Identification of key neuronal mechanisms triggered by dimethyl fumarate in SH-SY5Y human neuroblastoma cells through a metabolomic approach.

Dimethyl fumarate (DMF) is an old drug used for psoriasis treatment that has recently been repurposed to treat relapse-remitting multiple sclerosis, mostly due to its neuro- and immunomodulatory actions. However, mining of a pharmacovigilance database recently ranked DMF as the second pharmaceutical most associated with cognitive adverse events. To our best knowledge, the signaling mechanisms underlying its therapeutic and neurotoxic outcomes remain mostly undisclosed. This work thus represents the first-hand assessment of DMF-induced metabolic changes in undifferentiated SH-SY5Y human neuroblastoma cells, through an untargeted metabolomic approach using gas chromatography-mass spectrometry (GC-MS). The endometabolome was analyzed following 24 h and 96 h of exposure to two pharmacologically relevant DMF concentrations (0.1 and 10 µM). None of these conditions significantly reduced metabolic activity (MTT reduction assay). Our data showed that 24 h-exposure to DMF at both concentrations tested mainly affected metabolic pathways involved in mitochondrial activity (e.g., citric acid cycle, de novo triacylglycerol biosynthesis), and the synthesis of catecholamines and serotonin by changing the levels of their respective precursors, namely phenylalanine (0.68-fold decrease for 10 µM DMF vs vehicle), and tryptophan (1.36-fold increase for 0.1 µM DMF vs vehicle). Interestingly, taurine, whose levels can be modulated via Nrf2 signaling (DMF's primary target), emerged as a key mediator of DMF's neuronal action, displaying a 3.86-fold increase and 0.27-fold decrease for 10 µM DMF at 24 h and 96 h, respectively. A 96 h-exposure to DMF seemed to mainly trigger pathways associated with glucose production (e.g., gluconeogenesis, glucose-alanine cycle, malate-aspartate shuttle), possibly related to the metabolism of DMF into monomethyl fumarate and its further conversion into glucose via activation of the citric acid cycle. Overall, our data contribute to improving the understanding of the events associated with neuronal exposure to DMF.

Cardiotoxicity of cyclophosphamide's metabolites: an in vitro metabolomics approach in AC16 human cardiomyocytes.

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.

In vivo toxicometabolomics reveals multi-organ and urine metabolic changes in mice upon acute exposure to human-relevant doses of 3,4-methylenedioxypyrovalerone (MDPV).

3,4-Methylenedioxypyrovalerone (MDPV) is consumed worldwide, despite its potential to cause toxicity in several organs and even death. There is a recognized need to clarify the biological pathways through which MDPV elicits general and target-organ toxicity. In this work, a comprehensive untargeted GC-MS-based metabolomics analysis was performed, aiming to detect metabolic changes in putative target organs (brain, heart, kidneys and liver) but also in urine of mice after acute exposure to human-relevant doses of MDPV. Male CD-1 mice received binge intraperitoneal administrations of saline or MDPV (2.5 mg/kg or 5 mg/kg) every 2 h, for a total of three injections. Twenty-four hours after the first administration, target organs, urine and blood samples were collected for metabolomics, biochemical and histological analysis. Hepatic and renal tissues of MDPV-treated mice showed moderate histopathological changes but no significant differences were found in plasma and tissue biochemical markers of organ injury. In contrast, the multivariate analysis significantly discriminated the organs and urine of MDPV-treated mice from the control (except for the lowest dose in the brain), allowing the identification of a panoply of metabolites. Those levels were significantly deviated in relation to physiological conditions and showed an organ specific response towards the drug. Kidneys and liver showed the greatest metabolic changes. Metabolites related with energetic metabolism, antioxidant defenses and inflammatory response were significantly changed in the liver of MDPV-dosed animals, while the kidneys seem to have developed an adaptive response against oxidative stress caused by MDPV. On the other hand, the dysregulation of metabolites that contribute to metabolic acidosis was also observed in this organ. The heart showed an increase of fatty acid biosynthesis, possibly as an adaptation to maintain the cardiac energy homeostasis. In the brain, changes in 3-hydroxybutyric acid levels may reflect the activation of a neurotoxic pathway. However, the increase in metabolites with neuroprotective properties seems to counteract this change. Metabolic profiling of urine from MDPV-treated mice suggested that glutathione-dependent antioxidant pathways may be particularly involved in the compensatory mechanism to counteract oxidative stress induced by MDPV. Overall, this study reports, for the first time, the metabolic profile of liver, kidneys, heart, brain, and urine of MDPV-dosed mice, providing unique insights into the biological pathways of toxicity. Our findings also underline the value of toxicometabolomics as a robust and sensitive tool for detecting adaptive/toxic cellular responses upon exposure to a physiologically relevant dose of a toxic agent, earlier than conventional toxicity tests.

3,4-Methylenedioxymethamphetamine Hepatotoxicity under the Heat Stress Condition: Novel Insights from in Vitro Metabolomic Studies.

Hyperthermia has been extensively reported as a life-threatening consequence of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse. In this work, we used a sensitive untargeted metabolomic approach based on gas chromatography-mass spectrometry to evaluate the impact of hyperthermia on the hepatic metabolic changes caused by MDMA. For this purpose, primary mouse hepatocytes were exposed to subtoxic (LC01 and LC10) and toxic (LC30) concentrations of MDMA for 24 h, at 37 or 40.5 °C (simulating body temperature increase after MDMA consumption), and alterations on both intracellular metabolome and extracellular volatilome were evaluated. Multivariate analysis showed that metabolic patterns clearly discriminate MDMA treated cells from control cells, both in normothermic and hyperthermic conditions. The metabolic signature was found to be largely common to MDMA subtoxic and toxic concentrations, although with evident differences in the magnitude of response, with metabolic changes significantly more pronounced at 40.5 °C. Discriminant metabolites associated with MDMA-induced hepatotoxicity are mostly involved in the amino acid metabolism, aminoacyl tRNA biosynthesis, glutathione metabolism, tricarboxylic acid cycle, and pyruvate metabolism. Moreover, our metabolomic findings were corroborated by classical toxicity parameters, demonstrating the high sensitivity of this omic approach to assess molecular-level effects. Overall, this study indicates that MDMA triggers significant metabolic alterations on hepatic cells, even at low concentrations, that are clearly exacerbated at high temperatures. These findings provide new metabolic pieces to solve the puzzle of MDMA's hepatotoxicity mechanism and emphasize the increased risks of MDMA abuse due to the thermogenic action of the drug.

Metabolic signature of methylone in primary mouse hepatocytes, at subtoxic concentrations.

Methylone (3,4-methylenedioxymethcathinone) is one of the most popular new psychoactive drugs worldwide. Although advertised as a safe drug, its use has been associated to several cases of liver damage. In this work, a metabolomics approach based on gas chromatography-mass spectrometry (GC-MS) combined with chemometric analyses was used to characterize the disturbances occurring in the intra- and extracellular metabolome of primary mouse hepatocytes exposed to two subtoxic concentrations (LC01 and LC10) of methylone to better understand the early hepatotoxic events. Results showed a characteristic metabolic fingerprint for methylone, where aspartate, cysteine, 2-methyl-1-pentanol, 4-methylheptane, dodecane, 2,4-dimethyl-1-heptene, 1,3-di-tert-butylbenzene, acetophenone, formaldehyde and glyoxal levels were significantly changed at both concentrations tested. Furthermore, subtoxic concentrations of methylone caused profound changes in several biochemical pathways, suggesting adaptations in energy production processes (TCA cycle, amino acids metabolism and pyruvate metabolism), cellular antioxidant defenses (glutamate, cysteine and glutathione metabolism) and hepatic enzymes (associated to hydrocarbons, alcohols, aldehydes and ketones metabolism). This metabolic response to the initial methylone challenge most probably reflects the activation of protective mechanisms to restore cellular homeostasis. Overall, this study highlights the potential of untargeted metabolomic analysis to reveal the hepatic metabolic signature of methylone at subtoxic concentrations, and also provides clues to clarify the early mechanisms underlying the toxicity triggered by this new psychoactive substance, opening a new perspective for the study of toxicity mechanisms of new xenobiotics.

Volatile metabolomic signature of bladder cancer cell lines based on gas chromatography-mass spectrometry.

INTRODUCTION: Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers. OBJECTIVES: The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells. METHODS: VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC-MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies. RESULTS: Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype. CONCLUSIONS: We demonstrate that a GC-MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.

GC-MS metabolomics reveals disturbed metabolic pathways in primary mouse hepatocytes exposed to subtoxic levels of 3,4-methylenedioxymethamphetamine (MDMA).

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a well-known hepatotoxic drug. Although its toxicity has been thoroughly studied at high concentrations, there is still insufficient knowledge on possible alterations of cell function at subtoxic concentrations, which are in fact more representative concentrations of intoxication scenarios. In this study, a gas chromatography-mass spectrometry (GC-MS) metabolomics approach was used to investigate the metabolic changes in primary mouse hepatocytes (PMH) exposed to two subtoxic concentrations of MDMA (LC01 and LC10) for 24 h. Metabolomic profiling of both intracellular metabolites and volatile metabolites in the extracellular medium of PMH was performed. Multivariate analysis showed that the metabolic pattern of cells exposed to MDMA discriminates from the controls in a concentration-dependent manner. Exposure to LC10 MDMA induces a significant increase in some intracellular metabolites, including oleic acid and palmitic acid, and a decrease in glutamate, aspartate, 5-oxoproline, fumarate, malate, phosphoric acid, α-ketoglutarate and citrate. Extracellular metabolites such as acetophenone, formaldehyde, pivalic acid, glyoxal and 2-butanone were found significantly increased after exposure to MDMA, compared to controls, whereas 4-methylheptane, 2,4-dimethyl-1-heptene, nonanal, among others, were found significantly decreased. The panel of discriminatory metabolites is mainly involved in tricarboxylic acid (TCA) cycle, fatty acid metabolism, glutamate metabolism, antioxidant defenses and possibly changes in the liver enzyme machinery. Overall, these results highlight the potential of the intra- and extracellular metabolome to study alterations triggered by subtoxic concentrations of MDMA in hepatic cell functions, which represents a more realistic appraisal of early toxicity events posed by exposure to this drug. In addition, these results also revealed some metabolites that may be used as potential biomarkers indicative of early events in the hepatotoxicity induced by MDMA.

Metabolomic approaches in the discovery of potential urinary biomarkers of drug-induced liver injury (DILI).

Drug-induced liver injury (DILI) is a major safety issue during drug development, as well as the most common cause for the withdrawal of drugs from the pharmaceutical market. The identification of DILI biomarkers is a labor-intensive area. Conventional biomarkers are not specific and often only appear at significant levels when liver damage is substantial. Therefore, new biomarkers for early identification of hepatotoxicity during the drug discovery process are needed, thus resulting in lower development costs and safer drugs. In this sense, metabolomics has been increasingly playing an important role in the discovery of biomarkers of liver damage, although the characterization of the mechanisms of toxicity induced by xenobiotics remains a huge challenge. These new-generation biomarkers will offer obvious benefits for the pharmaceutical industry, regulatory agencies, as well as a personalized clinical follow-up of patients, upon validation and translation into clinical practice or approval for routine use. This review describes the current status of the metabolomics applied to the early diagnosis and prognosis of DILI and in the discovery of new potential urinary biomarkers of liver injury.

3,4-Methylenedioxypyrovalerone (MDPV): in vitro mechanisms of hepatotoxicity under normothermic and hyperthermic conditions.

Synthetic cathinones have emerged in recreational drug markets as legal alternatives for classical amphetamines. Though currently banned in several countries, 3,4-methylenedioxypyrovalerone (MDPV) is one of the most commonly abused cathinone derivatives worldwide. We have recently reported the potential of MDPV to induce hepatocellular damage, but the underlying mechanisms responsible for such toxicity remain to be elucidated. Similar to amphetamines, a prominent toxic effect of acute intoxications by MDPV is hyperthermia. Therefore, the present in vitro study aimed to provide insights into cellular mechanisms involved in MDPV-induced hepatotoxicity and also evaluate the contribution of hyperthermia to the observed toxic effects. Primary cultures of rat hepatocytes were exposed to 0.2-1.6 mM MDPV for 48 h, at 37 or 40.5 °C, simulating the rise in body temperature that follows MDPV intake. Cell viability was measured through the MTT reduction and LDH leakage assays. Oxidative stress endpoints and cell death pathways were evaluated, namely the production of reactive oxygen and nitrogen species (ROS and RNS), intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione, adenosine triphosphate (ATP) and free calcium (Ca(2+)), as well as the activities of caspases 3, 8 and 9, and nuclear morphological changes with Hoechst 33342/PI double staining. At 37 °C, MDPV induced a concentration-dependent loss of cell viability that was accompanied by GSH depletion, as one of the first signs of toxicity, observed already at low concentrations of MDPV, with negligible changes on GSSG levels, followed by accumulation of ROS and RNS, depletion of ATP contents and increases in intracellular Ca(2+) concentrations. Additionally, activation of caspases 3, 8, and 9 and apoptotic nuclear morphological changes were found in primary rat hepatocytes exposed to MDPV, indicating that this cathinone derivative activates both intrinsic and extrinsic apoptotic death pathways. The cytotoxic potential of MDPV and all the studied endpoints were markedly aggravated under hyperthermic conditions (40.5 °C). In conclusion, these data suggest that MDPV toxicity in primary rat hepatocytes is mediated by oxidative stress, subsequent to GSH depletion and increased ROS and RNS accumulation, mitochondrial dysfunction, and impairment of Ca(2+) homeostasis. Furthermore, the rise in body temperature subsequent to MDPV abuse greatly exacerbates its hepatotoxic potential.

The hallucinogenic world of tryptamines: an updated review.

In the area of psychotropic drugs, tryptamines are known to be a broad class of classical or serotonergic hallucinogens. These drugs are capable of producing profound changes in sensory perception, mood and thought in humans and act primarily as agonists of the 5-HT2A receptor. Well-known tryptamines such as psilocybin contained in Aztec sacred mushrooms and N,N-dimethyltryptamine (DMT), present in South American psychoactive beverage ayahuasca, have been restrictedly used since ancient times in sociocultural and ritual contexts. However, with the discovery of hallucinogenic properties of lysergic acid diethylamide (LSD) in mid-1900s, tryptamines began to be used recreationally among young people. More recently, new synthetically produced tryptamine hallucinogens, such as alpha-methyltryptamine (AMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), emerged in the recreational drug market, which have been claimed as the next-generation designer drugs to replace LSD ('legal' alternatives to LSD). Tryptamine derivatives are widely accessible over the Internet through companies selling them as 'research chemicals', but can also be sold in 'headshops' and street dealers. Reports of intoxication and deaths related to the use of new tryptamines have been described over the last years, raising international concern over tryptamines. However, the lack of literature pertaining to pharmacological and toxicological properties of new tryptamine hallucinogens hampers the assessment of their actual potential harm to general public health. This review provides a comprehensive update on tryptamine hallucinogens, concerning their historical background, prevalence, patterns of use and legal status, chemistry, toxicokinetics, toxicodynamics and their physiological and toxicological effects on animals and humans.

Raising awareness of new psychoactive substances: chemical analysis and in vitro toxicity screening of 'legal high' packages containing synthetic cathinones.

The world's status quo on recreational drugs has dramatically changed in recent years due to the rapid emergence of new psychoactive substances (NPS), represented by new narcotic or psychotropic drugs, in pure form or in preparation, which are not controlled by international conventions, but that may pose a public health threat comparable with that posed by substances listed in these conventions. These NPS, also known as 'legal highs' or 'smart drugs', are typically sold via Internet or 'smartshops' as legal alternatives to controlled substances, being announced as 'bath salts' and 'plant feeders' and is often sought after for consumption especially among young people. Although NPS have the biased reputation of being safe, the vast majority has hitherto not been tested and several fatal cases have been reported, namely for synthetic cathinones, with pathological patterns comparable with amphetamines. Additionally, the unprecedented speed of appearance and distribution of the NPS worldwide brings technical difficulties in the development of analytical procedures and risk assessment in real time. In this study, 27 products commercialized as 'plant feeders' were chemically characterized by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. It was also evaluated, for the first time, the in vitro hepatotoxic effects of individual synthetic cathinones, namely methylone, pentedrone, 4-methylethcathinone (4-MEC) and 3,4-methylenedioxypyrovalerone (MDPV). Two commercial mixtures ('Bloom' and 'Blow') containing mainly cathinone derivatives were also tested, and 3,4-methylenedioxymethamphetamine (MDMA) was used as the reference drug. The study allowed the identification of 19 compounds, showing that synthetic cathinones are the main active compounds present in these products. Qualitative and quantitative variability was found in products sold with the same trade name in matching or different 'smartshops'. In the toxicity studies performed in primary cultured rat hepatocytes, pentedrone and MDPV proved to be the most potent individual agents, with EC50 values of 0.664 and 0.742 mM, respectively, followed by MDMA (EC50 = 0.754 mM). 4-MEC and methylone were the least potent substances, with EC50 values significantly higher (1.29 and 1.18 mM, respectively; p < 0.05 vs. MDMA). 'Bloom' and 'Blow' showed hepatotoxic effects similar to MDMA (EC50 = 0.788 and 0.870 mM, respectively), with cathinones present in these mixtures contributing additively to the overall toxicological effect. Our results show a miscellany of psychoactive compounds present in 'legal high' products with evident hepatotoxic effects. These data contribute to increase the awareness on the real composition of 'legal high' packages and unveil the health risks posed by NPS.

Do microplastics influence the long-term effects of ciprofloxacin on the polychaete Hediste diversicolor? An integrated behavioral and biochemical approach.

Ciprofloxacin (CPX), the most commonly used fluoroquinolone antibiotic, and microplastics (MPs) are two classes of emerging contaminants with severe adverse impacts on aquatic organisms. Previous studies suggest that both CPX and MPs induce deleterious changes in exposed aquatic biota, but the characterization of a chronic and combined ecotoxicological response is not well known, especially in organisms from estuarine ecosystems. Thus, in this study, we investigated the behavioral and biochemical effects of environmentally relevant levels of CPX alone and in combination with polyethylene terephthalate (PET) microplastics over 28 days of exposure, using the polychaete Hediste diversicolor as a model. In addition to behavioral parameters, different biochemical endpoints were also evaluated, namely the levels of metabolic enzymes of phase I (7-ethoxy-resorufin-O-deethylase, EROD), and phase II (glutathione-S-transferase, GSTs), antioxidant defense (catalase, CAT; glutathione peroxidase, GPx; superoxide dismutase, SOD), oxidative damage (lipid peroxidation, by means of levels of thiobarbituric acid reactive substances [TBARS]) and acetylcholinesterase (AChE). Chronic exposure to ciprofloxacin caused a decrease in burrowing time and a significant increase in SOD activity. In animals exposed to the combination of CPX and PET MPs, effects on behavioral traits were also observed, with higher concentrations of MPs leading to a marked delay in the animals' burrowing time. In addition, these animals showed changes in their antioxidant defenses, namely, a significant increase in SOD activity, while GPx activity was severely compromised. For none of the experimental groups, significant alterations were observed in the metabolic enzymes, TBARS or AChE. These findings provide the first insights into the responses of H. diversicolor when exposed to the combination of CPX and PET MPs, highlighting that, although the here studied conditions, there was no evidence of oxidative damage or neurotoxicity, these organisms are not risk-free in co-exposure scenarios, even at low environmental relevant concentrations.

From data to insight: Exploring contaminants in different food groups with literature mining and machine learning techniques.

Food remains a major source of human exposure to chemical contaminants that are unintentionally present in commodities globally, despite strict regulation. Scientific literature is a valuable source of quantification data on those contaminants in various foods, but manually summarizing the information is not practicable. In this review, literature mining and machine learning techniques were applied in 72 foods to obtain relevant information on 96 contaminants, including heavy metals, polychlorinated biphenyls, dioxins, furans, polycyclic aromatic hydrocarbons (PAHs), pesticides, mycotoxins, and heterocyclic aromatic amines (HAAs). The 11,723 data points collected from 254 papers from the last two decades were then used to identify the patterns of contaminants distribution. Considering contaminant categories, metals were the most studied globally, followed by PAHs, mycotoxins, pesticides, and HAAs. As for geographical region, the distribution was uneven, with Europe and Asia having the highest number of studies, followed by North and South America, Africa and Oceania. Regarding food groups, all contained metals, while PAHs were found in seven out of 12 groups. Mycotoxins were found in six groups, and pesticides in almost all except meat, eggs, and vegetable oils. HAAs appeared in only three food groups, with fish and seafood reporting the highest levels. The median concentrations of contaminants varied across food groups, with citrinin having the highest median value. The information gathered is highly relevant to explore, establish connections, and identify patterns between diverse datasets, aiming at a comprehensive view of food contamination.

Toxicometabolomics: Small Molecules to Answer Big Toxicological Questions.

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 Induce Oxidative Stress and Apoptosis in Human Kidney Cells.

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.

Effect of temperature on 3,4-Methylenedioxypyrovalerone (MDPV)-induced metabolome disruption in primary mouse hepatic cells.

3,4-Methylenedioxypyrovalerone (MDPV) is one of the most popular cathinone derivatives worldwide and has recently been associated with several intoxications and deaths, in which, similarly to amphetamines, hyperthermia appears to play a prominent role. However, there remains a huge information gap underlying the mechanisms associated with its hepatotoxicity, namely under hyperthermic conditions. Here, we use a sensitive untargeted metabolomic approach based on gas chromatography-mass spectrometry (GC-MS) to investigate the effect of subtoxic and toxic concentrations of MDPV on the metabolic profile of primary mouse hepatocytes (PMH), under normothermic and hyperthermic conditions. For this purpose, hepatocytes were exposed to increasing concentrations of MDPV (LC01, LC10 and LC30) for 24 h, at 37 °C or 40.5 °C, and alterations on both intracellular metabolome and extracellular volatilome were evaluated. Multivariate analysis showed a clear separation between MDPV exposed cells and control cells in normothermic conditions, even at subtoxic concentrations (LC01 and LC10). In normothermia, there was a significant dysregulation of pathways associated with ascorbate metabolism, tricarboxylic acid (TCA) cycle and pyruvate metabolism. These metabolic changes were significantly increased at 40.5 °C, and several other pathways appear to be affected with the evolution of toxicity caused by MDPV under hyperthermic conditions, namely aspartate and glutamate metabolism, phenylalanine and tyrosine biosynthesis, aminoacyl-tRNA biosynthesis, butanoate metabolism, among others. Overall, our findings provide novel insights into the mechanism of hepatotoxicity triggered by MDPV and highlight the higher risks that may occur under hyperthermic conditions.

Volatilomics Reveals Potential Biomarkers for Identification of Renal Cell Carcinoma: An In Vitro Approach.

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.

Synthetic Cannabinoids JWH-122 and THJ-2201 Disrupt Endocannabinoid-Regulated Mitochondrial Function and Activate Apoptotic Pathways as a Primary Mechanism of In Vitro Nephrotoxicity at In Vivo Relevant Concentrations.

The widespread recreational use of synthetic cannabinoids (SCBs) represents a major public health issue, as reports of intoxications and deaths following SCB use rapidly mount up. Specifically, a direct link between SCB use and acute kidney injury (AKI) has been established, although the pathophysiologic mechanisms remain undefined. Here we assessed the in vitro nephrotoxicity of 3 commonly detected and structurally distinct SCBs-AB-FUBINACA, JWH-122, and THJ-2201-in human proximal tubule cells (HK-2), to ascertain potential similarities and/or differences regarding their nephrotoxicity signatures. We showed that 2 of the 3 SCBs tested, namely JWH-122 and THJ-2201, at in vivo relevant concentrations (1 nM-1 µM), triggered apoptotic cell death pathways, mainly through a shared mechanism involving the deregulation of mitochondrial function (ie, with mitochondrial membrane hyperpolarization and increased intracellular ATP levels), as the primary molecular signature of nephrotoxicity mechanism. Noteworthy, no SCB affected cell viability (MTT reduction, lactate dehydrogenase release, Neutral Red inclusion). Use of the cannabinoid receptor (CBR) antagonists SR141716A and SR144528, as well as HEK293T cells, which do not express CBRs, confirmed the involvement of these receptors in SCB-mediated mitochondrial membrane hyperpolarization but not on other events, suggesting an off-target action regulating SCB-induced kidney cell death. Our results further strengthen the relevance of the endocannabinoid system in maintaining mitochondrial function in kidney cells, as we demonstrate that HK-2 incubation with CBR antagonists or inhibitors of endocannabinoid biosynthesis (ie, methyl arachydonyl fluorophosphonate, tetrahydrolipstatin) alone produced deleterious effects similar to those now reported for SCBs. Overall, SCB-induced nephrotoxicity seems to be mainly regulated at the mitochondrial level, but the specific mechanisms involved require further clarification.

Hepatic Metabolic Derangements Triggered by Hyperthermia: An In Vitro Metabolomic Study.

BACKGROUND AND AIMS: Liver toxicity is a well-documented and potentially fatal adverse complication of hyperthermia. However, the impact of hyperthermia on the hepatic metabolome has hitherto not been investigated. METHODS: In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was applied to assess the in vitro metabolic response of primary mouse hepatocytes (PMH, n = 10) to a heat stress stimulus, i.e., after 24 h exposure to 40.5 °C. Metabolomic profiling of both intracellular metabolites and volatile metabolites in the extracellular medium of PMH was performed. RESULTS: Multivariate analysis showed alterations in levels of 22 intra- and 59 extracellular metabolites, unveiling the capability of the metabolic pattern to discriminate cells exposed to heat stress from cells incubated at normothermic conditions (37 °C). Hyperthermia caused a considerable loss of cell viability that was accompanied by significant alterations in the tricarboxylic acid cycle, amino acids metabolism, urea cycle, glutamate metabolism, pentose phosphate pathway, and in the volatile signature associated with the lipid peroxidation process. CONCLUSION: These results provide novel insights into the mechanisms underlying hyperthermia-induced hepatocellular damage.

Development and optimization of a HS-SPME-GC-MS methodology to quantify volatile carbonyl compounds in Port wines.

A method based on headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-triple quadrupole/mass spectrometry detection (GC-TQ/MS) with a prior derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was developed to quantify carbonyl compounds in different categories of Port wines. Optimal extraction conditions were obtained incubating 2 ml of wine with 2.3 g/l of PFBHA for 10 min and extracted during 20 min at 32 °C. The method was validated for 38 carbonyl compounds (alkanals, alkenals, Strecker aldehydes, dialdehydes, ketones and furan aldehydes) with regard to linearity, repeatability, inter and intra-day precision and accuracy, showing that the method is suitable for the determination of carbonyl compounds in wines. Tawny wines with 'indication of age' (10-40 years old) presented the highest levels of some carbonyl compounds, such as propanal, pentanal, hexanal, Strecker aldehydes, diacetyl, methyl glyoxal, 3-pentanone and 2-furfural, whereas Ruby wines were characterized by the highest amounts of some unidentified compounds.