Abstract
The aim of the present study was to investigate the effect of atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) on the left ventricle myocardium in juvenile/peripubertal male Wistar rats. Atrazine was administered orally at 50 or 200 mg/kg of body weight dose for 28 consecutive days. In order to assess possible structural alterations, tissue sections were examined histologically and then subjected to quantification analysis using stereological methods. The tissue specimens were routinely processed and stained with Mallory trichrome method in order to clearly distinguish muscle cells from the connective tissue components. A toluidine blue staining method was additionally used for the demonstration of mast cells. Statistically significant increase in length density and numerical density of capillaries were found at both the investigated doses of atrazine compared with the control. The increase in surface density and volume density of capillaries found at lower dosage of atrazine was significant in comparison with the control. The extensive mast cell degranulation was noted on the histological examination at both doses of the applied chemical. No significant changes were demonstrated for the stereological parameters of cardiomyocytes. Based on the available published data and the present results, it can be concluded that atrazine promoted angiogenesis in the rat myocardium, which might be partially mediated by mast cells.
Introduction
Atrazine (International Union of Pure and Applied Chemistry (IUPAC): 6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) is a selective pesticide used in weed control on crop fields and a nonselective agent for vegetation control in industrial areas. As a member of s-triazine class of herbicides, atrazine inhibits the process II or P680 of photosynthesis by blocking the electron transport system in plant species sensitive to this compound. The most extensive use of atrazine is associated with the production of corn, sugar cane and sorghum on a worldwide scale. The application of atrazine to soil is considered particularly relevant in terms of potential harmful effect on wildlife and humans due to its heavy use and persistence in the environment. Atrazine is commonly detected in surface and ground waters in agricultural areas and also in municipal drinking water (Loos et al., 2010; WHO, 2003).
The route of atrazine exposure is primarily oral ingestion. Atrazine can also enter the body through inhalation or dermal penetration during its application. Once enters the general circulation, atrazine is transported to and rapidly metabolized in liver into deisopropyl and dealkilated (DACT) metabolites that are ultimately excreted in urine together with about 2% of unchanged atrazine (Catenacci et al., 1993). Despite such estimated time course for the biotransformation and excretion, traces of atrazine and/or its metabolites were found in a number of tissues. Experimental study of atrazine retention in liver and kidneys in female rats orally gavaged with a single dose of atrazine of either 500 or 1000 mg/kg body weight (bw) demonstrated a higher concentration measured in liver (Scutaru et al., 1998). Atrazine was also detected in mice brain after oral exposure at doses of 5–250 mg/kg bw with maximal levels between 0.06 and 1.5 µM within 4 h after dosing, while the levels for DACT were detected 2 h after atrazine application in a range comparable to liver (1.5–50 µM) (Ross et al., 2009). In male rats exposed to a single oral dose of 30 mg/kg bw atrazine labeled with 14C, the radioactivity in skin and red blood cells were detected 72 h after administration (Timchalk et al., 1990). Atrazine was detected in human heart after the ingestion of herbicide mixture containing atrazine, although at a low concentration (Pommery et al., 1993).
A number of studies have been undertaken to investigate the potential harmful effects of atrazine particularly on reproductive outcomes (Friedmann, 2002; Stoker et al., 2000a; Victor-Costa et al., 2010), the endocrine system (Fraites et al., 2009; Rajkovic et al., 2010a) and its developmental toxicity (Greenlee et al., 2004; Rayner et al., 2004). Studies on atrazine effects on heart demonstrated an increase in the force of contraction of the isolated frog atria muscles during incubation with 1, 5 or 50 µM of atrazine (Papaefthimiou et al., 2003). In the same experiment, the incubation with 200 µM of atrazine exerted a gradual decrease in the force of muscle contraction, which returned to referent values after atrazine replacement with a physiological solution. Inhibition of twitch tensions was recorded in the atria isolated from adult rats as a response to atrazine treatment as well as a strong vasorelaxation of the isolated aorta (Chan et al., 2007). Atrazine exposure caused a reduction in heart rate in zebrafish embryos exposed to 30 or 40 mg/L of atrazine (Wiegand et al., 2001) and a reduction in developing heart with a distorted general shape of the heart in tadpoles exposed to 25 or 35 mg/L of atrazine (Lenkowski et al., 2008).
The aim of the present study was to investigate the potential effects of atrazine on the cardiac tissue of juvenile–peripubertal rats at the light microscopic level. The investigation involved the histological examination of cardiac tissue sections and the quantification analysis of cardiomyocytes and the connective tissue of the left ventricle based on stereological methods. The peroral administration has been chosen as a route of exposure to the pesticide, since the general population is most likely to be exposed to atrazine by oral ingestion via the polluted drinking water. The results of the study hereby reveal significant augmentation of myocardial capillary bed in exposed rats. According to the best knowledge of the authors, this is the first demonstration of structural alterations of the myocardium related to atrazine exposure.
Methods
Animals
Male Wistar rats aged 23 postnatal days (PNDs) were used for the experimental study. Animals were housed under laboratory conditions with 22 ± 2°C and subjected to a controlled photoperiod (14 h light and 10 h dark). Pelleted food and water were provided ad libitum. On the 22nd PND, rats were weighted and assigned to one of the experimental groups: (1) control group, (2) low-dose atrazine group or (3) high-dose atrazine group, each consisting of five animals with similar initial body weights. The study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. The investigation was made with the permission of the Ethical Committee on Animal Experiments of the University of Novi Sad, Serbia.
Atrazine treatments
The experiment was performed from PNDs 23 to 50. The animals of the two atrazine groups were orally gavaged with either 50 mg/kg bw (low dose) or 200 mg/kg bw (high dose) of atrazine. Atrazine (97% purity, a kind gift from Professor Sanja Lazic, Institute for Environmental and Plant Protection, Faculty of Agriculture, University of Novi Sad, Serbia) was dissolved in edible olive oil and administered daily in morning between 08.00 and 09.00 h. The control group was administered with olive oil only. All groups were treated for 28 consecutive days.
The doses of 50 and 200 mg/kg bw were selected based on previous studies on the effects of atrazine on premature rats (Stoker et al., 2000b). Animals were killed on PND 51, 24 h after the last atrazine treatment.
Sample preparation
After decapitation, samples of the whole left part of the heart were taken and fixed in 10% buffered formalin pH 7.0 for 24 h, dehydrated in graded series of ethanol, cleared in xylene, embedded in paraffin and sectioned vertically into 5-μm-thick sections. For the histological and stereological analysis, tissue sections were stained according to the Mallory trichrome method (Clark et al., 1973; hematoxylin: Merck, Darmstadt, Germany; acid fuchsin: Kemika, Zagreb, Croatia; Fast Light Green: Edward Gurr, London, UK). In order to demonstrate the connective tissue mast cells, the sections of cardiac tissue were stained with 0.5% toluidine blue (Clark et al., 1973; Merck, Darmstadt, Germany).
Stereological analysis
The stereological grid with 42 points (M42) placed in the ocular of a light microscope was used for quantification analysis of the sections stained with Mallory trichrome method and with toluidine blue.
Sections of the heart tissue stained with Mallory method were used for the quantification analysis of the cardiomyocytes and the connective tissue. The staining provided sufficient contrast of the muscle cells against the connective tissue and the ability to clearly distinguish the blood capillaries from the connective tissue fibers. Collectively, the staining method enabled a precise collection of the quantification data. The stereological analysis was performed on 25 subsequent fields of vision per animal using an oil immersion objective. The point-counting was performed on cross-sectioned cardiomyocytes of the left ventricle. The fields of visions involving blood capillaries were counted, while those covering larger blood vessels were omitted in order to obtain accurate data on myocardial capillaries. The stereological parameters determined involved the volume density (Vv) and the surface density (Sv) of cardiomyocytes; the Vv of the connective tissue as well as the Vv, Sv, length and numerical densities (Lv and Nv, respectively) of the blood capillaries (Weibel, 1979).
The toluidine blue histochemical method enabled the visualization of mast cells in the connective tissue of the left ventricle due to metachromatic staining of the proteoglycan molecules stored in the cytoplasmic secretory granules of these cells. The volume and numerical densities of the total mast cells (Vvm and Nvm, respectively) were determined on 100 fields of vision per animal under the total magnification of 400×. Additionally, the intact (resting) mast cells and the degranulated cells were analyzed. The latter cells were identified either by the decreased metachromatic staining or the altered cell morphology with the cytoplasmic granules released into the connective tissue surroundings. Correspondingly, the volume and numerical densities of the intact mast cells (VvmINT and NvmINT, respectively) and of the degranulated mast cells (VvmDEG and NvmDEG, respectively) were calculated.
All measurements and cell counts were performed on blind-coded sections. Data on cardiomyocytes, connective tissue and capillaries were statistically analyzed by the one-way analysis of variance followed by the Bonferroni’s post hoc test when needed. Calculated parameters for the mast cells were analyzed by the Kruskal–Wallis test. p < 0.05 was considered statistically significant.
Results
Effects of atrazine on cardiomyocytes and cardiac stroma
Histological examination of cardiac tissue sections revealed no treatment-related effects on cardiomyocytes (Figure 1(a) to (c)). No signs of myocardial fibrosis were found in both the groups that received different doses of atrazine. Correspondingly, results of the stereological analysis of the left myocardium demonstrated no statistically significant differences in the Vv of cardiomyocytes and the Vv of connective tissue between the experimental groups (p > 0.05) after 28 consecutive days of treatment with two investigated atrazine doses (Table 1). The Sv of cardiomyocytes increased in both the low (50 mg/kg bw) and the high (200 mg/kg bw) dose atrazine groups compared with the control, but did not reach statistical significance (p > 0.05; Table 1). The Vv (p = 0.0059), Sv (p = 0.0044), Lv (p = 0.000013) and Nv (p = 0.000028) of capillaries significantly increased in rats treated with the low dose of atrazine when compared with the control (Table 1). In the group treated with high-dose atrazine, the Lv and Nv of capillaries increased significantly (p = 0.049 and p = 0.0019, respectively) in comparison with the control, while the decreased Vv of capillaries and the increased Sv of capillaries versus the control were statistically insignificant (p > 0.05; Table 1).
Photomicrographs of cardiac tissue stained with histochemical methods of Mallory ((a)–(c)) and toluidine blue ((d)–(f)). The control ((a) and (d)), atrazine 50 mg/kg bw ((b) and (e)) and atrazine 200 mg/kg bw ((c) and (f)). Differences in the capillary network density in rats treated with atrazine (asterisks in (b) and (c)) and the control rat (asterisks in (a)) can be observed. Degranulated mast cells in atrazine-treated rats (arrows in (e) and (f)) and intact cells in the control rat (arrows in (d)). Photomicrographs ((a)–(c)) are taken under a total magnification of ×1000. Scale bar in (a) denotes 10 µm. The original magnification of photomicrographs ((d)–(f)) is ×600. Scale bar on (d) indicates 10 µm.
Mean values with SD of investigated stereological parameters for the cardiac tissue of control and atrazine-treated animals.a
bw: body weight; Lv: length density; Nv: numerical density; Sv: surface density; Vv: volume density.
aAll mean values designated with the same letter indicate homogenous groups (Bonferroni’s correction, p > 0.05).
Effects of atrazine on cardiac mast cells
Histological analysis of mast cells showed morphologically heterogeneous population in all experimental groups. The presence of mast cells in different functional status was observed, from intact to degranulated cells, in all animals (Figure 1(d) to (f)). Mast cells were often found in groups and localized in the proximity of blood vessels, endocardium and epicardium.
Stereological data point to an increased numerical and a decreased Vv of the myocardial mast cells in both the atrazine-treated groups compared with the control (Table 2). An exception from this tendency was observed for the Nv of intact mast cells in the group treated with 200 mg/bw of atrazine. The most prominent observation was an increase in total mast cell number in atrazine-treated groups compared with the control. Additionally, the proportion of intact-to-degranulated mast cells in atrazine-treated groups was in favor of the degranulated cells. In spite of the observed differences, no statistical significance was reached between atrazine-treated groups and the control regarding the Vv or Nv of Vvm, Nvm, VvmDEG, NvmDEG, VvmINT and NvmINT (Table 2). Nevertheless, the Nv of degranulated mast cells in the group treated with the low-dose atrazine was increased by 63.4% compared with the control and by the 62.8% in the group gavaged with the high-dose atrazine.
Median values with the lower and upper quartiles (in brackets) of all investigated stereological parameters for mast cells in the cardiac tissue of control and atrazine-treated animals.a
bw: body weight; Vvm: volume density of total mast cells; Nvm: numerical density of total mast cells; VvmINT: volume density of intact mast cells; NvmINT: numerical density of intact mast cells; VvmDEG: volume density of degranulated mast cells; NvmDEG: numerical density of degranulated mast cells.
aAll median values represent homogenous groups (Kruskal–Wallis, p > 0.05).
Discussion
The present study performed on juvenile–peripubertal male rats demonstrated a significant increase in numerical and length density of blood capillaries in the left ventricle after 28 consecutive days of exposure to atrazine at both doses. Mast cell degranulation in the left ventricle was found in both atrazine dose groups on histological examination. Results on the study group treated with the low-dose atrazine point to increased surface of the myocardial capillary network for gas and nutrient exchange, while a prominent increase in microvascular density in both the dose groups might be related to myocardial angiogenesis. Both these effects may represent compensatory response to atrazine treatment and have a physiological consequence of maintaining the net blood flow.
The coronary blood flow is controlled primarily by the myocardial oxygen demand as myocardium extracts a high percentage of oxygen. It is modulated by various paracrine vasoactive substances and neurotransmitters released from autonomic nerve endings. The smooth muscle tone of the myocardial resistance vessels (arterioles) is regulated by these respective substances maintaining, thus the balance between blood flow and oxygen requirements in the myocardium. However, under certain conditions, myocardial microvessels begin to sprout increasing the collateral blood flow. The formation of new blood vessels from existing ones or angiogenesis occurs under physiological conditions during embryogenesis and can also be initiated under various pathological conditions in adult tissue. The coronary angiogenic response is known to be related to ischemia, hypoxia and inflammation (reviewed in Maulik and Das, 2002a). The process of angiogenesis in the heart is associated with the paracrine action of various growth factors including the basic fibroblast growth factor (bFGF), acidic fibroblast growth factor, vascular endothelial growth factor (VEGF), platelet-derived growth factor and agiopoetin-1 (Li et al., 2001; Tao et al., 2011; Zhao et al., 2010). The referred factors are involved in different aspects of angiogenesis including proliferation and differentiation of vascular endothelial cells, recruitment of pericytes and induction of matrix metalloproteinases (MMPs). Furthermore, the involvement of reactive oxygen species (ROS) in the formation of new blood vessels has also been reported. Although ROS are prevalently linked to cell injury followed by apoptosis or even necrosis, the growing evidence suggest that ROS at low concentrations have a proangiogenic effect (reviewed in Maulik and Das, 2002b). Experimental studies have demonstrated that low concentrations of hydrogen peroxide stimulated angiogenesis in cultured endothelial cells through the enhancement of cell proliferation and migration, the increased formation of tubular structures and the increased level of ets-1 messenger RNA, a transcription factor that regulates the expression of MMPs (Yasuda et al., 1999). Recent studies investigating atrazine toxicity have focused on the investigation of its potential role as an oxidative stressor. Results of such studies demonstrated an increase in the activity of antioxidant enzymes in different cell types by atrazine exposure, pointing to cell response to oxidative stress (Bhatti et al., 2011; Singh et al., 2008). Accordingly, a significant augmentation of capillary density found in the present study might have resulted from atrazine-induced angiogenesis mediated through the ROS.
In order to determine a relative contribution of connective tissue cells to microvascular growth, the resident cardiac mast cells were further investigated. The focus on these cells was based on their known causal involvement in angiogenesis through granule-associated MMP release and subsequent MMP-evoked degradation of extracellular matrix, which is one of the principal features in the sequential events of new microvessel formation from parent microvessels (reviewed in Norrby, 2002). Furthermore, mast cells synthesize two potent angiogenic factors bFGF and VEGF responsible for the proliferation, differentiation, migration and survival of endothelial cells, hence playing a pivotal role in angiogenesis (Detoraki et al., 2009; Qu et al., 1998). It is well known that the population of cardiac mast cells is found in normal as well as in pathological conditions, suggesting their active participation during processes of heart tissue damage and subsequent remodeling (Facoetti et al., 2006). Numerous studies have focused on elucidating the role of cardiac mast cells in pathogenesis of the heart damage during myocardial ischemia, fibrosis and hypertensive heart disease (Estensen, 1985; Patella et al., 1998; Petrovic et al., 1999). It has been reported that mast cells degranulate during cardiac hypertrophy (Palladini et al., 2003; Shiota et al., 2003), cardiac transplantation and myocarditis (Fairweather et al., 2004; Li et al., 1992), while other studies detected an increased number of these cells in the myocardium during hypertension, infarction and tissue remodeling (Stewart et al., 2003).
The main observation in the present study in respect with myocardial mast cells was that atrazine treatments caused a substantial increase in the number of both mast cell subpopulations compared with the control, but particularly of degranulated cells, which was noticed by their altered cytoplasmic granule content. On the other hand, the Vv of mast cells in treated groups were decreased when compared with the control. Morphologically, mast cells in atrazine-treated groups had elongated shape, demasked nuclei with numerous granulations released, thus suggesting a process of ongoing degranulation and intensive cell activity. Conversely, the control group was characterized by a domination of rounded, fairly intact mast cells and small amount of granulations released, which is an indicator of a lower degree of mast cell activity. The present data are in agreement with our previous finding on increased cutaneous mast cell degranulation in juvenile–peripubertal male rats following 30 consecutive days of exposure to atrazine at 20 mg/kg bw dose (Rajkovic et al., 2010b). A statistically significant increase in numerical density of total and degranulated mast cells was also recorded (Rajkovic et al., 2010b).
The estimated numerical and volume parameters for mast cells in this study are in mutual correspondence suggesting an increased mast cell number and activity within the left ventricle in atrazine treatments. These findings were subsequently confirmed by the calculated stereological parameters but with no statistical significance. However, it should be emphasized that the number of degranulated mast cells increased by approximately 63% in both dose groups compared with the control. Collectively, the present evidence of the degree of mast cell degranulation indicates their possible involvement in myocardial capillary bed alterations in atrazine-treated groups. The mast cell-borne factor/factors relevant for angiogenesis or factors derived from a different source remains presently uncertain. A recently conducted in vitro experiment on atrazine and several other xenoestrogens on VEGF expression in two breast cancer cell lines have found that atrazine had no potency to modulate VEGF secretion in MELN cells (Buteau-Lozano et al., 2008). In the second studied cell line, the MELP, an increase in VEGF secretion has been recorded, which was, however, not statistically significant even when high concentrations of atrazine were applied (Buteau-Lozano et al., 2008). A potential causal involvement of atrazine in angiogenesis seems a promising field for future research involving both in vivo and in vitro experimental models.
In summary, the present findings demonstrate an augmentation of myocardial vascularization through atrazine-induced microvascular angiogenesis that might have emerged from an increased oxygen and nutrition demand by cardiomyocytes or direct effect of atrazine on microvessels. Histological observation of mast cell degranulation, although not statistically significant, appears to be biologically relevant and suggests an involvement of these cells in the response of cardiac tissue to atrazine effect.
Footnotes
Acknowledgements
The authors thank Zeljka Siladjev for skilful technical assistance.
Funding
This work was supported by the Serbia Ministry of Science and Technological Development (grant number III46001).
Conflict of interest
The authors declared no conflicts of interest.