Desorption Electrospray Ionization Mass Spectrometry Imaging Allows Spatial Localization of Changes in Acetaminophen Metabolism in the Liver after Intervention with 4-Methylpyrazole

Abstract

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the US, and hepatotoxicity is initiated by a reactive metabolite which induces characteristic centrilobular necrosis. The only clinically available antidote is N-acetylcysteine, which has limited efficacy, and we have identified 4-methylpyrazole (4MP, Fomepizole) as a strong alternate therapeutic option, protecting against generation and downstream effects of the cytotoxic reactive metabolite in the clinically relevant C57BL/6J mouse model and in humans. However, despite the regionally restricted necrosis after APAP, our earlier studies on APAP metabolites in biofluids or whole tissue homogenate lack the spatial information needed to understand region-specific consequences of reactive metabolite formation after APAP overdose. Thus, to gain insight into the regional variation in APAP metabolism and study the influence of 4MP, we established a desorption electrospray ionization mass spectrometry imaging (DESI-MSI) platform for generation of ion images for APAP and its metabolites under ambient air, without chemical labeling or a prior coating of tissue which reduces chemical interference and perturbation of small molecule tissue localization. The spatial intensity and distribution of both oxidative and nonoxidative APAP metabolites were determined from mouse liver sections after a range of APAP overdoses. Importantly, exclusive differential signal intensities in metabolite abundance were noted in the tissue microenvironment, and 4MP treatment substantially influenced this topographical distribution.

Keywords: 4-methylpyrazole; acetaminophen; desorption electro-spray ionization mass spectrometry imaging; fomepizole; liver; metabolites; mouse.

Fig. 1.. Early hepatic response to the range of APAP overdose:

Fasted male C57BL/6J mice received various doses of APAP ranging from 300–1200 mg/kg or 10 ml/kg saline (control) and were sacrificed 30 minutes later. Plasma alanine transaminase (ALT) (A), aspartate aminotransferase (AST) (B) and tissue glutathione (GSH) (C) were then measured. Data represent means ± SEM of n = 6 animals per group. *P < 0.05 (compared with control).

Fig. 2.. Dose-dependent change in APAP and its metabolites in whole liver tissue 30 minutes after overdose:

Fasted male C57BL/6J mice received various doses of APAP from 300–1200 mg/kg or 10 ml/kg saline (control) and were sacrificed 30 minutes later. Hepatic concentrations of the parent compound APAP (A), the non-oxidative metabolites APAP-Glucuronide (B), and APAP-Sulfate (C), as well as the oxidative metabolites APAP-Glutathione (D), APAP-Cysteine (E), APAP-N-Acetylcysteine (F) were measured by LC/MS/MS. Data represent means ± SEM of n = 4 animals per group. *P < 0.05 (compared to 300 mg/kg).

Fig. 3.. DESI-MS analysis of APAP and its non-oxidative and oxidative metabolites at 50 μm pixel resolution:

Fasted male C57BL/6J mice which received 600 mg/kg APAP were sacrificed 30 minutes later and liver sections used for DESI-MSI. DESI ion images of (A) APAP (m/z 152.0712) [M+H⁺], (B) APAP-GLUC (m/z 366.0591) [M+K⁺], (C) APAP-SULF (m/z 232.028) [M+H⁺], (D) APAP-GSH (m/z 457.1393) [M+H⁺], (E) APAP-CYS (m/z 2933.0362) [M+K⁺], (F) APAP-NAC (m/z 313.0858) [M+H⁺] at a resolution of 50 μm pixel size.

Fig. 4.. DESI-MSI visualization of the effect of 4MP on distribution of APAP and APAP-NAC following a moderate (300mg/kg) or severe (600mg/kg) APAP overdose at 50 μm pixel resolution:

Fasted male C57BL/6J mice received either 300 (A & B) or 600 (C & D) mg/kg APAP alone or along with 50 mg/kg 4MP (B & D) and were sacrificed 30 minutes later with liver sections used for DESI-MSI. Ion images of APAP (m/z 152.0712) [M+H⁺] + APAP (m/z 174.0531) [M+Na⁺], and APAP-NAC (m/z 313.0858) [M+H⁺] at 50 μm pixel size.

Fig. 5.. Spatial localization of hepatic APAP and APAP-NAC by DESI-MSI analysis at 50 μm pixel resolution after moderate (300 mg/kg) APAP overdose:

Fasted male C57BL/6J mice received 300 mg/kg APAP followed by sacrifice 30 minutes later. A representative photomicrograph of the liver section immunostained for Cyp2F2 after DESI-MSI is shown on the left. DESI ion images for APAP (A) and APAP-NAC (B) were then superimposed onto the corresponding Cyp2F2 immunostained image to generate merged images which are enlarged in panels on the left and rotated for easier visualization below (C).

Fig. 6.. DESI-MSI analysis for spatial localization of APAP and APAP-NAC in liver sections at 50 μm pixel resolution after severe (600 mg/kg) APAP overdose:

Fasted male C57BL/6J mice received 600 mg/kg APAP followed by sacrifice 30 minutes later. A representative photomicrograph of the cryo-section used for DESI-MSI and then immunostained for CYP2F2 is shown on the left. DESI ion images for APAP (A) and APAP-NAC (B) were then superimposed on the same liver cryo-section immunostained for CYP2F2 to generate merged images enlarged in panels on the right and expanded with rotation below (C).

Fig. 7.. Effect of 4MP on biodistribution of APAP and APAP-NAC captured with DESI-MSI analysis of liver tissue sections at 50 μm pixel resolution after severe (600 mg/kg) APAP overdose:

Fasted male C57BL/6J mice received 600 mg/kg APAP followed by sacrifice 30 minutes later. A representative photomicrograph of the cryo-section used for DESI-MSI and then immunostained for CYP2F2 is shown on left. DESI ion images of APAP (A) and APAP-NAC (B) were then overlayed onto the same liver immunostained for CYP2F2 to generate merged images enlarged in panels on the right and expanded with rotation below (C).