Samuele Sala

Clinical chemistry retains its position as a cornerstone of toxicological assessment, yet inter-laboratory variability in baseline values remains a challenge for the integration and interpretation of multisite datasets. This study leveraged a publicly available clinical chemistry database to assess the impact of inter-laboratory variability in response to hydrazine-induced steatosis. Seventeen clinical chemistry and physico-chemical parameters were evaluated in response to a single dose of hydrazine (at 30 mg/kg or 90 mg/kg) administered to Sprague-Dawley rats (n = 83) across five different pharmaceutical companies and compared with sham-dosed control animals. Hydrazine exposure produced a distinct and consistent biochemical signature at 48 h post-dose across the combined sample set from all laboratory sites, characterised by increased serum bilirubin and BUN and decreased serum protein concentrations, alongside atypical reductions in ALT and AST due to transaminase inhibition. Despite sizable inter-laboratory differences in response when considering single assays, multivariate analysis of the complete dataset was able to extract a core pathological response signature. Early changes at 24 h post-dose in AST, ALT, total protein, and calcium demonstrated strong predictive value for 48-h toxicity profiles (AUROC 0.98), underscoring the translational potential of early biomarkers. This study highlights both the robustness and contextual limitations of clinical chemistry data in toxicological studies. It underscores the importance of matched-control designs and multivariate approaches for multisite studies and advocates for the integration of early predictive modelling to optimise study design and align with the principles of the Replace, Reduce, and Refine initiative.

Accurate assignment of endogenous and exogenous constituents within complex biological matrices remains a central challenge in metabolomics. Phenylacetylglutamine (PAGln) and phenylacetylglycine (PAGly) are terminal metabolites of the host-microbial co metabolism of phenylalanine, predominantly observed in humans and rodents, respectively. Motivated by a pervasive misidentification within the literature, we demonstrate that PAGln in human urine has been erroneously identified as PAGly in 82 of 166 NMR-based studies, yielding an estimated misannotation rate of 49%. Herein, we provide definitive assignments for both PAGly and PAGln, and present a comprehensive statistical evaluation of the NMR resonances attributed to both species, alongside their quantitative determination via targeted line-fitting approaches in extensive human and rodent urine samples obtained from the INTERSALT cohort (N = 1589) and the historical COMET study (N = 4399) respectively. In agreement with canonical hepatic metabolic pathways, PAGln was found to be exclusively present in human urine, whereas PAGly was restricted to rodent (i.e., rat and mouse) urine. These findings underscore the need for systematic re-evaluation of prior metabolomic annotations involving these structurally related yet biochemically distinct metabolites.

Urban wetlands provide essential habitats for wildlife but face increasing contamination from anthropogenic sources, including but not limited to per- and polyfluoroalkyl substances (PFAS) and metal(loid)s. Accumulation of these contaminants are known to impair organism biological functions. This study quantified PFAS and metal(loid) concentrations in liver tissue of motorbike frogs (Ranoidea moorei) along a contamination gradient of conservation significant urban wetlands in Perth, Western Australia, and assessed relationships of liver concentrations with whole-organism health metrics and metabolomic profiles in four tissues to evaluate biological effects. Of 61 accepted PFAS in frog livers, 22 had at least one individual above reporting limit. Linear perfluorooctane sulfonate (PFOS) was the most abundant PFAS compound in frog livers and males had higher PFAS concentrations at larger body sizes. The total PFAS concentrations in motorbike frog livers (range: 1.3 - 145.7, outlier: 364.3 μg/kg) were relatively low compared to tertiary consumer species or those inhabiting heavily contaminated sites. Total PFAS concentrations, or interactions between total PFAS and metal(loid)s, were not significantly correlated with body condition, liver mass, or gross health indicators; however, there were subtle tissue-specific metabolic perturbations linked to PFAS exposure, particularly in males. Biological responses showed stronger associations with liver concentrations of Zn (range: 13.7 - 90 mg/kg), Se (range: <0.05 - 4.1 mg/kg), and Cu (range: 11.3 - 248.5 mg/kg). Zinc concentrations were negatively associated with body condition, and females showed greater muscle metabolic perturbation while males had more metabolic associations in fat tissue. Selenium concentrations exhibited a biphasic relationship with body condition, with males showing Se-associated metabolic changes in the liver and fat. Copper concentrations were inversely associated with liver mass, with females exhibiting higher Cu levels and greater metabolic disruption. This study integrates metabolomics with traditional health assessments to better understand how anthropogenic and naturally occurring contaminants influence amphibian health.

The Consortium for Metabonomic Toxicology (COMET) studies was designed to model metabolic responses to organ-and mechanism-specific toxins to predict acute drug toxicity in rats. A range of clinical chemical parameters were measured in 7-day toxicology studies for 86 toxins eliciting a range of organ-and mechanism-specific effects. Additionally, 21 surgical or physiological stressors were evaluated to identify physiological or metabolic responses that might confound the interpretation of observed toxicity profiles. From these studies on a total of 3473 rats measured at six pharmaceutical companies, we provide a set of 12 serum and 5 urine physical and clinical chemistry parameters. Samples were collected at 24 h, 48 h and 168 h post-dose for each animal and are presented as a downloadable database file. We also summarise the main observations based on the group response at the level of the individual toxin. We demonstrate that correlations between parameters, such as serum bilirubin and aspartate aminotransferase (AST), provide a more nuanced profile of organ-specific toxicity than consideration of individual parameters alone. In addition, we highlight the variability in the measured parameters across the dataset attributable to inter-laboratory differences, and the heterogeneity of metabolic responses to particular compounds or differences in temporal patterns of response. This clinical chemistry atlas of toxicity serves as a valuable reference tool for evaluating the potential toxicity of novel drug candidates.

In this study, we revise the structures of the marine sponge-derived pyrone-deoxyribosides tetillapyrone and nortetillapyrone, as well as the semisynthetic derivative tetillapyrone diacetate, to the nucleosides: thymidine, deoxyuridine, and thymidine diacetate, respectively. These revisions are proposed based on reanalysis of reported spectroscopic and mass spectrometric data, comparisons to authentic standards, and DFT calculations of NMR chemical shifts. We similarly propose revisions for the structurally related natural products ochraceopyronide and rhizoaspergillin A as well as for the semisynthetic derivative rhizoaspergillin A tripivalate, assigning them as spongouridine, kipukasin O, and kipukasin O tripivalate, respectively. Notably, the originally proposed structures of tetillapyrone and rhizoaspergillin A tripivalate were based on single-crystal X-ray diffraction experiments, widely regarded as the gold standard technique in small-molecule structure determination. Our findings highlight some of the key limitations of X-ray crystallographic analysis and emphasize the importance of cautious data interpretation, especially when discrepancies arise with spectroscopic or spectrometric results.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Two novel pyrroloiminoquinone alkaloids, 6-chlorodamirone A and 6-bromodamirone A, have been identified for the first time from the marine sponge Latrunculia sp. (order: Poecilosclerida: family Latrunculiidae), sourced from Western Australia. Alongside these new compounds, seven previously known metabolites were also isolated. Despite being obtained in submilligram quantities, the structures of these natural products were successfully elucidated using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. To confirm the structures of these newly discovered alkaloids, a semisynthetic approach was employed starting from the more abundant metabolite, damirone A, additionally, single crystal X-ray crystallography was used to validate our structural proposals. The semisynthetic studies suggest that the chlorinated alkaloids are likely formed through a nonenzymatic conjugate halide substitution reaction rather than an enzymatic process. This reactivity parallels that observed in related metabolites, such as the caulibugulones B and C. Furthermore, a biomimetic cascade reaction was attempted to synthesize the spirodienone moiety characteristic of the discorhabdin alkaloids, inspired by the nucleophilic substitution observed in the tricyclic damirone A system. Albeit unsuccessful, these findings provide valuable insight into the reactivity of halogenated pyrroloiminoquinones under various conditions.

Two novel free porphyrins, isabellins A and B, as well as the known compounds corallistin D and deuteroporphyrin IX were isolated from a marine sponge
sp. LC-MS analysis of the crude extract revealed that the natural products were present both as free porphyrins and iron(III) coordinated hemins, designated isabellihemin A, isabellihemin B, corallistihemin D and deuterohemin IX, respectively. Structures were determined via high-resolution mass spectrometry, UV-Vis spectroscopy and extensive NOESY NMR spectroscopic experiments. The type-I alkyl substitution pattern of isabellin A and isabellihemin A was assigned unambiguously by single crystal X-ray diffraction. Biological evaluation of the metabolites revealed potent cytotoxicity for isabellin A against the NS-1 murine myeloma cell line.

Objectives
The stratification of individuals suffering from acute and post-acute SARS-CoV-2 infection remains a critical challenge. Notably, biomarkers able to specifically monitor viral progression, providing details about patient clinical status, are still not available. Herein, quantitative metabolomics is progressively recognized as a useful tool to describe the consequences of virus-host interactions considering also clinical metadata.

Methods
The present study characterized the urinary metabolic profile of 243 infected individuals by quantitative nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography mass spectrometry (LC–MS). Results were compared with a historical cohort of noninfected subjects. Moreover, we assessed the concentration of recently identified antiviral nucleosides and their association with other metabolites and clinical data.

Results
Urinary metabolomics can stratify patients into classes of disease severity, with a discrimination ability comparable to that of clinical biomarkers. Kynurenines showed the highest fold change in clinically-deteriorated patients and higher-risk subjects. Unique metabolite clusters were also generated based on age, sex, and body mass index (BMI). Changes in the concentration of antiviral nucleosides were associated with either other metabolites or clinical variables. Increased kynurenines and reduced trigonelline excretion indicated a disrupted nicotinamide adenine nucleotide (NAD+) and sirtuin 1 (SIRT1) pathway.

Conclusions
Our results confirm the potential of urinary metabolomics for noninvasive diagnostic/prognostic screening and show that the antiviral nucleosides could represent novel biomarkers linking viral load, immune response, and metabolism. Moreover, we established for the first time a casual link between kynurenine accumulation and deranged NAD+/SIRT1, offering a novel mechanism through which SARS-CoV-2 manipulates host physiology.