Transcript CYP1A1 - Boston University

Oral benzo[a]pyrene, immunosuppression,
and tumors: role of the three CYP1 enzymes
Daniel W Nebert, MD
Department of Environmental Health
Department of Pediatrics, Division of Human Genetics
Center for Environmental Genetics
University of Cincinnati Medical Center
Boston University, March 5, 2oo7
OUTLINE of the TALK
Intro: the [Ah] gene battery in the mouse
CYP1 inducibility also in humans = AHR
The human CYP, mouse Cyp superfamily
Paradoxical studies in knockout mice
Route-of-administration, dose, target organ,
and cell-type-specific gene expression
(including metabolism)  are all critical in
environmentally-caused malignancies
Signal  Received by cell  Response
Tryptophan  Received in bacteria  Induction of
tryptophan
pyrrolase
80,000-fold
PERTURBATION
Polycyclic
aromatic
hydrocarbons
(PAHs) such as
benzo[a]pyrene
(BaP)
PHENOTYPE
Received
by the cell
Induction of an
enzyme that
metabolizes the
stimulus
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
“Resp”
3MC was one of
eight PAHs tested
“Nonresp”
50
25
50
75
CYP1A1 enzyme a ctivity per g wet weight
LUNG
LACK of
CYP1A1
induction
autosomal
recessive
50
25
50
75
CYP1A1 enzyme a ctivity per mg microsoma l protein
Genetics of “Ah-responsiveness”
AhbAhb x AhdAhd
AhbAhd x AhbAhb
AhbAhd
AhbAhb: AhbAhd
AhbAhd x AhbAhd
AhbAhd x AhdAhd
F1
F2
-
AhbAhb: AhbAhd:
AhbAhd: 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
Hundreds, if not thousands, of labs have also
entered AH receptor/CYP1 field of research
Numerous national and international awards
Xenobiotic-Metabolizing
Enzymes (XMEs)
DRUG-METABOLIZING ENZYMES
(DMEs)
?
XME
"DMEReceptors
Receptors"
Phase I
Phase II
CYP1’s
Environmental
pollutants
Drugs
Foodstuff
Heavy metals
Detoxication
Metabolic activation
OXYGENATED
INTERMEDIATES
Oxidative stress
Toxicity
Cancer
Mutations
CONJUGATED
PRODUCTS
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 ovarian toxicity; uroporphyria
PAH-induced athersclerosis; resistance to EtOH
Basis for identifying the AH receptor
––Crit Rev Toxicol 1989; 20: 153
2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD; “dioxin”)
TCDD is poorly metabolized, ~36,000 times
more potent than BaP in inducing rat
liver CYP1A1 activity (A. Poland, 1972)
In 1994 study,
B6 high-affinity
AHR: Pro-474;
D2 poor-affinity
AHR: Leu-474
4.9
B6
SPECIFIC CYP1A1 ACTIVITY
This 1974
study says the
Cyp1 structural
genes same, but
regulatory gene
(a receptor?)
is different?
3.5
D2
“Ah-Resp”
2.1
ED50
15-20X
different
“Ah-NonResp”
0.7
0
0.01
0.1
1.0
10
g TCDD/kg BODY WEIGHT
100
SEVEN TO TEN
OUT OF EVERY 100
CIGARETTE SMOKERS
DEVELOP LUNG CANCER
WHY DON’T
THE OTHER 90%+ SMOKERS
DEVELOP LUNG CANCER..?
Male Lung Cancer Incidence in
Pacific Island Populations
Incidence Rate per 100,000
100
>50-fold difference in lung cancer
susceptibility between cigarette
smokers of two ethnic groups..!!
80
60
40
20
0
Hawaii Fr. Polyn. Cook Is.
1978-82 1986-89 1978-83
Polynesians
Guam N. Caled.
Fiji
1978-88 1977-87 1986-89
Micronesians Melanesians
Fiji
1986-89
Indians
Could this be
like cigarette
smokers with
lung cancer?
4.9
SPECIFIC CYP1A1 ACTIVITY
B6
3.5
Highly Sensitive
(HS)
D2
Highly Resistant
(HR)
2.1
0.7
0
Could this be like
cigarette smokers who
don’t get cancer?
0.01
0.1
1.0
10
g TCDD/kg BODY WEIGHT
Cigarette-pack-years?
100
8
6
N = 78
4
Highest
affinity
Denver
Population
2
Probit
Dissociation constant
Kd = TCDD-binding
affinity, det’d by
Scatchard plot
analysis of HUMAN
placental cytosolic
samples (N=115)
0
0
5
10
15
8
6
Differences in affinity
also at least 15-20X
N= 37
4
2
Toronto
Population
0
0
5
10
Kd (nM)
15
Poorest
affinity
[Scheme, of course, fashioned after what was known of the ER]
Receptor
in cytosol
TCDD, BaP
a nd other
environmenta l
polluta nts
Inducer-receptor
comp lex in cytosol
Incorporation of
CYP1A1/1A2/1B1
into membra nes
Ba P
Inducer-receptor
com plex in nucleus
Transcription of
induction-specific
mRNAs
Tra nslation of inductionspecific proteins
Unknown
c ritic al target
Formation
of excreted
innocuous
products
Toxicity
and/or
ca ncer
Rea ctive
intermediate
Conjugate
Detoxication
Messa ge
rece ived
Binding
of reactive
intermediate
to critical
target
Metabolic activation
Critica l
target in
other cells
1979
TCDD
Prostaglandin G2
Tryptamine
Cl
Cl
Cl
Cl
Indirubin
Cl
Coplanar PCBs
Bilirubin
Omeprazole
Benzo[a]pyrene
NH2
7-Ketocholesterol
3-Methylcholanthrene
Classical AHR ligands
Naturally occurring
ligands?
NH2
1,5-Diaminonaphthalene
Non-classical ligands
“THE [Ah] GENE BATTERY”
AHRE
(XRE)
Ahr LOCUS
Gsta1
Ugt1a6
1976-92
Aldh3a1
ARNT
Nqo1
Cyp1b1
Cyp1a2
TCDD
BaP
E.L.
Cyp1a1
AH RECEPTOR
ACTH
EPRE (ARE)
AHR
NF1 C/CTF
(1A2; 1B1)
AHREs
+ TCDD
NFD Cyp1a1
R
BaP
EL
AHR
AHRR
ARNT
?
(?)
CYP1A1
enzyme
+
AHRE
EPRE
+
etc.
non-P450 GENES
non- P450 ENZYMES
EP
RO
BaPO
AHRR mRNA
Ahrr
+
AHRE
OXIDATIVE
STRESS
REDUCE OXIDATIVE STRESS
CYTOKINES
(IL-2, TNF)
1999
ENDOGENOUS FUNCTIONS of CYPs
Arachidonic acid cascade: >115 eicosanoids,
prostacyclin, thromboxane
Cholesterol, bile acid biosynthesis
Steroidogenesis
Vitamin D3 biosynthesis
Biogenic, neurogenic amines
Retinoic acid, (?)other morphogens
Still unknown functions
Family
CYP1
CYP2
CYP3
CYP4
CYP5
CYP7
CYP8
CYP11
CYP17
CYP19
CYP20
CYP21
CYP24
CYP26
CYP27
CYP39
CYP46
CYP51
Eicosanoids;
plants; drugs
CYP Gene Superfamily: 57 in human; 102 in mouse
Subfamilies
2
13
1
6
1
2
2
2
1
1
1
1
1
3
3
1
1
1
ancestral?
Members
(1A1, 1A2, 1B1)
16 ; 50
4; 8
12 ; 20
1
2
2
3
1
1
1
1
1
3
3 ; 2(+ ps)
1
1
1
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”
There are >115 eicosanoids
EpoxyEicosaTrienoic acids
HydroxyEicosaTriEnoic
ARACHIDONIC
HydroPeroxyEicosaTetraEnoic
ACID
prostaglandins, prostacyclins,
thromboxanes, leukotrienes (COX-1)
(COX-2)
CYP1
CYP2
CYP3
CYP4
PTGS1
PTGS2
PGD2
TXA2
CYP1
CYP2
CYP3
CYP4
PGE2
EETs
HETEs
HPETEs
Leukotrienes
Platelet
aggregation
CYP5A1
PGH2
CYP8A1
CYP1A1
PGI2
12-HHT
Platelet
disaggregation
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
Ubiquitous; expressed in utero, placenta (even in
mollusk, sea squirt, Drosophila, Caenorhabditis elegans)
Affects many pathways (EGFR, PKC, p21RAS, MAPK,
Src, Wnt/b-catenin, Myc, Myb, Fos, p27, p53, RB1 binding,
slowing at G1/S & G2/M boundaries)
Quite likely that AHR uses various endogenous
ligands (ELs) in different cell types
Ahr(-/-) k.o. mouse––lower viability, fertility;
defective (A-V) vasculature in liver, heart, kidney
PROPERTIES OF CYP1A1
Constitutive activity nil; PAH-inducible ;
metabolizes PAHs
Ubiquitous; expressed in utero, even 12-h ovum
No mutants of Cyp1a1 gene alter PAH activity
(steroid hydroxylases? eicosanoid metabolism?)
Cyp1a1(-/-) knockout mouse viable, fertile
PROPERTIES OF CYP1A2
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
Not detectable until neonatal period
In human, no DNA variant in CYP1A2 gene so
far can explain >60X differences in liver
Cyp1a2(-/-) knockout mouse viable, fertile
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)
Mutations in human CYP1B1 gene causes
primary congenital glaucoma (buphthalmos)
Cyp1b1(-/-) knockout mouse viable, fertile
(Glaucoma, when combined with ablation of Tyr gene)
OH
PAHs
CYP1A1
CYP1B1
O
HO
OH
O
Reactive
intermediates
O
N-C-CH3
N-C-CH3
CYP1A2
Aryl
amines
O
OH
O
C
N
CH3
C
N
OH
CH3
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
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]phenanthracene-induced CA
[as expected]
Cyp1a2 k.o. tumors, adducts: 4-aminobiphenyl
[ABP]; 2-NH2-1-Me-6-Phenl-ImidAz-Pyr [PhIP], 2-NH23-Me-imidazole-quinoline [IQ] paradoxical
effect..!
Cyp1a1 k.o. immunosuppression, tumors: with
oral BaP paradoxical
effect..!
BONE MARROW:
Cyp1a1(-/-) control
Cyp1(+/+) BaP
Cyp1a1(-/-) BaP
Oral BaP, 125 mg/kg/day for 18 days;
death 24-32 days for Cyp1a1(-/-)
Cyp1a1(-/-)
Cyp1a1(+/+)
BaP in blood (ng/ml)
300
200
Cyp1a1(-/-) knockout mice
have a greater BaP body
burden and slower clearance
rate than Cyp1(+/+) wild-type
100
0
0
5
10
15
Cyp1a1(-/-); TCDD
Cyp1a1(+/+); TCDD
600
400
200
0
0
5
10
Hours
15
TCDD pretreatment: BaP
clearance speeded up
Ma
Ma
Why does Cyp1a1(-/-) die,
"FIRST-PASS ELIMINATION KINETICS" AS THE
while wild-type
staysBENZO[a]PYRENE
so healthy?
CAUSE OF MARROW
TOXICITY BY ORAL
Cyp1a1(-/-)
w
rro
w
rro
Cyp1a1(+/+)
Oral BaP
BaP
LIVER
BPO
Oral BaP
BaP
BaP
BPO
LIVER
B aP
BPO
Bigger bolus of
BaP to marrow
BPO
O
BP
No CYP1A1
in GI tract or
liver
O
BP
BaP
BaP
GI TRACT
CYP1A1
inducible
GI TRACT
+
Metabolites
excreted
Metabolites
excreted
So, what about the Cyp1
double-knockouts..?
and the triple-knockout..?
loxP site in 3’ UTR
Mouse Cyp1a1_Cyp1a2 Locus, Head-to-Head Orientation
6.0 kb
---Mouse Chr 9---
 13.3 kb 
7.8 kb
Cyp1a1/1a2(-/-) line has been made
Cyp1a1 -- spacer region -- Cyp1a2
(6.2 kb)
(13,456 bp)
(6.7 kb)
loxP sites in 3' UTR of Cyp1a1 and Cyp1a2 genes
Cyp1a1/1a2(-/-) Cre-loxP excision of ~25 kb;
inter-chromosomal..!
Bone marrow
Oil
BaP
Cyp1(+/+)
Healthy
Cyp1a1(-/-)
Dies w/in
1 month
Double-k.o.
Cyp1a1/1b1(-/-)
+ Healthy !
Cyp1a1/1b1(-/-)
Cyp1a1(-/-)
Cyp1(+/+)
300
BaP in blood (ng/ml)
Cyp1a1/1b1(-/-)
has greater BaP
body burden but
shows less toxicity
than Cyp1a1(-/-)..!
-
TCDD
200
100
0
0
5
10
15
+ TCDD
600
Cyp1a1/1b1(-/-)
400
Cyp1a1(-/-)
200
0
0
5
10
Hours
15
BaP (ng/ml) in whole blood; 5 da oral BaP
Cyp1(+/+)
2.2 + 1.6
Cyp1a1(-/-) 1a1/1b1(-/-)
56 + 5.7
165 + 46
Cyp1a1(-/-) is 25X and Cyp1a1/1b1(-/-) is 75X more BaP in
blood, compared with Cyp1(+/+) wild-type..!!
Oral BaP,
125 mg/kg/day
Cyp1(+/+)
Cyp1b1(-/-)
Cyp1a2(-/-)
Cyp1a2/1b1(-/-)
Cyp1a1/1a2(-/-)
Cyp1a1(-/-)
Clinical outcome:
Healthy for
months, years
Die within
one month
Blood BaP levels
(ng/ml) after 5
days feeding:
1.5-6.0
Bone marrow,
thymus, spleen:
Normal
50-60
Severe
aplastic
anemia
1a1/1a2/1b1(-/-)
Cyp1a1/1b1(-/-)
Wild-type
phenotype
160-180
Near normal
Ma
Ma
WHY does lack of CYP1B1
"FIRST-PASS ELIMINATION KINETICS" AS THE
revert
back to BY
near-normal,
to wild-type?
CAUSECyp1a1(-/-)
OF MARROW TOXICITY
ORAL BENZO[a]PYRENE
Cyp1a1/1b1(-/-)
Cyp1a1(-/-)
w
rro
w
rro
Cyp1(+/+)
Cyp1a1(+/+)
Oral BaP
BaP
LIVER
BPO
Oral BaP
BaP
BaP
BPO
LIVER
B aP
BPO
Metabolism
by CYP1B1
required;
GREATER
BaP body
burden..!!
BPO
O
BP
BaP
O
BP
BaP
GI TRACT
CYP1A1
inducible
GI TRACT
No CYP1A1
in GI tract
or liver
+
Metabolites
excreted
Metabolites
excreted
CONCLUSIONS (oral BaP)
Oral BaP-induced CYP1A1 in GI tract
and/or liver is beneficial to the mouse
Cyp1a1(-/-) -mediated (oral) BaP problems
are largely ablated by lack of CYP1B1
(in spleen, thymus, bone marrow)
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
125 mg/kg/day  immunosuppression;
Cyp1a1(-/-) die within 1 month
12.5 mg/kg/day  immunosuppression still
seen; altered ALT, AST; BaP-DNA adducts;
Cyp1a1(-/-) dies within 4-6 months
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
Cyp1(+/+) wild-type
Outcome
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
Unique
“duodenal
intraepithelial
neoplasm”
(DIN) in lack
of CYP1A1
but presence
of CYP1B1
But: sometimes invasive; sometimes also in proximal jejunum
BaP, 12.5 mg/kg/day  die 4-6 mo instead of 1 month;
however, … (!!)
Genotype
Cyp1(+/+) wild-type
Outcome
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. … … … …
Generation of triple-k.o. pups shows embryolethality
7 combinations,
female left, male
right; A =
Cyp1a1/1a2 allele;
B = Cyp1b1
Expectd
2
C
number
value
of pups
Genetic
crosses
Total
number
of pups
Observ.
number
of pups
aabbCaaBb
aaBbCaabb
AabbCAaBb
AaBbCaaBb
AabbCaaBb
264
30
58.25
10.85
<0.001
11
11
11
0
1.0
P
value
AabbCAabb
aaBbCaaBb
aabbCaabb
Embryolethality – incomplete penetrance; survivors are fertile
Phenotype of Cyp1a1/1a2/1b1(-/-)
Embryolethality – incomplete penetrance
Hydrocephalus – incomplete penetrance
Cystic ovary – incomplete penetrance
Hermaphroditism – incomplete penetrance
Small in size, but some survive, are fertile;
smaller litters
Other details of Cyp1(-/-) triple k.o.
Metabonomics; cDNA microarray expression:
perturbations in “lipid, cell, cation, anion” genes
Decreased response to i.p. inflammatory
challenge by zymosan
What is the common denominator between
alterations in inflammatory response and
cation/anion homeostasis?
Arachidonic acid cascade: eicosanoids
CYP1 enzymes must be critical in formation and
degradation of specific eicosanoids
AHR must have as its E.L. one or several of
these >115 eicosanoids
Perturbations in eicosanoids also consistent
with A-V shunts, proliferation, apoptosis,
and development (& toxicity) of many types
hCYP1A1_CYP1A2 Locus in
Bacterial Artificial Chromosomes (BACs)
53 kb
23.3 kb
90 kb
180 kb
BAC-H
CYP1A1
CYP1A2
110 kb
BAC-D
130 kb
BAC-F
120 kb
BAC-E
60 kb
BAC-G
BAC-H: Normal Expression (basal; inducible) of hCYP1A1 & 1A2
mRNA/protein/enz. activity (liver, lung, kidney, intestine, brain, spleen,
pancreas, testis, ovary)
BAC-D: no expression of hCYP1A2 mRNA/protein/enz.activity
Other studies to be published
Now have the Cyp1a1/1a2(-/-) double and
Cyp1a1/1a2/1b1(-/-) triple k.o.  oral BaP
Now have a liver-specific Cyp1a1(-/-)
knockout line  i.p. versus p.o. 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
THE END
Any questions..?