Candidate biomarkers for the diagnosis and prognosis of drug-induced liver injury: An international collaborative effort.

Current blood biomarkers are suboptimal in detecting drug-induced liver injury (DILI) and predicting its outcome. We sought to characterize the natural variabilty and performance characteristics of 14 promising DILI biomarker candidates. Serum or plasma from multiple cohorts of healthy volunteers (n = 192 and n = 81), subjects who safely took potentially hepatotoxic drugs without adverse effects (n = 55 and n = 92) and DILI patients (n = 98, n = 28, and n = 143) were assayed for microRNA-122 (miR-122), glutamate dehydrogenase (GLDH), total cytokeratin 18 (K18), caspase cleaved K18, glutathione S-transferase α, alpha-fetoprotein, arginase-1, osteopontin (OPN), sorbitol dehydrogenase, fatty acid binding protein, cadherin-5, macrophage colony-stimulating factor receptor (MCSFR), paraoxonase 1 (normalized to prothrombin protein), and leukocyte cell-derived chemotaxin-2. Most candidate biomarkers were significantly altered in DILI cases compared with healthy volunteers. GLDH correlated more closely with gold standard alanine aminotransferase than miR-122, and there was a surprisingly wide inter- and intra-individual variability of miR-122 levels among healthy volunteers. Serum K18, OPN, and MCSFR levels were most strongly associated with liver-related death or transplantation within 6 months of DILI onset. Prediction of prognosis among DILI patients using the Model for End-Stage Liver Disease was improved by incorporation of K18 and MCSFR levels. Conclusion: GLDH appears to be more useful than miR-122 in identifying DILI patients, and K18, OPN, and MCSFR are promising candidates for prediction of prognosis during an acute DILI event. Serial assessment of these biomarkers in large prospective studies will help further delineate their role in DILI diagnosis and management.

Evaluation of Rat Acute Phase Proteins as Inflammatory Biomarkers for Vaccine Nonclinical Safety Studies.

The objectives were to characterize the kinetics of acute phase proteins (APPs) α-2 macroglobulin (A2M), α-1 acid glycoprotein (A1AGP), and fibrinogen (FIB), and injection site macroscopic and microscopic findings following intramuscular administration of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (TDaP; Adacel); adjuvants (aluminum phosphate [AlPO4]; aluminum hydroxide, Al[OH]3; CpG/Al[OH]3; or Quillaja saponaria 21 [QS-21]); or saline to female Wistar Han rats. Intravascular lipopolysaccharide (LPS) was a positive control. Injection sites and lymph nodes were evaluated microscopically, using hematoxylin and eosin (H&E) stained sections, 48 hours postdose (HPD) and compared with APP concentrations; A2M and A1AGP were measured using Meso Scale Discovery analyzer. Fibrinogen was measured on STA Compact analyzer. In a time-course study, APP peaked at 24 or 48 HPD. In a subsequent study at 48 HPD, injection site microscopic changes included inflammation and muscle degeneration/necrosis, which was different in severity/nature between groups. The APPs were not increased in rats administered saline, Al(OH)3, or AlPO4. Fibrinogen and A1AGP increased in rats administered CpG/Al(OH)3, QS-21, or TDaP; and A2M increased in rats administered QS-21. Fibrinogen, A2M, and A1AGP increased after LPS administration. Acute phase proteins can be used to monitor inflammatory responses to adjuvants; however, some adjuvants may induce inflammation without higher APPs.

Glutamate dehydrogenase as a biomarker for mitotoxicity; insights from furosemide hepatotoxicity in the mouse.

Glutamate dehydrogenase (GLDH) is a liver-specific biomarker of hepatocellular damage currently undergoing qualification as a drug development tool. Since GLDH is located within the mitochondrial matrix, it has been hypothesized that it might also be useful in assessing mitotoxicity as an initiating event during drug-induced liver injury. According to this hypothesis, hepatocyte death that does not involve primary mitochondrial injury would result in release of intact mitochondria into circulation that could be removed by high speed centrifugation and result in lower GLDH activity measured in spun serum vs un-spun serum. A single prior study in mice has provided some support for this hypothesis. We sought to repeat and extend the findings of this study. Accordingly, mice were treated with the known mitochondrial toxicant, acetaminophen (APAP), or with furosemide (FS), a toxicant believed to cause hepatocyte death through mechanisms not involving mitotoxicity as initiating event. We measured GLDH levels in fresh plasma before and after high speed centrifugation to remove intact mitochondria. We found that both APAP and FS treatments caused substantial hepatocellular necrosis that correlated with plasma alanine aminotransferase (ALT) and GLDH elevations. The plasma GLDH activity in both the APAP- and FS- treated mice was not affected by high-speed centrifugation. Interestingly, the ratio of GLDH:ALT was 5-fold lower during FS compared to APAP hepatotoxicity. Electron microscopy confirmed that both APAP- and FS-treatments had resulted in mitochondrial injury. Mitochondria within vesicles were only observed in the FS-treated mice raising the possibility that mitophagy might account for reduced release of GLDH in the FS-treated mice. Although our results show that plasma GLDH is not clinically useful for evaluating mitotoxicity, the GLDH:ALT ratio as a measure of mitophagy needs to be further studied.

The early effects of short-term dexamethasone administration on hepatic and serum alanine aminotransferase in the rat.

Dexamethasone (DEXA) administration has been associated with serum alanine aminotransferase (ALT) elevations that may result from enhanced ALT expression. The aim of our current study was to compare liver vs. serum ALT activity and to examine the onset of any hepatocellular changes. Groups of 4 male Sprague-Dawley rats were administered a single dose of DEXA or corn oil at 12, 16, and 24 h prior to euthanasia or once-daily for 2, 3, or 4 days. All (nonfasted) rats were necropsied together on Day 5. While DEXA incrementally increased liver ALT activity in the 1-, 2-, 3-, and 4-day treatment groups (maximal, 3.7-fold), liver aspartate aminotransferase (AST) never exceeded 1.4-fold over control. Significant hepatic glycogen elevations were detected after DEXA treatment, which correlated with microscopic observations. Serum ALT, AST, sorbitol dehydrogenase, and glutamate dehydrogenase (GLDH) increased after 2, 3, and 4 days of DEXA dosing (1.3-10.3-fold). DEXA-related necropsy findings included pale livers consistent with glycogen deposition. The relative percent liver to body weight was elevated in all DEXA-treated rats. Hepatocellular necrosis was observed in 1/4 rats at 12 h, 2/4 rats at 2 days, 4/4 rats at 3 days, and 3/4 rats at 4 days. DEXA treatment <2 days failed to produce consistent evidence of hepatic injury, as detected by serum biomarkers and pathology assessment. However, early DEXA treatment did correlate with apparent ALT induction. Ultimately, this may explain some early asymptomatic serum ALT elevations seen clinically.

Serum microRNA signatures as "liquid biopsies" for interrogating hepatotoxic mechanisms and liver pathogenesis in human.

MicroRNAs (miRNAs) released into the peripheral circulation upon cellular injury have shown a promise as a new class of tissue-specific biomarkers. We were first to demonstrate that next-generation sequencing analysis of serum from human subjects with acetaminophen-induced liver injury revealed a specific signature of circulating miRNAs. We consequently hypothesized that different types of hepatic liver impairments might feature distinct signatures of circulating miRNAs and that this approach might be useful as minimally invasive diagnostic "liquid biopsies" enabling the interrogation of underlying molecular mechanisms of injury in distant tissues. Therefore we examined serum circulating miRNAs in a total of 72 serum samples from a group of 53 subjects that included patients with accidental acetaminophen overdose, hepatitis B infection, liver cirrhosis and type 2 diabetes as well as gender- and age-matched healthy subjects with no evidence of liver disease. The miRNA signatures were identified using next-generation sequencing that provided analysis for the whole miRNome, including miRNA isoforms. Compared to the healthy subjects, a total of 179 miRNAs showed altered serum levels across the diseased subjects. Although many subjects have elevated alanine aminotransferase suggesting liver impairments, we identified distinct miRNA signatures for different impairments with minimum overlap. Furthermore, the bioinformatics analysis of miRNA signatures revealed relevant molecular pathways associated with the mechanisms of toxicity and or pathogenesis of disease. Interestingly, the high proportion of miRNA isoforms present in the respective signatures indicated a new level of complexity in cellular response to stress or disease. Our study demonstrates for the first time that signatures of circulating miRNAs show specificity for liver injury phenotypes and, once validated, might become useful for diagnosis of organ pathologies as "liquid biopsies".

The relationship among microsomal enzyme induction, liver weight, and histological change in cynomolgus monkey toxicology studies.

The purpose of this investigation was to examine the relationship among hepatic microsomal enzyme induction, liver weight, histological evidence of hepatic injury, and serum clinical chemistry markers of hepatic origin in the cynomolgus monkey. We report here the results from independent toxicology studies for 10 investigative drug candidates representing four therapeutic classes. Study conditions were selected to elicit target organ toxicity. We found that six of the 10 compounds altered cytochrome P450-associated activities in both male and female monkeys, two in females only, and one altered similar activities in males only. Frequently, significant treatment-related elevations in NADPH cytochrome c reductase and ethylmorphine N-demethylase were noted. When the results from all 10 studies were pooled, 14 cytochrome P450-associated activities were significantly increased and five were decreased in males compared to 15 significantly increased and three decreased in the females. Treatment-associated liver weight increases were noted in four studies. Except for hepatocellular hypertrophy in one study, no significant treatment-related microscopic changes in liver and no elevations of serum biomarkers commonly associated with liver toxicity were observed in any of the studies that demonstrated significant hepatic enzyme induction. Compared to parallel rat studies, one compound was an inducer only in monkeys and one was an inducer only in rats. Significant elevations of microsomal drug-metabolizing enzymes in the cynomolgus monkey liver are not accompanied by substantial hepatic changes except for hepatomegaly. These alterations in the hepatic drug-metabolizing enzyme system were benign based the absence of histopathological lesions and serum biomarkers of hepatobiliary toxicity.

Application of high-throughput sequencing to circulating microRNAs reveals novel biomarkers for drug-induced liver injury.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and the major reason for withdrawal of drugs from the market. Preclinical evaluation of drug candidates has failed to detect about 40% of potentially hepatotoxic compounds in humans. At the onset of liver injury in humans, currently used biomarkers have difficulty differentiating severe DILI from mild, and/or predict the outcome of injury for individual subjects. Therefore, new biomarker approaches for predicting and diagnosing DILI in humans are urgently needed. Recently, circulating microRNAs (miRNAs) such as miR-122 and miR-192 have emerged as promising biomarkers of liver injury in preclinical species and in DILI patients. In this study, we focused on examining global circulating miRNA profiles in serum samples from subjects with liver injury caused by accidental acetaminophen (APAP) overdose. Upon applying next generation high-throughput sequencing of small RNA libraries, we identified 36 miRNAs, including 3 novel miRNA-like small nuclear RNAs, which were enriched in the serum of APAP overdosed subjects. The set comprised miRNAs that are functionally associated with liver-specific biological processes and relevant to APAP toxic mechanisms. Although more patients need to be investigated, our study suggests that profiles of circulating miRNAs in human serum might provide additional biomarker candidates and possibly mechanistic information relevant to liver injury.

Emerging hepatotoxicity biomarkers and their potential to improve understanding and management of drug-induced liver injury.

Biomarkers of drug-induced liver injury (DILI) are essential for the diagnosis of severe cases of DILI in clinical trials and clinical practice, but the currently used biomarker paradigm detects damage after it has occurred and has limited prognostic value. The development of new biomarker strategies that improve the diagnosis of DILI by providing increased specificity and/or by identifying individual patients who are at risk for DILI is therefore crucial. See related Research, http://genomemedicine.com/content/5/9/86.

Development of blood biomarkers for drug-induced liver injury: an evaluation of their potential for risk assessment and diagnostics.

Drug-induced liver injury (DILI) remains a rare but serious complication in drug therapy that is a primary cause of drug failure during clinical trials. Conventional biomarkers, particularly the serum transaminases and bilirubin, serve as useful indicators of hepatocellular or cholestatic liver injury, respectively, but only after substantial and sometimes irreversible tissue damage. Ideally, more sensitive biomarkers that respond very early before irreversible injury has occurred would offer improved outcomes. Novel biomarkers are initially being developed in animal models exposed to intrinsically hepatotoxic stimuli. However, the eventual translation to human populations, even those with known risk factors that predispose the liver to drug toxicity, would be the fundamental goal. Ultimately, some might even be applicable for the early identification of individuals predisposed to idiosyncratic hepatotoxicity potential. This article reviews recent progress in the discovery and qualification of novel biomarkers for DILI and delineates the path to eventual utilization for risk assessment. Some major categories of plasma or serum biomarkers surveyed include proteins, cytokines, circulating mRNAs, and microRNAs.

Analysis of rat bone marrow by flow cytometry following in vivo exposure to cyclohexanone oxime or daunomycin HCl.

The purpose of these studies was to evaluate bone marrow from male CD rats following exposure to known hematotoxins using flow cytometry (FC) and a monoclonal antibody to the cell surface antigen CD71. Rats were treated with either CHO (300 mg/kg for 10 days) or DAUN (10 mg/kg for 1 day). Control groups received the appropriate vehicle. Half of the animals from each group were euthanized at the end of the dosing schedule and the remaining animals were euthanized after a recovery period. Hematology analyses were completed prior to the onset of each study and on the day of necropsy. Marrow was isolated from the tibia, stained with R-phycoerythrin-conjugated mouse anti-rat CD71 (transferrin receptor on proliferating cells) monoclonal antibody, and then analyzed by FC for myeloid:erythroid (M:E) ratios. FC determinations of myeloid and erythroid population percentages and M:E ratios from untreated rats were confirmed by microscopic examination of marrow cytospins and selected flow cell sorts. M:E ratios for control animals determined by FC were not significantly different between the two studies (1.83 vs 1.89). CHO treatment caused a significant (p < 0.01) decrease in M:E ratios (0.96 for CHO vs 1.48 for control) at day 11 due to increased erythroid cells. M:E ratios were significantly increased (p < 0.05) with DAUN treatment at day 3 (5.07 for DAUN vs 1.70 for control) and corresponded to generalized depletion of all marrow cell lines, especially erythroid cells. After recovery, M:E ratios of CHO and DAUN rats were similar to controls. Hematological values corroborated changes in marrow myeloid and erythroid populations evaluated by this FC technique. Using FC and a monoclonal antibody to the cell surface antigen CD71, this study confirmed the reversible selective toxicity on myeloid and erythroid marrow populations following in vivo exposure to CHO or DAUN. This FC procedure provides a rapid, sensitive method for bone marrow analysis compared to conventional cytological examination.