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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.

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Presentation on theme: "Oral benzo[a]pyrene, immunosuppression, and tumors: role of the three CYP1 enzymes Daniel W Nebert, MD Department of Pediatrics, Division of Human Genetics."— Presentation transcript:

1 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

2 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

3 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

4 Response of BaP Hydroxylase (CYP1A1) Activity to i.p. PAHs (e.g. benzo[a]pyrene) MouseControlTreated B ,000 D –– J Biol Chem ; 243: 6242 & 6250

5 LACK of CYP1A1 induction autosomal recessive “Resp” “Nonresp” MC was one of eight PAHs tested

6 Genetics of “Ah-responsiveness” Ah b Ah b x Ah d Ah d F 1 Ah b Ah d Ah b Ah d x Ah b Ah b Ah b Ah b : Ah b Ah d Ah b Ah d x Ah b Ah d F 2 Ah b Ah b : Ah b Ah d : - Ah b Ah d : Ah d Ah d Ah b Ah d x Ah d Ah d Ah b Ah d : Ah d Ah d

7 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 Numerous national and international awards Hundreds, if not thousands, of labs have also entered AH receptor/CYP1 field of research

8 Metabolic activation Detoxication CYP1’s XME Receptors Xenobiotic-Metabolizing Enzymes (XMEs) : CYPs are BAD

9 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

10 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)

11 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” ED X different

12 SEVEN TO TEN OUT OF EVERY 100 CIGARETTE SMOKERS DEVELOP LUNG CANCER WHY DON’T THE OTHER 90%+ SMOKERS DEVELOP LUNG CANCER..?

13 >50-fold difference in lung cancer susceptibility between cigarette smokers of two ethnic groups..!!

14 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)

15 Dissociation constant K d = TCDD-binding affinity, det’d by Scatchard plot analysis of HUMAN placental cytosolic samples (N=115) N= 37 N = 78 Highest affinity Poorest affinity Differences in affinity also at least 15-20X

16 DetoxicationMetabolic activation 1979 [Scheme, of course, fashioned after what was known of the ER]

17 TCDD Cl Coplanar PCBs Benzo[a]pyrene 3-Methylcholanthrene Classical AHR ligands Naturally occurring ligands? Non-classical ligands Bilirubin Prostaglandin G 2 7-Ketocholesterol NH 2 1,5-Diaminonaphthalene Tryptamine Indirubin Omeprazole

18 “THE [Ah] GENE BATTERY” (XRE) (ARE) TCDD BaP E.L.

19 1999 (1A2; 1B1)

20 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

21 CYP Gene Superfamily: 57 in human; 102 in mouse Family SubfamiliesMembers CYP1 2 (1A1, 1A2, 1B1) CYP ; 50 CYP3 1 4 ; 8 CYP ; 20 CYP5 1 1 CYP7 2 2 CYP8 2 2 CYP CYP CYP CYP CYP CYP CYP CYP ; 2(+ ps) CYP CYP CYP Eicosanoids; plants; drugs ancestral?

22 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”

23 (COX-1) (COX-2) There are >115 eicosanoids EpoxyEicosaTrienoic acids HydroxyEicosaTriEnoic HydroPeroxyEicosaTetraEnoic prostaglandins, prostacyclins, thromboxanes, leukotrienes

24 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

25 PROPERTIES OF AH RECEPTOR Affects many pathways ( EGFR, PKC, p21 RAS, MAPK, Src, Wnt/  -catenin, Myc, Myb, Fos, p27, p53, RB1 binding, slowing at G 1 /S & G 2 /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

26 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? )

27 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

28 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)

29 CYP1B1 PAHs Aryl amines Reactive intermediates

30 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

31 Toxicity or Tumors in Knockout Mouse Models Cyp1a2 k.o. tumors, adducts: 4-aminobiphenyl [ABP]; 2-NH 2 -1-Me-6-Phenl-ImidAz-Pyr [PhIP], 2-NH 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]

32 Cyp1a1(-/-) controlCyp1(+/+) BaPCyp1a1(-/-) BaP BONE MARROW: Oral BaP, 125 mg/kg/day for 18 days; death days for Cyp1a1(-/-)

33 Cyp1a1(-/-) knockout mice have a greater BaP body burden and slower clearance rate than Cyp1(+/+) wild-type TCDD pretreatment: BaP clearance speeded up

34 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

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

36 loxP site in 3 ’ UTR

37 6.0 kb  13.3 kb  7.8 kb Mouse Cyp1a1_Cyp1a2 Locus, Head-to-Head Orientation ---Mouse Chr 9---

38 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..!

39 Cyp1(+/+) Cyp1a1(-/-) Double-k.o. Cyp1a1/1b1(-/-) Healthy Dies w/in 1 month + Healthy !

40 Cyp1a1(-/-) Cyp1a1/1b1(-/-) Cyp1a1/1b1(-/-) has greater BaP body burden but shows less toxicity than Cyp1a1(-/-)..!

41 Cyp1(+/+) Cyp1a1(-/-) 1a1/1b1(-/-) 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..!!

42 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: Bone marrow, thymus, spleen: NormalNear normal Severe aplastic anemia

43 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

44 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

45 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

46 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

47 BaP, 12.5 mg/kg/day  die 4-6 mo instead of 1 month; however, at 6-9 weeks, … (!!) GenotypeOutcome Cyp1(+/+) wild-typeHealthy 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

48 Unique “duodenal intraepithelial neoplasm” (DIN) in lack of CYP1A1 but presence of CYP1B1

49 But: sometimes invasive; sometimes also in proximal jejunum

50 BaP, 12.5 mg/kg/day  die 4-6 mo instead of 1 month; however, … (!!) GenotypeOutcome Cyp1(+/+) wild-typeHealthy 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. … … … …

51 Genetic crosses Total number of pups Observ. number of pups Expectd number of pups  2 value P value aabb  aaBb aaBb  aabb Aabb  AaBb AaBb  aaBb Aabb  aaBb <0.001 Aabb  Aabb aaBb  aaBb aabb  aabb 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

52 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

53 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?

54 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

55 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

56 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

57 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

58 THE END Any questions..?


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