Dose Of APAP Research Articles

Endotoxin-Stimulated Hepatic Stellate Cells Augment Acetaminophen-Induced Hepatocyte Injury

Acetaminophen (APAP)-induced liver injury is influenced by inflammatory Gram-negative bacterial endotoxin [lipopolysaccharide (LPS)], mechanisms of which are not completely understood. Because LPS-stimulated perisinusoidal hepatic stellate cells (HSCs) produce cytokines that affect survival of hepatocytes, this study investigated their role in APAP-induced liver injury. Fed (nonstarved) rats were administered 5 mg/kg LPS or phosphate-buffered saline (PBS) vehicle, followed by 200 mg/kg APAP or PBS an hour later, and euthanized at 6 hours. Control rats received PBS at both time points. Both LPS and APAP caused mild hepatocyte injury (apoptosis), as assessed by histopathology, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and caspase-3 activation. The liver injury was augmented in rats administered LPS + APAP, in association with increased nuclear translocation of interferon-regulatory factor-1 (IRF1). Invitro, APAP augmented LPS/HSC-conditioned medium-induced inhibition of DNA and protein synthesis, apoptosis, and nuclear IRF1 in hepatocytes. LPS-stimulated HSCs produced interferon-β (IFN-β), and LPS/HSC + APAP-induced hepatocyte apoptosis was inhibited by anti-IFN-β antibody. Finally, HSC-depleted mice produced significantly lower IFN-β and tumor necrosis factor-α, exhibited less oxidative stress, and were protected from excessive injury due to high APAP dose (600 mg/kg), as well as LPS (5 mg/kg overnight) followed by APAP. In co-culture with or without LPS, HSCs increased expression of proinflammatory cytokines by Kupffer cells. These results suggest that HSCs play a critical role in APAP-induced liver injury without or with LPS preconditioning, and it involves INF-β-IRF1 signaling.

Crocin Possesses Excellent Hepatoprotective Effects Against Acetaminophen-Induced Hepatotoxicity in Mice

Background: Acetaminophen (APAP) is a common analgesic and antipyretic medicine that can lead to acute liver injury at high doses. Crocin, a Crocus sativus’ ingredient, has potent antioxidant effects. Objectives: This study examined the protective effects of crocin against APAP-induced oxidative stress in mice. Methods: In this study, 56 mice were randomly divided into seven groups (n = 8 per group), including the negative (normal saline, 10 mL/kg) and positive (oral normal saline for five days + a single dose of APAP (300 mg/kg) on day 6th) control groups. The third group (NAC) received normal saline for up to five days, and on the 6th day, immediately after the administration of acetaminophen, received NAC (50 mg/kg). Groups fourth to sixth received respectively 12.5, 25, and 50 mg/kg of crocin (orally for six days), followed by a single dose of APAP (300 mg/kg) on 6th day. The last group received crocin (50 mg/kg) for six days. Then 24 h after the last injection, the animals were sacrificed, and samples were collected for biochemical and histopathological evaluations. Results: The levels of ALT, AST, and MDA increased, and the activity of CAT, GSH, and GPX decreased in the APAP-treated group compared to the control group. In APAP-treated groups, the administration of crocin decreased the serum levels of AST, ALT, and MDA and increased the activity of CAT, GSH, and GPX. Histopathological evaluations confirmed the above findings. Conclusions: According to our results, it seems that crocin has a protective effect against acetaminophen-induced liver toxicity and can be used as a therapeutic agent to treat APAP-induced hepatotoxicity.

Formulating a New Pharmaceutical Drug; Acetaminophen Tablet Containing N-acetyl Cysteine, To Alleviate the Severity of Liver Damage in Rats: Phase I, Animal Study

Background and Aim: Acetaminophen (APAP) is a commonly used analgesic and also the leading cause of medication-induced liver damage. On the other hand, N-acetylcysteine (NAC) is a medication widely used to treat APAP overdose. Despite this interest, a few studies have investigated the co-administration effects of these medications. Therefore, this study aimed to evaluate the effects of NAC and APAP on renal and liver functions in rats when they use concurrently. Methods: Male Wistar rats were orally treated with a single dose of APAP (700 mg/kg) alone or in combination of NAC at the three different doses (200, 500, and 700 mg/kg). After 24 hours, the blood and liver samples were collected for biochemical and histopathological evaluations. Results: Liver damage was well established in the 700 mg/kg APAP-treated rats, as evidenced by elevated the plasma levels of aspartate transaminase (AST) and alanine transaminase (ALT). In addition, the plasma level of blood urea nitrogen (BUN) was significantly increased in the APPA group compared to the control group. Moreover, histological examinations revealed that liver degeneration was evident in APAP-treated animals. NAC only at the highest dose (700 mg/kg) could inhibit ALT elevation, but had no effect on AST and BUN levels. Interestingly, co-administration of NAC (700 mg/kg) with APAP (700 mg/kg) could slightly shift liver histological alterations from the irreversible stage (fibrosis) toward reversible lesions such as necrosis and hemorrhage. Conclusion: The study findings indicate that co-administration of NAC and APAP can reduce the severity of APAP-induced liver damage in rats. *Corresponding Author: Mehran Hosseini; Email: mehranhosseiny@bums.ac.ir; ORCID: https://orcid.org/0000-0002-6793-2035 Please cite this article as: Mehrpour O, Dastjerdi M, Nakhaee S, Amirabadizadeh A, Bijari B, Roomi H, Hosseini M. Formulating a New Pharmaceutical Drug; Acetaminophen Tablet Containing N-acetyl Cysteine, To Alleviate the Severity of Liver Damage in Rats: Phase I, Animal Study. Arch Med Lab Sci. 2021;7:1-8 (e14). https://doi.org/10.22037/amls.v7.35528

COMBINATION THERAPY WITH N-ACETYLCYSTEINE AND 4-METHYLPYRAZOLE FOR ACETAMINOPHEN OVERDOSE

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. We report a case of a patient who presented with acetaminophen overdose and was successfully treated with N-acetylcysteine (NAC) and 4-methylpyrazole (4-MP). CASE PRESENTATION: A 58-year-old female with history of alcohol abuse, hypertension, and hyperlipidemia presented with abdominal pain, nausea, and vomiting. Patient had fallen several days prior, resulting in rib fractures, and had been taking APAP for pain relief. Vital signs were within normal limits. Physical exam showed a lethargic, encephalopathic patient who only followed simple commands and had right upper quadrant abdominal tenderness. Labs revealed total bilirubin 6.4, AST 19816, ALT 5544, INR 3.7, acetaminophen level 58.3, lactic acid 12.2, creatinine 3.1, lipase 1187, and ammonia 82. Imaging was consistent with hepatic steatosis. Patient was treated with aggressive fluid resuscitation for pancreatitis; NAC and 4-MP for acetaminophen toxicity; lactulose and rifaximin for hepatic encephalopathy; vitamin K for coagulopathy; and albumin, octreotide, and midodrine for hepatorenal syndrome. Mental status and lab abnormalities gradually improved and acetaminophen level became undetectable. DISCUSSION: Our patient presented with unintentional APAP overdose. She likely had underlying chronic liver disease, as evidenced by hepatic steatosis on imaging, due to alcohol abuse. This likely made her more susceptible to liver injury, even with therapeutic doses of APAP.The pathogenesis APAP-induced hepatotoxicity starts with the conversion of APAP to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450 enzymes, which is detoxified by conjunction with glutathione.[1] When excessive amounts of APAP are ingested, hepatic glutathione levels are depleted, resulting in oxidant stress. NAC, the current standard of care and only clinically approved treatment for APAP overdose, attempts to limit this by accelerating glutathione synthesis and subsequent scavenging of NAPQI.[2]Recent literature has suggested that 4-MP may also be beneficial for attenuating liver injury in APAP overdose. 4-MP is classically known for its inhibition of alcohol dehydrogenase in methanol or ethylene glycol poisoning. However, it is unique in that it also directly inhibits CYP2E1, one of the major enzymes involved in the generation of NAPQI. 4-MP has been shown to be effective at preventing APAP-induced liver injury in mice and human hepatocytes and limiting oxidative metabolism in five human volunteers who ingested supratherapeutic APAP doses.[1,2] It may also protect against APAP-induced nephrotoxicity via a similar mechanism.[3] CONCLUSIONS: In patients presenting with APAP overdose, combination therapy with NAC and 4-MP may provide complementary protective effects through targeting of different therapeutic pathways. REFERENCE #1: Kang AM, Padilla-Jones A, Fisher ES, et al. The effect of 4-methylpyrazole on oxidative metabolism of acetaminophen in human volunteers. J Med Toxicol. 2020;16(2):169-176. REFERENCE #2: Akakpo JY, Ramachandran A, Kandel SE, et al. 4-Methylpyrazole protects against acetaminophen hepatotoxicity in mice and in primary human hepatocytes. Hum Exp Toxicol. 2018;37(12):1310-1322. REFERENCE #3: Akakpo JY, Ramachandran A, Orhan H, et al. 4-methylpyrazole protects against acetaminophen-induced acute kidney injury. Toxicol Appl Pharmacol. 2020;409:115317. DISCLOSURES: No relevant relationships by Travis Homan, source=Web Response No relevant relationships by Brittany Wichman, source=Web Response

Pulmonary implications of acetaminophen exposures independent of hepatic toxicity.

Both preclinical and clinical studies have demonstrated that exposures to acetaminophen (APAP) at levels that cause hepatic injury cause pulmonary injury as well. However, whether exposures that do not result in hepatic injury have acute pulmonary implications is unknown. Thus, we sought to determine how APAP exposures at levels that do not result in significant hepatic injury impact the mature lung. Adult male ICR mice (8-12 wk) were exposed to a dose of APAP known to cause hepatotoxicity in adult mice [280 mg/kg, intraperitoneal (ip)], as well as a lower dose previously reported to not cause hepatic injury (140 mg/kg, ip). We confirm that the lower dose exposures did not result in significant hepatic injury. However, like high dose, lower exposure resulted in increased cellular content of the bronchoalveolar lavage fluid and induced a proinflammatory pulmonary transcriptome. Both the lower and higher dose exposures resulted in measurable changes in lung morphometrics, with the lower dose exposure causing alveolar wall thinning. Using RNAScope, we were able to detect dose-dependent, APAP-induced pulmonary Cyp2e1 expression. Finally, using FLIM we determined that both APAP exposures resulted in acute pulmonary metabolic changes consistent with mitochondrial overload in lower doses and a shift to glycolysis at a high dose. Our findings demonstrate that APAP exposures that do not cause significant hepatic injury result in acute inflammatory, morphometric, and metabolic changes in the mature lung. These previously unreported findings may help explain the potential relationship between APAP exposures and pulmonary-related morbidity.

Intravenous Versus Oral Acetaminophen Use in Febrile Neurocritical Care Patients.

The use of intravenous (IV) acetaminophen (APAP) for fever has not been thoroughly studied in neurocritical care (NCC) patients, in whom a temperature of ≥38°C is associated with poor outcomes and treatment to normothermia is common practice. This retrospective study evaluated NCC patients admitted between May 1, 2012, and April 30, 2013, and received at least one dose of IV or oral (PO) APAP for a body temperature of ≥38°C. The primary aim of this study was to compare the reduction in body temperature (RIT) between IV and PO APAP, calculated as the change in temperature before and 0.5, 1, 2, 3, and 6 hours after administration. Descriptive statistics were used to assess use characteristics, and Kruskal-Wallis and Mann-Whitney U tests were used for between-group differences. There were 142 NCC patients who received a total of 405 IV APAP and 253 PO APAP doses. Seventy percent of all APAP doses resulted in a temperature of <38°C within 6 hours. The median oral body temperature before APAP was 38.8°C and 38.6°C for IV and PO APAP, respectively (p < 0.01). The median RIT at 0.5 (IV 0.25°C vs. PO 0.2°C), 1 (IV 0.4°C vs. PO 0.2°C), 2 (IV 0.7°C vs. PO 0.5°C), 3 (IV 0.9°C vs. PO 0.6°C), and 6 (IV 1°C vs. PO 0.8°C) hours was significantly greater for IV APAP than for PO APAP at all time points (p < 0.05). Patients with an acute ischemic stroke and patients with an intracerebral hemorrhage had a statistically significantly greater RIT with IV APAP therapy. IV APAP administered to febrile NCC patients was associated with a significantly greater RIT than PO, but 70% of all APAP doses resulted in a body temperature of <38°C within 6 hours. Further prospective studies are needed to determine if IV APAP improves clinical outcomes.

Exogenous phosphatidic acid reduces acetaminophen-induced liver injury in mice by activating hepatic interleukin-6 signaling through inter-organ crosstalk

We previously demonstrated that endogenous phosphatidic acid (PA) promotes liver regeneration after acetaminophen (APAP) hepatotoxicity. Here, we hypothesized that exogenous PA is also beneficial. To test that, we treated mice with a toxic APAP dose at 0 h, followed by PA or vehicle (Veh) post-treatment. We then collected blood and liver at 6, 24, and 52 h. Post-treatment with PA 2 h after APAP protected against liver injury at 6 h, and the combination of PA and N-acetyl-l-cysteine (NAC) reduced injury more than NAC alone. Interestingly, PA did not affect canonical mechanisms of APAP toxicity. Instead, transcriptomics revealed that PA activated interleukin-6 (IL-6) signaling in the liver. Consistent with that, serum IL-6 and hepatic signal transducer and activator of transcription 3 (Stat3) phosphorylation increased in PA-treated mice. Furthermore, PA failed to protect against APAP in IL-6-deficient animals. Interestingly, IL-6 expression increased 18-fold in adipose tissue after PA, indicating that adipose is a source of PA-induced circulating IL-6. Surprisingly, however, exogenous PA did not alter regeneration, despite the importance of endogenous PA in liver repair, possibly due to its short half-life. These data demonstrate that exogenous PA is also beneficial in APAP toxicity and reinforce the protective effects of IL-6 in this model.

Alterations of Cytochrome P450-Mediated Drug Metabolism during Liver Repair and Regeneration after Acetaminophen-Induced Liver Injury in Mice.

Acetaminophen (APAP)-induced liver injury (AILI) is the leading cause of acute liver failure in the United States, but its impact on metabolism, therapeutic efficacy, and adverse drug reactions (ADRs) of co- and/or subsequent administered drugs are not fully investigated. The current work explored this field with a focus on the AILI-mediated alterations of cytochrome P450-mediated drug metabolism. Various levels of liver injury were induced in mice by treatment with APAP at 0, 200, 400, and 600 mg/kg. Severity of liver damage was determined at 24, 48, 72, and 96 hours by plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), microRNA miR122, and tissue staining. The expression and activities of CYP3A11, 1A2, 2B10, 2C29, and 2E1 were measured. Sedation efficacy and ADRs of midazolam, a CYP3A substrate, were monitored after APAP treatment. ALT, AST, and miR122 increased at 24 hours after APAP treatment with all APAP doses, whereas only groups treated with 200 and 400 mg/kg recovered back to normal levels at 72 and 96 hours. The expression and activity of the cytochromes P450 significantly decreased at 24 hours with all APAP doses but only recovered back to normal at 72 and 96 hours with 200 and 400, but not 600, mg/kg of APAP. The alterations of cytochrome P450 activities resulted in altered sedation efficacy and ADRs of midazolam, which were corrected by dose justification of midazolam. Overall, this work illustrated a low cytochrome P450 expression window after AILI, which can decrease drug metabolism and negatively impact drug efficacy and ADRs. SIGNIFICANCE STATEMENT: The data generated in the mouse model demonstrated that expression and activities of cytochrome P450 enzymes and correlated drug efficacy and ADRs are altered during the time course of liver repair and regeneration after liver is injured by treatment with APAP. Dose justifications based on predicted changes of cytochrome P450 activities can achieve desired therapeutic efficacy and avoid ADRs. The generated data provide fundamental knowledge for translational research to drug treatment for patients during liver recovery and regeneration who have experienced AILI.

Modulation of NRF2/ARE pathway- and cell death-related genes during drug-induced liver injury.

Transcriptional factor NRF2 is an emerging tool in reviewing mechanistic behavior of drug-specific injury pathways. Drug-induced liver injury (DILI) represents a major clinical concern that often manifests oxidative stress and cell death. Despite the pivotal role of NRF2 pathway in liver pathologies, it is questioned whether NRF2 activation or regulatory efficiency could be hindered in by the severity of DILI and progression of cell death. In this study, we evaluate NRF2 as a biomarker to DILI in comparison to severity of injury as well as explore stress mediating factors affecting Nrf2 expression. In vivo DILI model was established in C57BL/6 mice by acetaminophen (APAP) at different toxic doses, confirmed by dose-dependent liver pathological changes and accompanied with in vitro time- and dose-dependent depletion of GSH and SOD in isolated primary mouse hepatocytes. Increase in liver NRF2 translocation and cytosolic content was observed in 70 mg/kg APAP-treated mice. At this subtoxic dose, liver Nrf2 transcription was increased in mice by 18.3-fold, a prominent downregulation was seen in ARE (antioxidant response element) genes; Hmox1, Nqo1 and Glcm, and apoptotic Bcl2 regulating genes. In addition, upregulation in necrosis inducer Parp2 was associated to downregulation in Hmgb1. Collectively, expression of genes related to cell survival were regulated at mild APAP hepatotoxicity. By increasing APAP dose, hemorrhagic necrosis and impaired genetic transcription in both Nrf2 and several other genes were evident. In conclusion, NRF2/ARE system and cell death modulation is halted by the increase of chemical stress and found directly associated with DILI severity.

Efficacy of a Fixed-Dose Combination of Ibuprofen and Acetaminophen Compared With Individual Monocomponents in Adult Male Subjects With Endotoxin-Induced Fever: A Randomized Controlled Trial

PurposeThis study evaluated antipyretic efficacy and onset of a novel fixed-dose combination (FDC) of ibuprofen (IBU; 250 mg) and acetaminophen (APAP; 500 mg) compared with placebo and IBU or APAP monocomponents. MetThis single-center, randomized, double-blind, placebo-controlled, full-factorial study was conducted in healthy males aged 18 to 55 years with pyrexia induced by intravenous administration of reference standard endotoxin (RSE). After attainment of an oral temperature ≥38.1°C, subjects were randomized 3:3:3:1 to a double-blind single oral dose of FDC IBU/APAP 250 mg/500 mg, APAP 500 mg, IBU 250 mg, or placebo. Oral temperature was measured every 10 minutes for 2 hours, then every 30 minutes until 8 hours postdose. Time-weighted sum of temperature differences from baseline to 8 hours (WSTD0–8) after study medication administration was the primary efficacy end point. Secondary end points included WSTD scores from 0 to 2 hours, 0 to 4 hours, 0 to 6 hours, and 6 to 8 hours; time to return to “normal” temperature; time to rescue medication use; and global drug evaluation. Safety was assessed via adverse events (AEs). FindingsTwo hundred ninety subjects were randomized; 273 were included in the primary efficacy analysis. WSTD0–8 was significantly better for FDC IBU/APAP 250 mg/500 mg (P = 0.002), IBU 250 mg (P = 0.030), and APAP 500 mg (P = 0.023) versus placebo; there were no significant differences between active treatments. For WSTD0–2, only the FDC was statistically significant versus placebo (P = 0.004). All active treatments were significantly better (P < 0.05) for WSTD0–4 and WSTD0–6 versus placebo; there were no differences in WSTD6–8 between cohorts. Temperature returned to normal during the 8-hour treatment period in ∼50% of subjects in each cohort. Only 1 subject (IBU cohort) took rescue medication. Post hoc analyses at early time points revealed significant treatment differences favoring FDC versus placebo and IBU for the WSTD from baseline during the 50- to 110-minute posttreatment window; for WSTD from baseline during the 80- to 110-minute posttreatment window, FDC provided significant treatment differences versus placebo and both monocomponents. Overall, 223 (76.9%) of 290 subjects experienced AEs related to RSE; only 2 subjects experienced treatment-related AEs (FDC, rash; placebo, ear pain). ImplicationsAlthough the primary end point was not met, these results suggest that FDC IBU/APAP 250 mg/500 mg provides effective antipyresis with a faster onset versus equal doses of IBU and APAP alone. ClinicalTrials.gov identifier: NCT02761980.

The effects of mitochondrial transplantation in acetaminophen-induced liver toxicity in rats

AimsAcetaminophen (APAP) toxicity is one of the leading causes of acute liver injury-related death and liver failure worldwide. In many studies, mitochondrial dysfunction has been identified as an important cause of damage in APAP toxicity. Therefore, our study aimed to investigate the possible effects of mitochondrial transplantation on liver damage due to APAP toxicity. Main methodsAPAP toxicity model was implemented by administering a toxic dose of APAP. To demonstrate the efficiency of mitochondria transplantation, it was compared with N-acetylcysteine (NAC) application, which is now clinically accepted. Mitochondrial transplantation was carried out by delivering mitochondria to the liver via the portal circulation, which was injected into the spleen. In our study, the rats were randomly divided into 6 groups as Sham, APAP, Control 1, APAP+mito, Control 2, and APAP+NAC. In the end of the experiment, histological and biochemical analysis were performed and the biodistribution of the transplanted mitochondria to target cells were also shown. Key findingsSuccessful mitochondrial transplantation was confirmed and mitochondrial transplantation improved the liver histological structure to a similar level with healthy rats. Moreover, plasma ALT levels, apoptotic cells, and total oxidant levels were decreased. It was also observed that NAC treatment increased GSH levels to the highest level among the groups. However, mitochondrial transplantation was more effective than NAC application in terms of histological and functional improvement. SignificanceIt has been evaluated that mitochondrial transplantation can be used as an important alternative or adjunctive treatment method in liver damage caused by toxic dose APAP intake.

Vitamin D deficiency exacerbates hepatic oxidative stress and inflammation during acetaminophen-induced acute liver injury in mice

Several experiments confirmed that vitamin D3 protected against acetaminophen (APAP)-induced acute liver injury (ALI). This research aimed to evaluate the influence of vitamin D deficiency (VDD) on APAP-induced ALI. In VDD and VDD + APAP groups, mice were fed with VDD diet. In APAP and VDD + APAP groups, mice were intraperitoneally injected with a sublethal dose of APAP (150 mg/kg). A sublethal dose of APAP caused a slight elevation of ALT and AST. Interestingly, APAP-induced elevation of ALT and AST was aggravated in VDD-fed mice. APAP-induced hepatic necrosis was exacerbated in VDD-fed mice. In addition, APAP-induced hepatocyte death, measured using TUNEL assay, was exacerbated in VDD-fed mice. Additional experiment showed that APAP-induced hepatic GSH depletion and lipid peroxidation were exacerbated in VDD-fed mice. Moreover, APAP-induced upregulation of antioxidant genes, such as hepatic heme oxygenase-1 (Ho-1), glutathione peroxidase (Gshpx), superoxide dismutase 1 (Sod1) and catalase enzymes (Cat), was aggravated in VDD-fed mice. Although a sublethal dose of APAP did not cause hepatic inflammation, hepatic proinflammatory cytokines and chemokines, such as Tnf-α, Kc, Mcp-1 and Mip2, were upregulated in VDD-fed mice treated with APAP. These results provide experimental data that VDD exacerbates hepatic oxidative stress and inflammation during APAP-induced ALI.

A n injured liver caused by overdose of acetaminophen has altered expression and activities of P450 enzymes and associated drug efficacy during liver recovery and regeneration

Acetaminophen (APAP)-induced liver injury (AILI) is the leading cause of acute liver failure in the United States. Alterations on therapeutic efficacy and adverse drug reactions (ADRs) in patients with AILI are not fully investigated. Our previous study showed that AILI could result in dose-dependent and immediate decreases in expression and activities of major hepatic drug-metabolizing cytochrome P450s (P450s) enzymes in mice. However, liver will go through regeneration after AILI. How P450-mediated drug metabolism alters during liver regeneration is unclear. The aim of the study was to investigate the alterations in expression and activities of major P450 enzymes and associated drug efficacy and ADRs during liver regeneration after AILI. Adult male C57BL6/J mice were treated with various doses of APAP at 0 (no damage control), 200 (mild damage and recoverable), 400 (severe damage but recoverable), and 600 mg/kg (life-threatening damage and unrecoverable). The mice were sacrificed at 24, 48, 72, and 96 hours after the APAP treatment. Severity of liver damage was detected by plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and miR122, with further confirmation by H & E staining in liver tissue sections. The expression and activities of CYP1A2, 2B10, 2C29, 2E1, and 3A11 were measured by RT-PCR (mRNAs) and MS/MS quantification of probe drugs, respectively. Sedation efficacy of midazolam was monitored at different time points after APAP treatment at different doses. The levels of ALT and AST significantly increased at 24 hours after APAP treatment at 200 and 400 mg/kg but dropped back to normal levels at 72 and 96 hours. No mice treated with 600 mg/kg APAP could survive to 72 hours. The magnitude of liver damage was further confirmed by H & E staining. The major examined P450 enzymes showed significant decreases at 24 hours in gene expression and activity levels and recovered back to normal with the doses of 200 and 400, but not 600 mg/kg of APAP. The activities of examined major P450s are correlated with the levels of ALT, AST, and mir122. The alterations of CYP3A11 resulted in correlated changes in sedation efficacy as reflected by sleeping times with midazolam treatment. These results indicated that P450 expression and function could recover after mild and even severe, but not lethal AILI. Overall, these results suggest that ALT, AST, and miR122 are potential biomarkers for predicting how alterations in P450 activities can justify dose selection of other drugs for desired efficacy. Further studies are needed to confirm these findings in human patient populations with AILI and to understand the underlying molecular mechanisms mediating recovery of hepatic P450s during AILI.

Diploid hepatocytes promote compensatory liver regeneration following acetaminophen induced acute liver injury

Background Acetaminophen (APAP) is a commonly used analgesic that can be safely used at doses under 4 g/day. When taken in excess, APAP causes acute liver injury, which leads to acute liver failure and death. APAP overdose leads to 56,000 emergency room visits and 26,000 hospitalizations annually and is the leading cause of acute liver failure in the United States. The only available pharmacological treatment, N-acetyl cysteine, is effective within hours of overdose. The only other treatment option is orthotopic liver transplantation, and this is limited by the availability of donor organs. Considering the paucity of therapeutic options, there is an urgent need to understand mechanisms of liver regeneration and repair. Most mammalian somatic cells are diploid and contain pairs of each chromosome, but there are also polyploid cells, such as hepatocytes that contain additional sets of chromosomes. Polyploid hepatocytes are among the best described, and in adult humans and mice comprise more than 25% and 90%, respectively, of the hepatocyte population. The cellular and molecular mechanisms that regulate polyploidy have been well-characterized; however, it is poorly understood if diploid and polyploid hepatocytes play specialized roles in liver injury and repair. Methods Our lab recently found that diploid hepatocytes are inherently more proliferative than polyploid hepatocytes. Additionally, we can specifically study diploid hepatocytes using mice lacking E2f7 and E2f8 in the liver (referred to as “LKO” mice), which are functionally normal, but livers are depleted of polyploid hepatocytes. Considering that liver regeneration affects the outcome of APAP-medicated injury, we asked if LKO mice enriched with highly proliferative diploid hepatocytes would respond differently than control livers. We fasted LKO and control mice for 16 hours, injected intraperitoneally with 300 mg/kg (the LD50 dose) APAP and harvested livers after 0.5, 3, 6, 12, 24, 48, 72 and 96 hours. We used these tissues to investigate differences in liver injury, necrosis, and proliferation. Results Liver injury enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were higher in control mice compared to LKO mice beginning at 3 hours. Necrotic liver tissue was abundant in control mice and was markedly reduced in LKO mice. We also measured more apoptotic, TUNEL positive hepatocytes in control mice. We next assessed the proliferation response after APAP-induced liver injury by staining tissues for PCNA which was induced by 6 hours in LKO mice compared to 24 hours in control mice. We treated wild-type 19-day old hepatocytes with 5 mM APAP for 12, 24, and 36 hours in vitro and found that diploid hepatocytes proliferate more readily compared to polyploids as marked by BrdU incorporation. Finally, we found higher doses of APAP eliminate the advantages seen in LKO mice. Conclusion Based on these data, diploid hepatocytes appear to be the main contributors of early-stage compensatory regeneration following APAP-mediated liver injury. These data suggest that, compared to polyploid hepatocytes, diploids initiate and drive liver healing/regeneration after acute injury.

Acute Liver Injury With Therapeutic Doses of Acetaminophen: A Prospective Study.

Because of the extensive use of this drug, further evaluation of acute liver injury (ALI) with therapeutic doses of acetaminophen (APAP; ≤6g/d) is required. We characterize ALI with therapeutic doses of APAP and determine the host factors associated with disease severity and the predictors of outcome. All patients admitted with severe APAP-related ALI in our center were included from 2002 to 2019, either attributable to therapeutic doses or overdose. ALI with therapeutic doses (ALITD) was defined as APAP intake <6g/d. Overall, 311 of 400 patients with APAP-related ALI had overdose and 89 had taken therapeutic doses. The host factors associated with ALITD were fasting ≥1day (47.5% of ALITD patients vs. 26% in overdose; P=0.001), excess drinking (93.3% vs. 48.5%; P<0.0001), and repeated APAP use (4 vs. 1day; P<0.0001). Patients with ALITD were older (44 vs. 30.7years; P<0.0001) and had more severe liver injury. In the overall population, the independent predictors of disease severity were older age, longer duration of APAP, and excess drinking. Thirty-day survival was lower in ALITD than in overdose (87.2±3.6% vs. 94.6±1.3%; P=0.02). Age and the presence of at least one of the King's College Hospital criteria were independent predictors of 30-day survival whereas the pattern of drug intoxication, excess drinking, and bilirubin were not. ALI with therapeutic doses of APAP is associated with more severe liver injury than overdose. It only occurs in patients with excess drinking and/or fasting. A warning should be issued about the repeated use of nontoxic doses of APAP in patients with those risk factors.

The developing murine lung is susceptible to acetaminophen toxicity.

Acetaminophen (n-acetyl-p-aminophenol, APAP) use in the neonatal intensive care unit is rapidly increasing. Although APAP-related hepatotoxicity is rarely reported in the neonatal literature, other end-organ toxicity can occur with toxic exposures. APAP-induced lung injury has been reported with toxic exposures in adults, but whether this occurs in the developing lung is unknown. Therefore, we tested whether toxic APAP exposures would injure the developing lung. Neonatal C57BL/6 mice (PN7, early alveolar stage of lung development) were exposed to a dose of APAP known to cause hepatotoxicity in adult mice (280 mg/kg, IP). This exposure induced significant lung injury in the absence of identifiable hepatic toxicity. This injury was associated with increased pulmonary expression of Cyp2e1, the xenobiotic enzyme responsible for the toxic conversion of APAP. Exposure was associated with increased pulmonary expression of antioxidant response genes and decreased pulmonary glutathione peroxidase activity level. Furthermore, we observed an increase in pulmonary expression of proinflammatory cytokines and chemokines. Lastly, we were able to demonstrate that this toxic APAP exposure was associated with a shift in pulmonary metabolism away from glycolysis with increased oxidative phosphorylation, a finding consistent with increased mitochondrial workload, potentially leading to mitochondrial toxicity. This previously unrecognized injury and metabolic implications highlight the need to look beyond the liver and evaluate both the acute and long-term pulmonary implications of APAP exposure in the perinatal period.

AMELIORATIVE EFFECTS OF HESPERIDIN AND MELATONIN AGAINST ACETAMINOPHEN-INDUCED NEPHROTOXICITY IN ADULT ALBINO RATS

ABSTRACT Background: Acetaminophen (APAP) is the most widely used analgesic antipyretic drug, either by prescription or over the counter. APAP overdose can damage multiple organs, especially the liver and kidney due to the formation and clearance of its toxic metabolites. Aim: To investigate and compare the ameliorative effects of hesperidin (HSP) and melatonin (MEL) in rats with APAP-induced nephrotoxicity. Methodology: This comparative experimental study was carried out on 72 adult male Wistar albino rats were divided into nine groups (eight/each): groups (1-5): controls “negative, solvent, N-acetyl cysteine (NAC), MEL and HSP respectively; group (6): APAP-nephrotoxicity “rats were given a single dose (900 mg/kg) of APAP intraperitoneally (i.p.); group (7): APAP+NAC (150mg/Kg); group (8): APAP+MEL (10mg/Kg) and group (9): APAP+HSP (200mg/Kg). All treated drugs (NAC, MEL, and HSP) were given i.p. 30 minutes before APAP administration. Renal blood flow (RBF), blood urea, serum creatinine, tissue malondialdehyde (MDA), reduced glutathione (GSH), tumor necrotic factor alfa (TNF-α), and interleukin-6 (IL-6) were estimated by colorimetric methods. Immunohistochemical expression of inducible nitric oxide synthase (iNOS) was detected and histopathological examination of renal tissue specimens was performed. Results: HSP; MEL and NAC partially improved APAP-induced nephrotoxicity, evidenced by biochemical, histopathological, and immunohistochemical findings. Conclusion: Both HSP and MEL alleviated APAP-induced nephrotoxicity, however, HSP was superior to MEL and it was as equal as NAC (which was also superior to MEL) as regards the tested parameters. The ameliorative effects of HSP and MEL may be at least in part due to their antioxidant and anti-inflammatory properties.

Renoprotective effects of cinnamon oil against APAP-Induced nephrotoxicity by ameliorating oxidative stress, apoptosis and inflammation in rats

Acetaminophen (APAP) is used as a primary medication in relieving moderate pain and fever. However, APAP is associated with toxic effects in renal tissue that appear because of its free radicals property. The principle goal of the present work is to assess the kidney damage by APAP and its restore antioxidative property of cinnamon oil (CO). Animals were distributed into six animals each in six groups. Rats were administered with three varying doses of CO from 50 to 200 mg/kg b.w. respectively and only a single dose of APAP. APAP induced an alteration in serum biochemical markers, imbalance in oxidative parameters, morphological changes in kidney tissue along with increased interleukins cytokines (IL-1β & 6) and caspase (3, 9) levels. CO administration significantly ameliorates all the parameters and histopathological changes were restored. Moreover, it also restored the activities of antioxidative enzymes. Our work proved that an variance of oxidative markers in the kidney by APAP is ameliorated by CO in rats. Thus, CO could be used in reducing APAP-induced nephrotoxicity.

Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice.

Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75mg/kg and 150mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.

Mitochondrial Membrane Potential Drives Early Change in Mitochondrial Morphology After Acetaminophen Exposure.

Mitochondrial morphology plays a critical role in regulating mitochondrial and cellular function. It is well established that oxidative stress and mitochondrial injury are central to acetaminophen (APAP) hepatotoxicity. However, the role of mitochondrial dynamics, namely the remodeling of mitochondrial morphology through fusion and fission, has largely gone unexplored. To investigate this, we used primary mouse hepatocytes treated with APAP which allowed for real-time visualization of mitochondrial morphology using mitotracker green. We found that alterations in mitochondrial morphology were dose dependent, with a biphasic response in mitochondrial shape at higher APAP doses. Importantly, these two distinct mitochondrial morphologies corresponded with differences in mitochondrial respiratory function and polarization. The early change in mitochondrial morphology can be reversible and appears to be an adaptive response caused by alterations in membrane potential, which ultimately help preserve mitochondrial function. The later delayed change in mitochondrial morphology is irreversible and is driven by loss of mitochondrial membrane potential, decreased canonical fusion proteins, and alterations in mitochondrial lipid composition. Collectively, these later changes tilt the scales toward mitochondrial fission resulting in fragmented mitochondria with reduced functionality. This work provides evidence of adaptive early changes in mitochondrial morphology, which results in functional consequences that are dictated by the severity of APAP overdose.