Jinchun Sun

Metabolomics approaches for discovering biomarkers of drug-induced hepatotoxicity and nephrotoxicity

Hepatotoxicity and nephrotoxicity are two major reasons that drugs are withdrawn post-market, and hence it is of major concern to both the FDA and pharmaceutical companies. The number of cases of serious adverse effects (SAEs) in marketed drugs has climbed faster than the number of total drug prescriptions issued. In some cases, preclinical animal studies fail to identify the potential toxicity of a new chemical entity (NCE) under development. The current clinical chemistry biomarkers of liver and kidney injury are inadequate in terms of sensitivity and/or specificity, prompting the need to discover new translational specific biomarkers of organ injury. Metabolomics along with genomics and proteomics technologies have the capability of providing translational diagnostic and prognostic biomarkers specific for early stages of liver and kidney injury. Metabolomics has several advantages over the other omics platforms such as ease of sample preparation, data acquisition and use of biofluids collected through minimally invasive procedures in preclinical and clinical studies. The metabolomics platform is reviewed with particular emphasis on applications involving drug-induced hepatotoxicity and nephrotoxicity. Analytical platforms for metabolomics, chemometrics for mining metabolomics data and the applications of the metabolomics technologies are covered in detail with emphasis on recent work in the field.

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS☆

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl-L-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.

Serum metabolomic profiles from patients with acute kidney injury: A pilot study

Low sensitivity of current clinical markers (serum creatinine and blood urea nitrogen (BUN)) in early stages of the development of acute kidney injury (AKI) limits their utility. Rapid LC/MS-based metabolic profiling of serum demonstrated in a pilot study that metabolomics could provide novel indicators of AKI. Metabolic profiles of serum samples from seventeen hospitalized patients with newly diagnosed AKI were compared with the profiles of serum from age-matched subjects with normal kidney function. Increases in acylcarnitines and amino acids (methionine, homocysteine, pyroglutamate, asymmetric dimethylarginine (ADMA), and phenylalanine) and a reduction in serum levels of arginine and several lysophosphatidyl cholines were observed in patients with AKI compared to healthy subjects. Increases in homocysteine, ADMA and pyroglutamate have been recognized as biomarkers of cardiovascular and renal disease, and acylcarnitines represent biomarkers of defective fatty acid oxidation. The results of this pilot study demonstrate the utility of metabolomics in the discovery of novel serum biomarkers that can facilitate the diagnosis and determine prognosis of AKI in hospitalized patients.

Evaluations of the trans-sulfuration pathway in multiple liver toxicity studies

Drug-induced liver injury has been associated with the generation of reactive metabolites, which are primarily detoxified via glutathione conjugation. In this study, it was hypothesized that molecules involved in the synthesis of glutathione would be diminished to replenish the glutathione depleted through conjugation reactions. Since S-adenosylmethionine (SAMe) is the primary source of the sulfur atom in glutathione, UPLC/MS and NMR were used to evaluate metabolites involved with the transulfuration pathway in urine samples collected during studies of eight liver toxic compounds in Sprague-Dawley rats. Urinary levels of creatine were increased on day 1 or day 2 in 8 high dose liver toxicity studies. Taurine concentration in urine was increased in only 3 of 8 liver toxicity studies while SAMe was found to be reduced in 4 of 5 liver toxicity studies. To further validate the results from the metabonomic studies, microarray data from rat liver samples following treatment with acetaminophen was obtained from the Gene Expression Omnibus (GEO) database. Some genes involved in the trans-sulfuration pathway, including guanidinoacetate N-methyltransferase, glycine N-methyltransferase, betaine-homocysteine methyltransferase and cysteine dioxygenase were found to be significantly decreased while methionine adenosyl transferase II, alpha increased at 24 h post-dosing, which is consistent with the SAMe and creatine findings. The metabolic and transcriptomic results show that the trans-sulfuration pathway from SAMe to glutathione was disturbed due to the administration of heptatotoxicants.

Translational biomarkers of acetaminophen-induced acute liver injury

Acetaminophen (APAP) is a commonly used analgesic drug that can cause liver injury, liver necrosis and liver failure. APAP-induced liver injury is associated with glutathione depletion, the formation of APAP protein adducts, the generation of reactive oxygen and nitrogen species and mitochondrial injury. The systems biology omics technologies (transcriptomics, proteomics and metabolomics) have been used to discover potential translational biomarkers of liver injury. The following review provides a summary of the systems biology discovery process, analytical validation of biomarkers and translation of omics biomarkers from the nonclinical to clinical setting in APAP-induced liver injury.

Metabolomics evaluation of the effects of green tea extract on acetaminophen-induced hepatotoxicity in mice.

Green tea has been purported to have beneficial health effects including protective effects against oxidative stress. Acetaminophen (APAP) is a widely used analgesic drug that can cause acute liver injury in overdose situations. These studies explored the effects of green tea extract (GTE) on APAP-induced hepatotoxicity in liver tissue extracts using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. Mice were orally administered GTE, APAP or GTE and APAP under three scenarios. APAP alone caused a high degree of hepatocyte necrosis associated with increases in serum transaminases and alterations in multiple metabolic pathways. The time of GTE oral administration relative to APAP either protected against or potentiated the APAP-induced hepatotoxicity. Dose dependent decreases in histopathology scores and serum transaminases were noted when GTE was administered prior to APAP; whereas, the opposite occurred when GTE was administered after APAP. Similarly, metabolites altered by APAP alone were less changed when GTE was given prior to APAP. Significantly altered pathways included fatty acid metabolism, glycerophospholipid metabolism, glutathione metabolism, and energy pathways. These studies demonstrate the complex interaction between GTE and APAP and the need to employ novel analytical strategies to understand the effects of dietary supplements on pharmaceutical compounds.

Identification of metabolite profiles of the catechol-O-methyl transferase inhibitor tolcapone in rat urine using LC/MS-based metabonomics analysis.

The process of drug metabolite identification is extremely important for drug efficacy, safety and pharmacokinetics. The traditional method usually involves using a drug with a radioactive labeled nuclei and/or isolating major drug metabolites by HPLC before applying MS and NMR analyses, which requires trained specialists to handle the radioactive compounds and is time consuming for offline-HPLC separation. A method using mass spectrometry-based metabonomics combined with multivariate statistical analysis was applied to rapidly identify metabolite profiles of tolcapone, a catechol-O-methyl transferase inhibitor for Parkinsons disease treatment. The tolcapone metabolites were identified based on the accurate mass measurement (<3 ppm) and MS(2) mass spectrum. In total, 16 tolcapone metabolites were detected and identified, 6 of which have not been reported previously. Our results indicate that the method has the capability to accelerate the process of identifying drug metabolites, ultimately reduce drug development costs, and make the process safer without requiring a drug with radioactive nuclei. Most importantly, the assay can detect the major and minor drug metabolites in a global view. Furthermore, since tolcapone has been associated with idiosyncratic drug induced liver toxicity the rapid detection of tolcapone-related metabolites can provide mechanistic toxicity information related to drug metabolism and the formation of reactive drug metabolites.

Metabolomics Techniques in Nanotoxicology Studies

The rapid growth in the development of nanoparticles for uses in a variety of applications including targeted drug delivery, cancer therapy, imaging, and as biological sensors has led to questions about potential toxicity of such particles to humans. High-throughput methods are necessary to evaluate the potential toxicity of nanoparticles. The omics technologies are particularly well suited to evaluate toxicity in both in vitro and in vivo systems. Metabolomics, specifically, can rapidly screen for biomarkers related to predefined pathways or processes in biofluids and tissues. Specifically, oxidative stress has been implicated as a potential mechanism of toxicity in nanoparticles and is generally difficult to measure by conventional methods. Furthermore, metabolomics can provide mechanistic insight into nanotoxicity. This chapter focuses on the application of both LC/MS and NMR-based metabolomics approaches to study the potential toxicity of nanoparticles.

Study of valproic acid‑induced endogenous and exogenous metabolite alterations using LC-MS ‑ based metabolomics

BACKGROUND Valproic acid (VPA; an anticonvulsant drug) therapy is associated with hepatotoxicity as well as renal toxicity. An LC-MS-based metabolomics approach was undertaken in order to detect urinary VPA metabolites and to discover early biomarkers of the adverse effects induced by VPA. RESULTS CD-1 mice were either subcutaneously injected with 600-mg VPA/kg body weight or vehicle only, and urine samples were collected at 6, 12, 24 and 48 h postinjection. A metabolomics approach combined with principal component analysis was utilized to identify VPA-related metabolites and altered endogenous metabolites in urine. Some VPA metabolites indicated potential liver toxicity caused by VPA administration. Additionally, some altered endogenous metabolites suggested that renal function might be perturbed by VPA dosing. CONCLUSION LC-MS-based metabolomics is capable of rapidly profiling VPA drug metabolites and is a powerful tool for the discovery of potential early biomarkers related to perturbations in liver and kidney function.

Systems Biology Investigation to Discover Metabolic Biomarkers of Acetaminophen-Induced Hepatic Injury Using Integrated Transcriptomics and Metabolomics

Background: Drug-induced hepatotoxicity is one of the major reasons for drug recall and hence it is of major concern to the FDA and consumers. Overdose of acetaminophen (APAP) can cause acute hepatic injury. The current clinical biomarkers of liver injury are insufficient in predicting the extent of injury; thus novel biomarkers are needed to integrate with the current biomarkers for better risk assessment during drug development and clinical use. Methods: Sprague-Dawley rats were orally gavaged with a single dose of 0.5% methylcellulose (control), 100 mg APAP/kg body weight or 1250 mg APAP/kg body weight. Urine, terminal blood samples and tissues were collected at 6, 24, 72, and 168 h for clinical chemistry and histopathology analyses. Based on the clinical chemistry data and histopathology, liver injury occurred in treated animals during the first 24 h, while recovery occurred during 72 to 168 h. A systems biology investigation of APAP-induced hepatic injury was conducted to elucidate novel metabolic biomarkers using an integrated transcriptomic and metabolomic approach. Both open metabolic profiling and broad metabolic profiling were utilized to examine metabolic changes in blood and open profiling was used to evaluate changes in the urinary metabolite profiles. Results: In total, 270 metabolites were evaluated in blood and/or urine. Metabolites involved in energy, urea and bile acid pathways were found to have strong correlations to hepatic necrosis scores and elevated alanine aminotransferase levels. The pathways associated with these metabolites were altered at the first 72 h but had generally recovered at 168 h. Changes in hepatic gene expression of the bile acid pathway supported the interpretation from the metabolomics data. Conclusion: The combination of the transcriptomics and metabolic profiling technologies discovered novel injury biomarkers (arginine, 2-oxoarginine, medium chain dicarboxylic acids, α-ketoglutarate and bile acids), which are involved in energy, bile acid, and arginine metabolism pathway.