Abstracts / Toxicology 226 (2006) 12–77 41

Table 1Response of PXR reporter gene constructs to known CYP3A transcriptional activators

Human Huh7 cell line Rat FaO cell line

Human Chimp Rat Human Chimp Rat

Clotrimazole 8.6@10 �M 3.5@10 �M 3@10 �M 1.6@25 �M – 2.6@5 �MRifampicin – 2.5@50 �M – – 3.5@100 �M 3.2@100 �MD

M g witha

vmstum

R

K

M

X

d

Ca

EN

1

aSW

teaisrie

l(

examethasone 2@1 �M 1.8@1 �M –

aximal PXR reporter gene activation for each chemical is given, alonctivation observed at any concentration within range examined.

ation, we demonstrate that both species-specific pro-oter sequences and cell-line specific expression of tran-

cription factors may play important roles in determininghe overall pool of PXR protein in the cell, and henceltimately the expression of PXR target genes includingany drug metabolizing/transporting proteins.

eferences

liewer, S.A., Moore, J.T., Wade., L., Staudinger, J.L., Watson, M.A.,Jones, S.A., McKee, D.D., Oliver, B.B., Willson, T.M., Zetter-strom, R.H., Perlmann, T., Lehmann, J.M., 1998. Cell 92, 73–82.

oore, L.B., Maglich, J.M., McKee, D.D., Wisley, B., Willson, T.M.,Kliewer, S.A., Lambert, M.H., Moore, J.T., 2002. Mol. Endocrinol.16, 977–986.

ie, W., Barwick, J.L., Downes, M., Blumberg, B., Simon, C.M., Nel-son, M.C., Neuschwander-Tetri, B.A., Brunt, E.M., Guzelian, P.S.,Evans, R.M., 2000. Nature 406, 435–438.

oi:10.1016/j.tox.2006.05.060

oordinate regulation of cellular proliferation andpoptosis in rodent liver

lizabeth Anderson 1, G. Gordon Gibson 1, Jon Lyon 2,ick Plant 1

Molecular Toxicology Group, School of Biomedicalnd Molecular Sciences, University of Surrey, Guildford,urrey GU2 7XH, UK; 2 GlaxoSmithKline, The Frythe,elwyn, Herts AL6 9AR, UK

The balance between cellular proliferation and apop-osis is a coordinated process. Rodent hepatocytes prolif-rate rapidly in response to non-genotoxic liver growthgents such as peroxisome proliferators, a result of bothncreased DNA synthesis and suppression of apopto-is (Roberts et al., 1995). This study used the rodentesponse to liver growth agents to identify genes exhibit-ng coordinate regulation, and the candidate regulatory

lements involved in this regulation.

The study used four compounds known to induceiver growth via different processes: cyproterone acetate60 mg/kg/day), dexamethasone (100 mg/kg/day), Wy-

7.8@50 �M 3.1@10 �M –

the concentration required to elicit such a response. (–) no significant

14,643 (50 mg/kg/day) and phenobarbital (50 mg/kg/day) for up to 4 days. Each compound, or vehicle, wasadministered to groups of five male rats, with animalsbeing sacrificed at time-points between 6 and 96 h post-dose. Total RNA from liver samples was processed andanalyzed by DNA microarray (Affymetrix). The expres-sion profile for each gene fragment across all time pointswas first compared across the four compounds tested,identifying 38 genes that could be clustered into groupsshowing similar expression profiles with at least threeof the compounds. From these 38 genes, one group ofsix genes showing coordinated expression profiles, wasselected for further analysis. For these genes the largestalteration in expression was observed after 72 h of dos-ing for all test chemicals, with the largest fold changebeing given in the table below.

Gene name Maximalfold change

Nob1p/nin one binding protein −7.8Sp3 −9.6EAR2/NR2F6/COUP� −6.0Hypertension-related, calcium regulated gene

(HCaRG)−4.3

Opioid growth factor receptor-like 1 (OGFRL1) −4.7Leucine-rich repeats and calponin homology

domain containing 4 (LRCH4)−3.8

The first kilobase of upstream promoter sequence ofeach gene was cloned into an SEAP reporter gene plas-mid and transfected into the FaO rat hepatoma cell line.Exposure of transfected cells to liver growth agents pro-duced a similar expression profile to that seen in theoriginal in vivo analysis, suggesting regulation of thegenes was at the transcriptional level. A deletion con-struct series was prepared from two of the reporter genes(HCaRG and Nob1p), and the transcriptional responselocalized to within the first 200 bp of the proximal pro-

moter. Using the Match database (TRANSFAC) we nextidentified potential regulatory elements within these twopromoter sequences, identifying common putative bind-ing sites for the Aryl hydrocarbon receptor (AhR) and

ology 2

42 Abstracts / Toxic

nuclear respiratory factor 2 (Nrf2). Exposure of the200 bp reporter gene constructs to 100 �M BNF, whichactivates both AhR and Nrf2, elicited a statistically sig-nificant two-fold decrease in reporter gene expression,similar to the decrease elicited by the liver growth agentsused in this study, suggesting that activation of AhRand/or Nrf2 may be responsible for the effects observedwith these chemicals as well.

Interestingly, AhR and Nrf2 have recently been sug-gested to interact during the coordination of transcrip-tome response to oxidative stress (Ma et al., 2004),providing a potential mechanistic rationale for the geneexpression changes observed in vivo following exposureto compounds such as Wy-14,643.

References

Ma, Q., Kinneer, K., Bi, Y., Chan, J.Y., Kan, Y.W., 2004. Biochem. J.377, 205–213.

Roberts, R.A., Soames, A.R., Gill, J.H., et al., 1995. Carcinogenesis16 (8), 1693–1698.

doi:10.1016/j.tox.2006.05.061

PXR and CAR are implicated in Oatp1a4 inductionin vivo and in vitro

David E. Cowie 1, Richard J. Weaver 2, Gabrielle M.Hawksworth 1

1 Department of Medicine & Therapeutics and Schoolof Medical Sciences, Polwarth Building, Foresterhill,Aberdeen University, Aberdeen AB25 2ZD, UK; 2 DrugMetabolism and Development, Servier SL3 6HH, UK

E-mail address: d.e.cowie@abdn.ac.uk (D.E. Cowie)

The organic anion transporting polypeptides (Oatps)are products of the solute carrier organic anion (Slco)transporter gene superfamily and constitute a major classof membrane solute carriers at the basolateral membraneof hepatocytes. Oatps are polyspecific and transport awide range organic solutes including bile salts, organicdyes, steroid conjugates and drugs from a range of ther-apeutic classes. There is evidence that the pregnane Xreceptor (PXR) has a role in the transcriptional regulationof Oatp1a4 (Guo et al., 2002) and indirect evidence thatthe constitutive androstane receptor (CAR) is implicatedin the transcriptional regulation of Oatp1a4 (Hagenbuch

et al., 2001). The induction of hepatic Oatps and microso-mal enzymes by nuclear hormone receptors may functionas a co-ordinately regulated mechanism for detoxifyingendogenous and exogenous substrates.

26 (2006) 12–77

The PXR ligands, pregnenolone-16�-carbonitrile(PCN) and dexamethasone, the CAR ligand, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), and theCAR activator, phenobarbital, resulted in a dose-dependent increase of Oatp1a4 protein levels in maleSprague Dawley rats. Transporter protein levels weredetermined by Western blot using antibodies specific toOatp1a1 and Oatp1a4 (Alpha Diagnostic). The intraperi-toneal doses resulting in a maximal response were-dexamethasone 150 mg/kg, phenobarbital 120 mg/kg,PCN 40 mg/kg and TCPOBOP 1 mg/kg (n = 3). Oatp1a1levels were not altered by administration of any of thesecompounds.

Hepatocyte isolation resulted in a substantial decline(>80% of intact liver level) in both Oatp1a1 and Oatp1a4levels compared with intact liver. Hepatocytes isolatedfrom untreated male Sprague Dawley rats were culturedin William’s E medium supplemented with 100 nM dex-amethasone and 1.2% (v/v) insulin, transferrin and Na+

selenite in either a MatrigelTM ‘sandwich’ or ‘singlelayer’ configuration. When cultured on a single layerof MatrigelTM Oatp1a4 expression was lost after 48 h.When hepatocytes were cultured in a ‘sandwich’ config-uration, Oatp1a4 levels were maintained for 12 h afterattachment, then declined to 10% of levels on attach-ment by 96 h. Oatp1a1 remained at low levels through-out the culture period (up to 96 h) irrespective of cul-ture conditions (n = 3). After 48 h, 1 �M TCPOBOP,10 �M dexamethasone or 25 �M PCN added to thecultures for 48 h resulted in a an increase (four-fold)in Oatp1a4 protein, whereas 1 mM phenobarbital wasneeded to produce an increase in Oatp1a4 protein lev-els (n = 3). Oatp1a4 induction was dependent on cellsbeing cultured in a ‘sandwich’ configuration, suggest-ing that hepatocyte phenotype may play a role in thetranscriptional upregulation of Oatp1a4. None of thesecompounds had an effect on Oatp1a1 levels (n = 3). Thefunction of Oatp1a4 was assessed by using the highlyspecific Oatp1a4 substrate, [3H]-digoxin. Three hourspost isolation, maximal uptake, Vmax, was 2.46 ± 0.12and 8.77 ± 0.35 pmol/mg/min after 96 h in culture (48 htreatment with Dex) and the Km for digoxin, was110 ± 20 nM after 3 h compared with 330 ± 40 nM afterDex treatment (n = 3), the increase in hepatic uptake cor-relating with the Oatp1a4 levels at these time points. Thedata provide evidence that both PXR and CAR regulatethe expression of Oatp1a4 in vivo and in vitro. The lackof nuclear receptor involvement in the regulation may be

partly explained by the differing physiological roles ofOatp1a1 and 1a4.