Interruption of bile acid uptake by hepatocytes after acetaminophen overdose ameliorates hepatotoxicity.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity.
METHODS: We performed intravital imaging to investigate BA transport in mice, quantified endogenous BA concentrations in the serum of mice and patients with APAP overdose, analyzed liver tissue and bile by mass spectrometry and MALDI-mass spectrometry imaging, assessed the integrity of the blood-bile barrier and the role of oxidative stress by immunostaining of tight junction proteins and intravital imaging of fluorescent markers, identified the intracellular cytotoxic concentrations of BAs, and performed interventions to block BA uptake from blood into hepatocytes.
RESULTS: Prior to the onset of cell death, APAP overdose causes massive oxidative stress in the pericentral lobular zone, which coincided with a breach of the blood-bile barrier. Consequently, BAs leak from the bile canaliculi into the sinusoidal blood, which is then followed by their uptake into hepatocytes via the basolateral membrane, their secretion into canaliculi and repeated cycling. This, what we termed 'futile cycling' of BAs, led to increased intracellular BA concentrations that were high enough to cause hepatocyte death. Importantly, however, the interruption of BA re-uptake by pharmacological NTCP blockage using Myrcludex B and Oatp knockout strongly reduced APAP-induced hepatotoxicity.
CONCLUSIONS: APAP overdose induces a breach of the blood-bile barrier which leads to futile BA cycling that causes hepatocyte death. Prevention of BA cycling may represent a therapeutic option after APAP intoxication. LAY
SUMMARY: Only one drug, N-acetylcysteine, is approved for the treatment of acetaminophen overdose and it is only effective when given within ∼8 hours after ingestion. We identified a mechanism by which acetaminophen overdose causes an increase in bile acid concentrations (to above toxic thresholds) in hepatocytes. Blocking this mechanism prevented acetaminophen-induced hepatotoxicity in mice and evidence from patients suggests that this therapy may be effective for longer periods after ingestion compared to N-acetylcysteine.

Introduction

Acetaminophen (APAP) intoxication is the single most common cause of acute liver failure in many countries and the mechanisms of APAP-induced hepatotoxicity have been intensively studied. APAP is metabolically activated in the liver by cytochrome P450 enzymes, mostly Cyp2e1, which forms protein adducts leading to oxidative stress, activates c-jun N-terminal kinase (JNK) in the cytoplasm, induces its translocation to mitochondria and further enhances oxidative stress, finally resulting in cell death. Despite extensive research on the mechanisms of APAP-induced hepatotoxicity, only one antidote, N-acetylcysteine, is approved for clinical application. N-acetylcysteine helps to restore glutathione (GSH) in hepatocytes, and is maximally effective only when given within 8 hours after APAP ingestion. However, patients often seek medical attention after the initial onset of symptoms, frequently ∼24 hours after APAP ingestion, which is often too late, and death generally follows 3-4 days after intoxication, or even in some cases after up to 14 days. Therefore, there is an urgent need for further therapies, ideally with a longer therapeutic window.

It is well-known that APAP overdose leads to increased bile acid (BA) concentrations in blood. The initial interpretation that elevated BA concentrations simply signify compromised liver functions has since been challenged.[5], [6], [7], [8] One hypothesis is that there is a breach of the blood-bile barrier, since BAs are present in the biliary tract in millimolar concentrations. Alternatively, it has been proposed that BA export from hepatocytes may be compromised. Differentiation of these mechanisms is important, since both would require different therapeutic strategies; however, due to technical challenges neither has yet been explored.

We have established intravital imaging techniques that facilitate the functional analysis of intact livers at subcellular resolution, including BA transport processes. Using these techniques, we demonstrate that APAP causes a breach of the blood-bile barrier at the apical hepatocyte membrane, and that futile BA cycling leads to accumulation of cytotoxic BA concentrations in hepatocytes.

Patients and methods

A detailed description including image analysis, biochemical assays and statistical analysis is available in the supplementary materials and methods and the supplementary CTAT table.

Patients with APAP overdose

Essen cohort: Serum samples of 10 patients with acute liver failure due to APAP intoxication, and no clinical indication for any co-factor, co-morbidity, or underlying chronic liver disease were selected from a consecutive cohort of patients with acute liver failure of various causes. Phoenix patient: Blood was collected time-dependently from a 19-year-old woman who purchased a bottle of APAP/diphenhydramine tablets, promptly ingested 86.5 g of APAP and 4.325 g of diphenhydramine and immediately sought care, arriving 70 minutes post-ingestion. The study was conducted in accordance with the ethical guidelines of the 1975 Helsinki Declaration and was approved by the ethics committee of the Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany. Informed consent was obtained.

Mice and induction of acute liver injury by APAP

Male 8–12-week-old C57BL/6N (Janvier Labs, France) or Oatp-deficient and corresponding wild-type mice (Taconic Biosciences, USA) were used. All experiments were approved by the local animal welfare committee (LANUV, North Rhine-Westphalia, Germany, application number: 84-02.04.2016.A279). To induce acute liver injury by APAP, a single dose of 300 mg/kg b.w. was intraperitoneally (i.p) injected after overnight fasting. For the intervention experiments in Oatp-deficient mice, a dose of 5 mg/kg b.w. Myrcludex B was administered intravenously simultaneously with the APAP injection.

Intravital imaging

Functional intravital imaging of mouse livers was performed using a two-photon microscope (Zeiss, Germany) as previously described . Details are provided in the supplementary materials and methods.

Staining of fixed liver tissue

H&E, immunohistochemistry, and TUNEL stainings were performed in 5 μm-thick paraformaldehyde (4%)-fixed paraffin-embedded liver tissue sections using the Discovery Ultra Automated Slide Preparation System (Roche, Germany), as described in the supplementary materials and methods.

Results

Transient cholestasis precedes hepatocyte death after APAP intoxication

The analysis of blood from patients with APAP overdose on admission to emergency departments revealed a heterogeneous pattern, with some patients presenting with increased BA concentrations and almost normal alanine aminotransferase (ALT) activity, others with both increased ALT and BAs, a third group with increased ALT and normal/close to normal BAs, and some patients with both normal BAs and ALT (Fig. 1A; Table S3; Datasheet S1A). Consequently, no significant correlation between BA and ALT levels was observed (Fig. 1B). Since the exact time point of intoxication was not documented for these patients, a time-resolved analysis of BAs and ALT was not possible. Therefore, we sampled blood at documented periods after overdose from a 19-year-old woman who ingested 86.5 g of APAP, resulting in blood concentrations of 3.7 mM and 2.2 mM at 1.7 h and 4 h post-ingestion, respectively. At the earliest analyzed time point post-ingestion (28.5 hours), the sum of BAs and ALT was only slightly elevated (Fig. 1C; Datasheet S1B). Interestingly, the increase in BA occurred before that of ALT and declined when ALT levels were still high (Fig. 1C).

Fig. 1

Blood BA concentrations and transaminase activities in relation to APAP-induced hepatotoxicity.

(A,B) ALT activity and sum of BA concentrations in plasma of APAP-intoxicated patients in whom the period between overdose and blood sampling was unkown. Patients were grouped by their combination of ALT and BA levels. (C) Time-resolved analysis of the sum of blood BA concentrations and ALT activity of a 19-year-old woman after APAP overdose. Dashed baselines: reference values in healthy individuals. (D) Experimental design. (E) Sum of BA concentrations and ALT in heart blood after APAP overdose in mice (mean and SE of 4 mice per time point). (F,G) Staining of Cyp2e1 plus TUNEL as well as cleaved caspase-3 in liver tissue after APAP overdose. ∗∗p <0.01, Dunnett's multiple comparisons test. ALT, alanine transaminase; Cl. Caspase-3, cleaved caspase-3; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling. (This figure appears in color on the web.)

To study the time-course under controlled conditions, a hepatotoxic dose of APAP was administered to mice (Fig. 1D) and BAs and ALT were analyzed in heart blood (Fig. 1E; Datasheet S1C). A strong transient increase in BAs was observed 2 hours after intoxication (Fig. 1E). ALT activity was only slightly above the control levels 2 h after APAP injection but increased at later timepoints (Fig. 1E; Fig. S1). In agreement, the macroscopically visible speckled pattern that indicates pericentral cell death was observed at 4 h, and more clearly at 8 h, after intoxication (Fig. 1F). Furthermore, only few TUNEL-positive hepatocytes were present 2 hours after intoxication; thereafter, the number of positive cells increased strongly in the Cyp2e1-positive area (Fig. 1F). In contrast, no increase in cleaved caspase-3 was observed (Fig. 1G; Fig. S1C), which is in line with the previously published death mechanism of programmed necrosis. Thus, the time-dependent analyses in humans and mice show that a transient increase in BA precedes cell death. However, in mice, this process occurs much faster with BAs reaching maximal concentrations after ∼2 h, compared to ∼76 h in the patient. The time-dependent analyses also help to elucidate the heterogeneous situation among patients admitted to emergency units without documented time of overdose (Fig. 1A), since group 1 (high BA, normal ALT) may correspond to an early stage (41-53 h after overdose), group 2 (high BA, high ALT) to an intermediate stage (53-88 h), and group 3 (high ALT, moderately increased/normal BA) to a late stage (later than 90 h).

Altered bile canalicular morphology after APAP intoxication

To study if the transient increase in BAs is associated with altered canalicular morphology, we applied a recently established intravital imaging toolbox that visualizes canalicular secretion of fluorescent BA analogues. For this purpose, 2 functional markers were used: CLF, a green-fluorescent BA analogue; and TMRE, a mitochondrial membrane potential marker. TMRE is more abundantly taken up by periportal hepatocytes, thereby outlining the lobular zone (Fig. 2A). Within 3 min after injection into control mice, CLF was rapidly taken up from the blood by the hepatocytes and secreted into the bile canaliculi (BC) (Fig. 2A). After 2 h of APAP intoxication, CLF was also taken up by hepatocytes and secreted into the BC; however, the diameter of the canaliculi was strikingly dilated (Fig. 2A). Image analysis demonstrated that the dilatation of BC occurred only in the pericentral (TMRE intensity bins 0-3) and not in the periportal (TMRE intensity bins 4-9) lobular zone (Fig. 2B). Moreover, hepatocytes in the pericentral zone enriched CLF (Fig. 2A). Next, time-lapse videos were recorded early after APAP administration to characterize the process leading to the alterations of canalicular morphology and the CLF enrichment in hepatocytes (Fig. 2C; Video S1). Recording began 85 min after APAP injection, when BC in the pericentral zone already appeared dilated, with a further increase in diameter over the next 2 h (Fig. 2C; Video S1). The dilated BC formed small CLF-containing protrusions (60 min) that grew larger over time. These canalicular alterations coincided with decreased TMRE-associated red fluorescence indicating a loss in mitochondrial potential, as well as CLF enrichment in hepatocytes (Fig. 2C; Video S1). Importantly, these canalicular alterations and CLF enrichment in pericentral hepatocytes precedes cell death as evidenced by propidium iodide exclusion (Video S1, minute 174). Analysis of the time-lapse videos showed a time-dependent increase in the number of canalicular protrusions (‘active blebs’) and in the total bleb area at the first 3 h after APAP intoxication (Fig. 2D). These changes coincided with an overall increase in CLF intensity that was more pronounced in the pericentral than the periportal lobular zone (Fig. 2E)

Fig. 2

Dilatation and blebbing of BC after APAP overdose.

(A) Intravital imaging after APAP intoxication. Red: mitochondrial activity (TMRE); green: bile acid analogue (CLF); blue: nuclear staining (Hoechst). (B) Quantification of the BC diameter in relation to its zonation in controls and mice 2 h after APAP overdose. (C) Stills from an intravital video that begins 85 min after APAP overdose to visualize the formation of BC blebs (corresponding to Video S1); (D) Quantification of number and area of active blebs; (E) CLF-associated intensity in pericentral and periportal hepatocytes. (F) Co-immunostaining of Cyp2e1 and CD13 before and after APAP overdose. (G) Quantification of the average BC diameter from immunostained sections (F) in the Cyp2e1 positive and negative lobular zones; ∗∗∗p <0.001, unpaired t test. (H) Canalicular blebs after APAP overdose visualized by immunostaining of CD13 of a mouse liver 2 h after APAP overdose. bc, bile canaliculi; CLF, Cholyl-Lysyl-Fluorescein; PI, propidium iodide; TMRE, Tetramethylrhodamine ethyl ester. (This figure appears in color on the web.)

The aforementioned imaging toolbox requires the administration of fluorescent markers, such as CLF; thus, controls are required to analyze if key observations can be confirmed without the administration of fluorescent markers. Therefore, we co-immunostained fixed mouse liver tissues at various time periods after APAP intoxication using antibodies against the BC marker CD13, and Cyp2e1 that is known to metabolically activate APAP. In line with the intravital imaging results, marked dilatation of BC was also observed in the Cyp2e1-positive zone at 1 h and 2 h after APAP injection (Fig. 2F, G; Fig. S2). Canalicular protrusions into the cytoplasm of hepatocytes resembled those observed by intravital imaging (Fig. 2H). Thus, the observation of compromised canalicular microanatomy from intravital imaging was confirmed by immunostaining of fixed liver tissue ex vivo.

Compromised blood-bile barrier early after APAP intoxication

The aforementioned observations led us to hypothesize that the blood-bile barrier may be compromised early on after APAP intoxication. To test this hypothesis, we performed a set of functional intravital imaging experiments investigating blood-bile barrier integrity (Fig. 3). First, BA transport was studied in untreated, as well as in APAP-intoxicated mice using CLF. After bolus intravenous injection into control mice, CLF appeared in the sinusoidal blood within seconds, was rapidly cleared by uptake into hepatocytes, and later secreted into the BC (Fig. 3A upper panel, B; Video S2A). Intravital recording starting 85 min after APAP administration showed similar CLF uptake by hepatocytes, as well as secretion into the BC as in the controls (Fig. 3A lower panel; Video S2B). However, as soon as CLF was secreted into the BC its signal also increased and remained high in the sinusoidal blood and in hepatocytes of the pericentral zone (Fig. 3B, lower panel; Video S2B), in contrast to the controls, where CLF was rapidly cleared from sinusoidal blood and hepatocytes.

Fig. 3

Enrichment of BAs in the pericentral lobular zone after APAP intoxication.

(A) Intravital imaging of CLF transport after bolus intravenous injection in healthy and in APAP-intoxicated (85 min after overdose corresponds to 0 min on the stills) mice (corresponding to Video S2). (B) Quantification of the CLF-associated signal in BC, hepatocytes, and sinusoids. Continuous lines indicate the pericentral and dashed lines the periportal lobular zones. (C) MALDI-MSI analysis of TCA superimposed onto Cyp2e1-immunostained adjacent liver tissue sections. (D) Quantification of the TCA intensity in the MALDI-MSI images. (E) TCA intensity in the periportal and pericentral lobular zones analyzed by MALDI-MSI. (F) Sum of BA concentrations in liver tissue at different intervals after APAP overdose. (G) Co-immunostaining of ZO1 and Cyp2e1. (H) Intravital imaging of livers of control and APAP-intoxicated mice after tail vein injection of fluorescein-coupled Dextran 70 kDa (green). The arrows indicate BC that appear green after APAP overdose but not in controls (corresponding to Video S3). (I) Intravital imaging of livers of control and APAP-intoxicated mice after bolus tail vein injection of CMFDA (corresponding to Video S4). (J) Quantification of the 5-CMF-associated signal in the sinusoids. ∗p <0.05, ∗∗∗p <0.001, Dunnett's multiple comparisons test (D, F). bc, bile canaliculi; CLF, Cholyl-Lysyl-Fluorescein; 5-CMF, 5-Chlormethylfluorescein; PI, propidium iodide; TCA, taurocholic acid; TMRE, Tetramethylrhodamine ethyl ester. (This figure appears in color on the web.)

The CLF experiments in APAP-intoxicated mice clearly showed an accumulation of CLF in the pericentral zone directly after secretion into the BC. A limitation of this approach is administration of an exogenous BA analogue. Therefore, we studied if imaging of endogenous BA by MALDI-mass spectrometry imaging (MALDI-MSI) leads to similar conclusions. Cyp2e1-immunostained frozen liver tissue sections at various time periods after APAP administration were superimposed with the MALDI signal for taurocholic acid (TCA) (Fig. 3C). The result showed a strong but transient TCA accumulation at 2 hours after APAP intoxication, which was more pronounced in the pericentral than the periportal regions (Fig. 3C-E). This was in line with the result of mass spectrometry-based analysis of BA in liver tissue homogenate (Fig. 3F). Thus, the accumulation of CLF in the pericentral zone after APAP intoxication (Fig. 3A, B) corresponds to the increased levels of endogenous BAs (Fig. 3C-F). Collectively, these data suggest that BAs leak from the BC into the sinusoidal blood in the pericentral zone.

To study the mechanism responsible for BA leakage from the canaliculi, immunostaining of the tight junction proteins, ZO1 and Claudin3 was performed. Both ZO1 and Claudin3 immunostaining showed considerably altered tight junction morphology at 2 h after APAP intoxication characterized by a strongly increased gap between neighboring hepatocytes (Fig. 3G; Fig. S3). These alterations occurred only in the Cyp2e1-positive zone. Next, we studied functional consequences of the observed morphological alterations of the blood-bile barrier. For this purpose, intravital imaging was performed in mice after tail vein injection of fluorescein-coupled dextran (70 kDa). In controls, dextran appeared in the sinusoids within seconds after injection, but did not pass from the blood into the BC during a 30 min imaging period (Fig. 3H; Video S3A; Fig. S4). In contrast, the same analysis 90 min after APAP intoxication demonstrated leakiness from the blood to the canaliculi (Fig. 3H; Fig. S4). The time-lapse videos show that fluorescent dextran first appeared in the sinusoids, after which it continuously increased in the BC (Video S3B,C); leakage was only observed in the pericentral region. Importantly, dextran-associated fluorescence was observed in the BC of hepatocytes with intact mitochondrial potential as evidenced by the vital dye TMRE, and green fluorescence did not increase in the cytoplasm of these cells (Video S3B,C), suggesting a paracellular passage from the blood to canaliculi.

To study if leakage also occurs in the opposite direction from the canaliculi into the sinusoidal blood, we used CMFDA as the intravital dye. The advantage of CMFDA compared to CLF is that it remains non-fluorescent in the blood before its passive uptake into hepatocytes, where it is cleaved by intracellular esterases to produce the green-fluorescent product, 5-CMF. The time-lapse videos of control mice show the cytoplasmic generation of green fluorescence, followed by secretion into canaliculi (Fig. 3I; Video S4A; Fig. S5), while the 5-CMF signal was initially not detectable in the sinusoidal blood. Interestingly, 80 min after APAP intoxication, green fluorescence was present at the interface of pericentral hepatocytes and sinusoids, where the space of Disse is located, almost simultaneously or shortly after secretion into the BC (Fig.3I, lower panel; Video S4B,C; Fig. S5). Subsequently, green fluorescence became detectable in the sinusoids. Quantification of green fluorescence in the sinusoidal blood after CMFDA injection resulted in only very low intensities in the control mice (Fig. 3J); in contrast, a strong increase was detected in APAP-intoxicated mice, particularly in the first 10 min after CMFDA injection (Fig. 3J). This demonstrates that 5-CMF leaks from the BC into the sinusoidal blood.

Spatio-temporal GSH depletion and cholestasis

Since the previous experiments suggested a zonated pattern of BA accumulation, MALDI-MSI was applied to study (i) GSH, (ii) the NAPQI adduct of GSH (APAP-GSH) and (iii) TCA on the same tissue sections. The MALDI signals were superimposed onto an adjacent Cyp2e1-immunostained section to identify the pericentral lobular zone. Experimental conditions were chosen where an APAP overdose transiently depleted GSH for 1-4 h after administration (Fig. 4A,B). Already 1 h after administration, APAP strongly decreased GSH in the pericentral lobular zone accompanied by increased APAP-GSH, while TCA was not or only slightly increased (Fig. 4C). In contrast, 2 h after APAP overdose, pericentral regions with depleted GSH and increased APAP-GSH coincided with strongly increased TCA. Quantification of the MALDI-MSI signals in pericentral and periportal zones confirmed a time-course where pericentral GSH depletion coincided with altered canalicular morphology (Fig. 2F) and preceded TCA accumulation (Fig. 4D-F).

Fig. 4

Zonation of APAP-induced canalicular alterations and BA accumulation.

(A) Experimental schedule. (B) Total GSH in homogenized liver tissue. (C) MALDI-MSI signals of GSH, APAP-GSH, and TCA superimposed onto an adjacent Cyp2e1-immunostained section at different periods after APAP overdose. (D-F) Quantification of MALDI-MSI signals in relation to the pericentral or periportal zone as evidenced by Cyp2e1 staining. Bar plots in B, D-F: mean and SE; dots indicate individual mice. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, unpaired t test. GSH, glutathione; TCA, taurocholic acid. (This figure appears in color on the web.)

A second intervention was performed with combined buthionine sulfoximine (BSO; blocks GSH synthesis) and diethyl maleate (DEM; depletes GSH) (Fig. 5A). Similar to APAP, BSO/DEM treatment also depleted GSH (Fig. 5B); however, depletion occurred throughout the entire lobule (Fig. S6A), in contrast to the pericentral GSH depletion by APAP. A strong increase in randomly distributed TUNEL-positive hepatocytes was only observed 8 h after BSO/DEM treatment (Fig. 5C), which coincided with increased transaminase activities and non-zonated dead cell areas (Fig. 5D; Fig. S6B, C). Like APAP, compromised canalicular morphology was observed after BSO/DEM treatment particularly at 2 h and later (Fig. 5E-H; Video S5), but dilatation occurred in both the Cyp2e1-positive and -negative regions (Fig. 5H); however, the BSO/DEM intervention did not induce leakage of CLF from canaliculi into sinusoidal blood (Fig. 5F), in contrast to the effect of APAP. Correspondingly, BA concentrations did not increase in the blood nor in the liver tissue (Fig. 5D, I-K). An explanation for this difference may be that APAP induces higher levels of oxidative stress, which has been shown to compromise tight junctions. Indeed, levels of the oxidative stress marker 4-hydroxynonenal analyzed in liver tissue homogenate were much higher after APAP than BSO/DEM administration (Fig. 6A). Also, intravital imaging with the oxidative stress indicator 2ʹ,7ʹ-dichlorofluorescein diacetate, that fluoresces green after oxidation, demonstrated oxidative stress predominantly in the pericentral region after APAP overdose, whereas oxidative stress after BSO/DEM was much weaker (Fig. 6B).

Fig. 5

GSH depletion and canalicular dilatation after administration of BSO/DEM. (A) Experimental schedule.

(B) Depletion of GSH in homogenized liver tissue. (C) Co-staining of Cyp2e1 and cell death by TUNEL. (D) ALT and sum of BA levels in blood. (E) Stills from an intravital video (Video S5) beginning 3 hours after BSO/DEM administration and quantification of the CLF signal (F) in sinusoids, hepatocytes, and canaliculi. (G) Co-staining of Cyp2e1, CD13 and the tight junction marker Claudin-3. (H) Quantification of the BC diameter in relation to the Cyp2e1-positive zone; p <0.001 (∗∗∗), unpaired t test. (I) Sum of BAs in liver tissue (mean and SE; dots indicate individual mice). (J) TCA signal of MALDI-MSI superimposed onto an adjacent Cyp2e1-stained section and quantification (K). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, Dunnett's multiple comparisons test (I, K). ALT, alanine transaminase; bc, bile canaliculi; CLF, Cholyl-Lysyl-Fluorescein; GSH, glutathione; TCA, taurocholic acid; TMRE, Tetramethylrhodamine ethyl ester; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling. (This figure appears in color on the web.)

Fig. 6

Oxidative stress in liver tissue and extrahepatic bile after intoxication with APAP or BSO/DEM in mice. (A) 4-HNE in liver tissue homogenate. (B) Intravital images visualizing oxidative stress in liver tissue based on green fluorescence of oxidized DCF. (C) Bile volume sampled by a catheter fixed in the extrahepatic bile duct. (D) BA concentration in the sampled bile and total excreted BA. (E) Total GSH concentrations of extrahepatic bile. Means and SE of 4 mice are given. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, Dunnett's multiple comparisons test. 4-HNE, 4-hydroxynonenal; BSO, buthionine sulfoximine; DEM, diethyl maleate; GSH, glutathione. (This figure appears in color on the web.)

A further hypothesis possibly explaining the widening of BC and BA leakage is a cholestatic block downstream of the pericentral canaliculi. However, both APAP and BSO/DEM treatments caused increased flow and total BA excretion (Fig. 6C, D) and strongly reduced the GSH concentration in bile (Fig. 6E). Thus, an obstruction downstream of the pericentral canaliculi is unlikely to explain the observed alterations.

Identification of intracellular cytotoxic concentration ranges of BAs

While the previous experiments demonstrated that the total BA concentrations in liver tissue increased after APAP overdose, it remained to be studied if the resulting intracellular BA concentrations exceed toxic thresholds. Therefore, we performed concentration-dependent incubations of cultivated mouse hepatocytes with mouse bile in the presence and absence of APAP in the culture medium (Fig. 7A). Individual BA concentrations in mouse bile were determined by mass spectrometry (Datasheet S1D) and concentrations in the culture medium of cultivated hepatocytes were adjusted based on the sum of BA concentrations. The EC50 of the sum of BAs in hepatocytes co-incubated with 0, 1 and 4 mM APAP were 542, 223 and 90 μM, respectively (Fig. 7B; Fig. S7). BA concentrations in homogenized hepatocytes after cultivation for 2 h at cytotoxic BA concentrations of 0, 10, 100 and 1,000 μM were ∼7, 170, 260, and 1,100 pmol/μg DNA, respectively (Fig. 7C). Analysis of homogenized liver tissue (Fig. 7D) and isolated hepatocytes (Fig. 7E) from the same mice 2 h after APAP intoxication showed an increase in BA concentrations. To enable a direct comparison of the in vitro- and in vivo-exposed hepatocytes, the sum of BA concentrations for both were analyzed and normalized to the DNA content. Interestingly, the sum of BA concentrations in hepatocytes of APAP-intoxicated mice ex vivo (∼400 pmol/μg DNA; Fig. 7E) reached similar levels as the in vitro-cultivated hepatocytes incubated with cytotoxic concentrations of BAs (Fig. 7C). It should be considered that, in vivo, it is mainly pericentral hepatocytes that are damaged by APAP and appear to undergo futile BA cycling; therefore, it is possible that ex vivo-analyzed BA concentrations in the pericentral hepatocytes are even higher than the ∼400 pmol/μg DNA obtained for all isolated hepatocytes, because the pericentral hepatocytes are diluted by less affected periportal cells. Thus, the extent of the APAP-induced increase in BA concentrations in hepatocytes in vivo is indeed high enough to explain the observed hepatotoxicity.

Fig. 7

Intracellular BA concentrations in relation to cytotoxicity. (A) Experimental design of cultivated mouse hepatocytes incubated with APAP and/or mouse bile. (B) Concentration-dependent cytotoxicity of BA in the presence of 0, 1 and 4 mM APAP. The inlay gives the EC10 and EC50 values of the fitted curves, the horizontal lines show the EC10 values and 95% confidence values. The open circles are data of 3 independent experiments obtained with hepatocytes from different mice, the closed circles represent mean values of the independent experiments. (C) Sum of intracellular BA concentrations in mouse hepatocytes incubated with extrahepatic mouse bile. (D) Sum of BA concentrations in homogenized liver tissue before and 2 h after APAP overdose. (E) Sum of BA concentrations in homogenized hepatocytes isolated from the same livers analyzed in D; the bar plots in C-E show mean values and SE; ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, unpaired t test. CTB, CellTiter-Blue, FCS, fetal calf serum. (This figure appears in color on the web.)

Therapeutic intervention by interruption of BAs cycling

Based on our proposed model of APAP-induced BA cycling (Graphical abstract), interruption of BA re-uptake should reduce BA concentrations in liver tissue and ameliorate APAP-induced hepatotoxicity. To test this hypothesis, we used Oatp knockout mice and additionally inhibited the Na+-taurocholate co-transporting polypeptide (NTCP, SLC10A1) pharmacologically by injecting Myrcludex B to block the BA uptake carriers in hepatocytes. APAP (300 mg/kg b.w.) was administered i.p. simultaneously with Myrcludex B and liver damage was evaluated 24 h later. This intervention protected the mice from APAP-induced liver damage as evidenced by macroscopic appearance, the extent of pericentral cell death, and liver enzymes (Fig. 8A-D; Fig. S8).

Fig. 8

Interruption of futile BA cycling by Oatp knockout and Myrcludex B ameliorates APAP-induced hepatotoxicity.

(A-D) Gross pathology, histopathology, quantification of the dead cell area, and Liver enzymes. The bar plots in C and D show mean values and SE; ∗∗p <0.01, ∗∗∗p <0.001, unpaired t test. (E) Cyp2e1 immunostaining in wild-type and Oatp knockout mice. (F) Similar glutathione levels in wild-type and Myrcludex B-treated Oatp knockout mice before and after APAP administration; data are mean values and SE; p <0.001 (∗∗∗), Tukey's multiple comparisons test. (G,H) Co-staining of Cyp2e1 and CD13 (upper panel) or Cyp2e1 and ZO1 (lower panel) in wild-type and Myrcludex B-treated mice before and 2 h after APAP administration. (I) MALDI-MSI showing reduced TCA signal in Myrcludex B-treated Oatp knockout mice 2 h after APAP overdose. (J,K) Sum of BA concentrations in liver tissues (J) and plasma (K) of wild-type and Myrcludex B-treated Oatp knockout mice with and without APAP overdose. Data in J and K are means and SE; ∗∗∗p <0.001, Sidak's multiple comparisons test and Tukey's multiple comparisons test, respectively. ALT, alanine transaminase; AST, aspartate transaminase; GSH, glutathione; KO, knockout; Myrcl. B, Myrcludex B; WT, wild-type. (This figure appears in color on the web.)

To further corroborate these data and to exclude possible alternative explanations, such as decreased metabolic activation or increased detoxification of APAP, we next analyzed Cyp2e1 expression and found no difference between Oatp knockout and WT mice prior to APAP administration (Fig. 8E; Fig. S9). In agreement, APAP clearance from blood, APAP metabolite and adduct levels did not show systematic differences between Myrcludex B-treated Oatp knockout and WT mice after APAP intoxication (Fig. S10). Moreover, no difference was observed in GSH levels in liver tissue prior to APAP injection, and similar depletion was detected afterwards (Fig. 8F).

Next, we investigated whether blocking BA uptake carriers ameliorated the APAP-induced alterations in canalicular and tight junction morphology. Although Myrcludex B plus the loss of Oatp ameliorated APAP-induced cell death, they did not prevent the compromised canalicular and tight junction morphology due to APAP overdose, as evidenced by CD13, ZO1, and Claudin3 immunostaining (Fig. 8G, H; Fig. S11). However, Myrcludex B plus Oatp knockout massively reduced BA concentrations in liver tissue as demonstrated by MALDI-MSI (Fig. 8I; Fig. S12) and mass spectrometry (Fig. 8J) analyses, while BA concentrations strongly increased in the blood (Fig. 8K), demonstrating the efficacy of BA uptake inhibition. Altogether, these data suggest that blocking the sinusoidal BA uptake carriers strongly ameliorates APAP-induced hepatotoxicity.

Discussion

In the liver, APAP is metabolically activated to NAPQI, which conjugates to and depletes GSH, binds to proteins, induces oxidative stress, JNK activation and mitochondrial dysfunction. The present study clearly demonstrates that this sequence of events has not yet considered an essential additional aspect that aggravates hepatocyte death after APAP overdose. Herein, we show that besides the aforementioned mechanisms, APAP compromises the blood-bile barrier, thereby causing leakage of bile with high BA concentrations from the canaliculi into the sinusoidal blood, from where BAs are transported into hepatocytes and secreted again into the BC. This futile cycling increases BA concentrations in the hepatocytes above cytotoxic levels and causes cell death. Interruption of futile cycling by blocking the uptake carriers strongly reduces intracellular BA concentrations and rescues hepatocytes from cell death. This finding does not exclude that APAP is also toxic without the synergistic effect of futile BA cycling, but higher doses may be required. The therapeutic intervention does not interfere with the leakiness of the blood-bile barrier. However, when BA cycling and accumulation in hepatocytes is prevented, the liver tissue recovers from APAP-induced stress.

It has already been reported that APAP and oxidative stress compromise tight junctions.[11], [12], [13] However, it has not yet been shown that APAP causes futile BA cycling after compromising the blood-bile barrier. Previous studies have addressed the question of whether “systemic increase in BA levels can be a direct cause of hepatotoxicity, or whether they are just an indicator of liver dysfunction”. One hypothesis is that elevation of BA concentrations in serum and liver tissue causes hepatocyte death.[14], [15], [16], [17] However, other studies have challenged this concept[5], [6], [7], [8] hypothesizing that “the source of the BA elevation must be due to either rupture of the biliary tract where BA are present in millimolar quantities, or direct inhibition of BA export from hepatocytes”. Here, we demonstrate that the first hypothesis proposing, “rupture of the biliary tract”, is correct but the concept of “inhibition of BA export” does not apply. However, the term “rupture of the biliary tracts” seems to be imprecise, and it is more likely a leakiness of the BC due to morphologically and functionally altered tight junctions. This insight was made possible by implementing intravital imaging. Using fluorescent high molecular weight dextran, we directly observed APAP-induced leakiness from sinusoidal blood to the lumen of the BC. Vice versa, leakiness from canaliculi to sinusoidal blood was demonstrated by using the intravital non-fluorescent probe CMFDA, which is taken up by hepatocytes and cleaved by intracellular esterases to release the fluorescent 5-CMF. This in turn is secreted into the BC and – only after APAP pretreatment – leaks into the sinusoidal blood. Moreover, administration of the green-fluorescent BA analogue CLF demonstrated that the compound is secreted into the BC, and after APAP overdose leaks from the canaliculi into the sinusoidal blood and accumulates in pericentral hepatocytes. This accumulation was almost completely prevented by inhibiting the sinusoidal BA uptake carriers. Since CLF is an exogenously administered BA analogue, we corroborated the key finding of APAP-induced BA accumulation and its rescue using MALDI-MSI to analyze endogenous BAs. Intravital imaging also enabled us to analyze BA secretion from hepatocytes into the BC. Interestingly, APAP had no influence on CLF secretion, and consequently the second hypothesis of Woolbright et al. – direct inhibition of BA export from hepatocytes – could be disproven.

MALDI-MSI allowed us to identify the sequence of metabolic changes and their spatial organization in liver tissue after APAP overdose. Initially, increased APAP-GSH adducts were observed in the pericentral zone which coincides with decreased GSH levels. Subsequently, a strong enrichment of TCA occurred in the GSH-depleted regions. The GSH-depleted and TCA-enriched zones coincide with the pericentral regions in which intravital imaging demonstrated dilated BC and bile-to-blood leakage. Since APAP is known to cause zonated liver damage due to pericentral metabolic activation by cytochrome P450 enzymes, we compared this scenario to intoxication with BSO/DEM which inhibits GSH synthesis and depletes GSH all over the lobule. In agreement with the non-zonated pattern of GSH depletion, dilated BC occurred throughout the entire lobule. However, no bile-to-blood leakage was observed after BSO/DEM in contrast to APAP. A likely explanation is that APAP induced much higher levels of oxidative stress (in the pericentral zone) than BSO/DEM. A limitation of the present study is that we did not study the molecular mechanisms by which oxidative stress compromises the tight junctions of BC, but this aspect has already been addressed previously. Taken together, spatio-temporal imaging demonstrated that the breach of the bile-blood barrier occurred in tissue regions with high oxidative stress and that GSH depletion with only mildly increased reactive oxygen species is not sufficient to cause this effect.

Blocking NTCP in mice has already been shown to be beneficial in selected cholestatic conditions. However, in mice, BAs are taken up by both NTCP and OATPs (SLC01B1 and SLC01B3), and as a result, inhibition of both uptake carriers is important to block BA uptake. This may be different in humans where BAs are predominantly taken up by NTCP; therefore, Myrcludex B treatment without using additional OATP inhibitors may be sufficient, but confirmation (e.g., in studies with human hepatocytes) is still required.

The mechanism discovered here could be of clinical relevance since futile BA cycling after APAP overdose can be interrupted. Our human data show that as in mice, the increase in BAs occurs before the increase of transaminases. Mechanistic studies in humans are challenging, because obtaining the blood of patients with documented time of overdose and repeated sampling is difficult. The present study was made possible by special circumstances, e.g. the time of APAP ingestion was narrowed down by the receipt of drug purchase, the patient sought immediate care and the dose (86.5 g) could be documented using remaining pill counts. It will be important to study further patients where the time of ingestion and dose are documented. Nevertheless, the present data show that the period of increased blood BAs in humans may last for at least 100 hours after APAP overdose. Thus, the time-window when pharmacological interruption of futile BA cycling seems to be indicated exceeds the ∼8 hour window within which N-acetylcysteine works effectively.

In conclusion, we identified futile BA cycling as a mechanism that follows the APAP-induced oxidative stress and breach of the blood-bile barrier and causes hepatocyte death. The study answers the long-standing question of whether systemic increases in BA concentrations represent a direct cause or consequence of hepatotoxicity.

Abbreviations

ALT, alanine transaminase; AST, aspartate transaminase; APAP, acetaminophen; BA(s), bile acid(s); BSO/DEM, buthionine sulfoximine/diethyl maleate; GSH, glutathione; MSI, mass spectrometry imaging; TCA, taurocholic acid; CLF, Cholyl-Lysyl-Fluorescein; TMRE, Tetramethylrhodamine ethyl ester; bc, bile canaliculi; FCS, fetal calf serum; CTB, CellTiter-Blue; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; PI, propidium iodide; 5-CMF, 5-Chlormethylfluorescein.

Financial support

A.G. was funded by the German-Research-Foundation (DFG; GH 276/1-1; project no.: 457840828). Work in the lab of T.L. was funded from the (ERC) through the ERC Consolidator Grant PhaseControl (Grant Agreement number 771083) and the German-Research-Foundation (LU 1360/3-2 and SFB CRC 1382). A.C. was funded by DFG; project no.: 267/13-3.

Authors’ contributions

AG and JGH: study concept and design, data acquisition, analysis and interpretation of data, manuscript writing, funding, study supervision; RH, ZH, LB, BBT, MM, AS, DG, WM: performed mouse experiment, tissue and blood analyses, contributed to manuscript writing; UH: bile acid assay, critical revision of the manuscript; JR, NO, SS, SZ: clinical chemistry, MALDI-MSI, critical revision of the manuscript; AF and SH: image analysis, contributed to manuscript writing; TB, FK, and JR: analysis of concentration-cytotoxicity relationships; CG, IMC, and RM: critical revision of the manuscript; MV, TL: performed analyses, critical revision of the manuscript, and obtained funding, histological evaluation; SU: provided Myrcludex B, critical revision of the manuscript. AC, TS, MT, JYA, MO, SCC, and JPS: provided clinical data and critical revision of the manuscript; HJ: contributed to study design, clinical data, analysis and interpretation of the clinical data and critical revision of the manuscript.

Data availability statement

The data presented in this study are available on request from the corresponding author.

Conflict of interest

S.U. is holder and inventor on patents protecting Myrcludex B (Hepcludex/bulevirtide). All other authors declare that they have no conflict of interest.

Please refer to the accompanying ICMJE disclosure forms for further details.