Whole-Cell Multiparameter Assay for Ricin and Abrin Activity-Based Digital Holographic Microscopy

Abstract

Ricin and abrin are ribosome-inactivating proteins leading to inhibition of protein synthesis and cell death. These toxins are considered some of the most potent and lethal toxins against which there is no available antidote. Digital holographic microscopy (DHM) is a time-lapse, label-free, and noninvasive imaging technique that can provide phase information on morphological features of cells. In this study, we employed DHM to evaluate the morphological changes of cell lines during ricin and abrin intoxication. We showed that the effect of these toxins is characterized by a decrease in cell confluence and changes in morphological parameters such as cell area, perimeter, irregularity, and roughness. In addition, changes in optical parameters such as phase-shift, optical thickness, and effective-calculated volume were observed. These effects were completely inhibited by specific neutralizing antibodies. An enhanced intoxication effect was observed for preadherent compared to adherent cells, as was detected in early morphology changes and confirmed by annexin V/propidium iodide (PI) apoptosis assay. Detection of the dynamic changes in cell morphology at initial stages of cell intoxication by DHM emphasizes the highly sensitive and rapid nature of this method, allowing the early detection of active toxins.

Keywords: abrin; digital holographic microscopy; holomonitor; intoxication; morphology; ricin.

Figure 1

Morphology features of ricin intoxication detected by digital holographic microscopy (DHM). Effect of ricin treatment on HeLa cells was tested using DHM. Cells were subjected to ricin (100 ng/mL), and digital holograms of four different areas in each well were recorded for 20 h at 10 min intervals. Untreated cells were used as a control. (A) Representative three-dimensional Images of treated and untreated cells as captured at 0, 3, 7, 11, and 17 h. Marker for optical thickness depicted on the right side. (B) Quantification of the relative changes in various morphological parameters was performed in parallel in the presence of neutralizing antibodies MH1 (monoclonal anti-subunit A), MH75 (monoclonal anti-subunit B), and polyclonal antibodies. * indicate p < 0.05 of intoxicated vs. untreated cells according to 2-tailed Student’s t-test.

Figure 2

The effect of ricin intoxication on cell viability. HeLa and Vero cells were incubated in the presence and absence of the toxin at concentrations of 10–100 ng/mL. (A) AlamarBlue viability assays were performed 17 h post-ricin exposure. The percentage of viable cells (mean ± SD) in treated cells was calculated relatively to untreated cells in each measurement. p < 0.05 of HeLa vs. Vero-treated cells was calculated according to 2-tailed Student’s t-test. (B) Scanning electron microscopy imaging of ricin intoxication on HeLa cells morphology was performed 5 h post 100 ng/mL ricin exposure. Panel 1 represents untreated cells, panel 2 represents ricin treated cells, and panel 3 represents higher magnification of the inset in panel 2.

Figure 3

Similarities in morphology features during ribosome inactivating proteins (RIPs) intoxication. Comparison of ricin and abrin intoxication on various morphology features in HeLa and Vero cell lines. HeLa (A–C) and Vero (B–C) were treated with ricin and abrin (100 ng/mL) and digital holograms of four different areas in each well were recorded every 10 min for 19 h. Untreated cells were used as a control. (A) Quantification of the relative changes in morphological parameters (mean ± SE) detected using DHM. (B) The table summarizes the time range for the detection of significant changes in the morphological features followed of intoxicated HeLa and Vero cells compared to untreated cells. p < 0.05 was calculated according to 2-tailed Student’s t-test. (C) Effect of abrin on viability of HeLa and Vero cells was performed 17 h post-toxin exposure (10–100 ng/mL) using AlamarBlue assay. The percentage of viable cells (mean ± SD) in treated cells were calculated relatively to untreated cells in each measurement. p < 0.05 of HeLa- vs. Vero-treated cells was calculated according to 2-tailed Student’s t-test. (D) Confluence, roughness, and optical thickness are representative parameters show neutralizing effect of anti-abrin polyclonal antibodies (pAbs) on abrin intoxicated HeLa cells detected by DHM.

Figure 4

Intoxication evaluation of live cells by DHM. HeLa cells were exposed to ricin 10–100 ng/mL. (A) Percentage of apoptotic cells (mean ± SE) during 8 h of ricin intoxication was determined using annexin V and PI apoptosis assays. (B) Live cell segmentation was performed using DHM cell tracking application according to roundness and thickness parameters in each frame. In each frame, 50–60 cells were tracked and analyzed. Treated and untreated tracked live cells were analyzed for morphological changes during 11 h post-ricin exposure (100 ng/mL). Shown are representative results out of three independent experiments (mean of 4 different areas in each well ± SE). (C) A representative DHM 2D image for presumed live cell segmentation analysis (cell margins are highlighted by colored lines).

Figure 5

Increased susceptibility of preadherent versus adherent HeLa cells to RIP intoxication. HeLa cells were expose to ricin or abrin in two modes of intoxication: precell adhesion and post-cell adhesion. The effect of the state of the cells on ricin and abrin intoxication was evaluated by two approaches: DHM and Annexin V/PI assays. (A) DHM analysis of the relative changes in morphological parameters in preadherent intoxicated HeLa cells (mean ± SE). (B) Table summarizes the time range for the detection of significant changes in the morphological features as presented in A. p < 0.05 was calculated according to 2-tailed Student’s t-test. (C) Annexin V and PI apoptosis assays at 8 h postintoxication (mean ± SE). p value of preadherent vs. adherents cells for ricin or abrin intoxication were calculated according to 2-tailed Student’s t-test.

Figure 6

A dose–response curve of ricin intoxication during cell adhesion. Preadherent HeLa cells were treated with 0.1–100 ng/mL ricin and (A) digital holograms of 4 different areas in each well were recorded every 10 min. A representative quantification of the relative changes in morphological parameters detected by DHM presented as mean ± SE. (B) Table summarizes the time ranges for the detection of significant changes (p < 0.05 according to 2-tailed Student’s t-test) in the morphological features followed of intoxicated HeLa cells compared to untreated cells. Values are taken from several independent experiments. N.S.-non significant.