EVALUATION OF HEPATOPROTECTIVE EFFECT OF PROPOLIS AGAINST SUB-CHRONIC DIAZINON INDUCED HEPATOTOXICITY

Nowadays, the puplic health faces a major proplem of increased pesticides use with appearing of various side effects. One of the most commonly used is diazinon. It exerts its effects on variable tissues and cells including hepatocytes. This work aimed to study the tendency of diazinon to cause hepatic affection in rats and study the possible propolis protective role. We used 30 rats, divided into 4 groups; group Ia (-ve control); group Ib (+ve control, corn oil group); group II (propolis group; 400mg /kg/day orally); group III (diazinon 20 mg/kg/day orally); group IV (diazinon 20mg/kg/day + propolis 400mg /kg/day orally) for 8 weeks. We studied biochemical parameters: serum aminotransferases (ALT and AST), tissue oxidative stress markers (hepatic MDA, hepatic catalase and hepatic GPx activities) . Liver will be examined by light microscope to evaluate histopathological changes and immunohistochemical reaction to caspase 3 to predict apoptosis. Results indicated: Significant increase in the serum levels of the ALT and AST as well as hepatic MDA of diazinon treated rats compared to control group, whereas both hepatic catalase and GPx activities were significantly decreased these results had significantly improved in both propolis and diazinon group. Light microscopic examination revealed disruption in hepatic histoarchitecture in diazinon group unlike that in group III which returned near normal; while immunohistochemical staining showed many positive reacted cells to active caspase 3 in diazinon group which became few in group III near to control. In conclusion, this study demonstrated that diazinon may have hepatotoxic effects which can be protected by co-administration of propolis in adult rats. Further studies about diazinon effect on liver and about propolis role in hepatoprotection are recommended.


INTRODUCTION
Environmental pollution regarding pesticide residues is a main concern as a result of their wide agricultural use.Organophosphorus insecticides (OPs) are one of the main classes of pesticides commonly used.Galloway and Handy, 2003 had revealed residuals of organophosphates in food stufs as fruits, vegetables, grains and others as well as water and soil.
Diazinon (DZN) is one of the excessevily utilized organophosphates agriculturally.Diazinon affects various organs ; such as liver, hematologic system, immune system, nervous system and kidney through cellular imbalance between over production of free radical and depleted tissue antioxidant mechanism (Galloway andHandy, 2003 andYilmaz et al., 2012).Previous studies had reported that DZN might cause hepatotoxicity (Shadnia et al., 2007).One of the potential mechanisms of DZN induced hepatic affection was the overproduction of reactive oxygen species (ROS) (Teimouri et al., 2006).
Priyadarsini and Nagini, 2012 defined oxidative stress as imbalance between the compensatory reaction of endogenous anti-oxidants with increased production of reactive oxygen species.
Bee glue, Propolis, had been much familiar in the world for human health in traditional medicine and food supplementation (Pereira et al., 2008).It was believed that it could cure diabetes, heart diseases and even cancer.Several biological properties of propolis including antioxidant, antibiotic, anti-inflammatory and tumor cell arrest had also been reported (Moreno et al., 2000).Kurek-G´orecka et al., 2014 andTapas et al., 2008 explained that the principal antioxidant mechanism of propolis polyphenols can be summarized in the significant ability of "scavenger" reactive oxygen species and radical reactive nitrogen species so that reduction of xanthine oxidase reaction; chelate ion metals implicated in the sequence of free radical production , interrupt the reactions cascade of the lipids peroxidation and synergistic action added to different antioxidants.
So this work aimed to study the tendency of diazinon to cause hepatic affection in rats and study the possible propolis protective role [B] Animals:

MATERIAL& METHODS
The study was done on 30 healthy adult male albino rats weighing 150-200g.Rats were got from the animal house of Zagazig Faculty of Medicine.We gave the rats balanced food consisting of milk, barley and bread, this food should be rich in all substances necessary to preserve their health before and during administration of the treatment.We gave them water in a different clean utunsiles.The experimental work was done according to the basic instructions advised through the Institutional Research Board for the use and care of experimental animals.
[C]Experimental Design: Rats were randomly divided into four groups: Group I a (negative control group): No intervention was done to rats in this group for adjusting the basic parameters, and allowed drinking water ad libitum.Group I b (positive control group): Rats received 1ml corn oil orally for 8 weeks.Group II (Propolis group): Rats received propolis (400mg/kg/day) dissolved in corn oil orally for 8 weeks (Rahvar et al., 2018).Group III (diazinon treated group): The group was Orally received diazinon dissolved in corn oil.A daily dose of 20 mg/kg body weight which equals 1/20 of DZN LD50 (Gokcimen et al., 2007).Group IV (diazinon and Propolis): The group was administered diazinon (20 mg/kg) and 400mg /kg Propolis orally once daily for 8 weeks.
* Methods: Twenty-four hours after the end of experimental duration, the rats were subjected to sampling of blood and liver tissue as the follows: -Collection of blood samples; Under light ether anesthesia, venous samples from the retro-orbital plexus were obtained by capillary glass tubes as described by Schemere (1967)

Biochemical results:
The results of this study had been appropriately summarized, tabulated and subjected to statistical analysis before completion and graphic illustration.ANOVA test was used in this study to compare between means of different groups.
* As regard the groups; group Ia (ve control), group Ib (+ve control) & group II (Propolis): There were a non significant differences (p > 0.05) between groups all over the the study period using test of ANOVA (Table 1) as regard: 3-MDA in hepatic tissues.So the negative control group was selected to be compared with the results of the treated groups; group III (diazinon ) and group IV (diazinon + Propolis).

DISCUSSION
Agricultural extensive use of pesticides and its resulting residuals is considered a universal bother.One of the main classes of pesticides used is organophosphorus compounds which were detected in food, soil and drinking water as residuals (Costa, 2006).
Diazinon is considerd one of the globally used organophosphates.Impact of low levels chronic exposure to DZN on human health was reported in only few studies.Hepatotoxicity had been reported to be induced by DZN In agreement with these results

Sarhan and Al-Sahhaf
(2011) reported that, AST and ALT showed a significant increase in serum of rabbits that received diazinon 20 mg/kg for 4 weeks.
In the existing study, we estimated hepatotoxicity resulting from chronic DZN exposure in rat and its possible mechanism as lipid peroxidation and apoptosis and the potential effect of propolis in its protection.
The present study also showed a significant rise in hepatic MDA in DZN treated group when correlated with control group while a significant decrease in its mean value was observed after administration of propolis with DZN when compared with the corresponding results in DZN administered group.
Also, the present study reported a significant decrease in hepatic GPx and catalase activities with significant increase in MDA in DZN treated group when compared with control group.On the other hand, the above mentioned biochemical results obtained in the present study had been supported by findings in light microscopic histopathologic examination and immunohistochemical staining of liver sections.
The histological results showed that DZN induced several histopathological alterations in the liver.Many hepatic lobules showed dilatation of the sinusoidal spaces, inflammatory cellular infiltration, congested veins, shrunken hepatocytes with darkly stained nuclei.
These findings were in agree with Sarhan and Al-Sahhaf (2011) who reported leucocytic infiltrations, congestion of veins, cytoplasmic vacuolation of the hepatocytes and fatty degeneration in liver samples of rats received DZN.
In the present work, adding propolis with DZN to rats showed improvement in the form of preserved normal hepatic lobular architecture and uncongested veins.Less dilated sinusoidal spaces and few cellular infiltration were observed.
Also, we studied the activated form of caspases-3.Our study showed that DZN induced apoptosis in hepatocytes of the rats treated with DZN through activation of caspases -3.
Caspases are deemed to be the key moderator of apoptosis.Caspase-3 is known to be the a final main implementer of apoptosis.It is responsible for the splitting of the key cellular proteins, leading to the known morphological modulations observed in apoptotic cells

CONCLUSION
Taking the previous findings together, it can be concluded that hepatic apoptosis through oxidative stress pathway can be induced by sub-chronic DZN exposure which can be alleviated by propolis coadministration.

RECOMMENDATIONS
Further studies about diazinon effect on liver and about propolis role in hepatoprotection are recommended.

Figure ( 1
Figure (1): A photomicrograph of a section from the liver of a control group showing hepatocytes (H) arranged in plates radiating from the central vein (V) and separated by blood sinusoids (S); hepatocytes are polygonal in shape, with central rounded vesicular nuclei and acidophilic cytoplasm.(H&E X 400)

Figure ( 4 )
Figure (4): photomicrograph of a section from the liver of a control group showing few positive reaction for activated caspase 3 in few hepatocytes (arrow) (Caspase 3 immunostaining X 400).

Figure ( 5
Figure (5): A photomicrograph of liver from diazinon treated group showing many positive reacted cells for activated caspase 3 (arrows) in cytoplasm of hepatocytes (H) (Caspase 3 immunostaining X 400).

Figure ( 6 )
Figure (6): photomicrograph of a section from the liver of diazinon and propolis group showing less positive reaction for activated caspase 3 in few hepatocytes (arrows) (Caspase 3 immunostaining X 400).

Table 1 :
Statistical comparison among groups (Ia & Ib & II) as regard serum ALT&AST, hepatic catalase, GPx activity and hepatic MDA along the period of the study by ANOVA test: N.B: Number of rats = 6 rats for each group.SD = Standard Deviation.p>0.05 = non significant * As regard the treated groups; group III (diazinon) & group IV (diazinon +

Table ( 2
): Statistical comparison among the negative control (Ia), diazinon (III) and diazinon and propolis (IV) groups as regard ALT & AST, hepatic GPx, and catalase enzymatic activity and MDA levels in hepatic tissues by ANOVA test.

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