The Toxmatrix: Chemo-Genomic Profiling Identifies Interactions That Reveal Mechanisms of Toxicity

Abstract

A chemical genomics "Toxmatrix" method was developed to elucidate mechanisms of cytotoxicity using neuronal models. Quantitative high-throughput screening (qHTS) was applied to systematically screen each toxicant against a panel of 70 modulators, drugs or chemicals that act on a known target, to identify interactions that either protect or sensitize cells to each toxicant. Thirty-two toxicants were tested at 10 concentrations for cytotoxicity to SH-SY5Y human neuroblastoma cells, with results fitted to the Hill equation to determine an IC₅₀ for each toxicant. Thirty-three toxicant:modulator interactions were identified in SH-SY5Y cells for 14 toxicants, as modulators that shifted toxicant IC₅₀ values lower or higher. The target of each modulator that sensitizes cells or protects cells from a toxicant suggests a mode of toxicant action or cellular adaptation. In secondary screening, we tested modulator-toxicant pairs identified from the SH-SY5Y primary screening for interactions in three differentiated neuronal human cell lines: dSH-SY5Y, conditionally immortalized dopaminergic neurons (LUHMES), and neural stem cells. Twenty toxicant-modulator pairs showed pronounced interactions in one or several differentiated cell models. Additional testing confirmed that several modulators acted through their primary targets. For example, several chelators protected differentiated LUHMES neurons from four toxicants by chelation of divalent cations and buthionine sulphoximine sensitized cells to 6-hydroxydopamine and 4-(methylamino)phenol hemisulfate by blocking glutathione synthesis. Such modulators that interact with multiple neurotoxicants suggest these may be vulnerable toxicity pathways in neurons. Thus, the Toxmatrix method is a systematic high-throughput approach that can identify mechanisms of toxicity and cellular adaptation.

Figure 1.

Example plots illustrating interactions between toxicant MAP and indicated modulators in SH-SY5Y cells: sensitization to cytotoxicity (A), no interaction (B), or protection (C). Green spots show that the modulators were not toxic at the three concentrations indicated, except the highest concentration of CoCl₂ which killed the cells. Black lines indicate toxicant MAP alone concentration-response; Dark blue-, Light blue-, and grey-, lines indicate MAP plus a modulator at low, medium, and high concentrations, respectively.

Figure 2.

Heatmap showing interactions between toxicants and modulators (columns) that alter cytotoxicity in SH-SY5Y cells. Maximal fold-change values are shown comparing IC₅₀ value with modulator divided by IC₅₀ value without modulator. Cell sensitization to a toxicant by a modulator is indicated by a positive IC₅₀ shift value if it exceeded 3-fold increase or decrease at the highest modulator concentration that did not cause ≥10% cytotoxicity. Red fill indicates sensitization to a toxicant, with increasing color saturation from 2 to 16-fold. Blue fill indicates a negative IC₅₀ shift hence protection from a toxicant, with increasing color saturation from −2 to −16-fold. White fill indicates no interaction. 32 toxicants were tested in 10 concentrations, or with DMSO vehicle, and with 3 concentrations of each of 70 modulators. Toxicant/modulator combinations that yielded significant interactions, are colored red for increased sensitivity, blue for protection by modulator, or white for no significant interaction. The maximum fold-change shift in IC₅₀ observed is shown in each colored square.

Figure 3.

Heatmap illustrating toxicant:modulator interactions in three differentiated neuronal models. Differentiated human cells are labeled “dSH-SY5Y, “dLUHMES”, or “dNSC”. Among toxicant:modulator pairs tested, 16 did not show IC₅₀ shifts exceeding 3-fold for any of the three differentiated cell types, and are omitted from this Figure. Cell colors are as described in the Figure 2 legend except extended to show 2- to 16-fold IC₅₀ shifts. Modulator abbreviations and drug targets are listed in Table 1. “NT” indicates the toxicant did not kill the cells at the highest concentration tested, 100 μM; and “TOX” indicates the toxicant killed the cells at the lowest concentration tested, 15 nM

Figure 4.

Sensitization of dLUHMES cells to 10 μM 6HD (A), or 4 μM MAP (B) by glutathione synthesis inhibitor, BSO, is partially relieved by 100 μM GSH or 100 μM NAC. BSO alone (open circles) was moderately cytotoxic to dLUHMES cells; whereas A) 10 μM 6HD + BSO (filled squares) were highly toxic. Addition of 100 μM NAC (grey filled circles) or 100 μM GSH (open triangles) to 6HD + BSO or MAP + BSO moderated cytotoxicity.

Figure 5.

Protection of dLUHMES cells from 6HD MAP by PHE, but not by M30. PHE alone (open circles) was not toxic to dLUHMES cells; whereas M30 alone (open squares) was moderately cytotoxic. Addition of PHE (filled circles) to 10 μM 6HD (A), or 4 μM MAP (B) moderated cytotoxicity; whereas addition of M30 (filled squares) increased cytotoxicity.