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Oral benzo[a]pyrene, immunosuppression,
and tumors: role of the three CYP1 enzymes
Daniel W Nebert, MD
Department of Pediatrics, Division of Human Genetics
Center for Environmental Genetics
Department of Environmental Health
University of Cincinnati Medical Center
Boston University, March 5, 2oo7
OUTLINE of the TALK
Route-of-administration, dose, target organ, and cell-type-specific gene expression (including metabolism) are all critical in environmentally-caused malignancies
CYP1 inducibility also in humans = AHR
Paradoxical studies in knockout mice
The human CYP, mouse Cyp superfamily
Intro: the [Ah] gene battery in the mouse
Signal Received by cell Response
Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP)
Received by the cell
Induction of an enzyme that metabolizes the stimulus
Tryptophan Received in bacteria Induction of tryptophan pyrrolase 80,000-fold
PHENOTYPE
PERTURBATION
Response of BaP Hydroxylase (CYP1A1) Activity to i.p. PAHs (e.g. benzo[a]pyrene)
Mouse Control Treated
B6 500 3,000
D2 500 500
––J Biol Chem 1968; 243: 6242 & 6250
LIVER
LUNG
C YP1A1 e nzym e a c tivity p e r m g m ic ro so m a l p ro te in
C YP1A1 e nzym e a c tivity p e r g we t we ig ht
50 5025 75
LACK of CYP1A1 induction autosomal recessive
“Resp”
“Nonresp”
50 5025 75
3MC was one of eight PAHs tested
Genetics of “Ah-responsiveness”
AhbAhb x AhdAhd
F1 AhbAhd
AhbAhd x AhbAhb
AhbAhb: AhbAhd
AhbAhd x AhbAhd
F2 AhbAhb: AhbAhd: - AhbAhd: AhdAhd
AhbAhd x AhdAhd
AhbAhd: AhdAhd
B6-D2 Difference in CYP1A1 Inducibility: the Shot Heard ’Round the World
Has resulted in >400 publications by DwN Lab
Top 1% “most cited” in pharmacology and toxicology field by ISI––since 1st survey 1964-78
Numerous national and international awards
Hundreds, if not thousands, of labs have also entered AH receptor/CYP1 field of research
C O N J U G AT E DP R O D U C T S
O X Y G E N AT E DIN T E R M E D IAT E S
P h a se II
" D M E R e c e p to rs"
P h a se I
O x id a tiv e s tre ssTo x ic ityC a n c e rM u ta t io n s
?D R U G -M E TA B O L IZ IN G E N Z Y M E S (D M E s)
E n v iro n m e n ta l p o llu ta n tsD r u g sF o o d stu ffH e a v y m eta ls
Metabolic activation
DetoxicationCYP1’s
XME Receptors
Xenobiotic-Metabolizing Enzymes (XMEs)
1968-99: CYPs are BAD
B6-D2 Difference in CYP1 Inducibility:
PAH-induced in utero lethality; teratogenesis
PAH-induced malignancies of certain tissues, following various routes-of-administration (ROAs)
PAH-induced mutagenesis (Ames test)
PAH-induced marrow toxicity; immunosuppression
PAH-induced athersclerosis; resistance to EtOH
Basis for identifying the AH receptor
PAH-induced ovarian toxicity; uroporphyria
––Crit Rev Toxicol 1989; 20: 153
2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD; “dioxin”)
TCDD is poorly metabolized, ~36,000 timesmore potent than BaP in inducing rat
liver CYP1A1 activity (A. Poland, 1972)
SPEC
IFIC
CYP
1A1
AC
TIVI
TY
g TC DD/kg BO DY WEIG HT
B6
D2
4.9
3.5
2.1
0.7
0
0.01 0.1 1.0 10 100
This 1974 study says the Cyp1 structural genes same, but regulatory gene (a receptor?) is different?
In 1994 study, B6 high-affinity AHR: Pro-474; D2 poor-affinity AHR: Leu-474
“Ah-Resp”
“Ah-NonResp”
ED50 15-20X different
SEVEN TO TEN OUT OF EVERY 100
CIGARETTE SMOKERSDEVELOP LUNG CANCER
WHY DON’T THE OTHER 90%+ SMOKERS
DEVELOP LUNG CANCER..?
0
20
40
60
80
100
FijiFijiN. Caled.GuamCook Is.Fr. Polyn. Hawaii
1986-891986-891977-871978-881978-831986-89 1978-82
Polynesians Micronesians Melanesians Indians
Inc
iden
ce
Rat
e p
er
100
,00
0
Male Lung Cancer Incidence in Pacific Island Populations
>50-fold difference in lung cancer susceptibility between cigarette smokers of two ethnic groups..!!
SPEC
IFIC
CYP
1A1
AC
TIVI
TY
g TC DD/kg BO DY WEIG HT
B6
D2
4.9
3.5
2.1
0.7
0
0.01 0.1 1.0 10 100
Could this be like cigarette smokers with lung cancer?
Could this be like cigarette smokers who don’t get cancer?
Cigarette-pack-years?
Highly Sensitive (HS)
Highly Resistant (HR)
D e n v e rP o p u la tio n
To ro n to P o p u la tio n
K d (n M )
8
6
4
2
00 5 1 0 1 5
Pro
bit
8
6
4
2
00 5 1 0 1 5
Dissociation constant
Kd = TCDD-binding
affinity, det’d by Scatchard plot analysis of HUMAN placental cytosolic samples (N=115)
N= 37
N = 78Highest affinity
Poorest affinity
Differences in affinity also at least 15-20X
Detoxication
Reactive intermediate
Inducer-receptor complex in cytosolTC DD, BaP
Ba P
C onjug ate
a nd other
enviro nm enta l
p o lluta nts
Formation of excreted innocuous products
Critical target in other cells
Induc er-rec ep tor c om plex in nuc leus
M essa ge
rec e ived
Unknown
Rec ep tor
in c y toso l
B in d in g
o f r e a c t iv e
i n te r m e d i a t e
to c r i ti c a l
ta r g e t
specific proteinsTranslation of induction-
Incorporation of CYP1A1/1A2/1B1 into membranes
c ritic a l ta rg et
Transcription of induction-specificmRNAs
Toxicity
cancerand/or
Metabolic activation 1979
[Scheme, of course, fashioned after what was known of the ER]
TCDD
Cl
Cl
Cl
Cl
Cl
Coplanar PCBs
Benzo[a]pyrene
3-Methylcholanthrene
Classical AHR ligands
Naturally occurringligands?
Non-classical ligands
Bilirubin
Prostaglandin G2
7-Ketocholesterol
NH2
NH21,5-Diaminonaphthalene
Tryptamine
Indirubin
Omeprazole
A h r L O C U S
A H R E C E P T O R
A R N T
E P R E
A H R E
A C T H
Cyp1a1
Cyp1a2
Cyp1b1
Nqo1
Aldh3a1
Ugt1a6
Gsta1
“THE [Ah] GENE BATTERY”
(XRE)
1976-92 (ARE)
TCDDBaPE.L.
+
NF1 C /C TF AHR etc .
O XIDATIVESTRESS
REDUC E O XIDATIVE STRESS
RO
(?)
R
?
+
AHREsEPRE
no n- P450 ENZYM ES
non-P450 G ENES
C YP1A1enzym e
Ba PO
Ba P
NFDAHRE
EP
EL
AHRR m RNA
AHRR ARNT
TC DD
C YTO KINES(IL-2, TNF )
Ahrr
AHR
+
+AHRE
C yp1a 1
1999
(1A2; 1B1)
ENDOGENOUS FUNCTIONS of CYPs
Arachidonic acid cascade: >115 eicosanoids, prostacyclin, thromboxane
Cholesterol, bile acid biosynthesis
Steroidogenesis
Vitamin D3 biosynthesis
Retinoic acid, (?)other morphogens
Still unknown functions
Biogenic, neurogenic amines
CYP Gene Superfamily: 57 in human; 102 in mouse
Family Subfamilies Members CYP1 2 (1A1, 1A2, 1B1)CYP2 13 16 ; 50CYP3 1 4 ; 8CYP4 6 12 ; 20CYP5 1 1CYP7 2 2CYP8 2 2CYP11 2 3CYP17 1 1CYP19 1 1CYP20 1 1CYP21 1 1CYP24 1 1CYP26 3 3CYP27 3 3 ; 2(+ ps)CYP39 1 1CYP46 1 1CYP51 1 1
Eic
osan
oid
s;
pla
nts
; d
rugs
ancestral?
Definition of EICOSANOID:
“Any of the many dozens of physiologically active substances derived from arachidonic acid––including the prostaglandins, leukotrienes, prostacyclins, and thromboxanes ––involved in many critically important life functions”
CYP8A1
ARACHIDONIC ACID
12-HHT
PGI2
TXA2
EETsHETEsHPETEsLe ukotrie ne s
CYP1CYP2CYP3CYP4
PTGS1PTGS2
PGH 2
CYP5A1
CYP1A1PGE 2
PGD2
CYP1CYP2CYP3CYP4
Plateletaggregation
Plateletdisaggregation
(COX-1)(COX-2)
There are >115 eicosanoids
EpoxyEicosaTrienoic acidsHydroxyEicosaTriEnoicHydroPeroxyEicosaTetraEnoicprostaglandins, prostacyclins,thromboxanes, leukotrienes
Arachidonic acid
EETs, HPETEs :bronchodilation, renal vasoconstriction, intestinal vasodilation, inhibit cyclooxygenase, stimulate c-Fos and c-Jun, mitogenesis, inhibit platelet aggregation, peptide hormone secretion, mobilize intracellular Ca+
+, electrolyte transport, M++ homeostasis during inflammation
HETEs; -, -1 alcohols :vasodilation, chemotaxis, vasoconstriction, bronchoconstriction, inhibit Na,K ATPase, stimulate Na,K-ATPase
PROPERTIES OF AH RECEPTOR
Affects many pathways (EGFR, PKC, p21RAS, MAPK, Src, Wnt/-catenin, Myc, Myb, Fos, p27, p53, RB1 binding, slowing at G1/S & G2/M boundaries)
Ubiquitous; expressed in utero, placenta (even in
mollusk, sea squirt, Drosophila, Caenorhabditis elegans)
Ahr(-/-) k.o. mouse––lower viability, fertility; defective (A-V) vasculature in liver, heart, kidney
Quite likely that AHR uses various endogenous ligands (ELs) in different cell types
PROPERTIES OF CYP1A1
Constitutive activity nil; PAH-inducible ; metabolizes PAHs
Ubiquitous; expressed in utero, even 12-h ovum
Cyp1a1(-/-) knockout mouse viable, fertile
No mutants of Cyp1a1 gene alter PAH activity (steroid hydroxylases? eicosanoid metabolism?)
PROPERTIES OF CYP1A2
Not detectable until neonatal period
High basal levels in liver; PAH-inducible in liver, lung, brain, GI tract, pancreas; VERY low in spleen, thymus; nil in kidney; metabolizes aryl and alkyl amines
Cyp1a2(-/-) knockout mouse viable, fertile
In human, no DNA variant in CYP1A2 gene so far can explain >60X differences in liver
PROPERTIES OF CYP1B1
High basal levels in blood vessels, GI tract, skin, all endocrine tissues, spleen, marrow, thymus, tumors; PAH-inducible; metabolizes PAHs
Expressed in placenta; in utero (adrenal cortex)
Cyp1b1(-/-) knockout mouse viable, fertile (Glaucoma, when combined with ablation of Tyr gene)
Mutations in human CYP1B1 gene causes primary congenital glaucoma (buphthalmos)
CN CH3
O
OH
OHHO
O
CN
CH3
O
O H
N-C-CH3
O
OH
N-C-CH3
O
C YP1A1
C YP1A2
CYP1B1
PAHs
Aryl amines
Reactive intermediates
Hypothesis:
If the Ahr gene, or any of the Cyp1a1, Cyp1a2, Cyp1b1 genes were genetically removed, the mice should be protected against chemical substrates that bind to AHR or that each of the enzymes metabolically activates
Thanks especially to: Tim Dalton, Shige Uno, Nadine Dragin
Toxicity or Tumors in Knockout Mouse Models
Cyp1a2 k.o. tumors, adducts: 4-aminobiphenyl [ABP]; 2-NH2-1-Me-6-Phenl-ImidAz-Pyr [PhIP], 2-NH2-
3-Me-imidazole-quinoline [IQ] paradoxical effect..!Cyp1a1 k.o. immunosuppression, tumors: with
oral BaP paradoxical effect..!
Ahr k.o. protected: TCDD-induced tox; BaP-induced skin CA; benzene-induced hematotoxicity [as expected]
Cyp1b1 k.o. protected: DMBA-induced lymphoma, marrow tox, leukemia, ovarian CA; dibenzo[a,l]phenan-thracene-induced CA [as expected]
Cyp1a1(-/-) control Cyp1(+/+) BaP Cyp1a1(-/-) BaP
BONE MARROW:
Oral BaP, 125 mg/kg/day for 18 days; death 24-32 days for Cyp1a1(-/-)
Hours
BaP
in b
lood
(ng
/ml)
0
100
200
300
Cyp1a1(-/-)Cyp1a1(+/+)
0
200
400
600
0 5 10 15
Cyp1a1(-/-); TCDDCyp1a1(+/+); TCDD
150 5 10
Cyp1a1(-/-) knockout mice have a greater BaP body burden and slower clearance rate than Cyp1(+/+) wild-type
TCDD pretreatment: BaP clearance speeded up
"FIRST-PASS ELIMINATION KINETICS" AS THECAUSE OF MARROW TOXICITY BY ORAL BENZO[a]PYRENE
Oral BaP
w
I TRACT
IVER
BaPBPO
BaP
BPO
Metabolitesexcreted
Oral BaP
w
I TRACT
IVER
BaPBPO
BaP
BPO
Metabolitesexcreted
Cyp1a1(+/+)
+
Cyp1a1(-/-)
No CYP1A1 in GI tract or liver
Bigger bolus of BaP to marrow
Why does Cyp1a1(-/-) die, while wild-type stays so healthy?
CYP1A1 inducible
Cyp1a1/1a2(-/-) line has been made
loxP sites in 3' UTR of Cyp1a1 and Cyp1a2 genes
Cyp1a1 -- spacer region -- Cyp1a2(6.2 kb) (13,456 bp) (6.7 kb)
Cyp1a1/1a2(-/-) Cre-loxP excision of ~25 kb;
inter-chromosomal..!
Bone marrowBaPOil
Cyp1(+/+)
Cyp1a1(-/-)
Double-k.o.Cyp1a1/1b1(-/-)
Healthy
Dies w/in 1 month
+ Healthy !
Hours
BaP
in b
lood
(ng
/ml)
0
100
200
300
0
200
400
600
0 5 10 15
150 5 10
Cyp1a1(-/-)Cyp1(+/+)
Cyp1a1/1b1(-/-)
TCDD-
TCDD+
Cyp1a1(-/-)
Cyp1a1/1b1(-/-)
Cyp1a1/1b1(-/-) has greater BaP body burden but shows less toxicity than Cyp1a1(-/-)..!
Cyp1(+/+)
Cyp1a1(-/-)
1a1/1b1(-/-)
2.2 + 1.6
56 + 5.7
165 + 46
BaP (ng/ml) in whole blood; 5 da oral BaP
Cyp1a1(-/-) is 25X and Cyp1a1/1b1(-/-) is 75X more BaP in blood, compared with Cyp1(+/+) wild-type..!!
Cyp1(+/+) Cyp1b1(-/-) Cyp1a2(-/-) Cyp1a2/1b1(-/-)
Cyp1a1/1a2(-/-)Cyp1a1(-/-)
1a1/1a2/1b1(-/-)Cyp1a1/1b1(-/-)
Oral BaP, 125 mg/kg/day
Clinical outcome:Healthy for months, years
Die within one month
Wild-type phenotype
Blood BaP levels (ng/ml) after 5 days feeding:
1.5-6.0 50-60 160-180
Bone marrow, thymus, spleen:
Normal Near normalSevere aplastic anemia
"FIRST-PASS ELIMINATION KINETICS" AS THECAUSE OF MARROW TOXICITY BY ORAL BENZO[a]PYRENE
Oral BaP
w
I TRACT
IVER
BaPBPO
BaP
BPO
Metabolitesexcreted
Oral BaP
w
I TRACT
IVER
BaPBPO
BaP
BPO
Metabolitesexcreted
Cyp1a1(+/+)
+
Cyp1a1(-/-)
Metabolism by CYP1B1 required; GREATER BaP body burden..!!
WHY does lack of CYP1B1 revert Cyp1a1(-/-) back to near-normal, to wild-type?
Cyp1a1/1b1(-/-)
Cyp1(+/+)
No CYP1A1 in GI tract or liver
CYP1A1 inducible
CONCLUSIONS (oral BaP)
Cyp1a1(-/-) -mediated (oral) BaP problems are largely ablated by lack of CYP1B1 (in spleen, thymus, bone marrow)
Oral BaP-induced CYP1A1 in GI tract and/or liver is beneficial to the mouse
BaP metabolism in vitro or cell culture studies DO NOT reflect what happens in the intact animal receiving oral BaP
GENERAL RULE OF BIG PHARMA
Any candidate drug that shows inducibility of CYP1A1/1A2/1B1 (“AHR activation”) is regarded as hazardous, potentially cancer-causing
Such candidate drugs––usually abandoned immediately, without further cost to the company
Oral BaP dosages
12.5 mg/kg/day immunosuppression still seen; altered ALT, AST; BaP-DNA adducts; Cyp1a1(-/-) dies within 4-6 months
125 mg/kg/day immunosuppression; Cyp1a1(-/-) die within 1 month
1.25 mg/kg/day immunosuppression still seen (lymphocytopenia); BaP-DNA adducts
BaP, 12.5 mg/kg/day die 4-6 mo instead of 1 month;however, at 6-9 weeks, … (!!)
Genotype Outcome
Cyp1(+/+) wild-type Healthy
Cyp1a1(-/-) Duodenum/jejunum CA
Cyp1a2(-/-) Healthy
Cyp1b1(-/-) Healthy
Cyp1a1/1a2(-/-) Duodenum/jejunum CA
Cyp1a1/1b1(-/-) Preputial gland CA
Cyp1a2/1b1(-/-) Healthy
Cyp1a1/1a2/1b1(-/-) Preputial gland CA
BaP, 12.5 mg/kg/day die 4-6 mo instead of 1 month;however, … (!!)
Genotype Outcome
Cyp1(+/+) wild-type Healthy
Cyp1a1(-/-) Duodenum/jejunum CA
Cyp1a2(-/-) Healthy
Cyp1b1(-/-) Healthy
Cyp1a1/1a2(-/-) Duodenum/jejunum CA
Cyp1a1/1b1(-/-) Preputial gland CA
Cyp1a2/1b1(-/-) Healthy
*Cyp1a1/1a2/1b1(-/-) Preputial gland CA
*But, … other problems exist in triple k.o. … … … …
Genetic crosses
Total number of pups
Observ. number of pups
Expectd number of pups
2 value
P value
aabbaaBb
aaBbaabb
AabbAaBb
AaBbaaBb AabbaaBb
264 30 58.25 10.85 <0.001
AabbAabb aaBbaaBb
aabbaabb 11 11 11 0 1.0
Generation of triple-k.o. pups shows embryolethality
7 combinations, female left, male right; A = Cyp1a1/1a2 allele; B = Cyp1b1
Embryolethality – incomplete penetrance; survivors are fertile
Phenotype of Cyp1a1/1a2/1b1(-/-)
Hydrocephalus – incomplete penetrance
Embryolethality – incomplete penetrance
Hermaphroditism – incomplete penetrance
Small in size, but some survive, are fertile; smaller litters
Cystic ovary – incomplete penetrance
Other details of Cyp1(-/-) triple k.o.
Decreased response to i.p. inflammatory challenge by zymosan
Metabonomics; cDNA microarray expression: perturbations in “lipid, cell, cation, anion” genes
What is the common denominator between alterations in inflammatory response and cation/anion homeostasis?
Arachidonic acid cascade: eicosanoids
AHR must have as its E.L. one or several of these >115 eicosanoids
CYP1 enzymes must be critical in formation and degradation of specific eicosanoids
Perturbations in eicosanoids also consistent with A-V shunts, proliferation, apoptosis, and development (& toxicity) of many types
CYP1A1 CYP1A2BAC-D
BAC-H
BAC-F
BAC-E
BAC-G
180 kb
130 kb
120 kb
60 kb
110 kb
hCYP1A1_CYP1A2 Locus in Bacterial Artificial Chromosomes (BACs)
BAC-D: no expression of hCYP1A2 mRNA/protein/enz.activity
BAC-H: Normal Expression (basal; inducible) of hCYP1A1 & 1A2 mRNA/protein/enz. activity (liver, lung, kidney, intestine, brain, spleen, pancreas, testis, ovary)
53 kb 23.3 kb 90 kb
Other studies to be published
Now have a liver-specific Cyp1a1(-/-) knockout line i.p. versus p.o. BaP
Now have the Cyp1a1/1a2(-/-) double and Cyp1a1/1a2/1b1(-/-) triple k.o. oral BaP
Now have a GI tract-specific Cyp1a1(-/-) knockout line i.p. versus p.o. BaP
Yes, hCYP1A1_1A2 behaves like mouse Cyp1a1/1a2(+/+) i.p. versus p.o. BaP
CONCLUSIONS (of the Entire Talk)
“Inducible” CYP1 act. not necessarily bad
The intact animal can exhibit different results from in vitro or cell culture studies
Route-of-administration, dose, target organ, and cell-type-specific gene expression (including metabolism) are all critical in environmentally-caused malignancies
Loss of all three Cyp1 genes, loss of AHR fundamental defects in eicosanoid action