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. 2014 Feb 25;111(8):3176-81.
doi: 10.1073/pnas.1322657111. Epub 2014 Feb 3.

TRPM2 channels mediate acetaminophen-induced liver damage

Ehsan Kheradpezhouh  1 , Linlin Ma, Arthur Morphett, Greg J Barritt, Grigori Y Rychkov
Affiliations

TRPM2 channels mediate acetaminophen-induced liver damage

Ehsan Kheradpezhouh et al. Proc Natl Acad Sci U S A. .

Abstract

Acetaminophen (paracetamol) is the most frequently used analgesic and antipyretic drug available over the counter. At the same time, acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. Acetaminophen overdose causes a multitude of interrelated biochemical reactions in hepatocytes including the formation of reactive oxygen species, deregulation of Ca(2+) homeostasis, covalent modification and oxidation of proteins, lipid peroxidation, and DNA fragmentation. Although an increase in intracellular Ca(2+) concentration in hepatocytes is a known consequence of acetaminophen overdose, its importance in acetaminophen-induced liver toxicity is not well understood, primarily due to lack of knowledge about the source of the Ca(2+) rise. Here we report that the channel responsible for Ca(2+) entry in hepatocytes in acetaminophen overdose is the Transient Receptor Potential Melanostatine 2 (TRPM2) cation channel. We show by whole-cell patch clamping that treatment of hepatocytes with acetaminophen results in activation of a cation current similar to that activated by H2O2 or the intracellular application of ADP ribose. siRNA-mediated knockdown of TRPM2 in hepatocytes inhibits activation of the current by either acetaminophen or H2O2. In TRPM2 knockout mice, acetaminophen-induced liver damage, assessed by the blood concentration of liver enzymes and liver histology, is significantly diminished compared with wild-type mice. The presented data strongly suggest that TRPM2 channels are essential in the mechanism of acetaminophen-induced hepatocellular death.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Acetaminophen and H2O2 activate Ca2+ entry and a nonselective cation current in rat hepatocytes. (A) Ca2+ entry in hepatocytes treated with 10 mM acetaminophen for 60 min under the conditions indicated in A (average data from three separate cell preparations). Both clotrimazole (50 μM) and ACA (10 μM) were applied to the bath 5 min before the addition of Ca2+. (B) I-V plots of membrane currents measured in control hepatocytes, and hepatocytes treated with 10 mM acetaminophen for 60 min in control bath solution, after replacement of 140 mM NaCl in the bath solution with 140 mM NMDG Cl and after the addition of 50 μM clotrimazole to the bath (n = 22 for each trace). Error bars are omitted for clarity here and all other I-V plots. (C) Ca2+ entry in hepatocytes treated with 0.5 mM H2O2 (n = 3). (D) I-V plots of membrane currents measured in control hepatocytes, and hepatocytes treated with 0.5 mM H2O2 in control bath solution, after replacement of 140 mM NaCl in the bath solution with 140 mM NMDG Cl and after the addition of 50 μM clotrimazole to the bath (n = 7). (E) Activation of membrane conductance in hepatocytes by 10 mM H2O2 applied to the bath. Each data point represents amplitude of the current at −100 mV. (F) I-V plots of membrane currents measured before application of H2O2 (control), and after full development of the H2O2-activated current in control bath solution (H2O2) and after replacement of 140 mM NaCl with 140 mM NMDG Cl (n = 5).
Fig. 2.
Fig. 2.
TRPM2 current in rat hepatocytes. (A) Activation of TRPM2 current in a rat hepatocyte in response to intracellular perfusion with 1 mM ADPR. The current was recorded in response to 100-ms voltage ramps between −120 and 120 mV and applied every 2 s. Current amplitude at −100 mV is plotted against time. Application of 10 μM ACA to the bath resulted in a 90% block of the current. (B) The dose–response curve for ADPR. TRPM2 current amplitude at −100 mV is plotted against ADPR concentration in the pipette. The data points are fitted with a Hill equation with slope 1 and EC50 of 480 μM. (C and D) I-V plots of fully developed TRPM2 currents activated by ADPR in control bath solution, after replacement of 140 mM NaCl in the bath solution with 140 mM NMDG Cl, and after application of 50 µM clotrimazole or 100 µM chlorpromazine to the control bath solution, respectively (n = 3).
Fig. 3.
Fig. 3.
The role of ADPR in activation of Ca2+ entry in hepatocytes. (A) Poly-ADPR–specific immunofluorescence in rat control hepatocytes (A, i) and hepatocytes treated with 10 mM acetaminophen (45 min in A, ii; 16 h in A, iv) or 1 mM H2O2 (45 min in A, iii). (B and C) Inhibition of H2O2 and acetaminophen-induced Ca2+ entry in hepatocytes by PARP inhibitor DPQ (10 μM). DPQ was added to the incubation medium 2 min before the addition of H2O2 or acetaminophen (n = 3).
Fig. 4.
Fig. 4.
The effect of TRPM2 knockdown on hepatocyte membrane currents and Ca2+ entry. (A) The effect of TRPM2 knockdown on the amplitude of membrane currents activated by intracellular ADPR, H2O2, and acetaminophen. The absolute amplitude of the current measured at −100 mV is shown (mean ± SEM). (B) Western blot of TRPM2 protein in hepatocytes treated with control siRNA (lane 2) and anti-TRPM2 siRNA (lane 3). (C) The effect of TRPM2 knockdown on Ca2+ entry in rat hepatocytes preincubated with 0.5 mM H2O2 for 30 min. (D) The effect of TRPM2 knockdown on Ca2+ entry in rat hepatocytes preincubated with 10 mM acetaminophen for 60 min.
Fig. 5.
Fig. 5.
H2O2- and acetaminophen-activated Ca2+ entry is attenuated in TRPM2 KO mouse hepatocytes. (A and B) Ca2+ entry in TRPM2 WT (A) and KO (B) mouse hepatocytes activated by the addition of H2O2 to the bath solution (n = 3). (C) Ca2+ entry in TRPM2 WT and KO mice hepatocytes treated with 10 mM acetaminophen for 60 min (n = 3). Both clotrimazole (50 μM) and ACA (10 μM) were applied to the bath 5 min before the addition of Ca2+.
Fig. 6.
Fig. 6.
TRPM2 KO mice are substantially protected against acetaminophen-induced damage. (A) Blood concentrations of the liver enzymes ALT and AST in TRPM2 KO, TRPM2 Het, and WT mice pretreated with acetaminophen or vehicle for 24 h. The results are the means ± SEM of the number of mice indicated. The degree of significance, determined using the one way ANOVA test was P < 0.007 for comparison of TRPM2 KO with each of TRPM2 WT and TRPM2 Het. (B) Representative bright field images of H&E-stained liver sections at 20× magnification. The light colored areas in the WT acetaminophen and KO acetaminophen images represent areas of necrosis.
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