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Experimental and Toxicologic Pathology 65 (2013) 751– 758

Contents lists available at SciVerse ScienceDirect

Experimental and Toxicologic Pathology

jo ur nal homepa ge: www.elsev ier .de /e tp

epatic metabolism of ibuprofen in rats under acute hypobaric hypoxia

hefali Gola, Gaurav Km. Keshri, Asheesh Gupta ∗

efence Institute of Physiology and Allied Sciences, DRDO, Lucknow Road, Timarpur, Delhi 110 054, India

r t i c l e i n f o

rticle history:eceived 18 May 2012ccepted 8 November 2012

eywords:cute hypobaric hypoxiaepatic metabolismharmacokineticsbuprofenYP2C9 level

a b s t r a c t

Hypobaric hypoxia induced at high altitude causes a subnormal oxygen concentration in cells whichaffects the drug metabolic and pharmacokinetic (PK) capacity of the body. The metabolism and PK of drugslike ibuprofen may be impaired under hypoxia and may require a different than usual therapeutic doseregimen to ensure safe therapy. The present investigation was undertaken to evaluate the effect of acutehypobaric hypoxia (AHH) on hepatic metabolism and PK of ibuprofen in rats. Animals were exposed tosimulated altitude of 7620 m (∼25,000 ft) for AHH exposure (6 and 24 h) in a decompression chamber andwere administrated with single dose of ibuprofen (80 mg/kg body weight, p.o.). The results showed thatGST activity was significantly reduced at 6 h (15%) and 24 h (23%) (p < 0.05) in hypoxic group as comparedto normoxic. A significant increase by 20–24% (p < 0.05) in AST level was observed after AHH exposure.LDH activity also exhibited significant increase (p < 0.05) after 24 h of AHH. A significant down-regulated

CYP2C9 level and mild histopathological changes were observed after 24 h of AHH. Furthermore, PKvariables viz. elimination half-life (T½) and mean residence time (MRT) of ibuprofen exhibited significantincrease by 42% and 51% (p < 0.05) respectively after 24 h of AHH. Thus, results suggest that AHH exposureof 24 h significantly affects phase II conjugation pathway, CYP2C9 level, AST level, liver histology andPK parameters. This asserts that AHH can impair disposition of ibuprofen however, it requires furtherinvestigation under chronic hypobaric hypoxic conditions.

© 2012 Elsevier GmbH. All rights reserved.

. Introduction

On induction to high altitude (HA) human body encountershe first environmental stress i.e. hypobaric hypoxia which causes

any pathophysiological effects to the individuals during their ini-ial days of induction and also following prolonged residency at HA.he normal physiological mechanism and metabolism in an indi-idual is hampered under hypoxia (Cymerman and Rock, 2009).t HA, the low barometric pressure of the atmosphere results iniminished alveolar oxygen tension and as a result, arterial par-ial pressure of oxygen (pO2) drops, in turn decreasing the oxygenaturation.

The functions and physiology of vital organs like liver, brain,eart and lungs get affected due to hypoxia interceded at HA dueo subnormal oxygen concentration in cells (Hoppeler and Vogt,

001; Muhlinga et al., 2006; Nakanishi et al., 1995; Pagani et al.,000; Sulkowska, 1997). Liver is responsible for regulation of aide variety of biochemical pathways including the metabolism

∗ Corresponding author. Present address: Wellman Centre for Photomedicine,assachusetts General Hospital, BAR 416, 40 Blossom Street, Boston, MA 02114,SA. Tel.: +91 11 23883314; fax: +91 11 23914790.

E-mail address: asheeshgupta2001@gmail.com (A. Gupta).

940-2993/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.etp.2012.11.001

of endogenous and exogenous compounds and detoxification. Forregulation of these metabolic processes liver requires more oxygenthan other tissues and is more prone to hypoxia mediated oxida-tive stress (Savransky et al., 2007; Berendsohn, 1962). The majorpathways of hepatic drug metabolism are dependent on oxygen.Oxidases and oxygenases are responsible for altered hypoxic func-tions due to deficient metabolic activities. As these enzymes havedifferent affinities for O2, it follows that their functional sensitivityto the severity of hypoxia also differs. Phase I hepatic enzymes usu-ally converts the parent lipophilic drug to a more polar metaboliteby introducing or unmasking a functional group. However in phaseII metabolism, the hydroxylated or other compounds produced inphase I are conjugated with glucuronic acid, sulphate, acetate, glu-tathione or certain amino acids or by methylation, etc. Phase IIenzyme systems are indirectly dependent on oxygen for the gen-eration of essential co-factors, such as NAD and ATP (Guengerich,1991). The cellular oxygen deficiency caused due to hypoxia at HAperturbs cellular metabolism.

The studies conducted earlier have provided the basis that drugmetabolism and PK get affected under hypoxia (Costa, 1990; Jones

et al., 1989; Jürgens et al., 2002; Shan et al., 1992; Woodrooffeet al., 1995). The metabolism and PK studies on various classesof drugs including corticosteroids, carbonic anhydrase inhibitors,antipyretic and anti-analgesic drugs like acetaminophen,

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52 S. Gola et al. / Experimental and To

cetazolamide, prednisolone, furosemide and lithium haveupported that hypoxia causes alterations in metabolism of drugArancibia et al., 2003, 2004, 2005; Aw et al., 1991; Ritschel et al.,998). Though there has been continued interest in the safetynd absorption, distribution, metabolism and elimination (ADME)roperties w.r.t. non-steroidal anti-inflammatory drugs (NSAIDs)or its implication as prophylactic and therapeutic intervention,till there is paucity of literature regarding the effect of hypoxia onSAIDs. Therefore, we have selected ibuprofen as a candidate drugf this class.

NSAIDs play crucial role as therapeutic agents for thereatment of pain and inflammatory diseases. Ibuprofen [2-(4-sobutylphenyl)-propionic acid], a member of NSAID family, is a

idely used and well-tolerated analgesic (Davies, 1998). Ibuprofens a known non-selective cyclooxygenase (COX) inhibitor, inhibit-ng both COX-1 and COX-2 forms. However its analgesic, antipyreticnd anti-inflammatory effects are principally due to COX-2 inhi-ition (Neupert et al., 1997). Ibuprofen is metabolized to formarboxy and hydroxy ibuprofen, as well as an acyl glucuronide,hich are excreted in urine (Mills et al., 1973).

CYP2C9 is a major cytochrome P450 isoform, which is responsi-le for metabolic clearance of a wide variety of therapeutic agents,

ncluding NSAIDs. It plays a major role (>70%) in the oxidativeetabolism of ibuprofen (Hamman et al., 1997; Leemann et al.,

993; McGinnity et al., 2000). In conditions related to oxygen ten-ion the expression and activity of several CYP proteins get affectedhich suggests that their expression and/or activity may be impli-

ated in hypoxic conditions. Previous in vitro and ex vivo studiesave reported decreased activity and expression of CYP450 iso-

orms i.e. CYP1A1, CYP1A2, CYP2B6, CYP2C9 and CYP2C19, CYP2E1nder hypoxic conditions (Fradette et al., 2002, 2007; Fradettend Du Souich, 2004; Michaelis et al., 2005). Along with effect ofypoxia on drug metabolism, hepatic damage is also caused underypoxia. Previous studies have also demonstrated liver damagender chronic intermittent hypoxia (Feng et al., 2011; Savranskyt al., 2007, 2009).

As a result of altered drug metabolism and PK, safe drug dosageegime may differ under HA induced hypoxia which establisheshe requirement for safe and effective treatment of HA related

aladies. Despite a number of studies conducted at HA evidentor usefulness of ibuprofen for treatment of HA induced headacheBerghold, 2000; Broome et al., 1994; Harris et al., 2003; Gertscht al., 2010), there is paucity of literature on ibuprofen w.r.t. itsltered metabolism and PK as well as therapeutic dosage underypoxia.

Hence, the present study was undertaken with the aim to inves-igate the effect of AHH stress on alteration of hepatic metabolismnd PK of ibuprofen, if any. For further understanding the potentialffect of hypoxia on hepatic metabolism extensive analysis ofeveral detoxification enzymes (phase I and II), liver pathology,YP2C9 protein level and PK was carried out using ibuprofen as aandidate drug.

. Materials and methods

.1. Chemicals

Chemicals including ibuprofen, horse heart cytochrome c,efenamic acid, UDP used in the study were procured from

igma–Aldrich Chemical Co., St. Louis, USA. Rabbit polyclonal anti-YP2C9 antibody was purchased from Abcam, MA, USA and all

ther chemicals of high analytical grade used in the experimentsere obtained from S.D. Fine chemical, and SISCO research labora-

ories, India. HPLC grade acetonitrile, methanol, ortho-phosphoriccid (OPA) and water were purchased from Merck, India.

gic Pathology 65 (2013) 751– 758

2.2. Experimental animals

Male Sprague-Dawley rats (180 ± 20 g), from the animal colonyof the Defence Institute of Physiology and Allied Sciences (DIPAS),Delhi were used for this study. The animals were maintained undercontrolled environment at the Institute’s animal house at 25 ± 1 ◦Cand 12-h light–dark cycle and had food and water ad libitum. Theexperiments were performed in accordance with the regulationsspecified by the Institute’s Animal Ethical Committee and conformto the national guidelines on the care and use of laboratory animals,India.

2.3. Induction to hypobaric hypoxia and experimental design

Experimental animals were randomly divided into four groupsof six animals each. Animals of the group I and III remained at sealevel atmospheric pressure within the same laboratory conditionsand were administered with single dose of ibuprofen (80 mg/kgbody weight, p.o.). Animals of the group II and IV were exposedto AHH for a duration of 6 and 24 h respectively in decompressionchamber (Seven Star, Delhi, India) at a simulated altitude of 7620 m(∼25,000 ft). The animals of hypoxia group II and IV were alsoadministered with single dose of with ibuprofen (80 mg/kg bodyweight, p.o.), immediately prior to hypoxia exposure. The hypo-baric hypoxia decompression chamber was maintained at 28 ± 2 ◦Ctemperature, 55 ± 5% humidity and air flow 9–10 lit/min during theexposure to prevent accumulation of exhaled gases.

2.4. Evaluation of phase I and II drug metabolizing enzymes

On completion of the stipulated period of hypoxia exposure (6or 24 h), the rats of both normoxic and hypoxic groups treatedwith ibuprofen were sacrificed by cervical dislocation. The liverwas perfused with cold saline, excised and weighed. One portion ofliver was used to prepare a 10% liver homogenate (w/v) in (0.15 MKCl + 5 mM Sod. EDTA) buffer for lactate dehydrogenase (LDH), ala-nine tranaminases (ALT) and aspartate transferases (AST) assays.Another portion of each liver was homogenized in 0.1 M potassiumphosphate buffer (pH 7.4) for microsome preparation. Microso-mal fractions were isolated with differential centrifugation method(Omura and Sato, 1964). Briefly, liver homogenate (prepared in0.1 M potassium phosphate buffer pH 7.4) was centrifuged at800 × g in refrigerated centrifuge for 10 min (to remove nuclei andcell debris). The post-nuclear supernatant was collected in separatetubes and centrifuged at 12,000 × g in refrigerated centrifuge for10 min (to remove mitochondria). Post-mitochondrial supernatantwas centrifuged at 1,05,000 × g in refrigerated ultracentrifuge for1 h and microsomal pellets obtained were re-suspended in 0.1 Mpotassium phosphate buffer (pH 7.4) containing 0.5 mM EDTA and20% (w/v) glycerol for further analysis of hepatic metabolizingenzyme (phase I and II) activities.

2.4.1. Total cytochrome P450 (CYP 450) contentTotal CYP 450 content in microsomes was determined

by carbon monooxide (CO)-difference spectrophotometry ofdithionite-reduced samples, using a molar extinction coefficient of91 cm−1 mM−1 as method described by Omura and Satto method(1962). Results were expressed in n moles/mg microsomal protein.

2.4.2. NADPH cyt c reductase assayThe assay of NADPH cyt c reductase was carried out by the

method as described previously by Phillip and Langdon (1962)

with some modifications. 0.50 mM horse heart cyt c (preparedin 10 mM potassium phosphate buffer) and 0.30 mM potassiumphosphate buffer were mixed with microsomal suspension andNADPH was added to initiate reaction. Then rate of reaction was

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ead at 550 nm using UV-Vis spectrophotometer (SmartSpecTM

000, Bio-Rad, USA). The activity was expressed in n moles cyt ceduced/min/mg protein.

.4.3. UDP-glucuronosyl transferase assayUDP-glucuronosyl transferase activity was measured in liver

icrosomes using 4-methylumbelliferone (4-MU) as a substrateAitio, 1974). A stock solution of 4-MU was prepared in 0.1 Mris–HCl buffer (pH 7.4). For measuring the activity of UDP-lucuronosyl transferase, liver microsomes were added to 50 timesiluted stock solution of 4-MU and 2 mM UDPGA was then added to

nitiate reaction. Fluorescence was monitored in a Perkin-Elmer flu-rimeter (LS-45) at excitation 355 nm, emission 460 nm and resultas expressed in n mole/min/mg microsomal protein.

.4.4. Glutathione S-transferase (GST) assayGST activity was estimated in cytosol (prepared after differen-

ial centrifugation of liver homogenate) according to the methods described previously by Habig et al. (1974). The cytosolic sam-le was mixed with 0.3 M KH2PO4 buffer and 7.5 mM GSH and-Chloro-2,4 dinitrobenzene (7.5 mM) was added to initiate reac-ion. Kinetics of the enzyme was read at 340 nm. The activity wasxpressed in units/mg cytosolic protein.

.4.5. Aminopyrine demethylase assayAminopyrine demethylase activity was estimated using colori-

etric procedure of Nash (1953) and Cochin and Axelrod (1959)ith some modifications. Briefly, post-mitochondrial supernatant

ample was incubated with 10 mM aminopyrine solution, co-actor mixture (containing 5 mM magnesium chloride, 50 mM G6P,.5 mM NADPH, 25 mM nicotinamide and 50 mM semicarbazide)nd 0.5 M sodium phosphate buffer pH 7.4 at 37 ◦C for 30 min.hen 15% zinc sulphate solution and barium hydroxide saturatedolutions were added to the incubation mixture and centrifugedt 3000 rpm for 10 min. To the supernatant, Nash reagent wasdded and incubated at 60 ◦C for 30 min. Then the solution wasead at 415 nm. Aminopyrine demethylase activity was expressedn n mole formaldehyde formed/min/mg protein.

.4.6. Aniline hydroxylase assayThe aniline hydroxylase assay was carried out according

o the method as described previously by Imai et al. (1966).ost-mitochondrial supernatant sample was incubated with

mM aniline, co-factor mixture (containing 1.3 units glucose-6-hosphate, 0.32 mM NADP and 0.05 M KH2PO4 buffer pH 7.4) and.05 M KH2PO4 buffer pH 7.4 at 37 ◦C for 60 min. Then 20% TCAolution was added to the incubation mixture and centrifuged at000 rpm for 10 min. To the supernatant 10% sodium carbonate and% phenol reagent were added and incubated at room temperatureor 30 min. Then the solution was read at 640 nm and activity wasxpressed in n moles/min/mg protein.

.5. Evaluation of alanine transaminases (ALT) and aspartateransferases (AST)

.5.1. ALT assayALT activity was estimated using colorimetric procedure of

eitman and Frankel (1957). Briefly, substrate was incubated for min at 37 ◦C and 10% liver homogenate sample was added andgain incubated at 37 ◦C for 30 min. Then DNPH reagent was added

o the incubation mixture and kept at room temperature for 20 min.o the above mixture 0.4 N NaOH was added and kept at roomemperature for 10 min. Then the solution was read at 540 nm andctivity was expressed in IU/l.

gic Pathology 65 (2013) 751– 758 753

2.5.2. AST assayAST activity was estimated using colorimetric procedure of

Reitman and Frankel (1957). Briefly, substrate was incubated for5 min at 37 ◦C and 10% liver homogenate sample was added andagain incubated at 37 ◦C for 60 min. Then DNPH reagent was addedto the incubation mixture and kept at room temperature for 20 min.To the above mixture 0.4 N NaOH was added and kept at roomtemperature for 10 min. Then the solution was read at 540 nm andactivity was expressed in IU/l.

2.5.3. Lactate dehyrogenase (LDH) assayLDH activity was estimated as described by Kornberg (1969)

with some modifications. Briefly, liver homogenate was centrifugedat 5000 rpm for 30 min and supernatant was collected for estima-tion. The supernatant was mixed with 0.1 M sodium phosphatebuffer and 10 mM sodium pyruvate. Then reaction was initiatedwith the addition of 4.22 mM NADH and rate of reaction was readat 340 nm. Results were expressed in n moles/mg protein.

2.6. Total protein

Total protein in liver homogenate and hepatic cellular frac-tions (post-nuclear supernatant, post-mitochondrial supernatantand microsomes) were assayed using Lowry et al. method (1951).

2.7. Histological studies

The injection of sodium pentobarbital (50 mg/kg, I.P.) was usedto anesthetise animals of both normoxic and hypoxic groups (sixanimals/group) and intracardial perfusion was done with 0.1 M PBS(pH 7.4) followed by 4% formaldehyde. Then liver was carefullydissected out and post-fixed in the same fixative for 48 h. Paraf-fin blocks were then prepared after dehydration, clearing and waximpregnation. 5-�m sections were prepared with a rotary micro-tome and deparaffinised in xylene. The histological sections werestained with haematoxylin and eosin (H&E) and observed underlight microscope for any morphological or pathological changes inliver architecture and photomicrographs were taken.

2.8. Immunoblotting of CYP2C9

The time dependent change in level of CYP2C9 protein onexposure to AHH (6 or 24 h) was determined by Western blot.The microsomal samples were mixed in sample buffer containing1% SDS, 2% 2-mercaptoethanol, and 10% glycerol and were heatreduced in boiling water bath, whereas gel and running buffercontained 0.1% and 0.2% SDS, respectively. The molecular weightwas determined by using standard protein markers (Broad range6.9–200 kDa, Bio-RaD). Then sample proteins were electrophoret-ically resolved on 4% (v/v) stacking and 8% (v/v) separationpolyacrylamide gels and electro-transferred onto a nitrocellulosemembrane pre-soaked in transfer buffer (20% methanol, 0.3% Trisand 1.44% glycine) using semi-dry transblot module (Bio-RaD, USA).The membranes were blocked with 5% non-fat milk, washed withPBST (0.01 M phosphate buffer saline, pH 7.4, 1 ml of 0.01%Tween-20) and incubated with primary rabbit polyclonal anti-CYP2C9antibody (1:1000 dilution) for 3 h at room temperature. The blotswere then washed extensively with PBST and incubated with sec-ondary antibody (Goat anti-rabbit IgG-HRP conjugated, Santa Cruz,CA, USA) diluted in 3% non-fat milk for 2 h at room temperature.Membranes were then finally washed with PBST and the bands

were developed on X-ray film using chemiluminescent substrate(Chemiluminescent peroxidise substrate-3, Sigma–Aldrich). Thebands thus obtained on the films were quantified by densitometry(ImageJ Software) to determine level of the protein.

7 xicologic Pathology 65 (2013) 751– 758

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Fig. 1. Effect of acute hypobaric hypoxia on glutathione S-transferase (GST) activityin ibuprofen treated rats. Value are mean ± SEM; N = 6 per group. *p < 0.05 comparedwith normoxic rats.

ular region. After exposure to AHH the general architecture of livertissue samples was intact and normal. However, slight histopatho-logical damage including sinusoidal dilation (+) in centrilobular

54 S. Gola et al. / Experimental and To

.9. Evaluation of pharmacokinetics

In another set of experiment, the effect of AHH on PK of ibupro-en was evaluated. Experimental animals of both normoxic andypoxic groups (six animals/group) were treated with single dosef ibuprofen (80 mg/kg body weight, p.o.), immediately after AHHxposure (6 or 24 h). Ibuprofen was administered after exposureecause pressure in the decompression chamber cannot be releasedepeatedly during hypobaric hypoxia exposure. The blood sam-les were collected pre-dose which served as 0 min time point andt 15 min, 30 min, 1, 2, 3, 4 and 6 h post-dose for each hypoxiaxposure period through orbital sinus. All blood samples weremmediately centrifuged, and the plasma was separated and storedt −20 ◦C until analyzed.

.9.1. Assay procedureThe plasma concentration of ibuprofen was estimated using

everse phase-high performance liquid chromatography (RP-HPLC)rocedure with ultraviolet detection at 220 nm using 20 �l plasmaample and mefenamic acid as internal standard as previouslyescribed (Shah and Jung, 1985). The HPLC system consisted ofaters (Milford, MA, USA) 600-solvent delivery pump, Waters

17plus auto sampler, Waters 2487 dual � Absorbance Detectornd Purospher® Star RP-18e (5 �m) (150 mm × 4.6 mm) analyti-al column. The mobile phase composed of 58% acetonitrile, 37%ater, 5% methanol and 0.05% OPA (v/v) filtered through nylonembrane filter and delivered in isocratic mode at a flow rate of

.0 ml/min. The concentration data obtained from analytical studyas entered in WinNonlin pharmacokinetic software Version 5.1,

cientific Consultants, USA for further analyzing the pharmacoki-etic variables viz. elimination half-life (T½), mean residence timeMRT), clearance (Cl) and volume of distribution (Vd).

.10. Statistical analysis

Data are expressed as mean ± SEM and statistical significanceetween control and hypoxic values was analyzed using Student t-est using Graph Pad Prism 2.01 (Graph Pad Software Inc., La Jolla,A, USA). A p-value < 0.05 was considered statistically significant.

. Results

.1. Effects of acute hypobaric hypoxia on phase I and II drugetabolizing enzymes

Drug metabolizing enzyme (phase I and II) activities were ana-yzed after exposure of rats to AHH for 6 or 24 h. The resultsndicated a significant reduction in GST dependent detoxificationystem. A significant reduction in glutathione conjugation wasbserved as evidenced by decreased GST activity at 6 and 24 hypoxia exposure by 15% and 23% (p < 0.05) respectively in cytosolic

raction of rat liver (Fig. 1). Other detoxification enzymes, involvedn metabolism of endogenous and exogenous substances, like totalYP450, NADPH cyt c reductase, UDP-glucuronosyl transferase,niline hydroxylase and aminopyrine demethylase showed no sig-ificant change after ibuprofen treatment under AHH (Table 1).

.2. Effects of acute hypobaric hypoxia on ALT, AST and LDH

AST activity was significantly increased by 20% and 24% (p < 0.05)n 6 and 24 h hypoxic rats, respectively (Fig. 2). Similarly ALT andDH also showed increased activity (p < 0.05) after exposure to AHHFigs. 3 and 4).

Fig. 2. Effect of acute hypobaric hypoxia on aspartate transferase (AST) activity inibuprofen treated rats. Value are mean ± SEM; N = 6 per group. *p < 0.05 comparedwith normoxic rats.

3.3. Effects of acute hypobaric hypoxia on liver histology

The 40× high power photomicrograph of liver sample from 6 h(Fig. 5A) and 24 h (Fig. 5B) normoxia animal revealed normal livercell architecture with normal hepatic parenchyma in the centrilob-

Fig. 3. Effect of acute hypobaric hypoxia on alanine transaminase (ALT) activity inibuprofen treated rats. Value are mean ± SEM; N = 6 per group. *p < 0.05 comparedwith normoxic rats.

S. Gola et al. / Experimental and Toxicologic Pathology 65 (2013) 751– 758 755

Table 1Effect of acute hypobaric hypoxia (6 h and 24 h exposure) on phase I and II hepatic metabolism of ibuprofen in rats.

Parameter Normoxia (6 h) Hypoxia (6 h) Normoxia (24 h) Hypoxia (24 h)

Total CYP 450 Content (nmol/mg protein) 0.77 ± 0.02 0.75 ± 0.01 0.78 ± 0.03 0.68 ± 0.02NADPH Cyt c reductase (nmol cyt c reduced/min) 180.95 ± 2.2 176.19 ± 2.9 187.50 ± 4.34 182.22 ± 1.61Aniline hydroxylase (nmol/min/mg protein) 2.53 ± 0.10 2.44 ± 0.50 2.48 ± 0.07 2.24 ± 0.17Aminopyrine demethylase (nmol/min/mg protein) 6.67 ± 0.30 6.52 ± 0.40 9.70 ± 0.05 9.30 ± 0.40UDP-glucuronosyl transferase (nmol/min/mg protein) 0.50 ± 0.02

Value are mean ± SEM; N = 6 per group.

Fig. 4. Effect of acute hypobaric hypoxia on lactate dehydrogenase (LDH) activity inibuprofen treated rats. Value are mean ± SEM; N = 6 per group. *p < 0.05 comparedw

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in 24 h hypoxic group (Fig. 8). It was also observed that clearance(Cl) calculated from blood plasma was insignificantly diminished

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egion was observed in the liver tissues of rats exposed to 6 hFig. 5C) of AHH. On the other hand, histological analyses of theiver tissue samples of rats exposed to 24 h (Fig. 5D) of AHH showed

urther damage including mild sinusoidal dilation (++) and vascularongestion (+) in centrilobular region. However no necrosis, fatty

ig. 5. Photomicrographs showing haematoxylin and eosin stained liver tissue of rats.

ormal liver cell architecture with normal hepatic parenchyma in the centrilobular regilight sinusoidal dilatation around the central vein. No fatty change was seen; (D) 24 h hild sinusoidal dilation and vascular congestion around the central vein. CV = Central Vei

0.49 ± 0.01 0.49 ± 0.01 0.46 ± 0.02

change or inflammation could be observed in any of the photomi-crographs.

3.4. Effects of acute hypobaric hypoxia on CYP2C9 level

Protein expression levels of CYP2C9 in microsomal proteinswere studied using Western blot (Fig. 6A and B). The results showeda significant decrease (11%, p < 0.05) in level of CYP2C9 proteinafter 24 h hypobaric hypoxic exposure (Fig. 6B), while no signifi-cant change was observed after 6 h exposure in rat liver microsomalpreparation (Fig. 6B).

3.5. Effects of acute hypobaric hypoxia on pharmacokinetics ofibuprofen

The PK studies showed no statistically significant difference inthe PK variables calculated in plasma of 6 h hypoxia exposed rats(Table 2). However in response to 24 h hypoxic exposure, elimi-nation half-life (T½) of ibuprofen increased significantly by 42%(p < 0.05) as compared to normoxic rats (Fig. 7). Further, there was asignificant increase in mean residence time (MRT) by 51% (p < 0.05)

and volume of distribution (Vd) insignificantly increased afterexposure to acute hypoxia of 24 h (Table 2).

(A) 6 h and (B) 24 h control group (normoxia with ibuprofen treatment) showingon. (C) 6 h hypoxia (with ibuprofen treatment) exposed group showing an area ofypoxia (with ibuprofen treatment) exposed group show further damage includingn (400×). Scale bar is 10 �m.

756 S. Gola et al. / Experimental and Toxicologic Pathology 65 (2013) 751– 758

Fig. 6. (A) Effect of acute hypobaric hypoxia (6 and 24 h) on CYP2C9 level as determined

software. N = 6 per group. *p < 0.05 compared with normoxic rats.

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. Discussion

Liver plays a vital role in detoxification mechanism. Hypoxiaeads to various pathophysiological conditions and is also associ-ted with the alteration in hepatic drug metabolism and toxicity,s it affects pathways and rate of drug detoxification and can also

ig. 8. Effect of acute hypobaric hypoxia on mean residence time (MRT) of ibuprofen.alue are mean ± SEM; N = 6 per group. *p < 0.05 compared with normoxic rats.

by immunoblotting. (B) Corresponding densitometric graph as analyzed by ImageJ

change the vulnerability of cells to injury. This occurs becauseoxygen is essential for drug metabolizing systems directly asa substrate for drug oxidations as a terminal electron acceptorthat controls other processes dependent upon the cellular redoxstate and as the terminal electron acceptor in the mitochondrialsynthesis of high energy bonds required for drug transport andconjugation reaction. Hypoxia affects activity of many enzymesresponsible for metabolism of drugs and it also hinders the dispo-sition of variety of drugs which can cause alteration in therapeuticregime (Costa, 1990; Jones et al., 1989; Jürgens et al., 2002; Shanet al., 1992; Woodrooffe et al., 1995). Due to lack of sufficient stud-ies regarding the altered hepatic metabolism, PK and therapeuticregime affected under hypoxic condition, the present study inves-tigates the effects of AHH on the hepatic drug metabolism and PKof ibuprofen.

As phase-II pathways of drug metabolism are dependent onfunctions of the mitochondria, hypoxic conditions cause effects onmitochondrial oxygenation characteristics which might have fur-ther impact on these detoxification systems (Aw et al., 1991). Asignificant decrease in GST activity in the liver during exposureto acute hypobaric hypoxia was observed in present study, whichsuggests that the glutathione conjugation gets impaired becauseof lower availability of essential co-factors, such as NAD and ATPunder hypoxic conditions. UDP-glucuronosyl transferase is crucialenzyme in detoxification system of ibuprofen that utilizes conjuga-tion reaction i.e. glucuronidation. Present study revealed that AHHled to an insignificant decrease in microsomal UDP-glucuronosyltransferase activity in 24 h hypoxia exposed rats compared to nor-moxic rats. Similarly, previous studies carried out by Aw and Jones

(1982, 1984) reported that glucuronide conjugation under hypoxiawas found to be decreased due to decrease in UTP and glucose forsynthesis of UDP-glucose and UDP-glucuronic acid. Thus, decreasedglucuronidation under oxygen deficiency is related to, decreased

Table 2Effect of acute hypobaric hypoxia (6 h and 24 h exposure) on pharmacokinetics ofibuprofen in rats.

Parameter Normoxia Hypoxia (6 h) Hypoxia (24 h)

Clearance (Cl) (ml/min) 2.62 ± 0.19 2.60 ± 0.33 2.19 ± 0.24Volume of distribution

(Vd) (lt)0.41 ± 0.03 0.44 ± 0.12 0.47 ± 0.03

Value are mean ± SEM; N = 6 per group.

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nergetic of the cell as a result of impaired cytochrome oxidaseunction. However, acute hypoxia for 6 and 24 h did not cause anyignificant change in total CYP 450 content, NADPH cyt c reduc-ase, aniline hydroxylase and aminopyrine demethylase activities.hough the organ/body weight ratio did not change significantlyfter AHH exposure (not shown), the altered hepatic metabolismndicated the effect of hypoxia on liver physiology under hypoxictress.

For investigating lysosomal destruction and cellular deteriora-ion ALT and AST were studied, which were observed to be elevatedndicating that at HA the disruption of lysosomes occurs concomi-antly with changes in cell membrane permeability and release ofhese enzymes. LDH activity was also found to be increased afterbuprofen treatment in 24 h hypoxia exposed group indicating LDHeakage which is also an effect of hypoxia per se.

Histological examinations of liver tissue samples were per-ormed to evaluate evidences of the effects of AHH on liverntegrity. Liver samples were collected from hypobaric hypoxiaxposed-ibuprofen treated and normoxic-ibuprofen treated ratsnd examined for the potential presence of histopathological indi-ations. The liver histology results indicated only milder effect likeild sinusoidal dilation (++) and vascular congestion (+) in cen-

rilobular region under AHH of 24 h. These results are similar tobservations of earlier studies conducted for assessing liver damagender chronic intermittent hypoxia which revealed that oxidativetress plays an important role in the mechanism (Feng et al., 2011;avransky et al., 2007, 2009). Because under lower O2 concentrationradient, the liver is perfused with partially deoxygenated por-al blood which make it prone to damage caused due to hypoxiconditions (Bonkovsky et al., 1986).

CYP2C9 forms the two metabolites of ibuprofen 3- and 2-ydroxyibuprofen (Davies and Anderson, 1997) and the 3-hydroxyetabolite formed is metabolized almost completely to the corre-

ponding carboxy derivative via cytosolic dehydrogenases (Davies,998; Hamman et al., 1997; Rudy et al., 1991). Our present study

ndicates the effect of AHH on the protein expression level ofYP2C9 in rat microsomal preparations. The protein expression

evel of CYP2C9 is observed to be down-regulated under 24 hypoxia exposure, which in turn reduces the biotransformation ofrugs like ibuprofen cleared by CYP2C9. These results were in accor-ance with the findings of biochemical analysis of the enzymesesponsible for hepatic metabolism of drugs and previously con-ucted studies (Fradette and Du Souich, 2004; Michaelis et al.,005). In the present study, we have shown that acute hypoxia canignificantly affect activities of hepatic detoxification enzymes inbuprofen metabolism and protein expression level of cytochrome450 isoform i.e. CYP2C9 due to dependence of drug metabolizingystem on oxygen concentration.

Furthermore, in the present study the influence of AHH onhe PK of ibuprofen has also been investigated. Although sig-ificant differences were not seen for all parameters measured,owever a significant increase was observed in T½ values after AHHxposure of 24 h as compared to normoxic group. There was a sig-ificant increase in mean residence time of ibuprofen after 24 hypoxia exposure. It has been reported earlier that the dispositionf drugs which are highly protein-bound and renally eliminated areost prone to physiological changes experienced by body at HA

Arancibia et al., 2004) and ibuprofen exhibit high plasma protein-inding tendency (98–99%) (Lockwood et al., 1983a, 1983b). Dueo which, the clearance, although not statistically significant, iseduced in 24 h hypoxia group. The observations of our study aren coherence with the previous PK studies conducted with drugs

ike lithium (Arancibia et al., 2003), acetazolamide (Ritschel et al.,998), prednisolone (Arancibia et al., 2005), furosemide (Arancibiat al., 2004), mepridine (Ritschel et al., 1996) at HA which indicatedltered disposition of these drugs.

gic Pathology 65 (2013) 751– 758 757

The results of present study indicate that AHH can impair dispo-sition of ibuprofen however, it requires further investigation underchronic hypobaric hypoxic conditions. These observations providea basis for study of disposition of array of therapeutic drugs and forintervention of drug therapies under hypoxic conditions.

5. Conclusion

The results of the present study suggest that hepatic drugmetabolizing mechanism and PK is prone to hypobaric hypoxiamediated changes which is indicated by impairment in conjugationpathway of ibuprofen metabolism, decreased level of cytochromeP450 isoform i.e. CYP2C9 protein along with enhanced AST, LDHlevels and altered PK. Exposure to AHH causes alteration in hep-atic drug metabolism and PK which requires further investigationunder chronic hypobaric hypoxia that this in turn may affect thedose regime for optimal effect and safe therapy of drug at HA. Suchknowledge will endow the basis for the study of disposition ofvarious therapeutic drugs and their safe and effective therapy inadverse environmental vagaries such as HA hypoxia.

Acknowledgements

This study was supported and funded by the Defence Institute ofPhysiology and Allied Sciences (DIPAS), DRDO, Ministry of Defence,Government of India. The authors are grateful to Director, DIPAS forproviding kind support and encouragement to the study.

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