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Highlights

• Protective influence of fisetin (dietary flavonoid) on hyperammonemia was studied. • Hyperammonemic rats showed raised oxida-

tive stress and glutamate level in brain. • Downregulated astrocytic markers were seen under hyperammonemia. • Fisetin assuages

these effects by antioxidant and cytoprotective actions.

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Please cite this article in press as: Perumal Subramanian, Murugesan Jayakumar, Muniyandi Singaravel, Dhanananajay Kumar, Priyoneel Basu, Jaime Jacqueline Jayapalan,Onn Haji Hashim, Fisetin, a dietary flavonoid, attenuates hyperammonemia and improves circadian locomotor deficits, redox balance, and astrocytic markers in rats, Journalof Functional Foods (2014), doi: 10.1016/j.jff.2014.11.025

Fisetin, a dietary flavonoid, attenuateshyperammonemia and improves circadianlocomotor deficits, redox balance, and astrocyticmarkers in rats

Perumal Subramanian a,*, Murugesan Jayakumar a,Muniyandi Singaravel b, Dhanananajay Kumar b, Priyoneel Basu b,Jaime Jacqueline Jayapalan c,d, Onn Haji Hashim c,d

a Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram,Tamil Nadu 608 002, Indiab Chronobiology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, Indiac University of Malaya Centre for Proteomics Research (UMCPR), Faculty of Medicine, University of Malaya,Kuala Lumpur 50603, Malaysiad Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia

A R T I C L E I N F O

Article history:

Received 27 May 2014

Received in revised form 23

November 2014

Accepted 27 November 2014

Available online

A B S T R A C T

Fisetin, a bioactive flavonoid, found in many fruits, was administered orally (50 mg/kg body

weight) to hyperammonemic rats (treated with ammonium chloride (AC) for 8 weeks (i.p.

injections, 100 mg/kg body weight). Treatment with AC augmented oxidative stress, levels

of ammonia, enzymes (alanine and aspartate transaminases and alkaline phosphatase) in

blood, glutamate and glutamine content in brain, caused disturbances in the circadian lo-

comotor rhythm with a reduction in amplitude and period and downregulated the expression

of soluble guanylyl cyclase (sGC) and glial fibrillary acidic protein (GFAP) in brain. Co-

treatment of fisetin treatment with AC, prevented the AC-induced elevation in oxidative stress,

normalized the levels of ammonia, transaminases and alkaline phosphatase in circula-

tion, glutamate and glutamine in brain, upregulated the expression of sGC and GFAP and

stabilized the circadian locomotor rhythm. Thus, fisetin assuages AC induced effects through

its antioxidant, anti-inflammatory, cytoprotective, neuroprotective and neurotrophic properties.

© 2014 Published by Elsevier Ltd.

Keywords:

Hyperammonemia

Fisetin

Circadian

Redox balance

1. Introduction

Fisetin (3, 3′, 4′ 7 – tetrahydroxyflavone), a flavonoid, exten-sively dispersed in fruits (grapes, apples, strawberries andpersimmons) at concentrations of 2–160 mg/g (Scalbert &

Williamson, 2000), has been reported to possess antioxidant(Sengupta, Banerjee, & Sengupta, 2004), cancer preventing (Sung,Pandey, & Aggarwal, 2007), allergy preventing (Kim et al., 2014),anti-inflammatory (Tago, Nakamura, Tago, Mashino, & Kasahara,2011), antidiabetic (Maher et al., 2011), neuroprotective (Chiruta,Schubert, Dargusch, & Maher, 2012), neurotrophic (Maher,

* Corresponding author. Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram, TamilNadu 608 002, India. Tel.: +91 94432 71346.

E-mail addresses: annamalai_rhythm@yahoo.co.in; psmanian1965@gmail.com (P. Subramanian).http://dx.doi.org/10.1016/j.jff.2014.11.0251756-4646/© 2014 Published by Elsevier Ltd.

j o u rna l o f f un c t i ona l f o od s ■ ■ ( 2 0 1 4 ) ■ ■ –■ ■

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Available online at www.sciencedirect.com

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2009), and antiangiogenic (Fotsis et al., 1997) properties. Fisetincounters osteoporosis and supplementation of fisetin sign-ificantly thwarts bone loss and inflammation in mice(Leotoing et al., 2013). Fisetin is reported to enhance memoryin rats (Maher, 2009), offers defensive capacity against oxida-tive glutamate toxicity and thus preventing peroxynitrite-mediated damages in primary rat neurons (Ishige, Schubert,& Sagara, 2001). Flavonoids, being structurally analogous tovitamin E, could mimic their antioxidant action in cell mem-branes (Acker, Schouten, Haenen, Vijgh, & Bast, 2000).The 3-OHgroup of fisetin has the lowest estimated BDE (bond dissocia-tion energy) value (O—H covalent bond) succeeded by its3′- and 4′-OH groups. A lower BDE value is attributed to a higherability to donate a hydrogen atom from the hydroxyl group andthereby to scavenge free radicals (Cao, Sotic, & Prior, 1997).Fisetin and its derivatives could enhance intracellular gluta-thione (GSH) levels and maintenance of GSH levels is linkedwith cell survival with a multiplicity of paradigms (Touil et al.,2011).

The prevalence of hyperammonemia and urea cycle di-sorders globally is estimated at 1 per 30,000 live births (Msall,Batshaw, Suss, Brusilow, & Mellits, 1984). Elevated ammoniaduring hyperammonemic conditions affects several impor-tant central nervous system (CNS) functions includingenergy metabolism, hemodynamics and neurotransmission(Bosoi & Rose, 2009). Liver failure or disease reduces the ca-pacity of the liver to convert ammonia into urea and leads tohyperammonemia. Hyperammonemia, in turn, causes alteredglutamatergic neurotransmission (Rodrigo & Felipo, 2006),hepatic encephalopathy (HE) and a spectrum of neuropsychi-atric and neurological symptoms with high mortality (Felipo& Butterworth, 2002).

Ammonia intoxication also impairs mitochondrial func-tion, decreases ATP synthesis and increases the formation offree radicals (Kosenko, Venediktova, Kaminsky, Montoliu, &Felipo, 2003). Pathogenesis of hyperammonemia is associ-ated with free radical induced oxidative/nitrosative damage(Kosenko et al., 2003). The potential association of oxidativestress in addition to nitrosative stress in contributing to thedeleterious effects of ammonia has been documented (Murthy,Rao, Bai, & Norenburg, 2001). Hyperammonemia elevates brainglutamine levels, which is responsible for pathophysiologicaland neurotoxic malfunctions (Rodrigo & Felipo, 2006).The func-tional foods including naturally occurring bioactive substancesand foods supplemented with bioactives (probiotics and an-tioxidants) could afford hepatoprotective effects (Al-Sherajiet al., 2013) and the present study validates the preventive actionof fisetin on hyperammonemia.

In addition, dysfunctional or abnormal circadian rhythmshave been associated with metabolic syndrome and liverdiseases. Investigations on circadian rhythms in normal andpathological states can be employed to improve the percep-tive of pathological processes and therapeutic approachesto diseases (Zelinski, Deibel, & McDonald, 2014). The cir-cadian rhythm of blood corticosterone is modified in hyper-ammonemic rats (Llansola et al., 2013). Locomotor activity isinterconnected to neuronal action and plasticity, which isrecurrently impaired in hyperammonemia induced hepaticencephalopathy (HE) (Apelqvist, Hindfelt, Andersson, &Bengtsson, 1997; Felipo & Butterworth, 2002). Patients with HE

showed impairment of the sleep–wake or rest–activity cycle(Wilkinson, Smeeton, & Watt, 2010).

Ammonia toxicity leads to astrocyte swelling, dysfunctionand brain edema (Norenberg et al., 1991; Ott, Clemmesen, &Larsen, 2005). Astrocyte swelling stimulates signal transduc-tion processes and hinders cellular metabolism. A deregulationof cell volume homeostasis due to increased glutamine in brainis a main pathogenic event of ammonia toxicity and HE (Zhok& Norenberg, 2000). Astrocytic swelling, further, may causebrainstem herniation and death (Clemmenson, Larsen, Kondrup,Hansen, & Ott, 1999).

In addition, hyperammonemia deregulates the glutamate–NO–cGMP pathway in brain neurons of rats, resulting in re-duced synthesis of cGMP owing to suppression of solubleguanylate cyclase (sGC) (Hermenegildo et al., 1998; Monfort,Munoz, Ayadi, Kosenko, & Felipo, 2002). Inhibition of sGC bynitric oxide (NO) and thereby the glutamate–NO–cGMP pathwaycould be accountable for the neurological alterations initi-ated in HE (Rodrigo et al., 2010). Glial fibrillary acidic protein(GFAP) is mainly expressed in astrocytes and constitutes aselective marker for normal and pathological conditionsand is also involved in the regulation of cell volume (Belanger,Desjardins, Chatauret, & Butterworth, 2002; Eng, Ghirnikar, &Lee, 2000). The specific expression of GFAP arbitrates shape,structure and motility of astrocytes (Eng et al., 2000). The lossof GFAP is also associated with astrocytic swelling (Belangeret al., 2002).

At present, the key disadvantages of numerous anti-hyperammonemic agents/therapies are the recurrence ofsymptoms and staid undesirable effects after their discon-tinuation (Braissant, McLin, & Cudalbu, 2013). Consequently,development and monitoring of drugs averting hypera-mmonemia is indispensable. We conjectured that fisetin(a bioactive antioxidant) could be valuable in avertinghyperammonemia, and evaluated the defensive effect offisetin by assessing (a) the levels of ammonia, urea, uricacid, creatinine, liver markers, lipid peroxidation productsand antioxidants in circulation, liver and brain, (b) circadianlocomotor activity rhythms, and levels of glutamine and glu-tamate in brain and (c) mRNA expression of sGC and GFAP inbrain.

2. Materials and methods

2.1. Animals and maintenance

Male rats (Wistar strain, body weight 190–210 g) obtained fromthe Animal House, Faculty of Medicine, Annamalai Univer-sity were used for investigations. The rats were dwelled inpolypropylene cages and were fed with standard diet pellets(Hindustan Lever Ltd., Bangalore, India) and drinking water adlibitum. Standard conditions of temperature (24 ± 2 °C) withlight:darkness (12:12 h) cycles were maintained throughout thestudy. The study protocols were endorsed by the ethical com-mittee (Approval No.: 737; dated 02/09/2010), AnnamalaiUniversity (Reg. No. 160/1999), compliant with the guiding prin-ciples of Indian Council of Medical Research (ICMR), New Delhi,India.

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Please cite this article in press as: Perumal Subramanian, Murugesan Jayakumar, Muniyandi Singaravel, Dhanananajay Kumar, Priyoneel Basu, Jaime Jacqueline Jayapalan,Onn Haji Hashim, Fisetin, a dietary flavonoid, attenuates hyperammonemia and improves circadian locomotor deficits, redox balance, and astrocytic markers in rats, Journalof Functional Foods (2014), doi: 10.1016/j.jff.2014.11.025

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2.2. Chemicals

Fisetin, ammonium chloride (AC), adenosine triphosphate (ATP),nicotinamide adenine dinucleotide (NAD) and nitroblue tet-razolium (NBT) were acquired from Sigma Chemical Company,St. Louis, MO, USA. Ethylenediaminetetraacetic acid (EDTA),butylated hydroxytoluene (BHT) and phosphate buffered salinewere obtained from S.D. Fine Chemicals Ltd., Mumbai, India.The remainder of the chemicals and biochemicals consumedwere purchased from Bangalore Genei, Bangalore, India andwere of analytical grade.

2.3. Induction of hyperammonemia andexperimental plan

Hyperammonemia was provoked in rats by ammoniumchloride injections (intraperitoneal, three times in a week,100 mg/kg b.w.) for the experimental period of 8 weeks (Subash& Subramanian, 2009; Subramanian, Jayakumar, Jayapalan, &Hashim, 2014).

Three studies were conducted systematically. In study I,the antihyperammonemic and protective influences of fisetinon liver and kidney markers, end products of lipid peroxidation(thiobarbiturica acid reactive substances, TBARS) and antioxi-dants were evaluated. In study II, the protective influences offisetin on circadian locomotor activity rhythms and levels ofglutamine and glutamate were investigated. In study III, mRNAexpression of sGC and GFAP by RT-PCR was examined. The ratswere segregated into four groups (n = 6/group) for all studies(group I – untreated control, group II – treated with fisetin (oral,50 mg/kg b.w.) (Touil et al., 2011), group III – injected with ACand group 4 – administered with AC and fisetin). At the endof the experimental period (8th week), the animals were sac-rificed and blood was collected (with and without anticoagulant)for biochemical determinations. Brain and liver tissues werecut into pieces and homogenized (20% homogenate, w/v) in ap-posite (0.1 M potassium phosphate) buffer in ice cold condition(pH 7.0). The homogenate was centrifuged at 5 °C and thesupernatant was utilized for further analyses.

2.4. Determination methods of ammonia, urea, uric acidand creatinine

Levels of ammonia in blood was determined (Wolheim, 1984)using Roche/Hitachi 912 kit (Roche Diagnostics, Holzheim,Germany). Triethanolamie (151 mM, 200 µl) buffered sub-strate (α-ketoglutarate) was added to blood (20 µl), mixedthoroughly and the absorbance was measured at 480 nm.Content of urea (in plasma) was estimated (Natelson, 1957)using Roche/Hitachi 912 kit. To 0.2 ml of plasma, 3.5 ml ofdouble-distilled water was added. Then 0.35 M sulphuric acid(0.4 ml) and 10% sodium tungstate (0.3 ml) were added andcentrifuged. To the supernatant (2.1 ml), diacetyl monoxime(0.5 ml), water (2 ml) and sulphuric acid–phosphoric acid mix(1.6 ml) were added, boiled for 30 min, cooled and the absor-bance was read at 480 nm.

Plasma uric acid was determined by adding tungstic acid(5.4 ml) to plasma (0.6 ml). The contents were centrifuged. To3 ml of supernatant, 20% sodium carbonate (0.6 ml) and 1%phosphotungstic acid reagent (0.6 ml) were added, incubated

at 25 °C for 10 min and the absorbance was read at 700 nm(Brown, 1945). Serum creatinine was determined by addingalkaline picrate (2 ml) to 3 ml of deproteinized supernatant(prepared by centrifugation of 0.2 ml of serum with 4.3 ml of10% TCA). The mixture was kept for 30 min (at 25 °C) and theabsorbance was read at 520 nm (Bonsnes & Taussky, 1945).

2.5. Assay of transaminases and alkaline phosphatase

Levels of aspartate transaminase (AST) and alanine transami-nase (ALT) were assayed by adding substrate mixture (1 ml,aspartate and α-ketoglutarate-AST; alanine and α-ketoglutarate-ALT) in phosphate buffer (pH 7.4) with serum (0.2 ml). Themixture was kept at 25 °C (60 and 30 min for AST and ALT, re-spectively) followed by the addition of 2, 4-dinitrophenylhydrazineand 0.4 M sodium hydroxide (1 ml) and was read at 540 nm(Reitman & Frankel, 1957). Alkaline phosphatase (ALP) activitywas assayed by adding serum (0.2 ml) with disodiumphenylphosphate (substrate) in bicarbonate buffer (0.12 M, pH10.5) followed by incubation for 15 min (at 25 °C) and additionof Folin-phenol reagent (1%).The contents were centrifuged, and10% sodium bicarbonate (2 ml) was added to the supernatant(1 ml) and was read at 680 nm (King & Armstrong, 1951).

2.6. Determination of oxidative stress markersand antioxidants

Tissue homogenate (1.2 ml)/0.6 ml of plasma was treated with2.5 ml of TBA–TCA–HCl reagent and mixed carefully to deter-mine the content of thiobarbituric acid reactive substances(TBARS) (Fraga, Leibovity, & Toppel, 1988). The contents werecentrifuged and the supernatant was used for absorbance mea-surement at 532 nm. Lipid hydroperoxides (HP) were quantifiedby mixing Fox reagent (1.8 ml) with tissue homogenate/plasma (0.2 ml). The mixture was incubated for 40 min at roomtemperature and was read at 560 nm (Jiang, Hunt, & Wolff, 1992).NO was estimated by mixing 0.5 ml of plasma with 0.5 ml ofsulphosalicylic acid. The mixture was centrifuged and to thesupernatant (200 µl), sodium hydroxide (10%, 30 µl), and Tris–HCl buffer (300 µl) were added and mixed. Griess reagent (530 µl)was added and the mixture was incubated in dark (15 min) andwas read at 540 nm (Green et al., 1982).

Activity of superoxide dismutase (SOD) was assayed (Kakkar,Das, & Viswanathan, 1984) by diluting issue homogenate orhemolysate (0.6 ml) to 1.4 ml with distilled water. Further,ethanol (3 ml) and chloroform (2 ml) were added and themixture was centrifuged. To the supernatant (2 ml), sodiumpyrophosphate buffer (1.2 ml), reduced NAD (0.2 ml), NBT(0.3 ml) and phenazine methosulphate (0.1 ml) were added andincubated at 25 °C for 60 s followed by the addition of aceticacid (1 ml).The contents were mixed well with n-butanol (4 ml)and the absorbance was read at 560 nm. To hemolysate/tissue homogenate (0.5 ml), hydrogen peroxide (0.4 ml) andphosphate buffer (0.9 ml) were added for catalase (CAT) assay(Sinha, 1972). After incubation (30 °C for 60 s) dichromate–acetic acid mixture (2 ml) was added and the tube was boiledfor 15 min and was read at 620 nm. Reduced glutathione (GSH)was estimated (Ellman, 1959) by adding tissue homogenate/plasma (0.5 ml) with trichloroacetic acid (5%, 2 ml) followed bycentrifugation.To the supernatant (1 ml) Ellman reagent (0.5 ml)

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Please cite this article in press as: Perumal Subramanian, Murugesan Jayakumar, Muniyandi Singaravel, Dhanananajay Kumar, Priyoneel Basu, Jaime Jacqueline Jayapalan,Onn Haji Hashim, Fisetin, a dietary flavonoid, attenuates hyperammonemia and improves circadian locomotor deficits, redox balance, and astrocytic markers in rats, Journalof Functional Foods (2014), doi: 10.1016/j.jff.2014.11.025

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and phosphate buffer (3 ml) were added and was read at420 nm. The protein content in serum/plasma/tissue homog-enate was quantified (Lowry, Rosenbrough, Farr, & Randall,1951).

2.7. Circadian locomotor rhythm analysis

The rats were initially acclimatized to laboratory conditions(light-proof chamber with temperature at 25 ± 2 °C). Light–dark cycles (12L:12D, light: 6 am–6 pm; darkness: 6 pm–6 am)were imposed using an electronic timer (Larsen and Toubro,Mumbai, India). Rats were given unrestricted access to loco-motor running wheels (Lafayette Instrument Co., Lafayette, LA,USA) (diameter 36 cm) with individually attached housing cages(41 × 51 × 21 cm) (Fig. 1) and provided with food and water adlibitum. Each revolution of running wheel was counted by a mag-netic reed-relay switch and recorded as running wheel counts/min by a computerized recording unit (ACT-5537 channelbreakout box with a CL-20056 channel interface).The actogramsgenerated were analyzed using Lafayette AWM (Version 11.07)and Clocklab (Coulbourn, Whitehall, PA, USA) softwares. Wheeland laboratory maintenance were done following standardguidelines (Jud, Schmutz, Hampp, Oster, & Albrecht, 2005).

2.8. Measurement of glutamine and glutamate

Glutamine and glutamate levels in brain were measured (Lund,1985). Glutamine estimation was done by adding acetate buffer(0.5 ml) and glutaminase solution (0.2 ml) with 0.25 ml of brainhomogenate (0.5 ml).The mixture was kept for 1 h at 30 °C, glu-tamate dehydrogenase (GLDH) (0.02 ml) was added, the contentswere mixed, kept at 30 °C for 40 min and was read at 340 nm.Glutamate content was estimated by adding Tris–EDTA–hydrazine buffer (1 ml), NAD solution (0.1 ml), adenosinediphosphate solution (0.01 ml), GLDH (0.02 ml) with brainhomogenate (0.25 ml). The contents were mixed well andabsorbance was read at 340 nm after 40 min of incubationat 30 °C.

2.9. mRNA expressions of GFAP and sGC

Total RNA extracted from brain tissues using Trizol reagent wasreverse transcribed into cDNA using AMV reverse transcriptaseand an oligo (dT) primer (Bangalore Genei, Bangalore, India). Poly-merase chain reaction was performed using PCR ready mix

(Bangalore Genei).The PCR conditions for the housekeeping gene,glyceraldehydes 3-phosphate dehydrogenase (GAPDH) were 24thermal cycles at 95 °C for 50, 56 °C for 50, and 73 °C for 70 s re-spectively and pursued by a final extension for 8 min at 73 °C.GFAP was subjected to 20 thermal cycles at 95 °C for 40, 60 °Cfor 40 s respectively, and 73 °C for 5 min followed by an a finalextension at 73 °C for 4 min. sGC was subjected to 35 thermalcycles at 95 °C for 30, 55 °C for 50, and 73 °C for 60 s, respec-tively and pursued by an extension at 73 °C for 9 min.The primersequence for GAPDH – forward (5′-ACCACAGTCCATGCCATCAC-3′) and reverse (5′-TCCACCACCCTGTTGCTGTA-3′); GFAP – forward(5′-CGACCTTGAGTCCTTGCG-3′) and reverse (5′-CCGTCTTTACCACGATGTT-3′) and sGCα2 subunit – forward (5′-ACCCTTATCTCCGCAGTCCT-3′) and reverse (5′-CGCCGTCAGGAGACTGATAC-3′) primers were used and they generated 485-bp,416-bp and 692-bp PCR products respectively. Amplificationreactions were accomplished in a final volume of 50 µl contain-ing 5 µl of cDNA, 250 µM of dNTPs, 150 pmol of primer, and 3.5 Uof Taq DNA polymerase (Bangalore Genei). PCR products (25 µl)were separated on a 1.8% agarose gel containing ethidiumbromide. Intensities of the bands were quantified using Gel Docsystem (Bio-Rad, Hercules, CA, USA) and Quantity on 1-D analy-sis software (Bio-Rad). The result was represented as relativemRNA expression of target gene product/GAPDH.

2.10. Statistical analysis

The data of diverse biochemical parameters were examinedusing two way analysis of variance (ANOVA) and Duncan’s mul-tiple range test (DMRT). Values were deemed statisticallysignificant when p < 0.05.

3. Results

3.1. Influence of fisetin on intermediates of proteinmetabolism, uric acid and liver markers

Levels of blood ammonia, serum creatinine and plasma uricacid were significantly (p < 0.05) elevated in AC-treated rats andthese levels were significantly (p < 0.05) plummeted when fisetinwas administered to hyperammonemic rats. On the other hand,the level of urea was decreased in AC-treated rats. Adminis-tration of fisetin significantly (p < 0.05) augmented the levelin hyperammonemic rats. Rats treated with fisetin only

Fig. 1 – Activity wheel for circadian locomotor rhythm analysis.

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Please cite this article in press as: Perumal Subramanian, Murugesan Jayakumar, Muniyandi Singaravel, Dhanananajay Kumar, Priyoneel Basu, Jaime Jacqueline Jayapalan,Onn Haji Hashim, Fisetin, a dietary flavonoid, attenuates hyperammonemia and improves circadian locomotor deficits, redox balance, and astrocytic markers in rats, Journalof Functional Foods (2014), doi: 10.1016/j.jff.2014.11.025

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exhibited no significant difference in the levels of ammonia,creatinine and uric acid in comparison with controls (Table 1).The levels of liver markers were found to be considerably(p < 0.05) elevated in the serum of AC-treated rats.

Hyperammonemic rats exhibited significantly higher levelsof ALT, AST and ALP than controls. Supplementation of fisetinto hyperammonemic rats significantly (p < 0.05) restored thelevels of liver markers. No significant difference in the livermarker enzymes was observed in normal and fisetin alonetreated rats (Table 1).

3.2. Inhibitory effect of fisetin on oxidative stress

The levels of TBARS and HP (indicators of lipid peroxidationand oxidative stress) were significantly elevated and accom-panied by a significant reduction of antioxidants (SOD, CAT andGSH) in hyperammonemic rats. The AC + fisetin treated grouphad elevation in the activities of antioxidative enzymes (SODand CAT) and non-enzymatic antioxidant (GSH) and a plummetin the markers of oxidative stress. The fisetin alone treatedgroup displayed more or less same levels of TBARS, HP and an-tioxidants as the control group (Table 1). Brain and liver tissuesshowed a similar trend in the redox balance (Table 1). NO levelsare significantly elevated in AC treated rats and were normal-ized in AC + fisetin treated group.

3.3. Circadian locomotor rhythm

Representative actograms of control, fisetin, AC and AC + fisetintreated rats are shown (Fig. 2A–D). Periodograms showingthe period of the locomotor rhythm with amplitude wererepresented. There is a significant reduction of period of thelocomotor rhythm in hyperammonemic rats (23.74 ± 0.08) ascompared to control rats (24.00 ± 0.03; p < 0.05). Fisetin andhyperammonemic rats treated with fisetin showed insignifi-cant differences from the period of 24 h. Further, AC-inducedhyperammonemic rats showed fragmentation of activity andsignificantly reduced amplitude (mean ± SD; 980 ± 275) as com-pared with amplitude of controls (3526 ± 652; p < 0.001).Administration of fisetin reduced the fragmentation and re-stored the circadian rest–activity pattern in hyperammonemicrats with amplitude of 1864 ± 377 (Fig. 2D). Insignificant changeswere observed in the control and fisetin alone treated rats (am-plitude – 3750 ± 453; p > 0.05) with an undisturbed activitypattern (Fig. 2A and B).

3.4. Glutamate and glutamine contents in brain

Significant (p < 0.05) elevation of glutamate and glutamine levelsin the brain of hyperammonemia induced rats was noticed incomparison to control group. Administration of fisetin to

Table 1 – Changes in experimental variables of hyperammonemic and fisetin treated rats.

Variable studied Control Fisetintreated

ACtreated

AC + fisetintreated

Ammonia (µmole/l) 83.70 ± 6.23a 81.94 ± 6.14a 369.48 ± 27.71c 118.46 ± 8.05b

Urea (mg/ml) 9.77 ± 0.74c 9.89 ± 0.75c 4.21 ± 0.30a 8.22 ± 0.57b

Uric acid (mg/dl) 1.56 ± 0.12a 1.50 ± 0.11a 2.95 ± 0.22c 1.78 ± 0.14b

Creatinine (mg/dl) 0.67 ± 0.05a 0.60 ± 0.05a 1.81 ± 0.14c 0.89 ± 0.07b

Aspartate transaminase (IU/l) 68.98 ± 5.18a 68.91 ± 5.17a 120.96 ± 9.07c 81.06 ± 6.08b

Alanine transaminase (IU/l) 23.21 ± 1.74a 23.04 ± 1.72a 65.48 ± 4.91c 27.90 ± 2.10b

Alkaline phosphatase (IU/l) 70.41 ± 5.28a 70.32 ± 5.28a 139.56 ± 10.48c 80.15 ± 6.02b

Thiobarbituric acid reactive substances (nmole/dl) 2.68 ± 0.20a 2.62 ± 0.19a 4.75 ± 0.35c 3.09 ± 0.23b

Hydroperoxides (×10−5 mmole/dl) 7.12 ± 0.60a 7.06 ± 0.60a 13.04 ± 1.05c 8.51 ± 0.69b

Nitric oxide (mmole/mg protein) 0.072 ± 0.005a 0.078 ± 0.006a 0.212 ± 0.016c 0.102 ± 0.008b

Superoxide dismutase (UA/mg Hb) 2.78 ± 0.20c 2.81 ± 0.21c 1.73 ± 0.13a 2.42 ± 0.17b

Catalase (UB/mg Hb) 2.14 ± 0.16c 2.19 ± 0.16c 1.30 ± 0.09a 1.96 ± 0.15b

Reduced glutathione (mg/dl) 26.94 ± 2.02c 27.15 ± 2.03c 14.28 ± 1.07a 22.40 ± 1.82b

(In brain)Thiobarbituric acid reactive substances (mmole/g tissue) 1.04 ± 0.07a 0.97 ± 0.07a 2.11 ± 0.16c 1.23 ± 0.09b

Hydroperoxides (mmole/g tissue) 103.95 ± 7.79a 103.87 ± 7.79a 155.28 ± 11.64c 117.86 ± 8.68b

Superoxide dismutase (UA/g tissue) 7.44 ± 0.55c 7.39 ± 0.55c 4.93 ± 0.36a 6.82 ± 0.51b

Catalase (UB/ g tissue) 3.18 ± 0.24c 3.24 ± 0.24c 0.79 ± 0.05a 2.94 ± 0.22b

Reduced glutathione (mg/g tissue) 38.18 ± 2.86c 38.67 ± 2.90c 20.17 ± 1.51a 31.20 ± 2.41b

Glutamine (mmole/g tissue) 4.43 ± 0.31a 4.31 ± 0.30a 13.04 ± 0.91c 6.98 ± 0.49b

Glutamate (mmole/g tissue) 9.23 ± 0.65a 9.47 ± 0.66a 17.9 ± 1.25c 11.71 ± 0.82b

(In liver)Thiobarbituric acid reactive substances (mmole/g tissue) 0.81 ± 0.06a 0.78 ± 0.06a 2.15 ± 0.16c 1.02 ± 0.07b

Hydroperoxides (mmole/g tissue) 61.97 ± 4.64a 61.84 ± 4.63a 99.89 ± 7.49c 74.15 ± 5.41b

Superoxide dismutase (UA/g tissue) 8.92 ± 0.66c 8.99 ± 0.67c 3.20 ± 0.24a 7.18 ± 0.54b

Catalase (UB/ g tissue) 85.98 ± 6.45c 86.32 ± 6.47c 35.34 ± 2.65a 72.82 ± 5.61b

Reduced glutathione (mg/g tissue) 52.26 ± 3.92c 52.60 ± 3.94c 22.25 ± 1.67a 43.36 ± 3.55b

Values are given as mean ± S.D from each group (n = 6).Values not sharing a common superscript alphabet vary significantly at p < 0.05 (DMRT).UA – one unit of enzyme activity which results in 50% inhibition of nitroblue tetrazolium reduction/minute.UB – µmole of hydrogen peroxide utilized/minute.

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AC-treated rats considerably (p < 0.05) diminished the levels ofglutamate and glutamine in comparison to AC treated rats. Fur-thermore, administration of fisetin to normal rats did notdemonstrate considerable variation in the levels of gluta-mate and glutamine (Table 1).

3.5. Expression of astrocytic markers

Significant downregulation (p < 0.05) of synthesis of sGC andGFAP mRNAs was noticed in brain of hyperammonemic rats(Fig. 2A and B). The expression levels of sGC and GFAP wereupregulated in fisetin treated hyperammonemic rats (p < 0.05)while remains unchanged in control and fisetin alone treatedrats (Fig. 3A and B).

4. Discussion

The reduction in ammonia, uric acid and creatinine levels, in-crease in urea, and stabilization of liver marker enzymesindicated antihyperammonemic nature of fisetin since it pos-sesses antioxidant (Sengupta et al., 2004), anti-inflammatory(Tago et al., 2011) and neuroprotective (Chiruta et al., 2012)

properties. The antioxidant nature of flavonoids may sustainthe body’s defences against the hyperammonemia mediatedfree radical reactions (Acker et al., 2000). When hepatocellu-lar membrane is damaged, an array of enzymes localized inthe cytosol of hepatocytes is discharged into circulation andtheir determination in blood is a valuable quantitative markerof the degree and type of damage (Recknagel, Glende, & Britton,1991). Results of this study demonstrated considerable in-crease in liver marker enzymes (transaminases and ALP)and thus indicate the liver damage in AC-induced hyper-ammonemia. Fisetin treatment, on the other hand, hadameliorated the liver damage in hyperammonemic rats.

An association involving oxidative stress and hyper-ammonemia has been well ascertained in many studies.The imbalanced ROS homeostasis and free radical pro-duction could be the major factors which entailed in themechanism of ammonia toxicity (Norenberg, 2003; Widmer,Kaiser, Engels, Jung, & Grune, 2007). In brain, activation ofN-methyl-D-aspartate (NMDA) receptors by excess ammoniais known to augment the formation of superoxide radicals(ElMlili et al., 2010). Blood and tissues (liver and brain) showedaugmented levels of TBARS and HP in AC induced rats whichcould be owing to ammonia stimulated free radical produc-tion, cell membrane damage, and cell lysis. Stressful conditionshave been reported to significantly augment blood nitrite levels,an indicator of NO production in rodents (Masood, Banerjee,

Fig. 2 – The representative double-plotted actograms showing circadian locomotor rhythm in (A) control, (B) fisetin, (C) ACand (D) AC + fisetin treated rats. Periodograms with amplitude are also represented.

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Vijayan, & Ray, 2013). Oxidative stress is involved in the mecha-nism of ammonia-activated nuclear factor kappa B (NF-κB)induction (Jayakumar, Panickar, Murthy, & Norenberg, 2006).Since NF-κB is a main transcriptional factor responsible forinducible nitric oxide synthase (iNOS) gene induction (Rao,Jayakumar, Tong, Alvarez, & Norenberg, 2010) this could be thecause for the increased NO levels in the hyperammonemic ratsin this study.

The significant decrease in enzymatic (SOD and CAT) andnon-enzymatic (GSH) antioxidants noticed in AC-inducedhyperammonemic rats in circulation and tissues (liver and brain)could be owing to NMDA receptor induced exicitotoxicityand oxidative insult caused by excess in ammonia levels. Inaddition to enzymatic antioxidants (SOD and CAT), glutathi-one (GSH) participates in key roles as free radical scavengerand antioxidant and its diminution would lead to oxidativestress and cellular degeneration (Meister & Anderson, 1983).Decline in glutamate uptake has been documented in am-monia-treated cultured astrocytes, synaptosomes, and inhyperammonemic rodent models (Chan, Zwingmann,Pannunzio, & Butterworth, 2003). Consequently the augmen-tation of extracellular glutamate causes decreased intracellular

uptake cystine, the precursor for GSH synthesis, cellulartoxicity, accrual of oxidants and cell death owing to the ulti-mate reduction in intracellular GSH (Cabrera-Pastor, Llansola,Reznikov, Boix, & Felipo, 2012).

To explore the antioxidant action of fisetin, via oxidativestress inhibition, we determined the levels of markers ofROS balance and the activity of the antioxidant enzymes.Fisetin is highly hydrophobic and it could readily passthrough the cell membranes and its further intracellularaccumulation protects the most susceptible brain and livercells from oxidative damage (Chiruta et al., 2012). Fisetincould increase intracellular glutathione levels via multiplemechanisms (Ehren & Maher, 2013). The present observa-tions clearly indicate that fisetin could exert potent anti-hyperammonemic effects hence, a more suitable agent fortreating hyperammonemia. Numerous reports describedthat a significant increase in the cerebral concentration offisetin could prevent and/or facilitate recovery from neu-rodegeneration by enhancing the levels of antioxidants(Ishige et al., 2001). The 3, 7-dihydroxyl groups of fisetincould block Ca2+ influx into nerve cells, which is one of thelast steps in the cell death cascade of NMDA-receptor

Fig. 3 – (A) sGC and GFAP mRNA expression in hyperammonemic and fisetin treated rats. (B) Relative mRNA expression ofsGC and GFAP.

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mediated oxidative stress (ElMlili et al., 2010). An increasein cerebral concentration and production of intracellularantioxidants, and the blockade of Ca2+ ion channels arealso considered as most favorable effects of fisetin and maybe used in alleviating hyperammonemia-mediated toxicity andcomplications. Furthermore, fisetin itself could act as an an-tioxidant (Sengupta et al., 2004). The essential mechanismsthrough which fisetin diminishes hyperammonemia could pos-sibly be ascribed to its free radical scavenging properties,preserving antioxidant reserve, cellular architecture and therebyaugmenting the detoxification of ammonia.

Hyperammonemia impairs adrenal function and thelevels of cortisol/corticosterone which is a unique factorresponsible for rest–activity of mammals which could lead toreduced feedback modulation of the hypothalamic–pituitary–adrenocortical (HPA)-axis (Llansola et al., 2013). If perturbationoccurs in HPA-axis, it might contribute to the impairment ofcircadian locomotor rhythms (Nader, Chrousos, & Kino, 2010).There are previous reports on impaired adrenal function andassociated disturbances on temporal locomotor and sleep–activity cycles in hyperammonemic rats with or without HE(Jayakumar & Subramanian, 2013; Llansola et al., 2013;Subramanian et al., 2014).The present representative actogramsof hyperammonemic rats demonstrated reduction in total ac-tivity counts, fragmentation of activity, significant reductionin amplitude and period of the rhythm. Administration of fisetinreduced the fragmentation, reinstated the circadian rest–activity pattern and period of the rhythm in hyperammonemicrats and this might be by preventing hyperammonemic con-ditions. The modulated glutamate–NO–cGMP pathway anddecreased cGMP levels could also alter circadian rhythms(Apelqvist et al., 1997) and hence the locomotor rhythms aremodified in hyperammonemia. To the best of our knowledgethis is the first report of this kind. Normalization of thelocomotor rhythm and the restitution of rest–activity to thelight–dark cycle by fisetin treatment is an indicator of thera-peutic efficacy of fisetin.

To facilitate its diverse functions in nerve cells, glutamatemust be compartmentalized into distinctive pools (Dahlberg,Ivanovic, & Hassel, 2014) via the glutamate transporters presentin various membranes. The elevation in brain ammonia levelsrestrains the intake of glutamate into neurons by blocking theglutamate receptors and their mRNA synthesis (Chan et al.,2003). Moreover, reduced intracellular glutamine synthesis mightcause an elevation in the extracellular glutamate duringhyperammonemic condition. The failure of neuronal and as-trocytic glutamate uptake in hyperammonemia hinderselimination of glutamate from extracellular milieu and even-tually implicated in the deleterious effect (exicitotoxicitymediated oxidative stress) of brain edema, a key complica-tion of liver failure with hyperammonemia (Zielinska, Popek,& Albrecht, 2014). Further, glutamine, which could generatefree radicals in astrocytes, has been associated with astro-cytic swelling (Zielinska et al., 2014). Enhanced glutamine uptakeinto mitochondria causes raised ammonia levels, increase inglutamate (by the activation of glutaminase in mitochon-dria), blockade in the respiratory oxidative enzymes andultimately reduction of ATP synthesis. In addition, ammonia,a competitive inhibitor of α-ketoglutarate dehydrogenase, in-stigates α-ketoglutarate accrual and synthesis of glutamate

in neurons and astrocytes (Zundorf, Kahlert, Bunik, & Reiser,2009). Therefore, the variations in the glutamine and gluta-mate levels evidenced in this study specify their homeostaticimbalance owing to stern changes and defects in their recep-tors and transporters under hyperammonemia. Fisetintreatment could prevent excess ammonia entry into the brain,initiate increased activity of glutamine synthetase, furtherreduce ammonia levels (by conversion of glutamate into glu-tamine) and thereby, could attenuate their levels in brain asevidenced in this study.

In astroglial cells of brain, hyperammonemia could causeincreased expression of inducible nitric oxide (iNOS) (Jayakumaret al., 2006). Nitric oxide is an essential signaling moleculefor normal physiological processes but elevated NO underhyperammonemia could lead to deleterious effects on lipidsand proteins of cell membranes. Elevated reactive nitrogenspecies (RNS) formation diminishes glutamine synthetaseactivity resulting in decreased glutamine and elevated ammoniasynthesis (in vivo) and oxidative stress mediated metabolicdefects (Jayakumar et al., 2006). Fisetin treatment in hyper-ammonemic rats showed a considerable decrease in NO levels(in circulation) and this could enhance glutamine synthetaseactivity and supports the amelioration of cyto/neurotoxicresponses.

AC-treated rats showed downregulated mRNA expressionof GFAP and sGC as compared to controls. These results cor-roborated with the earlier reports on destructive effects ofhyperammonemia and/or HE with a reduction in the expres-sion in astrocytic markers (Corbalan, Chatauret, Behrends,Butterworth, & Felipo, 2002). The NO-sensitive sGC is the bestknown target for NO and cGMP (Hanafy, Krumenacker, & Murad,2001).The glutamate–NO–cGMP pathway mediates essential ce-rebral processes (Hermenegildo et al., 1998) such as intercellularcommunication, circadian rhythms including the activity–rest cycle, several forms of learning and memory. Thesefunctions are impaired in cerebellum of rats with chronichyperammonemia (Llansola et al., 2013), ensuing reduced syn-thesis of cGMP. Liver failure with hyperammonemia is knownto diminish the level of sGC (via NO) in brain as demon-strated in liver cirrhotic patients (Corbalan et al., 2002).Diminished levels of sGC by NO and hence the glutamate–NO–cGMP pathway may be responsible for various neurologicalmodifications found in HE such as the altered circadian rhythms(Rodrigo & Felipo, 2006) as also evidenced in our study. However,upregulated sGC expression by fisetin could be by quenchingthe NO levels.

A considerable decrease in the synthesis of GFAP mRNA andprotein resulting from hepatic devascularization is well knownin hepatoencephalopathic patients with hyperammonemia(Belanger et al., 2002). Reduced GFAP gene expression oc-curred when brain ammonia concentrations reach 1–5 mMrange (Neary, Whittemore, Zhu, & Norenberg, 1994). We did notmeasure the brain ammonia concentrations, but the dose andduration we selected could approximate the ammonia levelsin brain between the ranges of 1–5 mM. Oxidative stress stimu-lated by ammonia plays a major role in the pathogenicity ofcultured astrocytes (Widmer et al., 2007). Upregulated expres-sion of GFAP by fisetin could be owing to reduction of NO levels,enhancement of antioxidants, and amelioration of ammoniatoxicity and thus, ultimately leading to neuro/cytoprotection.

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Please cite this article in press as: Perumal Subramanian, Murugesan Jayakumar, Muniyandi Singaravel, Dhanananajay Kumar, Priyoneel Basu, Jaime Jacqueline Jayapalan,Onn Haji Hashim, Fisetin, a dietary flavonoid, attenuates hyperammonemia and improves circadian locomotor deficits, redox balance, and astrocytic markers in rats, Journalof Functional Foods (2014), doi: 10.1016/j.jff.2014.11.025

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5. Conclusion

This work confirmed that fisetin mediated refurbishment ofintermediates of protein metabolism, liver markers, redoxbalance, circadian locomotor rhythm, glutamine, and levelsglutamate and expression of sCG and GFAP which might bethrough its antioxidant, anti-inflammatory, neurotrophic andneuroprotecting effects. Nevertheless its therapeutic action isneeded to be corroborated by comprehensive clinical studies.

Acknowledgements

Visiting Professorship to PS at Dept. of Molecular Medicine,University of Malaya is gratefully acknowledged.This study wassupported by research grants to PS (UGC, New Delhi, India) andto OHH (RP017-13HTM and HIR-MOHE H-20001-00-E000009).

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ARTICLE IN PRESS

Please cite this article in press as: Perumal Subramanian, Murugesan Jayakumar, Muniyandi Singaravel, Dhanananajay Kumar, Priyoneel Basu, Jaime Jacqueline Jayapalan,Onn Haji Hashim, Fisetin, a dietary flavonoid, attenuates hyperammonemia and improves circadian locomotor deficits, redox balance, and astrocytic markers in rats, Journalof Functional Foods (2014), doi: 10.1016/j.jff.2014.11.025

11j o u rna l o f f un c t i ona l f o od s ■ ■ ( 2 0 1 4 ) ■ ■ –■ ■

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