Histological and micrometric effects of diazinon exposure on adrenal medulla and cortex in mice

Abstract

Histopathological effects of Diazinon (DZ) were studied on adrenal gland in virgin young female mice divided into five groups (10 animals each): (1) Control (untreated); (2) and (3) 9 and 18 mg kg⁻¹ single exposure groups (given distilled water on day 1 and 2 and DZ on day 3); (4) and (5) 9 and 18 mg kg⁻¹ multiple exposure groups (given respective DZ doses on days 1–3). All doses were applied by gavage. Animals were euthanized by cervical dislocation on day 6. Adrenals were removed surgically for histological and micrometric assessments of medulla and cortex. Histological examination of adrenal medulla revealed apoptotic changes and granular depletion in chromaffin cells while cortex gave impression of increased thickness of Zona Fasciculata following multiple DZ exposures. Mean cell sizes in cortical and medullary regions showed dose and exposure dependent significant decline. In conclusion, DZ exposure caused granular depletion in chromaffin cells indicating a rapid release of adrenaline and increased fascicular thickness indicating an anticipated shift from adrenal sex steroids to glucocorticoids biosynthesis and release. Apoptotic changes and significant decline in mean cell sizes of cortical and medullary regions were considered as the outcomes of oxidative and biosynthetic stresses of DZ exposure.

Keywords:

• diazinon
• histopathology
• micrometry
• adrenal
• mice

Introduction

Organophosphate (OP) insecticides, containing esters of phosphoric or thiophosphoric acids, are one of the most commonly used insecticides in the world. Besides their intended effects on the target animals, these insecticides inflict toxicity to the non-target animals (particularly mammals) by means of phosphorylation of acetylcholinesterase leading to an accumulation of acetylcholine and hence repeated impulses in the post-synaptic neurons (Perry et al. 2011). Among OP insecticides parathion, malathion and Diazinon (DZ) are considered highly potent acetylcholinesterase inhibitors (Schrickel et al. 2009). Secondarily these insecticides have a common feature of endocrine toxicology through disruption of neuro-secretary centres in hypothalamus disturbing pituitary–thyroid, pituitary–gonadal and pituitary–adrenal axes (Clement 1985; Kokka et al. 1987; Peiris-John and Wickremasinghe 2008).

Adrenals are considered as the most important endocrine glands due to their involvement in major biological functions in animals including developmental, immune, inflammatory, osmoregulatory and metabolic activities. Adrenal hormones directly influence the metabolic adjustments of carbohydrates proteins and lipids; thus, influencing the functional status of all major body organs including heart, liver, kidney, bones, muscles and nervous system in animals (Harvey 2010). Stress related activities such as exposure to an insecticide trigger adrenal medulla and cortex for enhanced secretions of catecholamines and glucocorticoids, respectively (Jortner 2008).

Diazinon caused a dose-dependent increase in blood glucose levels in rodents (Seifert 2001). Similarly, dimethoate (Kamath and Rajini 2007), malathion (Abdollahi et al. 2004; Pournourmohammadi et al. 2005; Panahi et al. 2006) and monocrotophos (Joshi and Rajini 2010) induce an increased blood glucose level in rats. Oral exposure of acephate at 140 mg kg⁻¹ in rats has caused reversible hyperglycemia with peak blood glucose level (87% above Control) within 2 h of its administration (Joshi and Rajini 2009). Authors emphasized on the acephate exposure related increase of corticosterone (78% in this case) levels to be the reason for hyperglycemia. Lukaszewicz-Hussain (2010) opined that hyperglycemia following acute or chronic OP exposure is mediated through various mechanisms such as physiological or oxidative stress and disturbed tryptophan metabolism in liver in addition to their established stimulatory effect on the adrenals and inhibition of cholinesterase. In addition to its hyperglycemic effect, DZ exposure is reported to enhance lipid peroxidation (Teimouri et al. 2006) and oxidative and nitrosative stresses (Ghafour-Rashidi et al. 2007).

Diazinon exposure caused organ pathologies in mouse, such as necrotic degeneration of spleen and thymus, hyperplasia of thymus, spleen and lymph nodes, and sometimes haemorrhage from all tissues (Handy et al. 2002). In spite of the availability of a great deal of information on OP and particularly DZ related toxicological implications on adrenals, there are only a few studies dealing directly with histopathological effects of OPs on adrenal medulla and cortex (Yano et al. 2000; Akhtar et al. 2009). In a preliminary study, Sufiriyanto and Indradji (2002) made a reference of swollen cells and congestive cortical lesions in rats following DZ exposure at 100 ppm for 40 day in drinking water. It was thus decided to investigate the histopathological and morphometric changes in adrenals following DZ exposure in mice.

Materials and methods

Maintenance and feeding

Fifty young virgin female mice (Swiss Webster) aged 3–4 months, weighing 30–35 g were used in this study. These animals were born reared and used in this experimental work in animal house (Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan). The animals were kept in 12′′×18′′ metal gauzed steel cages under standard protocol of 12–12 h dark light cycles. Fine cuttings of fresh toilet paper were provided for bedding that was replaced on alternate days. Temperature and humidity were maintained at 24±2°C and 35–45%, respectively. Food and water were provided ad libitum. Required treatments of DZ in various groups were provided by gavage.

Experimental groups and insecticide treatments

Animals were randomly distributed in five (10 in each) groups; respectively named as Control, 0.9 mg kg⁻¹ single, 0.09 mg kg⁻¹ multiple, 18 mg kg⁻¹ single, 18 mg kg⁻¹ multiple DZ treatment groups. In multiple DZ groups animals received their respective doses on days 1, 2 and 3. The Animals in single DZ groups were provided their respective dose of diazinon only on day 3 while in first two days of the experiment they were given vehicle (distilled water) only. The animals in Control group received distilled water on first 3 days of the experiment. Dose volume was kept constant (0.1 ml) throughout while the required strength of insecticide was adjusted by appropriate aqueous dilution in each case according to the weight of animal at the time of dosing.

Organ recoveries, histology and photography

Adrenals (both intact) were recovered on day 6 of the study from each animal after cervical dislocation. The organs were fixed in ‘alcoholic Buoin's’ for 24 h and finally processed for wax embedding. Histological sections (6-µ thick) were obtained on a rotary microtome and stained with Hematoxylin and Eosin. Photomicrographs, at 100 and 400×, of 10 randomly selected sections from each animal (five from each adrenal) of various groups were obtained using Sony (Model No. DSC-W35) 7.2 MP digital camera mechanically attached with Labomed CXR2 trinocular microscope. Selected photographs were processed in CorelDRAW 11 for colour, contrast and on-snap labelling to highlight histological observations.

Digital morphometric data

Images of stage micrometer were obtained on optical and digital specifications fixed for photomicrography of histological sections of the tissues. These images were used in digital calibration for sizing the endocrine cells of adrenal medulla and cortex. To calculate the Cross-sectional Area (CA), diameter of 10 randomly selected cells (separately for adrenal medulla and cortex) from each digital image of all the animals (100 cells/tissue/animal) were obtained from two right angle positions and the values obtained were put into the following equation:

Mean CA for each animal (considered as a unit) of the groups were calculated. The data were subjected to statistical analysis [ANOVA and post hoc analysis of the means using Duncan's Multiple Range Test (DMRT)] and presented as Means±SEM as bar graphs.

Results

Adrenal cortex

A general dose and exposure dependent decline in mean cell size in the cortical region was observed (Figure 1). Statistical analysis of the variance for the cell size data among the groups indicate an overall highly significant difference (F calculated = 12.927, F critical = 2.69; P=3.12 E-06). Post hoc comparative analysis of mean between the groups [based on DMRT] indicated a significant decrease of the mean cell size in all DZ treated groups except 9 mg kg⁻¹ single exposure group to that of the Control (P ≤ 0.05) (Figure 1).

Figure 1.  Mean (values shown with in the histogram bars) cortical cell size in Control and DZ treated groups.

Note:+bars = SEM; uppercase letter (A, AB and B) above the + bars indicate the statistical comparison of the means among the experimental groups [any two groups not sharing a common uppercase letter show significant difference with each other (comparison of the means based upon Duncan's Multiple Range Test: P <0.05)]. Title bar shows the F, F critical and P values of ANOVA. 9S: 9 mg/kg Single DZ exposure; 9M: 9 mg/kg Multiple DZ exposure; 18S: 18 mg/kg Single DZ exposure; and 18M: 18 mg/kg Multiple DZ exposure groups.

Adrenal medulla

A trend of decline in the mean cell size was observed in the medullary region on DZ treatment. Analysis of variance indicate an over all significant variation among the data groups (F=3.103, F Critical = 2.689; P=0.029921). Post hoc comparative analysis of means (DMRT) indicate significant decline in mean cell size of 9 and 18 mg kg⁻¹ multiple exposure group to that of the Control group (P ≤ 0.05) (Figure 2).

Figure 2.  Mean (values shown with in the histogram bars) medullary cell size in Control and DZ treated groups.

Note:+bars = SEM; uppercase letter (A, AB and B) above the + bars indicate the statistical comparison of the means among the experimental groups [any two groups not sharing a common uppercase letter show significant difference with each other (comparison of the means based upon Duncan's Multiple Range Test: P <0.05)]. Title Bar shows the F, F critical and P values of ANOVA. 9S: 9 mg/kg Single DZ exposure; 9M: 9 mg/kg Multiple DZ exposure; 18S: 18 mg/kg Single DZ exposure; and 18M: 18 mg/kg Multiple DZ exposure groups.

Histopathological findings

The histological data revealed characteristic alterations in adrenal cortex and medulla in DZ treated groups to that of the Control. While the histological slides from the Control group show well placed adrenal medulla and cortex (Figure 3: Control) – with all three regions (Reticularis, Fasciculata and Glomerulosa) distinctly visible. Increase in the thickness of Zona Fasciculata, following DZ treatment in various groups, at the expense of Zona Reticularis was noted (Figure 3: 18M). Obvious signs of macro-vesicular steatosis were present exclusively in Zona Reticularis in 9 and 18 mg kg⁻¹ multiple treatments groups but not in single exposure groups (Figure 3: 18M). While clear signs of proliferation were seen in Zona Fasciculata in both multiple DZ exposure groups (Figure 3: C2). Chromaffin cells of the adrenal medulla show granular depletion. Apoptotic changes were seen in chromaffin cells of adrenal medulla in 9 and 18 mg kg⁻¹ multiple exposure groups only (Figure 3: M2).

Figure 3.  Control (100×) and 18M [18 mg kg⁻¹(100×)]; C1: 9 mg kg⁻¹ (400×) and C2: 18 mg kg⁻¹ (400×) – adrenal Cortex in DZ multiple exposure groups; M1: 9 mg kg⁻¹ (400×) and M2: 18 mg kg⁻¹ (400×) – adrenal Medulla in DZ Multiple exposure groups, showing various histological abnormalities.

Note: A: Medulla; B: Zona Reticularis; C: Zona Fasciculata; D: Zona Glomerulosa; E: Central vein; F: Fat droplets in Zona Reticularis; G: Chromaffin cells apoptosis; H: Shrunken cluster of chromaffin cells due to the depletion of granules; White arrow heads: Mitotic proliferation in Zona Fasciculata.

Discussion

The foremost stress response, as in the case of a sudden exposure to an insecticide, is usually mediated by rapid hormonal changes particularly catecholamines and glucocorticoids (Jortner 2008). Adrenal medulla synthesize, store and secrete catecholamines (epinephrine and nor-epinephrine) − the established hormones of emergency (Rady 2009); while a variety of steroid hormones are produced in adrenal cortex. Three distinct zones of adrenal cortex namely: Zona Glomerulosa, Zona Fasciculata and Zona Reticularis produce mineralocorticoids (the aldosterone), glucocorticoids (corticosterone and cortisol) and sex steroids (adrenal androgens), respectively (Rosol et al. 2001; Harvey 2010). These three major classes of adrenal steroids are respectively involved in homeostasis of osmotic and ionic regulations; metabolism of carbohydrates, lipids and proteins; and the suppression of inflammatory and immune responses under toxic and oxidative stress conditions (Salih 2010).

Organophosphorus pesticides are generally considered as endocrine disrupters (Jeong et al. 2006). In present study, we have seen depletion of cytoplasmic granules from the chromaffin cells of adrenal medulla and a significant decrease in mean cell size, particularly in DZ multiple exposure groups. The granular depletion clearly indicate hyper catecholamenergic situation causing size reduction of the chromaffin cells.

Under stressful conditions (DZ exposure) secretion of glucocorticoids (corticosterone and cortisol) increase (Buckingham 2008; Harvey 2010). Exposure of OP pesticides in general and DZ in particular has been related to the serum concentration of testosterone along with simultaneous pathologies of testis in rats and mice (Fattahi et al. 2009; Okamura et al. 2009). Decline in the thickness of Zona Reticularis of the adrenal cortex following DZ exposure seems to be linked partly to the anti-androgenic effect of DZ. A probable simultaneous transformative change of reticular cells to the fascicular cells to cope with the enhanced demand of glucocorticoids might have been responsible for a simultaneous increase in the thickness of Zona Fasciculata. The apparent of signs of macro-vesicular steatosis seen exclusively in Zona Reticularis, in 9 and 18 mg kg⁻¹ multiple treatments groups are considered as the indications of lipid peroxidation and storage of triglycerides under oxidative stress related impaired steroidogenesis in these cells on DZ exposure (Rosol et al. 2001; Shah and Iqbal 2010).

Hyperplasia of the adrenal cortex is relatively uncommon (Nyska and Maronpot 1999; Capen et al. 2002) nevertheless it has been proposed that exposure to exogenous steroid antagonists may block the action of steroid hormones leading to an augmented adrenocorticotropic hormone (ACTH) secretion from the pituitary that induce hyperplasia in the cortical region (Rosol et al. 2001). Very clear signs of proliferation in Zona Fasciculata following multiple DZ exposures in this study were considered to be a logical consequence of the augmented release of ACTH from the pituitary, partly under antagonistic effect on biosynthesis and release and partly because of the DZ induced increased demand of ‘Glucocorticoids’. Chromaffin cells in adrenal medulla release catecholamines under direct cholinergic sympathetic stimulations (Tischler et al. 2010). The hyper catecholamenergic situation enhance the secretion of aldosterone from cortical region. Thus, persistent cholenergic stimulation of multiple DZ treatment is still another expected reason for the proliferative response of Zona Fasciculate. Significant decline in mean cell size in adrenal cortex can be seen in conjuncture with excessive metabolic stress for the biosynthesis and release of glucocorticoids along with hyperplasia of the fascicular cells causing storage depletions in cytoplast along with the transformative change of the reticular cells to the fascicular cells under this DZ stressful exposure.

Conclusion

The present study gives an insight towards histopathological responses of adrenal medulla and cortex on DZ exposure. Otherwise an established endocrine disruptors of sex glands (Fattahi et al. 2009; Okamura et al. 2009) this OP insecticide also damage adrenal cortex and medulla. Our findings suggest that apart from its proven neuronal (Slotkin and Seidler 2007) and reproductive (Fattahi et al. 2009; Okamura et al. 2009) toxicological effects on the non-target animals DZ is also a profound disruptor of adrenal histology.