Characterization of Murine Hepatocarcinogenesis Induced by the - PowerPoint PPT Presentation

Characterization of Murine Hepatocarcinogenesis Induced by the Hepatocarcinogenesis Induced by the Nitrification Inhibitor Nitrapyrin: Mode of Action, Human Relevance Mode of Action, Human Relevance Framework, and Risk Assessment Implications RASS Webinar February 10, 2016 February 10, 2016 Kerry Hastings D Phil Kerry Hastings, D.Phil Risk Assessor Dow AgroSciences Risk Assessor, Dow AgroSciences Jessica LaRocca, PhD Toxicologist, Dow AgroSciences Matthew LeBaron, PhD Toxicologist, Dow Chemical 1 |

Agenda • Background/overview • Liver MoA evaluation Liver MoA evaluation • Application of the HRF • Risk assessment overview • Risk assessment overview 2 |

Nitrapyrin • (2-chloro-6-trichloromethyl pyridine) • Registered in the US since 1974 Registered in the US since 1974 • Nitrification inhibitor • Nitrogen stabilizer • Nitrogen stabilizer Cl N Cl Cl Cl Cl 3 |

Nitrate Loss Ammonium Nitrite Nitrate Nit Nitrosomonas Nit Nitrobacter b t NH 4 NO 2 NO 3 bacteria bacteria (+) positive (-) soil negative ( ) (-) negative g charge h charge h charge Nitrogen loss Nitrogen loss through denitrification Nitrogen loss through nitrate leaching through nitrate leaching 4 |

Nitrapyrin Stabilizes Nitrogen Ammonium Nitrite X X X X X X X X Nitrate Nitrosomonas Nitrobacter NH 4 NO 2 NO 3 bacteria bacteria 3 (+) positive charge (-) soil negative charge (-) negative charge STABILIZED NITROGEN Readily Available 5 |

Liver Tumors 250 No adverse 200 effects • 250 mkd: ↑ adenomas and adenomas/carcinomas in ♂ and ♀ g/day) Systemic toxicity 150 ↑ absolute and relative liver weights in ♂ and ♀ ↑ absolute and relative liver weights in ♂ and ♀ ose (mg/kg • 125 mkd: ↑ adenomas and adenomas/carcinomas in ♀ Tumors 100 ↑ absolute and relative liver weights in ♂ and ♀ Do 50 • 125/250 mkd: ↓ bw at 12 and 24 months in ♂ and ♀ • 250 mkd: ↓ bwg at 12 mo (29.6%), 24 mo (26.9%) in ♂ 0 0 Rat Mouse Mouse (1989) (1990) (1997) 6 |

What is the MoA for nitrapyrin-mediated mouse liver tumors and is it relevant to humans? 7 |

Approach: • Question 1: Can we assimilate/generate data to define an MoA for nitrapyrin- data to define an MoA for nitrapyrin mediated mouse liver tumors? • Question 2: Can we exclude other MoAs? • Question 3: Is the MoA relevant to humans? • Question 3: Is the MoA relevant to humans? 8 |

Question 1: MoA • Assessed potential MoAs by evaluating Assessed potential MoAs by evaluating previous toxicity data • Generated additional MoA data to rule in or rule out nuclear receptor activation p  Key events (NR activation, proliferation)  Recovery after removal of treatment Recovery after removal of treatment 9 |

Nitrapyrin Liver MoA Study 0, 75, 250, 400 mg/kg/day 0 mg/kg/day nitrapyrin (recovery) nitrapyrin Days of exposure 4 7 14 14 plus 21 day recovery Endpoints: • Gene expression of biomarkers of NR activation (AhR, CAR, PXR, PPAR- α ) • Protein and enzyme activity • Liver weight and histopathology • Hepatocellular proliferation (via BrdU osmotic pumps) • Assess recovery after treatment cessation Assess recovery after treatment cessation 10 |

Key Event #1: NR Activation 0 5 0 0 0 5 0 0 0 5 0 0 0 5 0 0 7 5 0 7 5 0 7 5 0 7 5 0 2 4 2 4 2 4 2 4 Cyp1a1 Cyp2b10 Cyp3a11 Cyp4a10 AhR CAR PXR PPAR - 11 |

Key Event #1: CAR Activation ( Cyp2b10 ) trol over con d change p2b10 fold Cyp mg/kg/day nitrapyrin 12 | N=6-9 mice/timepoint/dose

Key Event #1: Liver Weight Increases rol over contr ncrease o Percent In P N=6 9 mice/timepoint/dose N=6-9 mice/timepoint/dose mg/kg/day nitrapyrin Similar responses for liver hypertrophy 13 |

Cyp2b10 and Expected PROD Activity 7-Pentoxy-Resorufin O-Deethylation (PROD) activity (Cyp2b10-dependent) Activation downstream PB genes/pathways Cyp2b10 C b2b10 Cyb2b10 CAR Cyp2b10 14 |

Key Event #1: CYP Enzyme Induction 7-Pentoxy-Resorufin O-Deethylation (PROD) activity (Cyp2b10-dependent) ontrol e Over Co d Change Fold mg/kg/day nitrapyrin Cyp2b10 Western Blot Cyp2b10 Western Blot ← Cyp2b10 N=6 mice/timepoint/dose 15 |

Suicide Inhibition • Inhibition of cytochrome activity (irreversible) • Phenobarbital (PB)-induced liver microsomes used to investigate the role for suicide inhibition Inhibitor Binds irreversibly, removes active enzyme from system Enzyme Enzyme 16 |

Suicide Inhibition • Inhibition of cytochrome activity (irreversible) Cl Activation downstream N Cl Cl genes/pathways Cl Cyp2b10 C b2b10 Cyb2b10 CAR NR I hibi Inhibitor Cl N Cl Cl Cl Cl Cyp2b10 17 |

CYP Enzyme Induction/Suicide Inhibition Cl C N Cl PB Cl Cl Cyp2b10 Cyp2b10 Cyp2b10 Cyp2b10 enzymatic inhibition similar to what was seen in vivo 18 |

Key Event #2: Increased Hepatocellular Proliferation 4.3 Cells Per 1,000 Fold increases ive Cells P 4.9 4 9 2 3.5 rdU Positi 2.4 Br N=6-9 mice/timepoint/dose mg/kg/day nitrapyrin 19 |

Summary Key Events #1 and 2 • Nitrapyrin exposure in mice causes:  Key Event #1 – CAR activation ─ Cyp2b10 gene and protein expression ─ Liver weight increases ─ Liver hypertrophy ─ Suicide inhibition of PROD  Key Event #2 – Hepatocellular proliferation ─ BrdU Labeling Index 20 |

Question 1: Conclusion Can we assimilate/generate data to define an g MoA for nitrapyrin-mediated mouse liver tumors? tumors? YES Key events #1 (CAR) and #2 (Proliferation) y ( ) ( ) 21 |

Question 2 • Can we exclude other MoAs? • Is CAR necessary for nitrapyrin mediated • Is CAR necessary for nitrapyrin-mediated liver effects (proliferation)? • Addressed this question with a CAR-KO q mouse study 22 |

CAR-KO Mouse Study Design 0, 250 mkd nitrapyrin Endpoints: • Cyp2b10 C 2b10 WT CAR-KO • Liver weight increases, g histopathology • Hepatocellular Hepatocellular proliferation Days of Exposure 4 4 23 |

Gene expression in WT and CAR-KO Livers rol over contr change o Fold mg/kg/day nitrapyrin (mouse strain) N=6 mice/strain/dose 24 |

Relative liver weight increases ease rcent Incr Per mg/kg/day nitrapyrin (mouse strain) g g y py ( ) 25 |

Histopathologic changes in WT and CAR-KO Mice Vacuolization; Very slight V li ti Hypertrophy; Very slight or slight H t h V li ht V li ht li ht 6/6 6/6 6/6 5/6 ected ected Percent Affe Percent Affe P P 0/6 0/6 0/6 0/6 mg/kg/day nitrapyrin (mouse strain) mg/kg/day nitrapyrin (mouse strain) Increased number of mitotic figures Increased number of mitotic figures 5/6 Affected Percent A 1/6 0/6 0/6 0/6 mg/kg/day nitrapyrin (mouse strain) 26 |

Hepatocellular Proliferation in WT and CAR-KO Mice ol ver contro change ov Fold c mg/kg/day nitrapyrin (mouse strain) N=6 mice/strain/dose Nitrapyrin-induced proliferation requires CAR activation 27 |

Alternative MoAs Alternative MoAs were evaluated for plausibility and coherence by Bradford Hill Criteria: • DNA Reactivity •Not plausible •No coherence • AhR, PXR, PPAR α Activation •Not plausible •No coherence • Cytotoxicity (1 Wk – 12 Mo) •Plausible •No coherence: based on magnitude of effect, entirety of data • Increased Apoptosis •Not plausible •No coherence • Estrogens, Statins, Metals, Infectious •Not plausible •No coherence 28 |

Temporality Key Event 1 Key Event 2 Apical Endpoints: Apical Endpoints: Causal: CAR Increased Key Events Activation Hepatocellular Hepatocellular After Recovery (Cyp2b10 Tumors and Proliferation Dose (mkd) ( ) Transcript & p Altered Foci Protein) 14 Days Plus 21 4-14 Days 4-14 Days 2 Yrs Days Recovery - 5 Dose - 25 - - - - 75 +,+ # - 125 + + - + 250 - - 250 CAR KO + Indicates effect present, - indicates effect absent at indicated duration of treatment. Blank cell = No data. # Data only from 1-year interim sacrifice 29 |

Question 2: Conclusion Can we exclude other MoAs? YES CAR is necessary for nitrapyrin-induced hepatocellular proliferation hepatocellular proliferation 30 |

Question 3: Relevance to Humans? • CAR activation has been shown to be not relevant to humans: Key Events Evidence in Rodents Evidence in Humans Activation of CAR Yes Yes CYP Enzyme Yes; unclear if critical Yes; different enzymes #1 Induction step or indicator of induced compared to Associated Liver activity secondary to rodents Hypertrophy CAR activation No evidence of increased Hepatocellular cell proliferation in the #2 Yes Proliferation human liver (limited in vitro and in vivo data). vitro and in vivo data) Selective Clonal Yes No; none reported Expansion (Foci) Apical Endpoints Apical Endpoints O Occurrence of f No; based on Hepatocellular Yes epidemiological data Tumors 31 |

Relevance to Humans for Nitrapyrin? • Wanted to generate nitrapyrin-specific data • How?  Mouse vs. human hepatocyte proliferation study 32 |

Study Design • Mouse hepatocytes treated Mouse hepatocytes treated with 0, 1, 3, 10 µM nitrapyrin • Human hepatocytes treated p y with 0, 3, 10, 30, 100 µM nitrapyrin • Positive control EGF • DNA synthesis analyzed via EdU t i i EdU staining (fluorescent (fl t alternative to BrdU) 33 |