ENVR 132/TOXC 142/BIOC142ENVR 132/TOXC 142/BIOC142Biochemical & Molecular ToxicologyBiochemical & Molecular Toxicology

Induction of Metabolism by ToxicantsInduction of Metabolism by Toxicants

Instructor:Stephen S. Ferguson, Ph.D.e-mail: stephen.ferguson@lifetech.com

Induction: Definitions and PrinciplesInduction: Definitions and Principles

• The process of increasing the amount or the activity of a protein.

• A homeostatic mechanism for regulating enzyme production in a barrier organ, such as the liver, intestine, kidney.

• In enzymology, an inducer usually combines with and deactivates/activates a regulatory protein which leads to increased gene expression.

P450 Enzyme InductionP450 Enzyme Induction

• Induction can cause marked increases in P450 composition (>20-fold) and chemical clearance or bioactivation.

• As a result, induction can increase tolerance to some toxicants while enhancing the toxicity of others.

• Induction can decrease the therapeutic effect of drugs by increasing the rate and pattern of metabolism.

• Xenobiotics are known to induce enzymes that play a major or no role in their biotransformation (e.g., omeprazole vs. ethanol).

Invitrogen Proprietary & Confid6

Inhibition-Induction

TimeCo

ncen

trat

ion

Ineffective level

Therapeutic Window(drug efficacy)

Toxic / side-effect level

Why Is It Important to Assess Enzyme Why Is It Important to Assess Enzyme Induction?Induction?

• Failure of therapy (e.g. OC’s, epilepsy, HIV)

• Drug tolerance with auto-induction

• Xenobiotic toxicity potentiated

• Complicated dosing regimen

• Chemical carcinogenesis potentiated

• Perturbation of endogenous substrate metabolism/homeostasis

• Hepatomegaly & proliferation of cellular ER & peroxisomes

Internal Exposure to Natural andInternal Exposure to Natural andManMan--made Chemicalsmade Chemicals

• drugs• industrial chemicals • pesticides • pollutants • alkaloids• cigarette smoke

• cruciferous vegetables (indole-3-carbinol)

• secondary plant metabolites

• toxins produced by molds, plants, and animals

• pyrolysis products in cooked food

Types of P450 InducersTypes of P450 Inducers• Many “prototypical” inducers of specific families or

subfamilies of P450 enzymes– CYP1A inducers: 3-MC, BNF, omeprazole, TCDD– CYP3A inducers: rifampin, dexamethasone, troglitazone– CYP2B inducers: phenobarbital, PCBs, phenytoin– CYP4A inducers: fibrates– CYP2E1 inducers: ethanol, isoniazid

• Some overlap in “specificities” of inducers• An inducer for one family of enzymes may also suppress

another family (e.g., BNF)

Induction of Rat Liver P450 Enzymes by Induction of Rat Liver P450 Enzymes by Prototypical Inducers Prototypical Inducers In VivoIn Vivo

CLO

PCN

PB

BNF

Inducer

10,693 ± 620489 ± 52CYP4A

12,693 ± 2,2552,460 ± 780CYP3A

1,460 ± 18024 ± 4CYP2B

3,320 ± 183152 ± 27CYP1A

Induced ActivityControl Activity

In Vivo Induction in Male Rats

P450 Enzyme

CYP1A, EROD; CYP2B, PROD; CYP3A, testosterone 6β-hydroxylation;CYP4A, lauric acid 12-hydroxylation.

Induction and Inhibition of P450 in Mice Treated Induction and Inhibition of P450 in Mice Treated with PB or SKF525A: [with PB or SKF525A: [1414CC--methyl]aminopyrinemethyl]aminopyrine

Serr

umtri

azol

am (n

g/m

l)

Rifampin Effects on Triazolam DispositionRifampin Effects on Triazolam Disposition

Villikka et al., Clin Pharmacol Ther 1997;61:8-14.

RifampinPlacebo

Consequences of Cytochrome P450 Enzyme Induction

Consequences of Cytochrome Consequences of Cytochrome P450 Enzyme InductionP450 Enzyme Induction

• Increased toxic effect– Acetaminophen Alcohol, 3-MC– Bromobenzene, CCl4 Phenobarbital

• Increased bioactivation– Cyclophosphamide Macrolides, pesticides

• Increased tumor formation– Altered disposition of endogenous substrates

• Altered cellular and physiological function– proliferation of peroxisomes and SER– increased liver weight– endocrine disruption

• Porphyria, chloracne– PCDDs, azobenzenes, biphenyls (PCBs), naphthalene

Effects of Inducers on Rodent Liver Effects of Inducers on Rodent Liver Physiology and FunctionPhysiology and Function

Acetaminophen Metabolism and ToxicityAcetaminophen Metabolism and Toxicity

~60% ~35%

CYP2E*CYP1A CYP3A

NAPQIN-acetyl-p-benzoquinone imine

*induced by ethanol, isoniazid, phenobarbital

Protein adducts,Oxidative stress,Toxicity

HNCOCH 3

OH

HNCOCH 3

OSO 3H

HNCOCH 3

OO CO 2H

OHOH

HON

O

COCH 3

Endocrine Disruption Endocrine Disruption • Many xenobiotics can mimic certain hormones and

bind to target cellular sites receptive to natural hormones

• Modes of endocrine disruption can result from agonistic or antagonistic receptor binding affecting biosynthesis, transport, storage, release, and clearance of hormones

• Some pesticides have been identified as endocrine disruptors, in particular the thyroid hormone can be affected by: acetochlor, alachlor, fipronil (Frontline), heptachlor, maneb, methomyl, and zineb

• PCBs, mercury, pentachlorophenol are some of the thyroid hormone disruptors that are no longer used as pesticides

• DDT, dieldrin, lindane, methoxychlor, triadimefon are thought to be estrogenic-type environmental endocrine disruptors (EEDs) while atrazine, vinclozolin, and procymidone are thought to be andogenic EEDs

UGT1A

Molecular Mechanisms of P450 Molecular Mechanisms of P450 Enzyme InductionEnzyme Induction

General Mechanisms of P450 InductionGeneral Mechanisms of P450 Induction

• Receptor-mediated transcriptional activation

– Receptor • A macromolecule with which a

hormone, drug, or other chemical interacts to produce a characteristic effect.

– Two key features:• chemical recognition• signal transduction

– Ligand: A chemical that exhibits specific binding to a receptor.

• mRNA stabilization• Protein stabilization

Coordinates: Kumar R, Thompson EB (1999). "The structure of the nuclear hormone receptors". Steroids 64 (5): 310–9

Enzyme InductionEnzyme InductionGeneral mechanism of hepatic enzyme inductionGeneral mechanism of hepatic enzyme induction

proteinprotein

activityactivity

mRNAmRNA

Gene transcriptionGene transcription

XX

Nuclear ReceptorNuclear Receptor

XXRR cytosolcytosol XXRR nucleusnucleus

Phase1Phase1Phase 2 Phase 2

transporterstransporters

cytoplasm

nucleus

Hepatocyte

NRNR’’s and P450 Inductions and P450 Induction

CYP450 genePromoter XREM PBREM

RNA poly IITranscription P450

mRNA

Translation

P450

Increased Drug Metabolism

Drug-OH

Drug

TFs

PXRCAR

RXR NR

SRC-1I

Complex Transcriptional MachineryComplex Transcriptional Machinery

precursor mRNA

mature mRNA

mRNA degradation

micro RNA

protein translation

protein folding

protein degradation

CoCo--regulation of Target Genes by NRregulation of Target Genes by NR’’ss

• Complementary roles of NR’s in protection against xenobiotic exposure.

• Increased expression of the hepatic genes involved in drug metabolism and excretion (e.g., CYP’s, UGT’s, GST’s, transporter proteins).

• These target genes represent redundant but distinct layers of defense.

• There are overlapping similarities and distinct differences in species’ response to activators of NR’s.

Transcription factor

Dimerization partner

Examples of ligands

Genes Regulated

AHR ARNT Dioxins, non-ortho PCBs, some PAHs, bilirubin, etc.

CYP1A, CYP1B GST, UGT, NQO

CAR

RXR

Phenobarbital (PB), TCPOBOP, chlorinated pesticides, ortho-PCBs, androstanol/ androstenol (inhibits)

CYP2B, CYP3A GST, ABC transporters

PXR (SXR)

RXR

PB, ortho-PCBs, organochlorine pesticides, dexamethasone, pregnenalone, corticosterone, bile acids (lithocholic acid)

CYP3A, CYP2B, CYP7A (repression) GST, ABC transporters

PPAR

RXR

Fibrate drugs, phthalate esters, linoleic acid, arachidonic acid,

CYP4A, CYP7A (repression), CYP8B, LXR, HMGCS2

LXR RXR Cholesterol; (24 S)- hydroxycholesterol CYP7A, ABC transporters, LXR

FXR RXR Bile acids, chenodeoxycholic acid Represses CYP7A, BSEP (ABCB11), CYP8B, CYP27A

ER ER Structurally diverse xenoestrogens CYP19

Receptors Involved in the Regulation of Receptors Involved in the Regulation of CYP Gene ExpressionCYP Gene Expression

Modified from Kast, H. R. et al. J. Biol. Chem. 277:2908-2915, 2002

Coordinate Regulation of P450Coordinate Regulation of P450’’s, s, UGTUGT’’ss and and Transporters by NRTransporters by NR’’ss

UGT’s

MRP3

Role of CAR/PXR in lipid metabolism, Role of CAR/PXR in lipid metabolism, synthesis, and uptake & glucose homeostasissynthesis, and uptake & glucose homeostasis

Moreau et al. 2007 Mol. Pharmaceutics

Induction in cultures of primary Induction in cultures of primary human hepatocyteshuman hepatocytes

CYP2B6 Activity and mRNA with PB & RIF

• Saturable, sigmoidal responses

What is Relevant Induction?What is Relevant Induction?Potency and EfficacyPotency and Efficacy

Dose-Response ‘Window’

(Position → potency)

Magnitude of Response (Efficacy)

EC50

1. Efficacy (e.g. % of PC)

2. Potency (e.g. EC50)

Emax

Relationship between In Vitro Potency and Induction In VivoEC50 Cmax [Cmax]/EC50 Clinical

RelevanceNifedione 8 0.008 0.001 No knownLovastatin 1-6 0.008 0.008-0.002 No knownRosiglitazone 5-10 0.3-1.2 0.05-0.12 No knownSimvastatin 0.14 0.024 0.17 No knownTroglitazone 3-6 7 2.3 YesPhenytoin 25 80 3.2 YesAvasimibe 0.2 1-6 5-30 YesRifampicin 0.8 14 17.5 YesCarbamazepine 0.9 25 28 YesClotrimazole 1-5 Topical (Inhibition)

[Cmax]/EC50 < 0.1, induction not likely

1< [Cmax]/ EC50 < 0.1, induction possible

[Cmax]/ EC50 > 1, induction likely

Aryl Hydrocarbon Receptor Aryl Hydrocarbon Receptor (AhR)(AhR)

• Aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix (bHLH) protein belonging to the Per-Arnt-Sim (PAS) family of transcription factors

• It transcriptionally induces expression of hepatic CYP1A1, CYP1A2, and CYP1B1 , as well as several other genes, including some phase II metabolizing enzymes

• AHR ligands include PAHs and TCDD

AhR Signaling PathwayAhR Signaling Pathway

Cytoplasm Nucleus

9090

X

AhR

L

L

9090

X

L

9090

X

L

L

9090

X

L

or Arnt

From: Anne Mullen, Advanced Pharmacology, McMaster University, Ontario, CA

AhR Signaling PathwayAhR Signaling Pathway

XRE promoter gene (CYP1A1)

Translation

Increased expression CYP1A1 protein

Increased expression of other gene products

+

AhR/Arntheterodimer

mRNA

IC

+

TNGCGTG

Amino Carboxy

AF-1 DBD LBD AF-2

Modulators interactwith some cofactors

Binding to responseelements of target genes

Ligand and coactivatorbinding pockets

Translocaseactivity

5’ 3’ 5’ 3’ 5’ 3’

Monomers RXR Heterodimers Homodimers

LBD

DBD

NR-LBD RXR-LBD

DBD DBD DBD DBD

NR-LBD NR-LBD

RORTLXERRNGFI-B

PXRCARPPARLXRFXRRAR

GRERRXRCOUP-TFHNF4Rev-ErbGCNF

Nuclear Hormone ReceptorsNuclear Hormone Receptors

Nuclear Receptor PXRNuclear Receptor PXRPB

CA

R

PXR

cytoplasm

nucleus

HSP

90

PXR

RIF

RXR

PXR

RXR

XREM CYP3A

?translocation?-mouse-yes-human-?

Activator/Agonist CYP TargetHuman RIF CYP3A4Rat PCN CYP3A1/2Mouse PCN Cyp3a11

Nuclear Receptor CAR: PB InductionNuclear Receptor CAR: PB Induction--Constitutively ActiveConstitutively Active

CAR

cytoplasmnucleus

HSP

90

HSP

90

CAR

PP2A

PB

CCRP

RXR

CA

R

RXR

CCRP

OA

PBREM CYP2B

?

Activator/Agonist Inhibitor/Antagonist CYP TargetHuman CITCO, PB, DPH Clotrimazole?, Miclizine? CYP2B6Rat PB, TCPOBOP Androstenol CYP2B1Mouse PB, TCPOBOP Androstenol Cyp2b10

In cell lines spontaneously translocates to the nucleus

5’ 3’

nnn

DRn

IRn

ERn

CYP2B Response elements

CYP2B6 TGTACT n=4 TGACCCCYP2b10 TGTACT n=4 TGACCTCYP2B1 TCTACT n=4 TGACCT CYP2B2 TGTACT n=5 TGACCT

NR1s

CYP2B6 TGGACT n=4 TGAACCCYP2b10 TCAACT n=4 TGACACCYP2B1 TCAACT n=4 TGACAC CYP2B2 TCAACT n=4 TGACAC

NR2s

NR3CYP2B6 TGGACT n=4 TGACCC

CYP3A Response elements

CYP3A4 TGAACT n=3 TGACCCCYP3A2 TGACCT n=3 TGAGCTCYP3A23 TGACCT n=4 TGAGTT CYP3A2 TGAACT n=3 TGAACT

DRs

CYP3A4 TGAAAT n=6 GGTTCA CYP3A4 TGAACT n=6 AGGTCACYP3A23 TTAACT n=6 AGGTCA CYP3A5 TGAACT n=6 AGGTAACYP3A7 TTAACT n=6 AGGTCA CYP3A7 TGAAAT n=6 AGTTCA

ERs

Other GenesUGT1A1 TGAGTT n=4 TAACCT MDR1 TGAGAT n=6 AGTTCArMRP2 TGAACT n=8 AGTTCA CYP2C9 CAAACT n=4 TGACCT

2C9-1839 2C8-8806

21 3 4 5 6 7 21 3 4 5 6 7

1. RXR2. CAR3. PXR4. CAR/RXR5. CAR/RXR-20Xcc6. PXR/RXR7. PXR/RXR-20Xcc

PXR/RXRCAR/RXR

PXR Binding Sites

agTCAACTttgaTGACCCca

aaTGAACTtgc.TGACCCtc

2C8-8806

3A4-7733

NR Binding (PXR and CAR) to NR Binding (PXR and CAR) to Promoter Response ElementsPromoter Response Elements

NR Binding (PXR and CAR) to Promoter NR Binding (PXR and CAR) to Promoter Response Elements (CYP3A4)Response Elements (CYP3A4)

Goodwin et al., Mol. Pharmacol., 2001

Differential Binding of PXR and Differential Binding of PXR and CAR to Other Promoter RegionsCAR to Other Promoter Regions

NR3-2B6 ER6-3A4PXR + + + + + + CAR + + + + + +RXR + + + + + + + + + + + +

PXR/RXRCAR/RXR

GR/Dexamethasone Role in Basal & Induced Expression GR/Dexamethasone Role in Basal & Induced Expression via CAR/PXR (Master Regulator)via CAR/PXR (Master Regulator)

Pascussi et al. 2001, Eur J. Biochem, v. 268, p.6346

Wang & LeCluyse 2003, Clin Pharmacokin, v. 32, p.1331

Molecular Basis for the Species Molecular Basis for the Species Differences in Enzyme InductionDifferences in Enzyme Induction

Rabbit

Human

Rat

0.1%

DM

SO

5μM

PC

N

10μM

Rifa

mpi

cin

10μM

SR

1281

3

10μM

DTB

A

CYP3A6

CYP3A4

CYP3A23

Species Differences in the Regulation of CYP3A Enzymes

Species Differences in the Regulation Species Differences in the Regulation of CYP3A Enzymesof CYP3A Enzymes

Species Differences in CYP2B Species Differences in CYP2B Induction by PhenobarbitalInduction by Phenobarbital

Species Differences in CYP1A Species Differences in CYP1A Induction by XenobioticsInduction by Xenobiotics

CYP1A1/2 Activity in Rat Hepatocytes as a Function of Treatment with Drug 'X'

0

100

200

300

400

500

600

700

800

900

1000

Contro

l (0.1%

DMSO)

3-MC 1µ

M

Drug 'X

' 0.6µ

M

Drug 'X

' 2µM

Drug 'X

' 6µM

Drug 'X

' 20µ

M

Phen

acet

in O

-Dea

lkyl

atio

n (p

mol

/min

/mg

CYP1A Activity in Dog Hepatocytes as a Function of Treatment with Drug 'X'

0

100

200

300

400

500

600

Contro

l (0.1%

DMSO)

3-MC 2µ

M

Drug 'X

' 0.6µ

M

Drug 'X

' 2µM

Drug 'X

' 6µM

Drug 'X

' 20µ

M

Phen

acet

in O

-Dea

lkyl

atio

n (p

mol

/min

/mg)

CYP1A2 Activity in Human Hepatocytes as a Function of Treatment with Drug 'X'

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Contro

l (0.1% D

MSO)

3-MC 2µM

Drug 'X

' 0.2µM

Drug 'X

' 2µM

Drug 'X

' 6µM

Drug 'X

' 20µ

M

CY

P1A

2 A

ctiv

ity (n

mol

/min

/mg

Species Differences in CYP4A Species Differences in CYP4A Induction by Clofibric AcidInduction by Clofibric Acid

Rat Human

CTL 1 10 100 500 1000

CYP

4A1

Fold

Indu

ctio

n

0

10

20

30

40

50

60

Clofibric Acid (µM)

CTL 1 10 100 500 1000

CYP

4A11

Fol

d In

duct

ion

0

10

20

30

40

50

60

Clofibric Acid (µM)

CTL 1 10 100 500 1000

CYP

4A11

Fol

d In

duct

ion

0

1

2

3

4

Clofibric Acid (µM)

Rat Hepatocytes Human Hepatocytes

Laur

ic a

cid

12-h

ydro

xyla

tion

PAH Inducers in Rat vs. Human

Rat TCDD 1A1 mRNA

EC50 = 0.00767 +/- 0.00409

EC50 = 0.0107 +/- 0.043

CYP1A1 mRNA Hu497

Species difference in potency and efficacy

EC50 = 0.00767 +/- 0.00409

Observations and QuestionsObservations and Questions• Significant species differences are observed

in response to inducers.• All major subfamilies of inducible CYP’s

(CYP1A, CYP2B, CYP3A, CYP4A) exhibit this behavior.

• What is the molecular basis of the species-specific responses?

• What is the significance of these differences to predicting human toxicity?

PXR ExpressionPlasmid

RXR PXR

PXRE Reporter Gene

Drug

Reporter Plasmid

Transfection Assay for P450 Transfection Assay for P450 Enzyme InductionEnzyme Induction

CV-1HuH7 cell

PXRRXR

Differential Activation of Differential Activation of Human,Human, Rabbit,Rabbit,andand RatRat PXR by CYP3A InducersPXR by CYP3A Inducers

PCN

rifampicin

lovastatin

clotrimazole

Normalized Reporter Activity

0 20 40 60 80 100 300 350 400

OH

OHO

NN

NMe

NH

OO

O

HO

AcO

MeO

OHHO

N N

Cl

O

O

H

O

HO O

H

HO

O

HCN

PXR Sequence HomologyPXR Sequence Homology1 41 107 141 434

Human PXR1

Rat PXR11 38 104 138 431

Xenopus ONR11 37 102 136 386

Human VDR1 24 89 122 427

LigandDNA

96

69

63 37

76

42

Mouse PXR11 38 104 138 431

96 76

Rabbit PXR11 41 107 141 434

8294 Variation in ligand binding domain consistent with in vivo species differ-ences in response to inducers

Amino Acid Differences in the Amino Acid Differences in the Ligand Binding Domain of PXRLigand Binding Domain of PXR

Zhang et al., Arch. Biochem. Biophys., 1999

Ser187 Leu213 Asp266 Glu337 Ile417

Gly181 Leu206 Tyr263 His333

hPXR

Phe184 Leu210 Asp263 Lys334 Ser414

Gly178 Arg203 Tyr260 Arg333

mPXR

Val184 Val210 Glu263 Glu333 Thr414

Asp178 Ser203 His260 Arg333

rPXR

ATTTAAGGAAAgGGGTCAGACC------AACTAGGGTAaAGTTCAGTG

+1 (gene)

Rat CYP4A1

-2kb-10kb

-4466-4850384 bpDR1 (9/12) DR1 (9/12)

Rat CYP4A1 Response ElementsRat CYP4A1 Response Elements

Proximal PPRE Identified by Aldridge et. al. Biochem. J. 306, 473-479, 1995

Element 1 not functional Element 2 is a Functional PPRE

+1

Human CYP411

-2kb-5kb

AAACAAGGGAATAGCCCAAAAG

-4493DR1 (8/12)

-4472

-7kb-10kb

AAAAGTGGGCAAAGGATATGCADR1 (8/12)

-7238 -7217

Analysis of the Human CYP4A11 GeneAnalysis of the Human CYP4A11 Gene

Upstream analysis of the CYP4A11 gene located on chromosome 1 revealed two possible PPRE’s

Kawashima et. al., Archives of Biochemistry and Biophysics (2000) 378(2), 333-339Sequenced -2251 bp upstream of gene, no PPRE identified.

Gel Shift AssayGel Shift Assay

PPARα + - .5 1 2 + - .5 1 2 + - .5 1 2RXRα - + .5 1 2 - + .5 1 2 - + .5 1 2

Rat Human -4.5 kb Human -7.5 kb

PPRE/PPARα/RXR

SummarySummary• Induction of metabolism is caused by many

structurally unrelated xenobiotics.• Induction occurs mainly by transcriptional

regulation of metabolizing enzymes and transporter proteins.

• Nuclear receptors mediate the induction response by most xenobiotics.

• Amino acid differences in the ligand-binding domain of the receptors are mainly responsible for the species differences in the induction of CYP450 enzymes.

Additional ReadingAdditional Reading• Parkinson, A.: Biotransformation of xenobiotics. In: Casarett and Doull’s

Toxicology. The Basic Science of Poisons. Sixth edition (edited by C.D. Klaassen). McGraw Hill, New York, 2001.

• Wang, H. and Negishi, M. (2003) Transcriptional regulation of cytochrome p450 2B genes by nuclear receptors. Curr Drug Metab. 4(6):515-25.

• Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydowbackman, M., Ohlsson, R., Postlind, H., Blomquist, P. and Berkenstam, A. (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. USA.95:12208-12213.

• Blumberg, B., and Evans, R.M. (1998) Orphan nuclear receptors – new ligands and new possibilities. Genes Dev. 12:3149-3155.

• Geick A., Eichelbaum M., and Burk O. (2001) Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J Biol Chem.276(18):14581-14587.

• Moreau, A, Vilarem, MJ, Maurel, P; and Pascussi, JM. (2007) Xenoreceptors CAR and PXR Activation and Consequences on Lipid Metabolism, Clucose Homeostasis, and Inflammatory Response. Mol. Pharmaceutics 5(1):35-41

Additional ReadingAdditional Reading• Goodwin B., Hodgson E., and Liddle C. (1999) The orphan human

pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Mol Pharmacol56:1329-1339.

• Honkakoski P. and Negishi M. (1998) Regulatory DNA elements of phenobarbital-responsive cytochrome P450 CYP2B genes. J Biochem Mol Toxicol 12:3-9.

• Jones, S. A., Moore, L. B., Shenk, J. L., Wisely, G.B., Hamilton, G. A., McKee, D. D., Tomkinson, N. C. O., LeCluyse, E. L., Wilson, T. M., Kliewer, S. A. and Moore, J. T. 2000. The pregnane X receptor, a promiscuous xenobiotic receptor that has diverged during evolution. Mol. Endocrinol. 14: 27-39.

• Wang, H., and LeCluyse E. L. 2003. Role of orphan nuclear receptors in the regulation of drug metabolising enzymes. Clin. Pharmacokinet. 42: 1331-1357.