Biomarkers For Drug-induced Liver Injury Research Articles

Salivary metabolites are promising noninvasive biomarkers of drug-induced liver injury

BACKGROUND Drug-induced liver injury (DILI) is one of the most common adverse events of medication use, and its incidence is increasing. However, early detection of DILI is a crucial challenge due to a lack of biomarkers and noninvasive tests. AIM To identify salivary metabolic biomarkers of DILI for the future development of noninvasive diagnostic tools. METHODS Saliva samples from 31 DILI patients and 35 healthy controls (HCs) were subjected to untargeted metabolomics using ultrahigh-pressure liquid chromatography coupled with tandem mass spectrometry. Subsequent analyses, including partial least squares-discriminant analysis modeling, t tests and weighted metabolite coexpression network analysis (WMCNA), were conducted to identify key differentially expressed metabolites (DEMs) and metabolite sets. Furthermore, we utilized least absolute shrinkage and selection operato and random fores analyses for biomarker prediction. The use of each metabolite and metabolite set to detect DILI was evaluated with area under the receiver operating characteristic curves. RESULTS We found 247 differentially expressed salivary metabolites between the DILI group and the HC group. Using WMCNA, we identified a set of 8 DEMs closely related to liver injury for further prediction testing. Interestingly, the distinct separation of DILI patients and HCs was achieved with five metabolites, namely, 12-hydroxydodecanoic acid, 3-hydroxydecanoic acid, tetradecanedioic acid, hypoxanthine, and inosine (area under the curve: 0.733-1). CONCLUSION Salivary metabolomics revealed previously unreported metabolic alterations and diagnostic biomarkers in the saliva of DILI patients. Our study may provide a potentially feasible and noninvasive diagnostic method for DILI, but further validation is needed.

Pharmacogenomic Predictors of Antibiotic-Associated Drug-Induced Liver Injury in Critically Ill Children: Observational Study Results

INTRODUCTION. The pathogenesis of antibiotic-associated drug-induced liver injury (DILI) in children has not been fully elucidated to date. Certain genotypes in patients increase the probability of developing DILI. Therefore, the identification of pharmacogenetic markers associated with DILI in children is essential.АIM. This study aimed to identify pharmacogenetic biomarkers of new-onset DILI associated with tigecycline and meropenem in children.MATERIALS AND METHODS. This prospective observational study was conducted in the Morozov Children’s CityClinical Hospital from 1 February 2020 to 1 September 2021. The study analysed the incidence and types of antibiotic-associated adverse drug reactions (ADRs) in 100 critically ill children aged 0 to 17 years (44 boys and 56 girls). Pharmacogenetic testing was performed in children with ADRs (n=30) to identify potential mechanisms involved in the development of their ADRs. The authors isolated and tested DNA from buccal epithelium swabs using the Agena Bioscience iPLEX® PGx Pro-based VeriDose® Core Panel covering 68 single nucleotide polymorphisms (SNPs) or short insertions and deletions (INDELs) and 5 copy number variants (CNVs).RESULTS. The odds of developing DILI associated with meropenem and tigecycline were higher in carriers of the homozygous cytochrome genotype CYP3A5*3/*3 (OR: 12.6, 95% CI: 1.9–79.4, r=6.54, p=0.011) than in patients with the heterozygous genotype CYP3A5*1A/*3. The odds were even higher in patients not carrying the CYP3A5*1A/*3 genotype (OR: 17.14, 95% CI: 1.79–16.3, r=6.24, p=0.013). The detection of the CYP3A5*3/*3 genotype had a prognostic accuracy of 76.7%, a sensitivity of 82%, and a specificity of 74% in predicting the risk of DILI associated with meropenem and tigecycline. Moreover, children with DILI carried the heterozygous genotype SLCO1B1*1/*5 (rs4149056 polymorphism) more often than children with other adverse reactions  associated with meropenem and tigecycline (r=9.8, p=0.002).CONCLUSION. The results of this study prove the prognostic significance of the homozygous cytochrome genotype CYP3A*3/*3 as an indicator of a potential risk for developing DILI associated with meropenem and tigecycline in children in critical conditions.The study was registered at ClinicalTrials.gov under No. NCT04141657 on 24 October 2019.

A superoxide anion activatable near-infrared fluorescent probe with a large Stokes shift for imaging of drug-induced liver injury

Drug-induced liver injury (DILI) has become a common liver disease and a major public health problem. Superoxide anion (O2•-) is closely related to DILI, which can be employed as a biomarker of DILI. In this paper, based on a near-infrared fluorescent dye with quinolinium-xanthene structure and a large Stokes shift, a superoxide anion activatable near-infrared fluorescent probe (QL-3F) was designed and synthesized for imaging of drug-induced liver injury. QL-3F displayed a high sensitivity and an excellent selectivity to O2•-, and also the response mechanism of QL-3F to O2•- was verified by calculation of density functional theory (DFT). Moreover, QL-3F has been successfully applied to image endogenous O2•- in RAW264.7 cells and in HepG2 cells with drug-induced hepatotoxicity. More importantly, QL-3F has been used in monitoring O2•- in DILI mice with satisfactory results. The above results indicated that QL-3F is a useful tool to detect O2•- and diagnose drug-induced liver injury.

Simple, sensitive, and label-free miRNA analysis through strand displacement reaction integrating with G-quadruplex-based signal generation

AbstractMicroRNAs (miRNAs) play a significant role in the pathogenesis of various diseases throughout biological processes, and the accurate detection of miRNA biomarkers holds great potential for early stage disease diagnosis and treatment. In this study, a novel method is developed to detect miRNA-21, a biomarker for drug-induced liver injury, by combining target sequence recycling with G-quadruplex-based signal production. This approach is highly sensitive and does not require the use of labels. The target sequence facilitates the cyclic exposure of G-rich regions in the detection probe by toehold-mediated strand displacement processes, with the aid of the catalytic chain. The G-quadruplex sequences that have been produced subsequently interact with thioflavin T (ThT), resulting in a significant increase in its fluorescence intensity. This enhanced fluorescence is utilized for the purpose of detecting miRNA-21, with a remarkably low detection limit of 4.4 fM. The suggested technique also allows for the very specific identification of the target miRNA-21. Due to its non-label format, excellent selectivity, and sensitivity, this technology presents a straightforward and versatile approach for detecting a wide range of biomarkers in the early phases of illness detection.

Combined analysis of 16S rDNA sequencing and metabolomics to find biomarkers of drug-induced liver injury

Drug induced liver injury (DILI) is a kind of liver dysfunction which caused by drugs, and gut microbiota could affect liver injury. However, the relationship between gut microbiota and its metabolites in DILI patients is not clear. The total gut microbiota DNA was extracted from 28 DILI patient and 28 healthy control volunteers (HC) and 16S rDNA gene were amplified. Next, differentially metabolites were screened. Finally, the correlations between the diagnostic strains and differentially metabolites were studied.The richness and uniformity of the bacterial communities decreased in DILI patients, and the structure of gut microbiota changed obviously. Enterococcus and Veillonella which belong to Firmicutes increased in DILI, and Blautia and Ralstonia which belong to Firmicutes, Dialister which belongs to Proteobacteria increased in HC. In addition, these diagnostic OTUs of DILI were associated with the DILI damage mechanism. On the other hands, there were 66 differentially metabolites between DILI and HC samples, and these metabolites were mainly enriched in pyrimidine metabolism and steroid hormone biosynthesis pathways. Furthermore, the collinear network map of the key microbiota-metabolites were constructed and the results indicated that Cortodoxone, Prostaglandin I1, Bioyclo Prostaglandin E2 and Anacardic acid were positively correlated with Blautia and Ralstonia, and negatively correlated with Veillonella.This study analyzed the changes of DILI from the perspective of gut microbiota and metabolites. Key strains and differentially metabolites of DILI were screened and the correlations between them were studied. This study further illustrated the mechanism of DILI.

Study design for development of novel safety biomarkers of drug-induced liver injury by the translational safety biomarker pipeline (TransBioLine) consortium: a study protocol for a nested case–control study

A lack of biomarkers that detect drug-induced liver injury (DILI) accurately continues to hinder early- and late-stage drug development and remains a challenge in clinical practice. The Innovative Medicines Initiative’s TransBioLine consortium comprising academic and industry partners is developing a prospective repository of deeply phenotyped cases and controls with biological samples during liver injury progression to facilitate biomarker discovery, evaluation, validation and qualification.In a nested case–control design, patients who meet one of these criteria, alanine transaminase (ALT) ≥ 5 × the upper limit of normal (ULN), alkaline phosphatase ≥ 2 × ULN or ALT ≥ 3 ULN with total bilirubin > 2 × ULN, are enrolled. After completed clinical investigations, Roussel Uclaf Causality Assessment and expert panel review are used to adjudicate episodes as DILI or alternative liver diseases (acute non-DILI controls). Two blood samples are taken: at recruitment and follow-up. Sample size is as follows: 300 cases of DILI and 130 acute non-DILI controls. Additional cross-sectional cohorts (1 visit) are as follows: Healthy volunteers (n = 120), controls with chronic alcohol-related or non-alcoholic fatty liver disease (n = 100 each) and patients with psoriasis or rheumatoid arthritis (n = 100, 50 treated with methotrexate) are enrolled. Candidate biomarkers prioritised for evaluation include osteopontin, glutamate dehydrogenase, cytokeratin-18 (full length and caspase cleaved), macrophage-colony-stimulating factor 1 receptor and high mobility group protein B1 as well as bile acids, sphingolipids and microRNAs. The TransBioLine project is enabling biomarker discovery and validation that could improve detection, diagnostic accuracy and prognostication of DILI in premarketing clinical trials and for clinical healthcare application.

Mitochondria-targeted ruthenium(II)-based phosphorescent chemodosimeter for peroxynitrite detection in drug-induced liver injury

Peroxynitrite (ONOO−), as a strong oxidative biomolecule, is considered a potential biomarker of drug-induced liver injury (DILI) that could disturb biochemistry processes and cause a great variety of liver diseases. However, it remains a significant challenge to discover quickly and track accurately ONOO− in vivo environments even if these active substances have aggravated liver damage. Herein, we developed a peroxynitrite-activable mitochondria-targeted phosphorescent chemodosimeter (Ru-Mit) for DILI monitoring, which was designed by covalently linking ruthenium(II) complex with the strong electron-withdrawing malononitrile moiety. In the presence of ONOO−, the chemodosimeter hard to be oxidized exhibited excellent turn-on phosphorescent response at 610 nm with a longer lifetime (∼ 300 ns). The sensing mechanism indicated the malononitrile group in Ru-Mit was removed via the hydrolysis of C=C bond, accompanied by the change of electronic structure and the enhancement of radiative transitions. Further, reliable signals are capable of not only imaging exogenous and endogenous ONOO− in mitochondria, but also visualizing APAP-induced and tolcapone-induced ONOO− generation in HepG2 cells by confocal luminescence imaging. This work might provide a new molecular platform to design and develop ONOO− probes, and it might be used as a practical tool to detect ONOO− for the pathogenesis research of DILI.

Screening and optimization of a water-soluble near-infrared fluorescent probe for drug-induced liver injury monitoring

Peroxynitrite (ONOO−) is a potential biomarker of drug-induced liver injury (DILI) and is involved in the process of DILI. Therefore, developing a reliable detection method for ONOO− will greatly contribute to ensuring drug safety and improving treatment efficiency. Here, based on the previous work, two kinds of NIR fluorescence probes PN and SPN were developed with phenyl-hydrazine as the ONOO− recognition group, which based on two fluorophores RN and SRN that are stable to ONOO−. A sensitive NIR probe SPN with good water solubility, low detection limit and good biocompatibility was selected through in vitro spectral property screening. Further experimental results show that there is a good linear relationship between the response intensity of probe SPN to ONOO− and the concentration of ONOO−, and the detection limit can reach 19.7 nM. At the cellular level, probe SPN can achieve a good and specific response to endogenous and exogenous ONOO−. Also, the probe SPN can be used for imaging and detection of DILI in zebrafish level and small animal level, indicating that probe SPN can be used as a powerful tool for diagnosis of DILI and efficacy evaluation of therapeutic drugs.

Serum interleukin-4 is elevated in clinical drug-induced liver injury

Background Drug-induced liver injury (DILI) remains a challenging diagnosis due to an absence of specific biomarkers. DILI due to volatile anaesthetics (VA-DILI) is characterised by trifluoroacetyl and CYP2E1 antibodies, but may not be seen for weeks after injury. Interleukin-4 (IL-4) may be involved in the production of these antibodies and may serve as a clinically useful early biomarker of VA-DILI. Aim To prospectively compare serum IL-4 levels between patients who develop VA-DILI and controls following exposure to the volatile anaesthetic. Methods A nested case-control study of patients exposed to VA during surgery was conducted. Thirteen DILI cases were identified from the original cohort, and 26 controls were matched according to age, sex and VA agent. Serum samples were collected before and 48-96 h after VA exposure, and analysed for IL-4 using quantitative enzyme-linked immunosorbent assay techniques. Results There was a statistically significant difference in serum IL-4 in post-VA samples between DILI cases and controls (control: 0.030 pg/mL, IQR: 0.030 − 0.030 pg/mL vs DILI: 0.044 pg/mL, IQR: 0.030 − 0.061 pg/mL; p = 0.039). A greater proportion of DILI cases had post-VA IL-4 levels above the assay lower limit of detection compared to controls (control: 23% vs DILI: 69%; p = 0.013). Conclusion IL-4 is a potential biomarker of DILI. Clinical diagnosis and understanding of DILI disease mechanisms may be improved by further investigation of novel biomarkers, and this IL-4 signal in serum is important as proof of concept for prospective study designs.

Expression Analysis of Exosomal MicroRNAs in Chlorpromazine Treated Human Hepatic Spheroids: A Promising Tool for Novel Drug-Induced Liver Injury Biomarker Discovery

Introduction: Risk management and mitigation of new drug candidates that induce drug-induced liver injury (DILI) in the clinic is a pivotal task to protect patients and save money during the drug development process. Nevertheless, clinical DILI risk assessment with conventionally available liver biomarkers comes with high failure rates. MicroRNAs (miRNAs) are short noncoding RNA molecules known to regulate mRNA expression. Actively secreted miRNAs which are circulating in patient blood can provide important pathway information involving specific DILI-inducing mechanisms and, therefore, have potential as early diagnostic biomarkers. Materials and Methods: In this study, the exosomal miRNA response of human primary hepatic spheroids treated with toxic concentrations of Chlorpromazine for 3 or 8 days was analyzed. The miRNA response of the hepatic spheroids was analyzed by quantifying a total of 752 miRNAs in the culture medium by quantitative polymerase chain reaction. Results: Clustering and pathway enrichment analysis of the miRNA response revealed several differentially expressed miRNAs involved in the regulation of signaling pathways that are toxicologically relevant, including apoptosis. This exosomal miRNA response was validated by a caspase 3/7 activity assay which confirmed an induction of apoptosis in hepatic spheroids treated with Chlorpromazine. Conclusion: Our results suggest that human hepatic spheroids are a promising test system for the discovery of secreted miRNA signatures. This could become a novel tool for discovery of human in vivo relevant compound-specific DILI biomarkers, inducing understanding mechanisms. In addition, miRNA biomarkers which can be monitored directly in the patient blood can become valuable tools to assist clinicians in risk management.

Evaluation of diagnostic and prognostic candidate biomarkers in drug-induced liver injury vs. other forms of acute liver damage.

Detection and characterization of idiosyncratic drug-induced liver injury (DILI) currently rely on standard liver tests, which are suboptimal in terms of specificity, sensitivity and prognosis. Therefore, DILI diagnosis can be delayed, with important consequences for the patient. In this study, we aimed to evaluate the potential of osteopontin, cytokeratin-18 (caspase-cleaved: ccK18 and total: K18), α-glutathione-S-transferase and microRNA-122 as new DILI biomarkers. Serial blood samples were collected from 32 DILI and 34 non-DILI acute liver injury (ALI) cases and a single sample from 43 population controls without liver injury (HLC) and analysed using enzyme-linked immunosorbent assay (ELISA) or single-molecule arrays. All biomarkers differentiated DILI and ALI from HLC with an area under receiver operator characteristic curve (AUC) value of >0.75 but were less efficient in distinguishing DILI from ALI, with ccK18 (0.79) and K18 (0.76) demonstrating highest potential. However, the AUC improved considerably (0.98) for ccK18 when comparing DILI and a subgroup of autoimmune hepatitis cases. Cytokeratin-18, microRNA-122 and α-glutathione-S-transferase correlated well with traditional transaminases, while osteopontin correlated most strongly with the international normalized ratio (INR). ccK18 appears promising in distinguishing DILI from autoimmune hepatitis but less so from other forms of acute liver injury. Osteopontin demonstrates prognostic potential with higher levels detected in more severe cases regardless of aetiology.

Amplification-free electrochemical biosensor detection of circulating microRNA to identify drug-induced liver injury

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. There is a need for rapid diagnostic tests, ideally at point-of-care. MicroRNA 122 (miR-122) is an early biomarker for DILI which is reported to increase in the blood before standard-of-care markers such as alanine aminotransferase activity. We developed an electrochemical biosensor for diagnosis of DILI by detecting miR-122 from clinical samples. We used electrochemical impedance spectroscopy (EIS) for direct, amplification free detection of miR-122 with screen-printed electrodes functionalised with sequence specific peptide nucleic acid (PNA) probes. We studied the probe functionalisation using atomic force microscopy and performed elemental and electrochemical characterisations. To enhance the assay performance and minimise sample volume requirements, we designed and characterised a closed-loop microfluidic system. We presented the EIS assay's specificity for wild-type miR-122 over non-complementary and single nucleotide mismatch targets. We successfully demonstrated a detection limit of 50 pM for miR-122. Assay performance could be extended to real samples; it displayed high selectivity for liver (miR-122 high) comparing to kidney (miR-122 low) derived samples extracted from murine tissue. Finally, we successfully performed an evaluation with 26 clinical samples. Using EIS, DILI patients were distinguished from healthy controls with a ROC-AUC of 0.77, a comparable performance to qPCR detection of miR-122 (ROC-AUC: 0.83). In conclusion, direct, amplification free detection of miR-122 using EIS was achievable at clinically relevant concentrations and in clinical samples. Future work will focus on realising a full sample-to-answer system which can be deployed for point-of-care testing.

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. Here, we identify, confirm, and replicate the biomarker performance characteristics of candidate proteins in patients with DILI at onset (DO; n = 133) and follow-up (n = 120), acute non-DILI at onset (NDO; n = 63) and follow-up (n = 42), and healthy volunteers (HV; n = 104). Area under the receiver operating characteristic curve (AUC) for cytoplasmic aconitate hydratase, argininosuccinate synthase, carbamoylphosphate synthase, fumarylacetoacetase, fructose-1,6-bisphosphatase 1 (FBP1) across cohorts achieved near complete separation (range: 0.94–0.99) of DO and HV. In addition, we show that FBP1, alone or in combination with glutathione S-transferase A1 and leukocyte cell-derived chemotaxin 2, could potentially assist in clinical diagnosis by distinguishing NDO from DO (AUC range: 0.65–0.78), but further technical and clinical validation of these candidate biomarkers is needed.

Significant association between HLA-B*35:01 and onset of drug-induced liver injury caused by Kampo medicines in Japanese patients.

Drug-induced liver injury (DILI) is a severe and life-threatening immune-mediated adverse effect, occurring rarely among treated patients. We examined genomic biomarkers in the Japanese population that predict the onset of DILI after using a certain class of drugs, such as Kampo products (Japanese traditional medicines). A total of 287 patients diagnosed as DILI by hepatology specialists were recruited after written informed consent was obtained. A genome-wide association analysis and human leukocyte antigen (HLA) typing in four digits were performed. We found a significant association (p=9.41×10-10 ) of rs146644517 (G>A) with Kampo product-related DILI. As this polymorphism is located in the HLA region, we evaluated the association of HLA types and found that 12 (63.2%) of 19 Kampo-DILI patients contained HLA-B*35:01, whereas only 15.2% were positive for this HLA among healthy volunteers. The odds ratio was 9.56 (95% confidence interval 3.75-24.46; p=2.98×10-6 , corrected p=4.17×10-5 ), and it increased to 13.55 compared with the DILI patients not exposed to Kampo products. The individual crude drug components in the Kampo products, including Scutellaria root (ougon in Japanese), rhubarb (daiou), Gardenia fruit (sanshishi), and Glycyrrhiza (kanzou), were significantly associated with HLA-B*35:01. HLA-B*35:01 is a genetic risk factor and a potential predictive biomarker for Kampo-induced DILI in the Japanese population.

Liver Damage and microRNAs: An Update.

One of the major organs in the body with multiple functions is the liver. It plays a central role in the transformation of macronutrients and clearance of chemicals and drugs. The serum biomarkers often used to indicate liver damage are not specifically for drug-induced liver injury (DILI) or liver injury caused by other xenobiotics, nor for viral infection. In this case, microRNAs (miRNAs) could play an exciting role as biomarkers of specific liver damage. In this review, we aimed to update the current literature on liver damage induced by drugs, as acute conditions and viral infections mediated by the hepatitis B virus (HBV) linked these two conditions to advanced research, with a focus on microRNAs as early biomarkers for liver damage. The undoubtable evidence that circulating miR-122 could be used as a human biomarker of DILI came from several studies in which a strong increase of it was linked with the status of liver function. In infancy, there is the possibility of an early miRNA detection for hepatitis B virus infection, but there are a lack of solid models for studying the HVB molecular mechanism of infection in detail, even if miRNAs do hold unrealized potential as biomarkers for early detection of hepatitis B virus infection mediated by HBV.

Dual-targetable fluorescent probe for mapping the fluctuation of peroxynitrite in drug-induced liver injury model

Drug-induced liver injury (DILI) is one of the most common and serious adverse drug reactions which can cause acute liver failure or even death in severe cases. With the incidence rate increasing over the years, DILI has became a frequent clinical liver disease and a global public health problem. As a biomarker of DILI, the detection of peroxynitrite (ONOO−) has became a powerful tool for the early diagnosis of liver injury. Here, we synthesized five mitochondria-targetable probes, 1–5, for detecting endogenous ONOO−. Through dye-screening, probe 5 was stood out by its excellent performance. In the presence of ONOO−, the fluorescence signal of probe 5 reduced 40-fold in 19 s with a low detection limit (9.36 nM). At the same time, the transformation can be observed with the naked eye under sunlight or UV lamp without being affected by the other reactive species. Even better, with low toxicity and high biocompatibility, probe 5 could successfully detect endogenous ONOO− in the mitochondrion of cells. Finally, probe 5 could specifically target the liver, and can be employed for monitoring the therapeutic effect of hepatoprotective medicine after drug-induced hepatotoxicity in vivo. In brief, probe 5 has the practical application capability for diagnosing the severity of the liver injury and researching the therapeutic effect of antidote in complex bio-systems.

Development of peroxynitrite-responsive fluorescence probe for recognition of drug-induced liver injury

Peroxynitrite (ONOO−) as an active substance, is produced during normal physiological process, which plays an important role in maintaining cell REDOX balance and cell function. Moreover, the peroxynitrite is involved in many diseases and especially can be used as a biomarker of drug-induced liver injury (DILI). Therefore, in this work, we synthesized a fluorescent probe JQ-3 for detecting ONOO−. The results showed the probe JQ-3 possessed excellent selectivity, fast response time (10 min) and low detection limit (32 nM). The probe JQ-3 is almost unaffected by pH, showing the potential application in biological systems. Moreover, the probe JQ-3 can be successfully used for the detection of exogenous and endogenous ONOO− in living cells and zebrafish. At the same time, the DILI was successfully recognized by visualizing ONOO− with JQ-3 in living cells and zebrafish. Therefore, the probe JQ-3 provides a potential tool for detecting ONOO− to understand physiological and pathology processes of disease.

Ultra-performance chromatography-electrospray tandem mass spectrometry analysis of bile acid profiles in the enterohepatic circulation following geniposide and acetaminophen-induced liver injury

Bile acids (BAs) play an important role in pre-diagnosing drug-induced liver injury (DILI). However, in clinical practice, different types of liver injury are characterized by different pathogeneses and pathological manifestations. Therefore, whether BAs can be used as biomarkers across different DILIs remains unclear. In this study, an ultra-performance chromatography–mass spectrometry (MS)/MS-based technique was developed for the simultaneous quantitative analysis of 31 BAs in the serum, liver, feces, urine, and intestinal contents of rats treated with acetaminophen (APAP) and geniposide to induce liver injury. The total extraction recovery for representative analytes ranged between 80.60% and 99.23% in the serum, urine, liver, feces, and intestinal contents. The correlation coefficients for all standard curves of the different matrices were at least 0.99. Validation of the BA analytical method including selectivity, residue, lower limit of quantification, accuracy, precision, matrix effect, and stability conformed with the biospecimen quality control standards of the Chinese Pharmacopoeia (version 2020). Serum biochemical and pathohistological analyses revealed APAP- and geniposide-induced hepatocellular and cholestatic DILI, respectively, with different effects on BA profiles in the enterohepatic circulation. Metabolomics further revealed that the trends in BA changes in the serum, feces, urine, and intestinal tissues were consistent between the geniposide- and APAP-treated groups. However, in the liver, the total BAs (TBA) concentration increased by 1.70 fold in the geniposide group but decreased by 43% in the APAP group compared with the control group. Multivariate analysis revealed differentially expressed BAs, including TCA, CA, and GCA, which are potential biomarkers for DILI, in the serum, liver, and urine following treatment with geniposide. Interestingly, the differentially expressed BAs in the APAP group were similar to those in the control group. Additionally, the magnitude of changes in the TBA in the urine (3.3 fold and 15.5 fold in the APAP and geniposide groups, respectively) was higher than that in the blood (290 fold and 640 fold in the APAP and geniposide groups, respectively). However, given the BA profiles after geniposide- and APAP-induced liver injury, BAs were found to be more suitable as biomarkers for diagnosing cholestatic liver injury. Overall, the BA assay developed in this study is rapid, simple, accurate, validated, sensitive, and suitable for analyzing the levels and distribution of BAs in various parts of the enterohepatic circulation.

H2O2-Activated NIR-II Fluorescent Probe with a Large Stokes Shift for High-Contrast Imaging in Drug-Induced Liver Injury Mice.

Drug-induced liver injury (DILI) is the most common clinical adverse drug reaction, which is closely associated with the oxidative stress caused by overproduced reactive oxygen species. Hepatic H2O2, as an important biomarker of DILI, plays a crucial role in the progression of DILI. However, there remains a challenge to develop H2O2-activatable second near-infrared (NIR-II, 1000-1700 nm) small molecular probes with both a large Stokes shift and a long emission wavelength beyond 950 nm. Herein, we developed an activatable NIR-II fluorescent probe (IR-990) with an acceptor-π-acceptor (A-π-A) skeleton for real-time detection of H2O2 in vivo. In the presence of H2O2, nonfluorescent probe IR-990 was successfully unlocked by generating a donor-π-acceptor (D-π-A) structure and switched on intense NIR-II fluorescence, exhibiting a peak emission wavelength at 990 nm and a large Stokes shift of 200 nm. Moreover, it was able to detect H2O2 with high sensitivity and selectivity in vitro (LOD = 0.59 μM) and monitor the behavior of endogenous H2O2 in the HepG2 cell model of DILI for the first time. Notably, probe IR-990 was successfully applied in real-time imaging of endogenous H2O2 generation in the DILI mouse model, showing a high signal-to-background ratio of 11.3/1. We envision that IR-990 holds great potential as a powerful diagnosis tool for real-time visualization of H2O2 in vivo and revealing the mechanism of DILI in the future.

Characterization and Quantification of Oxidized High Mobility Group Box 1 Proteoforms Secreted from Hepatocytes by Toxic Levels of Acetaminophen.

The high mobility group box 1 (HMGB1), which is released during acute acetaminophen (APAP) overdose, is thought to mediate a subsequent immune response, particularly hepatic infiltration of macrophages. The redox behavior of HMGB1 and the proteoforms of HMGB1 present in oxidative environments has been the subject of a number of confusing and contradictory studies. Therefore, a stable isotope dilution two-dimensional nanoultrahigh-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry method was developed in order to characterize and quantify oxidative modifications to the cysteine (Cys) residues (Cys-23, Cys-45, and Cys-106) that are present in HMGB1. Disulfide linkages were determined using carbamidoethyl derivatization before and after reduction as well as by direct analysis of disulfide cross-linked peptides. A stable isotope labeled form of HMGB1 was used as an internal standard to correct for sample to sample differences in immunoaffinity precipitation, derivatization, and electrospray ionization. Four discrete HMGB1 proteoforms were found to be released from a hepatocarcinoma cell model of APAP overdose after 24 h. Fully reduced HMGB1 with all three Cys-residues in their free thiol state accounted for 18% of the secreted HMGB1. The proteoform with disulfide between Cys-23 and Cys-45 accounted for 24% of the HMGB1. No evidence was obtained for a disulfide cross-link between Cys-106 and the other two Cys-residues. However, 45% of the HMGB1 formed a cross-link with unidentified intracellular proteins via an intermolecular disulfide bond, and 12% was present as the terminally oxidized cysteic acid. Surprisingly, there was no evidence for the formation of HMGB1 disulfides with GSH or other low molecular weight thiols. Secreted plasma HMGB1 Cys-23/Cys45 disulfide proteoform together with the Cys-106/protein disulfide proteoforms could potentially serve as early biomarkers of hepatoxicity after APAP overdose as well as biomarkers of drug-induced liver injury.