Effect of Interleukin-10 on Tissue Damage Caused by Organophosphate Poisoning*

Authors


  • *

    The experiment was performed in Hakan Cetinsaya Experimental and Clinical Research Center, Kayseri, Turkey.

Author for correspondence: Ibrahim Ikizceli, Esenyurt Mh. Hangar Cd. Adiguzel Sk., Hokelekli Ap. 13/17, 38039, Melikgazi, Kayseri, Turkey (fax + 90 352 437 52 73, e-mail ikizceli@erciyes.edu.tr).

Abstract

Abstract:  Organophosphate poisoning is a common cause of severe morbidity and mortality among patients admitted to emergency departments. Tissue damages as a consequence of organophosphate poisoning are frequently reported, but preventing this potentially severe complication has not been the subject of much research. We tested whether interleukin-10, a cytoprotective agent, could prevent or diminish pathological signs of tissue damages caused by organophosphate poisoning. Thirty rats were divided into three equal groups (n = 10). Group 1 (sham) did not receive any agent during the experiment. Group 2 (control) received 0.8 g/kg of fenthion intraperitoneally, followed by 6 ml/kg of intraperitoneal normal saline 30 min. and 3 hr later. Group 3 (treatment) received 0.8 g/kg of fenthion intraperitoneally, followed by 2 µg/kg of interleukin-10 intraperitoneally 30 min. and 3 hr later. All rats were killed under anaesthesia after 6 hr and tissue samples were obtained from liver, kidneys and lungs. Even organophosphate poisonings do not cause significant clinical problems; several degrees of damages could be observed in liver, kidneys and lungs. These damages could be reduced by interleukin-10 treatment.

Millions of organophosphate poisoning cases are reported annually worldwide, mostly due to insecticide exposure [1]. The widespread use and easy accessibility of these compounds result in a huge number of poisoning cases [2]. The mode of exposure to organophosphate compounds includes dermal, gastrointestinal and inhalation. Poisoning occurs as a result of agricultural use, accidental exposure, suicide and, rarely, homicide [3].

The best-known effect of the organophosphate compounds is inhibition of acetylcholinesterase enzyme (AChE), which causes the accumulation of acetylcholine (ACh) in the body [4]. The inhibition of cholinesterase activity leads to accumulation of ACh at synapses, causing overstimulation and disruption of neurotransmission in both central and peripheral nervous systems [3]. Finally, acute organophosphate poisoning may result in serious life-threatening conditions, such as initial acute cholinergic crisis, and sometimes intermediate syndrome. For this reason, early recognition of such conditions is very important, especially to institute the appropriate treatment [5].

The mortality rate of organophosphate poisoning is high, fatal issue is often related to delay in diagnosis or improper management [3]. Acute treatment includes rapid administration of atropine, which blocks the muscarinic effects, and that of pralidoxime, which reactivates the AChE inhibited by the organosphosphate [6]. At present, the use of the traditional antidotes, atropine and oximes, has not significantly reduced the morbidity and mortality of organosphosphate poisonings, despite the great advances in patient monitoring and critical care medicine. The need to develop newer treatment regimes is urgent [4]. Interleukin-10 is a recently characterized cytoprotective agent, and it may be useful as an alternative or adjunctive therapy in organophosphate poisonings, but has been studied little in connection with organophosphate poisonings [7].

The aim of this experimental study was to investigate whether interleukin-10 could prevent or diminish pathological signs of tissue damages caused by organophosphate poisoning.

Materials and Methods

Chemicals.  Fenthion, an organophosphate compound (0,0-dimethyl 0–4-methylhio-m-tolyl phosphorothioate, Lebaycid®, Bayer CropScience, East Hawthorn, Victoria, Australia) and interleukin-10 (Recombinant Rat interleukin-10, Chemicon International, Temecula, CA, USA) were used for the study.

Test animals.  Female Wistar-Albino rats, weighing 160 to 280 g, were purchased from Hakan Cetinsaya Experimental and Clinical Research Center (Kayseri, Turkey), housed under standard bedding and allowed free access to standard food and water. These experiments were approved by the Institutional Animal Care and Use Committee of the University of Erciyes and were in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication, vol. 25, no. 28, 1996).

Experimental design.  Thirty rats were divided into three equal groups (n = 10). Group 1 (sham) did not receive any agent during the experiment. Group 2 (control) received 0.8 g/kg of fenthion intraperitoneally, followed by 6 ml/kg of intraperitoneal normal saline 30 min. and 3 hr later. Group 3 (treatment) received 0.8 g/kg of fenthion intraperitoneally, followed by 2 µg/kg of interleukin-10 intraperitoneally 30 min. and 3 hr later. All rats were killed under anaesthesia after 6 hr and tissue samples were obtained from the liver, kidneys and lungs.

Histopathological analysis.  All sections (5 µ) were examined with classical light microscopy techniques (with haematoxilin and eosin staining). The histopathological changes in the liver, kidney and lung after organophosphate poisoning and treatment were evaluated according to the criteria as defined in Fiorentino, Bond et al. [8] Fiorentino, Zlotnik et al. [9] and Gokcimen et al. [10] (tables 1–3).

Table 1. 
Histopathological evaluation of the liver [8].
GradeFindings
0No hepatocellular injury
1Minimal cellular change
2Only moderate degree central lobular injury
3Severe central lobular injury
4Moderate degree central lobular injury
5Severe central lobular and mid-zonal injury
6Total destruction of hepatocytes
Table 2. 
Histopathological evaluation of the kidney [9].
GradeFindings
0Normal
1Mitosis and necrosis in individual cells
2Necrosis in the cells near the proximal tubules, living cells around
3Restricted necrosis in the distal one-third of proximal tubules and bend shaped necrosis progressing to inner cortex
4Necrosis affected all of the three segments of proximal tubules
Table 3. 
Histopathological evaluation of the lung [10].
GradeFindings
1Alveolar congestion
2Haemorrhage
3Infiltration or aggregation of the neutrophils to vessel walls or air spaces
4Thickening of alveolar wall/hyaline membrane formation

Statistical analysis.  Histopathological results were compared to Kruskal–Wallis test. Significances of intergroup differences were determined by using Mann–Whitney U-test.

Results

Liver biopsy results.

Minimal and sometimes in patches, cellular damage was detected in the control and treatment groups being more obvious in the control group. The degree of damage in the control group was significantly higher than in the sham group (P < 0.05). There was no statistically significant difference between sham and treatment groups with respect to damage (P > 0.05). When we compared the control and treatment groups, we detected that the damage was significantly decreased in the treatment group (P < 0.05) (table 4, fig. 1).

Table 4. 
Mean (range) histopathological scores (see table 1; n = 10 in each group).
 ShamControlTreatedχ2P
  • *

    Statistically significant difference when compared to Group 1.

  • Statistically significant difference when compared to Group 2.

  • Statistically significant difference when compared to Group 3.

Liver0 (0–1)  1 (1–2)*0 (0–1)14.94<0.05
Kidneys0 (0–1)  1 (1–2)*0 (0–1)14.06<0.05
Lungs0 (0–0)2.5 (2–3)*1 (0–2)*24.20<0.05
Figure 1.

(A) Microscopic appearance of liver tissue from a rat in the sham group: Grade 0 normal liver tissue (haematoxylin and eosin staining, magnification ×400). (B) Microscopic appearance of liver tissue from a rat in the control group: Grade 2 moderate degree central lobular injury (haematoxylin and eosin staining, magnification ×400). (C) Microscopic appearance of liver tissue from a rat in the treated group: Grade 1 minimal cellular change (haematoxylin and eosin staining, magnification ×400).

Kidney biopsy results.

Minimal cellular changes and necrosis in patches near the proximal tubules were detected in the control and treatment groups being more obvious in the control group. Damage in the control group was significantly more severe than in the sham group (P < 0.05). There was no statistically significant difference between the sham and treatment groups with respect to damage (P > 0.05). When we compared the control and treatment groups, we detected that the damage was significantly reduced in the treatment group (P < 0.05) (table 4, fig. 2).

Figure 2.

(A) Microscopic appearance of kidney tissue from a rat in the sham group: Grade 0 normal renal tissue (haematoxylin and eosin staining, magnification ×400). (B) Microscopic appearance of kidney tissue from a rat in the control group: Grade 2 necrosis in the cells near the proximal tubules (haematoxylin and eosin staining, magnification ×400). (C) Microscopic appearance of kidney tissue from a rat in the treated group Grade 1 necrosis in individual cells (haematoxylin and eosin staining, magnification ×400).

Lung biopsy results.

Alveolar congestion, haemorrhage, neutrophil infiltration or aggregations in the vascular walls or air regions were detected in the control and treatment groups, more obvious in the control group. The damage was significantly increased in the control group when compared to the sham group (P < 0.05). There was a statistically significant difference between the sham and the treatment group with respect to damage (P < 0.05). When we compared the control and treatment groups, the damage was significantly reduced in the treatment group (P < 0.05) (table 4, fig. 3).

Figure 3.

(A) Microscopic appearance of lung tissue from a rat in the sham group: Grade 0 normal lung tissue (haematoxylin and eosin staining, magnification ×400). (B) Microscopic appearance of lung tissue from a rat in the control group: Grade 3 neutrophil infiltration to walls of air spaces (haematoxylin and eosin ×400). (C) Microscopic appearance of lung tissue from a rat in the treated group: Grade 2 haemorrhage in the lung tissues (haematoxylin and eosin ×400).

Discussion

Organophosphate poisoning causes three well-recognized clinical states: acute cholinergic syndrome, intermediate syndrome and delayed polyneuropathy. The cholinergic and intermediate syndromes are due to accumulation of ACh and changes at the neuromuscular junction, and carry a high risk of death in the absence of adequate treatment [4]. Time of death following a single exposure varies from 5 min. up to 24 hr or more, depending on agent, dose and duration of exposure [11].

The toxicity of organophosphates causes adverse effects on many organs. The systems that could be affected by an organophosphate intoxicant are the immune system, urinary system, reproductive system, pancreas, liver, lungs, due to haematological and biochemical changes [12–14]. Organophosphate compounds not only affect AChE but also may alter the liver, kidney and endocrine glands functions. [15,16].

Some studies reported that organophosphates cause liver damage [15,17,18]. Mikhail et al. [19] have demonstrated liver necrosis of mid-zonal type and fatty change at the periphery in animals treated with organophosphate compounds. Gokcimen et al. [18] observed that 200 mg/kg diazinon (an organophosphate compound) resulted in excessive hepatic necrosis. In our study, we found a similar result, namely mid-zonal type liver damage. The pathological changes can be explained with the fact that organophosphate compounds have deleterious effects on the cytochrome P450 system of hepatocytes, or it may also cause some changes in the membrane transport system of mitochondrions in hepatocytes [18].

Acute nephropathy after organophosphate poisoning appears to result mainly from proximal renal tubular damage with a benign and reversible clinical course [20]. Similarly, we found proximal renal tubular damage in our study. The exact aetiology of this is unclear, but in the light of recent literature it is likely that oxidative stress at the renal tubular level leading to renal tubular damage may be the explanation [21]. Another study suggested rhabdomyolysis as a well-known complication of severe poisonings appearing to be relatively frequent in severe organophosphate pesticide poisoning, including diazinon. In the acute phase, this may cause acute renal failure and in later stages paresis if not treated correctly [22].

Death from organophosphate poisoning is commonly due to respiratory paralysis. Organophosphate compounds affect the respiratory system by peripheral muscarinic actions on the airways, nicotinic actions on the muscles of respiration, effects on the medullary center in the brain and direct toxic effects on the alveolar-capillary membrane. Respiratory effects of high-dose organophosphate exposure include broncho-constriction, pulmonary oedema and respiratory muscle paralysis [13,23]. Morphologic changes in the tracheal epithelium, hyperplasia, thickening of the alveolar-capillary membrane, degeneration in alveoli, and alveolar ducts have been seen on histopathological investigation [13,24]. In our study, we observed similar findings such as alveolar congestion, haemorrhage, neutrophil infiltration or aggregations in the vascular walls or air spaces.

Treatment of organophosphate poisoning consists of general supportive measures, decontamination, intensive respiratory support and prevention of absorption. Specific therapy includes the administration of antidotes and is based on the degree of toxicity [25]. Treatment modalities aiming at preventing or diminishing tissue damage have not yet been reported.

Interleukin-10 was discovered in 1989 and was originally named ‘cytokine synthesis inhibitory factor’. It is produced by Th2 cells, macrophages/monocytes and B cells [26]. It is a recently characterized cytoprotective agent, a potent anti-inflammatory mediator that inhibits the production of other cytokines from activated macrophages [27–29]. Interleukin-10 reduces activation of macrophages and inhibits the production of reactive oxygen species [30] and pro-inflammatory cytokines [27].

In one study, exogenous interleukin-10 was used to prevent or decrease the organ damages caused by organophosphate poisoning. In this study, Ikizceli et al. [7] showed a decrease in pancreas damage histopathologically when using interleukin-10 for the pancreas damage caused by fenthion. In our study, we also notice that liver, kidney and lung damages at the histopathological level caused by fenthion could be reduced by interleukin-10 administration.

The results of the current investigation suggest that poisoning due to organophosphate compounds affects liver, kidneys and lungs. This condition may be related to the effects of ACh, direct effect of organophosphate compounds and oxidative stress caused by poisoning. However, interleukin-10 prevents histological damage to the liver, kidneys and lungs after organophosphate poisoning. More advanced studies are needed to clarify the relationship between organophosphate poisoning and interleukin-10 treatment.

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