What s in a Tipping Point?

Transcription

1 What s in a Tipping Point? Using Systems Biology to Characterize Adverse Oxidative Responses in Human Lung Cells Jenna M. Currier, Ph.D. ORISE Postdoctoral Fellow at U.S. EPA Mentors: Brian Chorley, Ph.D. and Rory Conolly, Sc.D. I n Vitro and A l t e r n a t i v e M e t h o d s S p e c i a l t y S e c t i o n We b i n a r : M a y 1 8,

2 Why study tipping points? Exposure Uptake/Delivery to Target Tissues Key event-based points of departure for adverse events can assist in the prediction of adverse health outcomes Biologic Inputs Perturbation Normal Biologic Function Early Cellular Changes Adverse Responses Adaptive Responses Cell Injury, Inability to Regulate Adverse Outcomes Modified from: Andersen, M. E. and D. Krewski (2009) Toxicol Sci 107(2): of 16

3 What mediates the switch between adaptive and adverse responses? Zn is sequestered and buffered to maintain normal cellular function Zn 2+ KEAP1 NRF2 Crosstalk between p53 and NRF2 coordinate the p53-initiated cell IV MDM2 p53 survival response E GSH H 2 O 2 E GSH H 2 O 2 Mechanisms of the shift from p53-mediated cellular adaption to programmed cell death are less understood 3 of 16

4 Zn 2+ Free / Zn Bound Ratio Fraction of Maximum Binding Using biological modeling to define the tipping point Saturation of Zn Binding Extracellular ZnPyr Intracellular ZnPyr Zn 2+ Pyr Zn Bound Zn 2+, M (with Pyr) Free Zn 2+ /Zn Bound Pyrithione (Pyr) a zinc specific ionophore that facilitates transport Pharmacodynamic model predicts saturation of intracellular Zn 2+ binding Zn 2+, M (with Pyr) 4 of 16

5 % of Control Does in vitro cytotoxicity correspond to computational predictions? % of Control h 40 h 48 h Zn 2+, µm BEAS-2B cells exposed to Zn 2+ in the presence of 1 µm pyrithione ATP content measured by luminescent CellTiter-Glo assay Cytotoxicity corresponds to concentrations predicted to saturate intracellular Zn 2+ buffering 5 of 16

6 Are markers of apoptosis altered after Zn 2+ exposure? Mean ± SD, n=3. p<0.01 compared with control () or each other (#) by two-way ANOVA and Holm-Sidak post hoc test. 6 of 16

7 Are markers of apoptosis altered after Zn 2+ exposure? Tipping point from adaptive, recoverable cellular Mean ± SD, n=3. p<0.01 compared with control () or each processes other (#) to by two-way an unrecoverable, ANOVA and Holm-Sidak post cytotoxic hoc test. response with exposures 2.5 µm Zn 2+ 7 of 16

8 -log(p-value) PC #2 (10%) -log(p-value) Exploring adaptive and apoptotic gene expression changes PC #3 (9.96%) PCA Mapping (39.2%) Controls [Zn 2+ ] Time DEGs PC #1 (19.3%) 2 µm 4 h µm 4 h µm 24 h µm 24 h µm 48 h µm 48 h 1864 Concentration and duration of Zn 2+ exposure mediate significant gene expression changes Pathway Enrichment p53 Signaling Time, h NRF2-Mediated Oxidative Stress Time, h 2 µm Zn 2+ 3 µm Zn 2+ 8 of 16

9 Can early gene expression changes differentiate cellular responses? Hierarchical Clustering 4 h Exposures 68 genes related to p53 and/or NRF2 Control NRF2 p53 Adaptive Cytotoxic A 154 gene biomarker set identified by two sample t- Gene expression changes are evident earlier than typically measured phenotypic endpoints of cytotoxicity or apoptosis test (p <0.01). Log 2 normalized probe intensity values are shifted to a mean of zero and scaled to a standard deviation of 1. 9 of 16

10 Can early gene expression changes differentiate cellular responses? Fold-Change (qpcr) MXD1 DUSP5 ID1 HSPA4L MCL1 # # # WEE1 MAP2K3 GADD45A GCLM MYC SOD2 CDKN1A # # # # # # # IL8 SRXN1 HIST1H4H SERPINE1 EEA1 ETS1 GNG12 PTGER4 TXNRD1 TGFB2 JAK1 2 µm Zn 2+ 3 µm Zn 2+ RB1 FILIP1L THBS1 EGR1 MBP TSC22D3 p53- and NRF2-related genes may mediate the switch between adaptation and apoptosis # NRF2 p53 10 of 16

11 Can p53 activation distinguish the tipping point? p53 activation, but no early distinction around tipping point Whole cell extracts after 4 h exposures were probed using a capillary electrophoresis-based Western system. Mean ± SD, n=3. p<0.05 compared with control () by one-way ANOVA and Holm-Sidak post hoc test. 11 of 16

12 Fold Over Control Can NRF2 activation distinguish the tipping point? # 2 µm Zn 2+ 3 µm Zn Time, h Increased NRF2 Nuclear Translocation. activation Mean of ± SD, NRF2 n=3. p <0.001 at the compared with control () or each other (#) by twoway adaptive, ANOVA and Holm-Sidak 2 µm post Zn hoc 2+ test. exposure 12 of 16

13 ^ What s the point? Zn 2+ Uptake in Bronchial Epithelial Cells p53- and NRF2-related genes are potential transcriptomic biomarkers of early adaptive and adverse cellular responses Time (h) Early Genomic Changes Moderate [Zn 2+ ] More robust NRF2 activation Adaptive Responses Increased expression of most p53 and NRF2- related genes Cell Survival Higher [Zn 2+ ] Reduced NRF2 activation Dampening of adaptive response gene expression Cytotoxicity Cell Death, Apoptosis 13 of 16

14 Extending the computational model Extracellular Intracellular ZnPyr ZnPyr Pyr Zn 2+ Zn Bound KEAP1 NRF2 Antioxidants ROS 14 of 16

15 Future Applications Identify transcriptomic-based biomarkers for screening and prioritization of chemical and environmental-based exposures Utilize high-throughput in vitro model systems Develop a computational model to predict adverse outcomes Assess complex chemical mixtures using both paradigms 15 of 16

16 Acknowledgements US EPA Mentors Brian Chorley Rory Conolly Gleta Carswell Wan-Yun Cheng Lisa Dailey Eugene Gibbs-Fluornoy Gail Nelson Jim Samet Natalia Ryan Philip Wages Genomics Research Core Other Collaborators Funding Daniel Menendez (NIEHS) Qiang Zhang (Emory University) Oak Ridge Institute for Science and Education Internship/Research Participation Program U.S. Environmental Protection Agency Air, Climate, and Energy Research Program Integrated Systems Toxicology Division 16 of 16