Metabolomics Simultaneously Derives Benchmark Dose Estimates and Discovers Metabolic Biotransformations in a Rat Bioassay.

Benchmark dose (BMD) modeling estimates the dose of a chemical that causes a perturbation from baseline. Transcriptional BMDs have been shown to be relatively consistent with apical end point BMDs, opening the door to using molecular BMDs to derive human health-based guidance values for chemical exposure. Metabolomics measures the responses of small-molecule endogenous metabolites to chemical exposure, complementing transcriptomics by characterizing downstream molecular phenotypes that are more closely associated with apical end points. The aim of this study was to apply BMD modeling to in vivo metabolomics data, to compare metabolic BMDs to both transcriptional and apical end point BMDs. This builds upon our previous application of transcriptomics and BMD modeling to a 5-day rat study of triphenyl phosphate (TPhP), applying metabolomics to the same archived tissues. Specifically, liver from rats exposed to five doses of TPhP was investigated using liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance spectroscopy-based metabolomics. Following the application of BMDExpress2 software, 2903 endogenous metabolic features yielded viable dose-response models, confirming a perturbation to the liver metabolome. Metabolic BMD estimates were similarly sensitive to transcriptional BMDs, and more sensitive than both clinical chemistry and apical end point BMDs. Pathway analysis of the multiomics data sets revealed a major effect of TPhP exposure on cholesterol (and downstream) pathways, consistent with clinical chemistry measurements. Additionally, the transcriptomics data indicated that TPhP activated xenobiotic metabolism pathways, which was confirmed by using the underexploited capability of metabolomics to detect xenobiotic-related compounds. Eleven biotransformation products of TPhP were discovered, and their levels were highly correlated with multiple xenobiotic metabolism genes. This work provides a case study showing how metabolomics and transcriptomics can estimate mechanistically anchored points-of-departure. Furthermore, the study demonstrates how metabolomics can also discover biotransformation products, which could be of value within a regulatory setting, for example, as an enhancement of OECD Test Guideline 417 (toxicokinetics).

Exploration of xenobiotic metabolism within cell lines used for Tox21 chemical screening.

The Tox21 Program has investigated thousands of chemicals with high-throughput screening assays using cell-based assays to link thousands of chemicals to individual molecular targets/pathways. However, these systems have been widely criticized for their suspected lack of 'metabolic competence' to bioactivate or detoxify chemical exposures. In this study, 9 cell line backgrounds used in Tox21 assays (i.e., HepG2, HEK293, Hela, HCT116, ME180, CHO-K1, GH3.TRE-Luc, C3H10T1/2 and MCF7) were evaluated via metabolite formation rates, along with metabolic clearance and metabolite profiling for HepG2, HEK293, and MCF-7aroERE, in comparison to pooled donor (50) suspensions of primary human hepatocytes (PHHs). Using prototype clinical drug substrates for CYP1A2, CYP2B6, and CYP3A4/5, extremely low-to-undetectable CYP450 metabolism was observed (24 h), and consistent with their purported 'lack' of metabolic competence. However, for Phase II metabolizing enzymes and metabolic clearance, surprisingly proficient metabolism was observed for bisphenol AF, bisphenol S, and 7-hydroxycoumarin. Here, comparatively low glucuronidation relative to sulfation was observed in contrast to equivalent levels in PHHs. Overall, while a lack of CYP450 metabolism was confirmed in this benchmarking effort, Tox21 cell lines were not 'incompetent' for xenobiotic metabolism, and displayed surprisingly high proficiency for sulfation that rivaled PHHs. These findings have implications for the interpretation of Tox21 assay data, and establish a framework for evaluating of 'metabolic competence' with in vitro models.

Benchmark Concentrations for Untargeted Metabolomics Versus Transcriptomics for Liver Injury Compounds in In Vitro Liver Models.

Interpretation of untargeted metabolomics data from both in vivo and physiologically relevant in vitro model systems continues to be a significant challenge for toxicology research. Potency-based modeling of toxicological responses has served as a pillar of interpretive context and translation of testing data. In this study, we leverage the resolving power of concentration-response modeling through benchmark concentration (BMC) analysis to interpret untargeted metabolomics data from differentiated cultures of HepaRG cells exposed to a panel of reference compounds and integrate data in a potency-aligned framework with matched transcriptomic data. For this work, we characterized biological responses to classical human liver injury compounds and comparator compounds, known to not cause liver injury in humans, at 10 exposure concentrations in spent culture media by untargeted liquid chromatography-mass spectrometry analysis. The analyte features observed (with limited metabolites identified) were analyzed using BMC modeling to derive compound-induced points of departure. The results revealed liver injury compounds produced concentration-related increases in metabolomic response compared to those rarely associated with liver injury (ie, sucrose, potassium chloride). Moreover, the distributions of altered metabolomic features were largely comparable with those observed using high throughput transcriptomics, which were further extended to investigate the potential for in vitro observed biological responses to be observed in humans with exposures at therapeutic doses. These results demonstrate the utility of BMC modeling of untargeted metabolomics data as a sensitive and quantitative indicator of human liver injury potential.

Cation recombination energy/coulomb repulsion effects in ETD/ECD as revealed by variation of charge per residue at fixed total charge.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) experiments in electrodynamic ion traps operated in the presence of a bath gas in the 1-10 mTorr range have been conducted on a common set of doubly protonated model peptides of the form X(AG)nX (X = lysine, arginine, or histidine, n = 1, 2, or 4). The partitioning of reaction products was measured using thermal electrons, anions of azobenzene, and anions of 1,3-dinitrobenzene as reagents. Variation of n alters the charge per residue of the peptide cation, which affects recombination energy. The ECD experiments showed that H-atom loss is greatest for the n = 1 peptides and decreases as n increases. Proton transfer in ETD, on the other hand, is expected to increase as charge per residue decreases (i.e., as n increases). These opposing tendencies were apparent in the data for the K(AG)nK peptides. H-atom loss appeared to be more prevalent in ECD than in ETD and is rationalized on the basis of either internal energy differences, differences in angular momentum transfer associated with the electron capture versus electron transfer processes, or a combination of the two. The histidine peptides showed the greatest extent of charge reduction without dissociation, the arginine peptides showed the greatest extent of side-chain cleavages, and the lysine peptides generally showed the greatest extent of partitioning into the c/z•-product ion channels. The fragmentation patterns for the complementary c- and z•-ions for ETD and ECD were found to be remarkably similar, particularly for the peptides with X = lysine.

Electron transfer dissociation of amide nitrogen methylated polypeptide cations.

Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process.

Transition metal complex cations as reagents for gas-phase transformation of multiply deprotonated polypeptides.

Triply deprotonated DGAILDGAILD was reacted in the gas-phase with doubly charged copper, cobalt, and iron metal complexes containing either two or three phenanthroline ligands. Reaction products result from two major pathways. The first pathway involves the transfer of an electron from the negatively charged peptide to the transition-metal complex. The other major pathway consists of the displacement of the phenanthroline ligands by the peptide resulting in the incorporation of the transition-metal into the peptide to form [M - 3H + X(II)](-) ions, where X is Cu, Co, or Fe, respectively. The extent to which each pathway contributes is dependent on the nature of transition-metal complex. In general, bis-phen complexes result in more electron-transfer than the tris-phen complexes, while the tris-phen complexes result in more metal insertion. The metal in the complex plays a large role as well, with the Cu containing complexes giving rise to more electron transfer than the corresponding complexes of Co and Fe. The results show that a single reagent solution can be used to achieve two distinct sets of products (i.e., electron-transfer products and metal insertion products). These results constitute the demonstration of novel means for the gas-phase transformation of peptide anions from one ion type to another via ion/ion reactions using reagents formed via electrospray ionization.

Biscationic bicephalic (double-headed) amphiphiles with an aromatic spacer and a single hydrophobic tail.

We report the synthesis and aggregation studies of a homologous family of biscationic "bicephalic" amphiphiles. Each of has a linear alkoxy chain and two trimethylammonium bromide headgroups connected to a benzene ring. Krafft temperatures (T(K)) in water were determined by differential scanning calorimetry (DSC) and conductivity. Critical micelle concentration (cmc) and ionization degree (alpha) values were determined by monitoring the conductivity of aqueous solutions as a function of concentration, and confirmed by monitoring the (1)H NMR chemical shift of the terminal methyl group as a function of concentration. Values for log(cmc) decrease linearly with increasing chain length, with a smaller dependence on chain length than for single-headed amphiphiles, consistent with literature reports on other bicephalic amphiphiles. Comparison to two related amphiphiles, each with a single headgroup reveals that the addition of a second head group results in an increase in cmc and a decrease of T(K). These effects are attributed to greater water solubility due to the incorporation of a second, hydrophilic headgroup. The effect on alpha is also discussed.