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Cyclophosphamide is a widely used anticancer and immunosuppressive prodrug that unfortunately causes severe adverse effects, including cardiotoxicity. Although the exact cardiotoxic mechanisms are not completely understood, a link between cyclophosphamide’s pharmacologically active metabolites, namely 4-hydroxycyclophosphamide and acrolein, and the toxicity observed after the administration of high doses of the prodrug is likely. Therefore, the objective of this study is to shed light on the cardiotoxic mechanisms of cyclophosphamide and its main biotransformation products, through classic and metabolomics studies. Human cardiac proliferative and differentiated AC16 cells were exposed to several concentrations of the three compounds, determining their basic cytotoxic profile and preparing the next study, using subtoxic and toxic concentrations for morphological and biochemical studies. Finally, metabolomics studies were applied to cardiac cells exposed to subtoxic concentrations of the aforementioned compounds to determine early markers of damage. The cytotoxicity, morphological and biochemical assays showed that 4-hydroxycyclophosphamide and acrolein induced marked cardiotoxicity at µM concentrations (lower than 5 µM), being significantly lower than the ones observed for cyclophosphamide (higher than 2500 μM). Acrolein led to increased levels of ATP and total glutathione on proliferative cells at 25 µM, while no meaningful changes were observed in differentiated cells. Higher levels of carbohydrates and decreased levels of fatty acids and monoacylglycerols indicated a metabolic cardiac shift after exposure to cyclophosphamide’s metabolites, as well as a compromise of precursor amino acids used in the synthesis of glutathione, seen in proliferative cells’ metabolome. Overall, differences in cytotoxic mechanisms were observed for the two different cellular states used and for the three molecules, which should be taken into consideration in the study of cyclophosphamide cardiotoxic mechanisms.

Serum prostate-specific antigen (PSA) is currently the most used biomarker in clinical practice for prostate cancer (PCa) detection. However, this biomarker has several drawbacks. In this work, an untargeted gas chromatography-mass spectrometry (GC-MS)-based metabolomic profiling of PCa cells was performed to prove the concept that metabolic alterations might differentiate PCa cell lines from normal prostate cell line. For that, we assessed the differences in volatile organic compounds (VOCs) profile in the extracellular medium (exometabolome) of four PCa cell lines and one normal prostate cell line at two pH values (pH 2 and 7) by GC-MS. Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal prostate cells. The most altered metabolites included ketones, aldehydes and organic acids. Among these, we highlight pentadecane-2-one and decanoic acid, which were significantly increased in PCa compared to normal cells, and cyclohexanone, 4-methylheptan-2-one, 2-methylpentane-1,3-diol, 4-methylbenzaldehyde, 1-(3,5-dimethylfuran-2-yl)ethanone, methyl benzoate and nonanoic acid, which were significantly decreased in PCa cells. The PCa volatilome was markedly influenced by the VOCs extraction pH, though the discriminant capability was similar. Overall, our data suggest that VOCs monitoring has the potential to be used as a PCa screening methodology.

Given the high biological impact of classical and emerging toxicants, a sensitive and comprehensive assessment of the hazards and risks of these substances to organisms is urgently needed. In this sense, toxicometabolomics emerged as a new and growing field in life sciences, which use metabolomics to provide new sets of susceptibility, exposure, and/or effects biomarkers; and to characterize in detail the metabolic responses and altered biological pathways that various stressful stimuli cause in many organisms. The present review focuses on the analytical platforms and the typical workflow employed in toxicometabolomic studies, and gives an overview of recent exploratory research that applied metabolomics in various areas of toxicology.

Gold nanoparticles (AuNPs) are highly attractive for biomedical applications. Therefore, several in vitro and in vivo studies have addressed their safety evaluation. Nevertheless, there is a lack of knowledge regarding their potential detrimental effect on human kidney. To evaluate this effect, AuNPs with different sizes (13 nm and 60 nm), shapes (spheres and stars), and coated with 11-mercaptoundecanoic acid (MUA) or with sodium citrate, were synthesized, characterized, and their toxicological effects evaluated 24 h after incubation with a proximal tubular cell line derived from normal human kidney (HK-2). After exposure, viability was assessed by the MTT assay. Changes in lysosomal integrity, mitochondrial membrane potential (ΔΨm), reactive species (ROS/RNS), intracellular glutathione (total GSH), and ATP were also evaluated. Apoptosis was investigated through the evaluation of the activity of caspases 3, 8 and 9. Overall, the tested AuNPs targeted mainly the mitochondria in a concentration-dependent manner. The lysosomal integrity was also affected but to a lower extent. The smaller 13 nm nanospheres (both citrate- and MUA-coated) proved to be the most toxic among all types of AuNPs, increasing ROS production and decreasing mitochondrial membrane potential (p ≤ 0.01). For the MUA-coated 13 nm nanospheres, these effects were associated also to increased levels of total glutathione (p ≤ 0.01) and enhanced ATP production (p ≤ 0.05). Programmed cell death was detected through the activation of both extrinsic and intrinsic pathways (caspase 8 and 9) (p ≤ 0.05). We found that the larger 60 nm AuNPs, both nanospheres and nanostars, are apparently less toxic than their smaller counter parts. Considering the results herein presented, it should be taken into consideration that even if renal clearance of the AuNPs is desirable, since it would prevent accumulation and detrimental effects in other organs, a possible intracellular accumulation of AuNPs in kidneys can induce cell damage and later compromise kidney function.

Long-term cognitive dysfunction, or “chemobrain”, has been observed in cancer patients treated with chemotherapy. Mitoxantrone (MTX) is a topoisomerase II inhibitor that binds and intercalates with DNA, being used in the treatment of several cancers and multiple sclerosis. Although MTX can induce chemobrain, its neurotoxic mechanisms are poorly studied. This work aimed to identify the adverse outcome pathways (AOPs) activated in the brain upon the use of a clinically relevant cumulative dose of MTX. Three-month-old male CD-1 mice were given a biweekly intraperitoneal administration of MTX over the course of three weeks until reaching a total cumulative dose of 6 mg/kg. Controls were given sterile saline in the same schedule. Two weeks after the last administration, the mice were euthanized and their brains removed. The left brain hemisphere was used for targeted profiling of the metabolism of glutathione and the right hemisphere for an untargeted metabolomics approach. The obtained results revealed that MTX treatment reduced the availability of cysteine (Cys), cysteinylglycine (CysGly), and reduced glutathione (GSH) suggesting that MTX disrupts glutathione metabolism. The untargeted approach revealed metabolic circuits of phosphatidylethanolamine, catecholamines, unsaturated fatty acids biosynthesis, and glycerolipids as relevant players in AOPs of MTX in our in vivo model. As far as we know, our study was the first to perform such a broad profiling study on pathways that could put patients given MTX at risk of cognitive deficits.

Synthetic cathinones (SCs), commonly referred to as “bath salts”, are a class of novel psychoactive substances (NPSs) that elicit amphetamine-like effects and severe cardiovascular outcomes, including myocardial infarction and sudden cardiac death. Despite these risks, the mechanisms underlying SC-induced cardiotoxicity remain poorly studied. This study investigated the in vitro cardiotoxicity of two prevalent SCs—methylone and 3,4-dimethylmethcathinone (3,4-DMMC)—in H9c2 rat cardiomyoblasts, focusing on oxidative stress and the potential protective role of antioxidants. Cells were exposed to methylone (0.01–4.0 mM) or 3,4-DMMC (0.0005–0.8 mM) for 24 and 48 h, and cytotoxicity was assessed by an MTT assay. Intracellular reactive oxygen/nitrogen species (ROS/RNS) were quantified by fluorescence, and antioxidant effects were evaluated using ascorbic acid, N-acetylcysteine, and Trolox. Both SCs caused concentration-dependent cytotoxicity, with 3,4-DMMC showing higher potency than methylone (IC50: 0.28 vs. 0.98 mM, p = 0.0013). ROS/RNS levels increased in a concentration- and time-dependent manner for both compounds, reflecting early and sustained redox imbalance. Of the antioxidants, only ascorbic acid significantly improved cell viability. Taken together, these findings demonstrate for the first time that methylone and 3,4-DMMC exert cardiotoxic effects in vitro, with oxidative stress as a key contributor. The protective effect of ascorbic acid highlights its potential as a therapeutic candidate against SC-induced cardiac injury.

Prostate cancer (PCa) is an important health problem worldwide. Diagnosis and management of PCa is very complex because the detection of serum prostate specific antigen (PSA) has several drawbacks. Metabolomics brings promise for cancer biomarker discovery and for better understanding PCa biochemistry. In this study, a gas chromatography–mass spectrometry (GC-MS) based metabolomic profiling of PCa cell lines was performed. The cell lines include 22RV1 and LNCaP from PCa with androgen receptor (AR) expression, DU145 and PC3 (which lack AR expression), and one normal prostate cell line (PNT2). Regarding the metastatic potential, PC3 is from an adenocarcinoma grade IV with high metastatic potential, DU145 has a moderate metastatic potential, and LNCaP has a low metastatic potential. Using multivariate analysis, alterations in levels of several intracellular metabolites were detected, disclosing the capability of the endometabolome to discriminate all PCa cell lines from the normal prostate cell line. Discriminant metabolites included amino acids, fatty acids, steroids, and sugars. Six stood out for the separation of all the studied PCa cell lines from the normal prostate cell line: ethanolamine, lactic acid, β-Alanine, L-valine, L-leucine, and L-tyrosine.

Consumers are regularly exposed to well-known food contaminants (FCs), which are typically assessed for risk on an individual basis. However, there is limited knowledge about the overall levels and combinations of these compounds depending on dietary choices. The goal of this study was to estimate the real-life mixtures of FCs in different dietary models by integrating extensive data from the scientific literature concerning the reliable quantification of FCs in foods. A FAIR database detailing the occurrence of 73 FCs in 16 foods commonly consumed was built. The data were integrated into an omnivorous and a vegetarian dietary model. A weighted estimate of the 25th, 50th, and 75th percentiles of FCs in both dietary models revealed that the omnivorous model presented slightly higher levels of FCs than the vegetarian. At the 25th percentile, the FC levels in both dietary models fall within the European Food Safety Authority (EFSA) reference exposure levels for chemical hazards, except for arsenic, lead, cadmium, fumonisin B1, and OTA. At the 75th percentile, the FC levels exceed the EFSA reference levels for those FCs and additional mycotoxins. Using in vitro models, the 25th percentile can mimic real-life FC exposure, while the 75th percentile simulates a possible worst-case scenario.

Previous studies have shown that metabolomics can be a useful tool to better understand the mechanisms of carcinogenesis; however, alterations in biochemical pathways that lead to bladder cancer (BC) development have hitherto not been fully investigated. In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was applied to unveil the metabolic alterations between low-grade and high-grade BC cultured cell lines. Multivariable analysis revealed a panel of metabolites responsible for the separation between the two tumorigenic cell lines. Significantly lower levels of fatty acids, including myristic, palmitic, and palmitoleic acids, were found in high-grade versus low-grade BC cells. Furthermore, significantly altered levels of some amino acids were observed between low- and high-grade BC, namely glycine, leucine, methionine, valine, and aspartic acid. This study successfully demonstrated the potential of metabolomic analysis to discriminate BC cells according to tumor aggressiveness. Moreover, these findings suggest that bladder tumorigenic cell lines of different grades disclose distinct metabolic profiles, mainly affecting fatty acid biosynthesis and amino acid metabolism to compensate for higher energetic needs.

The identification of noninvasive biomarkers able to detect renal cell carcinoma (RCC) at an early stage remains an unmet clinical need. The recognition that altered metabolism is a core hallmark of cancer boosted metabolomic studies focused in the search for cancer biomarkers. The present work aims to evaluate the performance of the volatile metabolites present in the extracellular medium to discriminate RCC cell lines with distinct histological subtypes (clear cell and papillary) and metastatic potential from non-tumorigenic renal cells. Hence, volatile organic compounds (VOCs) and volatile carbonyl compounds (VCCs) were extracted by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography–mass spectrometry (GC–MS). Multivariate and univariate analysis unveiled a panel of metabolites responsible for the separation between groups, mostly belonging to ketones, alcohols, alkanes and aldehydes classes. Some metabolites were found similarly altered for all RCC cell lines compared to non-tumorigenic cells, namely 2-ethylhexanol, tetradecane, formaldehyde, acetone (increased) and cyclohexanone and acetaldehyde (decreased). Furthermore, significantly altered levels of cyclohexanol, decanal, decane, dodecane and 4-methylbenzaldehyde were observed in all metastatic RCC cell lines when compared with the non-metastatic ones. Moreover, some alterations in the volatile composition were also observed between RCC histological subtypes. Overall, our results demonstrate the potential of volatile profiling for identification of noninvasive candidate biomarkers for early RCC diagnosis.