Acetaminophen overdose can cause liver injury. Hepatotoxicity results from N-acetyl-p-benzoquinone imine (NAPQI), generated via cytochrome P450-2E1 (CYP2E1). Acetylcysteine is the mainstay of treatment, but efficacy may be reduced with delayed administration or large ingestions. Fomepizole, a CYP2E1 inhibitor, reduces NAPQI when co-administered with acetaminophen, but its clinical relevance remains uncertain. The objective was to evaluate the effect of fomepizole administered 2 hours after acetaminophen ingestion in immediate- and modified-release overdose. In a randomized crossover study simulating overdose, healthy volunteers received ~≈80 mg/kg of either immediate- or modified-release acetaminophen followed by intravenous fomepizole or placebo 2 hours later. Volunteers crossed over after 2 weeks. Urine and serum were collected over 24 hours to measure acetaminophen, non-toxic metabolites (APAP-Glu, APAP-Sul), and NAPQI metabolites (APAP-Mer, APAP-Cys). The primary outcome was the proportion of NAPQI metabolites in urine relative to total excreted compounds. Secondary outcomes included 24-hour AUCs for metabolites. Five crossover pairs were completed per formulation. Median 24-hour urinary recovery was 92% (IQR: 89-95%). Fomepizole significantly reduced the mean percentage of urinary NAPQI metabolites for both immediate-release (4.92% [SD: 0.97] vs. 1.72% [SD: 0.3], mean difference [fomepizole-control]: -3.20%, 95% CI: -4.45 to -1.95%, P = 0.0021) and modified release acetaminophen (5.62% [SD: 1.1] vs. 1.43% [SD: 0.3], mean difference [fomepizole-control]: -4.19%, 95% CI: -5.23 to -3.15%, P = 0.0004). Mean serum NAPQI metabolite AUCs were lower with fomepizole: 89.3 [SD: 11.1] vs. 32.5 [SD: 3.3] μmol/L*h (mean difference [fomepizole-control]: -56.8, 95% CI: -72.6 to -41.0, P =0.0006) for immediate-release, and 96.7 [SD: 15.2] vs. 27.2 [SD:7.2] μmol/L*h (mean difference [fomepizole-control]: -69.5, 95% CI:-84.6 to -54.4, P = 0.0002) for modified-release acetaminophen. Fomepizole, administered 2 hours after an acetaminophen overdose, reduced NAPQI formation by 60-70%, supporting further evaluation as an adjunct in overdoses where acetylcysteine may be insufficient.

BACKGROUND:One-carbon metabolism supports methyl-donor availability for cellular methylation reactions. (6S)-5-methyltetrahydrofolate dicholine salt (5-MTHF-DCh) is a source of bioactive folate and choline, and a potential modifier of one-carbon metabolism and DNA methylation. However, despite the potential relevance of this dual folate-choline formulation to dietary supplement science, its effects on methyl-donor metabolism and downstream epigenetic endpoints remain insufficiently characterized. STUDY DESIGN:Here, we tested whether 5-MTHF-DCh modifies one-carbon metabolism endpoints and DNA methylation of selected repetitive elements (RE) in HepaRG cells. Differentiated HepaRG cells were treated with 5-MTHF-DCh (0, 1, 10, or 40 nM) for 96 h. Cellular S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and methionine were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). DNA methylation was assessed for five RE (L1PA2, L1PA13, L1PREC1, Alu, and HERVK11I long terminal repeat, LTR). Transcript levels were measured by quantitative real time PCR (qRT-PCR) for DNA methylation machinery and one-carbon enzymes as well as for selected RE. Dose effects were evaluated by ANOVA with Holm-adjusted comparisons. RESULTS:5-MTHF-DCh increased cellular SAM by 71% at 40 nM (p < 0.01). SAH decreased modestly at 1 nM (-31%, p < 0.01), while methionine was unchanged across doses. Among the RE, L1PA2 methylation increased at all doses with an inverted U-shaped pattern, while HERVK11I LTR methylation increased in a dose-dependent manner, with a 14-fold increase at 40 nM (p < 0.001). These changes were accompanied by a trend toward increased BHMT expression at 40 nM (2.1-fold; p = 0.074), while the mRNA levels of the DNA methylation machinery remained unchanged. In HepaRG cells, 5-MTHF-DCh increased SAM and was associated with selective increases in DNA methylation at specific RE, without detectable changes in core DNA methylation machinery transcripts. CONCLUSIONS:These findings support 5-MTHF-DCh as a modulator of methyl-donor status with measurable epigenetic endpoints in a human hepatic cell model, and provide mechanistic evidence relevant to further evaluation of this folate-choline ingredient.

Liver transplantation is often the only life-saving intervention in acute liver failure (ALF), so it is critical to identify all ALF patients who need a transplant to survive. However, transplantable organs are scarce and recipients face significant life-threatening risks, so it is also important to avoid transplantation when possible. Thus, prognostics that can predict death in ALF with both high sensitivity and specificity are needed. We previously demonstrated total lactate dehydrogenase (LDH) activity may be useful for this purpose. However, LDH is a tetramer of LDH-M and LDH-H, and proteomics indicated only the liver enzyme - LDH-M - increases in non-survivors. LDH-H decreased. Because total LDH comprises both, it is possible that LDH-M could have even better prognostic performance. To test that, we compared total LDH and LDH-M in a subset of samples from our prior study of survivors and non-survivors of acetaminophen (APAP)-induced ALF. The model for end-stage liver disease (MELD) score and the MELD-LDH score were also calculated. Both total LDH and LDH-M values were greater in non-survivors than survivors, but there was no significant difference in prognostic metrics between them. Overall, total LDH performed similarly to or modestly better than both LDH-M and the MELD. The MELD-LDH score also performed somewhat better than the MELD. See also the graphical abstract(Fig. 1).

Serum activities of alanine- and aspartate aminotransferases (ALT and AST) are considered the "gold standard" biomarkers of hepatocyte injury in clinical practice and drug development. However, due to the expression of ALT and AST in myocytes, the diagnosis of hepatocellular injury in patients with underlying muscle diseases, including drug-induced muscle injury, is severely limited. Thus, we proposed glutamate dehydrogenase (GLDH) as a liver-specific alternative to serum ALT and AST. In fact, our exploratory studies showed that GLDH has comparable performance to ALT for detecting hepatocyte injury without interference from concomitant muscle injury. Here, we report the results of studies confirming the reference intervals in a healthy human population and the sensitivity and specificity of GLDH for the detection of hepatocyte injury in human subjects. In human subjects, we could not perform liver biopsies due to ethical reasons; we also confirmed the relationship of GLDH and histopathologic lesions using 32 model toxicants in rats. Furthermore, we have shown that injury to tissues that are known to express appreciable levels of GLDH does not affect serum GLDH measurements, indicating excellent liver specificity of serum GLDH. Finally, we observed faster elimination of GLDH than ALT in humans, indicating that decreasing GLDH values could be considered an early sign of recovery. This study provides comprehensive evidence of excellent sensitivity and liver specificity of GLDH for diagnosis of hepatocellular injury, including evaluation of reference intervals, which is essential for the interpretation of serum GLDH in human subjects.

Macrophages displaying a pro-repair and anti-inflammatory polarization have been implicated in resolution of acute liver injury. Macrophage receptor with collagenous structure (MARCO) expression marks tolerogenic hepatic macrophages and is expressed by pro-resolution macrophages in the injured liver. We tested the hypothesis that MARCO promotes repair of the acetaminophen (APAP)-injured liver. Robust and sustained induction of MARCO mRNA and protein expression was evident in livers of mice challenged with a hepatotoxic dose of APAP (i.e. 300 mg/kg), whereas hepatic MARCO induction failed in mice with APAP-induced liver failure (i.e. 600 mg/kg). Serum proteomics identified a significant increase in serum MARCO levels in surviving acute liver failure (ALF) patients, but not in ALF patients who died. MARCO expression was high in F480+ liver macrophages, and MARCO deficiency reduced macrophage expression of pro-resolution markers such as Gpnmb and Mertk during the repair phase (i.e. 48 h). The results suggested a delay in necrosis resolution along with a trend toward increased mortality in APAP-challenged MARCO-/- mice. Notably, a robust increase in peak hepatic injury (i.e. 6- to 24-h post-APAP challenge) was evident in MARCO-/- mice, which could not be ascribed to differences in NAPQI/APAP-adduct generation nor changes in hepatic neutrophil/macrophage numbers. Interestingly, a reduction in hepatic CD11c+ cells, shown previously to limit APAP-induced liver injury, was evident 24 h after APAP challenge in MARCO-/- mice. The results indicate that MARCO deficiency worsens APAP-induced acute liver injury in mice and provide experimental and initial translational evidence linking MARCO induction to positive outcomes in acute liver injury.

Acetaminophen (APAP) is the most-used over-the-counter analgesic among pregnant women. However, concerns have arisen over the safety of APAP exposure during gestation. In particular, it's been speculated that the hepatotoxic metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), forms in the brain after maternal use of therapeutic APAP doses and leads to neurodevelopmental disorders (NDDs). However, APAP metabolism in the brain is understudied. Here, we tested the hypothesis that NAPQI can be generated in the brain by overdosing BTBR T+ Itpr3tf/J (common model of the NDD autism) and C57Bl/6J mice with APAP and measuring glutathione loss and APAP-protein adducts as two of the best markers of NAPQI available. Despite glutathione depletion and adducts in the liver, we saw none in the brain. We conclude NAPQI is unlikely to contribute to the pathophysiology of NDDs. It has been hypothesized that NAPQI formation in the brain provides biological plausibility for the purported link between APAP and NDDs. Our results cast doubt on that hypothesis.

Acute liver failure (ALF) is an uncommon but severe condition with high morbidity and mortality. Advances in supportive care have improved patient outcomes, but liver transplantation remains the only life-saving intervention in many cases. Unfortunately, healthy donor livers are in short supply. In addition, transplant recipients face several potentially fatal risks including organ rejection, biliary and vascular complications, and infection. It is therefore critical to accurately identify patients who need a new liver while sparing those who do not. This also needs to be done quickly, within the first few days of hospital admission, due to the rapid progression of ALF. Prognostic tools, like the Clichy criteria, the King's College Criteria (KCC), the model for end-stage liver disease (MELD) score, the Acute Liver Failure Study Group Prognostic Index (ALFSGPI), and others have been available for this purpose since at least the 1980s and are commonly used today, but their performance is imperfect, leading to many efforts over the last several decades - and especially in recent years - to identify new noninvasive biomarkers. This review begins with a description of the earliest liver function (e.g. the levulose [fructose] test) and liver injury (e.g. alkaline phosphatase [ALP] and alanine aminotransferase [ALT]) tests and continues through the most recent proposed biomarkers, with critical evaluation of the prognostic utility of each using the KCC and MELD as benchmarks for comparison. Overall, there is as-yet no single biomarker that clearly and consistently performs better than the latter tools, though many may modestly improve the performance of the KCC or MELD when used in combination with them. The search for a better, single biomarker is therefore likely to continue.

Basic science is critical for understanding fundamental biological processes and disease mechanisms [...]

Our understanding of the fundamental molecular mechanisms of acetaminophen (APAP) hepatotoxicity began in 1973 to 1974, when investigators at the US National Institutes of Health published seminal studies demonstrating conversion of APAP to a reactive metabolite that depletes glutathione and binds to proteins in the liver in mice after overdose. Since then, additional groundbreaking experiments have demonstrated critical roles for mitochondrial damage, oxidative stress, nuclear DNA fragmentation, and necrotic cell death as well. Over the years, some investigators have also attempted to translate these mechanisms to humans using human specimens from APAP overdose patients. This review presents those studies and summarizes what we have learned about APAP hepatotoxicity in humans so far. Overall, the mechanisms of APAP hepatotoxicity in humans strongly resemble those discovered in experimental mouse and cultured hepatocyte models, and emerging biomarkers also suggest similarities in liver repair. The data not only validate the first mechanistic studies of APAP-induced liver injury performed 50 years ago but also demonstrate the human relevance of numerous studies conducted since then. SIGNIFICANCE STATEMENT: Human studies using novel translational, mechanistic biomarkers have confirmed that the fundamental mechanisms of acetaminophen (APAP) hepatotoxicity discovered in rodent models since 1973 are the same in humans. Importantly, these findings have guided the development and understanding of treatments such as N-acetyl-l-cysteine and 4-methylpyrazole over the years. Additional research may improve not only our understanding of APAP overdose pathophysiology in humans but also our ability to predict and treat serious liver injury in patients.

Acetaminophen (APAP) overdose is one of the most common causes of acute liver injury. The current standard-of-care treatment for APAP hepatotoxicity, N-acetyl-l-cysteine, is highly effective when administered early after overdose, but loses efficacy in later-presenting patients. As a result, there is interest in the identification of new treatments for APAP overdose patients. Natural products are a promising source of new treatments because many are purported to have hepatoprotective effects. In fact, a great deal of research has been done to identify natural products that can protect against APAP-induced liver injury. However, serious concerns have been raised about the rigor and human relevance of these studies. Here, we systematically reviewed the APAP-natural product literature from 2013 to 2023 to determine the veracity of these concerns and the scope of the potential problem. The results substantiate the concerns that have been previously raised and point to concrete steps that can be taken to improve APAP-natural product research.

Cannabidiol (CBD) is a major phytocannabinoid from Cannabis sativa. It is currently widely available and widely used in the USA, but despite its rapid progress to market, the pharmacology and toxicology of both CBD and cannabidiol-rich cannabis extracts (CRCE) remain largely unknown. The goals of this study were to investigate the potential of a novel human microphysiological system to emulate CRCE-induced hepatotoxicity and pharmacological properties demonstrated in animal models. For this purpose, C57BL6/J male mice were subjected to dosing with either 0, 61.5, 184.5, or 615 mg/kg of CRCE for 10 days. The liver-on-chip system, incorporating human primary hepatocytes, sinusoidal endothelial cells, as well as Kupffer and stellate cells was subjected to 0, 300, 1,200, or 4,400 ng/mL of CRCE (8 h exposure followed by 16 h washout) for 5 days. Administration of CRCE in mice resulted in nearly 4-fold elevations of plasma ALT at 615 mg/kg (p < 0.01) and a dose-dependent decrease in intrahepatic miR-122. Elevated levels of ALT, paralleled by decreased intrahepatic and increased effluent levels of miR-122, were also observed in the liver-on-chip, although these results were not statistically significant. Exposure to CRCE resulted in a robust and dose-dependent induction of key cytochrome P450 enzymes, namely Cyp1a2, Cyp2b6 (CYP2B10), Cyp2e1, and Cyp2c9 (CYP2C19) in both mouse livers and liver-on-chip. The results of this study demonstrate the congruence between the responses observed in mouse and human liver-on-chip experimental systems and provide evidence of the potential microphysiological systems hold for translating animal data into clinical practice.

Tyrosine kinase inhibitors (TKIs) are increasingly popular drugs used to treat more than a dozen different diseases, including some forms of cancer. Despite having fewer adverse effects than traditional chemotherapies, they are not without risks. Liver injury is a particular concern. Of the FDA-approved TKIs, approximately 40% cause hepatotoxicity. However, little is known about the underlying pathophysiology. The leading hypothesis is that TKIs are converted by cytochrome P450 3A4 (CYP3A4) to reactive metabolites that damage proteins. Indeed, there is strong evidence for this bioactivation of TKIs in in vitro reactions. However, the actual toxic effects are underexplored. Here, we measured the cytotoxicity of several TKIs in primary mouse hepatocytes, HepaRG cells, and HepG2 cells with and without CYP3A4 modulation. To our surprise, the data indicate that CYP3A4 increases resistance to sorafenib and lapatinib hepatotoxicity. The results have implications for the mechanism of toxicity of these drugs in patients and underline the importance of selecting an appropriate experimental model.

Forensic laboratories need quick and simple technology to improve turnaround times, while delivering reliable results. The goal of this study is first to create a simplified workflow to meet new Academy Standards Board requirements for urine testing in drug-facilitated crime investigations and, second, to create "ready-to-go", "hands-free" testing technology to further streamline analytical procedures. A first of its kind, the ToxBox forensic test kit is used to validate a single analytical procedure for opioids, benzodiazepines, cannabinoids, antidepressants, and several other drug classes. Method performance indicators follow accreditation requirements and include accuracy, precision, measurement uncertainty, calibration models, reportable range, sensitivity, specificity, carryover, interference, ion suppression/enhancement, and analyte stability. "Hands-free" testing platforms require the use of new suspended-state technology to stabilize NIST-traceable standards premanufactured at precise concentrations in the presence of sample preparation reagents. By suspending all reaction components in the solid state, with air gaps between the phases, reference standards and process controls are built in a "ready-to-go" format and stabilized for long-term storage in the presence of a sample matrix, β-d-glucuronidase, and enzymatic buffers. "Hands-free" test kits are removed from storage, incubated at either ambient temperature or 60 °C, and assayed using validated methods. This is the first example of how complex forensic testing workflows can be streamlined with new "hands-free" testing strategies to meet analytical challenges associated with quantitative and confirmatory analyses.

Abstract Background Estimating the glomerular filtration rate (GFR) is essential for evaluating kidney function, which is part of routine medical care. Most clinical laboratories report an estimated glomerular filtration rate (eGFR) based on serum creatinine, but cystatin C is a potential alternative marker. Evidence suggests that using the creatinine-plus-cystatin C (the 2021 CKD-EPI creatinine-cystatin C equation) eGFR gives more accurate results than the eGFR based on creatinine alone. Here we compared two 2021 CKD-EPI equations for estimating GFR—one using standardized creatinine alone (Cre) and the other using cystatin C combined with standardized creatinine (Cre-Cys). We also explored the effect of the equations on the GFR category. Methods: We retrospectively analyzed 777 simultaneous values for eGFR calculated by the two 2021 CKD-EPI equations over a period of nine months, from the time our laboratory initiated the Cre-Cys equation to the present.Cystatin C was measured turbidometrically on the Optilite (Binding Site, Birmingham, UK) and the creatinine was measured by the DxC method (Beckman Coulter, California, USA). We excluded any patient with Cre eGFR >90 mL/min/1.73 m2 because we do not result any value above 90 for that parameter. Results Out of 777 patients (52% female and 48% male), we excluded 82 results with Cre GFR > 90. The mean ± SD for Cre-Cys eGFR and Cre eGFR were 37.3 ± 23.8 and 37.1 ± 23.1, respectively, with a non-statistical difference between both groups using the paired T-Test (t = −0.8249, P-value = 0.4097). The mean difference (Cre-Cys eGFR minus Cre eGFR) was 0.28, with a standard deviation of 9.027. The correlation coefficient between Cre eGFR and Cre-Cys eGFR was r = 0.93. Two hundred and seventy-four (35%) samples had a difference between the numerical value of the two eGFR equations significant enough to change the GFR classification. 128 (34.5%) of the males had different categories when both equations calculated the eGFR vs 146 (36%) females. 36.3% of the white and 34% of the black patients had a discrepancy. The calculations matched for the other 502 patients. Of the 274 samples with discordant category classifications, the Cre-Cys equation gave a lower result and a higher (worse) category in 144 (53%), and a higher result and lower (better) category in 130 (47%). Conclusion The use of Cystatin C is growing and has moved beyond the nephrology community. Furthermore, the 2012 KDIGO guidelines suggest using additional tests (such as cystatin C or a clearance measurement) for confirmatory testing in specific circumstances when eGFR based on serum creatinine is less accurate. Currently, cystatin C testing is only available M-F, 8 AM–4 PM at our institute, but increasing availability and decreasing cost will likely expand Cre-Cys eGFR testing in the future. However, understanding the relationships between eGFR values from the different equations and how they will affect patient management can help clinicians optimize their use for kidney function evaluation.

Acetaminophen (APAP) is a widely used drug, but overdose can cause severe acute liver injury. The first reports of APAP hepatotoxicity in humans were published in 1966, shortly after the development of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as the first biomarkers of liver injury as opposed to liver function. Thus, the field of liver injury biomarkers has evolved alongside the growth in APAP hepatotoxicity incidence. Numerous biomarkers have been proposed for use in the management of APAP overdose patients in the intervening years. Here, we comprehensively review the development of these markers from the 1960s to the present day and briefly discuss possible future directions.