Laura K. Schnackenberg

Deletion of LOX-1 Reduces Atherogenesis in LDLR Knockout Mice Fed High Cholesterol Diet

Atherosclerosis is associated with oxidative stress and inflammation, and upregulation of LOX-1, an endothelial receptor for oxidized LDL (oxLDL). Here, we describe generation of LOX-1 knockout (KO) mice in which binding of oxLDL to aortic endothelium was reduced and endothelium-dependent vasorelaxation preserved after treatment with oxLDL (P<0.01 versus wild-type mice). To address whether endothelial functional preservation might lead to reduction in atherogenesis, we crossed LOX-1 KO mice with LDLR KO mice and fed these mice 4% cholesterol/10% cocoa butter diet for 18 weeks. Atherosclerosis was found to cover 61±2% of aorta in the LDLR KO mice, but only 36±3% of aorta in the double KO mice. Luminal obstruction and intima thickness were significantly reduced in the double KO mice (versus LDLR KO mice). Expression of redox-sensitive NF-&kgr;B and the inflammatory marker CD68 in LDLR KO mice was increased (P<0.01 versus wild-type mice), but not in the double KO mice. On the other hand, antiinflammatory cytokine IL-10 expression and superoxide dismutase activity were low in the LDLR KO mice (P<0.01 versus wild-type mice), but not in the double KO mice. Endothelial nitric oxide synthase expression was also preserved in the double KO mice. The proinflammatory signal MAPK P38 was activated in the LDLR KO mice, and LOX-1 deletion reduced this signal. In conclusion, LOX-1 deletion sustains endothelial function leading to a reduction in atherogenesis in association with reduction in proinflammatory and prooxidant signals.

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Hepatotoxicity and nephrotoxicity are two major reasons that drugs are withdrawn post-market, and hence it is of major concern to both the FDA and pharmaceutical companies.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.

The Liver Toxicity Biomarker Study: Phase I Design and Preliminary Results

Drug-induced liver injury (DILI) is the primary adverse event that results in withdrawal of drugs from the market and a frequent reason for the failure of drug candidates in development. The Liver Toxicity Biomarker Study (LTBS) is an innovative approach to investigate DILI because it compares molecular events produced in vivo by compound pairs that (a) are similar in structure and mechanism of action, (b) are associated with few or no signs of liver toxicity in preclinical studies, and (c) show marked differences in hepatotoxic potential. The LTBS is a collaborative preclinical research effort in molecular systems toxicology between the National Center for Toxicological Research and BG Medicine, Inc., and is supported by seven pharmaceutical companies and three technology providers. In phase I of the LTBS, entacapone and tolcapone were studied in rats to provide results and information that will form the foundation for the design and implementation of phase II. Molecular analysis of the rat liver and plasma samples combined with statistical analyses of the resulting datasets yielded marker analytes, illustrating the value of the broad-spectrum, molecular systems analysis approach to studying pharmacological or toxicological effects.

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.

Metabonomics evaluations of age-related changes in the urinary compositions of male Sprague Dawley rats and effects of data normalization methods on statistical and quantitative analysis

BackgroundUrine from male Sprague-Dawley rats 25, 40, and 80 days old was analyzed by NMR and UPLC/MS. The effects of data normalization procedures on principal component analysis (PCA) and quantitative analysis of NMR-based metabonomics data were investigated. Additionally, the effects of age on the metabolic profiles were examined by both NMR and UPLC/MS analyses.ResultsThe data normalization factor was shown to have a great impact on the statistical and quantitative results indicating the need to carefully consider how to best normalize the data within a particular study and when comparing different studies. PCA applied to the data obtained from both NMR and UPLC/MS platforms reveals similar age-related differences. NMR indicated many metabolites associated with the Krebs cycle decrease while citrate and 2-oxoglutarate, also associated with the Krebs cycle, increase in older rats.ConclusionThis study compared four different normalization methods for the NMR-based metabonomics spectra from an age-related study. It was shown that each method of normalization has a great effect on both the statistical and quantitative analyses. Each normalization method resulted in altered relative positions of significant PCA loadings for each sample spectra but it did not alter which chemical shifts had the highest loadings. The greater the normalization factor was related to age, the greater the separation between age groups was observed in subsequent PCA analyses. The normalization factor that showed the least age dependence was total NMR intensity, which was consistent with UPLC/MS data. Normalization by total intensity attempts to make corrections due to dietary and water intake of the individual animal, which is especially useful in metabonomics evaluations of urine. Additionally, metabonomics evaluations of age-related effects showed decreased concentrations of many Krebs cycle intermediates along with increased levels of oxidized antioxidants in urine of older rats, which is consistent with current theories on aging and its association with diminishing mitochondrial function and increasing levels of reactive oxygen species. Analysis of urine by both NMR and UPLC/MS provides a comprehensive and complementary means of examining metabolic events in aging rats.

Global metabolic profiling and its role in systems biology to advance personalized medicine in the 21st Century

Systems biology attempts to elucidate the complex interaction between genes, proteins and metabolites to provide a mechanistic understanding of cellular function and how this function is affected by disease processes, drug toxicity or drug efficacy effects. Global metabolic profiling is an important component of systems biology that can be applied in both preclinical and clinical settings for drug discovery and development, and to study disease mechanisms. The metabolic profile encodes the phenotype, which is composed of the genotype and environmental factors. The phenotypic profile can be used to make decisions about the best course of treatment for an individual patient. Understanding the combined effects of genetics and environment through a systems biology framework will enable the advancement of personalized medicine.

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.

Studies of Acetaminophen and Metabolites in Urine and Their Correlations with Toxicity Using Metabolomics

A LC/MS-based metabolomic assay was utilized to investigate a drugs excretion kinetic profile in urine so that the drug toxicity information could be obtained. Groups of 10 male Sprague-Dawley rats per dose were orally gavaged with a single dose of 0.2% carboxymethylcellulose, 400 mg acetaminophen (APAP)/kg body weight or 1600 mg APAP/kg. UPLC/MS and NMR were used to evaluate the excretion kinetics of major drug metabolites. N-acetyl-L-cysteine acetaminophen (APAP-NAC) had statistically significant correlations with clinical chemistry data, endogenous metabolite concentrations and histopathology data. The potential toxicity of a drug can be assessed through the study of the drugs metabolite profiles.

Structural and optoelectronic properties of P3HT-graphene composites prepared by in situ oxidative polymerization

Poly(3-hexylthiophene) (P3HT)-graphene nanocomposites were synthesized via in situ oxidative polymerization of 3-hexylthiophene monomer in the presence of graphene. The main thrust was to investigate the structural and optoelectronic properties of P3HT-graphene nanocomposites with various graphene concentrations. NMR spectroscopy was used to determine the regioregularity of the polymer composites, whereas Fourier transform infrared spectroscopy and differential scanning calorimetry were used to study their structural and thermal properties. Moreover, cyclic voltammetry was employed to evaluate the HOMO levels of the nanocomposites, while optical spectrophotometry (UV-Vis-NIR) was utilized to determine the optical bandgap of the composites. The information from the aforementioned techniques was used to estimate the HOMO-LUMO energy levels. The results revealed changes in the optical bandgap of P3HT with increasing graphene content. Furthermore, an extensive study aiming at the effect of graphene content on...