Objectives More effective topical treatments remain an unmet need for the localized forms of cutaneous leishmaniasis (CL). The aim of this study was to evaluate the efficacy and safety of a topical berberine cream in BALB/c mice infected with Leishmania major parasites. Methods A cream containing 0.5% berberine-beta-glycerophosphate salt and 2.5% menthol was prepared. Its physicochemical and stability properties were determined. The cream was evaluated for its capacity to reduce lesion size and parasitic load as well as to promote wound healing after twice-a-day administration for 35 days. Clinical biochemical profile was used for estimating off-target effects. In vitro time-to-kill curves in L. major-infected macrophages and skin and plasma pharmacokinetics were determined, aiming to establish pharmacokinetic/pharmacodynamic relationships. Results The cream was stable at 40 degrees C for 3 months and at 4 degrees C for at least 8 months. It was able to halt lesion progression in all treated mice. At the end of treatment, parasite load in the skin was reduced by 99.9% (4 log) and genes involved in the wound healing process were up-regulated compared with untreated mice. The observed effects were higher than expected from in vitro time-to-kill kinetic and plasma berberine concentrations, which ranged between 0.07 and 0.22 mu M. Conclusions The twice-a-day administration of a topical berberine cream was safe, able to stop parasite progression and improved the appearance of skin CL lesions. The relationship between drug plasma levels and in vivo effect was unclear.

Liver S9 fraction is usually employed in mutagenicity/genotoxicity in vitro assays, but some genotoxic compounds may need another type of bioactivation. In the present work, an alternative S9 fraction from the kidneys was used for the genotoxicity assessment of 12 mycotoxins with the SOS/umu test. The results were compared with liver S9 fraction, and 2-4 independent experiments were performed with each mycotoxin. The expected results were obtained with positive controls (4-nitroquinoline-N-oxide and 2-aminoanthracene) without metabolic activation or with liver S9, but a potent dose-dependent effect with 4-nitroquinoline-N-oxide and no activity of 2-aminoanthracene with kidney S9 were noticed. Aflatoxin B1 was genotoxic with metabolic activation, the effect being greater with liver S9. Sterigmatocystin was clearly genotoxic with liver S9 but equivocal with kidney S9. Ochratoxin A, zearalenone and fumonisin B1 were negative in all conditions. Trichothecenes were negative, except for nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 and HT-2 toxins, which showed equivocal results with kidney S9 because a clear dose-response effect was not observed. Most of the mycotoxins have been assessed with kidney S9 and the SOS/umu test for the first time here. The results with the positive controls and the mycotoxins confirm that the organ used for the S9 fraction preparation has an influence on the genotoxic activity of some compounds.

In this study, the thermal behavior of a series of leishmanicidal selenocyanate and diselenide biological active compounds has been studied by means of differential scanning calorimetry, X-ray diffraction and thermogravimetry in order to establish thermal stability criteria and investigate their polymorphism. Moreover, stability under acid, alkaline and oxidative media was tested using high-performance liquid chromatography with fluorescence detection. The results of the experiments show that there are five types of polymorphic behaviors for the studied compounds. In addition, relationship is found among stability and a series of structural effects and stress conditions of compounds.

Compounds 1 and 2 (selenocyanate and diselenide derivatives, respectively) were evaluated for their potential use in vivo against visceral leishmaniasis (VL). Both entities showed low cytoxicity in vitro in Vero and Caco-2 cell lines. However, the compounds were not suitable for their oral administration, since they exhibited poor values of intestinal permeability in vitro. Microsomal stability assays did not show any metabolite for compound 1 after 120 min, whereas 2 was highly metabolized by the enzyme CYP450. Thus, the in vivo efficacy of compound 1 was assessed in a murine model of L. infantum VL. The daily i.v. administration of 1 mg/kg of compound 1 during 5 consecutive days reduced parasite load in liver, spleen and bone marrow (99.2%, 91.7% and 61.4%, respectively) compared to non-treated mice. To the best of our knowledge, this is the first time that a selenium compound has been tested in vivo against VL. Thus, this work evidences the possible usefulness of selenocyanate derivatives for the treatment of this disease.

In this study, we present, for the first time in Spain, the levels of 19 mycotoxins in plasma samples from healthy and sick children (digestive, autism spectrum (ASD), and attention deficit hyperactivity (ADHD) disorders) (n = 79, aged 2-16). The samples were analyzed by liquid chromatography-mass spectrometry (triple quadrupole) (LC-MS/MS). To detect Phase II metabolites, the samples were reanalyzed after pre-treatment with beta-glucuronidase/arylsulfatase. The most prevalent mycotoxin was ochratoxin A (OTA) in all groups of children, before and after enzyme treatment. In healthy children, the incidence of OTA was 92.5% in both cases and higher than in sick children before (36.7% in digestive disorders, 50% in ASD, and 14.3% in ADHD) and also after the enzymatic treatment (76.6 % in digestive disorders, 50% in ASD, and 85.7% in ADHD). OTA levels increased in over 40% of healthy children after enzymatic treatment, and this increase in incidence and levels was also observed in all sick children. This suggests the presence of OTA conjugates in plasma. In addition, differences in OTA metabolism may be assumed. OTA levels are higher in healthy children, even after enzymatic treatment (mean OTA value for healthy children 3.29 ng/mL, 1.90 ng/mL for digestive disorders, 1.90 ng/mL for ASD, and 0.82 ng/mL for ADHD). Ochratoxin B appears only in the samples of healthy children with a low incidence (11.4%), always co-occurring with OTA. Sterigmatocystin (STER) was detected after enzymatic hydrolysis with a high incidence in all groups, especially in sick children (98.7% in healthy children and 100% in patients). This supports glucuronidation as a pathway for STER metabolism in children. Although other mycotoxins were studied (aflatoxins B1, B2, G1, G2, and M1; T-2 and HT-2 toxins; deoxynivalenol, deepoxy-deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol; zearalenone; nivalenol; fusarenon-X; neosolaniol; and diacetoxyscirpenol), they were not detected either before or after enzymatic treatment in any of the groups of children. In conclusion, OTA and STER should be highly considered in the risk assessment of mycotoxins. Studies concerning their sources of exposure, toxicokinetics, and the relationship between plasma levels and toxic effects are of utmost importance in children.

Exposure to environmental contaminants might play an important role in neurodegenerative disease pathogenesis, such as Parkinson ' s disease (PD) and Alzheimer's disease (AD). For the first time in Spain, the plasmatic levels of 19 mycotoxins from patients diagnosed with a neurodegenerative disease (44 PD and 24 AD) and from their healthy companions (25) from La Rioja region were analyzed. The studied mycotoxins were aflatoxins B1, B2, G1, G2 and M1, T-2 and HT-2, ochratoxins A (OTA) and B (OTB), zearalenone, sterigmatocystin (STER), nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deepoxy-deoxynivalenol, neosolaniol, diacetoxyscirpenol and fusarenon-X. Samples were analyzed by LC-MS/MS before and after treatment with beta-glucuronidase/arylsulfatase in order to detect potential metabolites. Only OTA, OTB and STER were detected in the samples. OTA was present before (77% of the samples) and after (89%) the enzymatic treatment, while OTB was only detectable before (13%). Statistically significant differences in OTA between healthy companions and patients were observed but the observed differences might seem more related to gender (OTA levels higher in men, p-value = 0.0014) than the disease itself. STER appeared only after enzymatic treatment (88%). Statistical analysis on STER, showed distributions always different between healthy controls and patients (patients' group > controls, p-value < 0.0001). Surprisingly, STER levels weakly correlated positively with age in women (rho = 0.3384), while OTA correlation showed a decrease of levels with age especially in the men with PD (rho = -0.4643).

Leishmaniasis urgently needs new oral treatments, as it is one of the most important neglected tropical diseases that affects people with poor resources. The drug discovery pipeline for oral administration currently discards entities with poor aqueous solubility and permeability (class IV compounds in the Biopharmaceutical Classification System, BCS) such as the diselenide 2m, a trypanothione reductase (TR) inhibitor. This work was assisted by glyceryl palmitostearate and diethylene glycol monoethyl ether-based nanostructured lipid carriers (NLC) to render 2m bioavailable and effective after its oral administration. The loading of 2m in NLC drastically enhanced its intestinal permeability and provided plasmatic levels higher than its effective concentration (IC50). In L. infantum-infected BALB/c mice, 2m-NLC reduced the parasite burden in the spleen, liver, and bone marrow by at least 95% after 5 doses, demonstrating similar efficacy as intravenous Fungizone. Overall, compound 2m and its formulation merit further investigation as an oral treatment for visceral leishmaniasis.

Some epidemiological studies with different levels of evidence have pointed to a higher risk of Parkinson's disease (PD) after exposure to environmental toxicants. A practically unexplored potential etiological factor is a group of naturally-occurring fungal secondary metabolites called mycotoxins. The mycotoxin ochratoxin A (OTA) has been reported to be neurotoxic in mice. To further identify if OTA exposure could have a role in PD pathology, Balb/c mice were orally treated with OTA (0.21, 0.5 mg/kg bw) four weeks and left for six months under normal diet. Effects of OTA on the onset, progression of alpha-synuclein pathology and development of motor deficits were evaluated. Immunohistochemical and biochemical analyses showed that oral subchronic OTA treatment induced loss of striatal dopaminergic innervation and dopaminergic cell dysfunction responsible for motor impairments. Phosphorylated alpha-synuclein levels were increased in gut and brain. LAMP-2A protein was decreased in tissues showing alpha-synuclein pathology. Cell cultures exposed to OTA exhibited decreased LAMP-2A protein, impairment of chaperone-mediated autophagy and decreased alpha-synuclein turnover which was linked to miRNAs deregulation, all reminiscent of PD. These results support the hypothesis that oral exposure to low OTA doses in mice can lead to biochemical and pathological changes reported in PD.

Humans are widely exposed to a great variety of mycotoxins and their mixtures. Therefore, it is important to design strategies that allow prioritizing mycotoxins based on their toxic potential in a time and cost-effective manner. A strategy combining in silico tools (Phase 1), including an expert knowledge-based (DEREK Nexus(R)(,) Lhasa Limited, Leeds, UK) and a statistical-based platform (VEGA QSAR (c), Mario Negri Institute, Milan, Italy), followed by the in vitro SOS/umu test (Phase 2), was applied to a set of 12 mycotoxins clustered according to their structure into three groups. Phase 1 allowed us to clearly classify group 1 (aflatoxin and sterigmatocystin) as mutagenic and group 3 (ochratoxin A, zearalenone and fumonisin B1) as non-mutagenic. For group 2 (trichothecenes), contradictory conclusions were obtained between the two in silico tools, being out of the applicability domain of many models. Phase 2 confirmed the results obtained in the previous phase for groups 1 and 3. It also provided extra information regarding the role of metabolic activation in aflatoxin B1 and sterigmatocystin mutagenicity. Regarding group 2, equivocal results were obtained in few experiments; however, the group was finally classified as non-mutagenic. The strategy used correlated with the published Ames tests, which detect point mutations. Few alerts for chromosome aberrations could be detected. The SOS/umu test appeared as a good screening test for mutagenicity that can be used in the absence and presence of metabolic activation and independently of Phase 1, although the in silico-in vitro combination gave more information for decision making.

We report the methodology for the quantification of 19 mycotoxins in human plasma using high performance liquid chromatography-mass spectrometry (triple quadrupole). The studied mycotoxins were: deepoxy-deoxynivalenol, aflatoxins (B1, B2, G1, G2 and M1), T-2 and HT-2, ochratoxins A and B, zearalenone, sterigmatocystin, nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, diacetoxyscirpenol and fusarenon-X. Sample deproteinization and cleanup were performed in one step using Captiva EMR-lipid (3 mL) cartridges and acetonitrile (with 1% formic acid). The extraction step was simple and fast. Validation was based on the evaluation of limits of detection (LOD) and quantification, linearity, precision, recovery, matrix effect, and stability. LOD values ranged from 0.04 ng/mL for aflatoxin B1 to 2.7 ng/mL for HT-2, except for nivalenol, which was 9.1 ng/mL. Recovery was obtained in intermediate precision conditions and at three concentration levels. Mean values ranged from 68.8% for sterigmatocystin to 97.6% for diacetoxyscirpenol (RDS <= 15% for all the mycotoxins). Matrix effects (assessed at three concentration levels and in intermediate conditions) were not significant for most of the mycotoxins and were between 75.4% for sterigmatocystin and 109.3% for ochratoxin B (RDS <= 15% for all the mycotoxins). This methodology will be useful in human bio-monitoring studies of mycotoxins for its reliability.

Climate change constitutes an important driver affecting food sector, and consequently represents a public health issue that deserves particular attention. Under this context, mycotoxins emerge as a particular concern since their prevalence and concentrations in food and feed may vary due to climatic conditions. Aflatoxins, the most toxic mycotoxins, present a particular concern, taking into account the potential health effects arising from human and animal exposure. The present article aims to answer two main questions: Are aflatoxins a concern in the Iberian Peninsula? How the climate change could impact aflatoxins contamination and the risk of human exposure in Iberian Peninsula?