APAP Metabolites Research Articles

Human Target Proteins for Benzo(a)pyrene and Acetaminophen (And Its Metabolites): Insights from Inverse Molecular Docking and Molecular Dynamics Simulations

Acetaminophen (APAP) is a widely used analgesic and antipyretic, whereas benzo(a)pyrene (B[a]P) is a carcinogen with significant global health risks due to environmental exposure. While APAP is generally safe at therapeutic doses, co-exposure to B[a]P can exacerbate its toxicity. This study aimed to identify potential human target proteins for B[a]P and APAP through inverse molecular docking and molecular dynamics simulations. We performed inverse docking with B[a]P, APAP, and three APAP metabolites against 689 human proteins involved in various biological processes. Five proteins were selected based on high docking affinity and their involvement in multiple pathways. Molecular dynamics simulations revealed that B[a]P primarily interacted via hydrophobic and π-stacking interactions with proteins like LXR-β, HSP90α, HSP90β, and AKT1, while AM404 formed hydrogen bonds and hydrophobic interactions. The simulations confirmed that the complexes had high conformational stability, except for protein AKT1. These results provide insights into the potential impacts of B[a]P and AM404 on protein functions and their implications for understanding the toxic effects of combined exposure.

Bacterial bioaugmentation for paracetamol removal from water and sewage sludge. Genomic approaches to elucidate biodegradation pathway

Wastewater treatment plants (WWTPs) are recognized as significant contributors of paracetamol (APAP) into the environment due to their limited ability to degrade it. This study used a bioaugmentation strategy with Pseudomonas extremaustralis CSW01 and Stutzerimonas stutzeri CSW02 to achieve APAP biodegradation in solution in wide ranges of temperature (10-40oC) and pH (5-9), reaching DT50 values < 1.5hours to degrade 500mgL-1 APAP. Bacterial strains also mineralized APAP in solution (<30%), but when forming consortia with Mycolicibacterium aubagnense HPB1.1, mineralization significantly increased (up to 74% and 58% for CSW01+HPB1.1 and CSW02+HPB1.1, respectively), decreasing DT50 values to only 1 and 9 days. Despite the complete degradation of APAP and its high mineralization, residual toxicity throughout the process was observed. Three APAP metabolites were identified (4-aminophenol, hydroquinone and trans-2-hexenoic acid) that quickly disappeared, but residual toxicity remained, indicating the presence of other non-detected intermediates. CSW01 and CSW02 degraded also 100% APAP (50mgkg-1) adsorbed on sewage sludge, with DT50 values of only 0.7 and 0.3 days, respectively, but < 15% APAP was mineralized. A genome-based analysis of CSW01 and CSW02 revealed that amidases, deaminases, hydroxylases, and dioxygenases enzymes were involved APAP biodegradation, and a possible metabolic pathway was proposed. Environmental ImplicationThe wide use of paracetamol (APAP) involves ecological risks, being considered an emerging pollutant. APAP excreted by humans ends up in WWTPs, where its elimination is not complete, remaining in part in the effluent treated waters (discharged into surface waters used for soil irrigation) and in part adsorbed on sewage sludge (used as fertilizer in agricultural soils). APAP bioremediation in WWTPs is the unique opportunity to remove it from both matrices before its dispersion in the environment.The novelty of this study is the isolation of two bacterial strains from sewage sludge, P. extremaustralis CSW01 and S. stutzeri CSW02, that could degrade very high concentrations of APAP and their main formed metabolites, both in water and, for the first time, in sewage sludge, in very short periods of time, and even reach high percentages of APAP mineralization in water. Moreover, CSW01 and CSW02 were active under wide ranges of temperature and pH, and are therefore excellent candidates for the chance to achieve a reduction of APAP toxicity in the environment. In addition, by a genome-based analysis, enzymes possibly involved in APAP biodegradation have been identified and a possible degradation pathway has been proposed.

Elderly mice with history of acetaminophen intoxication display worsened cognitive impairment and persistent elevation of astrocyte and microglia burden

Acetaminophen (APAP) is a leading cause of acute liver failure. The effect of APAP metabolite’s effects in the periphery are well characterized; however, associated consequences in the brain remain poorly understood. Animal studies on this subject are few and reveal that frequent APAP intake can trigger cerebral abnormalities that vary depending on the subject’s age. Alarmingly, experimental efforts have yet to examine associated consequences in elderly hosts, who correspond to the highest risk of medication overload, impaired drug clearance, and cognitive deficits. Here, we interrogated the cerebral and peripheral pathology of elderly mice submitted to monthly episodes of APAP intoxication since a young adult age. We found that weeks after the final episode of recurrent APAP exposure, mice exhibited worsened non-spatial memory deficit whereas spatial memory performance was unaltered. Interestingly, one month after the period of APAP intoxication, these mice showed increased glial burden without associated drivers, namely, blood–brain barrier disruption, cholesterol accumulation, and elevation of inflammatory molecules in the brain and/or periphery. Our experimental study reveals how recurrent APAP exposure affects the cognitive performance and cellular events in elderly brains. These data suggest that APAP-containing pharmacological interventions may foreshadow the elevated risk of neuropsychiatric disorders that afflict elderly populations.

High-resolution mass spectrometry identifies delayed biomarkers for improved precision in acetaminophen/paracetamol human biomonitoring

Paracetamol/acetaminophen (N-acetyl-p-aminophenol, APAP) is a top selling analgesic used in more than 600 prescription and non-prescription pharmaceuticals. To study efficiently some of the potential undesirable effects associated with increasing APAP consumption (e.g., developmental disorders, drug-induced liver injury), there is a need to improve current APAP biomonitoring methods that are limited by APAP short half-life. Here, we demonstrate using high-resolution mass spectrometry (HRMS) in several human studies that APAP thiomethyl metabolite conjugates (S-methyl-3-thioacetaminophen sulfate and S-methyl-3-thioacetaminophen sulphoxide sulfate) are stable biomarkers with delayed excretion rates compared to conventional APAP metabolites, that could provide a more reliable history of APAP ingestion in epidemiological studies. We also show that these biomarkers could serve as relevant clinical markers to diagnose APAP acute intoxication in overdosed patients, when free APAP have nearly disappeared from blood. Using in vitro liver models (HepaRG cells and primary human hepatocytes), we then confirm that these thiomethyl metabolites are directly linked to the toxic N-acetyl-p-benzoquinone imine (NAPQI) elimination, and produced via an overlooked pathway called the thiomethyl shunt pathway. Further studies will be needed to determine whether the production of the reactive hepatotoxic NAPQI metabolites is currently underestimated in human. Nevertheless, these biomarkers could already serve to improve APAP human biomonitoring, and investigate, for instance, inter-individual variability in NAPQI production to study underlying causes involved in APAP-induced hepatotoxicity. Overall, our findings demonstrate the potential of exposomics-based HRMS approach to advance towards a better precision for human biomonitoring.

Detection of Distinct Distributions of Acetaminophen and Acetaminophen-Cysteine in Kidneys up to 10 μm Resolution and Identification of a Novel Acetaminophen Metabolite Using an AP-MALDI Imaging Mass Microscope.

Drug distribution studies in tissue are crucial for understanding the pharmacokinetics and potential toxicity of drugs. Recently, matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) has gained attention for drug distribution studies due to its high sensitivity, label-free nature, and ability to distinguish between parent drugs, their metabolites, and endogenous molecules. Despite these advantages, achieving high spatial resolution in drug imaging is challenging. Importantly, many drugs and metabolites are rarely detectable by conventional vacuum MALDI-MSI because of their poor ionization efficiency. It has been reported that acetaminophen (APAP) and one of its major metabolites, APAP-Cysteine (APAP-CYS), cannot be detected by vacuum MALDI-MSI without derivatization. In this context, we showed the distribution of both APAP and APAP-CYS in kidneys at high spatial resolution (25 and 10 μm) by employing an atmospheric pressure-MALDI imaging mass microscope without derivatization. APAP was highly accumulated in the renal pelvis 1 h after drug administration, while APAP-CYS exhibited characteristic distributions in the outer medulla and renal pelvis at both 30 min and 1 h after administration. Interestingly, cluster-like distributions of APAP and APAP-CYS were observed in the renal pelvis at 10 μm spatial resolution. Additionally, a novel APAP metabolite, tentatively coined as APAP-butyl sulfate (APAP-BS), was identified in the kidney, brain, and liver by combining MSI and tandem MSI. For the first time, our study revealed differential distributions of APAP, APAP-CYS (in kidneys), and APAP-BS (in kidney, brain, and liver) and is believed to enhance the understanding of the pharmacokinetics and potential nephrotoxicity of this drug.

Calcitriol Protects against Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) overdose is one of the major causes of acute liver failure. Severe liver inflammation and the production of oxidative stress occur due to toxic APAP metabolites and glutathione depletion. Growing evidence has proved that vitamin D (VD) exerts anti-inflammatory and antioxidative functions. Our objective was to explore the protective role of calcitriol (VD3) in acute APAP-induced liver injury. Methods: Adult male mice were randomized into three groups; control (n = 8), APAP (n = 8), and VD3 group (n = 8). All mice, except controls, received oral administration of APAP (400 mg/kg) and were sacrificed 24 h later. In the VD3 group, calcitriol (10 µg/kg) was injected intraperitoneally 24 h before and after exposure to APAP. Blood samples were collected to assess serum aminotransferase and inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)]. Liver tissues were analyzed for hepatic glutathione (GSH), malondialdehyde (MDA), and histopathology. Results: APAP administration significantly increased serum aminotransferase, inflammatory cytokines, and induced cellular inflammation and necrosis. APAP also depleted hepatic GSH and elevated oxidative stress, as indicated by high MDA levels. In the APAP group, 25% of the mice (two out of eight) died, while no deaths occurred in the VD3 group. Treatment with calcitriol significantly reduced serum aminotransferase, TNF-α, and IL-6 levels in the VD3 group compared to the APAP group. Additionally, VD3 effectively restored GSH reserves, reduced lipid peroxidation, and attenuated hepatotoxicity. Conclusions: These findings demonstrate that VD3 prevents APAP-induced acute liver injury and reduces mortality in mice through its anti-inflammatory and antioxidative activity. Thus, VD3 might be a novel treatment strategy for APAP-induced hepatotoxicity.

8-methoxypsoralen protects against acetaminophen-induced liver injury by antagonising Cyp2e1 in mice

This study aimed to investigate the effect and mechanism of 8-methoxypsoralen (8-MOP) on acetaminophen (APAP)-induced hepatotoxicity in mice. The study found that 1 h after intraperitoneal injection of 300 mg/kg APAP, treatment with 40 mg/kg, 80 mg/kg and 120 mg/kg 8-MOP could reduce serum transaminase level and histopathological liver necrosis area. Elevated mRNA expression of liver inflammatory mediators caused by excessive APAP was also reversed. 8-MOP significantly reduced APAP-induced hepatotoxicity dose-dependently, and the highest therapeutic dose of 8-MOP (120 mg/kg) had no harmful effects on the liver. Cocktail probe assay revealed that 8-MOP can inhibit Cyp2e1 enzymatic activities of mice, thereby reducing the production of acetaminophen-cysteine (APAP-CYS), a toxic metabolite of APAP. 8-MOP had no significant effect on the protein and gene expression of Cyp2e1. The three-dimensional structures of mouse Cyp2e1 were constructed by homologous modeling. Molecular docking showed that 8-MOP had a good binding effect on the enzyme activity site of Cyp2e1. In summary, 8-MOP dose-dependently attenuated APAP-induced hepatotoxicity by binding to Cyp2e1 and occupying the active center of the enzyme, thus competitively inhibiting the oxidative metabolism of APAP, and reducing the generation of toxic product APAP-CYS.

C-Jun-N Terminal Kinase-Mediated Degradation of γ-Glutamylcysteine Ligase Catalytic Subunit Inhibits GSH Recovery After Acetaminophen Treatment: Role in Sustaining JNK Activation and Liver Injury.

Aims: Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. Liver glutathione (GSH) depletion and sustained P-JNK (c-Jun-N-terminal kinase) activation are key modulators in the mechanism leading to hepatic necrosis. GSH depletion is directly related to the consumption of GSH by APAP metabolites N-acetyl-p-benzoquinone imine (NAPQI). We previously noticed that the glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in GSH synthesis, rapidly decreased at the same time P-JNK increased. Our aims were to determine if JNK was directly responsible for decreased GCLC causing impaired recovery of GSH and if this was an important factor in determining APAP hepatotoxicity. Results: Immunoprecipitation of JNK after APAP identified binding to GCLC. Expression of a site-directed mutated canonical JNK docking site in GCLC was resistant to degradation and led to rapid restoration of GSH and inhibited sustained JNK activation. The JNK-resistant GCLC markedly protected against necrosis and alanine aminotransferase (ALT) elevation. The proteolytic loss of GCLC was abrogated by inhibition of the proteasome, ubiquitination, or calpain. Innovation: Using mutated-GCLC resistant to JNK-induced degradation, the results allowed us to identify impaired GSH recovery as an important contributor to early progression of APAP toxicity after the metabolism of APAP and initial GSH depletion had occurred. Conclusion: Activated JNK interacts directly with GCLC and leads to proteolytic degradation of GCLC. Degradation of GCLC impairs GSH recovery after APAP allowing the continued activation of JNK. Conversely, rapid recovery of GSH inhibits the sustained activation of the mitogen-activated protein (MAP) kinase cascade and dampens APAP toxicity by suppressing the continued activation of JNK. Antioxid. Redox Signal. 38, 1071-1081.

Fluorine-thiol displacement probes for acetaminophen's hepatotoxicity.

Chemicals possessing reactive electrophiles can denature innate proteins leading to undesired toxicity, and the overdose-induced liver injury by drugs containing electrophiles has been one of the major causes of non-approval and withdraw by the US Food and Drug Administration (FDA). Elucidating the associated proteins could guide the future development of therapeutics to circumvent these drugs' toxicities, but was largely limited by the current probing tools due to the steric hindrance of chemical tags including the common "click chemistry" labels. Taking the widely used non-steroidal anti-inflammatory drug acetaminophen (APAP) as an example, we hereby designed and synthesized an APAP analogue using fluorine as a steric-free label. Cell toxicity studies indicated our analogue has similar activity to the parent drug. This analogue was applied to the mouse hepatocellular proteome together with the corresponding desthiobiotin-SH probe for subsequent fluorine-thiol displacement reactions (FTDRs). This set of probes has enabled the labeling and pull-down of hepatocellular target proteins of the APAP metabolite as validated by Western blotting. Our preliminary validation results supported the interaction of APAP with the thioredoxin protein, which is an important redox protein for normal liver function. These results demonstrated that our probes confer minimal steric perturbation and mimic the compounds of interest, allowing for global profiling of interacting proteins. The fluorine-thiol displacement probing system could emerge as a powerful tool to enable the investigation of drug-protein interactions in complex biological environments.

Analgesic Action of Acetaminophen via Kv7 Channels

The mechanism of acetaminophen (APAP) analgesia is at least partially unknown. Previously, we showed that the APAP metabolite N-acetyl-p-benzoquinone imine (NAPQI) activated Kv7 channels in neurons in vitro, and this activation of Kv7 channels dampened neuronal firing. Here, the effect of the Kv7 channel blocker XE991 on APAP-induced analgesia was investigated in vivo. APAP had no effect on naive animals. Induction of inflammation with λ-carrageenan lowered mechanical and thermal thresholds. Systemic treatment with APAP reduced mechanical hyperalgesia, and co-application of XE991 reduced APAP's analgesic effect on mechanical pain. In a second experiment, the analgesic effect of systemic APAP was not antagonized by intrathecal XE991 application. Analysis of liver samples revealed APAP and glutathione-coupled APAP indicative of metabolization. However, there were no relevant levels of these metabolites in cerebrospinal fluid, suggesting no relevant APAP metabolite formation in the CNS. In summary, the results support an analgesic action of APAP by activating Kv7 channels at a peripheral site through formation of the metabolite NAPQI.

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

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.

Exposure assessment of acetaminophen use in pregnancy: a study comparing self-reported intake with maternal and newborn biomarker measures

Background: Acetaminophen (APAP) is the most commonly used medication during pregnancy. Increasingly, studies have reported potential adverse associations between maternal prenatal use of APAP and offspring asthma, adverse reproductive outcomes, and neurodevelopmental disorders. However, most studies have relied on self-reported maternal exposure which has the potential for exposure misclassifications. We conducted this study to compare maternal self-reported APAP intake and three acetaminophen metabolites measured in urine collected during pregnancy and umbilical cord serum samples. Methods We used maternal interview data and biospecimens from the Nutrition in Pregnancy cohort of 2291 pregnant women recruited from Connecticut and Western Massachusetts during 1996-2000. We selected three exposure groups including 10 mothers who reported frequent intake (14 days+) of APAP in the first 3 months of pregnancy, 10 who reported frequent intake in the last 3 months of pregnancy, and 10 who reported never using APAP in these two periods from the cohort. We analyzed three APAP metabolites (parent compound, acetaminophen glucuronide, and 3-(N-acetyl-l-cystein-S-yl)-acetaminophen) levels in the maternal urine (avg. 24 gestational weeks) and the umbilical cord serum samples. Results The parent APAP compound and 3-(N-acetyl-l-cystein-S-yl)-acetaminophen) were detected in all urine and serum samples analyzed (100%), while acetaminophen glucuronide was less detectable in the cord serum (40% vs. 100% in urine). APAP metabolites in maternal urine and cord serum were correlated (r=0.43-0.70) within mother-child pairs. All three urinary and cord serum APAP metabolite levels were more strongly correlated with the reported number of days of APAP intake in late pregnancy (r=0.57-0.73; p-values &amp;#x3c;0.03, rank tests), and less with early pregnancy intake (r=0.18-0.34; p-values 0.21-0.60, rank tests). Conclusions Our findings suggest detailed intake data collected in maternal interviews is an efficient and valid measure to indicate exposure to APAP in pregnancy, while biomarker measures can be used to represent a specific exposure period of interest.

Mitochondrial Glrx2 Knockout Augments Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) is one of the most widely used drugs with antipyretic and analgesic effects, and thus hepatotoxicity from the overdose of APAP becomes one of the most common forms of drug-induced liver injury. The reaction towards thiol molecules, such as GSH by APAP metabolite, N-acetyl-p-benzo-quinonimine (NAPQI), is the main cause of APAP-induced hepatotoxicity. However, the role of many other thiol-related regulators in toxicity caused by APAP is still unclear. Here we have found that knockout of the Glrx2 gene, which encodes mitochondrial glutaredoxin2 (Grx2), sensitized mice to APAP-caused hepatotoxicity. Glrx2 deletion hindered Nrf2-mediated compensatory recovery of thiol-dependent redox systems after acetaminophen challenge, resulting in a more oxidized cellular state with a further decrease in GSH level, thioredoxin reductase activity, and GSH/GSSG ratio. The weakened feedback regulation capacity of the liver led to higher levels of protein glutathionylation and thioredoxin (both Trx1 and Trx2) oxidation in Glrx2−/− mice. Following the cellular environment oxidation, nuclear translocation of apoptosis-inducing factor (AIF) was elevated in the liver of Glrx2−/− mice. Taken together, these results demonstrated that mitochondrial Grx2 deficiency deteriorated APAP-induced hepatotoxicity by interrupting thiol-redox compensatory response, enhancing the AIF pathway-mediated oxidative damage.

The concerted elevation of conjugation reactions is associated with the aggravation of acetaminophen toxicity in Akr1a-knockout mice with an ascorbate insufficiency

AimsAcetaminophen (APAP) is a relatively safe analgesic drug, but overdosing can cause acute liver failure. Ingested APAP is detoxified by metabolic conversion through conjugation reactions with glucuronate, sulfate, or glutathione (GSH). The consumption of GSH through conjugation as well as mitochondrial dysfunction is considered to be responsible for the increased susceptibility to APAP-induced hepatotoxicity. Compared to wild-type (WT) mice, Akr1a-knockout (KO) mice are vulnerable to developing hepatotoxicity due to the fact that ascorbate synthesis is attenuated. We used such KO mice to investigate how these conjugation reactions are involved in the hepatotoxicity caused by an overdose of APAP under ascorbate-deficient conditions. Main methodsAPAP (400 mg/kg) was intraperitoneally administered to WT mice and KO mice. In addition to histological and blood biochemical analyses, metabolites in the liver, blood plasma, and urine were measured at several time points by liquid chromatography-mass spectrometry. Key findingsLiver damage occurred earlier in the KO mice than in the WT mice. The levels of APAP-Cys, a final metabolite of GSH-conjugated APAP, as well as glucuronidated APAP and sulfated APAP were all higher in the KO mice compared to the WT mice. Treatment of the APAP-administered KO mice with N-acetylcysteine or supplementation of ascorbate suppressed the conjugation reactions at 6 h after APAP had been administrated, which mitigated the degree of liver damage. SignificanceAn ascorbate deficiency coordinately stimulates conjugation reactions of APAP, which, combined with the mitochondrial damage caused by APAP metabolites, collectively results in the aggravation of the acute liver failure.

Fenton-reaction-triggered metabolism of acetaminophen for enhanced cancer therapy

Acetaminophen (APAP), a classic nonsteroidal anti-inflammatory drug (NSAID), has attracted much attention due to the overdose-induced hepatotoxicity in the past several decades. N-Acetyl-p-benzoquinone imine (NAPQI), the P450-dependent metabolism of APAP, leads to GSH depletion, protein binding, mitochondrial oxidative stress, and eventually the liver injury. Herein, we develop a Fe-based metal-organic framework (MOF) to deliver and transform acetaminophen into toxic “chemo” drug through the cascade reaction for enhanced cancer therapy. In the acidic tumor microenvironment, the Fe-based MOF collapses and releases abundant Fe ions to generate hydroxyl radicals (•OH) via Fenton reaction, subsequently catalyzing nontoxic APAP into toxic NAPQI. Meanwhile, NAPQI depletes intracellular glutathione (GSH) rapidly, leading to alleviating the antioxidant ability of cancer cells and amplifying Fenton activity. The intracellular oxidative stress and the toxic metabolite of APAP can provide a synergistic effect on antitumor activity.

Hepatoprotective Effect of Oplopanax elatus Nakai Adventitious Roots Extract by Regulating CYP450 and PPAR Signaling Pathway.

O. elatus Nakai is a traditional medicine that has been confirmed to exert effective antioxidant and anti-inflammatory functions, and is used for the treatment of different disorders. However, its potential beneficial effects on drug induced hepatotoxicity and relevant molecular mechanisms remain unclear. This study investigated the protective effect and further elucidated the mechanisms of action of O. elatus on liver protection. O. elatus chlorogenic acids-enriched fraction (OEB), which included chlorogenic acid and isochlorogenic acid A, were identified by HPLC-MS/MS. OEB was administrated orally daily for seven consecutive days, followed by a single intraperitoneal injection of an overdose of APAP after the final OEB administration. The effects of OEB on immune cells in mice liver were analyzed using flow cytometry. APAP metabolite content in serum was detected using HPLC-MS/MS in order to investigate whether OEB affects CYP450 activities. The intestinal content samples were processed for 16 s microbiota sequencing. Results demonstrated that OEB decreased alanine aminotransferase, aspartate aminotransferase contents, affected the metabolism of APAP, and decreased the concentrates of APAP, APAP-CYS and APAP-NAC by inhibiting CYP2E1 and CYP3A11 activity. Furthermore, OEB pretreatment regulated lipid metabolism by affecting the peroxisome proliferator-activated receptors (PPAR) signaling pathway in mice and also increased the abundance of Akkermansia and Parabacteroides. This study indicated that OEB is a potential drug candidate for treating hepatotoxicity because of its ability to affect drug metabolism and regulate lipid metabolism.

The Role of the Optic Atrophy 1 (OPA1) Protein in Drug‐Induced Liver Injury

The liver is the metabolic hub, and is responsible for the myriad of processes including the nutrient homeostasis and detoxification. Mitochondria of liver are critical for these functions. The detoxification process in liver, when severe, often results in liver damage through causing oxidative stress. Mitochondria are the main source of ROS and are also vulnerable to oxidant damage. Therefore, mitochondrial dysfunction is one of the prominent causes for drug‐induced liver injury.Mitochondrial fission and fusion, the main processes of mitochondrial dynamics, determine mitochondrial shape, and are important for functional maintenance of mitochondria. However, the role of mitochondrial dynamics in drug‐induced liver injury is poorly understood. In the current study, we examined the role of the optic atrophy 1 (OPA1) protein in drug‐induced liver injury. OPA1 is associated with mitochondrial inner membrane (IM) and mediates IM fusion. OPA1 also regulates cristate structure, and is required for proper electron transport and ATP production. To investigate the OPA1’s role, we used liver‐specific OPA1‐knockout (OPA1‐LKO) mice with acetaminophen (APAP) administration as a model for drug‐induced liver injury.We generated OPA1‐LKO mice by crossing OPA1 flox mice with mice carrying the Cre recombinase under the albumin promoter. Whereas whole body KO of OPA1 causes embryonic lethality, OPA1‐LKO mice appeared healthy and showed normal growth and behavior. Although the OPA1 gene in the liver was disrupted, OPA1‐LKO mice showed approximately 30% of OPA1 remaining in the liver, presumably due to less efficient albumin promoter‐mediated Cre expression and from other cell types of the liver. Liver histology revealed that OPA1‐KO livers have disorganized hepatic cords with enlarged hepatocytes. Despite a reduced OPA1 level, mitochondria in OPA1‐KO liver show near intact cristae structure and respiration, suggesting that a low level of OPA1 would support mitochondrial function in liver.We then tested the effect of OPA1 LKO on liver function under APAP stress. In APAP overdose, excess APAP metabolite depletes GSH in hepatocytes, which causes mitochondrial oxidative stress, leading to mitochondrial permeability transition, mitochondrial dysfunction, ATP depletion, and ultimately necrotic cell death. Upon administration of excess APAP, we found that OPA1‐LKO mice were more sensitive to APAP‐induced liver injury compared with the control mice. Histological analyses showed significantly more expanded focal centrilobular necrosis with vacuolization, cell swelling, and nuclear disintegration in OPA1‐KO livers. Alanine aminotransferase levels, as a clinical chemistry parameter, were higher in OPA1‐LKO mice than in control mice with APAP overdose. Furthermore, phospho‐JNK levels were higher in OPA1‐KO livers, indicating increased initial oxidative stress. However, depletion of hepatic glutathione (GSH) contents and reduction of GSH/GSSG ratio upon APAP treatment were similar between the LKO and control liver. Together, our experimental results indicate that although liver is tolerant to a reduced level of OPA1, OPA1 depletion lowers the stress threshold and makes hepatocytes more sensitive to APAP‐induced liver injury.

Acetaminophen treatment evokes anticontractile effects in rat aorta by blocking L-type calcium channels.

Acetaminophen (APAP) is the most widely used analgesic and antipyretic in the world. However, in high or continuous doses, it can cause serious side effects including blood pressure variability and cardiovascular injuries, which are barely explored. This study aimed to evaluate the acute effect of APAP treatment on vascular tone focused on the blocking of Ca2+ channels. Rats were treated with APAP orally by gavage (500mg/kg/single dose). After 12h, the aorta was isolated for vascular reactivity studies in an isolated organ bath. Vascular contraction and relaxation were measured after different stimuli. Moreover, molecular docking studies were performed to evaluate the action of NAPQI (APAP metabolite) on L-type calcium channels. Phenylephrine-induced maximal vascular contraction was reduced in the APAP group (138.4 ± 9.2%) compared to the control group (172.2 ± 11.1%). APAP treatment significantly reduced contraction induced by Ca2+ influx stimulated with phenylephrine or KCl and reduced contraction mediated by Ca2+ released from the sarcoplasmic reticulum induced by caffeine. There was no difference in vascular relaxation induced by acetylcholine or sodium nitroprusside. Computational molecular docking demonstrated that NAPQI is capable of blocking L-type Ca2+ channels (Cav1.2), which would limit the influx of Ca2+. These results suggest that APAP treatment causes an anticontractile effect in rat aorta, possibly by blocking the influx of Ca2+ through L-type channels (Cav1.2).

Metabolite alteration analysis of acetaminophen-induced liver injury using a mass microscope.

Acetaminophen (APAP)-induced liver injury (APAP-ILI), which occurs during APAP overdose, has been extensively studied. The production of N-acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of APAP, primarily contributes to liver injury. However, the mechanism underlying APAP-ILI has not been fully characterized. For further clarification, it is important to consider drug localization and endogenous substances in the injured liver. Herein, we show the localization of NAPQI metabolites and the injury site-specific changes in endogenous substances in the rat liver following APAP overdose using a mass microscope. Our results of on-tissue derivatization matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) showed that the glutathione metabolite of APAP, a detoxified metabolite of NAPQI, localized in the damaged central vein region in the rat liver following APAP administration. Moreover, in the conventional MALDI-MSI, the intensities of some phospholipids, phosphocreatine, and ceramides decreased or increased in the damaged regions compared with those in non-damaged regions. Phosphocreatine was localized in the damaged cells, whereas its related mitochondrial creatine kinase was localized in the non-damaged cells. These results are expected to contribute to further elucidation of the mechanisms underlying APAP-ILI. Our findings illustrate the localization of NAPQI-related metabolites and endogenous molecules associated with APAP-ILI, which may be related to apoptosis or metabolic adaptation ultimately protecting the cells. As MALDI-MSI can analyze and differentiate regions with tissue damage, it is a valuable tool for analyzing the mechanism underlying drug-induced liver injury to identify novel biomarkers.

Optimization of gradient reversed phase high performance liquid chromatography analysis of acetaminophen oxidation metabolites using linear and non-linear retention model

Acetaminophen (paracetamol, APAP) is one of the most widely used drugs worldwide. Unfortunately, its overdose, which is caused by predominant oxidation of APAP, can lead to acute liver injury. In liver, oxidized APAP is conjugated with glutathione, leading to APAP-glutathione conjugate, which is metabolized to APAP-cysteine and APAP-N-acetylcysteine conjugates. Thus, all of those compounds could be used to monitor APAP metabolism in the overdosed patients. To date, only a limited number of rapid and accurate methods have been reported for the assessment of APAP oxidation metabolites using simple instrumentation, and thus this work was aimed at developing a fast and convenient gradient HPLC-UV/MS method. For this purpose, APAP conjugates with glutathione, cysteine, and N-acetylcysteine were synthesized, purified by preparative liquid chromatography, and characterized by NMR and high-resolution mass spectrometry. The gradient elution conditions were optimized using the window diagram approach and the effects of mobile phase composition and additives on separation and detection sensitivity were evaluated using two, i.e., linear and non-linear isocratic retention models. Quantitative parameters of the developed method were evaluated and the effectiveness, sensitivity, and specificity of the method were demonstrated on the analysis of human kidney HK-2 cell lysates, confirming the suitability of the method for routine use in studies on APAP toxicity.