ENVR 132/TOXC 142/BIOC142 Biochemical & Molecular Toxicology Induction of Metabolism by Toxicants...
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Transcript of ENVR 132/TOXC 142/BIOC142 Biochemical & Molecular Toxicology Induction of Metabolism by Toxicants...
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: [email protected]
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 & Confidential6
Inhibition-Induction
TimeCon
cent
rati
on
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 andMan-made ChemicalsMan-made 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
P450 Enzyme Inducer
In Vivo Induction in Male Rats
Control Activity Induced Activity
CYP1A BNF 152 27 3,320 183
CYP2B PB 24 4 1,460 180
CYP3A PCN 2,460 780 12,693 2,255
CYP4A CLO 489 52 10,693 620
CYP1A, EROD; CYP2B, PROD; CYP3A, testosterone 6-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: [1414C-methyl]aminopyrineC-methyl]aminopyrine
Ser
rum
tria
zola
m (
ng/m
l)
Rifampin Effects on Triazolam DispositionRifampin Effects on Triazolam Disposition
Villikka et al., Clin Pharmacol Ther 1997;61:8-14.
Rifampin Placebo
Consequences of Cytochrome Consequences of Cytochrome P450 Enzyme InductionP450 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
HN
COCH 3
OH HN
COCH 3
OSO 3H
HN
COCH 3
OO CO 2H
OH
OHHO
N
O
COCH 3
Endocrine Disruption Endocrine Disruption 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
NR’s and P450 InductionNR’s and P450 Induction
CYP450 gene Promoter 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
Co-regulation of Target Genes by NR’sCo-regulation of Target Genes by NR’s
• 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 P450’s, UGT’s and Coordinate Regulation of P450’s, UGT’s and Transporters by NR’sTransporters by NR’s
UGT’s
MRP3
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
PAH Inducers in Rat vs. Human
Rat TCDD 1A1 mRNA
EC50 = 0.00767 +/- 0.00409
EC50 = 0.0107 +/- 0.043
CYP1A1 mRNA Hu497
EC50 10X Difference
EC50 = 0.00767 +/- 0.00409
Relationship between In Vitro Potency and Induction In Vivo
EC50 Cmax [Cmax]/EC50 Clinical Relevance
Nifedione 8 0.008 0.001 No known
Lovastatin 1-6 0.008 0.008-0.002 No known
Rosiglitazone 5-10 0.3-1.2 0.05-0.12 No known
Simvastatin 0.14 0.024 0.17 No known
Troglitazone 3-6 7 2.3 Yes
Phenytoin 25 80 3.2 Yes
Avasimibe 0.2 1-6 5-30 Yes
Rifampicin 0.8 14 17.5 Yes
Carbamazepine 0.9 25 28 Yes
Clotrimazole 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/Arnt heterodimer
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
5’ 3’
n
n
n
DRn
IRn
ERn
CYP2B Response elements
CYP2B6 TGTACT n=4 TGACCC CYP2b10 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
NR3
CYP2B6 TGGACT n=4 TGACCC
CYP3A Response elements
CYP3A4 TGAACT n=3 TGACCC CYP3A2 TGACCT n=3 TGAGCT CYP3A23 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 Genes
UGT1A1 TGAGTT n=4 TAACCT MDR1 TGAGAT n=6 AGTTCA rMRP2 TGAACT n=8 AGTTCA CYP2C9 CAAACT n=4 TGACCT
Nuclear Receptor PXRNuclear Receptor PXRPB
CA
R
PXR
cytoplasm
nucleus
HS
P90
PXR
RIF
RXR
PX
R
RX
R
XREM CYP3A
?translocation?
-mouse-yes
-human-no
Activator/Agonist CYP TargetHuman RIF CYP3A4Rat PCN CYP3A1/2Mouse PCN Cyp3a11
Nuclear Receptor CAR: PB Induction-Constitutively ActiveNuclear Receptor CAR: PB Induction-Constitutively Active
CAR
cytoplasmnucleus
HS
P90
HS
P90
CAR
PP2A
PB
CCRP
RX
R
CA
R
RX
R
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
Similar Binding of PXR and CAR Similar Binding of PXR and CAR to Promoter Response Elementsto Promoter Response Elements
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 ER6-3A4
PXR + + + + + + CAR + + + + + +RXR + + + + + + + + + + + +
PXR/RXRCAR/RXR
GR/Dex Role in Basal & Induced P450 GR/Dex Role in Basal & Induced P450 Expression via CAR/PXR (master regulator)Expression via CAR/PXR (master regulator)
Role of CAR/PXR in lipid metabolism, Role of CAR/PXR in lipid metabolism, synthesis, and uptakesynthesis, and uptake
Moreau et al. 2007 Mol. Pharmaceutics
PXR & CAR role in Glucose HomeostasisPXR & CAR role in Glucose Homeostasis
Molecular Basis for the Species Molecular Basis for the Species Differences in Enzyme InductionDifferences in Enzyme Induction
Rabbit
Human
Rat
0.1%
DM
SO
5M
PC
N
10M
Rifa
mpi
cin
10M
SR
1281
3
10M
DT
BA
CYP3A6
CYP3A4
CYP3A23
Species Differences in the Regulation Species Differences in the Regulation of CYP3A Enzymesof 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
% D
MSO)
3-M
C 1µM
Drug '
X' 0.6µ
M
Drug '
X' 2µM
Drug '
X' 6µM
Drug '
X' 20µ
M
Ph
enac
etin
O-D
ealk
ylat
ion
(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
% D
MSO)
3-M
C 2µM
Drug '
X' 0.6µ
M
Drug '
X' 2µM
Drug '
X' 6µM
Drug '
X' 20µ
M
Phe
nace
tin
O-D
ealk
ylat
ion
(pm
ol/m
in/m
g)
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 2
µM
Drug 'X
' 0.2µM
Drug 'X
' 2µM
Drug 'X
' 6µM
Drug 'X
' 20µ
M
CY
P1A
2 A
ctiv
ity
(nm
ol/m
in/m
g)
Species Differences in CYP4A Species Differences in CYP4A Induction by Clofibric AcidInduction by Clofibric Acid
Rat Human
CTL 1 10 100 500 1000
CY
P4A
1 F
old
Ind
uct
ion
0
10
20
30
40
50
60
Clofibric Acid (µM)
Rat Hepatocytes Human Hepatocytes
Lauri
c aci
d 1
2-h
ydro
xyla
tion
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 Inducers PXR 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
H
CN
PXR Sequence HomologyPXR Sequence Homology1 41 107 141 434
Human PXR1
Rat PXR11 38 104 138 431
Xenopus ONR11 37 102 136 386
Human VDR 1 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
AAAAGTGGGCAAAGGATATGCA
DR1 (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
PPAR + - .5 1 2 + - .5 1 2 + - .5 1 2 RXR - + .5 1 2 - + .5 1 2 - + .5 1 2
Rat Human -4.5 kb Human -7.5 kb
PPRE/PPAR/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.
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 Pharmacol 56:1329-1339.
• 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.