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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.
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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 Induction of an enzyme that metabolizes the stimulus Received by the cell
Response of BaP Hydroxylase (CYP1A1) Activity to i.p. PAHs (e.g. benzo[a]pyrene) Mouse Control Treated B65003,000 D2 500500 ––J Biol Chem 1968; 243: 6242 & 6250
“Resp” 3MC was one of eight PAHs tested “Nonresp” 50 50 25 75 LACK of CYP1A1 induction autosomal recessive 50 50 25 75
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) XME Receptors Detoxication CYP1’s Metabolic activation 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)
This 1974 study says the Cyp1 structural genes same, but regulatory gene (a receptor?) is different? “Ah-Resp” ED50 15-20X different “Ah-NonResp” In 1994 study, B6 high-affinity AHR: Pro-474; D2 poor-affinity AHR: Leu-474
SEVEN TO TEN OUT OF EVERY 100 CIGARETTE SMOKERSDEVELOP LUNG CANCER WHY DON’T THE OTHER 90%+ SMOKERSDEVELOP LUNG CANCER..?
>50-fold difference in lung cancer susceptibility between cigarette smokers of two ethnic groups..!!
Could this be like cigarette smokers with lung cancer? Highly Sensitive (HS) Highly Resistant (HR) Could this be like cigarette smokers who don’t get cancer? Cigarette-pack-years?
Dissociation constant Kd = TCDD-binding affinity, det’d byScatchard plot analysis of HUMAN placental cytosolic samples (N=115) N = 78 Highest affinity Poorest affinity N= 37 Differences in affinity also at least 15-20X
[Scheme, of course, fashioned after what was known of the ER] Detoxication Metabolic activation 1979
TCDD 1,5-Diaminonaphthalene Indirubin Cl NH2 Cl Cl Bilirubin Cl NH2 Cl Omeprazole Benzo[a]pyrene Coplanar PCBs Tryptamine Prostaglandin G2 7-Ketocholesterol 3-Methylcholanthrene Naturally occurring ligands? Classical AHR ligands Non-classical ligands
“THE [Ah] GENE BATTERY” (XRE) TCDD BaP E.L. 1976-92 (ARE)
(1A2; 1B1) 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
CYP Gene Superfamily: 57 in human; 102 in mouse FamilySubfamiliesMembers CYP1 2 (1A1, 1A2, 1B1) CYP2 13 16 ; 50 CYP3 1 4 ; 8 CYP4 6 12 ; 20 CYP5 1 1 CYP7 2 2 CYP8 2 2 CYP11 2 3 CYP17 1 1 CYP19 1 1 CYP20 1 1 CYP21 1 1 CYP24 1 1 CYP26 3 3 CYP27 3 3 ; 2(+ ps) CYP39 1 1 CYP46 1 1 CYP51 1 1 Eicosanoids; plants; drugs 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”
There are >115 eicosanoids EpoxyEicosaTrienoic acids HydroxyEicosaTriEnoic HydroPeroxyEicosaTetraEnoic prostaglandins, prostacyclins, thromboxanes, leukotrienes (COX-1) (COX-2)
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 levelsin 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)
PAHs CYP1B1 Reactive intermediates Aryl amines
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]phenan-thracene-induced CA [as expected] 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 BaPparadoxical 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(-/-) knockout mice have a greater BaP body burden and slower clearance rate than Cyp1(+/+) wild-type TCDD pretreatment: BaP clearance speeded up
Why does Cyp1a1(-/-) die, while wild-type stays so healthy? Bigger bolus of BaP to marrow CYP1A1 inducible No CYP1A1 in GI tract or liver
So, what about the Cyp1 double-knockouts..? and the triple-knockout..?
Mouse Cyp1a1_Cyp1a2 Locus, Head-to-Head Orientation 7.8 kb 6.0 kb 13.3 kb ---Mouse Chr 9---
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..!
Healthy Cyp1(+/+) Dies w/in 1 month Cyp1a1(-/-) + Healthy! Double-k.o. Cyp1a1/1b1(-/-)
Cyp1a1/1b1(-/-)has greater BaP body burden but shows less toxicity than Cyp1a1(-/-)..! Cyp1a1/1b1(-/-) Cyp1a1(-/-)
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(-/-) Oral BaP, 125 mg/kg/day Cyp1a1/1a2(-/-) Cyp1a1(-/-) 1a1/1a2/1b1(-/-) Cyp1a1/1b1(-/-) Healthy for months, years Die within one month Wild-type phenotype Clinical outcome: Blood BaP levels (ng/ml) after 5 days feeding: 1.5-6.0 50-60 160-180 Bone marrow, thymus, spleen: Severe aplastic anemia Normal Near normal
WHY does lack of CYP1B1 revert Cyp1a1(-/-) back to near-normal, to wild-type? Cyp1(+/+) Cyp1a1/1b1(-/-) Metabolism by CYP1B1 required; GREATER BaP body burden..!! CYP1A1 inducible No CYP1A1 in GI tract or liver
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, … (!!)
Unique “duodenal intraepithelial neoplasm” (DIN) in lack of CYP1A1 but presence of CYP1B1
BaP, 12.5 mg/kg/day die 4-6 mo instead of 1 month; however, … (!!) *But, … other problems exist in triple k.o. … … … …