Autophagy (but not metabolism) is a key event in mitoxantrone-induced cytotoxicity in differentiated AC16 cardiac cells.

Mitoxantrone (MTX) is an antineoplastic agent used to treat advanced breast cancer, prostate cancer, acute leukemia, lymphoma and multiple sclerosis. Although it is known to cause cumulative dose-related cardiotoxicity, the underlying mechanisms are still poorly understood. This study aims to compare the cardiotoxicity of MTX and its' pharmacologically active metabolite naphthoquinoxaline (NAPHT) in an in vitro cardiac model, human-differentiated AC16 cells, and determine the role of metabolism in the cardiotoxic effects. Concentration-dependent cytotoxicity was observed after MTX exposure, affecting mitochondrial function and lysosome uptake. On the other hand, the metabolite NAPHT only caused concentration-dependent cytotoxicity in the MTT reduction assay. When assessing the effect of different inhibitors/inducers of metabolism, it was observed that metyrapone (a cytochrome P450 inhibitor) and phenobarbital (a cytochrome P450 inducer) slightly increased MTX cytotoxicity, while 1-aminobenzotriazole (a suicide cytochrome P450 inhibitor) decreased fairly the MTX-triggered cytotoxicity in differentiated AC16 cells. When focusing in autophagy, the mTOR inhibitor rapamycin and the autophagy inhibitor 3-methyladenine exacerbated the cytotoxicity caused by MTX and NAPHT, while the autophagy blocker, chloroquine, partially reduced the cytotoxicity of MTX. In addition, we observed a decrease in p62, beclin-1, and ATG5 levels and an increase in LC3-II levels in MTX-incubated cells. In conclusion, in our in vitro model, neither metabolism nor exogenously given NAPHT are major contributors to MTX toxicity as seen by the residual influence of metabolism modulators used on the observed cytotoxicity and by NAPHT's low cytotoxicity profile. Conversely, autophagy is involved in MTX-induced cytotoxicity and MTX seems to act as an autophagy inducer, possibly through p62/LC3-II involvement.

Ecstasy metabolites and monoamine neurotransmitters upshift the Na+/K+ ATPase activity in mouse brain synaptosomes.

3,4-Methylenedioximethamphetamine (MDMA; "ecstasy") is a psychotropic drug with well-known neurotoxic effects mediated by hitherto not fully understood mechanisms. The Na+- and K+-activated adenosine 5'-triphosphatase (Na+/K+ ATPase), by maintaining the ion gradient across the cell membrane, regulates neuronal excitability. Thus, a perturbation of its function strongly impacts cell homeostasis, ultimately leading to neuronal dysfunction and death. Nevertheless, whether MDMA affects the Na+/K+ ATPase remains unknown. In this study, we used synaptosomes obtained from whole mouse brain to test the effects of MDMA, three of its major metabolites [α-methyldopamine, N-methyl-α-methyldopamine and 5-(glutathion-S-yl)-α-methyldopamine], serotonin (5-HT), dopamine, 3,4-dihydroxy-L-phenylalanine (L-Dopa) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the Na+/K+ ATPase function. A concentration-dependent increase of Na+/K+ ATPase activity was observed in synaptosomes exposed to the tested compounds (concentrations ranging from 0.0625 to 200 µM). These effects were independent of protein kinases A and C activities. Nevertheless, a rescue of the compounds' effects was observed in synaptosomes pre-incubated with the antioxidant N-acetylcysteine (1 mM), suggesting a role for reactive species-regulated pathways on the Na+/K+ ATPase effects. In agreement with this hypothesis, a similar increase in the pump activity was found in synaptosomes exposed to the chemical generator of superoxide radicals, phenazine methosulfate (1-250 µM). This study demonstrates the ability of MDMA metabolites, monoamine neurotransmitters, L-Dopa and DOPAC to alter the Na+/K+ ATPase function. This could represent a yet unknown mechanism of action of MDMA and its metabolites in the brain.

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.

Study of the intestinal uptake and permeability of gold nanoparticles using both in vitro and in vivo approaches.

Gold nanoparticles (AuNPs) are highly attractive to biomedical applications. Here, we investigated the effects of (i) ca. 15 nm spherical AuNPs capped with citrate or 11-mercaptoundecanoic acid (MUA) and (ii) ca. 60 nm spherical citrate-capped AuNPs, and ca. 60 nm MUA-capped star-shaped AuNPs on the cytotoxicity, cellular uptake and permeability, using media supplemented or not with 1% fetal bovine serum (FBS) on caucasian colon adenocarcinoma Caco-2 cells. In addition, the colloidal stability of the nanoparticles in media (supplemented or not) was assessed after 24 h-incubations at 60 µM. The 60 nm gold nanospheres and stars were administrated orally to Wistar rats in order to evaluate their systemic absorption and biodistribution after 24 h. At non-supplemented media settings, citrate-capped gold nanoparticles seem to be more toxic than their MUA-capped counterparts. Also, smaller nanoparticles show higher toxicity than larger ones. The use of cell culture media with 1% FBS not only increased the stability of all AuNPs, as also significantly reduced their cytotoxicity. In the uptake studies, higher AuNPs incorporation was noticed in serum supplemented media, this effect being particularly significant for the 60 nm nanoparticles. Cellular incorporation depended also on the capping agent and size. None of the tested samples crossed the in vitro intestinal barrier. Confirming the in vitro results, the in vivo biodistribution study of the 60 nm AuNPs orally given to rats showed that their systemic absorption is low and that they are mainly eliminated through the faeces. Altogether, these preliminary results suggest that our novel AuNPs have high potential to be considered promising candidates for application in diagnostics or drug delivery at the intestinal level, showing high biocompatibility. However, unless it is desired that these nanomaterials avoid systemic absorption upon oral administration, additional functionalization should be sought to increase their low bioavailability.

Identification of a biomarker panel for improvement of prostate cancer diagnosis by volatile metabolic profiling of urine.

BACKGROUND: The lack of sensitive and specific biomarkers for the early detection of prostate cancer (PCa) is a major hurdle to improve patient management. METHODS: A metabolomics approach based on GC-MS was used to investigate the performance of volatile organic compounds (VOCs) in general and, more specifically, volatile carbonyl compounds (VCCs) present in urine as potential markers for PCa detection. RESULTS: Results showed that PCa patients (n = 40) can be differentiated from cancer-free subjects (n = 42) based on their urinary volatile profile in both VOCs and VCCs models, unveiling significant differences in the levels of several metabolites. The models constructed were further validated using an external validation set (n = 18 PCa and n = 18 controls) to evaluate sensitivity, specificity and accuracy of the urinary volatile profile to discriminate PCa from controls. The VOCs model disclosed 78% sensitivity, 94% specificity and 86% accuracy, whereas the VCCs model achieved the same sensitivity, a specificity of 100% and an accuracy of 89%. Our findings unveil a panel of 6 volatile compounds significantly altered in PCa patients' urine samples that was able to identify PCa, with a sensitivity of 89%, specificity of 83%, and accuracy of 86%. CONCLUSIONS: It is disclosed a biomarker panel with potential to be used as a non-invasive diagnostic tool for PCa.

l-proline supplementation improves nitric oxide bioavailability and counteracts the blood pressure rise induced by angiotensin II in rats.

We evaluated whether l-proline (Pro) supplementation improves redox status and nitric oxide (NO) bioavailability and prevents or delays angiotensin II (AngII)-induced hypertension. Male Sprague-Dawley rats were distributed to four experimental groups: Pro + AngII (Pro-Ang), Pro + Saline (Pro-Sal), Vehicle + AngII (Veh-Ang) and Veh + Saline (Veh-Sal). Pro solution (2 g.kg-1·day-1) or water (vehicle) were orally administered, from day 0 to day 21. AngII (200 ng.kg-1.min-1) or saline were infused (s.c.) from day 7 to day 21. Systolic blood pressure (SBP) was measured by the tail-cuff method. From day 20-21, animals were kept on metabolic cages for 24h-urine collection. On day 21, urine and blood were collected for further quantification of redox status biomarkers, NO-related markers (urinary nitrates and nitrites, U-NOx; plasma asymmetric dimethylarginine, P-ADMA), metabolic and renal parameters. Pro prevented the AngII-induced SBP rise [mean (95% CI), Day 19: Pro-AngII, 137 (131; 143) vs. Veh-AngII, 157 (151; 163) mm Hg, P < 0.001]. Pro-AngII rats also had increased values of U-NOx, systemic and urinary total antioxidant status (TAS), urinary H2O2 and plasma urea, as well as reduced P-ADMA and unaltered urinary isoprostanes. Plasma Pro was inversely correlated with P-ADMA (r = -0.52, p = 0.0009) and positively correlated with urinary TAS (r = 0.55, p = 0.0005) which, in turn, was inversely correlated with P-ADMA (r = -0.56, p = 0.0004). Furthermore, urinary H2O2 values decreased across P-ADMA tertiles (p for linear trend = 0.023). These results suggest that Pro reduces P-ADMA levels and improves redox status, thereby increasing NO bioavailability and counteracting the AngII-induced SBP rise. H2O2 and TAS modulation by Pro may contribute to the reduced P-ADMA concentration.

An effective antidotal combination of polymyxin B and methylprednisolone for α-amanitin intoxication.

Amanita phalloides is one of the most toxic mushrooms worldwide, and it is involved in the majority of human fatal cases of mushroom poisoning. α-Amanitin, the most deleterious toxin of A. phalloides to humans, inhibits RNA polymerase II (RNAPII), causing hepatic and renal failure. Previously, we have shown that polymyxin B (polB) reverts α-amanitin inhibition of RNAPII, although it was not able to guarantee the full survival of α-amanitin-intoxicated mice or prevent α-amanitin pro-inflammatory effects. α-Amanitin is also a substrate of the organic-anion-transporting polypeptide 1B3 (OATP1B3) and Na(+)-taurocholate cotransporter polypeptide (NTCP) transporters. Therefore, in the present work, we used a combination of polB [(2.5 mg/kg intraperitoneal (i.p.)] with the anti-inflammatory and NTCP inhibitor drug, methylprednisolone (MP) (10 mg/kg i.p.), as an attempt to fully revert α-amanitin-induced toxicity (0.33 mg/kg i.p.) in CD-1 mice. Results showed that the administration of the polB + MP combination, 4 h after α-amanitin, led to the full survival of the intoxicated animals, with a significant attenuation of α-amanitin-induced renal and hepatic necrosis. Also, the combination polB + MP led to a decrease of aminotransferase plasma levels, of the renal myeloperoxidase activity and of renal inflammatory cell infiltrate promoted by α-amanitin, although not preventing any of the hepatic pro-inflammatory effect of the toxin. The obtained results indicate that this combination may represent an important and valuable therapeutic approach to be used against α-amanitin intoxication.

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.

Biomarkers in bladder cancer: A metabolomic approach using in vitro and ex vivo model systems.

Metabolomics has recently proved to be useful in the area of biomarker discovery for cancers in which early diagnostic and prognostic biomarkers are urgently needed, as is the case of bladder cancer (BC). This article presents a comprehensive review of the literature on the metabolomic studies on BC, highlighting metabolic pathways perturbed in this disease and the altered metabolites as potential biomarkers for BC detection. Current disease model systems used in the study of BC metabolome include in vitro-cultured cancer cells, ex vivo neoplastic bladder tissues and biological fluids, mainly urine but also blood serum/plasma, from BC patients. The major advantages and drawbacks of each model system are discussed. Based on available data, it seems that BC metabolic signature is mainly characterized by alterations in metabolites related to energy metabolic pathways, particularly glycolysis, amino acid and fatty acid metabolism, known to be crucial for cell proliferation, as well as glutathione metabolism, known to be determinant in maintaining cellular redox balance. In addition, purine and pyrimidine metabolism as well as carnitine species were found to be altered in BC. Finally, it is emphasized that, despite the progress made in respect to novel biomarkers for BC diagnosis, there are still some challenges and limitations that should be addressed in future metabolomic studies to ensure their translatability to clinical practice.

Hepatotoxicity of piperazine designer drugs: up-regulation of key enzymes of cholesterol and lipid biosynthesis.

The piperazine derivatives most frequently consumed for recreational purposes are 1-benzylpiperazine, 1-(3,4-methylenedioxybenzyl) piperazine, 1-(3-trifluoromethylphenyl) piperazine and 1-(4-methoxyphenyl) piperazine. Generally, they are consumed as capsules, tablets or pills but also in powder or liquid forms. Currently, the precise mechanism by which piperazine designer drugs induce hepatotoxicity and whether they act by a common pathway is unclear. To answer this question, we performed a gene array study with rat hepatocytes incubated with the four designer drugs. Non-cytotoxic concentrations were chosen that neither induce a decrease in reduced glutathione or ATP depletion. Analysis of the gene array data showed a large overlap of gene expression alterations induced by the four drugs. This 'piperazine designer drug consensus signature' included 101 up-regulated and 309 down-regulated probe sets (p < 0.05; FDR adjusted). In the up-regulated genes, GO groups of cholesterol biosynthesis represented a dominant overrepresented motif. Key enzymes of cholesterol biosynthesis up-regulated by all four piperazine drugs include sterol C4-methyloxidase, isopentyl-diphosphate-Δ-isomerase, Cyp51A1, squalene epoxidase and farnesyl diphosphate synthase. Additionally, glycoprotein transmembrane nmb, which participates in cell adhesion processes, and fatty acid desaturase 1, an enzyme that regulates unsaturation of fatty acids, were also up-regulated by the four piperazine designer drugs. Regarding the down-regulated probe sets, only one gene was common to all four piperazine derivatives, the betaine-homocysteine-S-methyltransferase 2. Analysis of transcription factor binding sites of the 'piperazine designer drug consensus signature' identified the sterol regulatory element binding protein (SREBP-1) as strongly overrepresented in the up-regulated genes. SREBP transcription factors are known to regulate multiple genes of cholesterol metabolism. In conclusion, the present study shows that piperazine designer drugs act by up-regulating key enzymes of cholesterol biosynthesis which is likely to increase the risk of phospholipidosis and steatosis.

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.

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B.

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. α-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to α-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of α-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by α-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal α-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to α-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-α-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with α-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in α-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.

Determination of amatoxins and phallotoxins in Amanita phalloides mushrooms from northeastern Portugal by HPLC-DAD-MS.

Amanita phalloides is a toxic mushroom responsible for the majority of deaths occurring after mushrooms ingestion, mainly due to amatoxins. In the present study the contents and distribution of the major amatoxins and phallotoxins in different tissues of A. phalloides from two different sites of Portugal were analyzed by liquid chromatography (LC) coupled to diode array (DAD) and mass spectrometry (MS) detection. The main toxins were separated by LC and its chemical structures confirmed by MS. α-Amanitin contents in caps, stipe and volva tissues were quantified by RP-HPLC. The results show that caps have the highest content of amatoxins, whereas the volva was richest in phallotoxins. Moreover variability in the toxins composition from different geographic sites was also observed. This study provides for the first time the content of toxins in A. phalloides from Portugal.

Khat and synthetic cathinones: a review.

For centuries, 'khat sessions' have played a key role in the social and cultural traditions among several communities around Saudi Arabia and most East African countries. The identification of cathinone as the main psychoactive compound of khat leaves, exhibiting amphetamine-like pharmacological properties, resulted in the synthesis of several derivatives structurally similar to this so-called natural amphetamine. Synthetic cathinones were primarily developed for therapeutic purposes, but promptly started being misused and extensively abused for their euphoric effects. In the mid-2000's, synthetic cathinones emerged in the recreational drug markets as legal alternatives ('legal highs') to amphetamine, 'ecstasy', or cocaine. Currently, they are sold as 'bath salts' or 'plant food', under ambiguous labels lacking information about their true contents. Cathinone derivatives are conveniently available online or at 'smartshops' and are much more affordable than the traditional illicit drugs. Despite the scarcity of scientific data on these 'legal highs', synthetic cathinones use became an increasingly popular practice worldwide. Additionally, criminalization of these derivatives is often useless since for each specific substance that gets legally controlled, one or more structurally modified analogs are introduced into the legal market. Chemically, these substances are structurally related to amphetamine. For this reason, cathinone derivatives share with this drug both central nervous system stimulating and sympathomimetic features. Reports of intoxication and deaths related to the use of 'bath salts' have been frequently described over the last years, and several attempts to apply a legislative control on synthetic cathinones have been made. However, further research on their pharmacological and toxicological properties is fully required in order to access the actual potential harm of synthetic cathinones to general public health. The present work provides a review on khat and synthetic cathinones, concerning their historical background, prevalence, patterns of use, legal status, chemistry, pharmacokinetics, pharmacodynamics, and their physiological and toxicological effects on animals and humans.

Comparative In Vitro Study of the Cytotoxic Effects of Doxorubicin's Main Metabolites on Cardiac AC16 Cells Versus the Parent Drug.

Doxorubicin (DOX; also known as adriamycin) serves as a crucial antineoplastic agent in cancer treatment; however, its clinical utility is hampered by its' intrinsic cardiotoxicity. Although most DOX biotransformation occurs in the liver, a comprehensive understanding of the impact of DOX biotransformation and its' metabolites on its induced cardiotoxicity remains to be fully elucidated. This study aimed to explore the role of biotransformation and DOX's main metabolites in its induced cardiotoxicity in human differentiated cardiac AC16 cells. A key discovery from our study is that modulating metabolism had minimal effects on DOX-induced cytotoxicity: even so, metyrapone (a non-specific inhibitor of cytochrome P450) increased DOX-induced cytotoxicity at 2 µM, while diallyl sulphide (a CYP2E1 inhibitor) decreased the 1 µM DOX-triggered cytotoxicity. Then, the toxicity of the main DOX metabolites, doxorubicinol [(DOXol, 0.5 to 10 µM), doxorubicinone (DOXone, 1 to 10 µM), and 7-deoxydoxorubicinone (7-DeoxyDOX, 1 to 10 µM)] was compared to DOX (0.5 to 10 µM) following a 48-h exposure. All metabolites evaluated, DOXol, DOXone, and 7-DeoxyDOX caused mitochondrial dysfunction in differentiated AC16 cells, but only at 2 µM. In contrast, DOX elicited comparable cytotoxicity, but at half the concentration. Similarly, all metabolites, except 7-DeoxyDOX impacted on lysosomal ability to uptake neutral red. Therefore, the present study showed that the modulation of DOX metabolism demonstrated minimal impact on its cytotoxicity, with the main metabolites exhibiting lower toxicity to AC16 cardiac cells compared to DOX. In conclusion, our findings suggest that metabolism may not be a pivotal factor in mediating DOX's cardiotoxic effects.

Doxorubicin-induced neurotoxicity differently affects the hippocampal formation subregions in adult mice.

Doxorubicin (DOX) is an anthracycline used to treat a wide range of tumours. Despite its effectiveness, it is associated with a long range of adverse effects, of which cognitive deficits stand out. The present study aimed to assess the neurologic adverse outcome pathways of two clinically relevant cumulative doses of DOX. Adult male CD-1 mice received biweekly intraperitoneal administrations for 3 weeks until reaching cumulative doses of 9 mg/kg (DOX9) or 18 mg/kg (DOX18). Animals were euthanized one week after the last administration, and biomarkers of oxidative stress and brain metabolism were evaluated in the whole brain. Coronal sections of fixed brains were used for specific determinations of the prefrontal cortex (PFC) and hippocampal formation (HF). In the whole brain, DOX18 tended to disrupt the antioxidant defences, affecting glutathione levels and manganese superoxide dismutase expression. Considering the regional analysis, DOX18 increased the volume of all brain areas evaluated, while GFAP-immunoreactive astrocytes decreased in the dentate gyrus (DG) and increased in the CA3 region of HF, both in a dose-dependent manner. Concerning the apoptosis pathway, whereas Bax increased in the DOX9 group, it decreased in the DOX18 group. Only in the latter group did Bcl-2 levels also decrease. While p53 only increased in the CA3 region of the DOX9 group, AIF increased in the PFC and DG of DOX18. Finally, phosphorylation of Tau decreased with the highest DOX dose in DG and CA3, while TNF-α levels increased in CA1 of DOX18. Our results indicate new pathways not yet described that could be responsible for the cognitive impairments observed in treated patients.

The metabolic profile of mitoxantrone and its relation with mitoxantrone-induced cardiotoxicity.

Mitoxantrone (MTX) is an antitumor agent that causes cardiotoxicity in 18 % patients. The metabolic profile of MTX was assessed after incubation of 100 µM MTX with hepatic S9 fraction isolated from rats. The presence of MTX and its metabolites was also assessed in vivo through the analysis of liver and heart extracts of MTX-treated rats. The cytotoxic effects of MTX and MTX metabolites were evaluated in the H9c2 cells after 24-h incubation with MTX alone and MTX + metabolites. The influence of CYP450- and CYP2E1-mediated metabolism for the cytotoxicity of MTX was assessed after 96-h incubation with MTX (100 nM and 1 µM) in the presence/absence of CYP450 or CYP2E1 inhibitors. After 4-h incubation in supplemented S9 fraction, the MTX content was 35 % lower and 5 metabolites were identified: an acetoxy ester derivative (never described before), two glutathione conjugates, a monocarboxylic acid derivative, and the naphtoquinoxaline, the later commonly related to MTX pharmacological effects. The presence of MTX and naphtoquinoxaline metabolite was evidenced in vivo in liver and heart of MTX-treated rats. The cytotoxicity caused by MTX + metabolites was higher than that observed in the H9c2 cells incubated with non-metabolized MTX group. The co-incubation of MTX with CYP450 and CYP2E1 inhibitors partially prevented the cytotoxicity observed in the MTX groups incubated with H9c2 cells, highlighting that the metabolism of MTX is relevant for its undesirable effects. The naphtoquinoxaline metabolite is described in heart and liver in vivo, highlighting that this metabolite accumulates in these tissues. It was demonstrated that MTX P450-mediated metabolism contributed to MTX toxicity.

Influence of the surface coating on the cytotoxicity, genotoxicity and uptake of gold nanoparticles in human HepG2 cells.

The toxicological profile of gold nanoparticles (AuNPs) remains controversial. Significant efforts to develop surface coatings to improve biocompatibility have been carried out. In vivo biodistribution studies have shown that the liver is a target for AuNPs accumulation. Therefore, we investigated the effects induced by ~20 nm spherical AuNPs (0-200 µM Au) with two surface coatings, citrate (Cit) compared with 11-mercaptoundecanoic acid (11-MUA), in human liver HepG2 cells. Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release assays after 24 to 72 h of incubation. DNA damage was assessed by the comet assay, 24 h after incubation with the capped AuNPs. Uptake and subcellular distribution of the tested AuNPs was evaluated by quantifying the gold intracellular content by graphite furnace atomic absorption spectrometry (GFAAS) and transmission electron microscopy (TEM), respectively. The obtained results indicate that both differently coated AuNPs did not induce significant cytotoxicity. An inverse concentration-dependent increase in comet tail intensity and tail moment was observed in Cit-AuNPs- but not in MUA-AuNPs-exposed cells. Both AuNPs were internalized in a concentration-dependent manner. However, no differences were found in the extent of the internalization between the two types of NPs. Electron-dense deposits of agglomerates of Cit- and MUA-AuNPs were observed either inside endosomes or in the intercellular spaces. In spite of the absence of cytotoxicity, DNA damage was observed after exposure to the lower concentrations of Cit- but not to MUA-AuNPs. Thus, our data supports the importance of the surface properties to increase the biocompatibility and safety of AuNPs.

Safety and efficacy of a feed additive consisting of Lentilactobacillus diolivorans (formerly Lactobacillus diolivorans) DSM 33625 as a silage additive for all animal species (Lactosan GmbH & Co.KG).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on Lentilactobacillus diolivorans (formerly Lactobacillus diolivorans) DSM 33625 when used as a technological additive to improve ensiling of forage. The additive is intended for use with all forages and for all animal species at a proposed minimum concentration of 1 × 108 colony forming units (CFU)/kg forage. The bacterial species L. diolivorans is considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. As the identity of the strain has been clearly established and no acquired antimicrobial resistance determinants of concern were detected, the use of the strain as a silage additive is considered safe for livestock species, for consumers of products from animals fed the treated silage and for the environment. The additive is not a skin or an eye irritant. In the absence of data, no conclusion can be drawn on the skin sensitisation of the additive. Given the proteinaceous nature of the active agent, the additive should be considered a respiratory sensitiser. The FEEDAP Panel concluded that Lentilactobacillus diolivorans DSM 33625 at a minimum concentration of 1 × 108 CFU/kg forage may extend the aerobic stability of silage prepared from easy and moderately difficult to ensile forage material with a DM range of 32-65%.