Shelli J. Schomaker

The current state of serum biomarkers of hepatotoxicity

The level of serum alanine aminotransferase (ALT) activity reflects damage to hepatocytes and is considered to be a highly sensitive and fairly specific preclinical and clinical biomarker of hepatotoxicity. However, an increase in serum ALT activity level has also been associated with other organ toxicities, thus, indicating that the enzyme has specificity beyond liver in the absence of correlative histomorphologic alteration in liver. Thus, unidentified non-hepatic sources of serum ALT activity may inadvertently influence the decision of whether to continue development of a novel pharmaceutical compound. To assess the risk of false positives due to extraneous sources of serum ALT activity, additional biomarkers are sought with improved specificity for liver function compared to serum ALT activity alone. Current published biomarker candidates are reviewed herein and compared with ALT performance in preclinical and on occasion, clinical studies. An examination of the current state of hepatotoxic biomarkers indicates that serum F protein, arginase I, and glutathione-S-transferase alpha (GSTalpha) levels, all measured by ELISA, may show utility, however, antibody availability and high cost per run may present limitations to widespread applicability in preclinical safety studies. In contrast, the enzymatic markers sorbitol dehydrogenase, glutamate dehydrogenase, paraxonase, malate dehydrogenase, and purine nucleoside phosphorylase are all readily measured by photometric methods and use reagents that work across preclinical species and humans and are commercially available. The published literature suggests that these markers, once examined collectively in a large qualification study, could provide additional information relative to serum ALT and aspartate aminotransferase (AST) values. Since these biomarkers are found in the serum/plasma of treated humans and rats, they have potential to be utilized as bridging markers to monitor acute drug-induced liver injury in early clinical trials.

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

The purpose of this study was to determine what histological changes, if any, accompany liver enlargement and microsomal enzyme induction in rats administered high doses of therapeutic agents in preclinical toxicology studies. This was accomplished by evaluating a database derived from a series of 11 induction studies in rats with 10 novel compounds comprising five therapeutic classes. Results from serum enzyme chemistry analyses, gross organ weight changes, and histological analyses of the liver sections were evaluated and compared with the magnitude and extent of hepatic cytochrome P450 induction. All compounds were administrated via oral intubation once a day for the duration of the study using multiple doses, each proportionally based on body weight. During the course of these studies, serum clinical chemistry data and clinical observations were recorded. After necropsy, histopathology observations were made, and hepatic microsomes were assayed for cytochrome P450 content and associated drug-metabolizing enzymes. In some cases, cyanide-insensitive beta-oxidation of palmitoyl CoA was also assayed. Liver weight increases of 20% or greater were associated with histological evidence of hypertrophy, but neither the severity of hypertrophy nor the magnitude of liver weight increase correlated with the magnitude of drug-metabolizing enzyme elevations. Hypertrophy alone was not associated with serum enzyme increases. While there was a correlation between the incidence of increased liver weights and microsomal enzyme induction, the magnitudes of these increases were not related. Decreased serum triglycerides were often associated with elevated beta-oxidation attributed to hepatic peroxisome proliferation. It was concluded that, while slight ALT elevations occasionally were observed, hepatic microsomal enzyme induction was generally not accompanied by substantial morphological changes or elevated serum enzyme levels considered indicative of liver injury.

Assessment of Emerging Biomarkers of Liver Injury in Human Subjects

Hepatotoxicity remains a major challenge in drug development. Although alanine aminotransferase (ALT) remains the gold standard biomarker of liver injury, alternative biomarker strategies to better predict the potential for severe drug-induced liver injury (DILI) are essential. In this study, we evaluated the utility of glutamate dehydrogenase (GLDH), purine nucleoside phosphorylase (PNP), malate dehydrogenase (MDH), and paraxonase 1 (PON1) as indicators of liver injury in cohorts of human subjects, including healthy subjects across age and gender, subjects with a variety of liver impairments, and several cases of acetaminophen poisoning. In the healthy subjects, levels of GLDH and MDH were not affected by age or gender. Reference ranges for GLDH and MDH in healthy subjects were 1-10 and 79-176U/L, respectively. In contrast, the levels of PON1 and PNP were not consistent across cohorts of healthy subjects. Furthermore, GLDH and MDH had a strong correlation with elevated ALT levels and possessed a high predictive power for liver injury, as determined by ROC analysis. In contrast, PON1 and PNP did not detect liver injury in our study. Finally, evaluation of patients with acetaminophen-induced liver injury provided evidence that both GLDH and MDH might have utility as biomarkers of DILI in humans. This study is the first to evaluate GLDH, MDH, PON1, and PNP in a large number of human subjects and, and it provides an impetus for prospective clinical studies to fully evaluate the diagnostic value of GLDH and MDH for detection of liver injury.

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.

Evaluation of Serum Bile Acid Profiles as Biomarkers of Liver Injury in Rodents

Bile acids (BAs) have been studied as potential biomarkers of drug-induced liver injury. However, the relationship between levels of individual BAs and specific forms of liver injury remains to be fully understood. Thus, we set out to evaluate cholic acid (CA), glycocholic acid (GCA), and taurocholic acid (TCA) as potential biomarkers of liver injury in rodent toxicity studies. We have developed a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay applicable to rat and mouse serum and evaluated levels of the individual BAs in comparison with the classical biomarkers of hepatotoxicity (alanine aminotransferase [ALT], aspartate aminotransferase [AST], glutamate dehydrogenase (GLDH), alkaline phosphatase, total bilirubin, gamma-glutamyl transferase, and total BAs) and histopathology findings in animals treated with model toxicants. The pattern of changes in the individual BAs varied with different forms of liver injury. Animals with histopathologic signs of hepatocellular necrosis showed increases in all 3 BAs tested, as well as increases in ALT, AST, GLDH, and total BAs. Animals with histopathologic signs of bile duct hyperplasia (BDH) displayed increases in only conjugated BAs (GCA and TCA), a pattern not observed with the other toxicants. Because BDH is detectable only via histopathology, our results indicate the potential diagnostic value of examining individual BAs levels in serum as biomarkers capable of differentiating specific forms of liver injury in rodent toxicity studies.

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.

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.

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

The present study represents a retrospective analysis of hepatic microsomal enzyme induction data collected over a period of years for the beagle dog. Comparisons were completed for up to six enzyme activities and P450 content versus histopathological examination of the liver for hepatic changes and serum chemistry data analysis for markers indicative of hepatic injury. In addition, qualitative comparisons were made for these compounds to data reported in the rat by the same authors. In this analysis of canine study data for nine different compounds comprising five different pharmacological classes, significant elevations in several microsomal enzyme activities were observed under study conditions that did not result in liver weight increases, histological changes or serum chemistry changes that would be indicative of hepatocellular or hepatobiliary damage. Despite some species differences in cytochrome P450 homologues, for this compound set, there was clearly a general association between the response in dog liver and that of the rat liver. Compounds that elicited significant increases in more than one canine P450 endpoints were also likely to produce an inductive response in rat liver; however, the magnitude of the response and the P450 endpoint involved were not always identical. We conclude that hepatic drug-metabolizing enzyme induction in the beagle dog liver is typically a benign adaptive response, which parallels that reported previously in the rat.

Ethylmorphine N-demethylase activity as a marker for cytochrome P450 CYP3A activity in rat hepatic microsomes

The purpose of this study was to evaluate the selectivity and sensitivity of ethylmorphine N-demethylase (EMD) as an indicator of chemically-induced cytochrome P450 CYP3A activity in liver microsomes of rats following treatment with selective enzyme inducers. Male and female Sprague-Dawley (CD) rats were dosed with either pregnenolone-16alpha-carbonitrile (PCN; 50 mg/kg per day for 5 days), phenobarbital (PB; 100 mg/kg per day for 4 days), beta-naphthoflavone (betaNF; 100 mg/kg per day for 3 days), clofibrate (CF; 300 mg/kg per day for 14 days), isoniazid (ISO; 100 mg/kg per day for 3 days), or dexamethasone (DEX; 50 mg/kg per day for 4 days). Microsomes were isolated, frozen and subsequently assayed for protein, cytochrome P450 content and EMD activity. In males, significant elevations (P < 0.01) in EMD activity were observed in microsomes from PB-, DEX- and PCN-dosed animals compared with untreated controls. Microsomes from ISO- and betaNF-dosed males showed a reduction (P < 0.05) in EMD activity when compared with control microsomes, and CF was without effect. In females, EMD activities were significantly increased in microsomes from PCN, DEX and PB-dosed but not betaNF, ISO, or CF-dosed animals. As expected on the basis of sex-related differences in gene expression, EMD activities in untreated animals were considerably higher in males than females, attributable to constitutive CYP3A and CYP2C11 activities. The selectivity of EMD for induced CYP3A was confirmed on the basis of inhibition studies with selected steroid substrates of CYP3A, polyclonal anti-CYP3A1 antibodies and triacetyloleandomycin (TAO), a selective inhibitor of CYP3A. In conclusion, for both sexes, the greatest elevations (approximately 3-13-fold) in EMD activity were observed in microsomes from rats dosed with DEX, a potent archetypal inducer with lesser but significant increases noted for PCN and PB, indicating that EMD is a reliable indicator of induced rat hepatic cytochrome P450 CYP3A activity.

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.