Download Key Events Dose-Response Analysis. Part 2: Application to

Key Events Dose-Response
Analysis.
Part 2: Application to Nutrients,
Pathogenic Microorganisms, and
Food Allergens

SOT RASS Teleconference
February 10, 2010
Elizabeth Julien (Consultant)
Mary Alice Smith (University of Georgia)
Steve Gendel (FDA/CFSAN)
Steve Olin (ILSI Research Foundation)
ILSI Research Foundation
1
Dose-Response and Thresholds

• Recognition of the centrality of doseresponse concept in life sciences
• QUESTION: Can our increasing
understanding of modes of action provide
insights for characterizing dose-response
relationships at low doses (including
thresholds) ?
• Not only for chemicals but also for other
bioactive agents.
ILSI Research Foundation
2
ILSI RF Threshold Working Group

• Characterizing fundamental biology of
human health effects for chemicals,
pathogens, allergens, nutrients
• Implications for dose-response, practical
thresholds, public health standards
• Fostering cross-disciplinary discussion
• → Key Events Dose-Response Framework
ILSI Research Foundation
3
ILSI RF Threshold Working Group

• Chemical Group: Alan Boobis (Imperial College
London), George Daston (Procter & Gamble), and
Julian Preston (EPA).
• Nutrient Group: Sanford Miller (U Maryland), Joseph
Rodricks (ENVIRON), Ian Munro (CANTOX), A.
Catharine Ross (Pennsylvania State), Robert Russell
(Tufts), and Elizabeth Yetley (retired NIH).
• Pathogen Group: Bob Buchanan (U Maryland), Arie
Havelaar (RIVM), Mary Alice Smith (U Georgia), and
Richard Whiting (Exponent).
• Allergen Group: Steven Gendel (FDA CFSAN), Geert
Houben (TNO), and Steve Taylor (U Nebraska).
ILSI Research Foundation
4
Work Products and Next Steps

•
•
5 papers – Crit Rev Food Sci Nutr, 49 (8), Sept 2009
(Overview, Chemicals, Nutrients, Pathogens,
Allergens) – open access.
Next Steps
•
•
•
•
Encourage the development of additional case studies
illustrating and evaluating the utility of the Framework
Organize small meetings and workshops to work through
specific examples
Explore application and integration of the Framework into
MOA analysis for risk assessment
CONTACT: Steve Olin ([email protected])
ILSI Research Foundation
5
The Key Events Analytical Framework:
A case study with retinol (Vitamin A)
Beth Julien, Ph.D.
SOT RASS Telecon, Feb 10 2010
6
Acknowledgements
This presentation describes the work of the ILSI Threshold
Project “Nutrient Group”:
Catharine Ross and Robert Russell
Sanford Miller, Ian Munro, Joseph Rodricks, Elizabeth Yetley
…. and incorporates ideas developed by the entire
Threshold Project Working Group. See Crit Rev Food
Sci Nutr, 49 (8), Sept 2009
7
Increasing refinement in approach
INITIAL DOSE
(Exposure or Intake)
Ultimate Effect
of Concern
INITIAL DOSE
(Exposure or Intake)
Target Tissue
Dose, Adjustment
Factors, etc.
Ultimate Effect
of Concern
INITIAL DOSE
(Exposure or Intake)
Multiple events,
Multiple dose levels;
Multiple d-r relationships
Ultimate Effect
of Concern
8
Overall KEDRF Concept
INITIAL DOSE (exposure or intake)
key event(s) (e.g., absorption, inhalation)
key event(s) (e.g., transport to target tissue)
key event(s) (interaction in target tissue)
At various events,
homeostatic mechanisms
may affect progression along
pathway
ultimate effect of concern
9
Looking at the whole pathway of events …
• Which events may be “control points” where
mechanisms exist to maintain homeostasis? Are any
control points especially vulnerable (readily
overwhelmed by dose? readily modified by host
factors? )
• Is any particular key event a possible “determining
event”? – i.e., Its outcome disproportionately affects
the probability of seeing the outcome of interest?
• Does any particular key event appear to drive the
slope or shape of the overall dose-response
relationship?
10
Examining an individual key event
Factors that combine to determine outcome of individual
events:

Dose (level and frequency)

Homeostatic mechanisms (e.g., repair, immune,
response, compensatory pathways)

Host factors (life-stage, disease state, genetic
makeup, nutritional status, co-exposure)
11
Host factors
Homeostatic
mechanisms
Dose
Event (Process or Interaction)
↓ likelihood of
effect of concern
Progression toward
effect of concern
12
Looking at an individual event
(esp. “control points” or “determining” events)
Can we characterize dose-response at this event?
 If not, what data are needed?
 Is there evidence for threshold?
 What homeostatic mechanisms exist?
 What host factors come into play?
How can this information be used for practical purposes
toward informing public health standards?
 Human relevance?
 Identifying susceptible sub-populations?
 Quantifying variability?
13
Applying the KEDRF to nutrients
 Typically, for a given nutrient there will be long-term
intake, with a dose that varies day to day
 Homeostatic controls exist to regulate blood and tissue
levels despite daily intake variation. Control via:
 One or more kinetic events
 One or more of dynamic events
• Various intake patterns may lead to adverse effects: acute
excess intake, chronic excess intake, chronic deficiency.
14
For certain nutrients, two types
of thresholds exist

An intake level that must be exceeded (usually on a
regular basis) to provoke a toxic effect

A minimum intake level required on a regular basis to
support health and prevent deficiency
A range of safe and sufficient intake levels is situated
between these two thresholds
15
Retinol (vitamin A)
A range of clinically-evident effects, depending on dose
level and dose frequency.
Very High – Extremely
High Acute Intake
Teratogenicity –
Severe toxicity/lethality
Moderately High
Chronic Intake
Organ damage,
affecting metabolism
Chronic Inadequate
Intake
Visual abnormalities,
impaired fertility, ↓
immune response,
↓bone growth
Focus of case study: upper levels of intake
16
Overview of Retinol Pathway
Uptake from Lumen
dose
Intestinal Metabolism; Distribution, Elimination
dose
Uptake into Liver
dose
Liver Metabolism, Storage, Release
dose
Uptake into Extrahepatic Tissues
dose
Target Tissue Interactions
17
Analysis of Events I
UPTAKE FROM LUMEN
Highly efficient hydrolysis RE → R; Carrier mediated passive
absorption; ~ 70% R absorbed. Not down-regulated with
↑Intake or with VA status. Not a control point.
dose
INTESTINAL METABOLISM; DISTRIBUTION, EXCRETION
Almost all R is re-esterified, packaged into chylomicrons (CM)
for transport. No evidence of regulation. Not a control point.
dose
UPTAKE INTO LIVER
Most CM remnants rapidly taken into liver;
Retinol is passively assimilated into hepatocytes.
No evidence for homeostatic regulation. Not a control point.
18
Analysis of Events II
LIVER METABOLISM AND STORAGE:
R → RE by LRAT for storage.
Under normal intake, LRAT activity correlates with circulating VA levels.
LRAT activity is reduced in states of low VA; increased with ↑ intake.
With high intake levels, LRAT activity ↑ only slightly.
 LRAT may become saturated
 Liver’s storage capacity is not inexhaustible; threshold may be
signaled by accumulation of circulating retinoid products.
Conclusion: LRAT activity is a regulated event - a control point.
Saturation of LRAT may be a “determining event” .
19
Analysis of Events III
RELEASE OF RETINOL FROM LIVER STORAGE
Possible feedback loop: Circulating retinoid metabolites (?)
may signal liver to release stored RE, and convert it to R.
R binds RBP and is released into circulation, where it forms a
trimolecular complex with transthyretin (R-RBP-T).
Plasma retinol concentration is nearly constant in a given individual.
Only when liver storage goes below or above a wide normal range (~ < 20
µg or >300 µg), do circulating levels change.
Plasma retinol levels are not a good indicator of VA status.
Conclusion: Not a mechanism for control of circulating retinol
when there is high intake.
20
Analysis of Events IV
UPTAKE INTO EXTRAHEPATIC TISSUES:
Mechanism unknown. R may dissociate from R-RBP-T, diffuse into cell.
TARGET TISSUE METABOLISM AND ACTIVITY :
R metabolite (RA) binds CRABPs, forms complex with RAR/RXR
receptors. Binding to RARE (or RXRE) on DNA, affects transcription.
 In cell nucleus, RA binding proteins bind specific isomers of RA and
regulate activity of retinoid responsive genes.
 Expression of a subset of binding proteins can be induced in some
tissues by the metabolite all-trans retinoic acid.
Conclusion: Binding activity appears to be only a partially regulated
event; not a control point for regulating RA levels.
21
Dietary Vitamin A
oxidative inactivation
LRAT
RE
Retinol
RA
storage
oxidative activation
and
release
polar metabolites
of RA
conjugation and
excretion
Excess dietary
Vitamin A
LRAT
RE
Retinol
RA CYPs polar metabolites
of RA
22
Case Study Conclusions
 Overwhelmed LRAT capacity, leading to excessive RA
levels is likely a “determining event”
Research question: how high must RA (or its
metabolites) rise in order to cause effect? How long must
it remain high?
Research need: study induction of Cyp26 and
accumulation of polar metabolites of RA in blood and
urine as potential early signals of toxicity .
23
General lessons
•
KEDRF is an analytical framework that facilitates a
systematic evaluation of multiple elements that
combine to determine overall dose-response
•
It complements empirical, mechanistic and modeling
approaches to dose-response
•
It supports a practical use: strengthens connection
between regulatory standards for a population (RfD,
ULs) and the underlying biology
24
Application of the Key Events
Dose Response Framework to:
Pathogenic Microorganisms
Working Group:
Robert L. Buchanan
Arie H. Havelaar
Mary Alice Smith
Richard C. Whiting
Elizabeth Julien
25
Current Approaches and Practice

“Infectious Dose” or “Minimum Infectious
Dose” - traditionally used to describe the ability
of a pathogenic microorganism to cause illness
and disease
Concept presumes a threshold dose
 Microbiological equivalent to NOAEL in toxicology

26
Microbial Pathogen Categories

Toxigenic bacteria – Threshold assumed
 Toxins are preformed in food
 Clostridium botulinum, Staphylococcus aureus

Toxicoinfectious bacteria – No threshold assumed
 Colonize GI tract, not invasive
 Toxins act locally (Vibrio parahemoliticus) and/or in distant tissues (Escherichia coli
O157:H7)
Invasive bacteria – No threshold assumed
 Colonize GI tract and disseminate in host
 Intercellular spread
 in mucosa (Salmonella enterica),
 to lymphoid system (Yersinia enterocolitica)
 to bloodstream (Salmonella Typhi)


Intracellular spread
 to fetus (Listeria monocytogenes)
27
Data Sources for Current Understanding of
Microbial Dose-Response

Expert elicitation (experience)

In vitro studies


Cell, tissue or organ cultures
Non-living experimental systems (fermenters, model intestinal
systems, test tubes); predictive microbiology: mechanistic models

Animal studies

Human studies
 Volunteer feeding studies
 Outbreak investigations
 Surveillance and annual health statistics
 Biomarkers
28
Current Basis of Microbial Dose-Response
Modeling





Conditional chain of events
Exposure  Infection  Illness
Single hit—One microorganism has a probability
Independent action by each microorganism
No threshold
All single hit models are approximately linear at low doses
(a mathematical property)
Haas, 1983; Teunis et al., 1996; FAO/WHO 2003
29
Dose -Response Models
1
Prob. Illness
0.8
0.6
Data
0.4
0.2
0
10
1
0
1
2
3
4
5
6
7
8
9
Prob. Illness
0.1
0.01
0.001
Public health and
regulatory concern
0.0001
0.00001
0.000001
0.0000001
0
2
4
log Dose
6
8
10
30
Pathogen-Host Interactions

The interactions between the pathogen and the
host can be very complex
Immune or adaptive response of host
 Homeostatic mechanisms of host
 Pathogens can evolve mechanisms to use host
resources to help with survival and growth
 Host characteristics such as age, health status,
immune status can also effect interactions

31
Key Events Dose-Response
Framework: Listeriosis





Exposure to L. monocytogenes via readyto-eat foods (soft cheeses, deli meats,
smoked fish, pates)
Invasive, infects spleen, liver and CNS
Risk groups: fetus and neonate, elderly,
immunocompromised
Rare but high case-fatality ratio (~20%)
Pregnant women at greatly increased risk:
spontaneous abortion, stillbirth, neonatal
meningitis
32
Key Events Pathway: L. monocytogenes intake and
potential fetal death
Interplay of
host
and pathogen
can influence
progression at
various events
Intake of contaminated food
1-P
Do not survive
P
Pathogens survive in upper GI tract
Do not establish, etc
Establish; attach; taken up into epithelial cells
Do not escape
Escape from phagosomes; transfer to phagocytes
Do not transfer
Cross placenta
Do not grow, no mortality
Growth; results in fetal mortality
33
Key Event 1: Survival in Stomach

Microbial death rate affected by
 digestive enzymes
 the food matrix
 quantity and composition/acidity of foods consumed
 general level of acidity (may be reduced by advanced
age, antacids consumption, achlorhydria)
• Adaptation by L. monocytogenes to acid environment

Research--Measure whether probability of survival is
proportional to number ingested, adaptation, strain & host
differences
34
Key Event 2: Establish; attach; taken up into epithelial
cells

Current knowledge - InlA on L. monocytogenes and Ecadherin receptors in host

Research



Does growth correspond to number internalized?
Role of host innate immune response
Gene control of InlA and E-cadherin (alleles, quantities)
35
Key Event 3: Escape from phagosomes; transfer
to phagocytes

Current knowledge

L. monocytogenes synthesizes listeriolysin O (LLO)
which forms small pores in phagosome and ultimately
L. monocytogenes escapes from phagosome
 Uses host actin to move to membrane-membrane
interface with adjacent cells
 Spread to other enterocytes and/or phagocytes which
disseminate pathogen to other organs including
placenta
Research
 Strain and host differences
 Model responses (quantitative)
36
Key Event 4: Transfer of Pathogen
across Placenta

Mechanism by which L. monocytogenes crosses placenta is
not known, but 2 mechanisms hypothesized:



Invasion of endothelial cells via In1A and E-cadherin
interaction (Lecuit et al 1999, 2004)
Actin-mediated cell-to-cell transfer from infected phagocytes
to placental endothelial cells (Drevets et al, 1995).
Knowledge of this step could potentially provide
method to prevent passage to the fetus.
37
Key Event 5: Pathogen Growth Leading to
Fetal Morbidity and Mortality

Once across placenta, gain entry to fetal circulation and
spread to fetal liver and brain



Immature fetal immune system puts fetus at great risk of
infection
Asymptomatic maternal infection but may result in
spontaneous abortion, delivery of stillborn infant or infected
infant.
Unknowns: is fetal death a reaction of maternal system
to fetal infection, loss of placental integrity, infection of
fetus directly, or some combination?
38
Key Events Dose-Response
Framework for Pathogens

For L. monocytogenes:
Some events appear probabilistic in nature (survival
through GI, attachment to intestinal epithelium)
 Other events engage host mechanisms and may have
a finite capacity that can be overcome. These would
likely be non-linear.


Other pathogens may be very different

pH tolerance, quorum sensing, toxin production, etc,
may affect the dose response relationship.
39
Key Events Dose-Response Framework
for Pathogens - Conclusions





Provides a structure for systematically considering
complex factors influencing dose response
Highlights research needs
Generates new hypotheses and focused research
Ultimately provides new data to refine dose response
Basis for iterative improvement in microbial doseresponse assessment
40
Potential Application of the
Key Events Dose Response
Framework to Food
Allergens
Allergen Working Group
Steven Gendel
Steve Taylor
Geert Houben
41
Food Allergy – What is it?
• An immunologic reaction to a food
• Usually IgE mediated
• IgE antibodies bind to one or more
proteins in a food
• Two step process
• Sensitization
• Elicitation
42
What Can Happen?
• Gastrointestinal
• nausea
• vomiting
• abdominal pain
• diarrhea
• Cutaneous
• urticaria
• angioedema
• atopic dermatitis
• Respiratory
• rhinitis
• laryngeal edema
• asthma
• Systemic
• anaphylactic
shock
43
How Much of a Problem Is It?
• 30,000 ER visits/ 2500
hospitalizations/ 150 deaths/yr
• Up to 2-3% of adults & 6-8% of
children have true food allergies
• Over 150 foods implicated; 8-10
commonly allergenic foods
• No cure, avoidance of allergenic
food is critical
44
What is different about
food allergens?
• Allergic response is to a food
component that is nutritious for
most of the population
• Sensitivity and severity
(biological endpoints) have large
range in the population
• No animal models or in vitro tests
for dose/response modeling
45
The Immunology of an Allergic
Response
Step 1 –
Sensitization
Step 2 –
Elicitation
46
Food Allergic Responses –
Application of The Key Event
Approach
47
Sensitization



Very poorly understood
May be breakdown of oral
tolerance
No data on thresholds for
sensitization
48
Elicitation




More data on elicitation process
Clinical evidence for thresholds
Thresholds may change over time
in an individual
Cross-reactivity and crosssensitivity can lead to reactions to
different foods
49
Major Steps in Elicitation
Ingestion
Digestion
Uptake
Cellular Events
Signs and Symptoms
50
Major Steps in Elicitation
Ingestion
Each of these is
a key event
Digestion
Uptake
Cellular Events
Signs and Symptoms
51
Major Steps in Elicitation
Ingestion
Digestion
But we don’t know
which is the
determining key
event
Uptake
Cellular Events
Signs and Symptoms
52
Major Steps in Elicitation
Ingestion
We do know some of
the biochemical and
physiological factors
that are important at
each step
Digestion
Uptake
Cellular Events
Signs and Symptoms
53
Ingestion
• Critical Factors
• Dose (amount of allergenic protein)
• Condition (effects of processing,
food matrix)
54
Digestion
• Critical Factors
• Stability of allergenic proteins to
proteases, pH
• Digestive capacity of individual
55
Uptake and Distribution
• Critical Factors
• Transporter system in intestines
• Capacity
• Kinetics
• Affinity
• Non-transport uptake systems
• Distribution of allergenic proteins or
breakdown products after uptake
56
Cellular Events
• Critical Factors
• Number of and types of effector cells
•
•
Basophils
Mast Cells
• Location of effector cells
• Number of IgE molecules
• Localized on effector cells
• Circulating
• Kinetics of IgE binding
• Number and type of mediator molecules
released by effector cells after cross
linking
57
Effector Interactions with
Tissues - Signs and Symptoms
• Critical Factors
• Site of mediator release
• Distribution of mediators after
release
• Concentration of mediators in
tissues
• Number and distribution of mediator
receptors
58
Applying the Key Events Approach to
Food Allergy – Future Directions


Identification of determining key
event in pathway
– Possible identification of factors that
affect individual differences
– Possible identification of factors that
affect nature of a reaction
– Possible identification of factors that
change over time in an individual
Provide basis for extrapolation from
high dose studies to estimate the
probability of low dose reactions
59
Applying the Key Events
Approach to Food Allergy

The Good News
– Relevant human data exist on
overall dose responses

The Bad News
– Dose response data do not exist for
the intermediate steps
60
Identification of the key event can be easy
61