HONEY ENHANCES THE HEPATIC MULTI-FUNCTIONAL DETOXIFICATION PROCESS

Heba Barakat * and Amira AbdEl-Rhman. Department ofBiochemistry and Nutrition, Women's College for Arts, Science and Education, Ain Shams University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 4 (9), 630-639 631 Liver is a major organ attacked by ROS. Parenchymal cells are primary cells subjected to oxidative stress induced injury in the liver. The mitochondrion, microsomes and peroxisomes in parenchymal cells can produce ROS, regulating on PPARα, which is mainly related to the liver fatty acid oxidation gene expression. Moreover, Kupffer cells, hepatic stellate cells and endothelial cells are potentially more exposed or sensitive to oxidative stress-related molecules (Sanchez-Valle et al., 2012).
A variety of cytokines like TNF-α can be produced in Kupffer cells induced by oxidative stress, which might increase inflammation and apoptosis. With regard to hepatic stellate cells, the proliferation and collagen synthesis of hepatic stellate cells is triggered by lipid peroxidation caused by oxidative stress (Cichoz-Lach and Michalak, 2014). When the ROS is excessive, the homeostasis will be disturbed, resulting in oxidative stress, which is not only triggers hepatic damage by inducing irretrievable alteration of lipids, proteins and DNA contents but also bymodulating pathwaysthatcontrolnormalbiologicalfunctions. Sincethesepathwaysregulategenestranscription, protein expression, cell apoptosis, and hepatic stellate cell activation. Moreover the oxidative stress is regarded as oneofthepathologicalmechanismsthatresultsininitiationandprogressionofvariousliverdiseases,suchaschronicviralhepat itis,alcoholicliverdiseasesandnon-alcoholicsteatohepatitis (Singal et al., 2011).
Honey is a natural product formed from nectar by honeybees. Its composition is rather depending on the floral source and other external factors, such as seasonal and environmental conditions and processing (Alvarez-Suarez et al., 2013). It consists of primarily sugars such as monosaccharides, disaccharides, oligosaccharides and polysaccharides. It contains enzymes such as glucose oxidase, diastase, invertase, catalase and peroxidase (Bogdanov et al., 2008). Honey also contains other bioactive constituents such as organic acids, trace elements, vitamins, amino acids, proteins (Erejuwaet al., 2012a) as well as a wide range of polyphenolic phytochemicals The main aim of this study was to investigate the potential of honey as an improvement factor on the hepaticdetoxification via its three phases.

Materials and methods:-
Materials:-Honey was purchased from Al-Safa for bees breeding and honey production, El-Menia, Egypt. Filled up to Egyptian Standard Specifications number 355/2005,4,5(2/1,3/1) Animals and treatment:-Thirty six adult male albino rats "Sprague Dawely" 186-210 g were kept in stainless steel cages in the wellventilated animal house of the Medical Research Center, Faculty of Medicine, Ain Shams University. The rats had been kept in the room for 1 week prior to the beginning of the experiment for acclimatization. They had access to 12h cycle of light/dark and provided with standard diet prepared by AIN (1993) and tap water ad libitum.
The animals were divided into the following three groups: Group 1, control was supplemented with plain water; group 2,was supplemented with honey 2.5g/Kg body weight and group 3, was supplemented with honey 5g/Kg body weight. Both honey and water were given orally using oral gavage for 30 days (Yao et al., 2011).

Sample collection:-
At the end of the experimental period, animals were sacrificed under ether anesthesia. Livers were excised, washed, dried and stored at -20 o C till analysis is done.
Hepatic biochemical assays:-Phase I enzyme activity assay:-The hepatic activity of cytochrome P450 2E1 (CYP2E1) was extracted by homogenizing the tissues in PBS on ice. The resulting suspensions were subjected to two freeze-thaw cycles to further break the cell membrane. After that; the homogenates were centrifuged for 5 min at 5000 x g for extracting the supernatants which were used in the 632 determination using the enzyme-linked immunosorbent assay kit (Cloud-Clone Corp., USA)according to themethod of Tonkiri et al. (2014).

Phase II enzyme activities assay:-
The hepatic activity of uridine-5 \ -diphosphate glucuronosyltransferase-1 (UGT) was extracted from liver tissue by homogenizing the tissues in PBS on ice. The resulting suspensions were subjected to two freeze-thaw cycles to further break the cell membrane. Then; the homogenates were centrifuged for 5 min at 5000 x g for extracting the supernatants which were used in the determination of the enzyme activity using the enzyme-linked immunosorbent assay kit (Cloud-Clone Corp., USA)according to themethod of Miyagi and Collier (2011).
The hepatic epoxide hydrolase-1 (EPH) was extracted from liver tissue by homogenization in PBS and stored overnight at -20 o C. After two freeze-thaw cycles were performed to break the cell membrane. The homogenates were centrifuged for 5 min at 5000 x g, 2-8 o C. The supernatants were used in the determination of the enzyme activity usingthe sandwich enzyme immunoassay technique byCusabio Kit, USA according to the method of Gill et al. (1982).
The hepatic glutathione-S-transferase (GST) was extracted from tissues by homogenizationin PBS pH 6.5.Then the suspensions were centrifuged at 10000g for 30 min at 4°C. The activity of the enzyme was assayed in the supernatant calorimetrically using Sigma Kits, USA according to the method of Pour et al. (2014).

Phase III enzyme activity assay:-
The hepatic activity of nuclear factor erythroid derived 2 like protein (Nrf2) was extracted from liver tissue by homogenization in PBS on ice. The resulting suspensions were subjected to two freeze-thaw cycles to further break the cell membrane. Then; the homogenates were centrifuged for 5 min at 5000 x g for extracting the supernatants which were used in the determination of the enzyme activity using the enzyme-linked immunosorbent assay kit (Cloud-Clone Corp., USA)according to themethod of Ebihara et al. (2016).

Statistical analysis:-
Results were expressed as mean ± Standard deviation (S.D.) of the mean. Differences among means were tested for statistical significance by one-way analysis of variance using SPSS package version 16. Statistical significance was considered when P <0.05

Results:-
The activity of hepatic CYP2E1activity represented in figure (1) showed that oral administration of 2.5g honey/Kg body weight honey solution resulted in a significant increase (P<0.05) (49.07± 1.88) in enzyme activity compared with the control group that record (43.92±2.00) of enzyme activity. While the group that administered 5g honey/Kg body weight honey solution showed a non-significant decrease (42.58±3.10) at P<0.05 compared to control.
Oral administration of honey solution at dose 2.5g honey/Kg body weight also showed a significant increase in the activity of phase II hepatic EPH by 106.02% compared with control group as listed in table (1), while dose of 5g honey/Kg body weight showed a non-significant decrease (P<0.05) in activity of enzyme as showed in figure (2) compared with control.
The activity of the two transferases enzymes of phase II detoxification(UGT and GST) are increased significantly (P<0.05) by the administration of each of the honey doses comparing with the control group as showed in table (1) and figures (3 and 4).Moreover; administration of the 5 g honey/Kg body weight exerted more potent increase in the activity as showed by its higher % of change from control than the other dose (table 1).      There is no significant difference between means have the same letter in the same row.

Discussion:-
Honey is claimed to be of therapeutic benefits in the management of chronic diseases commonly associated with oxidative stress even if it is administered alone or in combination with conventional therapy (Erejuwa et al., 2012a). The action of honey has been related to the agents' abilities to enhance the activities of the drug metabolizing enzymes (DMEs) (Alvarez-Suarez et al., 2013).
The DMEs play central roles in the metabolism, elimination and/or detoxification of xenobiotic or exogenous compounds introduced into the body. In general; DMEs protect the body against the potential harmful exposure to xenobiotic from the environment as well as certain endobiotics. In order to minimize the potential injury caused by these compounds,most of the tissues and organs are well equipped with DMEs including phase I, phase II metabolizing enzymes as well as phase III transporters which are present in abundance either at the basal uninduced level, and/or inducible at elevated level after xenobiotic exposure (Rushmore andKong, 2002 andLeCluyse, 2003). In the present study honey enhanced hepatic CYP2E1 activity which is one of the microsomal cytochrome P450 (CYP450) enzymes superfamily, indicating that honey has an effect for initiating the first phase in detoxification. CYP2E1 which represents 6.6% of total CYP450 in human liver (Vermeulen, 1996), plays crucial roles in hepatic metabolism and elimination of xenobiotic and drugs (Rannug et al., 1995, Lewis, 2003and Pascussi et al., 2003. CYP2E1is induced by ethanol, carbon tetrachloride, dimethylnitrosamine, acetone and isoniazid and some drugs as chlorzoxazone,enflurane and halothane (Liska, 1998). In a typical phase I reaction, a CYP450 enzyme uses oxygen and, as a cofactor, NADH, to add a reactive group, such as hydroxyl radical. As a consequence of this step in detoxification, reactive molecules which may be more toxic than the parent molecules are produced, which must be metabolized by phase II conjugation to avoid damage to proteins, RNA and DNA within the cell (Garg et al., 2008).
Interestingly, data in the present study demonstrated that honey solution administration-mediated enhancement in the activities of phase II enzymes;EPH, UGT and GST indicating the beneficial and powerful detoxifying role of honey in liver via eliminating the toxic effect of many substrates either from its original state or which produced from CYP450 reactions. As previously stated by Erejuwa et al. (2010) that, honey administration to diabetic rats increased significantly GST compared with diabetic-non treated rats. Moreover; Erejuwa et al. (2012b) concluded that honey supplementation restored the elevated renal GST of the hypertensive rats.
The increased hepatic phase II enzymes activities are explained according to the bioactive components of honey. Once honey is absorbed by the intestinal epithelium and before crossing into the bloodstream, flavonoids undergo some degree of phase II metabolism with the generation of different conjugated products, predominantly sulphates, glucuronides and methylated derivatives through the action of sulfotransferases ( In general; conjugation with phase II DMEs generally increasehydrophilicity and thereby enhance excretion in the bile and/or the urine and consequently a detoxification effect (Rushmore and Kong, 2002). EPH catalyzes epoxides by the reaction with water forming intermediates can be excreted from body (Liska, 1998). However; GST catalyzed the conjugation of reactive electrophiles with glutathione (GSH), forming reactive intermediates in particular when GSH levels in the cells are attenuated, consequently resulting in toxicological effects (Bolton and Chang, 2001). On the other hand; UGT catalyzes glucuronidation, may play roles in the conjugation and ultimately excretion and elimination of many drugs and xenobiotic containing hydroxyl functional group either present in the parent structure as phenols, thiols, amines and carboxylic acids and/or after biotransformation by the phase I enzymes (Xu et al., 2005).
The present study indicated that, honey administration increased the hepatic activity of Nrf2 as one of the receptors of phase III detoxification. This finding revealed that, honey administration exerted a closed detoxifying chain via Nrf2 activation beside the activation of each of the CYP2E1, EPH, UGT and GST, even if its degree of activation is changed by the dose administered. It was previously reported that reduced or impaired Nrf2 activity or expression contributes to increased susceptibility of kidney to oxidative stress in rats with chronic renal failure (Kim and Vaziri, 2010) or hypertension (Erejuwa et al., 2011 andErejuwa et al., 2012b).Honey exerted a beneficial ameliorationof the oxidative stress via up-regulation of Nrf2 activity or expression (Erejuwa et al., 2009, Erejuwa et al., 2011and Erejuwa et al., 2012b. This elevation in the Nrf2 activity as a result of honey administration may be due to quercetin, one of its flavonoids, as reported by Panchal et al. (2012). Theyreported that rats supplemented with quercetin presented a higher protein expression of Nrf2 compared with the control group with decrease in oxidative stress and inflammation. Nrf2 is a basic leucine zipper redox sensitive transcriptional factor which is responsible for one of the physiological important stress response mechanisms (Motohashi and Yamamoto, 2004). Under basal conditions, Nrf2 binds to kelch-like ECH associated protein1 (keap1) in the cytoplasm. However under oxidative stress conditions; 637 electrophiles and oxidative stress disrupt the Nrf2-keap1 complex, translocate it to the nucleus (Nakata et al., 2006). Then Nrf2 binds to antioxidant responsive element with heterodimeric combinations with other basic leucine zipper proteins, such as small Mafs, and regulates gene expression of detoxifying enzymes, such as GST, NAD(P)H:quinone oxidoreductase-1, UGT, γ-glutamylcysteinesynthetase and heme oxygenase-1 (Jaiswal, 2004, So et al., 2006and Shen and Kong, 2009).The transcription of these genes subsequently induces free radical scavenging enzymes and other detoxifying enzymes which swiftly neutralize, detoxify and eliminate the oxidants or xenobiotics (Kobayashi et al., 2009).

Conclusion:-
The present study revealed that honey administration is beneficial natural detoxifying via eliminating of some specific toxic agents and oxidative radicals through activation of a chain initiating from phase I (CYP2E1) passing through phase II (EPH, UGT and GST) then ending with phase III (Nrf2). Moreover; the lower dose of honey is more effective, hence it plays significant roles in each of the three phases than the higher dose.