Exposure to chemicals including agro-pesticides and various environmental toxicants, has been reported to cause damage of cellular macro-molecules such as lipids, proteins and DNA through induce of oxidative stress, which result from an imbalance between radical generating and radical scavenging system of cell, via enhancement production of reactive oxygen species (ROS) [Yang et al ., 2018 and Verma et al .,2007].
Conazoles are azole antifungal agents used in agricultural and pharmaceutical .products (Zarn et al ,2003).The fungicidal properties of conazole are due to their abilities to inhibit ergosterol biosynthesis which is an essential component of fungal membranes, consequently its inhibition lead to cell death [Vanden Bossche et al .,1989, Ronis et al., 1994 and Debeljak et al .,2003].
In mammalian systems, conazole modulate many CYP enzymes involved in the metabolism of xenobiotics sterol, steroids and other xenobiotics (Osman et al., 2011).
Recently, considerable attention has been focused on medicinal plants, which play a vital role for medication and to determine the active principles responsible for protecting or preventing the development of oxidative stress, which is an important cause of many chronic diseases.
Therefore, the present study carried to evaluate the adverse effects of difenoconazole fungicide and attempt of the possible anti-hepatotoxic of Cinnamon cassia against difenoconazole induced hepatotoxicity in male rats.
Phytochemical Screening of Cinnamon cassia.
The phytochemical investigation of Cinnamon cassia revealed the presence of total phenolic, flavonoids and tannis as shown in Table (1).
Cinnamon is known to have contain of phenolic compound and flavonoids which act as potent antioxidants due to their ability to scavenge free radical and form relatively inert phenoxy radical intermediates [Ferguson, 2001].
The phenolic compounds ,such as flavonoids ,phenolic acid and tannins posses divers biological activities , and they are responsible for antioxidant action as well as a potent scavenger of hydrogen peroxide( H2O2),nitric oxide (NO)and lipid peroxide free radicals [Aravid et al .,2012].
In addition ,the antioxidant activities of phenolic compounds is mainly due to their redox properties such as adsorbing and neutralizing free radicals as well as quenching single and triple oxygen or decomposing peroxides [Yashin et al .,2017].
With regard to flavonoids , they are typical phenolic compounds, which in turn ,act as potent metal-chelators and free radical scavengers.
Generally, they have higher antioxidant activity against peroxy radical than phenolic acid due to multiple hydroxyl group [Yashin et al .,2017].
Our results in agreement with the previous findings reported by [Pogngothan ,2011 and Benmehdi et al .,2012] who found that Cinnamon composed of many bioactive compounds such as polyphenols and flovanoids.
Changes in Body Weight and Liver Index.
In general, an increase or decrease in the body weight of an animal has been used as indicator of an adverse effect of drugs and chemical (Teo et al., 2002).
Our results demonstrated that oral administration of difenoconazole slightly but significant increased in the final body weight of rats.
Meanwhile, the body weight of rats received only the aqueous extract of cinnamon (AEC) did not show any significant change when compared with corresponding group of control. This is indicative of absence of general toxicity. The increase in body weight of difenoconazole-treated rats could be attributed to the ability of difenoconazole to stimulate the accumulation of triacylglycerol in adiapocyte [Masuro et al .,2005 and Wada et al.,2007] and so .the organs involved in metabolic syhlrom [Met-S][Marmugi et al .,2012] or due to hypothyrodism [Moriyama et al .,2002] .
There was a significant decrease in the levels of T4 and T3 in rats treated with difenoconazole for 28 days (data not shown).
In addition, difenoconazole could be induce accelerated differentiation of immature adipocytes into mature cells and may disrupt the lipid metabolism of difenoconazole-treatment rats [EFSAb].
The results of our study agree with the results of work done by Heba Hamdi et al (2019) who found that treated rats with epoxiconazole at doses of 8, 24, 40 and 56 mg/kg B.W ,induced a significant increase in the body weight after 28 days of treatment.
On the other hand, Allen et al (2006) and Wolf et al (2006) reported that propiconazole and triademofen treatment produced a significant decrease in the body weight at the end of experiment (90 days).
In contrast, supplemented rats AEC alone within the experimental period, did not induce any significant change in the body weight.
However, pre-treatment of AEC induced insignificant decrease in the body weight of rats treated with low dose of difenoconazole , this may be due to the ability of AEC to decrease fat mass percent [Ziegenfuss et al .,2006].
Lopes et al (2015) who found that oral administration of Cinnamon extract (400mg/kg BW) to healthy rats led to a lower body mass.
On the other hand, Kim et al (2006) demonstrated that treatment rats with Cinnamon extract at 200mg /kg BW did not induce any significant change in the final body weight , whereas this extract caused a significant decrease in the liver index of diabetic mice after12 weeks of treatment.
Concerning the liver weights when expressed as absolute or relative to body weight ,there was a significantly increase liver weight (hepatomegaly) in rats following expouse to difenoconazole. This could be explain by the induction of several xenobiotics metabolizing enzyme (FFSAb, 2008).
A hepatomegaly often accompany with increase in hepatic –derived enzymes (i.e ., Transaminase ,ALP and GGT),that may appear in the plasma following liver enlargement
Soliman et al. (2012) noticed that AEC did not induce any anti-obesty effects although noticeable decrease in liver fat and glycogen deposition was recorded.
our finding were consistent with previous studies have evaluated the toxic effect of some trizoles compounds like propicanozole ,myclobutanil ,triademofen ,epoxicanazole and bromucanazole ,which in turn, induced a significant increase in the liver weight in rodent animals .[Allen et al ., 2006, Martin et al .,2007 , Nesnow et al ., 2011 and Heba Hamdi et al .,2019].
Meanwhile, the absolute and relative weight of liver rats supplemented only with AEC comparable to control and no significant differences were found in their values .
However, it was observed that co-administration of AEC with difenoconazole, attenuated the increase weight of the liver following treatment with difenoconazole and consequently caused recovery towards normalization comparable to the control group. This indicated that AEC could exert in vivo hepatoprotective effect.
Previous study have shown that cinnamon zeylanicum treatment reduced the relative weight of the liver [Shan et al .,1998].
Changes in Serum hepato –specific markers.
Assessment of liver injury by difenoconazole is usually made by determination of serum enzymes level of ALT, AST, ALP and GGT, which are important markers to evaluate hepato-billary injuries.
In the current ,an increase significantly in the activities of serum ALT and AST was observed following treatment with difenoconazole, which have been attributed to leakage of cellular enzymes in circulation as a result of loss function integrity of cell membrane of hypatocytes [ Navaro et al .,1993 and Ozer et al .,2008]
A number of chemical including agro-pesticides and various environmental pollutanta can severe cellular damage in different organs in the body .This occurred through metabolic activation to highly reactive substances such as free radicals (Satpute et al., 2017).
In addition, there was also a significant elevation in the activities of ALP and GGT in difenoconazole treated rats, this induction of ALP, may be a attributed to presence of increasing of billiary pressure rather than increased release from damage cells .and this elevation may be occurred during liver regeneration [Moss and butterworth , 1974]. However, enhancement of ALP activity may be due to elevation of bile acids concentration during cholestasis is apparently necessary for the release and transport of solubilized hepatic ALP to serum.
Also, an induction in the activity of GGT was detected in rats treated with difenoconazole within the experimental period.
ALP and GGT activities are important biomarkers to evaluate hepatobilliary injuries or bile duct lesion in the rat liver, where ALP is primary marker of hepato-biliary effects and cholestasis (moderate to marked elevation (Ramaiah , 2007)
GGT is a cell surface enzyme that cleaves Y-glutamyl bonds (Godwin et al 1992).It is localized to the luminal surface of ducts and glands through out the body (Hanigan . 2014).
However, GGT is abiliary enzyme that is especially useful in the diagnosis of intra-hepatic cholestasis and obstructive Jaundice (Stein et al ., 1989).
In addition, GGT is more responsive to biliary obstruction than alkaline phosphatase , where GGT has no origin in bone or placenta ,and hence, GGT is not increased in bone disorders as is ALP .
In rats, GGT activity is considered a reliable marker for cholestasis compared to alkaline phosphatase activity (Ozer et al ., 2008). In addition, the rat GGT assay detects bile duct hyperplasia and necrosis [Leonard et al ., 1984].
The biochemical markers in the liver viz., ALT, AST and ALP were significantly elevated in rats treated with expoxicanazole at doses 8, 24, 40, 56 mg/kg BW for 28 days [Heba Hamdi et al .,2019].
Also, there are findings were reported previously by Abd-El Rhaman et al ., 2014 showed that treatment rats with difenoconazole at dose of 10mg /kg BW (representing of LOEL dose) caused a significant increase in ALT activity and total protein level, whereas a significant decrease in the activity of GGT and insignificant change in the AST activity were observed after 28 days of treatment.
In contrast ,rats supplemented with AEC at selected doses of 200 and 400 mg /kg B.W did not induce any significant change in serum liver markers (I.e .ALT,AST ,ALP and GGT),in addition to serum protein pattern and A/G ratio.
It is worth mentioning that C.cassia was able to ameliorate all the altered hepatic biomarkers post-treatment with difenoconazole ,this suggest that the vital role of Cinnamon extract in protecting the integrity and the function of cells and tissue .
Co-administration cinnamon with difenoconozole showed significant decrement in the activities of ALT ,AST,ALP and GGT compared to difenoconazole-treated rats , this indicate that the extract may reduce hepatocellular damage.
Co-administration of cinnamon extract in doses of 200 and 400mg/kg B.W, attenuated the increased levels of serum enzymes[i.e., ALT,AST and ALP] produced by difenoconazole and caused subsequent recovery towards normalization comparable to the control group .this in turn ,indicate that AEC could reduce liver injury –induced by difenoconazole . Hence, cinnamon extract its hepatoprotective effects as a result of presence of wide range of bioactive compounds including phenolics.
Cinnamon extract at doses of 200 and 400mg/kg B.W when orally given to obese diabetic rats ,significantly lowered the high serum levels of ALT,AST and ALP enzymes ,when compared to the positive control groups [Shalaby and Saifan ,2014].
The hepatoprotective effect of Cinnamon extract was evident from the significant decrease in serum activities of liver enzymes (ALT,AST&ALP)in obese diabetic rats[Shatwan et al .,2013].
Moselhy and Junbi (2010) observed that the elevated serum AST and ALT activities induced by CCL4, were restored towards normalization significantly by orally administration of 200mg/kg cinnamaldehyde (CNN) once a daily for 7 days when compared to control rats.
Our biochemical finding were confirmed with histopathological findings of rat liver sections in the current study.
An elevation in the serum total protein concentration and globulin levels was observed in rat treated with high dosage level of difenoconazole, whereas no significant differences in the concentration of albumin.
In addition, the albumin /globulin ratio (A/G ratio) was significantly decreased in the groups of rats treated with difenoconazole .This could be attributed to increase of serum globulin level as the result of the immune response of toxic effect.
Meanwhile, in the present study co-administration of AEC lowered the levels of TP and globulin and their values were found comparable to control values.
Changes in the markers of oxidative stress and antioxidant status .
It is well-established that the liver is a target organ for xenobiotics substances and it is play crucial role in the detoxification process. Therefore, any injury or impairment of its function produces hepatotoxicity on living organism.
Oxygen free radical (superoxide, O2.) and hydroxyl radical (OH.)and hydrogen peroxide (H2O2) called reactive oxygen species, which play a significant role in oxidative stress and they are also capable presenting a toxic action on self tissues causing lipid peroxidation [Sharmanov et al .,1990 and Baxter et al .,1983].
The elevation of oxidative stress can be monitored by several markers .The monlondialdehy (MDA) measure is considered as an indicator of lipid peroxidation (LPO) [De-Zwart et al ,. 1990].
Difinoconazole-treated induced lipid peroxidation in hepatic tissue, which observed by marked elevation in the level of MDA, this suggest that it mediated free radical induced lipid peroxidation in hepatocytes was strongly associated with tissue injuries.
In the present study, enhancement of MDA level in liver homogenate, suggest enhanced LPO leading to increase the preambility of the cell membrane of hepatocytes, consequently release of transaminases (ALT, AST) into circulation as shown in our result.
Therefore, the remarkable elevation of ALT and MDA in the current study supported that difenoconazole mediated lipid peroxidation in liver rat as a result of reducing the antioxidant potential and hence accelerating the oxidative damage of hepatocyte.
The MDA is by-product of lipid peroxidation ,while the glutathione (GSH)content could be used to evaluate the antioxidant status of cellular system[Dall-Donn et al .,2007,Circu and Aw,2008 and Pallarda et al ., 2009].
The glutathione (GSH) is non-enzymatic antioxidant and it is one of the most abundant tri-peptide and it present in the liver and its essential function are mainly concerned with maintain structure and function integrity of cell via removal of free radical. Therefore, it playa vital role in production against oxidative stress [Halliwell and Gutterdge 1990].
Our results revealed that difenoconazole treatment induce a significant deplection of GSH content in hepatic tissue and production of antioxidant enzymes viz , SOD and CAT.
In contrast, there was a significant elevation in the activity of hepatic GGT in difenoconazole –treated rats.
Induction of GGT expression it is considered as an adaptive response or as part of natural hepatocyte protective mechanism for GSH turnover (repletion) in hepatic tissue .The body's antioxidant defeneces undergo consumptive depletion following oxidative injury. Therefore, low antioxidant defense status are also correlated with elevated GGT, particularly reduced levels of GSH. However, GGT is needed to metabolize glutathionylated xenobiotics in liver and hence its elevation association with increased exposure to xenobiotics [Koening and Seneff ,2015].
Previous studies have shown that usually the deleterious effects of oxidative stress are counteracted by endogenous antioxidative enzymes, which provide a major defensive mechanism against free radical damage .The most important antioxidative enzymes i.e ., SOD and CAT, when ROS begin to accumulate in the cells, exhibits defensive mechanism using various antioxidant enzymes .The main detoxifying system for peroxides are CAT and GSH
However, superoxide dismutase (SOD), a common enzyme play an important protective role by catalyzing the removal of superoxide radical (O2.), and converted to hydrogen peroxide (H2O2) and hence (H2O2) is degraded by catalase (CAT) which catalyzes the reaction between two hydrogen peroxide molecules (H2O2) and this reaction results in water and O2 production and prevent form a highly reactive OH in presence of Iron as catalyst [Turner and Lysiad ,2008].
Previous reports have shown that treatment animals with propiconazole or bromucanazole induced a significant elevation in the level of MDA and depletion of GSH level in liver rat .Meanwhile, there was a decline in the activities of SOD and CAT following treatment rats with bromuconazole for 3 months [Nesnow et al .,2011 and Osmen et al .,2011].
Also, Abd-Alraheman et al. (2014) reported that treatment rats with difenoconazole at dose of 10mg/kg BW caused a significant elevation in the level of serum MDA and SOD activity , whereas a significant decline in the level of SH protein was detected after 28 days of treatment .
Our findings reveled that SOD activity was decreased in the hepatic tissue .The reason of the decreased SOD could be attributed to excessive ROS generation[Scott et al ., 2008].
Also this decrease could be due to a feedback inhibition or oxidative inactivation of enzyme protein due to excess ROS formation .
However, the reduction of the activity of CAT reflect inability of hepatic tissue to eliminate H2O2 produced the activity of CYP and inactivation of this enzyme as a result of excess of ROS production and /or suppressing of heme biosynthes [Pigeolet et al ., 1990].
Recently, considerable attention has been focused on some medicinal plants like cinnamon which are posses diverse biological activities.
The total phenolic might be the main bio-active compounds which, a potent scavenger of hydrogen peroxide( H2O2),nitric oxide (NO)and lipid peroxide [Aravind et al .,2012].The antioxidant activities of phenolic compounds is due to their redox properties.
In addition, the phenolic hydroxyl groups is shown to denote electron to oxygen radicals and also reduce ferrious ion to ferric ion, thereby suppressing the oxidation [Morel et al .,1993].
However ,it is found that cinnamon contain proteins ,carbohydrates .vitamins (A,C,K and B3 ) and minerals like Calcium ,Iron ,Manganes (Mn+2), Magnesim (Mg+2) Phophorus and Zink (Zn+2)[Vanalapati et al .,2012].
The polyphenolic polymers found in Cinnamon and they have antioxidants activity and reduce oxidative stress through inhibition of 5-lipoxygenase enzyme [Anderson et al .,2004].
The hepatoprotective potential of Cinnamon cassia against the oxidative stress induced by difinoconazole was evaluated in this study.
Apparently, the intracellular defenses based on glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) are insufficient to inhibit these pro-oxidant reaction, but the nature antioxidant supplementation impart protection against oxidative stress, via increased the intracellular antioxidant defenses,which can be overwhelmed .
In the current study, remarkable elevation of SOD and CAT activities was observed following co- administration of AEC with difenoconazole.
It is probable that initial increase in hepatic SOD and CAT level was occurred as adapative nature of the defense system against damage effect of superoxide radicals (O2.) in liver brought about by difenoconazole treatment.
Since, polyphenol effectively removes oxygen radicals, and diminished degradation or inactivation of antioxidants enzymes, partly explains increased SOD activity during phenol–rich intervention [Casten-miller et al ., 1999].
Moreever, Cinnamon oil (Co) exhibited SOD like activity of pyrogallol auto-oxidation this catalyzed by the superoxide radical [Kim et al .,1995].
Also, Murcia et al . (2004) reported that cinnamon extract exhibited protection effect against irradiation–induced LPO in liposome and quenched hydroxyl radical (OH.) and H2O2.
Furthermore, the protective action of Cinnamon extract against radiation induced oxidative stress and inflammatory damages was attributed to inhibiting effect on No production through inhibition of NF-Kb activation as a result of presence of phenolic and flavoniods compounds.[Lu et al .,2005 , Azab et al ., 2011 and Morgen et al .,2014].
Bej et al . (2018) found that treatment rats with Cinnamon for 4 weeks significantly increased to near normal levels of SOD, CAT and GPX in the pancreatic tissue of diabetic rats.
The major histopathlogical finding were observed in the liver of rats treated with high dose of difenoconazole as shown in Table (6) and Fig (1).
These findings might further confirm that hepatic injury induced by difenoconazole is associated with generation of free-oxygen radicals and the biochemical results supported these finding. The cell injury cause the release of cytokines, especially Tumor Necrosis factor-alpha [TNF-α] which generate ROS from the tissues, and consequently produce lipid peroxidation [Lachleitner et al .,2000].
The degeneration condition observed in liver of difenoconazole-treated rats correlated with the detected biochemical alteration wherein, an increase in the level of ALT, AST, ALP , GGT and LPO was noticed .
However, histopathological observation in AEC-supplemented rats confirmed with biochemical results showing the normal cellular architecture ,in particular low dose of AEC (200mg /kg B.W) this in turn ,substantiate the safety profile of the cinnamon extract at low dosage level.
In addition, our result revealed that rats received cinnamon extract prior to difenoconazole-treatment showed improvements in the histology of hypatocytes in comparison to difenoconazole-treated rats. This suggest that cinnamon has a role in counteracting the oxidative damage-induced by generation of free radicals via difenoconazole treatment.
Indeed, administration rats with low dose of AEC did not induce any degenerative effect, although this extract at high dose (400mg/kg B.W) showed degenerative change.
In such cases, low dose of AEC could not damage mitochondria and antioxidant compounds was not oxidized and could scavenge free radicals.
In contrast, high dose level of AEC could damage and permeablize and could react as pro-oxidant damaging rat hepatocytes. It seems that switch from anti-proxidant reactions occur at low antioxidant concentration in a very narrow rang.
Moreover, some phenolic components of essential oils are oxidized by contact with ROS ,consequently producing very reactive phenoxyl radical which add to ROS released by mitochondria and these type of radical reactions enhanced by the presence of cell trasition metal ions such as Fe+2 ,Cu+2,Zn+2and Mg+2 [Stadler et al .,1995 and Sakihama et al .,2002].
In addition, our results demonstrated that AEC has anti-fibrotic effect, as seen in Table (5) and Fig (1). This could be attributed to diminishing the oxidative stress, which occurred through difenoconazole-treatment. Thus, AEC supplementation lead to reduce inhibitors of metalo protinease [which are a group of zinc-dependant endopeptidases] in tissues ,consequently produce an increase in cellagenase level which degraded of fibrous tissue .
Lin et al .(2003) reported that Cinnamon cassia powder reduced significantly the expression of alpha-smooth muscle actin (α -SMA),which play an important role in fibrogensis and connective tissue growth factor (CTGF), Transforming growth factor beta-1 ,and tissue inhibitor of metallo Proteinse-1 ,which elevated by oxidative stress in Sprague-Dawly rats with acute liver injury –induced by dimethyl-nitrosamine.