Abstract
The occurrence of idiosyncratic drug-induced liver injury (IDILI) is a leading cause of post-marketing safety warnings and withdrawals of drugs. Carbamazepine (CBZ), widely used as an antiepileptic agent, could cause rare but severe idiosyncratic liver injury in humans. Although recent studies have shown that inflammasome is implicated in CBZ-induced hepatocellular injury in vitro, the precise pathogenesis of hepatotoxicity remains largely unexplored. Here we report that CBZ causes idiosyncratic liver injury through promoting specific stimuli-induced NLRP3 inflammasome activation. CBZ (40 μM) enhances NLRP3 inflammasome activation triggered by adenosine triphosphate (ATP) or nigericin, rather than SiO2, monosodium urate crystal or intracellular lipopolysaccharide (LPS). In addition, CBZ has no effect on NLRC4 or AIM2 inflammasome activation. Mechanistically, synergistic induction of mitochondrial reactive oxygen species (mtROS) is a crucial event in the enhancement effect of CBZ on ATP- or nigericin-induced NLRP3 inflammasome activation. Moreover, the “C=C” on the seven-membered ring and “C=O” on the nitrogen of CBZ may be contribute to NLRP3 inflammasome hyperactivation and hepatotoxicity. Notably, in vivo data indicate that CBZ (50 mg/kg) causes liver injury in an LPS (2 mg/kg)-mediated susceptibility mouse model of IDILI, accompanied by an increase in caspase-1 activity and IL-1β production, whereas the combination of CBZ and LPS does not exhibit the effect in NLRP3-knockout mice. In conclusion, CBZ specifically promotes ATP- or nigericin-induced NLRP3 inflammasome activation and causes idiosyncratic liver injury. Our findings also suggest that CBZ may be avoided in patients with NLRP3 inflammasome activation-related diseases that are triggered by ATP or nigericin, which may be risk factors for IDILI.
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Abbreviations
- IDILI:
-
Idiosyncratic drug-induced liver injury
- ATP:
-
Adenosine triphosphate
- CBZ:
-
Carbamazepine
- MSU:
-
Monosodium urate crystal
- LPS:
-
Lipopolysaccharide
- mtROS:
-
Mitochondrial reactive oxygen species
- PAMPs:
-
Pathogen-associated molecular patterns
- DAMPs:
-
Damage-associated molecular patterns
- PMA:
-
Phorbol-12-myristate-13-acetate
- BMDMS :
-
Bone-marrow-derived macrophages
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- ALT:
-
Alanine aminotransferase
- AST:
-
Aspartate transaminase
- LDH:
-
Lactate dehydrogenase
- HBSS:
-
Hank’s balanced salt solution
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Acknowledgements
This work has been supported by grants from National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2017ZX09301022, 2018ZX09101002-001-002), National Natural Science Foundation of China (81874368, 81630100, 81903891), Beijing Nova Program (Z181100006218001), Innovation Groups of the National Natural Science Foundation of China (81721002).
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Supplementary Fig. 1
CBZ promotes the activation of NLRP3 inflammasome triggered by ATP, an effect that is not present in NLRP3-/- BMDMs. (a) BMDMs or LPS-primed BMDMs were treated with ATP, CBZ, or ATP plus CBZ. The secretion of TNF-α was detected in SN. (b) LPS-primed BMDMs were treated with various doses of CBZ and then stimulated with ATP. The secretion of TNF-α was detected in SN. (c-g) LPS-primed wild type (WT) BMDMs or NLRP3 knock out (NLRP3-/-) BMDMs were treated with ATP in the presence or absence of CBZ. Western blot analysis of IL-1β (p17), caspase-1 (p20) in SN and pro-IL-1β, caspase-1 (p45), NLRP3, ASC in WCL (c). Caspase-1 activity (d), secretion of IL-1β (e), LDH (f), TNF-α (g) in SN from WT BMDMs and NLRP3-/- BMDMs described in (c). Data are expressed as mean ± SEM (n=3) from three independent experiments with biological duplicates in (a, b, d-g). Statistics differences were analyzed using an unpaired Student’s t-test: **P < 0.01, ***P < 0.001 vs. the WT ATP group. Supplementary Fig. 2 CBZ has no influence on the cell viability in BMDMs and cultured supernatant ALT and AST in L02 cells. (a) BMDMs were incubated at 37°C followed by treatment with CBZ for 24 h, then these cells were cultured with CCK-8 for 30 min. The optical density values at the wavelength of 450 nm were determined. (b, c) L02 cells were seeded in 96-well growth-medium plate overnight at 1×105 cells/well. Next, the cells were incubated by CBZ treatment for 24 h, then the cultured supernatant ALT (b) and AST (c) were determined. APAP, acetaminophen. Statistics differences were analyzed using one-way ANOVE: ***P < 0.001 vs. the control group. Supplementary Fig. 3 CBZ promotes NLRP3 inflammasome activation stimulated by nigericin in BMDMs and THP-1 cells. (a, b) LPS-primed BMDMs were treated with various doses of CBZ and then stimulated with nigericin. The secretion of LDH (a), TNF-α (b) were detected in SN. (c) PMA-primed THP-1 cells were stimulated with nigericin after CBZ treatment. Western blot analysis of IL-1β (p17), caspase-1 (p20) in SN and pro-IL-1β, caspase-1 (p45), NLRP3, ASC in WCL. (d-g) Caspase-1 activity (d), secretion of IL-1β (e), LDH (f), TNF-α (g) in SN from THP-1 cells described in (c). Data are expressed as mean ± SEM (n=3) from three independent experiments with biological duplicates in (a, b, d-g). Statistics differences were analyzed using one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001 vs. the LPS or PMA plus nigericin group. Supplementary Fig. 4 CBZ has no effect on the activation of NLRP3 inflammasome induced by MSU, SiO2 and intracellular LPS, as well as AIM2 and NLRC4 inflammasome. (a) LPS-primed BMDMs were treated with various doses of CBZ and then stimulated with MSU or ATP. The secretion of TNF-α was detected in SN. (b) LPS-primed BMDMs were treated with CBZ and then stimulated with SiO2 or ATP. The secretion of TNF-α was detected in SN. (c) Pam3CSK4-primed BMDMs were treated with various doses of CBZ and then stimulated with LPS, or LPS-primed BMDMs were treated with CBZ and then stimulated with ATP. The secretion of TNF-α was detected in SN. (d) LPS-primed BMDMs were treated with CBZ and then stimulated with ATP, poly(dA:dT) or Lfn-Flic. Western blot analysis of IL-1β (p17), caspase-1 (p20) in SN and pro-IL-1β, caspase-1 (p45), NLRP3, ASC in WCL. (e-g) Caspase-1 activity (e), secretion of IL-1β (f), TNF-α (g) in SN described in (d). Data are expressed as mean ± SEM (n=3) from three independent experiments with biological duplicates in (a-c, f-h). Statistics differences were analyzed using an unpaired Student’s t-test: **P < 0.01, ***P < 0.001 vs. the ATP group. Supplementary Fig. 5 CBZ has no effect on intracellular potassium. (a, b) Qualification of potassium efflux in LPS-primed BMDMs treated with CBZ and then stimulated with different stimuli. Supplementary Fig. 6 CBZ facilitates ATP/nigericin-induced mitochondrial reactive oxygen species (mtROS) production. LPS-primed BMDMs were treated with CBZ before stimulated by ATP, nigericin or SiO2. For mtROS measurement, BMDMs were loaded with MitoSOX red mitochondrial superoxide indicator (Ex/Em: 510/580 nm). After staining and washing, flow cytometry was conducted to test mtROS. Supplementary Fig. 7 The effect of H2O2 on NLRP3 inflammasome activation triggered by ATP, nigericin, and SiO2. (a-c) LPS-primed BMDMs were treated with H2O2 and then stimulated with ATP (a), nigericin (b) or SiO2(c). Western blot analysis of IL-1β (p17), caspase-1 (p20) in SN. Data are expressed as mean ± SEM (n=3) from three independent experiments with biological duplicates. Statistics differences were analyzed using one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001 vs. the ATP or nigericin group (PDF 1217 kb)
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Wang, Z., Xu, G., Zhan, X. et al. Carbamazepine promotes specific stimuli-induced NLRP3 inflammasome activation and causes idiosyncratic liver injury in mice. Arch Toxicol 93, 3585–3599 (2019). https://doi.org/10.1007/s00204-019-02606-3
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DOI: https://doi.org/10.1007/s00204-019-02606-3