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

So, what about the Cyp1 double-knockouts..? and the triple-knockout..?

loxP site in 3’ UTR

6.0 kb 13.3 kb 7.8 kb

Mouse Cyp1a1_Cyp1a2 Locus, Head-to-Head Orientation

---Mouse Chr 9---

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

Unique “duodenal intraepithelialneoplasm” (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 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

THE END

Any questions..?