Investigating the applicability domain of the hiPSC-based PluriLum assay: an embryotoxicity assessment of chemicals and drugs.

To meet the growing demand for developmental toxicity assessment of chemicals, New Approach Methodologies (NAMs) are needed. Previously, we developed two 3D in vitro assays based on human-induced pluripotent stem cells (hiPSC) and cardiomyocyte differentiation: the PluriBeat assay, based on assessment of beating differentiated embryoid bodies, and the PluriLum assay, a reporter gene assay based on the expression of the early cardiac marker NKX2.5; both promising assays for predicting embryotoxic effects of chemicals and drugs. In this work, we aimed to further describe the predictive power of the PluriLum assay and compare its sensitivity with PluriBeat and similar human stem cell-based assays developed by others. For this purpose, we assessed the toxicity of a panel of ten chemicals from different chemical classes, consisting of the known developmental toxicants 5-fluorouracil, all-trans retinoic acid and valproic acid, as well as the negative control compounds ascorbic acid and folic acid. In addition, the fungicides epoxiconazole and prochloraz, and three perfluoroalkyl substances (PFAS), PFOS, PFOA and GenX were tested. Generally, the PluriLum assay displayed higher sensitivity when compared to the PluriBeat assay. For several compounds the luminescence readout of the PluriLum assay showed effects not detected by the PluriBeat assay, including two PFAS compounds and the two fungicides. Overall, we find that the PluriLum assay has the potential to provide a fast and objective detection of developmental toxicants and has a level of sensitivity that is comparable to or higher than other in vitro assays also based on human stem cells and cardiomyocyte differentiation for assessment of developmental toxicity.

Toxicity Mechanisms of Gadolinium and Gadolinium-Based Contrast Agents-A Review.

Gadolinium-based contrast agents (GBCAs) have been used for more than 30 years to improve magnetic resonance imaging, a crucial tool for medical diagnosis and treatment monitoring across multiple clinical settings. Studies have shown that exposure to GBCAs is associated with gadolinium release and tissue deposition that may cause short- and long-term toxicity in several organs, including the kidney, the main excretion organ of most GBCAs. Considering the increasing prevalence of chronic kidney disease worldwide and that most of the complications following GBCA exposure are associated with renal dysfunction, the mechanisms underlying GBCA toxicity, especially renal toxicity, are particularly important. A better understanding of the gadolinium mechanisms of toxicity may contribute to clarify the safety and/or potential risks associated with the use of GBCAs. In this work, a review of the recent literature concerning gadolinium and GBCA mechanisms of toxicity was performed.

Age-Related Changes in Clinical and Analytical Variables in Chronic Hemodialyzed Patients.

Worldwide, the number of elderly individuals receiving chronic hemodialysis is rising. The aim of our study was to evaluate several clinical and analytical biomarkers in chronically dialyzed patients and analyze how they change with age. A cross-sectional study was performed by evaluating 289 end-stage renal disease patients undergoing dialysis. We evaluated the hemogram, adipokines, the lipid profile, and several markers related to inflammation, endothelial function/fibrinolysis, nutrition, iron metabolism, and cardiac and renal fibrosis. Clinical data and dialysis efficacy parameters were obtained from all patients. The relationships between studied biomarkers and age were assessed by a statistical comparison between younger (adults with age < 65 years) and older (age ≥ 65 years) patients and by performing regression analysis. Participants presented a mean age of 68.7 years (±13.6), with 66.8% (n = 193) being classified as older. Compared to younger patients, older patients presented the following: (a) significantly lower values of diastolic blood pressure (DBP) and ultrafiltration volume; (b) lower levels of phosphorus, uric acid, creatinine, and albumin; and (c) higher circulating concentrations of tissue-type plasminogen activator (tPA), D-dimer, interleukin-6, leptin, N-terminal pro B-type natriuretic peptide, and tissue inhibitor of metalloproteinase-1. In the multiple linear regression analysis, DBP values, tPA, phosphorus, and D-dimer levels were independently associated with the age of patients (standardized betas: -0.407, 0.272, -0.230, and 0.197, respectively; p < 0.001 for all), demonstrating relevant changes in biomarkers with increasing age at cardiovascular and nutritional levels. These findings seem to result from crosstalk mechanisms between aging and chronic kidney disease.

An Exemestane Derivative, Oxymestane-D1, as a New Multi-Target Steroidal Aromatase Inhibitor for Estrogen Receptor-Positive (ER+) Breast Cancer: Effects on Sensitive and Resistant Cell Lines.

Around 70-85% of all breast cancer (BC) cases are estrogen receptor-positive (ER+). The third generation of aromatase inhibitors (AIs) is the first-line treatment option for these tumors. Despite their therapeutic success, they induce several side effects and resistance, which limits their efficacy. Thus, it is crucial to search for novel, safe and more effective anti-cancer molecules. Currently, multi-target drugs are emerging, as they present higher efficacy and lower toxicity in comparison to standard options. Considering this, this work aimed to investigate the anti-cancer properties and the multi-target potential of the compound 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (Oxy), also designated by Oxymestane-D1, a derivative of Exemestane, which we previously synthesized and demonstrated to be a potent AI. For this purpose, it was studied for its effects on the ER+ BC cell line that overexpresses aromatase, MCF-7aro cells, as well as on the AIs-resistant BC cell line, LTEDaro cells. Oxy reduces cell viability, impairs DNA synthesis and induces apoptosis in MCF-7aro cells. Moreover, its growth-inhibitory properties are inhibited in the presence of ERα, ERß and AR antagonists, suggesting a mechanism of action dependent on these receptors. In fact, Oxy decreased ERα expression and activation and induced AR overexpression with a pro-death effect. Complementary transactivation assays demonstrated that Oxy presents ER antagonist and AR agonist activities. In addition, Oxy also decreased the viability and caused apoptosis of LTEDaro cells. Therefore, this work highlights the discovery of a new and promising multi-target drug that, besides acting as an AI, appears to also act as an ERα antagonist and AR agonist. Thus, the multi-target action of Oxy may be a therapeutic advantage over the three AIs applied in clinic. Furthermore, this new multi-target compound has the ability to sensitize the AI-resistant BC cells, which represents another advantage over the endocrine therapy used in the clinic, since resistance is a major drawback in the clinic.

Inflammatory biomarkers in staging of chronic kidney disease: elevated TNFR2 levels accompanies renal function decline.

BACKGROUND: Inflammation is a common feature in the pathogenesis of chronic kidney disease (CKD), regardless of the disease cause. Our aim was to evaluate the potential of several inflammatory biomarkers in CKD diagnosis and staging. METHODS: A total of 24 healthy controls and 92 pre-dialysis CKD patients with diverse etiologies, were enrolled in this study and grouped according to their CKD stage. We analysed the circulating levels of inflammatory molecules, C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), tumor necrosis factor receptor 2 (TNFR2), pentraxin 3 (PTX3) and leptin, as well as the hemogram. We studied their association with parameters of kidney function and kidney injury, to evaluate their potential as early markers of the disease and/or of its worsening, as well as their interplay. RESULTS: Compared to controls, patients in CKD stages 1-2 presented significantly higher IL-6 and TNFR2 levels, and higher neutrophil-to-lymphocyte ratio. All inflammatory cytokines and acute-phase proteins showed a trend to increase up to stage 3, stabilizing or declining thereafter, save for TNFR2, which steadily increased from stage to stage. All inflammatory molecules, apart from PTX3, were negatively and significantly correlated with eGFR, with a remarkable value for TNFR2 (r = - 0.732, p < 0.001). CONCLUSION: TNFR2 might be useful for an early detection of CKD, as well as for disease staging/worsening. Still, the potential value of this biomarker in disease progression warrants further investigation.

Sunitinib versus Pazopanib Dilemma in Renal Cell Carcinoma: New Insights into the In Vitro Metabolic Impact, Efficacy, and Safety.

Sunitinib and pazopanib are tyrosine kinase inhibitors (TKIs) used as first-line therapy for metastatic renal cell carcinoma (RCC). Although these TKIs are associated with similar survival outcomes, some differences have been reported in their safety profiles. In this work, traditional toxicological endpoints (cell viability and growth, oxidative stress, and nuclear morphology) and 1H NMR spectroscopy-based metabolomics analysis were used to provide new insights into the cytotoxicity and metabolic mechanisms underlying sunitinib and pazopanib treatments. Tumoral (Caki-1) and non-tumoral (HK-2) human renal cells were exposed to clinically relevant concentrations of sunitinib (2 µM) or pazopanib (50 µM). Sunitinib showed selectivity for cancer cells, inhibiting proliferation, and inducing apoptotic death of Caki-1 cells, whereas pazopanib had a similar cytotoxic effect in both tumoral and non-tumoral cells. 1H-NMR metabolomics unveiled a higher impact of sunitinib on the levels of intracellular metabolites of Caki-1 cells (seven dysregulated metabolites), suggesting dysregulations on amino acid, glutathione and glycerophospholipid metabolisms. In contrast, pazopanib had a higher impact on the levels of extracellular metabolites of Caki-1 cells (seven dysregulated metabolites in culture medium), unveiling alterations on amino acid and energetic metabolisms. In HK-2 cells, sunitinib caused only a minor increase in intracellular isoleucine levels, whereas pazopanib induced several alterations on the intracellular (three dysregulated metabolites) and extracellular (three dysregulated metabolites) compartments suggesting changes on amino acid, glycerophospholipid, and energy metabolisms. Our results demonstrate that these TKIs elicit distinct cellular and metabolic responses, with sunitinib showing better in vitro efficacy against target RCC cells and lesser nephrotoxic potential than pazopanib.

Beyond faith: Biomolecular evidence for changing urban economies in multi-faith medieval Portugal.

OBJECTIVES: During the Middle Ages, Portugal witnessed unprecedented socioeconomic and religious changes under transitioning religious political rule. The implications of changing ruling powers for urban food systems and individual diets in medieval Portugal is poorly understood. This study aimed to elucidate the dietary impact of the Islamic and Christian conquests. MATERIALS AND METHODS: Radiocarbon dating, peptide mass fingerprinting (ZooMS) and stable isotope analysis (δ13 C, δ15 N) of animal (n = 59) and human skeletal remains (n = 205) from Muslim and Christian burials were used to characterize the diet of a large historical sample from Portugal. A Bayesian stable isotope mixing model (BSIMM) was used to estimate the contribution of marine protein to human diet. RESULTS: Early medieval (8-12th century), preconquest urban Muslim populations had mean (±1SD) values of -18.8 ± 0.4 ‰ for δ13 C 10.4 ± 1 ‰ for δ15 N, indicating a predominantly terrestrial diet, while late medieval (12-14th century) postconquest Muslim and Christian populations showed a greater reliance on marine resources with mean (±1SD) values of -17.9 ± 1.3‰ for δ13 C and 11.1 ± 1.1‰ for δ15 N. BSIMM estimation supported a significant increase in the contribution of marine resources to human diet. DISCUSSION: The results provide the first biomolecular evidence for a dietary revolution that is not evidenced in contemporaneous historical accounts. We find that society transitioned from a largely agro-pastoral economy under Islamic rule to one characterized by a new focus on marine resources under later Christian rule. This economic change led to the naissance of the marine economy that went on to characterize the early-modern period in Portugal and its global expansion.

From street to lab: in vitro hepatotoxicity of buphedrone, butylone and 3,4-DMMC.

Synthetic cathinones are among the most popular new psychoactive substances, being abused for their stimulant properties, which are similar to those of amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Considering that the liver is a likely target for cathinones-induced toxicity, and for their metabolic activation/detoxification, we aimed to determine the hepatotoxicity of three commonly abused synthetic cathinones: butylone, α-methylamino-butyrophenone (buphedrone) and 3,4-dimethylmethcathinone (3,4-DMMC). We characterized their cytotoxic profile in primary rat hepatocytes (PRH) and in the HepaRG and HepG2 cell lines. PRH was the most sensitive cell model, showing the lowest EC50 values for all three substances (0.158 mM for 3,4-DMMC; 1.21 mM for butylone; 1.57 mM for buphedrone). Co-exposure of PRH to the synthetic cathinones and CYP450 inhibitors (selective and non-selective) proved that hepatic metabolism reduced the toxicity of buphedrone but increased that of butylone and 3,4-DMMC. All compounds were able to increase oxidative stress, disrupting mitochondrial homeostasis and inducing apoptotic and necrotic features, while also increasing the occurrence of acidic vesicular organelles in PRH, compatible with autophagic activation. In conclusion, butylone, buphedrone and 3,4-DMMC have hepatotoxic potential, and their toxicity lies in the interference with a number of homeostatic processes, while being influenced by their metabolic fate.

Emerging club drugs: 5-(2-aminopropyl)benzofuran (5-APB) is more toxic than its isomer 6-(2-aminopropyl)benzofuran (6-APB) in hepatocyte cellular models.

New phenylethylamine derivatives are among the most commonly abused new psychoactive substances. They are synthesized and marketed in lieu of classical amphetaminic stimulants, with no previous safety testing. Our study aimed to determine the in vitro hepatotoxicity of two benzofurans [6-(2-aminopropyl)benzofuran (6-APB) and 5-(2-aminopropyl)benzofuran (5-APB)] that have been misused as 'legal highs'. Cellular viability was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, following 24-h drug exposure of human hepatoma HepaRG cells (EC50 2.62 mM 5-APB; 6.02 mM 6-APB), HepG2 cells (EC50 3.79 mM 5-APB; 8.18 mM 6-APB) and primary rat hepatocytes (EC50 964 µM 5-APB; 1.94 mM 6-APB). Co-incubation of primary hepatocytes, the most sensitive in vitro model, with CYP450 inhibitors revealed a role of metabolism, in particular by CYP3A4, in the toxic effects of both benzofurans. Also, 6-APB and 5-APB concentration-dependently enhanced oxidative stress (significantly increased reactive species and oxidized glutathione, and decreased reduced glutathione levels) and unsettled mitochondrial homeostasis, with disruption of mitochondrial membrane potential and decline of intracellular ATP. Evaluation of cell death mechanisms showed increased caspase-8, -9, and -3 activation, and nuclear morphological changes consistent with apoptosis; at concentrations higher than 2 mM, however, necrosis prevailed. Concentration-dependent formation of acidic vesicular organelles typical of autophagy was also observed for both drugs. Overall, 5-APB displayed higher hepatotoxicity than its 6-isomer. Our findings provide new insights into the potential hepatotoxicity of these so-called 'safe drugs' and highlight the putative risks associated with their use as psychostimulants.

The new psychoactive substance 3-methylmethcathinone (3-MMC or metaphedrone) induces oxidative stress, apoptosis, and autophagy in primary rat hepatocytes at human-relevant concentrations.

3-Methylmethcathinone (3-MMC or metaphedrone) has become one of the most popular recreational drugs worldwide after the ban of mephedrone, and was recently deemed responsible for several intoxications and deaths. This study aimed at assessing the hepatotoxicity of 3-MMC. For this purpose, Wistar rat hepatocytes were isolated by collagenase perfusion, cultured and exposed for 24 h at a concentration range varying from 31 nM to 10 mM 3-MMC. The modulatory effects of cytochrome P450 (CYP) inhibitors on 3-MMC hepatotoxicity were evaluated. 3-MMC-induced toxicity was perceived at the lysosome at lower concentrations (NOEC 312.5 µM), compared to mitochondria (NOEC 379.5 µM) and cytoplasmic membrane (NOEC 1.04 mM). Inhibition of CYP2D6 and CYP2E1 diminished 3-MMC cytotoxicity, yet for CYP2E1 inhibition this effect was only observed for concentrations up to 1.3 mM. A significant concentration-dependent increase of intracellular reactive species was observed from 10 µM 3-MMC on; a concentration-dependent decrease in antioxidant glutathione defences was also observed. At 10 µM, caspase-3, caspase-8, and caspase-9 activities were significantly elevated, corroborating the activation of both intrinsic and extrinsic apoptosis pathways. Nuclear morphology and formation of cytoplasmic acidic vacuoles suggest prevalence of necrosis and autophagy at concentrations higher than 10 µM. No significant alterations were observed in the mitochondrial membrane potential, but intracellular ATP significantly decreased at 100 µM. Our data point to a role of metabolism in the hepatotoxicity of 3-MMC, which seems to be triggered both by autophagic and apoptotic/necrotic mechanisms. This work is the first approach to better understand 3-MMC toxicology.

Long Pentraxin 3 as a Broader Biomarker for Multiple Risk Factors in End-Stage Renal Disease: Association with All-Cause Mortality.

Persistent inflammation in end-stage renal disease (ESRD) patients is known to underlie the progression of chronic kidney disease and to be associated with multiple risk factors including malnutrition, atherosclerosis, and cardiovascular disease (CVD). The acute-phase protein pentraxin 3 (PTX3) has a proven potential as a local inflammatory biomarker, but its clinical utility in ESRD remains unclear. Circulating levels of PTX3 and classical inflammatory mediators, including the clinical prototypical C-reactive protein (CRP), were assessed in 246 ESRD patients on dialysis and analysed in relation to the lipid profile, adipokine levels, and nutritional, cardiac, and renal fibrosis markers. Occurrence of deaths was recorded for the following year. Contrarily to the classical inflammatory markers, PTX3 levels were negatively correlated with nutritional markers and associated with a less atherogenic lipid profile. Levels of the cardiac and renal fibrosis markers and of the oxidized LDL/LDL-C ratio were found to be independent determinants of PTX3 concentration. When comparing inflammatory mediators, the increase in the PTX3 levels was the only predictor of all-cause mortality in dialysis patients in a survival model adjusted to all markers under study, other than the inflammatory ones, besides common confounding factors in dialysis. Data support the clinical applicability of PTX3 as a broader inflammatory biomarker than the classical ones, presenting a close association with inflammation, malnutrition, CVD, and renal fibrosis and a great potential to predict all-cause mortality in dialysis patients. The pleiotropic character of PTX3 may be of clinical relevance, and it could be targeted to ameliorate the high morbidity and mortality associated with ESRD.

Hepcidin and diabetes are independently related with soluble transferrin receptor levels in chronic dialysis patients.

Background: Soluble transferrin receptor (sTfR) is a biomarker of erythropoiesis, which is often impaired in dialysis patients. The aim of our study was to evaluate sTfR levels in chronically dialyzed patients and assess potential determinants of its levels. Methods: We performed a cross-sectional study by evaluating 246 end-stage renal disease patients undergoing dialysis and 32 healthy controls. Circulating levels of interleukin (IL)-6, C-reactive protein (CRP), tumor necrosis factor (TNF)-α, hepcidin, sTfR, growth differentiation factor 15 (GDF15), and traditional iron metabolism markers were measured, as well as hemogram parameters. Clinical data was obtained from all patients. Results: Compared to controls, patients presented similar values of sTfR, reticulocytes and reticulocyte production index (RPI), and significantly higher levels of IL-6, CRP, ferritin, hepcidin, TNF-α, and GDF15. Iron, transferrin, hemoglobin levels, erythrocyte count, mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) values were significantly lower in dialysis group. Within patients, sTfR values were higher in diabetic patients and were positively and significantly correlated with reticulocytes and erythrocytes, RPI, and therapeutic doses of erythropoiesis stimulating agents (ESA) and intravenous iron; and inversely and significantly correlated with circulating iron, ferritin, transferrin saturation, hepcidin, MCH, and MCHC. In multiple linear regression analysis, ESA dose, RPI, serum iron, diabetes, and hepcidin levels were independently associated with sTfR levels in dialysis patients and, thus, with erythropoiesis. Conclusion: Our data suggest that, besides RPI and ESA dose, diabetes and hepcidin are closely related to erythropoiesis in dialysis patients. The influence of diabetes on sTfR levels deserves further investigation.

In vitro hepatotoxicity of 'Legal X': the combination of 1-benzylpiperazine (BZP) and 1-(m-trifluoromethylphenyl)piperazine (TFMPP) triggers oxidative stress, mitochondrial impairment and apoptosis.

N-Benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP) are two synthetic phenylpiperazine analogues that have been frequently commercialized in combination as an alternative to ecstasy ('Legal X'). Despite reports of several clinical complications following the use of these drugs in association, few studies have been conducted so far to elucidate their combined toxicity. The present study was aimed at clarifying the cytotoxic effects of mixtures of BZP and TFMPP in vitro. Human-derived HepaRG cells and primary rat hepatocytes were exposed to the drugs, individually or combined at different mixture ratios, and cytotoxicity was assessed by the MTT assay. Mixture additivity expectations were calculated by the independent action and the concentration addition (CA) models and compared with the experimental outcomes. To delineate the mechanisms underlying the elicited effects, a range of stress endpoints was evaluated, including oxidative stress, energetic imbalance, and metabolic interactions. It was observed that primary rat hepatocytes are more sensitive than HepaRG cells to the toxicity of BZP (EC50 2.20 and 6.60 mM, respectively) and TFMPP (EC50 0.14 and 0.45 mM, respectively). For all BZP-TFMPP combinations tested, CA was the most appropriate model to predict the mixture effects. TFMPP proved to act additively with BZP to produce significant hepatotoxicity (p < 0.01). Remarkably, substantial mixture effects were observed even when each drug was present at concentrations that were harmless individually. In primary hepatocytes, a small deviation from additivity (antagonism) was observed toward the upper range of the concentration-response curve. GC/MS data suggest that a metabolic interaction may be at a play, as the mixture favors the metabolism of both substances, to a significant extent in the case of BZP (p < 0.05). Also, our results demonstrate the influence of oxidative stress and energetic imbalance on these effects (increase in RNS and ROS production, decrease in intracellular GSH/GSSG, ATP depletion and mitochondrial Δψm disruption). The present work clearly demonstrates that potentially harmful interactions among BZP and TFMPP are expected when these drugs are taken concomitantly.

Methylone and MDPV activate autophagy in human dopaminergic SH-SY5Y cells: a new insight into the context of ß-keto amphetamines-related neurotoxicity.

Autophagy has an essential role in neuronal homeostasis and its dysregulation has been recently linked to neurotoxic effects of a growing list of psychoactive drugs, including amphetamines. However, the role of autophagy in ß-keto amphetamine (ß-KA) designer drugs-induced neurotoxicity has hitherto not been investigated. In the present study, we show that two commonly abused cathinone derivatives, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxypyrovalerone (MDPV), elicit morphological changes consistent with autophagy and neurodegeneration, including formation of autophagic vacuoles and neurite retraction in dopaminergic SH-SY5Y cells. Methylone and MDPV prompted the formation of acidic vesicular organelles (AVOs) and lead to increased expression of the autophagy-associated protein LC3-II in a concentration- and time-dependent manner. Electron microscopy confirmed the presence of autophagosomes with typical double membranes and autolysosomes in cells exposed to both ß-KA. The autophagic flux was further confirmed using bafilomycin A1, a known inhibitor of the late phase of autophagy. Moreover, we showed that autophagy markers were activated before the triggering of cell death and caspase 3 activation, suggesting that ß-KA-induced autophagy precedes apoptotic cell death. To address the role of oxidative stress in autophagy induction, we also investigated the effects of antioxidant treatment with N-acetyl-L-cysteine (NAC) on autophagy and apoptotic markers altered by these drugs. NAC significantly attenuated methylone- and MDPV-induced cell death by completely inhibiting the generation of reactive oxygen and nitrogen species, and hampering both apoptotic and autophagic activity, suggesting that oxidative stress plays an important role in mediating autophagy and apoptosis elicited by these drugs.

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.

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.

Impaired resolution of inflammation in human chronic heart failure.

BACKGROUND: Lipoxins (LXs) are proresolving and anti-inflammatory eicosanoids whose role in chronic heart failure (CHF) pathogenesis has never been investigated. This study evaluated levels of LXs in CHF patients, its relationship with disease severity and correlation with established CHF biomarkers. The effect of low-dose aspirin [acetylsalicylic acid (ASA)] on the levels of LXs was also studied. MATERIALS AND METHODS: Lipoxin A4 (LXA4 ), 15-epi-lipoxin A4 (15-epi-LXA4 ) and myeloperoxidase (MPO) concentration and activity were evaluated by immunoenzymatic and spectrophotometric assays in 34 CHF patients [New York Heart Association (NYHA) functional class I to IV]. B-type natriuretic peptide (BNP), troponin, myoglobin, C-reactive protein (CRP) and uric acid (UA) were also analyzed. RESULTS: Patients were stratified into mild-to-moderate CHF (NYHA, classes I and II) and severe CHF (NYHA classes III and IV). Severe patients had lower plasma LXA4 (0·262 ± 0·034 vs. 0·362 ± 0·039 ng/mL, P < 0·05) and decreased urinary 15-epi-LXA4 levels (2·28 ± 0·44 vs. 4·88 ± 1·03 µg/day, P < 0·05) besides exhibiting increased plasma BNP (1464 ± 442 vs. 555 ± 162 pg/mL, P < 0·05) and MPO activity (45·15 ± 11·56 vs. 15·90 ± 2·80 µmol/min/mg protein, P < 0·05). Plasma LXA4 was inversely correlated with BNP, troponin, myoglobin, CRP, UA and MPO activity. ASA treatment was associated with higher urinary excretion of 15-epi-LXA4 (7·70 ± 1·48 vs. 2·06 ± 0·30 µg/day, P < 0·05) in mild-to-moderate CHF patients and lower BNP levels in both groups. CONCLUSIONS: Higher severity of CHF is associated with reduced levels of LXs. Plasma LXA4 appears to be a valuable marker for risk stratification in CHF. Furthermore, the ASA-related increase in urinary 15-epi-LXA4 suggests enhanced renal synthesis of this eicosanoid and may represent a disregarded benefit of ASA.

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.

Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies.

BACKGROUND: Breast cancer therapy has been facing remarkable changes. Classic treatments are now combined with other therapies to improve efficacy and surpass resistance. Indeed, the emergence of resistance demands the development of novel therapeutic approaches. Due to key estrogen signaling, estrogen receptor-positive (ER+) breast cancer treatment has always been focused on aromatase inhibition and ER modulation. Lately, the effects of phytocannabinoids, mainly Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), have been evaluated in different cancers, including breast. However, Cannabis sativa contains more than 120 phytocannabinoids less researched and understood. METHODS: Here, we evaluated, both in silico and in vitro, the ability of 129 phytocannabinoids to modulate important molecular targets in ER+ breast cancer: aromatase, ER, and androgen receptor (AR). RESULTS: In silico results suggested that some cannabinoids may inhibit aromatase and act as ERα antagonists. Nine selected cannabinoids showed, in vitro, potential to act either as ER antagonists with inverse agonist properties, or as ER agonists. Moreover, these cannabinoids were considered as weak aromatase inhibitors and AR antagonists with inverse agonist action. CONCLUSIONS: Overall, we present, for the first time, a comprehensive analysis of the actions of the phytocannabinoids in targets of ER+ breast tumors, pointing out their therapeutic potential in cancer and in other diseases.

Shrouded in history: Unveiling the ways of life of an early Muslim population in Santarém, Portugal (8th- 10th century AD).

In around 716 AD, the city of Santarém, Portugal, was conquered by the Berber and Arab armies that swept the Iberian Peninsula and went on to rule the region until the 12th century. Archaeological excavations in 2007/08 discovered an Islamic necropolis (Avenida 5 de Outubro #2-8) that appears to contain the remains of an early Muslim population in Santarém (8th- 10th century). In this study, skeletal material from 58 adult individuals was analysed for stable carbon (δ13Ccol; δ13Cap), nitrogen (δ15N) and sulphur (δ34S) isotope ratios in bones, and stable oxygen (δ18O), carbon (δ13Cen) and radiogenic strontium (87Sr/86Sr) isotopes in tooth enamel. The results of this study revealed a dietary pattern of predominantly C3-plant and domestic C3-fed herbivore consumption during adulthood (δ13Ccol and δ15N, respectively) but a higher proportion of C4-plant input during childhood (δ13Cen) for some individuals-interpreted as possible childhood consumption of millet porridge, a common practice in North Africa-in those with unorthodox burial types (Groups 1 and 2) that was not practiced in the individuals with canonical burials (Group 3). In this first mobility study of a medieval Muslim population in Portugal, δ18ODW values revealed greater heterogeneity in Groups 1 and 2, consistent with diverse origins, some in more humid regions than Santarém when compared to regional precipitation δ18O data, contrasting the more homogenous Group 3, consistent with the local precipitation δ18O range. Ancient DNA analysis conducted on three individuals revealed maternal (mtDNA) and paternal (Y-chromosome) lineages compatible with a North African origin for (at least) some of the individuals. Additionally, mobility of females in this population was higher than males, potentially resulting from a patrilocal social system, practiced in Berber and Arab communities. These results serve to offer a more detailed insight into the ancestry and cultural practices of early Muslim populations in Iberia.