Activation of Adaptive Cellular Activation of Adaptive Cellular Networks and Hormetic Dose Networks and Hormetic Dose Networks and Hormetic Dose Networks and Hormetic Dose

Response RelationshipsResponse Relationships

Melvin AndersenMelvin AndersenThe Hamner Institutes for Health SciencesThe Hamner Institutes for Health Sciences

Research Triangle Park NC 27709 Research Triangle Park NC 27709 Research Triangle Park, NC 27709 Research Triangle Park, NC 27709 BELLE Conference BELLE Conference

Amherst, MAAmherst, MAMay 1May 1--3, 2007 3, 2007

Toxicity of hypochlorous acid in Mouse RAW cellsToxicity of hypochlorous acid in Mouse RAW cells

150)150

ControltBHQ

100

iabi

lity

Con

trol)

100

tBHQHOCl

iabi

lity

cont

rol)

50

Cel

l v(%

of C

50Cel

l vi

(% o

f c

0.0 2.5 5.0 7.50

0.0 2.5 5.0 7.50

In vitro results with various cell types show a region In vitro results with various cell types show a region of increased viability before regions of toxicityof increased viability before regions of toxicityy g yy g y

Pi et al. (2007)Pi et al. (2007)

OutlineOutlineOutlineOutline

Ri k t h ll f i it t i Risk assessment challenges for irritant gases in developing Reference Concentrations (RfCs)

Biological models of thresholds, dose-dependent transitions and hormesis versus safety factors

Choosing prototype irritants for study - e.g., irritant gases, hepatic enzyme inducers

Moving ideas of hormesis into the risk assessment processp

Reference Concentrations (RfCs) Reference Concentrations (RfCs) –– the Processthe Process

RfC = Benchmark Concentration x Duration x DAFRfC = Benchmark Concentration x Duration x DAF

UF1 UF2 X UF3UF1 UF2 X UF3UF1 x UF2 X UF3…………UF1 x UF2 X UF3…………

For Chlorine:For Chlorine:To develop an RfC that takes into account:

(1) Tissue dosimetry of HCl and HOCl within the respiratory tract

(2) Dose dependent modes of action of chlorine in these tissues, and

(3) Cell response modeling of activation of adaptive cell response pathways

(4) Places U-shaped from in vitro responses in a risk assessment contextin a risk assessment context

Why chlorine?Why chlorine?yy

It is a common water disinfectant a synthetic It is a common water disinfectant, a synthetic intermediate for many commodity chemicals, and a possible target for terrorist use as a weapon.

Good traditional toxicity data base, including an inhalation bioassay and a clear mode of action as a ycellular oxidant

Mechanistic Approaches to Irritant Gas Mechanistic Approaches to Irritant Gas R k R k

MechanisticMechanistic

Risk AssessmentsRisk Assessments

MechanisticMechanisticIncidenceIncidence--Dose Dose

ModelsModels

Mode of Action StudiesMode of Action StudiesTissue DosimetryTissue Dosimetry

PBPK and/or CFD modelsPBPK and/or CFD modelsSystems Biology of Systems Biology of

Affected Signaling PathwaysAffected Signaling Pathways

Hypothesis:Hypothesis: Oxidative stress from HOCl is the predominant mode of action for chlorine irritancy in the

Chlorine rapidly hydrolyzes in aqueous conditions

predominant mode of action for chlorine irritancy in the respiratory tract

Chlorine rapidly hydrolyzes in aqueous conditions

Cl2 + H2O HOCl + HCl

Nasal responses observed at several ppm Cl2compared to 100 ppm for HClcompared to 100 ppm for HCl

What does Chlorine do to tissues?What does Chlorine do to tissues?

NN

NN

AA1

AA2

O

O

O

OH

2

NN

NN

AA1O OHOCl

OHCl

ClAA2O

C

Reactions of HOCl with tissue produces a variety of idi d d hl i t d d t i l di hl i t d oxidized and chlorinated products, including chlorinated

aromatic amino acids. These products serve as a local biomarker for the presence of HOCl in tissues (Jarabek p (and Sochaski)

ol)

ol) How do tissues respond?How do tissues respond?

1500of C

ontr

oof

Con

tro

*Treatment of RAW Treatment of RAW

ll l l i d ll l l i d

500

1000

ein

(%

oein

(%

o *

*

cells clearly induces cells clearly induces Nrf2, the primary Nrf2, the primary mediator of antimediator of anti--

0

500

Nrf

2 pr

ote

Nrf

2 pr

ote

* *oxidant stress signaling.oxidant stress signaling.

N f2

NN

0 0.35 0.7 1.4 2.8HOCl (mM)

Nrf2Sam68

Pi et al.

C d S ifi

Then, as concentrations increase,Then, as concentrations increase,Compound Specific

Studies based on work of Andre Nel and others (Science, 2006)

Mechanistic Hierarchical Dose Response ModelMechanistic Hierarchical Dose Response Model

Tissue Phase ReactionsDosimetryDosimetryCl2 HOCl + HCl

yyInhaled Stressor

(1) (2) (3)

Normal Epithelial

AdaptiveState

StressedSt t

PathologyNecrosisEpithelial

CellState State Atrophy

A model for oxidative stress in Pathway AssistA model for oxidative stress in Pathway Assist--automated model building and dose responseautomated model building and dose response

Dose-Responses1

0.9

0.8

0.7

0.6

0.5

0.4

0 3

Response

1001010.10.01

0.3

0.2

0.1

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dose

Zhang et al. (in progress)

Controlling Cell AntiControlling Cell Anti--oxidant Synthesisoxidant Synthesis

300

400 0.14 mM1.4 mM

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l) 600 0.14 mM1.4 mM

etas

etro

l)

100

200

300

GC

LC%

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400

SH

Syn

the

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GS (

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ar G

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seon

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race

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Normal adaptive feedback processes, based on negative feedback with feed forward control. Homeostasis

Consequences of Feedback Loop on Consequences of Feedback Loop on St L l i C llSt L l i C llStressor Levels in CellsStressor Levels in Cells

ble

-Y In absence of up-regulation

l Var

iab

Toxicity

nter

nal

Region of Regulationincludin lt r d n xpr ssi n

External Stressor Level External Stressor Level -- SS

I including altered gene expression

External Stressor Level External Stressor Level -- SS

Incidence Incidence Dose Curves in vivoDose Curves in vivo

S b t d S b t d

Incidence Incidence –– Dose Curves in vivoDose Curves in vivo

Subsequent dose response Subsequent dose response curves for toxicity in the curves for toxicity in the intact animal will have an intact animal will have an initial threshold for initial threshold for initial threshold for initial threshold for activation of the stress activation of the stress response and then a response and then a controlled region before controlled region before controlled region before controlled region before transitioning to overt transitioning to overt toxicity. Utoxicity. U--shaped shaped responses possible due to responses possible due to p pp paltered energy uses with altered energy uses with upup--regulation of batteries regulation of batteries of antiof anti--stress factors in stress factors in tissuestissues

Prior exposure protects against HOCl exposures Prior exposure protects against HOCl exposures Prior exposure protects against HOCl exposures Prior exposure protects against HOCl exposures in vitro and enhances Uin vitro and enhances U--shaped dose responseshaped dose response

200 0.14 mMSH in) 150 Control

tBHQ)

100

1501.4 mM

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ility

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50

100

ntra

cell

( µm

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0 4 80

10 20

In (

0.0 2.5 5.0 7.50

Mechanistic Dose Response Model with Genomic DataMechanistic Dose Response Model with Genomic Data

Tissue Phase ReactionsCl2 Pr-OH

DosimetryDosimetryInhaled Formaldehyde

(1) (2) (3)

Pathology

(1) (2) (3)

Normal Epithelial

Cell

AdaptiveState

StressedState

PathologyNecrosisAtrophy

Use specific in vivo studies to develop a dose response model for Use specific in vivo studies to develop a dose response model for activation of oxidative oxidative stress pathways following formaldehyde exposure and differentiate dose regionsdifferentiate dose regions that activate adaptive responses at low concentrations and inflammatory and necrotic p yprocesses as concentrations increase

It has to be interdisciplinaryIt has to be interdisciplinary

In vivo exposures – limited genomic evaluations, oxidative markers, tissue/organ responses (Ms. Jarabek)markers, tissue/organ responses (Ms. Jarabek)

Genomic studies in vitro with epithelial cells in culture (Dr. Yin Chen)

Confirmation of and mechanistic studies of anti-oxidant response activation (Dr. Jingbo Pi)

Pathway modeling of activation of Nrf-2 signaling ( Dr. Qiang Zhang)

Functional Genomic mapping of Nrf2 signaling (Dr. Courtney Woods, Exxon-Mobil Post-Doctoral Fellow)

ACC-LRI supported programs at The Hamner Institutes

ConclusionsConclusions

Hormesis and thresholds are likely related to activation of adaptive pathways, i.e., to regions of homeostasisadaptive pathways, i.e., to regions of homeostasis

Use in risk assessment requires interdisciplinary development of several compelling, mechanistic prototypes - such as with p g p ypirritant gases or with some hepatic enzyme inducers

In the absence of such well-developed examples showing basis f h i d i i k for hormesis and non-monotonic responses, risk assessments will hold fast to threshold and low-dose linear methodologies that are now favored

Cellular responses to stressors are Cellular responses to stressors are ppfrequently dichotomousfrequently dichotomous

Chubb et al. (2004)

With Binary rather than Graded ResponsesWith Binary rather than Graded Responses

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Population Distribution – Cells with GFP

MAPMAP--Kinase Kinase modules provide modules provide cellular switchescellular switches

Stimulus PDGF Integrin OxidativeStress IL-1

TRAF6

PG

Activator

MKKK

RasGTP

C-Raf1

Rac1

MEKK1

Src

MEKK2

TRAF6-TAB1/2

TAK1

Activator

MOS

MKK

MAPK

MEK

MAPK

MKK4

JNK1

MKK5

ERK5

MKK6

p38

MEK1

p42

Substrate TFs

JNK

C-Jun

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MNK1

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