United States Environmental Protection Agency Office of Research and Development -1 0 -9 -8 -7 -6 -5 -4 0 5 0 1 0 0 1 5 0 2 0 0 h N O G - R O C K In h . - Y 2 7 6 3 2 -1 0 -9 -8 -7 -6 -5 -4 -3 0 5 0 1 0 0 1 5 0 2 0 0 A p o p to s is - N o tc h In h . - D A P T % C o n C a s p a s e 3 /7 -G lo % C o n C e llT ite r-G lo -1 0 -9 -8 -7 -6 -5 -4 -3 0 5 0 1 0 0 1 5 0 2 0 0 A p o p to s is - R O C K In h . - Y 2 7 6 3 2 -1 0 -9 -8 -7 -6 -5 -4 -3 0 5 0 1 0 0 1 5 0 2 0 0 A p o p to s is - E R K 1 /2 In h . - S C H 7 7 2 9 8 4 -1 0 -9 -8 -7 -6 -5 -4 -3 0 5 0 1 0 0 1 5 0 2 0 0 A p o p to s is - P K C In h . - B IS -1 BMPR Notch EGFR Wnt PPAR-y COX-2 mTORC PDGFR PKC RHO EP1-4 CREB AKT ETC I EGFR NO-cGMP ROCK Protein HDAC PARP1 ERK/MAPK p38/MAPK hNP1 Apoptosis Active Selective hNP1 Proliferation Active/Cytotoxic h. Neurite Outgrowth Inactive r. Neurite Outgrowth Awaiting Data Cortical Synap. To be tested Network Formation 7 3 4 5 6 8 9 1 2 -1 0 -9 -8 -7 -6 -5 -4 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 1 5 1 1 6 M K -2 2 0 6 m T O R C In h . -1 0 -9 -8 -7 -6 -5 -4 0 5 0 1 0 0 1 5 0 rN O G - C O X -2 In h . - C e le c o x ib -1 0 -9 -8 -7 -6 -5 -4 0 5 0 1 0 0 1 5 0 rN O G - E R K 1 /2 In h . - S C H 7 7 2 9 8 4 P331 %control %control %control %control %control %control %control %control -1 0 -9 -8 -7 -6 -5 -4 0 5 0 1 0 0 1 5 0 rN O G - A K T A c t. - S C 7 9 • The DNT battery captures some important and highly conserved signaling pathways associated with various processes involved in neurodevelopment. • Specifically, we demonstrated that: • Wnt, mTORC, PKC, and PDGFR pathway modulators have selective effects on neurite outgrowth. • ERK1/2, AKT, and COX-2 pathway modulators demonstrated non-selective effects on neurite outgrowth. • To date, ROCK, NO-cGMP, and COX-2 do not seem to play a significant role in our assays. Future Directions: • Ten chemicals remain to be tested in the battery. • After all chemicals have been tested, a concentration-response analysis will be performed using the ToxCast pipeline. • If we find that developmentally relevant biology is not being adequately recapitulated in the battery, we may explore incorporating other assays that include these pathways. Preliminary ToxCast Analysis Discussion References • These results work in conjunction with the full OECD DNT battery developed in collaboration with global partners. • Our work expands upon previous in vitro DNT pathway characterizations especially when it comes to neurite outgrowth¹, demonstrating that our battery is sensitive to Wnt, PKC, PDGFR, and mTORC disruption. • A more robust characterization of the biology captured by our assays will aid in the interpretation of results gathered using the DNT battery. * The views expressed in this poster are those of the author(s) and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency. • The current NOG data (15 chemicals) has been analyzed using ToxCast R package tcplfit2 (v0.1.6). The computed AC₅₀ for each chemical and endpoint are given in the figure below. • The color scale indicates selectivity, which is determined as the ratio between the cytotoxic AC₅₀ and the AC₅₀ for the endpoint of interest. • Eventually, the AC₅₀ for each chemical and endpoint in the DNT battery will be computed, showing which pathways are best covered by the DNT battery and where improvements are needed. rNOG Neuron count Neurite count per neuron Neurite length per neuron Branch points per neuron DAPT-Notch Inh. ODQ-NO-cGMP Inh. Pioglitazone-HCl PPAR-y IWP2-Wnt Inh. Y-27632 ROCK KG-501-CREB Inh. AG1478-EGFR Inh. Celecoxib-COX-2 Inh. SCH772984-ERK1/2 Inh. SC79-AKT Act. MK-2206-mTORC Inh. CP-673451-PDGFR Inh. MHY1485-mTORC Act. BIS-1/BIM-1-PKC Inh. CHIR99021-Wnt Act. Computed 𝑨𝑨𝑨𝑨𝟓𝟓𝟓𝟓 AC₅₀: Concentration (µM) at which 50% maximum activity is observed. Endpoint Selectivity Ratio Selective Non-selective 3 1 Conclusions Selectivity Ratio = 𝐴𝐴𝐴𝐴50𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐴𝐴𝐴𝐴50𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 ≥ 3, Selective Effect 1. Masjosthusmann