Research Article

Received: 6 January 2012, Revised: 10 April 2012, Accepted: 11 April 2012 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.2774

Modulation of aryl hydrocarbon receptor-regulated genes by acute administration ofammonium metavanadate in kidney, lung andheart of C57BL/6 miceGhada Abdelhamid, Issa E.A. Amara, Anwar Anwar-Mohamed andAyman O.S. El-Kadi*

ABSTRACT: We recently reported that vanadium (V5+) was able to decrease the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 and Nqo1 at mRNA, protein and catalytic activity levels in mouse hepatoma Hepa 1c1c7and human hepatoma HepG2 cells. However, little is known regarding the in vivo effects. Thus, the objective of this studywas to investigate whether similar effects would occur at the in vivo level. Therefore, we examined the effect of exposureto V5+ (5mgkg�1) with or without TCDD (15mgkg�1) on the AhR-regulated genes in kidney, lung and heart of C57BL/6 Jmice. Our results demonstrated that V5+ alone significantly decreased Cyp1b1 protein and catalytic activity levels in kidneyat 24h. Moreover, it significantly potentiated Nqo1 and Gsta1 gene expression in the heart, and only Gsta1 gene expressionin the lung. Upon co-exposure, we found that V5+significantly inhibited the TCDD-mediated induction of Cyp1a1, Cyp1a2 andCyp1b1 mRNA, protein and catalytic activity levels in the kidney at 24 h. On the other hand, V5+ significantly potentiated theTCDD-mediated induction of Nqo1 and Gsta1 protein and activity levels in the kidney. Cyp1a1, Cyp1b1, Nqo1 mRNA, proteinand catalytic activity levels in the lung were significantly potentiated at 6 h. Interestingly, all tested genes in the heartwere significantly decreased at 6 h with the exception of Gsta1 mRNA. The present study demonstrates that V5+ modulatesTCDD-induced AhR-regulated genes. Furthermore, the effect on one of these enzymes could not be generalized to otherenzymes even if it was in the same organ. Copyright © 2012 John Wiley & Sons, Ltd.

Keywords: aryl hydrocarbon receptor; C57B1/6 mouse; vanadium; carcinogenesis

*Correspondence to: Ayman O.S. El-Kadi, Faculty of Pharmacy and PharmaceuticalSciences, 2142 J Katz Group-Rexall Centre for Pharmacy and Health Research,University of Alberta, Edmonton, Alberta, Canada T6G 2E1.E-mail: aelkadi@pharmacy.ualberta.ca

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta,Edmonton, Alberta, Canada T6G 2N8

INTRODUCTIONPolyhalogenated aromatic hydrocarbons, including dioxins anddioxin-like polychlorinated biphenyls, induce a wide variety ofeffects in mammals, birds and fish such as immunotoxicity,carcinogenicity and metabolic changes. These compounds bindto an intracellular receptor, known as the aryl hydrocarbonreceptor (AhR) (Sonneveld et al., 2007). AhR is a ligand-activatedcytoplasmic transcription factor that belongs to the basic-helix-loop-helix protein family (Nebert et al., 1984). In the absence of aligand, AhR is associated with 90 kDa heat shock protein-90(HSP90), the 23 kDa heat shock protein (p23) and hepatitis B virusX-associated protein 2 (XAP2). Following ligand binding, AhRtranslocates to the nucleus, dissociates from the complex, andforms a heterodimerwith the AhR nuclear translocator (Hankinson,1995). The whole complex then acts as a transcription factor thatbinds to a specific DNA recognition sequence, termed thexenobiotic responsive element, located in the promoter region ofa number of AhR-regulated genes. Among the AhR-regulatedgenes are those encoding a number of xenbiotic metabolizingenzymes, including four phase I enzymes [cytochrome P450 1A1(CYP1A1), CYP1A2, CYP1B1 and CYP2S1] and four phase II enzymes[NAD(P)H: quinone oxidoreductase-1 (NQO1), glutathione-S-transferase A1 (GSTA1), cytosolic aldehyde dehydrogenase-3and UDP-glucuronosyltransferase 1A6 (UGT1A6) (Nebert andDuffy, 1997; Rivera et al., 2002).

J. Appl. Toxicol. (2012) Copyright © 2012 John

Phase I reactions include oxidation, reduction, hydrolysis,cyclization and decyclization. If the metabolites of phase Ireactions are sufficiently polar, they may be excreted. However,many phase I products are not eliminated rapidly and canundergo a subsequent reaction. Several AhR ligands are not onlyagonists of AhR, but also substrates for the induced phase Ienzymes with the exception of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD). The conversion of these AhR ligands into diol epoxidecompounds by CYP1A1 and CYP1A2 results in the formation ofcovalent adducts when these genotoxic metabolites react withguanines in critical genes, potentially initiating tumorigenesisand other toxic responses (Spink et al., 2002). It is well known thatthe induction of phase II enzymes serves as a detoxificationmechanism for many mutagens, carcinogens and other toxiccompounds; these are phase II reactions, usually known asconjugation reactions, where an endogenous substrate combineswith the newly incorporated functional group to form a highlypolar conjugate. The coupling of phase II enzymes such as NQO1

Wiley & Sons, Ltd.

G. Abdelhamid et al.

and GSTA1 to these phase I enzymes is necessary to detoxify thegenerated toxic metabolites prior to their excretion (Nebert andDalton, 2006).

Vanadium (V5+) is a poorly understood trace element that hasbecome a subject of interest among scientists since environ-mental contamination by V5+ has dramatically increased. Thishas occurred especially in the most developed countries owingto the widespread use of fossil fuels, many of which liberate fineparticulates of V5+ to the atmosphere during combustion (Baran,2008). Although many biochemical and physiological functionshave been suggested for this element, V5+ still does not have awell-known role in the higher forms of life (Baran, 2008). In vitroand in vivo studies have demonstrated that V5+ compoundsexert protective effects against chemical-induced carcinogen-esis, mainly through modifying various xenobiotic metabo-lizing enzymes (Evangelou, 2002). Yet, it has been seenthat high V5+ concentrations are found in tissue samples ofactual tumors as compared with those in normal tissues(Evangelou, 2002).

Humans and other species may be exposed to V5+ through theatmosphere, food and water. Food intake of V5+ in adults hasbeen estimated to range from 10–60 mg/day (Evangelou, 2002).In addition, the highest level of V5+ supplements in multivitaminproducts reaches 25mg per tablet or capsule. Furthermore, weighttraining athletes are reported to use up to 18.6mg per day as abody-building supplement (Barceloux, 1999). An estimate of thetotal body pool of V5+ in healthy individuals is from 100 to200mg (Byrne and Kosta, 1978). Samples from the drinking watersupplies in the USA showed that the concentration of V5+ canreach 10mg l�1 (Craun et al., 1981). Moreover, concentrationranges have been reported at 4.1–13mg l�1 for the rural UK(Galloway et al., 1982) and 0.12–0.65mg l�1 in Switzerland (Atteia,1994) . Levels in rain ranged from 1.1 to 46mg l�1 for rural andurban sites in North America and Europe (Galloway et al., 1982).Based on these reported concentrations, Mamane and Pirrone(1998) calculated total deposition rates of vanadium at 0.1–10 kgha�1 per year for urban sites, and 0.01– 0.1 kgha�1 per year forrural sites (Mamane and Pirrone, 1998). If we take into considerationthe fact that heavy metals such as V5+ are significantly deposited inhepatocytes and kidneys, the concentrations used in the currentstudy are of great relevance to humans.

The toxicological effect of individual AhR ligands is wellestablished (Germolec et al., 1996; Volotinen et al., 2009; Brauzeet al., 2006). Moreover, few studies have examined thecombined toxicological effects of these ligands with heavymetals such as V5+. At the in vitro level, we recently reported thatV5+ was able to decrease the TCDD-mediated induction ofCyp1a1 and Nqo1 at mRNA, protein and catalytic activity levelsin the mouse hepatoma Hepa 1c1c7 cells, which were derivedfrom BW7756 mouse hepatoma that arose in a C57BL/6 mouse(Anwar-Mohamed and El-Kadi, 2008, 2009) and in humanhepatoma HepG2 cells, which were derived from the liver tissueof a 15-year-old Caucasian Americanmale with a well-differentiatedhepatocellular carcinoma (Abdelhamid et al., 2010a, 2010b).However, to the best of our knowledge, there has been noprevious attempt to examine the effect of V5+ on the regulationof AhR-regulated genes in the presence and absence of TCDD inextrahepatic tissues in vivo. Therefore, we hypothesize that V5+

may alter the capacity of AhR-regulated genes, which in turn willinfluence the AhR ligands mediated toxicities. Thus, the presentstudy aims to address the possible effect of V5+ on AhR-regulatedgenes, in the kidney, lung and heart.

Copyright © 2012 Johnwileyonlinelibrary.com/journal/jat

MATERIALS AND METHODS

Materials

TRIzol reagent was purchased from Invitrogen (Carlsbad, CA,USA). A high-capacity cDNA reverse transcription kit, SYBR GreenSuperMix and 96-well optical reaction plates with opticaladhesive films were purchased from Applied Biosystems (FosterCity, CA, USA). Real-time PCR primers were synthesized byIntegrated DNA Technologies Inc. 1-Chloro-2,4-dinitrobenzene,2,6-dichlorophenolindophenol, 7-ethoxyresorufin, 7-methoxyresorufin, anti-goat IgG peroxidase secondary antibody, dicou-marol, protease inhibitor cocktail and ammonium metavanadate(NH4VO3) were purchased from Sigma Chemical Co. (St Louis,MO, USA). TCDD, >99% pure, was purchased from CambridgeIsotope Laboratories (Woburn, MA, USA). Chemiluminescencewestern blotting detection reagents were from GE HealthcareLife Sciences (Piscataway, NJ, USA). Nitrocellulose membranewas purchased from Bio-Rad Laboratories (Hercules, CA, USA).Cyp1a1 mouse polyclonal primary antibody, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) rabbit polyclonal antibodyand anti-rabbit IgG peroxidase secondary antibody werepurchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA,USA). Antimouse IgG peroxidase secondary antibody waspurchased from R&D Systems, Inc. (Minneapolis, MN, USA). Allother chemicals were purchased from Fisher Scientific (Toronto,Ontario, Canada).

Mouse Handling

Male C57BL6 mice, aged 10–12weeks (Charles River Labora-tories) were used in this study. Mice were group-housedunder standard conditions, three per cage with food andwater available ad libitum, and were maintained on a 12 hlight–dark cycle. Mice were treated in compliance with Uni-versity of Alberta Health Sciences Animal Policy and WelfareCommittee guidelines. All experiments included matchingnumbers of male mice.

Biohazard Precaution

TCDD is toxic and a likely human carcinogen. All personnel wereinstructed as to safe handling procedures. Lab coats, gloves andmasks were worn at all times, and contaminated materials werecollected separately for disposal by the Office of EnvironmentalHealth and Safety at the University of Alberta.

Animal Treatment

Male C57BL/6 J (22–30 g) mice were injected intraperitoneally(i.p.) with V5+ (dissolved in saline) at 5mg kg�1, in the presenceand absence of 15mg kg�1 TCDD (TCDD was dissolved in DMSO,followed by further dilution in corn oil). The mice were segre-gated into four groups. The first group (n=12) comprised con-trol mice and received saline plus corn oil. The second group(n=12) comprised V5+-treated mice and received V5+ dissolvedin saline plus corn oil. The third group (n= 12) comprisedTCDD-treated mice and received TCDD dissolved in corn oil plussaline. The fourth group (n= 12) comprised V5+ plus TCDD-treated mice and received V5+ dissolved in saline plus TCDD dis-solved in corn oil. Thereafter, the animals were euthanized aftera single injection at 6 h (n= 6) or 24 h (n= 6) by cervical

J. Appl. Toxicol. (2012)Wiley & Sons, Ltd.

Differential modulation of metabolizing enzymes by vanadium

dislocation. Kidney, lung and heart tissues were excised, imme-diately frozen in liquid nitrogen, and stored at �80 �C untilanalysis.

RNA Extraction and cDNA Synthesis

Total RNA from the frozen tissues was isolated using TRIzol reagent(Invitrogen) according to the manufacturer’s instructions andquantified by measuring the absorbance at 260 nm. RNA qualitywas determined by measuring the 260/280 ratio. Thereafter, first-strand cDNA synthesis was performed by using the high-capacitycDNA reverse transcription kit (Applied Biosystems) according tothe manufacturer’s instructions. Briefly, 1.5mg of total RNAfrom each sample was added to a mix of 2.0ml of 10� reversetranscriptase buffer, 0.8ml of 25� dNTP mix (100mM), 2.0ml of10� reverse transcriptase random primers, 1.0ml of MultiScribereverse transcriptase and 3.2ml of nuclease-free water. The finalreaction mix was kept at 25 �C for 10min, heated to 37 �C for120min, heated for 85 �C for 5 s, and finally cooled to 4 �C.

Quantification by Real-time PCR

Quantitative analysis of specific mRNA expression wasperformed using real-time polymeras chain reaction (PCR) bysubjecting the resulting cDNA to PCR amplification using 96-welloptical reaction plates in the ABI Prism 7500 System (AppliedBiosystems). The 25ml reaction mix contained 0.1 ml of 10mMforward primer and 0.1ml of 10mM reverse primer (40 nM finalconcentration of each primer), 12.5ml of SYBR Green UniversalMastermix, 11.05ml of nuclease-free water and 1.25ml of cDNAsample. The primers used in the current study are listed inTable 1. Assay controls were incorporated onto the same plate,namely, no-template controls, to test for the contamination ofany assay reagents. After sealing the plate with an opticaladhesive cover, the thermocycling conditions were initiated at95 �C for 10min, followed by 40 PCR cycles of denaturation at95 �C for 15 s and annealing/extension at 60 �C for 1min. Themelting curve (dissociation stage) was performed by the endof each cycle to ascertain the specificity of the primers and thepurity of the final PCR product.

Real-time PCR Data Analysis

The real time-PCR data were analyzed using the relative geneexpression i.e. (ΔΔCT) method as described in Applied BiosystemsUser Bulletin No. 2 and explained further by Livak and Schmittgen(2001). Briefly, the ΔCT values were calculated in every sample foreach gene of interest as follows: CT gene of interest – CT reporter gene,

Table 1. Primers sequences used for real-time PCR reactions

Gene Forward primer

B-actin 5′-TAT TGG CAA CGA GCG GTT CC-3′Cyp1a1 5′-GGT TAA CCA TGA CCG GGA ACT-3′Cyp1a2 5′-TGG AGC TGG CTT TGA CAC AG-3′Cyp1b1 5′-AAT GAG GAG TTC GGG CGC ACA-3′Gsta1 5′-CCC CTT TCC CTC TGC TGA AG-3′Nqo1 5′-GGA AGC TGC AGA CCT GGT GA-3′

J. Appl. Toxicol. (2012) Copyright © 2012 John

with Gapdh as the reporter gene. Calculation of relative changesin the expression level of one specific gene (ΔΔCT) was performedby subtraction ofΔCT of control (untreated control) from theΔCT ofthe corresponding treatment groups. The values and ranges givenin different figures were determined as follows: 2–ΔΔCT with ΔΔCT +SE and ΔΔCT – SE, where SE is the standard error of the mean ofthe ΔΔCT value.

Preparation of Microsomal and Cytosolic Protein Fractions

Tissue-specific microsomes were prepared using a previouslypublished method (Barakat et al., 2001; Elsherbiny et al., 2010).Briefly, all tissues were washed in ice-cold KCl (1.15%, KCl w/v),cut into pieces and homogenized separately in ice-cold sucrosesolution (1 g of tissue in 25ml of 0.25 M sucrose). Tissue homoge-nates were centrifuged at 600 g for 8min. The supernatant wasthen centrifuged at 12 000 g for 10min. Thereafter, supernatantsresulting from the previous step were mixed with 8mM CaCl2and centrifuged at 27 000 g for 15min. The consequential pelletswere suspended in 0.15 M KCl and recentrifuged at 27 000 g for15min. Final pellets were re-suspended in cold sucrose andsupernatant, cytosol, were stored at �80 �C. Thereafter,microsomal and cytosolic protein concentrations weredetermined by the Lowry method using bovine serum albuminas a standard (Lowry et al., 1951).

Western Blot Analysis

Western blot analysis was performed using a previouslydescribed method (Sambrook and Maniatatis, 1989). Briefly, themicrosomal or cytosolic proteins were dissolved in 1� samplebuffer, boiled for 5min, separated by 10% sodium dodecylsulfate–polyacrylamide gel (SDS–PAGE), and then electrophoret-ically transferred to nitrocellulose membrane. Protein blots werethen blocked overnight at 4 �C in blocking solution containing0.15 M sodium chloride, 3mM potassium chloride, 25mM Tris-base (TBS), 5% skim milk, 2% bovine serum albumin and 0.5%Tween-20. After blocking, the blots were incubated with thefollowing antibodies: polyclonal mouse anti-rat Cyp1a1/1a2,polyclonal rabbit anti-rat Cyp1b1, polyclonal goat anti-mouseNqo1, polyclonal goat anti-rat Gsta1 and polyclonal rabbit anti-mouse actin. Incubation with a peroxidase-conjugated goatanti-rabbit IgG secondary antibody for Nqo1, Cyp1b1 and actinor goat anti-mouse IgG secondary antibody for Cyp1a1/1a2, orrabbit anti-goat IgG secondary antibody for Gsta1 was carriedout. The bands were visualized using the enhanced chemilumi-nescence method according to the manufacturer’s instructions(GE Healthcare Life Sciences, Piscataway, NJ, USA). The intensity

Reverse primer

5′-GGC ATA GAG GTC TTT ACG GAT GTC-3′5′-TGC CCA AAC CAA AGA GAG TGA-3′5′-CGT TAG GCC ATG TCA CAA GTA GC-3′5′-GGC GTG TGG AAT GGT GAC AGG-3′5′-TGC AGC TTC ACT GAA TCT TGA AAG-3′5′-CCT TTC AGA ATG GCT GGC A-3′

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

G. Abdelhamid et al.

of the protein bands were quantified, relative to the signalsobtained for actin, using ImageJ software (National Institutes ofHealth, Bethesda, MD, http://rsb.info.nih.gov/ij.).

Microsomal Incubation and Measuring EROD and MRODCatalytic Activities

Cyp1a and Cyp1b activity in the microsomes were assessedusing 7-ethoxyresorufin O-deethylase activity (EROD) and 7-methoxyresorufin O-demethylase (MROD), respectively (Sinaland Bend, 1996). Microsomes from different tissues and differenttreatments (1mg protein ml�1) were incubated in the incuba-tion buffer (5mM magnesium chloride hexahydrate dissolved in0.5 M potassium phosphate buffer pH = 7.4) at 37 �C in a shakingwater bath (50 rpm). A pre-equilibration period of 5min wasperformed. The reaction was initiated by the addition of 1mM

NADPH. The concentrations of substrate were 2 mM for ERODand MROD. After incubation at 37 �C (5min for EROD, and10min for MROD assays), the reaction was stopped by adding0.5ml of cold methanol. The precipitated protein was removedby centrifugation for 5min at 120 g. Thereafter, the fluorescentproduct, resorufin, was measured in the supernatant using theBaxter 96-well fluorescence plate reader using excitation andemission wavelengths of 545 and 575 nm, respectively. Forma-tion of resorufin was linear with incubation time and proteinamount. Enzymatic activities were expressed as picomole ofresorufin formed per minute and per milligram of microsomalproteins.

Determination of Nqo1 ENZYMATIC activity

Nqo1 activity was determined by the continuous spectrophotomet-ric which quantities the reduction of 2,6-dichlorophenolindophenol(DCPIP) (Preusch et al., 1991; Ernster, 1967). Briefly, 20 mg ofcytosolic protein was incubated with 1ml of the assaybuffer [40 mM DCPIP, 0.2mM NADPH, 25mM Tris–HCl, pH 7.8,0.1% (v/v) Tween 20, and 0.7mgml�1 bovine serum albumin,0 or 30mM dicoumarol]. The rate of DCPIP reduction wasmonitored over 1.5 min at 600 nm with an extinction coefficient(e) of 2.1mM

–1 cm–1. The Nqo1 activity was calculated as thedecrease in absorbance per minute per milligram of total proteinof the sample, which quantitates the dicoumarol-inhibitablereduction of DCPIP.

Figure 1. Effect of co-exposure to V5+ and TCDD on Cyp1a1 mRNA inthe kidney, lung, and heart of C57BL/6 mice. Animals were injected i.p.

Determination of Gst Activity

Glutathione-S-transferase (Gst) activity was determined spectro-photometrically using 1-chloro-2,4-dinitrobenzene (CDNB) as asubstrate according to the method of Habig et al. (1974). Briefly,20 mg of cytosolic or microsomal protein was incubated with1mM CDNB, 1mM lower glutathione in 0.1 M potassium phos-phate buffer, pH 6.5 at 25 �C in a total volume of 1ml. Gst activitywas measured as the amount of CDNB conjugate formed by re-cording the absorbance at 340 nm for 1.5min with an extinctioncoefficient of 9600 M

�1 cm�1. The enzyme activity was expressedas nmoles per minute per milligram protein.

with 5mgkg�1 V5+ with or without 15mg kg�1 TCDD for 6 h (A) and24 h (B). Duplicate reactions were performed for each experiment, andthe values presented are the means of six independent experiments. +

Significant difference at P< 0.05, compared with control (C; untreatedanimals); * significant difference at P< 0.05, compared with respectiveTCDD (T) treated mice.

Statistical Analysis

Data are presented as means� standard error of the mean.Control and treatment measurements were compared using

Copyright © 2012 Johnwileyonlinelibrary.com/journal/jat

Student’s t test. Comparative gene expression across tissueswas analyzed using a one-way analysis of variance followed bya Student–Newman–Keuls post hoc comparison. A result wasconsidered statistically significant where P< 0.05.

RESULTS

Effect of Co-exposure toV5+ and TCDDonCyp1a1, Cyp1a2 andCyp1b1 mRNA in the Kidney, Lung and Heart of C57BL/6 Mice

In the kidney, lung and heart, V5+ alone did not cause any significantchanges in Cyp1a1 mRNA levels at 6 and 24h (Fig. 1A and B).Treatment with TCDD alone at 6h significantly induced Cyp1a1 inthe kidney, lung and heart by 983-, 218- and 284-fold, respectively(Fig. 1A). Similarly, treatment with TCDD for 24h caused a significantinduction of Cyp1a1 gene expression in the kidney, lung andheart by 1071-, 66- and 51-fold, respectively (Fig. 1A and B). Uponco-exposure to V5+ and TCDD, V5+ at 6h did not cause anysignificant change in TCDD-mediated induction of Cyp1a1 at themRNA levels in the kidney. V5+ at 6h significantly potentiated thelung Cyp1a1 mRNA levels by 1.85-fold. Alternatively, V5+ signifi-cantly inhibited the TCDD-mediated induction of Cyp1a1 mRNA inthe heart by 0.46-fold, compared with TCDD alone. At 24h, V5+

caused a significant inhibition of TCDD-mediated induction ofCyp1a1 mRNA in the kidney by 0.47-fold, compared withTCDD alone. However, V5+ was unable to significantly affect theTCDD-mediated induction of Cyp1a1 mRNA in the lung andheart (Fig. 1B).

Figure 2 shows the effect of co-exposure to V5+ and TCDD onCyp1a2 mRNA in the kidney, lung and heart. At 6 and 24 h, V5+

alone did not cause any significant changes in Cyp1a2 mRNA

J. Appl. Toxicol. (2012)Wiley & Sons, Ltd.

Figure 3. Effect of co-exposure to V5+ and TCDD on Cyp1b1 mRNA inthe kidney, lung, and heart of C57BL/6 mice. Animals were injected i.p.with 5mgkg�1 V5+ with or without 15mg kg�1 TCDD for 6 h (A) and24 h (B). Duplicate reactions were performed for each experiment, andthe values presented are the means of six independent experiments.+ Significant difference at P< 0.05, compared with control (C; untreatedanimals); * significant difference at P< 0.05, compared with respectiveTCDD (T) treated mice.

Figure 2. Effect of co-exposure to V5+ and TCDD on Cyp1a2 mRNA inthe kidney, lung, and heart of C57BL/6 mice. Animals were injected i.p.with 5mg kg�1 V5+ with or without 15mg kg�1 TCDD for 6 h (A) and24 h (B). Duplicate reactions were performed for each experiment,and the values presented are the means of six independent experiments.+ Significant difference at P< 0.05, compared with control (C; untreatedanimals); * significant difference at P< 0.05, compared with respectiveTCDD (T) treated mice.

Differential modulation of metabolizing enzymes by vanadium

levels in kidney, lung or heart (Fig. 2A and B). Furthermore, TCDDalone significantly induced Cyp1a2 mRNA levels at 6 h in thekidney, and heart by 37.6- and 10.5-fold, respectively. In thesame way, 632-, 2.3- and 5.5-fold were induced in the kidney,lung and heart at 24 h, respectively (Fig. 2A and B). Whenanimals were co-exposed to V5+ and TCDD, V5+ at 6 h significantlyinhibited the TCDD-mediated induction of Cyp1a2 mRNA inthe heart by 0.44-fold, compared with TCDD alone, while V5+

was unable to significantly affect the TCDD-mediatedinduction of Cyp1a2 mRNA in the kidney and lung (Fig. 2A).Conversely, V5+ at 24h significantly inhibited the kidney Cyp1a2mRNA by 0.09-fold, compared with TCDD alone, while V5+ wasunable to significantly affect the TCDD-mediated induction ofCyp1a2 mRNA in the lung and heart (Fig. 2B).

With regards to Cyp1b1, V5+ alone did not cause any signifi-cant changes in Cyp1b1 mRNA levels in the kidney, lung or heartat 6 and 24 h (Fig. 3A and B). Moreover, TCDD alone significantlyinduced Cyp1b1 mRNA levels at 6 h in the kidney, lung and heartby 3.3-, 70- and 66.7-fold, respectively. Similarly, 4.7-, 62.4- and4.9-fold were induced at 24 h, respectively (Fig. 3A and B). Whenanimals were co-exposed to V5+ and TCDD, V5+ at 6 h potenti-ated the TCDD-mediated induction of Cyp1b1 mRNA in the lungby 2.46-fold; on the other hand, V5+ significantly inhibited theTCDD-mediated induction of Cyp1b1 mRNA in the heart by0.25-fold, compared with TCDD alone. V5+ was unable to signif-icantly affect the TCDD-mediated induction of Cyp1b1 mRNAin the kidney (Fig. 3A). Also, V5+ at 24 h significantly inhibitedthe kidney Cyp1b1 mRNA levels by 0.7-fold. On the contrary,V5+ significantly potentiated the TCDD-mediated induction ofCyp1b1 mRNA in the lung by 1.73-fold, compared with TCDDalone. However, no changes were observed in TCDD-mediatedinduction of Cyp1a2 mRNA in the heart (Fig. 3B).

J. Appl. Toxicol. (2012) Copyright © 2012 John

Effect of Co-exposure to V5+ and TCDD on Cyp1a andCyp1b1 Protein Expression, EROD and MROD CatalyticActivity Levels in the Kidney and Lung of C57BL/6 Mice

Our results showed that, in the presence of V5+ alone, nochanges were observed in the kidney Cyp1a, and lung Cyp1aand Cyp1b1. On the other hand, V5+ significantly inhibited thekidney Cyp1b1 protein expression by 0.75-fold (Fig. 4B). TCDDalone significantly induced the kidney and lung Cyp1a proteinexpression levels by 22- and 7.2-fold, respectively. In the sameway the kidney and lung Cyp1b1 protein expression levels wereinduced by 2.66- and 8.9-fold, respectively (Fig. 4A and B). Uponco-exposure to V5+ and TCDD, V5+ significantly inhibited TCDD-mediated induction of the kidney Cyp1a and Cyp1b1 proteinexpression levels by 0.36- and 0.18-fold, respectively. On theother hand, V5+ significantly potentiated the TCDD-mediatedinduction of lung Cyp1a and Cyp1b1 protein expression levelsby 1.52- and 1.25-fold, respectively, compared with TCDD alone(Fig. 4A and B).At the catalytic activity levels, V5+ alone significantly inhibited

the kidney EROD and MROD activity by 0.57- and 0.51-fold,respectively, while V5+ alone did not significantly affect the lungEROD and/or MROD activities (Fig. 5A and B). Moreover, TCDDalone significantly induced kidney EROD and MROD activitiesby 14.4- and 17-fold, respectively. Similarly, TCDD also inducedthe lung EROD and MROD activities by 14.4- and 1.8-fold, respec-tively (Fig. 5A and B). Upon co-exposure to V5+ and TCDD, V5+

significantly inhibited TCDD-mediated induction of the kidneyEROD and MROD catalytic activities by 0.04- and 0.03-fold,while V5+ potentiated the TCDD-mediated induction of lungEROD and MROD by 1.6 and 1.43-fold, compared with TCDDalone (Fig. 5A and B).

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

Figure 4. Effect of co-exposure to V5+ and TCDD on Cyp1a and Cyp1b1 protein expression levels in the kidney and lung of C57BL/6 mice. Kidney andlung microsomal proteins were isolated after 24 h of treatment. 30mg microsomal proteins were separated on a 10% SDS–PAGE. The graph representsthe relative amount of protein normalized to b-actin signals (mean� SEM, n=6), and the results are expressed as percentage of the control valuestaken as 100%. + Significant difference at P< 0.05, compared with control (C; untreated animals); * significant difference at P< 0.05, compared withrespective TCDD (T) treated mice.

G. Abdelhamid et al.

The limitation of minimal protein quantities that could beextracted from heart samples hindered us from measuringprotein expression and catalytic activities of AhR-regulatedgenes in this organ.

Figure 5. Effect of co-exposure to V5+ and TCDD on EROD and MRODcatalytic activity levels in the kidney and lung of C57BL/6 mice. Kidneyand lung microsomal protein was isolated after 24 h of treatment. Valuesare presented as mean� SEM (n=6). + Significant difference at P< 0.05,compared with control (C; untreated animals); * significant difference atP< 0.05, compared with respective TCDD (T) treated mice.

Copyright © 2012 Johnwileyonlinelibrary.com/journal/jat

Effect of Co-exposure to V5+ and TCDD on Nqo1 and Gsta1mRNA in the Kidney, Lung and Heart of C57BL/6 Mice

At 6 and 24 h, V5+ alone did not cause any significant changes inNqo1 mRNA levels in kidney, lung or heart, with the exception ofheart Nqo1 at 24 h. V5+ significantly potentiated Nqo1 mRNA inthe heart by 2-fold (Fig. 6A and B). Furthermore, TCDD alone

Figure 6. Effect of co-exposure to V5+ and TCDD on Nqo1 mRNA in thekidney, lung, and heart of C57BL/6 mice. Animals were injected i.p. with5mgkg�1 V5+ with or without 15mg kg�1 TCDD for 6 h (A) and 24 h (B).Duplicate reactions were performed for each experiment, and the valuespresented are the means of six independent experiments. + Significantdifference at P< 0.05, compared with control (C; untreated animals);* significant difference at P< 0.05, compared with respective TCDD (T)treated mice.

J. Appl. Toxicol. (2012)Wiley & Sons, Ltd.

Differential modulation of metabolizing enzymes by vanadium

significantly induced Nqo1 mRNA levels at 6 h in the kidney,lung and heart by 2.2-, 6.6- and 1.7- fold, respectively. Similarinductions were observed in the kidney, lung, and heart by2.7-, 12.6- and 2.2-fold at 24 h, respectively (Fig. 6A and B). Uponco-exposure to V5+ and TCDD, we found that V5+ at 6 h did notcause any significant changes in the kidney Nqo1, while V5+

significantly potentiated the TCDD-mediated induction ofNqo1 mRNA in the lung by 1.76-fold. At the same time, V5+

significantly inhibited the TCDD-mediated induction of heartNqo1 gene expression levels by 0.6-fold, compared with TCCDalone (Fig. 6A). V5+ at 24 h significantly potentiated the kidneyNqo1 mRNA levels by 1.78-fold, compared with TCCD alone,but V5+ did not alter TCDD-mediated induction of Nqo1 mRNAin the lung and heart (Fig. 6B).

Regarding Gsta1 mRNA, V5+ alone did not cause any signifi-cant changes in Gsta1 mRNA levels in the kidney and lung at6 h, while V5+ alone significantly potentiated Gsta1 mRNA inthe heart at 6 h to 1.8-fold (Fig. 7A). At 24 h, V5+ alone did notcause any significant changes in Gsta1 mRNA levels in thekidney. Nevertheless, V5+ significantly potentiated Gsta1 mRNAin the lung and heart to 3.6- and 2-fold, respectively (Fig. 7B).Moreover, TCDD alone significantly induced Gsta1 mRNA levelsat 6 h in the kidney, lung and heart by 3.1-, 1.7- and 2.5-fold,respectively (Fig. 7A). In the same way at 24 h, TCDD alonesignificantly induced Gsta1 mRNA levels in kidney, lungand heart by 6.2-, 3.6- and 2.7-fold, respectively (Fig. 7B). Uponco-exposure to V5+ and TCDD, V5+ at 6 h significantly potentiatedthe TCDD-mediated induction of Gsta1 mRNA in the lungby 1.89-fold, compared with TCCD alone, while V5+ did notsignificantly alter Gsta1 mRNA in the kidney and heart (Fig. 7A).No changes were observed at 24 h in the kidney, lung and heartGsta1 mRNA levels (Fig. 7B).

Figure 7. Effect of co-exposure to V5+ and TCDD on Gsta1 mRNA in thekidney, lung, and heart of C57BL/6 mice. Animals were injected i.p. with5mg kg�1 V5+ with or without 15mg kg�1 TCDD for 6 h (A) and 24 h (B).Duplicate reactions were performed for each experiment, and the valuespresented are the means of six independent experiments. + Significantdifference at P< 0.05, compared with control (C; untreated animals);* significant difference at P< 0.05, compared with respective TCDD (T)treated mice.

J. Appl. Toxicol. (2012) Copyright © 2012 John

Effect of Co-exposure to V5+ and TCDD on Nqo1 and Gsta1Protein Expression and Catalytic Activity Levels in theKidney and Lung of C57BL/6 Mice

V5+ alone did not cause any significant changes in kidney andlung Nqo1 and Gsta1 protein expression levels (Fig. 8A and B).Relating to TCDD alone, TCDD significantly induced kidney andlung Nqo1 protein expression levels by 1.9- and 3.4-fold, respec-tively. Similar induction was found in the kidney and lung Gsta1protein expression levels to 1.6- and 3.1-fold, respectively (Fig. 8Aand B). Upon co-exposure to V5+ and TCDD, V5+ significantlypotentiated the TCDD-mediated induction of the kidney and lungNqo1 protein expression levels by 2.19- and 1.86-fold, respectively,and the kidney Gsta1 protein expression level by 1.32-fold,compared with TCDD alone. On the other hand, V5+ did not alterthe lung Gsta1 protein expression level (Fig. 8A and B).At the catalytic activity levels, V5+ alone did not cause any

significant changes in the kidney and lung Nqo1 and Gsta1activities (Fig. 9A and B). Additionally, TCDD alone significantlyinduced lung Nqo1 activity by 1.5-fold. On the other hand, nochanges were observed in the kidney Nqo1 activity (Fig. 9A).Furthermore, TCDD alone significantly induced kidney and lungGsta1 activities by 9.5- and 1.8-fold, respectively (Fig. 9B). Uponco-exposure to V5+ and TCDD, V5+ significantly potentiated theTCDD-mediated induction of the kidney and lung Nqo1 catalyticactivities by 1.63- and 1.46-fold, respectively. As well, V5+ signifi-cantly potentiated the TCDD-mediated induction of kidney Gsta1catalytic activity by 1.33-fold, compared with TCDD alone. On theother hand, V5+ did not significantly alter the TCDD-mediatedinduction of the lung Gsta1 catalytic activity (Fig. 9B).

DISCUSSIONTo better understand the effect of co-exposure to V5+ and TCDD,animals were treated with 5mg kg�1 V5+ with or without 15mgkg�1 TCDD. Thereafter, the animals were euthanized after asingle injection at 6 or 24 h via cervical dislocation. Heart, lungand kidney tissues were excised. These organs were specificallychose to be tested as previous studies have demonstrated thatthe highest concentrations of V5+ initially appear in the kidney,liver and lungs of exposed animals (Barceloux, 1999; Ivancsitset al., 2002; Edel et al., 1984). The concentrations of V5+ andTCDD utilized in this work were selected based on previousstudies (Uno et al., 2008; Roman et al., 1981; Dafnis et al., 1992;Beyhl and Mayer, 1983). The results of the current study havebeen summarized in Table 2.In the current work, V5+ alone did not significantly change

mRNA levels of all tested genes at both time points investigated.At protein and catalytic activity levels, V5+ alone was not able toaffect the Cyp proteins in the kidney and lung with the excep-tion of Cyp1b1 protein in the kidney at 24 h. Regarding theeffect of V5+ on Cyp1b1 protein, it has been previously reportedthat there are at least two regulatory pathways that control Cyp1b1gene expression. The first is the hormonal regulation thatmaintains the constitutive expression of Cyp1b1 in steroidal tissues(Bhattacharyya et al., 1995); the second is the AhR-dependentpathway that governs the Cyp1b1 gene expression in response toenvironmental pollutants. Moreover, these effects were translatedto the catalytic activity levels in which V5+ significantly inhibitedthe kidney but not the lung EROD and MROD catalytic activities.The magnitude of inhibition of kidney Cyp1b1 protein, EROD andMROD catalytic activities by V5+ did not match the observed effect

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

Figure 8. Effect of co-exposure to V5+ and TCDD on Nqo1 and Gsta1 protein expression levels in the kidney and lung of C57BL/6 mice. Kidney andlung cytosolic proteins were isolated after 24 h of treatment. 30mg cytosolic proteins were separated on a 10% SDS–PAGE. The graph represents therelative amount of protein normalized to Gapdh signals (mean� SEM, n=6), and the results are expressed as percentage of the control values taken as100%. + Significant difference at P< 0.05, compared with control (C; untreated animals); * significant difference at P< 0.05, compared with respectiveTCDD (T) treated mice.

Figure 9. Effect of co-exposure to V5+ and TCDD on Nqo1 and Gsta1catalytic activity levels in the kidney and lung of C57BL/6 mice. Kidneyand lung cytosolic proteins were isolated after 24 h of treatment. Valuesare presented as mean� SEM (n=6). + Significant difference at P< 0.05,compared with control (C; untreated animals); * significant difference atP< 0.05, compared with respective TCDD (T) treated mice.

G. Abdelhamid et al.

at the level of gene expression, suggesting that this inhibition islikely to occur at translational and/or post-translational levels. Thusthe effect of V5+ on kidney Cyp1b1 cannot be generalized toinclude lung Cyp1b1 or even kidney Cyp1a1.

Treatment of the animals with TCDD alone significantlyincreased the Cyp1a1, Cyp1a2, and Cyp1b1 mRNA in the kidney,lung and heart at 6 and 24 h with the exception of Cyp1a2

Copyright © 2012 Johnwileyonlinelibrary.com/journal/jat

mRNA in the lung at 6 h. This result was in agreement with otherinvestigators, who reported that b-naphthovlavone was not ableto induce Cyp1a2 mRNA in the lung of C57BL/6 mice and maleSprague–Dawely rats (Shimada et al., 2003; Elsherbiny et al.,2010). In this work, the differences in the fold of induction ofCyp genes by TCDD could be explained based on the preferen-tial factors: the distribution of TCDD in fat-containing organssuch as kidney (Diliberto et al., 1995). In addition, the levels ofexpression of the AhR co-regulatory proteins such as co-activatorsand co-repressors in different tissues may affect the expression ofAhR-regulated genes across tissues and even within the sametissue (Nishimura and Naito, 2006; Nishimura et al., 2003). At theprotein and activity levels, TCDD significantly induced Cyp1a1and Cyp1b1 protein expression levels in the kidney and lung,which were subsequently translated to their EROD and MRODcatalytic activity levels.

Previous reports from our laboratory demonstrated that V5+ wasable to decrease the TCDD-mediated induction of Cyp1a1 andNqo1 at mRNA, protein, and catalytic activity levels in the mousehepatoma Hepa 1c1c7 cells (Anwar-Mohamed and El-Kadi, 2009;Anwar-Mohamed and El-Kadi, 2008) and in human hepatomaHepG2 cells (Abdelhamid et al., 2010a, 2010b). However, it wasnot reported previously if V5+ could affect TCDD-mediatedinduction of Cyp1a1, Cyp1a2 and Cyp1b1 mRNA, protein orcatalytic activity levels in the kidney, lung or heart of C57BL/6mice.Therefore, in the current study we examined the effect of V5+ on theTCDD-mediated induction of Cyp1a1, Cyp1a2 and Cyp1b1 at themRNA, protein and catalytic activity levels. We demonstrated thatV5+ after 6h of treatment significantly inhibited the TCDD-mediatedinduction of Cyp1a1, Cyp1a2 and Cyp1b1 in the heart, while itsignificantly potentiated their levels in the lung with theexception of Cyp1a2. Interestingly, V5+ did not significantly alterthe TCDD-mediated induction of tested Cyp in the kidney. Thedifferential effect of TCDD alone or co-treatment of V5+ andTCDD on lung Cyp1a2 can be explained by the fact that

J. Appl. Toxicol. (2012)Wiley & Sons, Ltd.

Table

2.Su

mmaryof

theeffectsof

V5+an

dTC

DDon

theexpression

ofAhR

-reg

ulated

gene

sin

kidn

ey,lun

gan

dhe

art

Treatm

ent

Gen

eTissue

s

Kidn

eyLu

ngHeart

6h

24h

6h

24h

6h

24h

V+5

Cyp

1a1

↔mRN

A↔

mRN

A,↔

protein,

#activity

↔mRN

A↔

mRN

A,↔

protein,

↔activ

ity↔

mRN

A↔

mRN

ACyp

1a2

↔mRN

A↔

mRN

A,↔

protein,

#activity

↔mRN

A↔

mRN

A,↔

protein,

↔activ

ity↔

mRN

A↔

mRN

ACyp

1b1

↔mRN

A↔

mRN

A,#

protein,

#activity

↔mRN

A↔

mRN

A,↔

protein,

↔activ

ity↔

mRN

A↔

mRN

ANqo

1↔

mRN

A↔

mRN

A,↔

protein,

↔activ

ity↔

mRN

A↔mRN

A,↔

protein,

↔activ

ity↔

mRN

A"m

RNA

GST

Ya↔

mRN

A↔

mRN

A,↔

protein,

↔activ

ity↔

mRN

A"m

RNA,↔

protein,

↔activ

ity"m

RNA

"mRN

ATC

DD

Cyp

1a1

"mRN

A"m

RNA,"

protein,

"activity

"mRN

A"m

RNA,"

protein,

"activity

"mRN

A"m

RNA

Cyp

1a2

"mRN

A"m

RNA,"

protein,

"activity

↔mRN

A"m

RNA,"

protein,

"activity

"mRN

A"m

RNA

Cyp

1b1

"mRN

A"m

RNA,"

protein,

"activity

"mRN

A"m

RNA,"

protein,

"activity

"mRN

A"m

RNA

Nqo

1"m

RNA

"mRN

A,"

protein,

↔activ

ity"m

RNA

"mRN

A,"

protein,

"activity

"mRN

A"m

RNA

GST

Ya"m

RNA

"mRN

A,"

protein,

"activity

"mRN

A"m

RNA, "

protein,

"activity

"mRN

A"m

RNA

V+5an

dTC

DD

Cyp

1a1

↔mRN

A#m

RNA,#

protein,

#activity

"mRN

A↔

mRN

A,"

protein,

"activity

#mRN

A↔

mRN

ACyp

1a2

↔mRN

A#m

RNA,#

protein,

#activity

↔mRN

A↔

mRN

A,"

protein,

"activity

#mRN

A↔

mRN

ACyp

1b1

↔mRN

A#m

RNA,#

protein,

#activity

"mRN

A"m

RNA,"

protein,

"activity

#mRN

A↔

mRN

ANqo

1↔

mRN

A"m

RNA,"

protein,

"activity

"mRN

A↔

mRN

A,"

protein,

"activity

#mRN

A↔

mRN

AGST

Ya↔

mRN

A↔

mRN

A,"

protein,

"activity

"mRN

A↔

mRN

A,↔

protein,

↔activ

ity↔

mRN

A↔

mRN

A

",Increase;#,d

ecrease;↔

,nochan

ge.

Differential modulation of metabolizing enzymes by vanadium

J. Appl. Toxicol. (2012) Copyright © 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

G. Abdelhamid et al.

regulatory pathways for CYP1A1 and CYP1A2 expression maydiffer (Tamaki et al., 2005). At 24 h of V5+ treatment, V5+ signifi-cantly inhibited the TCDD-mediated induction of kidneyCyp1a1, Cyp1a2 and Cyp1b1 mRNA levels. Interestingly, V5+

significantly potentiated the TCDD-mediated induction of lungCyp1b1 mRNA levels, while it did not significantly alter theTCDD-mediated induction of heart Cyp1a1, Cyp1a2 andCyp1b1, and lung Cyp1a1 and Cyp1a2 at mRNA levels. At theprotein and catalytic activity levels, V5+ significantly inhibitedthe TCDD-mediated induction of kidney Cyp1a, and Cyp1b1protein expression levels, whereas it significantly potentiatedtheir levels in the lung. These effects were translated to theirEROD and MROD catalytic activity levels.

The differences observed between the tissues examined inresponse to V5+ might be due to the kinetic factor that couldplay a role in explaining the effect of V5+, especially at the geneexpression levels. Firstly, the kinetic factor previously showedthat i.v. and orally administered V5+ was mainly distributed andaccumulated in the kidney (Heinemann et al., 2003). Thiswas supported by an earlier study demonstrating that theconcentration average of V5+ in the kidney was about threetimes higher than that of the liver and spleen (Ramanadhamet al., 1991). In addition to the rodent, the highest concentrationof V5+ in the kidney was also found in catfish tissue, inthat the amount of V5+ was found in the following order:kidney> liver> intestine (Ray et al., 1990). Our results demon-strated that the effect of V5+ on the kidney tissue differs fromthat in the lung tissue. In agreement with our study, it has beenpreviously demonstrated that there is a differential response ofA549 (lung carcinoma) and HTB44 (kidney carcinoma) cell linesto sodium vanadate exposure (Klein et al., 2008). According toour results, the effect of V5+ on lung tissue appeared at 6 h treat-ment, and no changes were observed in the lung at 24 h, whichwas in agreement with a previous study, where it was found thatthe pulmonary clearance of V5+ was rapid (80–85% of the V5+

was removed within 3 h (Rhoads and Sanders, 1985). Anotherstudy reported that the physicochemical properties of V5+ playan important role in the pulmonary clearance, in which sodiumvanadate (soluble form) was rapidly removed from lung ratherthan vanadium pentoxide (less soluble) (Sharma et al., 1987).

With regards to AhR-regulated phase II enzymes, Nqo1 andGsta1, we observed their differential modulation across tissues.V5+ alone, after 6 and 24 h treatment, did not cause any signifi-cant changes in Nqo1 and Gsta1 mRNA levels in the kidneyand lung tissues. However, it significantly induced the Gsta1mRNA in the heart at 6 h and in the lung at 24 h. In addition tothe Gsta1, V5+ significantly increased the heart Nqo1 mRNA at24 h. It is well known that V5+ has a cardioprotective effect, yetthe mechanism by which V5+ produce this effect is not fully clear(Bhuiyan and Fukunaga, 2009; Bhuiyan et al., 2009; Barrio andEtcheverry, 2010). Therefore, we are suggesting that the effectsof V5+ on phase II AhR-regulated genes may play a role in itscardioprotective mechanism. In the current study, we havedemonstrated that V5+ alone did not cause any significantchanges in Nqo1 and Gsta1 mRNA, protein, and activity levelsin the kidney and lung tissues. In contrast to the previousstudies, it was found that V5+ showed a high activity as GSTinducer in the liver, kidney and lung (Bishayee and Chatterjee,1993, 1995; Bishayee et al., 2010; Siegers et al., 1987). TCDD alonesignificantly induced Nqo1 and Gsta1 mRNA levels in the kidney,lung and heart at two different time points. At protein andcatalytic activity levels, TCDD significantly induced kidney and

Copyright © 2012 Johnwileyonlinelibrary.com/journal/jat

lung Nqo1 and Gsta1 protein expression. These effects weretranslated to the catalytic activity levels of Nqo1 and Gsta1with the exception of kidney Nqo1 activity. When animals wereco-exposed to V5+ and TCDD, V5+ at 6 h did not significantlychange the TCDD-mediated induction of Nqo1 and Gsta1 inthe kidney, while it significantly potentiated their levels in thelung. In contrast, V5+ significantly inhibited the TCDD-mediatedinduction of Nqo1 gene expression in the heart. On the otherhand, at 24 h V5+ did not alter the TCDD-mediated induction ofNqo1 and Gsta1 mRNA in the kidney, lung and heart with theexception of kidney Nqo1 mRNA levels. At protein and activitylevels, V5+ significantly potentiated the TCDD-mediatedinduction of kidney and lung Nqo1 protein levels, while V5+

potentiated the Gsta1 protein level in the kidney but not in the lung.In conclusion, the present study demonstrates that V5+ modu-

lates constitutive and TCDD-induced AhR-regulated genes in atime-, tissue- and AhR-regulated enzyme-specific manner.Furthermore, the effect on one of these enzymes could not begeneralized to other enzymes, despite the fact that it is anAhR-regulated enzyme, as there are multiple factors that couldaffect the gene expression to cause differential effects.

Acknowledgments

This work was supported by Natural Sciences and EngineeringResearch Council of Canada Discovery Grant RGPIN 250139–07to A.O.S. I.E.A is the recipient of Libyan Government Scholarship.A.A-M. is the recipient of Alberta Ingenuity Graduate Scholarshipand Izaak Walton Killam Memorial Graduate Scholarship.

REFERENCESAbdelhamid G, Anwar-Mohamed A, Badary OA, Moustafa AA, El-Kadi AO.

2010a. Transcriptional and posttranscriptional regulation of CYP1A1by vanadium in human hepatoma HepG2 cells. Cell Biol. Toxicol.26(5): 421–434.

Abdelhamid G, Anwar-Mohamed A, Elmazar MM, El-Kadi AO. 2010b.Modulation of NAD(P)H:quinone oxidoreductase by vanadium inhuman hepatoma HepG2 cells. Toxicol. in Vitro 24(6): 1554–1561.

Anwar-Mohamed A, El-Kadi AO. 2008. Down-regulation of the carcinogen-metabolizing enzyme cytochrome P450 1a1 by vanadium. Drug Metab.Dispos. 36(9): 1819–1827.

Anwar-Mohamed A, El-Kadi AO. 2009. Down-regulation of the detoxifyingenzyme NAD(P)H:quinone oxidoreductase 1 by vanadium in Hepa1c1c7 cells. Toxicol. Appl. Pharmacol. 236(3): 261–269.

Atteia O. 1994. Major and trace elements in precipitation on westernSwitzerland. Atmosph. Environ. 28: 3617–3624.

Barakat MM, El-Kadi AO, du Souich p. 2001. L-NAME prevents in vivothe inactivation but not the down-regulation of hepatic cyto-chrome P450 caused by an acute inflammatory reaction. Life Sci.69(13): 1559–1571.

Baran EJ. 2008. Vanadium detoxification: chemical and biochemicalaspects. Chem. Biodivers. 5(8): 1475–1484.

Barceloux DG. 1999. Vanadium. J. Toxicol. Clin. Toxicol. 37(2): 265–278.Barrio DA, Etcheverry SB. 2010. Potential use of vanadium compounds in

therapeutics. Curr. Med. Chem. 17(31): 3632–3642.Beyhl FE, Mayer DG. 1983. Effects of ammonium metavanadate on rat

liver enzymes. Toxicol. Lett. 19(1–2): 81–85.Bhattacharyya KK, Brake PB, Eltom SE, Otto SA, Jefcoate CR. 1995.

Identification of a rat adrenal cytochrome P450 active in polycyclichydrocarbon metabolism as rat CYP1B1. Demonstration of a uniquetissue-specific pattern of hormonal and aryl hydrocarbon receptor-linked regulation. J. Biol. Chem. 270(19): 11595–11602.

Bhuiyan MS, Fukunaga K. 2009. Cardioprotection by vanadium com-pounds targeting Akt-mediated signaling. J. Pharmacol. Sci. 110(1):1–13.

Bhuiyan MS, Shioda N, Shibuya M, Iwabuchi Y, Fukunaga K. 2009. Activa-tion of endothelial nitric oxide synthase by a vanadium compoundameliorates pressure overload-induced cardiac injury in ovariecto-mized rats. Hypertension 53(1): 57–63.

J. Appl. Toxicol. (2012)Wiley & Sons, Ltd.

Differential modulation of metabolizing enzymes by vanadium

Bishayee A, Chatterjee M. 1993. Selective enhancement of glutathioneS-transferase activity in liver and extrahepatic tissues of ratfollowing oral administration of vanadate. Acta Physiol. Pharmacol.Bulg. 19(3): 83–89.

Bishayee A, Chatterjee M. 1995. Time course effects of vanadiumsupplement on cytosolic reduced glutathione level and glutathioneS-transferase activity. Biol. Trace Elem. Res. 48(3): 275–285.

Bishayee A, Waghray A, Patel MA, Chatterjee M. 2010. Vanadium in thedetection, prevention and treatment of cancer: the in vivo evidence.Cancer Lett. 294(1): 1–12.

Brauze D, Widerak M, Cwykiel J, Szyfter K, Baer-Dubowska W. 2006. Theeffect of aryl hydrocarbon receptor ligands on the expression ofAhR, AhRR, ARNT, Hif1alpha, CYP1A1 and NQO1 genes in rat liver.Toxicol. Lett. 167(3): 212–220.

Byrne AR, Kosta L. 1978. Vanadium in foods and in human body fluidsand tissues. Sci. Total Environ. 10(1): 17–30.

Craun GF, Greathouse DG, Gunderson DH. 1981. Methaemoglobin levelsin young children consuming high nitrate well water in the UnitedStates. Int. J. Epidemiol. 10(4): 309–317.

Dafnis E, Spohn M, Lonis B, Kurtzman NA, Sabatini S. 1992. Vanadatecauses hypokalemic distal renal tubular acidosis. Am. J. Physiol.262(3 Pt 2): F449–453.

Diliberto JJ, Akubue PI, Luebke RW, Birnbaum LS. 1995. Dose–responserelationships of tissue distribution and induction of CYP1A1and CYP1A2 enzymatic activities following acute exposure to2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. Toxicol. Appl.Pharmacol. 130(2): 197–208.

Edel J, Sabbioni RP, Marafante E, Springer A, Ubertalli L. 1984. Dispositionof vanadium in rat tissues at different age. Chemosphere 13: 87–93.

Elsherbiny ME, El-Kadi AO, Brocks DR. 2010. The effect of beta-naphthoflavone on the metabolism of amiodarone by hepaticand extra-hepatic microsomes. Toxicol. Lett. 195(2–3): 147–154.

Ernster L. 1967. DT diaphorase. Meth. Enzymol. 10: 309–317.Evangelou AM. 2002. Vanadium in cancer treatment. Crit. Rev. Oncol.

Hematol. 42(3): 249–65.Galloway JT, Norton J, Volchok S, MacLean HR. 1982. Trace metals in

atmospheric deposition: a review and assessment. Atmos. Environ.16: 1677–1700.

Germolec DR, Henry EC, Maronpot R, Foley JF, Adams NH, Gasiewicz TA,Luster MI. 1996. Induction of CYP1A1 and ALDH-3 in lymphoid tissuesfrom Fisher 344 rats exposed to 2,3,7,8-tetrachlorodibenzodioxin(TCDD). Toxicol. Appl. Pharmacol. 137(1): 57–66.

Habig WH, Pabst MJ, Jakoby WB. 1974. Glutathione S-transferases. Thefirst enzymatic step in mercapturic acid formation. J. Biol. Chem.249(22): 7130–7139.

Hankinson O. 1995. The aryl hydrocarbon receptor complex. Annu. Rev.Pharmacol. Toxicol. 35: 307–340.

Heinemann G, Fichtl B, Vogt W. 2003. Pharmacokinetics of vanadium inhumans after intravenous administration of a vanadium containingalbumin solution. Br. J. Clin. Pharmacol. 55(3): 241–245.

Ivancsits S, Pilger A, Diem E, Schaffer A, Rudiger HW. 2002. Vanadateinduces DNA strand breaks in cultured human fibroblasts at dosesrelevant to occupational exposure. Mutat. Res. 519(1–2): 25–35.

Klein A, Holko P, Ligeza J, Kordowiak AM. 2008. Sodium orthovanadateaffects growth of some human epithelial cancer cells (A549, HTB44,DU145). Folia Biol. (Krakow) 56(3–4): 115–121.

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression datausing real-time quantitative PCR and the 2(�Delta Delta C(T))method. Methods 25(4): 402–408.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol. Chem. 193(1): 265–275.

Mamane Y, Pirrone N. 1998. Vanadium in the Atmosphere. Wiley: NewYork.

Nebert DW, Dalton TP. 2006. The role of cytochrome P450 enzymes inendogenous signalling pathways and environmental carcinogenesis.Nat. Rev. Cancer 6(12): 947–960.

Nebert DW, Duffy JJ. 1997. How knockout mouse lines will be used to studythe role of drug-metabolizing enzymes and their receptors during

J. Appl. Toxicol. (2012) Copyright © 2012 John

View publication statsView publication stats

reproduction and development, and in environmental toxicity, cancer,and oxidative stress. Biochem. Pharmacol. 53(3): 249–254.

Nebert DW, Eisen HJ, Hankinson O. 1984. The Ah receptor: bindingspecificity only for foreign chemicals? Biochem. Pharmacol. 33(6):917–924.

Nishimura M, Naito S. 2006. Tissue-specific mRNA expression profiles ofhuman phase I metabolizing enzymes except for cytochrome P450and phase II metabolizing enzymes. Drug Metab. Pharmacokinet.21(5): 357–374.

Nishimura M, Yaguti H, Yoshitsugu H, Naito S, Satoh T. 2003. Tissue distri-bution of mRNA expression of human cytochrome P450 isoformsassessed by high-sensitivity real-time reverse transcription PCR.Yakugaku Zasshi 123(5): 369–375.

Preusch PC, Siegel D, Gibson NW, Ross D. 1991. A note on the inhibitionof DT-diaphorase by dicoumarol. Free Radic. Biol. Med. 11(1): 77–80.

Ramanadham S, Heyliger C, Gresser MJ, Tracey AS, McNeill JH. 1991. Thedistribution and half-life for retention of vanadium in the organs ofnormal and diabetic rats orally fed vanadium(IV) and vanadium(V).Biol. Trace Elem. Res. 30(2): 119–124.

Ray D, Banerjee SK, Chatterjee M. 1990. Bioaccumulation of nickel andvanadium in tissues of the catfish Clarias batrachus. J. Inorg. Biochem.38(3): 169–173.

Rhoads K, Sanders CL. 1985. Lung clearance, translocation, and acutetoxicity of arsenic, beryllium, cadmium, cobalt, lead, selenium,vanadium, and ytterbium oxides following deposition in rat lung.Environ. Res. 36(2): 359–378.

Rivera SP, Saarikoski ST, Hankinson O. 2002. Identification of a noveldioxin-inducible cytochrome P450. Mol. Pharmacol. 61(2): 255–259.

Roman RJ, Bonventre JV, Silva P, Lechene C. 1981. Sodium orthovanadatediuresis in rats. J. Pharmacol. Exp. Ther. 218(1): 168–174.

Sambrook JFE, Maniatatis T. 1989. Molecular Cloning. A LaboratoryManual. Cold Spring Harbor Laboratory Press: Plainview, NY.

Sharma RP, Flora SJ, Drown DB, Oberg SG. 1987. Persistence of vanadiumcompounds in lungs after intratracheal instillation in rats. Toxicol. Ind.Health 3(3): 321–329.

Shimada T, Sugie A, Shindo M, Nakajima T, Azuma E, Hashimoto M, InoueK. 2003. Tissue-specific induction of cytochromes P450 1A1 and 1B1by polycyclic aromatic hydrocarbons and polychlorinated biphenylsin engineered C57BL/6J mice of arylhydrocarbon receptor gene.Toxicol. Appl. Pharmacol. 187(1): 1–10.

Siegers CP, Schenke M, Younes M. 1987. Influence of cadmium chloride,mercuric chloride, and sodium vanadate on the glutathione-conjugatingenzyme system in liver, kidney, and brain of mice. J. Toxicol. Environ.Health 22(2): 141–148.

Sinal CJ, Bend JR. 1996. Kinetics and selectivity of mechanism-basedinhibition of guinea pig hepatic and pulmonary cytochrome P450by N-benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole. Drug Metab. Dispos. 24(9): 996–1001.

Sonneveld E, Jonas A, Meijer OC, Brouwer A, van der Burg B. 2007.Glucocorticoid-enhanced expression of dioxin target genes throughregulation of the rat aryl hydrocarbon receptor. Toxicol. Sci. 99(2):455–469.

Spink DC, Katz BH, Hussain MM, Spink BC, Wu SJ, Liu N, Pause R,Kaminsky LS. 2002. Induction of CYP1A1 and CYP1B1 in T-47D humanbreast cancer cells by benzo[a]pyrene is diminished by arsenite. DrugMetab. Dispos. 30(3): 262–269.

Tamaki H, Sakuma T, Uchida Y, Jaruchotikamol A, Nemoto N. 2005.Activation of CYP1A1 gene expression during primary culture ofmouse hepatocytes. Toxicology 216(2–3): 224–231.

Uno S, Dragin N, Miller ML, Dalton TP, Gonzalez FJ, Nebert DW. 2008.Basal and inducible CYP1 mRNA quantitation and protein localizationthroughout the mouse gastrointestinal tract. Free Radic. Biol. Med.44(4): 570–583.

Volotinen M, Maenpaa J, Kankuri E, Oksala O, Pelkonen O, Nakajima M,Yokoi T, Hakkola J. 2009. Expression of cytochrome P450 (CYP)enzymes in human nonpigmented ciliary epithelial cells: inductionof CYP1B1 expression by TCDD. Invest. Ophthalmol. Vis. Sci. 50(7):3099–3105.

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat