
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


• 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


• 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 (solin@ilsi.org)
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)
INITIAL DOSE
(Exposure or Intake)
Ultimate Effect of Concern
INITIAL DOSE
(Exposure or Intake)
Target Tissue
Dose, Adjustment
Factors, etc.
Multiple events,
Multiple dose levels;
Multiple d-r relationships
Ultimate Effect of Concern
Ultimate Effect of Concern
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Overall KEDRF Concept
INITIAL DOSE (exposure or intake) key event(s) (e.g., absorption, inhalation) key event(s) (e.g., transport to target tissue)
At various events, homeostatic mechanisms may affect progression along pathway key event(s) (interaction in target tissue) 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)
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Host factors Dose
Homeostatic mechanisms
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
Moderately High
Chronic Intake
Chronic Inadequate
Intake
Teratogenicity –
Severe toxicity/lethality
Organ damage, affecting metabolism
Visual abnormalities, impaired fertility, ↓ immune response,
↓bone growth
Focus of case study: upper levels of intake
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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
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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 .
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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” .
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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.
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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
RE
LRAT oxidative inactivation
Retinol RA storage and release oxidative activation polar metabolites of RA conjugation and excretion
RE
Excess dietary
Vitamin A
LRAT
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

Working Group:
Robert L. Buchanan
Arie H. Havelaar
Mary Alice Smith
Richard C. Whiting
Elizabeth Julien
25


“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
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
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)
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
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
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




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
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10
1
0.1
0.01
0.001
0.0001
0.00001
0.000001
0.0000001
0
0
1
0.8
0.6
0.4
0.2
0
1
2
2 3 4 5
4 log Dose
6
6 7 8
8 10
9
Data
Public health and regulatory concern
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
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






Exposure to L. monocytogenes smoked fish, pates) via readyto-eat foods (soft cheeses, deli meats,
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
Intake of contaminated food
1 - P
Do not survive
P
Pathogens survive in upper GI tract
Interplay of host and pathogen can influence progression at various events
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


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



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



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






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

Allergen Working Group
Steven Gendel
Steve Taylor
Geert Houben
41

• 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

• Gastrointestinal
• nausea
• vomiting
• abdominal pain
• diarrhea
• Cutaneous
• urticaria
• angioedema
• atopic dermatitis
• Respiratory
• rhinitis
• laryngeal edema
• asthma
• Systemic
• anaphylactic shock
43

• 30,000 ER visits/ 2500 hospitalizations/ 150 deaths/yr
• Over 150 foods implicated; 8-10 commonly allergenic foods
voidance of allergenic food is critical
44

• 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

Step 1 –
Sensitization
Step 2 –
Elicitation
46

47




Very poorly understood
May be breakdown of oral tolerance
No data on thresholds for sensitization
48





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

Ingestion
Digestion
Uptake
Cellular Events
Signs and Symptoms
50

Ingestion
Digestion
Each of these is a key event
Uptake
Cellular Events
Signs and Symptoms
51

Ingestion
Digestion
But we don’t know which is the determining key event
Uptake
Cellular Events
Signs and Symptoms
52

Ingestion
Digestion
We do know some of the biochemical and physiological factors that are important at each step
Uptake
Cellular Events
Signs and Symptoms
53

• Critical Factors
• Dose (amount of allergenic protein)
• Condition (effects of processing, food matrix)
54

• Critical Factors
• Stability of allergenic proteins to proteases, pH
• Digestive capacity of individual
55

• Critical Factors
• Transporter system in intestines
• Capacity
• Kinetics
• Affinity
• Non-transport uptake systems
• Distribution of allergenic proteins or breakdown products after uptake
56

• 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

• Critical Factors
• Site of mediator release
• Distribution of mediators after release
• Concentration of mediators in tissues
• Number and distribution of mediator receptors
58



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



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