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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 Fiji 1978-88 1977-87 1986-89 1986-89 Micronesians Melanesians 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 Highest affinity 4 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 ? (?) etc. CYP1A1 enzyme AHRE EPRE + 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 CH3 OH 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..?