ENDOCRINE DISTURBANCES INDUCED BY LOW-DOSE ORGANOPHOSPHATE EXPOSURE IN MALE WISTAR RATS

Abstract

Background

Organophosphate exposure induces many endocrine effects.

Aim

In this study we observed the effects of acute stress induced by cholinesterase inhibition on the main hormonal axes.

Materials and Methods

We included thirteen weanling Wistar rats that were subjected to organophosphate exposure. They were first tested for baseline levels of butyrylcholinesterase, cortisol, free triiodothyronine, thyroxine, thyroid-stimulating hormone and prolactin. Secondly, chlorpyrifos was administered. Next samples were taken to determine the level of all the above-mentioned parameters.

Results

Butyrylcholinesterase was significantly decreased after exposure (p<0.001). Cortisol levels were significantly higher after clorpyrifos administration (358.75±43 vs. 241.2±35 nmoL/L)(p<0.01). Although prolactin had a growing trend (450.25±24.65 vs. 423±43.4 uI/mL), the results were not statistically significant. Both free triiodothyronine and thyroxine were significantly higher after exposure. Surprisingly, thyroid-stimulating hormone level almost doubled after exposure with high statistical significance (p<0.001), suggesting a central stimulation of thyroid axis. Butyrylcholinesterase level was proportional with thyroid-stimulating hormone level (p=0.02) and thyroxine level was inversely correlated to the cortisol level (p=0.01). Acute cholinesterase inhibition may induce high levels of cortisol, free triiodothyronine, thyroxine and thyroid-stimulating hormone. From our knowledge this is the first study dedicated to the assessment of acute changes of hormonal status in weanling animals after low-dose organophosphate exposure.

Conclusion. Acute cholinesterase inhibition may cause acute phase hormonal disturbances specific to shocked patients.

INTRODUCTION

Organophosphates are the most widely used pesticides in the world and despite World Health Organization’s (WHO) restrictions on limiting its use, the number of cases of intoxication remains very high (1). Although the incidence of acute cholinergic syndrome caused by these chemicals is decreasing considering the latest restrictions, a sub-acute exposure occurs (2). A low dose organophosphate exposure was frequently associated with numerous metabolic disturbances like obesity and diabetes mellitus type 2 (3).

The main mechanism of toxicity associated with organophosphates is the inhibition of cholinesterase in the neuromuscular system, blood and glandular parenchyma (4). In addition to this well-established mechanism, it has been shown that their toxicity causes changes in the release of neuro-transmitters, such as GABA or glutamate, inhibition of cellular metabolism, but also stimulation of mast cell degranulation and histamine release (4).

The systemic changes produced by acute organophosphate exposure led to the classification of this type of intoxication as an acute stress situation like those identified in trauma or sepsis, such as sympathetic activation and stimulation of the hypothalamic-pituitary-adrenal (HPA) axis (5).

If the HPA axis has been extensively studied, thyroid damage under stress had benefited from a much lower interest (6). Beside acute stress-related thyroid dysfunction, organophosphate exposure, even in low doses, may cause thyroid hypofunction which is typically described as a euthyroid ‘sick syndrome’ (7,8). Moreover, histopathological derangements caused by organophosphate exposure were identified in various experimental studies (7).

As a stress hormone implicated in inflammatory response modulation, prolactin may be affected by organophosphate exposure through increased acetylcholine level on the dopaminergic neurons and pituitary (8). However, longer duration of exposure is necessary in order to obtain a significant increase in prolactin levels (8).

Considering the lack of data regarding the endocrine disturbances generated by acute cholinesterase inhibition resulting from low dose organophosphate exposure we designed the present study aiming to characterize the status of the main stress hormones after acute intoxication with low dose of organophosphates.

MATERIALS AND METHODS

We performed an experimental study in order to evaluate the hormonal status under conditions of acute stress caused by acute organophosphate intoxication in weanling rats, according to ARRIVE guidelines 2.0 and to Declaration of Helsinki principles. The study included 13 weanling Wistar rats (three-weeks-old) from an accredited laboratory, in order to evaluate the effects of low-dose organophosphate exposure on pediatric population, taking into account that this age in rats corresponds to a human population of one to two years old (13.8 rat days equals 1 human year)(9).

The study lasted for two months and included only male rats to avoid possible hormonal changes related to sex.

The 13 rats were divided into two groups - Group A represented the study group and included 8 rats, and Group B - represented the control group and consisted of 5 rats. During the study, all rats benefited from specific food and water ad libitum. They were housed at an average temperature of 22°C and a light / dark cycle of 12 hours.

In the study group, chlorpyrifos (ReldanTM 22 EC, Dow Agrosciences) was administered at a dose of 100 mg / kg via an orogastric tube. Considering that the lethal dose 50 (LD50) for chlorpyrifos is 229 mg / kg, the administered dose is equivalent to half the LD50 (220). After 4 hours, blood samples were taken by puncturing the vein from the tail with a 29 G needle. In the control group, saline solution was administered by gavage and the blood samples were collected in the same manner.

In both cases, blood samples were taken according to the protocol of the Institutional Animal Care and Use Committee (IACUC), according to which the maximum amount of blood that can be collected is calculated based on the formula: Blood quantity = body weight x1.5%. In addition, both blood sampling and gavage were performed under general anesthesia according to the same IACUC protocol, using 1% isoflurane and ketamine / xylazine 40/5 mg / kg.

Blood samples from both groups were transported to an authorized private medical laboratory, where plasma levels of butyrylcholinesterase, corticosterone, free T3, free T4, TSH and prolactin were measured. Before transporting to the laboratory, every blood sample was number-coded in order to blind the source. Determination of plasma levels for butyrylcholinesterase was performed by spectrophotometric technique, while plasma hormone values were performed using the ELISA sandwich technique for quantitative detection, using ELISA kits for rats with the following identification codes: corticosterone – ABIN368134, fT3 - ABIN2685560, fT4 - ABIN365197, TSH - ABIN2685829, prolactin – ABIN3222302.

Ethical issues

The study project received the approval of the Bucharest Emergency Clinical Hospital Ethics Committee on 31.01.2017, with registration number 2180. Throughout the study, the ethical norms in force related to the experimental studies on laboratory animals were respected.

Statistical analysis

The data obtained from the analysis of biochemical samples was included in a database and was statistically analyzed using the MedCalc 14.1. In the study, a threshold of statistical significance corresponding to a p <0.05 was considered.

The statistical test used to compare the two groups was the t test, and the results obtained were expressed as means, together with standard deviations (SD). T test for independent variables was used considering that analyzed data had a normal distribution in both groups, excepting T4 values. Taking into account that the control group included a small number of rats, Anderson-Darling and D’Agostino-Pearson tests could not be performed. However, Shapiro-Wilk and Kolmogorov-Smirnov tests indicated a normal distribution. In addition, for each parameter the coefficient of variation was analyzed, as the ratio between standard and average deviation. This coefficient provides information on the existing variability in the study group, in relation to the average of those in the group.

ROC curves were used in order to establish the hormone levels threshold after acute exposure since low doses of organophosphate were used, which are usually associated with a clinically silent evolution. These analyses were conducted taking into consideration a gold standard that differentiated between the exposed and non-exposed group. Based on the ROC curves, we developed a model of hormonal changes induced by the acute exposure to organophosphates. Cut-off values were obtained by calculating the area under the curve (AUC) and 95% confidence intervals. It was considered that an AUC value ≥ 0.9 shows an increased accuracy, between 0.9-0.7 moderate and an AUC situated between 0.7-0.5 suggested poor accuracy. Sensitivity and specificity were also analyzed, considering that high sensitivity corresponded to high negative predictive value and high specificity indicated a high positive predictive value.

Pearson correlation was also performed in order to evaluate the relations between measured hormone levels.

RESULTS

The mean value of butyrylcholinesterase in the study group was 12.7 ± 7.3U/L, while in the control group its level was 1471 ± 389U/L (Fig. 1a). The difference between the two measurements was 1458U/L and showed a strong statistical significance (p <0.0001). In addition, these data were supported by an increased coefficient of variation of 142%.

Figure 1.

a. Butyrylcholinesterase level after organophosphate exposure; b. Corticosterone level after organophosphate; c. fT3 level after organophosphate exposure; d. fT4 level after organophosphate exposure; e. TSH level after organophosphate exposure; f. Prolactin level after organophosphate exposure.

The corticosterone level in the study group was on average 358.75 ± 43 nmol / L, and in the control group 241.2 ± 35 nmol / L (Fig. 1b). The calculated difference between the two groups was 23.23 nmol/L, fulfilling the threshold of statistical significance (p = 0.0004). The coefficient of variation calculated for this analysis is 33.96%.

Regarding the evolution of thyroid hormones, the plasma level of fT3 in the group with intoxicated rats was 6.14 ng / mL ± 0.36, while fT3 in the control group was 3.82 ± 0.47 ng / mL (Fig. 1c).

A difference of 2.32 was calculated between the two measurements, which has the strongest statistical significance after butyrylcholinesterase (p <0.0001). Also, the value of the coefficient of variation is increased – 34.43%.

A similar evolution was recorded in the case of fT4, which had an average value of 48.37 ± 7.48 ng/ mL in group A and an average level of 34.6 ± 4.5 ng/ mL in the group of non-intoxicated rats (Fig. 1d). The difference between the two determinations was also statistically significant (p = 0.0036). The coefficient of variation in this case was 29.73%.

Figure 1e shows the plasma level of TSH measured in the study group (4.96 ± 0.43 mU/L) and in the control group (2.58 ± 0.4 mU/L), respectively. As in the case of fT3, the difference between the two means showed a strong statistical significance (p<0.0001). The coefficient of variation is also very high, respectively 47.13%.

However, the calculated values and as represented graphically (Fig. 1f) show a tendency for prolactin to increase in group A (Group A 450.25 ± 24.65 ng/mL vs. Group B 423 ± 43.4 ng/mL). The difference between the two measurements (27.25 ng/mL) was not statistically significant (p = 0.16).

Cut-off values for the studied hormones in the study group were performed based on the ROC curve analysis. In the case of corticosterone, the calculated limit was 280 ng / mL (p <0.0001), with a specificity and sensitivity of 100% and the AUC = 1 (Fig. 2a).

Figure 2.

a. Corticosterone ROC curve; b. fT3 ROC curve; c. fT4 ROC curve.; d. TSH ROC curve; e. Prolactin ROC curve.

All intoxicated subjects had a fT3 value greater than 4.4 ng / mL (p <0.0001), with a specificity and sensitivity of 100%, and the AUC = 1 (Fig. 2b).

The analysis of data obtained from fT4 level measurements led to two conclusions: on the one hand that 87.5% of intoxicated rats showed values above 41 ng/mL, and on the other hand if the fT4 value does not increase to a minimum value of 41 ng/mL, most likely the subject is not intoxicated (Fig. 2c).

The cut-off value for TSH was 3.2 mU/L, with a sensitivity and specificity of 100%. Given both AUC = 1 and the value of p <0.0001, so that we can confidently say that an intoxicated subject has a TSH value of more than 3.2 mU/L (Fig. 2d).

As we have demonstrated in the previous statistical analysis, prolactin does not undergo marked changes in conditions of acute organophosphate intoxication (p = 0.22)(Fig. 2e). However, we can assert that all intoxicated rats had prolactin values above 400 IU / mL. The statistical analysis suggests that if a minimum value of 400 ng / mL is not reached, 60% of the subjects will not experience acute intoxication. This makes prolactin not particularly useful in assessing the profile of the intoxicated rats. Practically, the statistical program estimates that there are rats intoxicated with prolactin below 400 ng / mL and non-intoxicated rats with prolactin above 400 ng / mL.

A proportional relationship was observed between the level of butyrylcholinesterase and TSH (r = 0.78), which also met the threshold of statistical significance (p = 0.021)(Fig. 3a). Another statistically significant correlation (p = 0.01) but negative this time was detected in the case of fT4 and corticosterone (r = -0.83). In other words, as shown in Figure 3b, the higher the corticosterone value, the lower the fT4 value.

Figure 3.

a. Correlation between butyrylcholinesterase and TSH; b. Correlation between fT4 and corticosterone; c. Correlation between fT4 and prolactin.

Following the statistical analysis, an inversely proportional correlation of fT4 and prolactin was also demonstrated (r = -0.70), which was very close to meeting the statistical significance threshold (Fig. 3c). No significant correlation between corticosterone and prolactin was detected in the study.

DISCUSSION

Organophosphates are currently the most widely used pesticides in the world, despite regulations imposed by the WHO(10). Although the number of acute severe cases of organophosphate poisoning decreased significantly worldwide, low-dose exposure occurs frequently in urban as well as in rural areas (2,11). Nowadays the exposure to the most frequently used organophosphates is associated with subclinical symptoms which are frequently overlooked (12). Increased availability and extensive use of these pesticides was proven to cause a continuous low-dose exposure which is responsible for a variety of cardiovascular, neuropsychiatric, fertility, metabolic and hormonal illnesses (3,12–14).

Currently, the most utilized method of detecting acute organophosphate exposure is the measurement of butyrylcholinesterase plasma levels(4). In the present research, the biochemical confirmation of intoxication was also performed based on butyrylcholinesterase, whose value was significantly lower in the study group than in the control group. This diagnostic method has been shown to have certain limitations due to individual variations of butyrylcholinesterase level, lack of sensitivity under repeated exposure conditions and lack of correlation between plasma levels and the severity of clinical manifestations (15). Still, it remains the most used diagnosis tool for organophosphate intoxication, considering its increased availability as well as the low cost (4,15).

Since the early 1980s, Clement et al. have considered the classification of acute organophosphate intoxication as a generalized stress response (16).

Beyond the best known mechanism of toxicity of organophosphate compounds, represented by the inhibition of acetyl cholinesterase and butyrylcholinesterase, further research has revealed a number of alternative mechanisms of toxicity responsible for the occurrence of acute or chronic side effects (17). Besides the acute stress-related hormonal changes, endocrine disturbances may occur also due to the inhibition of acetyl cholinesterase (18,19).

At present, the consequences of organophosphate toxicity and the intoxication related to acute stress in the pediatric population are a global concern, considering that it has already been stated by WHO that they have higher exposure levels and vulnerability even at low doses (20,21). Nevertheless prolonged exposure to organophosphate, even in low doses, proved to interfere with later development of young children (22,23). Extensive residential use of pesticides, proximity to agricultural fields and contaminated food are reported to be the most important source of exposure to pediatric population. Considering that the research on newborns and children are nowadays strictly limited, using young age laboratory animals is an attempt to evaluate toxic-induced changes on pediatric population (20). For this reason, the present study evaluated the endocrine effects of organophosphates exposure on weanling rats. Comparative studies of species-specific assessments, however, indicate a number of differences in the ability of liver metabolism of toxins between weanling rats and children (20). In the present research, the acute changes of the tested hormones are not influenced by the degree of maturation of liver microsomal enzymes.

Activation of the hypothalamic-pituitary-adrenal axis in acute stress conditions involves the stimulation of neurons in the paraventricular nucleus of the hypothalamus, which leads to the release of corticotropin-releasing hormone (CRH) and vasopressin (24). They stimulate the release of adreno-corticotropic hormone (ACTH) from the pituitary gland, which in turn promotes the release of gluco-corticoids, mineralocorticoids and catecholamines from the adrenal level (24). The action of cortisol under stress is considered vital given its effects of ensuring a level of glucose appropriate to the needs, to stimulate the cardiovascular system and brain activity (25). Increased levels of cortisol are responsible for maintaining the vasomotor tone, ensuring the energy substrate by gluconeogenesis or maintaining fluid homeostasis (26). The increased acetylcholine levels secondary to organophosphate exposure favor the release of cortisol secondary to the enhanced ACTH secretion (27). In the study group, intoxicated rats showed a significant increase in corticosterone levels compared to those in the control group. Similar data were obtained in the experimental research conducted by Joshi et al. (28). After monocrotophos exposure there was a significant increase in corticosterone levels, later improved in the group treated with atropine (28). Rezg and colleagues also revealed that exposure to malathion led to an increase in corticosterone levels and thus to an inhibition of CRH secretion (29).

The hypothalamic-pituitary-thyroid axis is one of the main factors involved in regulating energy metabolism, so improving the secretion of thyroid hormones under conditions of acute stress can be considered an adaptive mechanism (26). Changes in the hypothalamic-pituitary-thyroid axis under conditions of acute stress are specific to non-thyroid systemic impairment, in which there is mainly a decrease in triiodothyronine (T3) levels (30,31). This condition, identified in the literature as ‘low T3 syndrome’, ‘sick euthyroid syndrome’ or ‘non-thyroid impairment syndrome’, can be established early in critical conditions and is associated with an unfavorable prognosis (30). Although stress is traditionally thought to cause a decrease in thyroid hormones, there is conflicting data in the literature that certain stressful events lead to their increase (eg. loud noise)(6). It is also considered that the thyroid hormonal axis is directly related to the glucocorticoid axis, so that increasing cortisol levels causes decreased thyroid activity by altering the transformation of thyroxine (T4) to T3, but also by inhibiting the release of thyrotrophin-releasing hormone (TRH) at the hypothalamic level (6). In the initial phase of an acute stress, the secretion of thyroid hormones is briefly increased and shortly after the onset of the disease, a considerable persistent decrease is registered (26). In the present research, two hours after chlorpyrifos intoxication, an increase in plasma levels of fT3, fT4 and TSH was detected compared to the control group. Tseng et al. demonstrated in their observational study that patients intoxicated with organophosphate had an acute hyperthyroid status (32). In a study conducted by Satar et al., blood samples taken eight minutes after the administration of lethal doses of methamidophos indicated a decrease in the level of thyroid hormones typically to a sick euthyroid syndrome (33). However, we consider that the administration of a lethal dose of organophosphate compound may cause a completely different clinical situation than in the current study in which half of LD50 of chlorpyrifos was administered, LD50 for chlorpyrifos being in the range of 100-300 mg/kgc (34). Moreover, the fact that metamidophos has a higher toxicity than chlorpyrifos must be taken into account, being classified by the WHO as risk class IA, compared to chlorpyrifos (Class II) (35). In another study led by El-Sheikk et al., the assessment of thyroid hormone plasma levels was performed 24 hours after the administration of chlorpyrifos (1/20 of LD50) and a decrease of thyroid hormones in the study group was detected(36). Considering this data, we can conclude that in the present study the two-hour interval between intoxication and blood sampling was not sufficient to produce changes in thyroid hormones specific to “low T3 syndrome” following administration of chlorpyrifos. Angelier and colleagues also identified that a short period of time between the application of a stressor and sampling causes the thyroid hormone levels to remain unchanged (37).

The connection between the hypothalamic-pituitary-cortico-adrenal and thyroid axes is proven based on experimental data in which the administration of glucocorticoids causes a decrease in thyroid hormones (37). In the present research, we identified an inversely proportional correlation between plasma corticosterone levels and fT4, similar to those obtained in the study of Ferlazzo et al. in horses, in which increased levels of glucocorticoid hormones during physical stress are associated with decreased thyroid hormone levels (38). The mechanisms by which the two hormonal axes interact are represented by the inhibition of messenger ribonucleic acid (mRNA) for TRH at the hypothalamic level and the impairment of deiodination of thyroid hormones in the periphery by cortisol (6).

Prolactin is currently considered a stress hormone, whose activity is responsible for modulating the stress response, immune system stimulation and metabolic regulation (37,39). However, the mechanism by which prolactin exerts its effects is not currently fully elucidated (39). Moreover, reports of its behavior under stress are contradictory (39). Thus, it has been observed that it may increase or decrease its level depending on the type of stress to which the body is subjected (40). Dopamine is the main inhibitory factor, which controls the secretion of prolactin (41). However, there are other factors that contribute to the regulation of prolactin release, such as acetylcholine, which also has an inhibitory effect (40). The action of acetylcholine on prolactin release can be mediated by either nicotinic, somatostatin or GABA receptors (40). Although the release of prolactin and thyroid stimulating hormone (TSH) was anticipated to be associated given the stimulatory effects of hormone replacement therapy (HRT) on the two hormones, their secretion was found to be more rapidly dissociated, with prolactin being significantly increased by stress compared to TSH (40). The increase in plasma prolactin levels in the acute response to stress most likely promotes the activation of the immune system, this hormone being classified as a mediator between the central nervous system, the endocrine system and immunity (40). More recent data even classify the prolactin receptor as part of the cytokine receptor superfamily (41). In our study group there was an increase in the level of prolactin compared to the control group, however the threshold of statistical significance was not met. Similar results were obtained in the research conducted by Gahali and colleagues in which they detected an increased level of prolactin following their exposure to heat stress (42). In the study conducted by Dutta et al., patients intoxicated with organophosphate insecticides showed an increased level of prolactin which subsequently decreased gradually (8). It should be noted that in the present study, besides the acute stress caused by intoxication, acetyl cholinesterase inhibition occurred. Given the regulatory mechanisms of prolactin secretion, in which the main inhibitor of its release is dopamine, which in turn is stimulated by the accumulation of acetylcholine under conditions of organophosphate exposure, the evolution of prolactin in the study group was unexpected(43). However, recent data regarding prolactin secretion regulation may indicate that dopamine in low concentrations may even stimulate lactotrophic cells from lactating rats (40). The relevance of these data is somewhat unclear, given that repeated research on cells from male and female rats did not register similar results (40). Currently it is considered that the modulation of prolactin release is made by dopamine together with a number of other adjuvants (40). Somatostatin, another factor that inhibits prolactin secretion, is inhibited by cholinergic overstimulation secondary to organophosphate insecticide intoxication, a mechanism that may explain the increase in the prolactin secretion in the study group (44).

The relationship between the hypothalamic-pituitary-thyroid axis and prolactin is proven by the stimulatory effects of TRH on prolactin (40). Moreover, there are studies that suggest that an important decrease in dopamine levels may intensify the stimulatory effects of TRH (40). In the current study a negative correlation was obtained between the two hormonal lines, illustrated by the inversely proportional increase in fT4 value to that of prolactin.

Not unusual at all, this study, like many other studies that aimed to assess hormonal changes in the acute stress phase, failed to identify a correlation between corticosterone and prolactin levels, given that the effects of increased glucocorticoid on prolactin occur under conditions of prolonged exposure to organophosphates (37).

Based on the ROC curves we were able to develop a hormonal profile of the subject with acute organophosphate intoxication. Thus, according to our data, the intoxicated subject presented the following parameters, as summarized in Table 1.

Table 1.

Hormonal profile of the intoxicated subject in the study group

Butyrylcholinesterase	<28 U / L
Corticosterone	> 280 nmoli / L
fT3	> 4.4 mU/L
TSH	> 3.2 mU/L

Study limitations

A limitation of the present study is the lack off multiple blood samplings which would have provided additional information about the dynamics of hormone levels. However, it should be noted that the study group consisted of weanling rats and according to the IACUC experimental research guideline, further blood sampling would have been possible at two weeks distance. In addition, a complete evaluation of the hormonal lines, as well as the investigation of the changes of the studied somatotropic axis would have provided a clearer picture of the changes that occur in conditions of acute intoxication.

Although ROC curves analyses indicated in every case a specificity and sensitivity of 100% atypical aspects of the curves were observed. This limitation derives from a modest sample size in both groups. However, considering experimental research guidelines a minimum sample size should be used in order to demonstrate an effect.

Taking into account the extensive use of organophosphates in daily life, these complex hormonal changes are recurrently expected. Considering the insufficient development of pediatric population, a higher impact of this silent exposure is to be foreseen.

From our knowledge this is the first study dedicated to the assessment of acute changes of hormonal status in weanling animals after low-dose organophosphate exposure.

In conclusion, organophosphate exposure may cause acute phase hormonal disturbances specific to shocked patients. Following oral organophosphate poisoning, an increase in corticosterone levels was found at two hours, results that are consistent with available literature. Regarding the hypothalamic-pituitary-thyroid axis, in the study group the intoxication with organophosphates generated a hyperthyroid status, compared to the control group. Although data from the literature rather indicates the occurrence of a “low T3 syndrome”, determinations of plasma levels of thyroid hormones are usually taken at a greater distance from the time of exposure than in the present study. In the study group, prolactin increased two hours after intoxication, although the statistical significance threshold was not met.

The positive correlation between corticosterone and fT4 confirms the interdependence relationship between the hypothalamic-pituitary-adrenal and thyroid axes already described in the literature. The negative correlation between prolactin and T4 and the elevated plasma levels in the study group opposes existing physiological data.

Conflict of interest

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

Funding

The authors did not receive support from any organization for the submitted work and all the research expenses were assured through personal funds.

Ethics approval

Approval was obtained from the ethics committee of the Clinical Emergency Hospital Bucharest.