Dynamic cytotoxic profiles of sulfur mustard in human dermal cells determined by multiparametric high-content analysis

Abstract

Sulfur mustard (SM) is a well known chemical warfare agent that poses a major threat to military personnel and also populace. It targets multiple macromolecules, and its toxic effects are mediated by complex mechanisms. However, the sequence and manner of SM-induced cellular and molecular events underpinning the pathological processes are not fully elucidated. Effective therapeutic agents against SM poisoning are also lacking. The present study aimed to determine the dynamic cytotoxic profiles of SM in primary cultured human epidermal keratinocytes-fetal (HEK-f) and human dermal fibroblasts-adult (HDF-a) by establishing a high content analysis (HCA)-based multiparametric toxicity assay panel. SM was found to produce multiple, concentration-dependent cellular responses, including abnormal cellular morphology, cycle arrest, apoptosis, necrosis, mitochondrial membrane potential imbalance, increased membrane permeability, oxidative stress, DNA damage, and lysosome impairment. Time-course analysis indicated that the cellular and molecular responses related to the highly reactive targets of SM, such as glutathione depletion, reactive oxygen species release, DNA and lysosomal damage, and actin microfilament architecture modification, were congenerous initial events for SM injury. Moreover, this study demonstrated a novel finding that SM induced autophagy, and it was closely related to lysosome alterations in both cell types. Higher susceptibility of HEK-f cells to SM was associated with early lysosomal damage and decreased autophagy activity. Multiparametric HCA also revealed the concentration-dependent cytoprotective effect of hydroxychloroquine in HDF-a cells. The above results provided overall and objective evidence for elucidating the cytotoxic mechanism of SM, and also a good scientific base for further research on countermeasures against SM injury.

High‐content analysis boosts identification of the initial cause of triptolide‐induced hepatotoxicity

“…Data analyses for all HCA were conducted as reported previously (Long et al, ). All data from the in vitro experiments were expressed as the means ± SEs of three independent experiments.…”

Procaspase-3-activating compound 1 stabilizes hypoxia-inducible factor 1α and induces DNA damage by sequestering ferrous iron

“…Fluorescence images were all acquired with an IN CELL Analyzer 1000/2000 (GE Healthcare) under a ×10 objective lens except that in supplement Fig. 6 , as reported previously 10 , 11 . At least five fields and more than 500 cells/well were analyzed using the Multi Target Analysis Module of the IN CELL Analyzer Workstation 3.5.…”

WX-132-18B, a novel microtubule inhibitor, exhibits promising anti-tumor effects

“…HCA multiparametric analysis was conducted as previously described [ 41 ]. For cytoskeleton multiparametric analysis, A549 cells seeded at a density of 8 × 10 3 cells/well into a 96-well black plate (Costar 3603, USA) were cultured overnight and then treated with test compounds for 24 h. The cells were then fixed and cellular nuclei, actin, and microtubules were stained with Hoechst 33342, Alexa Flour 488 phalloidin, and α-tubulin antibody; the cells were then incubated with Alexa Flour 546 labeled secondary antibody.…”

“…The output parameters and their implications are shown in Table 2 . The other detection and assay conditions are the same as described in our previous paper [ 41 ]. In all experiments, cell culture medium containing 0.2% DMSO was designated as the blank control, and was included in each plate to allow data normalization and plate quality control.…”

“…Data analysis for all HCA were conducted as reported previously [ 41 ]. All data from the in vitro experiments were expressed as means ± SD of three independent experiments.…”