Individuals experiencing acute liver failure (ALF) or acute liver injury (ALI) exhibit massive hepatocyte necrosis. If untreated, it can lead to multi-organ damage and death. In many countries, the leading cause of ALI/ALF is acetaminophen (APAP) overdose [
1]. APAP and other hepatotoxic agents, such as thioacetamide (TAA), are primarily metabolized by cytochrome P450-2E1 (CYP2E1), which is known to produce oxidative stress and post-translational protein modifications (PTMs), contributing to immune activation and mitochondrial and cellular dysfunction [
2]. These sequential processes culminate in cell death and injury in multiple tissues. However, critical unanswered questions remain about the causal factors driving ALF. These include the potential contribution of oxidative PTMs in gut damage/leakiness and ALI/ALF, in which the molecular mechanisms underlying intestinal barrier dysfunction in promoting ALF remain elusive. A recent study found evidence of extensive analysis on single-cell transcriptomic and gut microbiome alterations following acute APAP or TAA exposure in ALFmodel mice [
3]. However, since these analyses were performed on tissues collected 20 hours post-insult, these changes likely reflect emergent consequences and compensatory responses to drug insult rather than causal drivers of ALF. Thus, we aimed to study the roles of oxidative PTMs and gut damage/leakiness in promoting ALF partly through the gut-liver axis by focusing on the changes in the early time points (e.g., 1, 2, 4, & 7 hours post-drug challenge).
We hypothesized that oxidative PTMs and gut damage/leakiness, occurring rapidly after drug-exposure, initiate a cascade of molecular changes, ultimately resulting in ALF usually observed later (e.g., 16–24 hours). To test this hypothesis, we treated young mice on 129X1/SvJ background (n≥4–6/group) with a single dose of APAP (350 mg/kg, i.p.) or TAA (300 mg/kg, i.p.), as similar as reported [
3]. Control mice were administered vehicle (same volume of normal saline, i.p.). To demonstrate the time-dependent changes in gut and hepatic injury, the blood, intestinal enterocytes, and liver tissues were collected at 1, 2, 7, and 24 h after APAP or 2, 4, 7, and 24 h after TAA exposure, as shown in
Supplementary Figure 1A, and stored at –80°C until further experiments. Gut and hepatic tissues collected at different time points were evaluated by hematoxylin & eosin (H&E)-stained histology, TUNEL-stained cell death assays, and biochemical characterizations, including immunoblot and proteomics analysis.
In a parallel study, global
Cyp2e1-KO mice on 129X1/SvJ background were also exposed to the same APAP dose to perform proteomics analysis to identify differentially expressed proteins in gut enterocytes. Serum collected from each mouse from different groups was used to determine the time-dependent changes in the levels of the blood markers of gut leakiness and liver injury enzymes, as detailed in the
Supplementary materials. All other methods, including densitometric comparison of each immunoblot, statistical analysis, and usage of intestinal organoids cocultured with hepatocytes to evaluate the effects of APAP or endotoxin lipopolysaccharide (LPS) on the rates of permeability change and cell deaths, are also described in the
Supplementary materials.
We observed that APAP or TAA caused gut damage/leakiness, as early as 1 hour after exposure, followed by signs of early hepatotoxicity starting from 2 hours and clear damage at later hours (
Fig. 1A and
Supplementary Fig. 1B). We found intestinal structural deformation, TUNEL-stained gut damage (
Fig. 1B), and significantly elevated serum endotoxin (LPS) and FITC-dextran-4kDa (FITC-D4), markers of intestinal permeability [
4,
5], from 1–2 hours (
Fig. 1C and
Supplementary Fig. 1C). Acute liver injury was evidenced at 7–24 hours at multiple levels of analyses, including necrotic histology by H&E, TUNEL-stained hepatocyte damage (
Fig. 1A, 1B and
Supplementary Fig. 1B), elevated serum transaminases (ALT and AST) (
Fig. 1C and
Supplementary Fig. 1C), and increased oxidative PTMs in the liver from APAP- and TAA-exposed mice (
Fig. 1D and
Supplementary Fig. 1D, 1J). Consistently, the levels of liver injury markers and cell death-related proteins were also elevated in APAP- and TAA-exposed mice (
Fig. 1D and
Supplementary Fig. 1D, 1J). Taken together, these results suggest that APAP or TAA exposure rapidly induces gut damage/leakiness and endotoxemia prior to the progression of ALI, observed at later hours (
Fig. 1A–1D and
Supplementary Fig. 1B–1D).
Prior studies, including from our laboratory, have shown that oxidative PTMs, especially nitration, acetylation (Ac-Lys), and JNK-mediated phosphorylation, of cellular proteins, lead to mitochondrial dysfunction and cell death [
4-
7]. To further examine the mechanisms underlying drug-induced gut damage/leakiness, we analyzed enterocyte extracts from APAP- or TAA-exposed mice compared with those of the vehicle controls. Our results showed that acute APAP and TAA exposure increased the levels of oxidative PTMs at early time points (1–2 hours) and decreased the levels of gut tight junction and adherens junction proteins (TJ/AJs) (
Fig. 1D and
Supplementary Fig. 1D, 1J). The reduced amounts of intestinal TJ/AJ proteins were substantiated by mass-spectral proteomics analysis of gut enterocytes (
Supplementary Fig. 1F and
Table 1). Furthermore, the mass-spectrum results also showed the decreased amounts of various mitochondrial and antioxidant proteins with increased levels of proteins associated with cell death, inflammation, and ubiquitin-related proteasome components in APAP-treated wild-type mice compared with mice deficient in CYP2E1, a major source of APAP-mediated oxidative PTMs (
Supplementary Fig. 1G–1I). These findings support a molecular mechanism by which increased oxidative PTMs of TJ/AJs lead to their degradation, activation of the cell death pathways, resulting in enterocyte damage and gut leakiness, all of which occur earlier than the significant liver cell damage in ALF mice. The resultant changes could then lead to translocation of endotoxin and other damage-associated molecules into the circulation, promoting ALI and eventually causing ALF.
To further investigate the direct, causal interaction between gut and liver in APAP-induced ALF, we prepared mouse intestinal organoids and cultured them in Transwell® system (
Fig. 1E), although this organoid culture system may not exactly resemble the architecture and physiological function of the intestines. On the apical side, intestinal organoid monolayers were treated with a single dose of APAP and/or LPS (as a positive control, representing a major gut-derived endotoxin, which can damage a variety of cells and/or tissues via increased oxidative PTMs, including nitration [
8], proinflammatory cytokines [
9], and cell death signaling pathways) [
10], while on the basolateral side, murine AML12 hepatocytes were plated without any treatment. As compared to the control baseline, we found that APAP or LPS induced intestinal cell permeability beginning 1 hour after treatment and persistently thereafter, with additive effects of co-treatment. At 2 hours post-drug, we detected increases in hepatocyte death, which were accentuated from 3 hours and later time points (
Fig. 1F and
Supplementary Fig. 1E). These results using the
in vitro co-culture system also demonstrate that intestinal cell permeability preceded and potentially caused subsequent hepatotoxicity. This provides further supporting evidence for the contributing role of intestinal damage/leakiness in promoting ALI in a time-dependent manner. Based on the changes noticed, especially during the early time points, recently described transcriptomic and microbial alterations occurring 20 hours post-drug [
3] are likely sequelae and/or compensatory responses, rather than causal drivers, of ALF/ALI. Furthermore, our results (observed in both APAP- and TAA-exposed mice) emphasize the critical importance of studying the early changes to identify causal drivers and understand the crosstalk mechanisms of multi-organ damage, as previously reported in the cases of APAP-mediated ALI/ ALF [
11,
12].
By carefully studying the biochemical and histological changes in very early time points after APAP or TAA exposure (
Fig. 1A–1F and
Supplementary Fig. 1B–1I), we demonstrate the contributing or causal roles of oxidative PTMs and gut damage/leakiness in drug-induced ALF and elucidate the underlying mechanisms for the early events prior to fulminant liver injury. However, our study has a few limitations. Although there were reports that APAP exposure caused endotoxemia in human ALF patients [
13] and in rodents with acute hepatotoxicity [
11,
12], we used well-established mouse models of ALF [
3,
11,
12,
14] but did not evaluate clinical specimens. Additionally, we did not study the potential roles of tissue-specific drug metabolism and abnormalities in enterohepatic circulation in APAP- or TAA-induced ALI/ALF. Furthermore, other factors, such as gut microbiota change [
12] and other intestinal cell types, including goblet and Paneth cells, might play a role in drug-induced gut leakiness, leading to ALF/ALI. These limitations should be considered in future studies.
Despite numerous clinical and mechanistic studies on drug-induced ALF/ALI in patients and experimental models, as described [
13-
15], very few studies were conducted to demonstrate the importance of gut damage/permeability as an early event in promoting ALI. In fact, our recent PubMed search results with the keywords of ‘drug-induced acute liver injury’, ‘acetaminophen or thioacetamide’, and ‘gut leakiness’ revealed only one report and none in APAP- and TAA-induced ALI/ALF, respectively. One report revealed the importance of chemokine (C-C motif) ligand 7-related inflammation, leading to gut leakiness (observed at 3 hours) and ALI observed 24 hours after APAP exposure [
11]. Here, our results demonstrate that oxidative stress-mediated changes in gut damage and intestinal hyper-permeability not only reflect another underlying mechanism but also underscore the important role of gut barrier dysfunction (1–2 hours in our study), consistent with the previous report [
11], in drug-induced ALF. Consequently, maintaining proper gut health should also be considered in the efficient management or treatment of ALF patients. Thus, it would be of interest whether similar mechanisms of oxidative stress-mediated gut damage/leakiness exist in ALF/ALI caused by many other clinically used drugs, such as various antibiotics and anticancer agents.
Our preclinical
in vivo and
in vivo results provide novel evidence that APAP and TAA initially trigger oxidative PTMs, leading to gut TJ/AJ proteolysis and enterocyte damage, followed by gut leakiness and endotoxemia. Importantly, these changes occur rapidly after drug insult, suggesting them as critical steps priming later occurrence of acute liver injury, hepatocyte death, and ultimately ALF (
Fig. 1G). Collectively, our findings raise the prospect of targeting the metabolic activation of APAP or TAA and preventing oxidative PTMs-mediated gut leakiness as an early therapeutic intervention to prevent or efficiently manage drug-induced ALF.
FOOTNOTES
-
Authors’ contribution
W.R. investigation, methodology, data analysis, and original draft preparation. Y.Q., A.H., and N.K. critical reviews and editing. B.-J.S. conceptualization, supervision, resources, review and editing. All the authors have approved the manuscript.
-
Acknowledgements
This research was supported by the Intramural Research Fund to Byoung-Joon Song from the National Institute on Alcohol Abuse and Alcoholism at the National Institutes of Health. We are also grateful to Dr. David Lovinger and Mr. Leon Ruiter Lopez for critical reading of the manuscript.
The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.
-
Conflicts of Interest
The authors have no conflicts to disclose.
SUPPLEMENTARY MATERIAL
Supplementary material is available at Clinical and Molecular Hepatology website (
http://www.e-cmh.org).
Supplementary Figure 1.
(A) Experimental designs of APAP or TAA exposure in mice. (B) Representative H&E-stained images of the intestine (top, 20× magnification) and liver (bottom, 10× magnification) of mice treated with TAA for 2 or 24 h versus control. (C) Timedependent changes in the levels of markers of gut leakiness (i.e., serum LPS and FITC-D4) and liver injury (i.e., serum ALT and AST) in TAA-exposed mice. (D) Immunoblot analyses of the respective oxidative PTMs and liver injury markers, and the respective intestinal TJ/AJs compared with GAPDH loading control, as indicated. (E) Intestinal organoid permeability to FITC-D4 and liver cell viability via MTT assay at different time points, as indicated. (F) Procedures for enterocyte proteomics analysis, (G) volcano plot and heatmap, and (H–I) STRING analysis of enterocyte proteomes decreased and increased, respectively, in APAP-exposed WT mice. (J) The density of each of all indicated protein bands relative to GAPDH used as a loading control was determined by iBright 1500FL (Life Technologies) and ImageJ software with significance of P-value at *P<0.05 and **P<0.01.
Figure 1.Acetaminophen or thioacetamide stimulated oxidative PTMs and gut leakiness in promoting ALF progression. (A, B) Representative (A) H&E- and (B) TUNEL-stained images of the intestine (top, 20× magnification) and liver (bottom, 10× magnification) of mice treated with APAP for 1, 2, 7, and 24 h. (C) Time-dependent changes in the levels of markers of gut leakiness (i.e., serum LPS and FITC-D4) and liver injury (i.e., serum ALT and AST). (D) Immunoblot analyses of the respective oxidative PTMs and liver injury markers and the indicated oxidative PTMs and respective intestinal TJ/AJs compared with GAPDH loading control. (E) Experimental designs for control (C) intestinal organoids, treated with APAP (A), LPS (L), or APAP+LPS (A+L), and AML12 murine hepatocytes, without any treatment, in the Transwell® system (top), and images of intestinal organoids cultured at 2 and 6 days (bottom). (F) Intestinal organoid permeability to FITC-D4 and liver cell viability via MTT assay at different time points, as indicated. Statistical significance was determined by the methods described in the Supplementary materials and the data are shown as a mean±SD, with significance of *P<0.05 and **P<0.01. (G) Summary diagram of APAP- or TAA-induced oxidative PTMs and gut leakiness in promoting ALF.
Abbreviations
post-translational protein modifications
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