characterization of murine hepatocarcinogenesis
TRANSCRIPT
Characterization of Murine Hepatocarcinogenesis Induced by theHepatocarcinogenesis Induced by the
Nitrification Inhibitor Nitrapyrin: Mode of Action, Human RelevanceMode of Action, Human Relevance Framework, and Risk Assessment
Implications
RASS WebinarFebruary 10, 2016February 10, 2016
Kerry Hastings D Phil Risk Assessor Dow AgroSciencesKerry Hastings, D.Phil Risk Assessor, Dow AgroSciencesJessica LaRocca, PhD Toxicologist, Dow AgroSciencesMatthew LeBaron, PhD Toxicologist, Dow Chemical
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Agenda• Background/overview• Liver MoA evaluationLiver MoA evaluation• Application of the HRF• Risk assessment overview• Risk assessment overview
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Nitrapyrin• (2-chloro-6-trichloromethyl pyridine)• Registered in the US since 1974Registered in the US since 1974• Nitrification inhibitor• Nitrogen stabilizer• Nitrogen stabilizer
ClN Cl
ClCl
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Cl
Nitrate Loss
Ammonium Nitrite NitrateNit Nit b t
NH4(+) positive
h
NO2(-) soil negative
h
NO3(-) negative
Nitrosomonasbacteria
Nitrobacterbacteria
Nitrogen loss
charge charge( ) g
charge
Nitrogen lossthrough
denitrification
Nitrogen lossthrough nitrate leaching
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through nitrate leaching
Nitrapyrin Stabilizes Nitrogen
Ammonium
NH4
Nitrite
NO2
Nitrate
NO3
Nitrosomonasbacteria
NitrobacterbacteriaX X XX(+) positive charge (-) soil negative charge
3(-) negative charge
X X XX
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STABILIZED NITROGENReadily Available
Liver Tumors250
150
200 No adverse effects
Systemic toxicity
g/da
y)• 250 mkd: ↑ adenomas and adenomas/carcinomas in ♂and♀↑ absolute and relative liver weights in ♂and♀
100 Tumors
ose
(mg/
kg
↑ absolute and relative liver weights in ♂and♀• 125 mkd: ↑ adenomas and adenomas/carcinomas in ♀
↑ absolute and relative liver weights in ♂and♀
0
50Do
• 125/250 mkd: ↓ bw at 12 and 24 months in ♂and♀• 250 mkd: ↓ bwg at 12 mo (29.6%), 24 mo (26.9%) in ♂
Rat(1989)
Mouse (1990)
Mouse (1997)
0
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What is the MoA for nitrapyrin-mediated mouse liver tumors and is it relevant to
humans?
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Approach:• Question 1: Can we assimilate/generate
data to define an MoA for nitrapyrin-data to define an MoA for nitrapyrinmediated 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?
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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 activationpKey events (NR activation, proliferation) Recovery after removal of treatmentRecovery after removal of treatment
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Nitrapyrin Liver MoA Study0, 75, 250, 400 mg/kg/day
nitrapyrin0 mg/kg/day nitrapyrin (recovery)
14Days of exposure 7 14 plus 21 day recovery4
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
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Assess recovery after treatment cessation
Key Event #1: NR Activation
0 75 250
400 0 75 250
400 0 75 250
400 0 75 250
400
Cyp1a1AhR
Cyp2b10CAR
Cyp3a11PXR
Cyp4a10 PPAR-
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Key Event #1: CAR Activation (Cyp2b10)tr
olov
er c
ond
chan
ge
p2b1
0fo
ldC
yp
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mg/kg/day nitrapyrin
N=6-9 mice/timepoint/dose
Key Event #1: Liver Weight Increasesro
lov
er c
ontr
ncre
ase
oPe
rcen
t In
P
N=6 9 mice/timepoint/dose
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mg/kg/day nitrapyrin N=6-9 mice/timepoint/dose
Similar responses for liver hypertrophy
Cyp2b10 and Expected PROD Activity7-Pentoxy-Resorufin O-Deethylation (PROD) activity (Cyp2b10-dependent)
Activation downstream genes/pathways
Cyp2b10C b2b10
PB
CARCyb2b10
Cyp2b10
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Key Event #1: CYP Enzyme Induction on
trol
7-Pentoxy-Resorufin O-Deethylation (PROD) activity (Cyp2b10-dependent)e
Ove
r Co
d C
hang
e
Cyp2b10Western Blot
Fold
mg/kg/day nitrapyrin
←Cyp2b10
Cyp2b10 Western Blot
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N=6 mice/timepoint/dose
Suicide Inhibition• Inhibition of cytochrome activity (irreversible)• Phenobarbital (PB)-induced liver microsomes used to
investigate the role for suicide inhibition
Inhibitor
Enzyme
Binds irreversibly, removes active enzyme from system
Enzyme
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Suicide Inhibition• Inhibition of cytochrome activity (irreversible)
Activation downstream genes/pathways
Cyp2b10C b2b10
N ClCl
ClCl
I hibi
CAR NRCyb2b10
Inhibitor
N ClCl
ClClCl
Cyp2b10
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CYP Enzyme Induction/Suicide InhibitionPB
Cyp2b10
C
Cyp2b10
N ClCl
ClCl
Cyp2b10
| 18Cyp2b10 enzymatic inhibition similar to what was seen in vivo
Key Event #2: Increased Hepatocellular Proliferation C
ells 4.3
Per 1
,000
4 9Fold increases
ive
Cel
ls P
2
4.9
3.5
rdU
Posi
ti
2.4
Br
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mg/kg/day nitrapyrin N=6-9 mice/timepoint/dose
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
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Question 1: Conclusion
Can we assimilate/generate data to define an gMoA for nitrapyrin-mediated mouse liver
tumors?tumors?
YESKey events #1 (CAR) and #2 (Proliferation)y ( ) ( )
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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 qmouse study
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CAR-KO Mouse Study Design
0, 250 mkd nitrapyrinEndpoints:
C 2b10• Cyp2b10
• Liver weight increases, CAR-KOWT
ghistopathology
• Hepatocellular
4Days of Exposure
Hepatocellular proliferation
4
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Gene expression in WT and CAR-KO Liversro
lov
er c
ontr
chan
ge o
Fold
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mg/kg/day nitrapyrin (mouse strain)N=6 mice/strain/dose
Relative liver weight increasesea
serc
ent I
ncr
Per
mg/kg/day nitrapyrin (mouse strain)
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g g y py ( )
Histopathologic changes in WT and CAR-KO Mice H t h V li ht li ht V li ti V li ht
6/6 6/6
ecte
d
Hypertrophy; Very slight or slight
5/66/6
ecte
d
Vacuolization; Very slight
Per
cent
Affe
Per
cent
Affe
Increased number of mitotic figures
0/6 0/6P
mg/kg/day nitrapyrin (mouse strain)
0/6 0/6P
mg/kg/day nitrapyrin (mouse strain)Increased number of mitotic figures
Affe
cted
5/6
Per
cent
A
1/60/6 0/6
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0/6
Hepatocellular Proliferation in WT and CAR-KO Miceol
ver c
ontr
och
ange
ov
Fold
c
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mg/kg/day nitrapyrin (mouse strain)N=6 mice/strain/doseNitrapyrin-induced proliferation requires CAR activation
Alternative MoAsAlternative MoAs were evaluated for plausibility and coherence by Bradford Hill Criteria:
•DNA Reactivity•Not plausible•No coherence
•AhR, PXR, PPARα 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
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Key Event 1 Key Event 2Apical Endpoints:
Temporality
Dose (mkd)
Key Events After Recovery
Apical Endpoints:Increased
Hepatocellular Tumors and Altered Foci
Causal: CAR Activation(Cyp2b10
Transcript &
Hepatocellular Proliferation( ) p
Protein)
4-14 Days 4-14 Days 14 Days Plus 21 Days Recovery 2 Yrs
5 -25 -
Dose
75 - - - -125 +,+# -250 + + - +
250 CAR KO - -
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+ Indicates effect present, - indicates effect absent at indicated duration of treatment. Blank cell = No data.
#Data only from 1-year interim sacrifice
Question 2: Conclusion
Can we exclude other MoAs?
YESCAR is necessary for nitrapyrin-induced
hepatocellular proliferationhepatocellular proliferation
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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
#1CYP Enzyme
InductionAssociated Liver
Hypertrophy
Yes; unclear if critical step or indicator of
activity secondary to CAR activation
Yes; different enzymes induced compared to
rodents
#2 Hepatocellular Proliferation Yes
No evidence of increased cell proliferation in the human liver (limited in vitro and in vivo data)vitro and in vivo data).
Apical Endpoints
Selective Clonal Expansion (Foci) Yes No; none reported
O f
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Apical Endpoints Occurrence of Hepatocellular
TumorsYes No; based on
epidemiological data
Relevance to Humans for Nitrapyrin?• Wanted to generate nitrapyrin-specific data
• How?Mouse vs. human hepatocyte proliferation study
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Study Design
• Mouse hepatocytes treatedMouse hepatocytes treated with 0, 1, 3, 10 µM nitrapyrin
• Human hepatocytes treated p ywith 0, 3, 10, 30, 100 µM nitrapyrin
• Positive control EGF• DNA synthesis analyzed via
EdU t i i (fl tEdU staining (fluorescent alternative to BrdU)
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Hepatocyte Proliferation in Mice and HumansMouse Hepatocytes Human Hepatocytes
* p<0.05*
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N=2 human donors, 2-3 technical replicates/donor/dose
N=5-7 technical replicates
Hepatocyte Proliferation in Mice and HumansMouse Hepatocytes Human Hepatocytes
* p<0.05
*
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N=2 human donors, 5-6 technical replicates/donor/dose
N=5-7 technical replicatesConclusion: Nitrapyrin does not increase hepatocellular proliferation in human hepatocytes
Conclusions: Nitrapyrin MOA and Relevance to HumansPOD =75 mg/kg/day
spon
se Hepatocellulartumors in mice
Res
N t di t d i
Nitrapyrin Dose
No tumors predicted in CAR-KO mice
No relevance to humansNitrapyrin Dose
Key event #1 Mice
Key event #1
Key event #2 Mice
Key event #2 Mouse Hepatocytes
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Key event #1 CAR-KO mice
Key event #2 CAR-KO mice
Key event #2Human Hepatocytes
ILSI/IPCS Mode-of-Action/Human Relevance Framework
Q1: Is the weight of evidence sufficient to establish the MoA in animals?
Yes No
Assume MoA
Assume
Q2: Fundamental qualitativedifferences in key events?
Relevant to Humans
Assume MoA NOT Relevant
NoYes
to HumansNo
Q3: Fundamental quantitative differences in
key events?
| 37As described by Meek et al. (2003) and revised by Seed et al.(2005).
key events?
Nitrapyin MoA/HRF• Data support CAR activation as MoAKey Event #1 CAR ActivationKey Event #1 – CAR ActivationKey Event #2 – Hepatocellular Proliferation
• Alternative MoAs can be excluded
D t lit ti diff M A f• Due to qualitative differences, MoA for nitrapyrin is not relevant to humans
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Conclusion – Part #1
Questions on the MoA/HRFQuestions on the MoA/HRF evaluation?
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Regulatory Reviews • EPACPRC (1992) - Not classifiableCPRC (1992) - Not classifiable CARC (2000) - LikelyCARC (2005) Lik lCARC (2005) - LikelyCARC (2012) - Suggestive EvidenceCARC (2017) – Under Review
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Dietary Risk Assessment• Dietary exposure is estimated using
Food consumption data (from NHANES surveys)
P t ti l l f ti id id i diff t f d (t l ) Potential values for pesticide residues in different foods (tolerances)
NHANES = National Health and Nutrition Examination Survey
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NHANES National Health and Nutrition Examination Survey
nitrapyrin tolerances
Dietary Risk Assessment
• Diet Residues of concern in food:
nitrapyrin 6-chloropicolinic acid
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Dietary Risk Assessment
• Diet Dietary cancer risk assessment:
nitrapyrin 6-chloropicolinic acid
No residues ever detected in food commodities
[Former] Cancer endpoint only relevant to nitrapyrin; not 6-CPA
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Dietary Risk Assessment Chronic RfD = 0.03mg/kg bw/day (based on NOAEL of 3mg/kg/day
(chronic feeding – dog) and uncertainty factor = 100; FQPA = 1)cPAD RfD÷FQPA 0 03mg/kg bw/day cPAD = RfD÷FQPA = 0.03mg/kg bw/day Exposure ≤1% cPAD for US population and all subgroups p p g p
Table 3 extracted from Nitrapyrin Chronic Dietary
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Exposure Assessment for the Registration Eligibility
Decision. D. Soderburg, EPA 2004. D299299
Dietary Risk Assessment
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Occupational Risk Assessment
• Occupational Potential exposure through mixing, loading and application:
Exposure is calculated using unit exposure values from specific studies (such as those conducted by the Agricultural Handlers Exposure Task Force).p )
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Occupational Risk Assessment
Exposure = AR (lb ai/acre) x AT(acre/day) x unit exposure (mg/lb ai) body weight (kg)
AR = application rateAR = application rateAT = area treated
Exposure (in mg/kg/day) is used to assess risk in two ways:
Cancer and non-cancer
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Occupational Risk Assessment
Cancer: Lifetime Average Daily Dose (LADD) calculated from daily exposure LADD used to calculate risk:exposure. LADD used to calculate risk:
LADD = total exposure x no. of days exposed per year* x 35 working years
365 days per year 70 year lifetime
*3 day per year for private applicators; 30 day per year for commercial applicators
Risk = Q1* [4.25 x 10-2 (mg/kg/day)-1 human equivalents] x LADD.
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Occupational Risk Assessment Cancer risks marginally higher than LOC (LOC = 1 x 10-6) but risk vs. benefit
recommends no additional mitigation beyond long pants, long sleeves and gloves for mixing/loading
(extracted from occupational t 2005)
C b d i k t t i d i 2012 ( l ifi ti )
exposure assessment, 2005)
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Cancer-based risk assessment not required since 2012 (re-classification)
Occupational Risk Assessment
Non-cancer: the MOE is calculated:
MOE = NOAEL (mg/kg/day) Need MOE ≥ 100 for pass Exposure (mg/kg/day)
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(extracted from occupational exposure assessment, 2005)
Occupational Risk Assessment
All MOEs ≥ 100 when long pants, long sleeves and gloves worn for mixing/loading
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AcknowledgementsDow AgroSciences Human Health Assessment
Dow Toxicology & Environmental Research & Consulting (TERC) Laboratories
Reza RasoulpourB Bhaskar Gollapudi
Johnson ThomasLynea Murphy B. Bhaskar Gollapudi
Dave Eisenbrandt H. Lynn Kan
Lynea Murphy Kamin Johnson Nico Viscontiy
Melissa Schisler Val Marshall
Lindsay Sosinski Dave DeLine
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