SEACSAFETY & ENVIRONMENTAL ASSURANCE CENTRE

SAFETY SCIENCE IN THE 21ST CENTURYFor more information visit www.tt21c.org

A systems biology model of oxidative stress that distinguishes

adaptive and adverse cellular responses

• Oxidative stress, an imbalance between free radical generation and its quenching, is one of the key mechanism of

toxicity.

• Nuclear transcription factors like Nrf2 and NFκB replenish cellular antioxidant reserves at low chemical exposure

• NFκB interferes with the sustained activation of JNK and prevents cell death.

• Excessive free radical overwhelms the cellular defenses leading to structural and functional damage to

macromolecules such as proteins and lipids resulting in adverse downstream consequences.

• Identifying the tipping point from adaptive to adverse effect is important in decision making for chemical safety risk

assessment.

2. Building a Systems Biology Model of Oxidative Stress

Objective: Develop mechanistic models that can bridge in vitro datasets and in vivo exposure scenarios

Gaurav Jain1, Andrew White2, Paul Carmichael2, Narasimha M K3, Nalini R3, Kas Subramanian3, Sarah Cooper2, Sonali Das3,

Jayasujatha Vethamanickam1, Alistair Middleton2, Stephen Glavin2, Abhinandan Raghavan2

1SEAC, Unilever, India 2SEAC , Unilever, UK 3Strand Life Sciences, Bangalore, India

• We present a mathematical model of the oxidative stress pathway regulated by Nrf2 and NFκB that cross-talks with

JNK-caspase pathway of cell death.

• The model simulates cellular mechanisms that follow low exposures and high exposure of free radicals and the

adaptive changes to restore oxidative stress homeostasis through Nrf2 and NFκB activation.

• The model has potential to predict the tipping point of adaptive and adverse changes when combined with

Pharmacokinetic models and in vitro measurements.

t=5min T=20hrs

3. Validation and Integration of Model into Chemical Safety Risk Assessment Framework

Model Tuning

Model Prediction

/Testing

In vitro Assays

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utputSimulatedOdParameterLabMeasureonCostFuncti

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The Model Processes Modeled Simulation Overview

1. Nrf2 Regulation on γ-GCS and GSH 2. NFkB Prevents Lipid Peroxidation and Interrupts Persistent JNK Activation

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Comparison with literature-Without Priming

Experiment Simulation

Altered

Biological

Function

Nrf2, NFkB Adaptive

Response

Biological

Inputs

Cell

Dysfunction

Adverse

Health

Outcomes

Exposure

Tissue Dose

Pathway

Perturbation

MIE-ROS/Electrophile

Signature of Adaptive and

Adverse Response

Free radical generation at multiple-site

Generation of secondary radicals, Haber-Weiss,

Fenton reactions

The GSH metabolism

Lipid peroxidation and recovery

Protein oxidation and recovery

Glycolysis and oxidative phosphorylation

Mitochondrial pore opening

ASK1, JNK-caspase activation

Cell death: apoptosis, necrosis

Model has been tuned to achieve homeostasis as well

as mimic perturbed oxidative stress.

We discuss 4 case studies of perturbation

1. Nrf2 activation and upregulation of γ-GCS and GSH

2. NFkB prevents lipid peroxidation and interrupts

persistent JNK activation

3. Adaptation by Nrf2 activation

4. Excess oxidative stress leads to adverse changes

3. Adaptation by Nrf2 Activation with H2O2 Priming 4. Excess Oxidative Stress Leads to Adverse Changes

Effect of excess ROS on activation of caspase-8 and subsequently cell death has been tuned as per

experiments in Jurkat cells by Hampton et. al., 1997 in the following way:

• Increase in caspase-8 activity till 50 μM H2O2, followed by its decline for higher H2O2 concentrations due to

oxidation of caspase itself

• Increase in cell death with a shift in profile: increase in necrosis (both absolute and relative to apoptosis) with

higher concentrations of H2O2

Effect of NFκB deficit due to

IκB excess was modelled as

per experiments in ewing

sarcoma cells by Mergny M

et. al., 2004.

When challenged with

TNFα, the lipid peroxidation

and cell death were higher

in cells with an excess of

IκB than the normal cells.

• The model, in combination with a set of in vitro assays, aims to

recapitulate the known core feedback mechanisms related to

oxidative stress to predict whether a specific exposure scenario

will lead to an adverse or an adaptive response.

• Each module has been developed and tuned in isolation to

recapitulate specific perturbations.

• Work is currently underway to fit the fully coupled model to the

experimental data in HepG2 and test it with case study chemicals.

• The approach of developing quantitative systems models

describing the causal relationships of cellular alterations and how

they link to higher order physiological responses aligns with

current proposed frameworks for mechanistic safety assessment.

1. Oxidative Stress Pathway Perturbation

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Keap1 Knockout

Experiment Simulation

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Experiment Simulation

Effects of H2O2 pre-pulse (priming) on 4HNE and JNK

have been modelled as per studies in human leukemia

K562 cells and HL-60 cells by Cheng et. al., 2001.

• Cells primed with H2O2 adapt to a subsequent

exposure of H2O2 and show lower rise in 4HNE,

lower JNK activation and a reduced fall in GSH

levels.

• Model shows that activation of Nrf2 could be one of

the mechanisms of adaptation.

Nrf2 regulation on γ-GCS and hence on GSH has been modelled based on

studies in mice hepatocytes by Wu et. al., 2011.

• Active Nrf2 in nucleus was matched with the experimental value.

• The fold increase and decrease in γ-GCS and GSH following Keap1 KO/KD

and Nrf2 KO respectively was comparable to reported values.

4HNE

GSH

Minutes

H2O2

Input

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Effect of Priming-Simulated values

Without Priming With Priming

Active

JNK

Minutes

Active

NFkB

Model Validation Integration of Model into Risk Assessment Framework

QSPR/in vitro

physicochemical

parameters

Biokinetic

model

(QIVIVE)

Computational

systems biology

models

In vivo human

safety estimate

(mg/kg/day)

In vitro adversity,

point of departure (POD/BPAD)

concentration determination

Chemical ingredient with ‘significant’ human exposure

Chemical profiling (chemo-informatics)In vitro HTS (pathway inference)

Defined tox-pathway(s) of concern

In vitro biokinetics & free concentration

In vitro

concentration

response

in appropriate

assays

TT21C

FRAMEWORK

Djavaheri-Mergny M, Javelaud D, Wietzerbin J,

Besancon F. NFκB activation prevents apoptotic

oxidative stress via an increase of both thioredoxin

and MnSOD levels in TNFα-treated Ewing

sarcoma cells. FEBS Lett 2004; 578:111-115

Ji-Zhong Cheng et al, Stress Response of Cells to Heat and Oxidative RLIP76 and hGST5.8 Is an Early Adaptive 4-Hydroxynonenal through Induction of J. Biol.

Chem. 2001, 276:41213-41223

Mark B. Hampton, Sten Orrenius, Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis, FEBS Letters 414 (1997) 552-556

Kai Connie Wu et al, Beneficial Role of Nrf2 in Regulating NADPH Generation and Consumption, Toxicological

Sciences 123(2), 590–600 (2011)

With normal NFκB activation, JNK recedes faster, thus contributing to a lesser amount of caspase 8 activation, Whereas,

with IKB excess, a more intense and prolonged/sustained JNK activation leads to excessive caspase 8 activation.

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Lipid Peroxidation

Experiment Simulation

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Experiment Simulation

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H2O2 CONCENTRATION (UM)

Experiment

Apoptosis Necrosis Total Cell Death Caspase Activity

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Simulation

Apoptosis Necrosis Total Cell Death Caspase Activity

Response to TNFα challenge under IκB Normal

IκB Excess

ROS

Quenching-

GSH,

Enzymatic

Adaptive

Feedback-

Nrf2, NFkB

Downstream

Damage-

Lipid, Protein

Adverse

Response-

Determinants of

Cell Damage