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. Author manuscript; available in PMC: 2024 Apr 3.
Published in final edited form as: Clin Toxicol (Phila). 2023 Apr 3;61(4):212–222. doi: 10.1080/15563650.2023.2185125

Figure 2. Mechanisms of azide toxicity; reversal by cobinamide.

Figure 2.

(A, B) COS-7 cells (A) or A549 cells (B) were incubated in glucose-free medium containing 25 mM galactose for 24 hours. The next day, cells were exposed for 24 hours to 0.1 mM NaN3 with or without 25 μM aquohydroxocobinamide. The cells were extracted in situ and ATP was measured in the extracts. Data represent the mean ± SEM of six (A) and five independent experiments (B), respectively. (C) A549 cells were incubated for 24 hours with 0.1 mM and 1 mM NaN3, in the absence or presence of 25 μM aquohydroxocobinamide and the intracellular malondialdehyde (MDA) content was measured 24 hours later. Data represent the mean ± SEM of five independent experiments and were analyzed by one-way ANOVA. (D, E) A549 cells were incubated for 24 hours with 0.1 mM and 1 mM NaN3, in the absence or presence of 25 μM aquohydroxocobinamide and induction of apoptosis based on caspase-3 cleavage was measured by immunofluorescence. (D) Fluorescence microscopy images of cleaved caspase-3 positive cells (green); Hoeschst stain (blue) indicates the DNA of cells. Images were taken at 20x magnification and each scale bar represents 100 μm. (E) Quantification of the data in (D); the data are the mean ± SEM of three to four independent experiments. All experiments were analyzed by one-way ANOVA. *P≤0.05, **P<0.01, and ****P≤0.0001. Cbi = cobinamide; NaN3 = sodium azide; ns = non-significant.