Ratiometric Fluorescent Sensors Illuminate Cellular Magnesium Imbalance in a Model of Acetaminophen-Induced Liver Injury

Abstract

Magnesium(II) plays catalytic, structural, regulatory, and signaling roles in living organisms. Abnormal levels of this metal have been associated with numerous pathologies, including cardiovascular disease, diabetes, metabolic syndrome, immunodeficiency, cancer, and, most recently, liver pathologies affecting humans. The role of Mg²⁺ in the pathophysiology of liver disease, however, has been occluded by concomitant changes in concentration of interfering divalent cations, such as Ca²⁺, which complicates the interpretation of experiments conducted with existing molecular Mg²⁺ indicators. Herein, we introduce a new quinoline-based fluorescent sensor, MagZet1, that displays a shift in its excitation and emission wavelengths, affording ratiometric detection of cellular Mg²⁺ by both fluorescence microscopy and flow cytometry. The new sensor binds the target metal with a submillimolar dissociation constant─well suited for detection of changes in free Mg²⁺ in cells─and displays a 10-fold selectivity against Ca²⁺. Furthermore, the fluorescence ratio is insensitive to changes in pH in the physiological range, providing an overall superior performance over existing indicators. We provide insights into the metal selectivity profile of the new sensor based on computational modeling, and we apply it to shed light on a decrease in cytosolic free Mg²⁺ and altered expression of metal transporters in cellular models of drug-induced liver injury caused by acetaminophen overdose.

Scheme 1. Synthesis of the Sensor MagZet1

Figure 1

Response of MagZet1 to Mg²⁺. Absorption (A) and fluorescence emission (B) spectra of a 10 μM solution of MagZet1 in aqueous buffer at pH 7.0, treated with increasing concentrations of MgCl₂. (C) Binding isotherm at 25 °C. Mg²⁺ dissociation constant was determined from nonlinear fit (red curve) of the ratio of fluorescence at 500 and 530 nm (F₅₃₀/F₅₀₀) upon excitation at 390 nm.

Figure 2

Metal selectivity profile of MagZet1. Fluorescence ratio of 1 μM MagZet1 in aqueous buffer at pH 7.0, 25 °C (blue bars); in the presence of biologically relevant divalent cations (1 mM Mg²⁺, 50 μM Ca²⁺, or 1 μM for other metals, red bars); in the presence of Mg²⁺ (1 mM) and competing cations (yellow bars); or in the presence of saturating Mg²⁺ (50 mM) and competing cations (green bars). Error bars correspond to the standard deviation of triplicate experiments (λ_exc = 390 nm).

Figure 3

Calculated molecular structures for MagZet1 complexes. (A) Ground state geometry-optimized structures (DFT/M062X) of MagZet1 complexes with Mg²⁺ and Ca²⁺. Hydrogen atoms are omitted for clarity. (B) Overlay of structures, showing the structural distortion of the carboxylate in the 8-position for the Ca²⁺ complex (cyan). Aqua ligands and hydrogen atoms are omitted for clarity. (C) Selected bond lengths and angles on the calculated structures and histograms of Ca–O distances and Ca–O–C–O dihedral angles for all crystallographically characterized monodentate Ca²⁺–acetate complexes in the CSD. Values corresponding to the calculated MagZet1·Ca²⁺ complex are shown in red.

Figure 4

Effect of pH on the fluorescence MagZet1. (A) Fluorescence ratio of a solution of 10 μM MagZet1 as a function of pH in the presence (red circles) and absence (blue squares) of 1 mM MgCl₂ at 25 °C, λ_exc = 390 nm. Values represent the average of three independent measurements with error bars representing the standard deviation. (B) Absorption profile of MagZet1 at 10 μM. (C) Fluorescence emission profile of the same solution.

Figure 5

(A) Fluorescence response of a solution of 20 μM MagZet1 to increasing concentrations of MgATP in aqueous buffer at pH 7, 25 °C. λ_exc = 390 nm. (B) Binding isotherm at 25 °C. The data could be fitted using a model that includes both binary and ternary complexes, the latter with a weak binding constant.

Figure 6

MagZet1 can be used to detect Mg²⁺ in live cells. (A) Fluorescence images of HeLa cells stained with 5 μM MagZet1-AM before and after treatment with ionophore 4-Br-A-23187 and EDTA. Box plot shows the change in the fluorescence ratio from a decrease in intracellular Mg²⁺ concentration. t test, n = 10. (B) Flow cytometry histograms of fluorescence ratio of HeLa cells stained with MagZet1-AM in the presence of 50 mM Mg²⁺ and 10 μM ionophore 4-Br-A-23187 (pink) or coloaded with 1 mM EDTA-AM (blue) vs untreated controls (yellow). Dashed lines mark the median fluorescence ratio. p-values were calculated from χ² values corresponding to comparisons of each population to the vehicle-treated control.

Figure 7

Changes in Mg²⁺ levels and transporters in liver cells treated with acetaminophen, APAP. (A) Flow cytometry histograms of the MagZet1 fluorescence ratio in THLE-2 cells treated with DMSO vehicle (yellow) or 10 mM APAP for 1 (blue), 3 (red), or 6 h (green). Dashed lines represent the median fluorescence ratio. p-values were calculated from χ² values corresponding to comparison of the corresponding population to the vehicle-treated control. (B) Changes in mRNA levels of genes encoding Mg²⁺ transporters TRPM7, TRPM6, MRS2, MMgT1, MagT1, and CNNM1–4 in THLE-2 cells under exposure to 10 mM APAP for 1, 3, or 6 h vs DMSO vehicle. (C) Protein levels of CNNM4 (Western blot) in THLE-2 cells under exposure to 10 mM APAP for 1, 3, or 6 h vs DMSO vehicle control group.