenesis in male rats.⁴⁾ They have also shown dopaminergic neurodegeneration if rechallenged during adulthood in rats.⁵⁾ IMP is a systemic insecticide, a neurotoxin (neonicotinoids) that acts on the central nervous system. IMP is the most widely used insecticide worldwide that acts only through contact and ingestion; interestingly, it is selectively more toxic to insects than to mammals.⁶⁾ Abandoned upland farms attract numerous crop pests to the green fields, thus necessitating the application of pesticides for increased yield and domestic pest control in spite of problems associated with usage.⁷⁾

Pesticide use is known to cause serious environmental problems, especially in the dry season, when there is increased risk of high concentrations of toxic chemicals both to surface and ground water during a critical period for many animals, especially those in the wild, uplands, and northern part of Nigeria. Contamination of water by pesticides either directly or indirectly, can lead to the death of aquatic animals of nutritional value to man, reduced aquatic food productivity, or elevated concentrations of undesirable chemicals in edible aquatic animal tissue, which can affect the health of humans eating them in addition to polluting the water and coming into direct contact with farm produce. Also, acute and chronic exposures of humans to pesticides could occur during commercial production and usage.⁸⁾ Effluents from pesticide industries ultimately empty through rivers and streams, which end up in bigger aquatic environments. In spite of the upsurge in the use of pesticides in Nigeria for domestic, public health, agricultural, and industrial purposes, there has been little or no user awareness of the probable hazards to the environment and end users.

Exposure to organophosphates leads to a change in the membrane permeability of erythrocytes.⁹⁾ Erythrocytes are excellent models for studying of the interactions of xenobiotics with biomembranes. It is sometimes difficult to measure the effects of pesticide pollution, especially if the poisoning is low-level, chronic, or intermittent. There may be measurable biochemical changes that are useful as short-term biomarkers, such as the inhibition of acetylcholinesterase activity. In the present study, the aforementioned challenges led us to evaluate the impact on adult male rats of short-term exposure of one-fourth of the LD₅₀ of some commonly used pesticides (DDVP, LMB, CPM and IMP) in the Nigerian market. The impact of these pesticides was also evaluated on rat erythrocyte antioxidant enzymes as well as the tissue histopathology of a few target organs.

Materials and Methods

1. Chemicals

Dichlorvos, lambda-cyhalothrin, cypermethrin and imidacloprid were purchased from the agricultural market in Dugbe, Ibadan, Oyo State. 1-Chloro 2,4-dinitro benzene (CDNB), 5,5′-dithiobis-(2-dinitrobenzene) (DTNB), Adrenaline, Thiobarbituric acid (TBA), Reduced glutathione (GSH) and bovine serum albumin (BSA) were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). A Randox diagnostic kit was used for the alanine amino transferase (ALT) and the aspartate amino transferase (AST), while a Dialab production and Vertrieb von Chemisch-technischen kit was used for the urea and creatinine. Other reagents used were of the purest quality available locally.

2. Experimental animals

Forty male albino rats (Wistar strain) weighing between 175 g and 200 g were used for the experiment. All animals were purchased from the animal house of the Department of Biochemistry primate colony, College of Medicine, University of Ibadan, Ibadan, Nigeria, and were housed in the Animal House of the Biochemistry Department of the University of Ibadan at normal room temperature. The rats were acclimatized for three weeks to standard laboratory conditions, at temperature of 25–35°C, under a 12 hr light/dark cycle in standard rat cages. The rats were placed on a diet of palletized Guinea feed (purchased from Guinea Feed, Ibadan, Nigeria) containing grains, animal protein, vegetable protein, limestone, minerals and vitamins. The animals were allowed free access to food and water ad libitum. Animals were picked at random during the period of acclimatization for pilot studies. All experiments however, were carried out according to the guidelines for the care of experimental animals. After three weeks of acclimatization, rats were randomly distributed into five groups of eight animals each. Control Group (normal control rats on only rat chow and water), Group DDVP (dichlorovos, 20 mg/kg of body weight), Group LMP (lambda-cyhalothrin, 20 mg/kg of body weight), Group CPM (cypermethrin, 62.5 mg/kg of body weight) and Group IMP (Imidachlopril, 112.5 mg/kg of body weight). Toxicity was induced by administering one-fourth of the lethal dose (LD₅₀) daily for 21 days. Water was used as the vehicle for administering pesticides; it was given orally for 3 weeks, during which time the animals were observed daily regarding their feeding habits, psychomotor changes, sleep patterns and mortality.

3. Sample collection

Blood was collected with capillary tubes by ocular puncture and immediately transferred into clean heparinized tubes to prevent coagulation. Blood cells were separated from plasma by subjecting the whole blood collected in dried heparinized tubes to centrifugation at 3,000 g for 15 min. Plasma was carefully withdrawn without removing any erythrocytes and stored in sample bottles for analysis while the erythrocyte hemolysate was prepared according to the method of McCord and Fridovich.¹⁰⁾ Briefly, after removing the plasma, the erythrocytes are suspended in distilled water in a volume corresponding to the initial volume of the whole blood, followed by 10-fold dilution with 0.1 M phosphate buffer, pH 7.4, and then frozen in order to lyse the erythrocytes. After thawing, the suspension was centrifuged at 3,000 g for 15 min at 4°C using a cold centrifuge. The supernatant obtained was collected into a clean sample container and used for the different assays.

4. Biochemical analysis

Protein concentrations of the various samples were examined by means of the Biuret method as described by Gornal et al. (1949),¹¹⁾ with some modifications. The estimation of acetylcholinesterase activity was determined according to the method described by Elman et al. (1961).¹²⁾ Reduced glutathione level was determined by measuring the rate of formation of the chromophoric product in a reaction between DTNB (5,5′-dithiobis-(2-nitrobenzoic acid) and free sulfhydryl groups at 412 nm as described by Beutler et al. (1963),¹³⁾ and glutathione-S-transferase activity was determined using CDNB at 340 nm, according to the method of Habig et al. (1974).¹⁴⁾ The activities of the G6PD in the erythrocyte hemolysate were determined by the modified method of Dawson et al. (1958).¹⁵⁾ Lipid peroxidation was assayed by measuring thiobarbituric acid reactive substances (TBARS) by colorimetric reaction of the lipid peroxidation product, malondialdehyde, with thiobarbituric acid (TBA) to form a pink precipitate, which was read at 532 nm by spectrophotometry, as described by Vashney and Kale (1970).¹⁶⁾ AST and ALT activities were determined according to the principle described by Reitman and Frankel (1957).¹⁷⁾ Urea estimation was done using kit supplies by Dialab Production and Vertrieb Von Chemisch-technischen. Urease hydrolysis of urea to ammonia and carbon dioxide causes the ammonia to react with the alkaline hypochlorite and sodium salicylate to produce a colored complex that is measured spectrophotometrically at 546 nm, according to the principle described by Fawcett et al. (1960).¹⁸⁾ Plasma creatinine was determined according to the principle described by Bartels et al. (1972).¹⁹⁾

5. Histopathology examinations

At the time of sacrifice, the liver and kidney tissues were removed and fixed in a 10% formaldehyde solution and sent to the Veterinary Anatomy Department, University of Ibadan, for histopathological examination. Briefly, the tissues were washed by dehydration in increasing gradients of ethanol and finally cleared in toluene. The tissues were then embedded in molten paraffin wax. Sections with a thickness of 5 µm were cut and stained with hematoxylin and eosin. The slides were photographed with an Olympus UTU1X-2 camera connected to an Olympus CX41 microscope (Tokyo, Japan) and the results and photomicrographs were provided.

6. Statistical analysis

All values were expressed as the mean±S.D. of eight animals. Data were analyzed using a one-way analysis of variance (ANOVA) followed by a post-hoc Duncan multiple test for the analysis of biochemical data using SPSS (10.0) statistical software. p values≤0.05 were considered statistically significant.

Results

1. Body weight and protein levels

There was a statistically significant (p<0.05) decrease in the body weight of rats subjected to these pesticides (Table 1) as compared to the control group drinking clean bottled water. Rats in the control group showed a significant (p<0.05) increase in body weight. Table 1 shows the results of both the plasma and erythrocyte protein concentrations. There was a decrease in plasma proteins in rats on pesticides as compared to the control, while the erythrocyte protein levels of rats on DDVP and LMP were significantly increased as compared to control.

Table 1. Effect of one-fourth of the LD₅₀ of the pesticides DDVP, LMB, CPM and IMP on Body weight and plasma protein level in male Wistar rats (Mean±S.D., n=8)

	Final weight	% Increase	Erythrocyte protein level	Plasma protein level
Control	211±8.9	19.20%	1.20±0.14	2.74±0.18
DDVP	185±7.9*	−5.10%	1.39±0.16*	2.19±0.22
LMB	184±4.73*	−6.60%	1.51±0.29*	2.23±0.23
CPM	183±6.46*	−6.15%	1.19±0.05	2.20±0.10
IMP	187±2.73*	−5.56%	1.21±0.10	2.29±0.26

Values are mean of eight animals±S.D; * Significantly different compared to control (p<0.005).

2. Plasma and erythrocyte bound antioxidants

Data obtained in this study for both plasma and erythrocyte bound antioxidants of lipid peroxidation, glucose-6-phosphate dehydrogenase, reduced GSH, GST, and acetylcholinesterase activities are shown in Table 2. In vivo treatment of pesticides in the present study resulted in decreased acetylcholinesterase activity in the erythrocytes of rats treated with DDVP and LMB. Treatment using DDVP and LMP resulted in decreased acetylcholinesterase activity of approximately 6% and 16%, respectively. The decrease was found to be statistically significant (p<0.05). There was, however, no significant change in the acetylcholinesterase activity of rats treated with CPM and IMP. DDVP- and LMB-treated rats showed decreased glucose-6-phosphate dehydrogenase activity of erythrocytes of approximately 14% and 21% respectively. The decrease was found to be statistically significant (p<0.05). There was, however, no significant change in the glucose-6-phosphate dehydrogenase activity of erythrocytes in CPM- and IMP-treated rats. Plasma glucose-6-phosphate dehydrogenase activity was insignificant in all of the pesticide-treated groups. As shown in Table 2, GST activity was found to be reduced significantly (p<0.05) in the erythrocytes of rats treated with dichlorvos for 21 days as compared to the control. There was an insignificant increase in the GST activity of rats treated with the pesticides lambda-cyhalothrin, cypermethrin, and imidacloprid. Treatment with pesticides resulted in decreased GSH content in the rats exposed to dichlorvos, lambda-cyhalothrin, and imidacloprid. The decreased GSH level in rats treated with dichlorvos and imidacloprid was statistically significant (p<0.05), while there was increase in administered cypermethrin. Still, as indicated in Table 2, administration of the pesticide dichlorvos, lambda-cyhalothrin, cypermethrin and imidacloprid for 21 days caused increases in lipid peroxidation of approximately 190%, 96%, 139% and 244%, respectively. The increase in rats administered dichlorvos, cypermethrin, and imidacloprid was statistically significant (p<0.05).

Table 2. Effect of one-fourth of the LD₅₀ of the pesticides DDVP, LMB, CPM and IMP on plasma and erythrocyte membrane bound antioxidant enzymes of male Wistar rats (Mean±S.D., n=8)

	Ery LPO	Plasma LPO	GSH	GST	ERY G6PD	Plasma G6PD	ACH
Control	2.82±4.60	4.59±0.78	71±3.50	2.44±0.24	500±51.10	218±17.45	1.46±0.07
DDVP	8.17±1.12*	6.01±1.14*	62±5.70	1.81±0.51*	428±45.00*	278±25.42*	1.38±0.12*
LMB	0.12±0.01	4.27±1.79	70±1.15	3.00±0.82	396±73.20*	364±25.47*	1.23±0.23*
CPM	6.73±4.44*	4.38±1.51	72±2.31	2.57±0.44	497±42.99	262±3.25*	1.48±0.05
IMP	8.38±0.79*	4.02±1.23	62±6.00*	2.79±0.57	496±43.14	275±15.48*	1.48±0.13

Values are mean of eight animals±S.D; * Significantly different compared to control (p<0.005).

3. Markers of kidney and liver tissue toxicity

The results of AST, ALT, urea, and creatinine in the plasma of rats exposed to one-fourth of the LD₅₀ of the pesticides for three weeks in this study are shown in Table 3. There was a significant (p<0.05) increase in the ALT and AST activity in the plasma of rats treated with dichlorvos for 21 days. There was no significant change in ALT and AST in rats treated with the pesticides lambda-cyhalothrin, cypermethrin and imidacloprid as compared to the control.

Table 3. Effect of one-fourth of the LD₅₀ of the pesticides DDVP, LMB, CPM, and IMP on Plasma AST, ALT activities, urea and creatinine levels in male Wistar rats (mean±S.D., n=8)

	Plasma AST	Plasma ALT	Plasma urea	Plasma creatinine
Control	43.67±7.37	77±12.19	13.83±0.36	0.24±0.22
DDVP	68.50±14.18*	89±4.95*	14.25±1.37	0.31±0.36
LMB	44.75±8.46	83±7.14	14.19±2.23	0.25±0.17
CPM	31.00±11.31	82±7.42	14.44±1.05	0.27±0.11
IMP	42.40±7.02	78±14.32	14.00±0.71	0.24±0.03

Values are mean of eight animals±S.D; * Significantly different compared to control (p<0.005).

4. Histopathological results of liver and kidney tissue

As shown in supplemental Figs. S1 and S2, the histopathological results for the livers of rats showed no visible lesions in the control group. Similarly, rats administered one-fourth of the LD₅₀ of the pesticides lambda-cyhalothrin and cypermethrin had no visible lesions in their liver. However, rats treated with one fourth of the LD₅₀ of dichlorvos showed diffuse vacuolar degeneration of hepatocytes with mild periportal cellular infiltration by mononuclear cells. Imidacloprid-treated rats showed diffuse vacuolar degeneration of hepatocytes. Histopathological results for kidneys in all groups indicated that there were no visible lesions in rats treated with the pesticides under study.

Discussion

Pesticides are widely used worldwide for industrial, agricultural, domestic, and public health purposes; in Nigeria, the pesticides are freely available for commercial use. We have examined an experimental model of laboratory rats exposure to drinking water contaminated with four commonly used pesticides. In a pilot study before the commencement of this study, doses higher than one-fourth LD₅₀ of the pesticides resulted in a high mortality rate, highly reduced psychomotor activity, and wasting; this was used as a guide in selecting the dosage used in this study. At the dose and study duration used in this study, there was no mortality; however, there was a marked reduction in feed intake and reduced psychomotor activity in rats on DDVP as compared to control, whereas the reductions in the others were milder. As seen in Table 1, the increase in the body weight of rats in the control group could be attributed to the proper metabolism of dietary intake while the decrease in body weight of the tested groups could be due to pesticide-induced oxidative stress that could have affected various metabolic processes. Pesticides have been shown to cause a decrease in the body weight of rats,^20–22) thus the observed weight loss is supported by earlier documentation.

Erythrocytes are excellent models for studying of the interactions of xenobiotics with biomembranes,²³⁾ due to alteration in membrane permeability, hence, the use of both plasma and erythrocytes in this study. Also, polyunsaturated fatty acids in erythrocyte membranes, the presence of iron in the heme and oxygen enhance the production of oxidative stress.²⁴⁾ Pesticide poisoning could be very low-level chronic toxicity or intermittent in nature. However, measurable biochemical changes in short-term biomarkers, such as the inhibition of acetylcholinesterase activity, could be helpful. Acetylcholinesterase activity is useful as an indicator of inhibition by pesticides, as it is responsible for neurotransmitter degradation at the cholinergic synapse and is the target of most pesticides.²⁵⁾ Treatment with of dichlorvos and lambda-cyhalothrin resulted in a statistically significant decrease in acetyl cholinesterase activity in each case; the inhibition of acetylcholinesterase activity might be due to direct interaction of the pesticide with enzymes.²⁶⁾

Glucose-6-phosphate dehydrogenase is an important enzyme of the hexose monophosphate shunt; its functions in mature red blood cells (erythrocytes) is to generate NADPH, which is required for the conversion of oxidized glutathione (GSSG) to GSH, thus protecting the cell from oxidative damage, which, in turn, is necessary for the membrane integrity of erythrocyte membranes.²⁷⁾ This might be the reason for the increased fragility of erythrocytes upon treatment with different pesticides. GST is one of several detoxification enzymes and is the most important phase II drug-metabolizing enzyme,²⁸⁾ which can be found in the cytosol or associated with membranes.²⁹⁾ The decrease in the activity of the enzyme GST could be attributed to the inactivation of this detoxifying enzyme as a result of free radical production by DDVP as compared to the control group. The insignificant increase in GST activity by lambda-cyhalothrin, cypermethrin, and imidacloprid may be attributed to variations in the respective LD₅₀s of these pesticides, the active constituent of the pesticides, and due to increased in activity of this enzyme in response to oxidative stress. Effects of the administration of the pesticides dichlorvos, lambda-cyhalothrin, cypermethrin, and imidacloprid on erythrocytes’ GSH content (Table 2) showed that treatment with pesticides resulted in decreased GSH content in dichlorvos, lambda-cyhalothrin, and imidacloprid. Decreased GSH content in dichlorvos- and Lambda-cyhalothrin-treated rats, in particular, might be attributed to the decreased glucose-6-phosphate dehydrogenase activity of the erythrocytes observed in the present study. Decreased glucose-6-phosphate dehydrogenase activity results in the decreased synthesis of NADPH, which in turn results in the low level of GSH. It has been well documented in the literature that in vivo administration of various xenobiotics results in decreased GSH content of erythrocytes.³⁰⁾ The decrease in the GSH level in rats treated with DDVP and IIMP was statistically significant, while there was an increase (p<0.05) in cypermethrin-administered rats. This may be attributed to varying lethal doses (LD₅₀s) of the pesticides administered and the constituent chemical moieties present in the different pesticides. The increase in the glutathione level may also be attributed to an increase in its synthesis in response to oxidative stress. Glutathione is usually the first line of defense against oxidative stress.³¹⁾ It has been shown that certain environmental toxicants can elicit oxidative stress by converting the parent compound to its reactive metabolites.³²⁾ The accumulation of MDA, which is one of the end products of lipid peroxidation (LPO), is an index of lipid peroxidation. The increased level of MDA could be attributed to an increase in the production of free radicals caused by pesticide intake that could attack biological membranes. However, there was a decrease of approximately 96% in MDA in the lambda-cyhalothrin-treated rats; on the other hand, the plasma LPO level increased significantly (p<0.05) only in the dichlorvos-treated rats.

Several studies have shown the effect of oxidative stress on liver and kidney function.³³⁾ The liver is an important internal organ because it detoxifies the body of many toxins and synthesizes a wide range of proteins. Liver function tests (LFTs) are groups of blood assays that provide information about the state of the liver. Hepatotoxicity involves only mild symptoms initially; therefore, early detection is vital.³⁴⁾ ALT and AST are known markers for liver damage.³⁵⁾ Elevated activity of the enzymes ALT and AST in plasma could indicate their leakage from the intracellular store as a result of possible liver damage. No significant change in ALT and AST was observed for rats treated with the pesticides lambda-cyhalothrin, cypermethrin, and imidacloprid, thus, indicating their safety at the dosage used in this study; however, this was very different for the organophosphorous pesticide dichlorovos which elicited elevated levels in ALT and AST implying tissue damage.

Plasma urea and creatinine levels were determined in the present study. There was however, no significant change in the levels of urea and creatinine in all treated groups relative to the control. Many pesticides can cause some toxic and adverse effects on kidney tissues.³⁶⁾ Urea and creatinine levels are kidney function parameters.^23,37) Pesticides can alter plasma urea and creatinine levels.³⁸⁾ Urea is the end product of protein catabolism. Increased blood urea is correlated with an increased protein catabolism in the mammalian body and/or referred kidney dysfunction. The levels of urea in the plasma of rats are tested as indicators for kidney functions.³⁹⁾ Also, high levels of blood urea result from the increased breakdown of tissue or impaired excretion. In this study, the decreased urea level may be due to no toxic effects of one-fourth LD₅₀ of the pesticides administered (Table 3).

Creatinine excretion is almost dependent on the process of glomerular filtration. A previous study reported that a significant rise in the serum creatinine level may be due to the impairment of the glomerular function and tubular damage in the kidneys.²³⁾ The creatinine level is a good risk marker for chronic renal insufficiency. Increased creatinine levels indicate damage of the glomerular function and tubular damage in the kidneys.^40,41) Pesticides have been shown to cause various histopathological changes in the kidney tissue of experimental animals⁴²⁾; however, in the present study, at the dose and duration studied, there was no evidence of kidney damage.

Conclusion

Based on these results, it can be suggested that the pesticides administered exert differential effects on the activity of the various antioxidant and membrane bound enzymes present in the erythrocytes which may be useful in their toxicological evaluation. The organophosphorous pesticide elicited more oxidative stress and tissue damage and the neonicotinoid IMP showed the least adverse effects in this study. Awareness is needed for using these pesticides, and, when consumers choose which pesticides to purchase, education is needed for considerations that go beyond cost that will ensure personal and environmental safety and maintain a friendlier environment for all. Therefore, protective methods and public enlightenment concerning the laws guiding the use of pesticides should be carried out regularly by the necessary organizations to prevent pesticide-induced toxicity.

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