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
0
dose
Zhang et al. (in progress)
Controlling Cell AntiControlling Cell Anti--oxidant Synthesisoxidant Synthesis
300
400 0.14 mM1.4 mM
C tro
l) 600 0.14 mM1.4 mM
etas
etro
l)
100
200
300
GC
LC%
of c
ont
200
400
SH
Syn
the
% o
f Con
t
0 8 16 240
(%
0 8 16 240
GS (
150
200 0.14 mM1.4 mM
ar G
SH
prot
ein)
300
4000.14 mM1.4 mM
ucta
seon
trol)
50
100nt
race
llula
( µm
ol/g
of p
100
200
GS
H re
du(%
of C
o
0 4 80
10 20
I
0 8 16 240
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
lula
r GS
of p
rote
i
100
tBHQHOCl
viab
ility
cont
rol)
50
100
ntra
cell
( µm
ol/g
50Cel
l v(%
of c
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
0.14
0.12
0.1
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y, y
1
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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
E K
MEF2
p
MNK1
p
Rsk1,2