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Elucidating mechanisms of toxicity using phenotypic data from primary human cell systems--a chemical biology approach for thrombosis-related side effects.

Berg EL, Polokoff MA, O'Mahony A, Nguyen D, Li X - Int J Mol Sci (2015)

Bottom Line: In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system.Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms.These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.

View Article: PubMed Central - PubMed

Affiliation: BioSeek, a Division of DiscoveRx Corp., 310 Utah Ave., Suite 100, South San Francisco, CA 94080, USA. eberg@bioseekinc.com.

ABSTRACT
Here we describe a chemical biology approach for elucidating potential toxicity mechanisms for thrombosis-related side effects. This work takes advantage of a large chemical biology data set comprising the effects of known, well-characterized reference agents on the cell surface levels of tissue factor (TF) in a primary human endothelial cell-based model of vascular inflammation, the BioMAP® 3C system. In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system. Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms. As a result, mechanisms for increasing (AhR, histamine H1 receptor, histone deacetylase or HDAC, hsp90, nuclear factor kappa B or NFκB, MEK, oncostatin M receptor, Jak kinase, and p38 MAPK) and decreasing (vacuolar ATPase or V-ATPase) and mTOR) TF expression levels were uncovered. These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.

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Heat map showing the effect of mechanisms on the cell surface protein expression levels of biomarker endpoints in the BioMAP 3C system. Mechanisms causing an increase in the level of the indicated biomarker are shown in green, and mechanisms resulting in decreased levels are shown as red. Black indicates that the levels were unchanged (no missing data). Data were generated from multiple compounds and an average profile representing the mechanism class was generated. The data used to generate this figure are taken from reference [21] and are included for convenience in Supplemental Table S1.
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ijms-16-01008-f001: Heat map showing the effect of mechanisms on the cell surface protein expression levels of biomarker endpoints in the BioMAP 3C system. Mechanisms causing an increase in the level of the indicated biomarker are shown in green, and mechanisms resulting in decreased levels are shown as red. Black indicates that the levels were unchanged (no missing data). Data were generated from multiple compounds and an average profile representing the mechanism class was generated. The data used to generate this figure are taken from reference [21] and are included for convenience in Supplemental Table S1.

Mentions: The BioMAP 3C system consists of human primary endothelial cells stimulated for 24 h with a cocktail of proinflammatory cytokines (IL-1β + TNFα + IFNγ), followed by measurement of cell surface levels of MCP-1, VCAM-1, TF, TM, IL-8, MIG, HLA-DR, E-selectin and uPAR; total protein levels (measured by sulforhodamine B (SRB) staining, see Experimental Section below and a more detailed description in Supplemental Text, Materials and Methods); and cell proliferation. We have been running this model system for over 10 years, testing various drugs, biologics and experimental tool compounds or test agents [1,16,17,18,19,20,21,22,23,24,25]. In a recent study, we reported on the evaluation of a set of 88 reference agents representing 28 pathway/mechanism classes, profiled in a panel of 8 BioMAP systems, including the BioMAP 3C system [21]. Figure 1 shows a heat map indicating the effect of each of 28 mechanism classes on the biomarker endpoint levels in the BioMAP 3C system, with significantly increased levels (>10% effect size) shown in green, and decreased levels shown in red (with no change indicated in black). As shown in this figure, each pathway mechanism evaluated in the BioMAP 3C system displays a specific pattern of biomarker changes, and correspondingly, each biomarker displays a distinguishable pattern of regulation by the various mechanism classes. Studying multiple endpoints simultaneously allows non-specific effects, for example, due to overt cell death, to be identified, preventing such confounding results from interfering with data interpretation. In Figure 1, treatment conditions leading to overt cell death were excluded prior to analysis [21].


Elucidating mechanisms of toxicity using phenotypic data from primary human cell systems--a chemical biology approach for thrombosis-related side effects.

Berg EL, Polokoff MA, O'Mahony A, Nguyen D, Li X - Int J Mol Sci (2015)

Heat map showing the effect of mechanisms on the cell surface protein expression levels of biomarker endpoints in the BioMAP 3C system. Mechanisms causing an increase in the level of the indicated biomarker are shown in green, and mechanisms resulting in decreased levels are shown as red. Black indicates that the levels were unchanged (no missing data). Data were generated from multiple compounds and an average profile representing the mechanism class was generated. The data used to generate this figure are taken from reference [21] and are included for convenience in Supplemental Table S1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307287&req=5

ijms-16-01008-f001: Heat map showing the effect of mechanisms on the cell surface protein expression levels of biomarker endpoints in the BioMAP 3C system. Mechanisms causing an increase in the level of the indicated biomarker are shown in green, and mechanisms resulting in decreased levels are shown as red. Black indicates that the levels were unchanged (no missing data). Data were generated from multiple compounds and an average profile representing the mechanism class was generated. The data used to generate this figure are taken from reference [21] and are included for convenience in Supplemental Table S1.
Mentions: The BioMAP 3C system consists of human primary endothelial cells stimulated for 24 h with a cocktail of proinflammatory cytokines (IL-1β + TNFα + IFNγ), followed by measurement of cell surface levels of MCP-1, VCAM-1, TF, TM, IL-8, MIG, HLA-DR, E-selectin and uPAR; total protein levels (measured by sulforhodamine B (SRB) staining, see Experimental Section below and a more detailed description in Supplemental Text, Materials and Methods); and cell proliferation. We have been running this model system for over 10 years, testing various drugs, biologics and experimental tool compounds or test agents [1,16,17,18,19,20,21,22,23,24,25]. In a recent study, we reported on the evaluation of a set of 88 reference agents representing 28 pathway/mechanism classes, profiled in a panel of 8 BioMAP systems, including the BioMAP 3C system [21]. Figure 1 shows a heat map indicating the effect of each of 28 mechanism classes on the biomarker endpoint levels in the BioMAP 3C system, with significantly increased levels (>10% effect size) shown in green, and decreased levels shown in red (with no change indicated in black). As shown in this figure, each pathway mechanism evaluated in the BioMAP 3C system displays a specific pattern of biomarker changes, and correspondingly, each biomarker displays a distinguishable pattern of regulation by the various mechanism classes. Studying multiple endpoints simultaneously allows non-specific effects, for example, due to overt cell death, to be identified, preventing such confounding results from interfering with data interpretation. In Figure 1, treatment conditions leading to overt cell death were excluded prior to analysis [21].

Bottom Line: In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system.Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms.These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.

View Article: PubMed Central - PubMed

Affiliation: BioSeek, a Division of DiscoveRx Corp., 310 Utah Ave., Suite 100, South San Francisco, CA 94080, USA. eberg@bioseekinc.com.

ABSTRACT
Here we describe a chemical biology approach for elucidating potential toxicity mechanisms for thrombosis-related side effects. This work takes advantage of a large chemical biology data set comprising the effects of known, well-characterized reference agents on the cell surface levels of tissue factor (TF) in a primary human endothelial cell-based model of vascular inflammation, the BioMAP® 3C system. In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system. Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms. As a result, mechanisms for increasing (AhR, histamine H1 receptor, histone deacetylase or HDAC, hsp90, nuclear factor kappa B or NFκB, MEK, oncostatin M receptor, Jak kinase, and p38 MAPK) and decreasing (vacuolar ATPase or V-ATPase) and mTOR) TF expression levels were uncovered. These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.

Show MeSH
Related in: MedlinePlus