The Effect of Parathion-methyl and Antidotes on Parotid and Pancreatic Glands: A Pilot Experimental Study

Abstract

The objective of this study is to investigate the functions of parotid and pancreatic glands in response to intoxication with parathion-methyl (PM) and the effects of treatment in rats. Seventy-five male Wistar rats were divided equally into five groups: Group I, control; group II, received atropine and pralidoxime (2-PAM) for 24 h, but no PM; group III, oral PM but no atropine and 2-PAM; group IV, PM and atropine for 24 h and 2-PAM; group V, PM and atropine for 96 h and 2-PAM. After the administration of the chemicals, blood samples were drawn to test for amylase, lipase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), while pancreatic and parotid glands of each rat were removed for light microscopic examination. Amylase levels were found significantly elevated in groups II, III, IV, and V, whereas lipase levels were supranormal in groups III, IV, and V. The blood levels of AChE were decreased in groups III and IV and BChE were decreased in II, III, IV, and V. No evidence of pancreatitis and parotitis was identified in the histopathologic evaluation in any group in 96 h; however, hyperchromasia, irregularity in nuclei, and binuclear cells were observed in all parotid glands in group V. Parotitis and pancreatitis were not evident; however, hyperamylasemia and hyperlipasemia were found, whereas various histologic changes in parotid glands were documented in the groups that were administered organophosphate and treatment.

Organophosphates (OPs) are widely used as domestic and agricultural pesticides. After toxic exposure, these agents bind irreversibly to acetylcholinesterase (AChE), with subsequent manifestations of increased cholinergic activity. Irreversible anticholinesterase intoxication causes accumulation of acetylcholine (ACh) at postsynaptic receptors and results in profound, sustained stimulation of skeletal muscle, the central nervous system, and the autonomic nervous system (Sofer and Weizman 1992). OPs lead to many well-defined complications such as cholinergic crisis, intermediate syndrome, and acute pancreatitis (Arneson et al. 1979; Borner et al. 1982; Bornman et al. 1997; Brand et al. 1990; Colardyn et al. 1991; Conradi et al. 1988; Fowler et al. 2004). But some other complications such as rhabdomyolysis, acute renal failure, and parotitis have been encountered less commonly (Acikalin, Gokel, and Gulalp 2002: Dusing et al. 1990; Flomenbaum et al. 1998). Acute pancreatitis and its mechanism following exposure to OPs have already been described in experimental studies in literature. However, OP effects in pancreas and parotis with treatment effects in an experimental model have not been studied yet. The aim of this study is to evaluate pancreatic and parotid glands in rats treated due to OP poisoning in the acute period.

MATERIALS AND METHODS

Seventy-five male Wistar rats, aged 4 months, provided by the Central Vivarium of the University, and weighing approximately 250 g were randomly assigned into five equal groups and of 15 rats each. All experiments were conducted with permission of the institutional review board.

The rats were in Nalgene metabolic cages maintained at 19°C to 23°C and 45% to 60% moisture. These cages were in brightness for 12 h and in darkness for 12 h in a day. They were fed with 16 mm standard rat food.

Serving as the control, group I received no medication. Group II was treated with a total of 1 mg/kg atropine in divided doses and 40 mg/kg/day pralidoxime (2-PAM) (Brydon et al. 1987) for 24 h intraperitoneally and did not receive parathion-methyl (PM). At the end of 24 h, analysis of blood and removal of organs were carried out as in group I. 40 mg/kg/day PAM dose given in one time to the intraperitoneal cavity once a day. Table 1 summarizes the research protocol. The timetable of the study is described in Table 2.

Group III received 0.12 mg/kg PM orally. The rats were intoxicated orally with gavage tube one by one for only one time. All rats given PM started to show intoxication symptoms, fasciculation, tremors, lethargy, and weakness, after 15 min. Anesthesia was used only before taking the samples and for sacrifice of surviving rats. All rats that were administered PM without treatment were sacrificed after 45 min with anesthesia. After the symptoms of intoxication, such as fasciculation, convulsion, and secretory increases (salivation), had been noted, analysis of blood and removal of organs were undertaken as in group I.

Group IV received 0.12 mg/kg PM orally. After the symptoms of intoxication such as fasciculation and secretory increases (salivation) had been identified, the rats were treated with a total of 1 mg/kg atropine in divided doses and 40 mg/kg/day 2-PAM intraperitoneally for 24 h. The time to treatment with antidotes to the intoxicated group is 15 min after the emergence of the intoxication symptoms. The researchers were present throughout the experiments and observed the whole treatment of the rats continuously through the treatment period. All the rats that received treatment survived, but all these rats were sacrificed with anesthesia in the form of 2.5 mg/kg intramuscular (IM) xylazine and 60 mg/kg IM ketamine. Approximately 10 min after the anesthesia, blood samples were drawn from the heart; 15 min later all the tissues had been extracted.

Group III were studied at first to observe the clinical findings of the rats. This was followed by the control group, groups IV and V to investigate the treatment doses of antidotes, and finally, group II was studied. Group V received 0.12 mg/kg PM orally. After the symptoms of intoxication had been apparent, the rats were treated with a total of 1 mg/kg atropine in divided doses in the first 24 h and 40 mg/kg/day 2-PAM intraperitoneally for 96 h. The rats were studied one by one at the same time within the same experimental group. In all groups, subsequent to drawing blood, serum amylase and lipase were analyzed using the dried biochemical method (Van 1985). AChE and butyrylcholinesterase (BChE) were analyzed by the Modified-Ellman method (Elder et al. 1981). Pancreas and both parotid glands were removed from each rat. Pancreatic and parotid glands were fixed in 10% neutral-buffered formalin for light microscopic examination. The control group values were taken as base values. Mann-Whitney U test and chi-square test were performed for statistical analysis and significance was established at the p < .05 level.

RESULTS

OP effects were remarkable in 15 min and released completely in 60 min following the treatment. Serum amylase, lipase, AchE, and BChE levels in all groups are shown in Table 3. There was a significant increase in the serum amylase values in groups II, III, IV, and V, with respect to the control group (Mann-Whitney U test; p = .01, p = 0.0001, p = 0.005, p = 0.0001, respectively). Serum lipase values in groups III, IV, and V were significantly higher than the control group (p = 0.0001, p = 0.05, p = 0.0001, respectively). The slight increase in lipase of group II was insignificant compared to the values of the control group (p = 0.8). The AChE level in groups III and IV were lower than that of the control group (p = 0.0001, p = 0.002, respectively). The decrease in the BChE level from control appeared to be significant for the groups III, IV, and V (p = 0.0001, p = 0.0001, p = 0.0001, respectively).

Histologic Changes in Parotid Gland

Increased secretions and ductal dilatation, inflammatory cell infiltration, interstitial hemorrhage, interstitial edema, nuclear hyperchromasia, enlargement, irregularity, and binuclear cells (Figure 1) were found in the parotis. The results are summarized in Table 4.

Histologic Changes in Pancreatic Gland

Frequencies of increased secretions and ductal dilatation (Figure 2), inflammatory cell infiltration, interstitial hemorrhage, and interstitial edema were identified in the pancreas, as shown in Table 5.

DISCUSSION

Acute pancreatitis (Arneson et al. 1979; Borner et al. 1982; Bornman et al. 1997; Brand et al. 1990; Conradi et al. 1988; Sofer and Weizman 1992) and acute parotitis (Dusing et al. 1990; Flomenbaum et al. 1998) resulting from OP poisoning are documented. In literature, there is not an experimental report showing interstitial hemorrhage in pancreas induced by OP, except for Lankisch and colleagues who reported two cases with hemorrhagic pancreatitis (Brand et al. 1990). Interstitial hemorrhage, interstitial edema, ductal dilatation, and inflammation were noted in pancreas. With the exception of interstitial hemorrhage, our histological observations (interstitial edema, ductal dilatation, and mononuclear cell infiltration) confirm the observations of the experimental reports in literature (Borner et al. 1982). In the present study, interstitial edema of parotid gland was noted in all rats in group III, and in nine rats in group IV. Nuclear hyperchromasia, enlargement, irregularity, and binuclear cells were noted in nine rats in group IV and in all rats in group V. There has not been a pilot experimental study available in literature on the effect of OP on parotid gland.

Reported serum enzyme elevations may be associated with ductular hypertension and cellular changes (Borner et al. 1982). Accumulation of ACh due to poisoning increases amylase secretion in pancreas and parotis (Dusing et al. 1990). In the literature, reported studies stated that atropine provides protection, as evidenced by stable serum enzyme levels and absent structural changes (Borner et al. 1982). A previous study demonstrated that cholinergic agents are weak stimulants of pancreatic flow and bicarbonate secretion, with more marked effect on enzyme secretion (Saad, Valenzuela, and Weiner 1986).

Group I was studied at first, before administering intoxicants to the other groups. The AChE and BChE levels were low, maybe as contamination of intoxication solutions as they were prepared in the same study room before drawing the blood and tissues of control group.

Blood amylase and lipase levels were significantly higher in group V, comparable to the groups receiving PM and these are taken as hyperamylasemia and hyperlipasemia due to poisoning effect (Chang et al. 1998) Amylase levels were increased in group II and it is an unexpected result.

Scoring table is not obtained for each rat as a function of fasciculation, secretion, convulsion, and number of them in a group and this is a deficiency of the study. AChE and BChE were higher in 96 h and 24 h, with effect of treatment on the intoxicated group, and these were not increased in the control group in this period. In this study, both atropine and 2-PAM were ineffective in regulating the levels of amylase and lipase enzymes sufficiently 96 h following administration of PM. This shows the severity of and related to the amount of the organophosphates. Our paper demonstrates that sublethal dose of PM induces the elevation of amylase and lipase and supports the previous papers about acute pancreatitis. The present findings pointed out a slight increase in levels of amylase and lipase along with a decrease in levels of AChE and BChE in the rats that received these two compounds in the absence of the insecticide; this result was unexpected and needs further investigation. This group may have been affected via inhalation and contamination of intoxicants.

Atropine and oximes are classically used in the treatment of OP poisonings. Administration of atropine depresses the pancreatic response in intact pancreas (Grossman, Singer, and Solomon 1980). Inhibited AChE can be reactivated only by strong nucleophilic compounds such as oximes, whereas treatment with 2-PAM can be associated with hepatotoxicity and cholinesterase inhibition (Altuntas et al. 2004). There are no studies on the effect of 2-PAM on pancreatic and parotid glands in the literature. Likewise, the effects of the treatment (atropine, 2-PAM) have not yet been identified clearly. The treatment may support the improvement of increasing AChE and BChE and decreasing amylase and lipase in OP poisoning.

Consequently, the mechanism of interstitial edema in parotid gland in this study is thought to be ductal hypertension and stimulation of exocrine secretion from the parotid glands by PM-induced cholinergic stimulation as in OP-induced pancreatitis. But the present study is unable to clarify the mechanism of nuclear changes in the parotid gland. Similarly, the interstitial edema in both glands noted in one rat in group II could not be explained.

The present study shows that PM intoxication induces nonspecific histologic changes, except acute pancreatitis and parotitis, in 96 h, with increased levels of amylase and lipase and decreased AChE and BChE. However, nuclear hyperchromasia, enlargement, irregularity, and binuclear cells in parotis and other specific treatment effects of OP would be enlightened with further studies.