Micotoxinas

Autores: Rodriguez Garraus, Adriana (Autor de correspondencia); Alonso Jauregui, María; Gil Royo, Ana Gloria; et al.

Título: Genotoxicity and Toxicity Assessment of a Formulation Containing Silver Nanoparticles and Kaolin: An In Vivo Integrative Approach

Revista: NANOMATERIALS

ISSN: 2079-4991 Vol.13 N° 1 2023 págs. 3

Resumen

A new material composed of a kaolin base with silver nanoparticles (AgNPs) attached to its surface was developed, as an alternative to antibiotics used as supplements in animal feed. As part of its safety assessment, an in vivo geno-toxicological evaluation of this material was conducted in rats. First, a preliminary dose finding study was carried out to decide the doses to be tested in the main study: 50, 300 and 2000 mg/kg b.w. For the main study, a combined strategy composed of the MN test (TG 474) and the comet assay (TG 489), integrated in a repeated dose 28-day oral toxicity study (TG 407), was performed. A No Observed Adverse Effect Level (NOAEL) of 2000 mg of the silver-kaolin formulation/kg b.w. by oral route, for 28 days, was determined. The silver-kaolin formulation did not induce micronuclei in bone marrow, or DNA strand breaks (SBs) or alkali labile sites (ALS) in liver, spleen, kidney or duodenum at any dose. The modified Fpg comet assay did not reveal oxidized bases in the same tissues at the dose of 2000 mg/kg b.w. Silver was quantified by ICP-MS in all the target organs, confirming the negative results obtained under these conditions.
Autores: Sanz Serrano, Julen (Autor de correspondencia); Garayoa Poyo, Roncesvalles; Vitas Pemán, Ana Isabel; et al.

Título: In vitro genotoxicity assessment of French fries from mass catering companies: a preliminary study

Revista: MUTAGENESIS

ISSN: 0267-8357 Vol.38 N° 1 2023 págs. 51 - 57

Resumen

It is generally assumed that French fries are likely to have weak in vitro mutagenic activity, but most studies thereof have only assessed gene mutations. In this article, the genotoxicity of 10 extracts of French fries was assessed using the in vitro micronucleus test (following the principles of the OECD 487 guidelines). Each sample was obtained from a different mass catering company in Navarra (Spain). This assay, together with the Ames test, is recommended in the basic in vitro phase included in the European Food Safety Authority Opinion on Genotoxicity Testing Strategies Applicable to Food and Feed Safety Assessment. Eight of 10 samples from mass catering companies induced chromosomal aberrations in the in vitro micronucleus test. Moreover, French fries deep-fried in the laboratory for different periods of time (0, 3, 5, 10, 20, 30 min) were assessed using the in vitro micronucleus test. Genotoxicity was observed in all time periods from 3 min on. The biological relevance of these results must be further explored.
Autores: Domenech, J.; Rodriguez Garraus, Adriana; López de Cerain Salsamendi, Adela; et al.

Título: Genotoxicity of graphene-based materials

Revista: NANOMATERIALS

ISSN: 2079-4991 Vol.12 N° 11 2022 págs. 1795

Resumen

Graphene-based materials (GBMs) are a broad family of novel carbon-based nanomaterials with many nanotechnology applications. The increasing market of GBMs raises concerns on their possible impact on human health. Here, we review the existing literature on the genotoxic potential of GBMs over the last ten years. A total of 50 articles including in vitro, in vivo, in silico, and human biomonitoring studies were selected. Graphene oxides were the most analyzed materials, followed by reduced graphene oxides. Most of the evaluations were performed in vitro using the comet assay (detecting DNA damage). The micronucleus assay (detecting chromosome damage) was the most used validated assay, whereas only two publications reported results on mammalian gene mutations. The same material was rarely assessed with more than one assay. Despite inhalation being the main exposure route in occupational settings, only one in vivo study used intratracheal instillation, and another one reported human biomonitoring data. Based on the studies, some GBMs have the potential to induce genetic damage, although the type of damage depends on the material. The broad variability of GBMs, cellular systems and methods used in the studies precludes the identification of physico-chemical properties that could drive the genotoxicity response to GBMs.
Autores: Rodriguez Garraus, Adriana; Azqueta Oscoz, Amaya (Autor de correspondencia); Laborda, F.; et al.

Título: In vitro genotoxicity evaluation of an antiseptic formulation containing kaolin and silver nanoparticles

Revista: NANOMATERIALS

ISSN: 2079-4991 Vol.12 N° 6 2022 págs. 914

Resumen

Worldwide antimicrobial resistance is partly caused by the overuse of antibiotics as growth promoters. Based on the known bactericidal effect of silver, a new material containing silver in a clay base was developed to be used as feed additive. An in vitro genotoxicity evaluation of this silver-kaolin clay formulation was conducted, which included the mouse lymphoma assay in L5178Y TK+/- cells and the micronucleus test in TK6 cells, following the principles of the OECD guidelines 490 and 487, respectively. As a complement, the standard and Fpg-modified comet assays for the evaluation of strand breaks, alkali labile sites and oxidative DNA damage were also performed in TK6 cells. The formulation was tested without metabolic activation after an exposure of 3 h and 24 h; its corresponding release in medium, after the continuous agitation of the silver-kaolin for 24 h was also evaluated. Under the conditions tested, the test compound did not produce gene mutations, chromosomal aberrations or DNA damage (i.e., strand breaks, alkali labile sites or oxidized bases). Considering the results obtained in the present study, the formulation seems to be a promising material to be used as antimicrobial in animal feed.
Autores: Etxebeste Mitxeltorena, Mikel; Durán Benito, Adrián; Sanmartín Grijalba, Carmen (Autor de correspondencia); et al.

Título: Thermal characterization and stability evaluation of leishmanicidal selenocyanate and diselenide derivatives

Revista: JOURNAL OF THERMAL ANALYSIS AND CALORIMETRY

ISSN: 1388-6150 Vol.147 N° 4 2022 págs. 3127 - 3139

Resumen

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.
Autores: Alonso Jauregui, María; González Peñas, María Elena; López de Cerain Salsamendi, Adela; et al.

Título: Genotoxicity of 12 mycotoxins by the SOS/umu test: comparison of liver and kidney S9 fraction

Revista: TOXINS

ISSN: 2072-6651 Vol.14 N° 6 2022 págs. 400

Resumen

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.
Autores: Moller, P. (Autor de correspondencia); Bankoglu, E. E.; Stopper, H.; et al.

Título: Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies

Revista: MUTAGENESIS

ISSN: 0267-8357 Vol.36 N° 3 2021 págs. 193 - 212

Resumen

DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.
Autores: Muruzábal Gambarte, Damián; Collins, A.; Azqueta Oscoz, Amaya (Autor de correspondencia)

Título: The enzyme-modified comet assay: past, present and future

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.147 2021 págs. 111865

Resumen

The enzyme-modified comet assay was developed in order to detect DNA lesions other than those detected by the standard version (single and double strand breaks and alkali-labile sites). Various lesion-specific enzymes, from the DNA repair machinery of bacteria and humans, have been combined with the comet assay, allowing detection of different oxidized and alkylated bases as well as cyclobutane pyrimidine dimers, mis-incorporated uracil and apurinic/apyrimidinic sites. The enzyme-modified comet assay has been applied in different fields - human biomonitoring, environmental toxicology, and genotoxicity testing (both in vitro and in vivo) - as well as in basic research. Up to now, twelve enzymes have been employed; here we describe the enzymes and give examples of studies in which they have been applied. The bacterial formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIll) have been extensively used while others have been used only rarely. Adding further enzymes to the comet assay toolbox could potentially increase the variety of DNA lesions that can be detected. The enzyme-modified comet assay can play a crucial role in the elucidation of the mechanism of action of both direct and indirect genotoxins, thus increasing the value of the assay in the regulatory context.
Autores: Sanz Serrano, Julen; Vettorazzi Armental, Ariane; Muruzábal Gambarte, Damián; et al.

Título: In vitro genotoxicity assessment of functional ingredients: betaine, choline, and taurine

Revista: FOODS

ISSN: 2304-8158 Vol.10 N° 2 2021 págs. 339

Resumen

This article focuses on a complete in vitro genotoxicity assessment of three nutrients widely used as functional ingredients in the European market: betaine, choline, and taurine. The European Food Safety Authority (EFSA) tiered approach for food additives in concordance with the safety assessment of chemicals in food developed by Food and Agriculture Organization/World Health Organization (FAO/WHO) was followed; the miniaturized Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of Organization for Economic Co-operation and Development (OECD) 471), and the micronucleus test (OECD 487) in TK6 cells were performed. In addition, the in vitro standard and enzyme-modified (human 8-oxoguanine DNA glycosylase 1 (hOGG), endonuclease III (EndoIII), 3-alkyladenine DNA glycosylase (hAAG)) comet assay (S9-/S9+) was conducted in order to assess the potential premutagenic lesions in TK6 cells. None of the compounds produced any signs of genotoxicity in any of the conditions tested. This article increases the limited evidence available and complements the EFSA recommendations for the in vitro genotoxicity testing of nutrients.
Autores: Pastor Castro, Laura; Vettorazzi Armental, Ariane (Autor de correspondencia); Guruceaga Martínez, Elisabet; et al.

Título: Time course of renal transcriptomics after subchronic exposure to ochratoxin A in Fisher rats

Revista: TOXINS

ISSN: 2072-6651 Vol.13 N° 3 2021 págs. 177

Resumen

The mycotoxin ochratoxin A (OTA) is a potent nephrocarcinogen, mainly in male rats. The aim of this study was to determine the time course of gene expression (GeneChip(R) Rat Gene 2.0 ST Array, Affymetrix) in kidney samples from male and female F344 rats, treated daily (p.o) with 0.50 mg/kg b.w. (body weight) of OTA for 7 or 21 days, and evaluate if there were differences between both sexes. After OTA treatment, there was an evolution of gene expression in the kidney over time, with more differentially expressed genes (DEG) at 21 days. The gene expression time course was different between sexes with respect to the number of DEG and the direction of expression (up or down): the female response was progressive and consistent over time, whereas males had a different early response with more DEG, most of them up-regulated. The statistically most significant DEG corresponded to metabolism enzymes (Akr1b7, Akr1c2, Adh6 down-regulated in females; Cyp2c11, Dhrs7, Cyp2d1, Cyp2d5 down-regulated in males) or transporters (Slc17a9 down-regulated in females; Slco1a1 (OATP-1) and Slc51b and Slc22a22 (OAT) down-regulated in males). Some of these genes had also a basal sex difference and were over-expressed in males or females with respect to the other sex.
Autores: Amrane, D.; Arnold, C. S.; Hutter, S.; et al.

Título: 2-Phenoxy-3-Trichloromethylquinoxalines are antiplasmodial derivatives with activity against the apicoplast of Plasmodium falciparum

Revista: PHARMACEUTICALS

ISSN: 1424-8247 Vol.14 N° 8 2021 págs. 724

Resumen

The malaria parasite harbors a relict plastid called the apicoplast. Although not photosynthetic, the apicoplast retains unusual, non-mammalian metabolic pathways that are essential to the parasite, opening up a new perspective for the development of novel antimalarials which display a new mechanism of action. Based on the previous antiplasmodial hit-molecules identified in the 2-trichloromethylquinoxaline series, we report herein a structure-activity relationship (SAR) study at position two of the quinoxaline ring by synthesizing 20 new compounds. The biological evaluation highlighted a hit compound (3i) with a potent PfK1 EC50 value of 0.2 mu M and a HepG2 CC50 value of 32 mu M (Selectivity index = 160). Nitro-containing (3i) was not genotoxic, both in the Ames test and in vitro comet assay. Activity cliffs were observed when the 2-CCl3 group was replaced, showing that it played a key role in the antiplasmodial activity. Investigation of the mechanism of action showed that 3i presents a drug response by targeting the apicoplast and a quick-killing mechanism acting on another target site.
Autores: Sanz Serrano, Julen; Garayoa Poyo, Roncesvalles; Vitas Pemán, Ana Isabel; et al.

Título: In vitro mutagenicity assessment of fried meat-based food from mass catering companies

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.156 2021 págs. 112494

Resumen

The current article aimed to evaluate the in vitro mutagenicity of ten fried meat-based food extracts obtained from different catering companies from Navarra (Spain). A miniaturized 6-well version of the Ames test in Salmonella typhimurium TA98, and the in vitro micronucleus test (OECD TG 487) in TK6 cells were performed. None of the ten extracts of fried meat-based food induced gene mutations in S. typhimurium TA98 with or without metabolic activation, but five induced chromosomal aberrations after 24 h treatment of TK6 without metabolic activation. More studies are needed to check the biological relevance of these in vitro studies.
Autores: Garayoa Poyo, Roncesvalles (Autor de correspondencia); Sanz Serrano, Julen; Vettorazzi Armental, Ariane; et al.

Título: Practices of deep-frying processes among food handlers in social food services in Navarra, Spain

Revista: INTERNATIONAL JOURNAL OF GASTRONOMY AND FOOD SCIENCE

ISSN: 1878-450X Vol.26 2021 págs. 100432

Resumen

Deep frying is one of the most used worldwide methods in food preparation, but controlling the oil quality (temperature and formation of polar compounds) is crucial. The main objective of this work was to assess the practices of food handlers with regard to the frying processes in social food services located in Navarra (a region of northern Spain). The study was performed in two phases: in the first one, a self-administrable questionnaire regarding the usual practices on food deep-frying processes was sent to the food services through the main social catering companies of Navarra participating in the study. In the second one, in situ monitoring of the frying practices was performed as verification tools of frying practices reported by food services and to check the oil quality. Almost half of the fryers exceeded the maximum recommended temperature to avoid the formation of toxic compounds (175 degrees C). Despite only one the fryers exceeded the maximum limit of polar compounds established by current Spanish regulation, the obtained values indicated that the oil had begun to degrade in 20% of the fryers. Oil temperature is an important factor that affects the quality of the oil. In addition, significant differences were found between the different frequencies of change or types of oils. We have noticed a lack of knowledge in relation to the risks associated to the bad management of frying oil. Therefore, it would be desirable to improve food handlers training in relation to this matter. Defining a periodic frequency of oil change according to its use and periodic controls of temperature and polar compounds (as part of the Hazard Analysis and Critical Control Point system), could be adequate tools to improve management of frying oil in food services.
Autores: Bonassi, S. (Autor de correspondencia); Ceppi, M.; Moller, P.; et al.

Título: DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death

Revista: SCIENTIFIC REPORTS

ISSN: 2045-2322 Vol.11 N° 1 2021 págs. 16793

Resumen

The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan-Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06-1.90) for overall mortality, and 1.94 (1.04-3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.
Autores: Fernández-Bertólez, N.; Azqueta Oscoz, Amaya; Pásaro, E.; et al.

Título: Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

Revista: ARCHIVES OF TOXICOLOGY

ISSN: 0340-5761 Vol.95 N° 6 2021 págs. 2179 - 2187

Resumen

Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.
Autores: Alonso Jauregui, María; Font Arellano, María; González Peñas, María Elena; et al.

Título: Prioritization of mycotoxins based on their genotoxic potential with an in silico-in vitro strategy

Revista: TOXINS

ISSN: 2072-6651 Vol.13 N° 10 2021 págs. 734

Resumen

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.
Autores: Arce-Lopez, B.; Alvarez-Erviti, L.; De Santis, B.; et al.

Título: Biomonitoring of mycotoxins in plasma of patients with Alzheimer's and Parkinson's disease

Revista: TOXINS

ISSN: 2072-6651 Vol.13 N° 7 2021 págs. 477

Resumen

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).
Autores: Izco, M.; Vettorazzi Armental, Ariane; de Toro, M.; et al.

Título: Oral sub-chronic ochratoxin A exposure induces gut microbiota alterations in mice

Revista: TOXINS

ISSN: 2072-6651 Vol.13 N° 2 2021 págs. 106

Resumen

Gut microbiota plays crucial roles in maintaining host health. External factors, such as diet, medicines, and environmental toxins, influence the composition of gut microbiota. Ochratoxin A (OTA) is one of the most prevalent and relevant mycotoxins and is a highly abundant food and animal feed contaminant. In the present study, we aimed to investigate OTA gut microbiome toxicity in mice sub-chronically exposed to low doses of OTA (0.21, 0.5, and 1.5 mg/kg body weight) by daily oral gavage for 28 days. Fecal microbiota from control and OTA-treated mice was analyzed using 16S ribosomal RNA (rRNA) gene sequencing followed by metagenomics. OTA exposure caused marked changes in gut microbial community structure, including the decrease in the diversity of fecal microbiota and the relative abundance of Firmicutes, as well as the increase in the relative abundance of Bacteroidetes at the phylum level. At the family level, six bacterial families (unclassified Bacteroidales, Porphyromonadaceae, unclassified Cyanobacteria, Streptococcaceae, Enterobacteriaceae, Ruminococcaceae) were significantly altered by OTA exposure. Interestingly, OTA-induced changes were observed in the lower-dose OTA groups, while high-dose OTA group microbiota was similar to control group. Our results demonstrated that sub-chronic exposure at low doses of OTA alters the structure and diversity of the gut microbial community.
Autores: Muruzábal Gambarte, Damián; Sanz Serrano, Julen; Sauvaigo, S.; et al.

Título: Validation of the in vitro comet assay for DNA cross-links and altered bases detection

Revista: ARCHIVES OF TOXICOLOGY

ISSN: 0340-5761 Vol.95 N° 8 2021 págs. 2825 - 2838

Resumen

Mechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.
Autores: Arce-López, B.; Lizarraga Pérez, Elena; López de Mesa, R.; et al.

Título: Assessment of exposure to mycotoxins in Spanish children through the analysis of their levels in plasma samples

Revista: TOXINS

ISSN: 2072-6651 Vol.13 N° 2 2021 págs. 150

Resumen

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.
Autores: Izco, M.; Vettorazzi Armental, Ariane (Autor de correspondencia); Forcén, R.; et al.

Título: Oral subchronic exposure to the mycotoxin ochratoxin A induces key pathological features of Parkinson's disease in mice six months after the end of the treatment

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.152 2021 págs. 112164

Resumen

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.
Autores: Sanz Serrano, Julen; Vettorazzi Armental, Ariane; Muruzábal Gambarte, Damián; et al.

Título: In vitro genotoxicity assessment of functional ingredients: DHA, rutin and alpha-tocopherol

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.153 2021 págs. 112237

Resumen

The in vitro genotoxicity of three compounds widely used as functional ingredients, docosahexaenoic acid (DHA), rutin and alpha-tocopherol, was assessed. A miniaturized version of the Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of OECD 471), and the in vitro micronucleus test in TK6 cells (OECD 487) were performed. This strategy is recommended by the European Food Safety Authority for the in vitro genotoxicity assessment of food and feed. In addition, this approach was complemented with the in vitro standard and enzyme-modified comet assay (S9-/S9+) using hOGG1, EndoIII and hAAG in order to assess potential premutagenic lesions in TK6 cells. Rutin showed an equivocal response in the in vitro micronucleus test and also was a potent Salmonella typhimurium revertant inductor in the Ames test. DHA showed equivocal results in the in vitro micronucleus test. In this regard, DHA and rutin seemed to interact with the DNA at a chromosomal level, but rutin is also capable of producing frameshift mutations. No genotoxicity was observed in cells treated with alpha-tocopherol. This article complements the evidence already available about the genotoxicity of these compounds. However, more studies are needed in order to elucidate the consequences of their use as functional ingredients in human health.
Autores: Vettorazzi Armental, Ariane; Izco, M.; González Peñas, María Elena; et al.

Título: Is ochratoxin A a potential neurodegenerative toxin?: mechanistic in vitro and in vivo studies

Revista: TOXICOLOGY LETTERS

ISSN: 0378-4274 Vol.350 2021 págs. S138 - S139
Autores: Vettorazzi Armental, Ariane (Autor de correspondencia); López de Cerain Salsamendi, Adela; Sanz Serrano, Julen; et al.

Título: European Regulatory Framework and Safety Assessment of Food-Related Bioactive Compounds

Revista: NUTRIENTS

ISSN: 2072-6643 Vol.12 N° 3 2020

Resumen

A great variety of functional foods, nutraceuticals, or foods with bioactive compounds are provided nowadays to consumers. Aware of the importance of the safety aspects, the food industry has to comply with different legal requirements around the world. In this review, the European regulatory framework for food-related bioactive compounds is summarized. The term 'bioactive compound' is not defined in the European regulations, however, since they can be part of food supplements, fortified foods, or novel food, they are included within the legal requirements of those corresponding types of foods or supplements. Lists of authorized compounds/foods appear in the correspondent regulations, however, when a new compound/food is going to be launched into the market, its safety assessment is essential. Although the responsibility for the safety of these compounds/foods lies with the food business operator placing the product on the market, the European Food Safety Authority (EFSA) carries out scientific evaluations to assess the risks for human health. To facilitate this procedure, different guidelines exist at the European level to explain the tier toxicity testing approach to be considered. This approach divides the evaluation into four areas: (a) toxicokinetics; (b) genotoxicity; (c) subchronic and chronic toxicity and carcinogenicity; and (d) reproductive and developmental toxicity.
Autores: Sanz Serrano, Julen; López de Cerain Salsamendi, Adela (Autor de correspondencia); Garayoa Poyo, Roncesvalles; et al.

Título: Genotoxicity evaluation of fried meat: A comprehensive review

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.136 2020

Resumen

Some years ago, the IARC published the carcinogenic potential of processed and red meat. It is known that frying meat can produce genotoxic substances. A systematic review of the literature was conducted to evaluate in vitro and in vivo genotoxicity of fried meat. A total of 31 scientific articles were retrieved and analyzed. The meat extraction methods have been grouped into 6 types based on their similarity to an initially described method or on the general methodology used (solid-liquid extraction or others). The in vitro mutagenic results have been summarised by type of meat studied (beef, pork, others), cooking conditions (method, time and temperature), extraction method, and test used, with or without S9. Most articles assessed the mutagenicity of the extracts using the Ames test. Meat extracts were consistently positive in strains TA98/TA1538 with metabolic activation. In the in vitro studies with meat from restaurants, positive results were always found with variations in the number of His(+) revertants between samples or between restaurants. The few in vivo studies retrieved show evidence of induced DNA damage in colon cells and chromosome aberrations in bone marrow cells after daily treatment with fried red meat for 4 weeks or longer.
Autores: Arce-López, B.; Lizarraga Pérez, Elena (Autor de correspondencia); Vettorazzi Armental, Ariane; et al.

Título: Human Biomonitoring of Mycotoxins in Blood, Plasma and Serum in Recent Years: A Review

Revista: TOXINS

ISSN: 2072-6651 Vol.12 N° 3 2020
Autores: Azqueta Oscoz, Amaya; Ladeira, C.; Giovannelli, L.; et al.

Título: Application of the comet assay in human biomonitoring: an hCOMET perspective

Revista: MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH

ISSN: 1383-5742 Vol.783 2020 págs. UNSP 108288

Resumen

The comet assay is a well-accepted biomonitoring tool to examine the effect of dietary, lifestyle, environmental and occupational exposure on levels of DNA damage in human cells. With such a wide range of determinants for DNA damage levels, it becomes challenging to deal with confounding and certain factors are inter-related (e.g. poor nutritional intake may correlate with smoking status). This review describes the effect of intrinsic (i.e. sex, age, tobacco smoking, occupational exposure and obesity) and extrinsic (season, environmental exposures, diet, physical activity and alcohol consumption) factors on the level of DNA damage measured by the standard or enzyme-modified comet assay. Although each factor influences at least one comet assay endpoint, the collective evidence does not indicate single factors have a large impact. Thus, controlling for confounding may be necessary in a biomonitoring study, but none of the factors is strong enough to be regarded a priori as a confounder. Controlling for confounding in the comet assay requires a case-by-case approach. Inter-laboratory variation in levels of DNA damage and to some extent also reproducibility in biomonitoring studies are issues that have haunted the users of the comet assay for years. Procedures to collect specimens, and their storage, are not standardized. Likewise, statistical issues related to both sample-size calculation (before sampling of specimens) and statistical analysis of the results vary between studies. This review gives guidance to statistical analysis of the typically complex exposure, co-variate, and effect relationships in human biomonitoring studies. (C) 2019 Elsevier B.V. All rights reserved.
Autores: Almeida, T. P. ; Ramos, A. A. ; Ferreira, J.; et al.

Título: Bioactive Compounds from Seaweed with Anti-Leukemic Activity: A Mini-Review on Carotenoids and Phlorotannins

Revista: MINI-REVIEWS IN MEDICINAL CHEMISTRY

ISSN: 1389-5575 Vol.20 N° 1 2020 págs. 39 - 53

Resumen

Chronic Myeloid Leukemia (CML) represents 15-20% of all new cases of leukemia and is characterized by an uncontrolled proliferation of abnormal myeloid cells. Currently, the first-line of treatment involves Tyrosine Kinase Inhibitors (TKIs), which specifically inhibits the activity of the fusion protein BCR-ABL. However, resistance, mainly due to mutations, can occur. In the attempt to find more effective and less toxic therapies, several approaches are taken into consideration such as research of new anti-leukemic drugs and "combination chemotherapy" where different drugs that act by different mechanisms are used. Here, we reviewed the molecular mechanisms of CML, the main mechanisms of drug resistance and current strategies to enhance the therapeutic effect of TKIs in CML. Despite major advances in CML treatment, new, more potent anticancer drugs and with fewer side effects are needed. Marine organisms, and particularly seaweed, have a high diversity of bioactive compounds with some of them having anticancer activity in several in vitro and in vivo models. The state-of-art suggests that their use during cancer treatment may improve the outcome. We reviewed here the yet few data supporting anti-leukemic activity of some carotenoids and phlorotannins in some leukemia models. Also, strategies to overcome drug resistance are discussed, particularly the combination of conventional drugs with natural compounds.
Autores: Rodriguez Garraus, Adriana; Azqueta Oscoz, Amaya (Autor de correspondencia); Vettorazzi Armental, Ariane; et al.

Título: Genotoxicity of Silver Nanoparticles

Revista: NANOMATERIALS

ISSN: 2079-4991 Vol.10 N° 2 2020 págs. E251

Resumen

Silver nanoparticles (AgNPs) are widely used in diverse sectors such as medicine, food, cosmetics, household items, textiles and electronics. Given the extent of human exposure to AgNPs, information about the toxicological effects of such products is required to ensure their safety. For this reason, we performed a bibliographic review of the genotoxicity studies carried out with AgNPs over the last six years. A total of 43 articles that used well-established standard assays (i.e., in vitro mouse lymphoma assays, in vitro micronucleus tests, in vitro comet assays, in vivo micronucleus tests, in vivo chromosome aberration tests and in vivo comet assays), were selected. The results showed that AgNPs produce genotoxic effects at all DNA damage levels evaluated, in both in vitro and in vivo assays. However, a higher proportion of positive results was obtained in the in vitro studies. Some authors observed that coating and size had an effect on both in vitro and in vivo results. None of the studies included a complete battery of assays, as recommended by ICH and EFSA guidelines, and few of the authors followed OECD guidelines when performing assays. A complete genotoxicological characterization of AgNPs is required for decision-making.
Autores: Muruzábal Gambarte, Damián; Sanz Serrano, Julen; Sauvaigo, S.; et al.

Título: Novel approach for the detection of alkylated bases using the enzyme-modified comet assay (vol 330, pg 108, 2020)

Revista: TOXICOLOGY LETTERS

ISSN: 0378-4274 Vol.335 2020 págs. 98 - 98
Autores: González Peñas, María Elena

Título: Mycotoxins in Beverages

Revista: BEVERAGES

ISSN: 2306-5710 Vol.6 N° 4 2020
Autores: Muruzábal Gambarte, Damián; Sanz Serrano, Julen; Sauvaigo, S.; et al.

Título: Novel approach for the detection of alkylated bases using the enzyme-modified comet assay

Revista: TOXICOLOGY LETTERS

ISSN: 0378-4274 Vol.330 2020 págs. 108 - 117

Resumen

The enzyme-modified comet assay is widely used for the detection of oxidized DNA lesions. Here we describe for the first time the use of the human alkyladenine DNA glycosylase (hAAG) for the detection of alkylated bases. hAAG was titrated using untreated and methyl methanesulfonate (MMS)-treated TK-6 cells. The hAAG-modified comet assay was compared to the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay, widely used to detect oxidized lesions but that also detects ring-opened purines derived from some alkylated lesions, using cells treated with potassium bromate (oxidizing agent) or MMS. Moreover, neutral and alkaline lysis conditions were used to determine the nature of detected lesions. When alkaline lysis was employed (condition normally used), the level of hAAG-sensitive sites was higher than the Fpg-sensitive sites in MMS-treated cells and hAAG, unlike Fpg, did not detect oxidized bases. After neutral lysis, Fpg did not detect MMS-induced lesions; however, results obtained with hAAG remained unchanged. As expected, Fpg detected oxidized purines and imidazole ring-opened purines, derived from N7-methylguanines under alkaline conditions. It seems that hAAG detected N7-methylguanines, the ring-opened purines derived at high pH, and 3-methlyladenines. Specificity of hAAG towards different DNA lesions was evaluated using a multiplex oligonucleotide-cleavage assay, confirming the ability of hAAG to detect ethenoadenines and hypoxanthine. The hAAG-modified comet assay is a new tool for the detection of alkylated bases.
Autores: Parets, S.; Irigoyen Barrio, Ángel; Ordinas, M.; et al.

Título: 2-Hydroxy-Docosahexaenoic Acid Is Converted Into Heneicosapentaenoic Acid via alpha-Oxidation: Implications for Alzheimer's Disease Therapy

Revista: FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY

ISSN: 2296-634X Vol.8 N° 164 2020

Resumen

Alzheimer's disease (AD) is a neurodegenerative disease with as yet no efficient therapies, the pathophysiology of which is still largely unclear. Many drugs and therapies have been designed and developed in the past decade to stop or slow down this neurodegenerative process, although none has successfully terminated a phase-III clinical trial in humans. Most therapies have been inspired by the amyloid cascade hypothesis, which has more recently come under question due to the almost complete failure of clinical trials of anti-amyloid/tau therapies to date. To shift the perspective for the design of new AD therapies, membrane lipid therapy has been tested, which assumes that brain lipid alterations lie upstream in the pathophysiology of AD. A hydroxylated derivative of docosahexaenoic acid was used, 2-hydroxy-docosahexaenoic acid (DHA-H), which has been tested in a number of animal models and has shown efficacy against hallmarks of AD pathology. Here, for the first time, DHA-H is shown to undergo alpha-oxidation to generate the heneicosapentaenoic acid (HPA, C21:5, n-3) metabolite, an odd-chain omega-3 polyunsaturated fatty acid that accumulates in cell cultures, mouse blood plasma and brain tissue upon DHA-H treatment, reaching higher concentrations than those of DHA-H itself. Interestingly, DHA-H does not share metabolic routes with its natural analog DHA (C22:6, n-3) but rather, DHA-H and DHA accumulate distinctly, both having different effects on cell fatty acid composition. This is partly explained because DHA-H alpha-hydroxyl group provokes steric hindrance on fatty acid carbon 1, which in turn leads to diminished incorporation into cell lipids and accumulation as free fatty acid in cell membranes. Finally, DHA-H administration to mice elevated the brain HPA levels, which was directly and positively correlated with cognitive spatial scores in AD mice, apparently in the absence of DHA-H and without any significant change in brain DHA levels. Thus, the evidence presented in this work suggest that the metabolic conversion of DHA-H into HPA could represent a key event in the therapeutic effects of DHA-H against AD.
Autores: Moller, P. (Autor de correspondencia); Azqueta Oscoz, Amaya; Boutet-Robinet, E.; et al.

Título: Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results

Revista: NATURE PROTOCOLS

ISSN: 1754-2189 Vol.15 N° 12 2020 págs. 3817 - 3826

Resumen

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers. Here, members of the hCOMET COST Action program provide a consensus statement on the Minimum Information for Reporting Comet Assays (MIRCA).
Autores: Arce López, Beatriz; Lizarraga Pérez, Elena; Irigoyen Barrio, Ángel; et al.

Título: Presence of 19 Mycotoxins in Human Plasma in a Region of Northern Spain

Revista: TOXINS

ISSN: 2072-6651 Vol.12 N° 12 2020 págs. 750

Resumen

This study was conducted to investigate human exposure to 19 compounds (mycotoxins and their metabolites) in plasma samples from healthy adults (n = 438, aged 19-68 years) from Navarra, a region of northern Spain. Samples were analyzed by LC-MS/MS, before and after enzymatic hydrolysis for the detection of possible glucuronides and/or sulfates (Phase II metabolites). The most prevalent mycotoxin was ochratoxin A (OTA), with an incidence of 97.3%. Positive samples were in the concentration range of 0.4 ng/mL to 45.7 ng/mL. After enzymatic treatment, OTA levels increased in a percentage of individuals, which may indicate the presence of OTA-conjugates. Regarding ochratoxin B, it has also been detected (10% of the samples), and its presence may be related to human metabolism of OTA. Sterigmatocystin was detected with a high incidence (85.8%), but only after enzymatic hydrolysis, supporting glucuronidation as a pathway of its metabolism in humans. None of the other studied mycotoxins (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) were detected in any of the samples, neither before nor after enzymatic treatment. To the best of our knowledge, this is the first report carried out in Spain to determine the exposure of the population to mycotoxins and some of their metabolites using plasma, and the obtained results
Autores: Moller, P. (Autor de correspondencia); Muruzábal Gambarte, Damián; Bakuradze, T. ; et al.

Título: Potassium bromate as positive assay control for the Fpg-modified comet assay

Revista: MUTAGENESIS

ISSN: 0267-8357 Vol.35 N° 4 2020 págs. 341 - 347

Resumen

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37 degrees C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control.
Autores: Collins, A.; Vettorazzi Armental, Ariane; Azqueta Oscoz, Amaya (Autor de correspondencia)

Título: The role of the enzyme-modified comet assay in in vivo studies

Revista: TOXICOLOGY LETTERS

ISSN: 0378-4274 Vol.327 2020 págs. 58 - 68

Resumen

The in vivo comet assay is an established genotoxicity test, with an OECD test guideline, but in its standard form it measures only DNA strand breaks. Including in the assay an additional step, in which the DNA is incubated with a lesion-specific enzyme, can provide important information about the nature of the DNA damage. Formamidopyrimidine DNA glycosylase, 8-oxoguanine DNA glycosylase or endonuclease III are commonly used in the in vitro genotoxicity test and in human biomonitoring to detect oxidised bases, but in vivo applications are rarer. A systematic literature search has identified a total of 60 papers that report such in vivo experiments, testing a variety of agents. In many cases, strand breaks were not seen, but significant levels of enzyme-sensitive sites were induced - indicating a mechanism of action involving oxidative stress. Compounds such as methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS) could be used as positive controls in both the standard and the enzyme-modified in vivo comet assays.
Autores: Chaccour Diaz, Carlos Javier (Autor de correspondencia); Abizanda Sarasa, Gloria María; Irigoyen Barrio, Ángel; et al.

Título: Nebulized ivermectin for COVID-19 and other respiratory diseases, a proof of concept, dose-ranging study in rats

Revista: SCIENTIFIC REPORTS

ISSN: 2045-2322 Vol.10 N° 1 2020 págs. 17073

Resumen

Ivermectin is a widely used antiparasitic drug with known efficacy against several single-strain RNA viruses. Recent data shows significant reduction of SARS-CoV-2 replication in vitro by ivermectin concentrations not achievable with safe doses orally. Inhaled therapy has been used with success for other antiparasitics. An ethanol-based ivermectin formulation was administered once to 14 rats using a nebulizer capable of delivering particles with alveolar deposition. Rats were randomly assigned into three target dosing groups, lower dose (80-90 mg/kg), higher dose (110-140 mg/kg) or ethanol vehicle only. A toxicology profile including behavioral and weight monitoring, full blood count, biochemistry, necropsy and histological examination of the lungs was conducted. The pharmacokinetic profile of ivermectin in plasma and lungs was determined in all animals. There were no relevant changes in behavior or body weight. There was a delayed elevation in muscle enzymes compatible with rhabdomyolysis, that was also seen in the control group and has been attributed to the ethanol dose which was up to 11 g/kg in some animals. There were no histological anomalies in the lungs of any rat. Male animals received a higher ivermectin dose adjusted by adipose weight and reached higher plasma concentrations than females in the same dosing group (mean Cmax 86.2 ng/ml vs. 26.2 ng/ml in the lower dose group and 152 ng/ml vs. 51.8 ng/ml in the higher dose group). All subjects had detectable ivermectin concentrations in the lungs at seven days post intervention, up to 524.3 ng/g for high-dose male and 27.3 ng/g for low-dose females. nebulized ivermectin can reach pharmacodynamic concentrations in the lung tissue of rats, additional experiments are required to assess the safety of this formulation in larger animals.
Autores: Muñoz Solano, Borja; González Peñas, María Elena (Autor de correspondencia)

Título: Mycotoxin Determination in Animal Feed: An LC-FLD Method for Simultaneous Quantification of Aflatoxins, Ochratoxins and Zearelanone in This Matrix

Revista: TOXINS

ISSN: 2072-6651 Vol.12 N° 6 2020 págs. 374

Resumen

Mycotoxins are toxic compounds for humans and animals that are produced by fungi. Mycotoxin contamination in feed is a global safety concern and effective control of these compounds in this matrix is needed. This study proposes a simple, cost-effective analytical method based on liquid chromatography coupled with a fluorescence detector, which is suitable for the routine monitoring of some of the most important mycotoxins in feed: aflatoxins (G2, G1, B2, and B1), zearalenone, and ochratoxins A and B. Mycotoxin extraction, chromatographic separation and quantification are carried out simultaneously for all mycotoxins. The extraction procedure is performed using acetonitrile, water and orthophosphoric acid (80:19:1). Purification of the extract is carried out using an OASIS PRIME HLB solid-phase extraction cartridge followed by a dispersive liquid-liquid microextraction procedure. Aflatoxins G1 and B1 are derivatized post-column (photochemical reactor at 254 nm) to increase their signal. The method has been validated in feed for pigs, cows, sheep, and poultry with very satisfactory results. The detection limits are 2 mu g/kg for aflatoxins B1 and G1, 0.64 mu g/kg for aflatoxins B2 and G2, 42 mu g/kg for zearalenone, and 5 mu g/kg for ochratoxins A and B. These values are low enough to allow for monitoring of these mycotoxins in feed. Global recovery values were between 73.6% and 88.0% for all toxins with a relative standard deviation (RSD) % < 7%. This methodology will facilitate laboratory control and analysis of mycotoxins in feed.
Autores: Vodenkova, S.; Azqueta Oscoz, Amaya; Collins, A.; et al.

Título: An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity

Revista: NATURE PROTOCOLS

ISSN: 1754-2189 Vol.15 N° 12 2020 págs. 3844 - 3878

Resumen

This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d. This protocol describes a comet-based in vitro assay for detecting base and nucleotide excision repair activity for use in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues.
Autores: Fersing, C. ; Boudot, C. ; Paoli-Lombardo, R.; et al.

Título: Antikinetoplastid SAR study in 3-nitroimidazopyridine series: Identification of a novel non-genotoxic and potent anti-T.b. brucei hitcompound with improved pharmacokinetic properties

Revista: EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY

ISSN: 0223-5234 Vol.206 N° 112668 2020

Resumen

To study the antikinetoplastid 3-nitroimidazo[1,2-alpyridine pharmacophore, a structure-activity relationship study was conducted through the synthesis of 26 original derivatives and their in vitro evaluation on both Leishmania spp and Tiypanosoma brucei brucei. This SAR study showed that the antitrypanosomal pharmacophore was less restrictive than the antileishmanial one and highlighted positions 2, 6 and 8 of the imidazopyridine ring as key modulation points. None of the synthesized compounds allowed improvement in antileishmanial activity, compared to previous hit molecules in the series. Nevertheless, compound 8, the best antitrypanosomal molecule in this series (EC50 = 17 nM, SI = 2650 & E degrees = -0.6 V), was not only more active than all reference drugs and previous hit molecules in the series but also displayed improved aqueous solubility and better in vitro pharmacokinetic characteristics: good microsomal stability (T-1/2 > 40 min), moderate albumin binding (77%) and moderate permeability across the blood brain barrier according to a PAMPA assay. Moreover, both micronucleus and comet assays showed that nitroaromatic molecule 8 was not genotoxic in vitro. It was evidenced that bioactivation of molecule 8 was operated by T. b. brucei type 1 nitroreductase, in the same manner as fexinidazole. Finally, a mouse pharmacokinetic study showed that 8 displayed good systemic exposure after both single and repeated oral administrations at 100 mg/kg (NOAEL) and satisfying plasmatic half-life (T-1/2 = 7.7 h). Thus, molecule 8 appears as a good candidate for initiating a hit to lead drug discovery program. (C) 2020 Elsevier Masson SAS. All rights reserved.
Autores: Arce-Lopez, Beatriz; Lizarraga Pérez, Elena; Flores Flores, Myra Evelyn; et al.

Título: Development and validation of a methodology based on Captiva EMR-lipid clean-up and LC-MS/MS analysis for the simultaneous determination of mycotoxins in human plasma

Revista: TALANTA

ISSN: 1873-3573 Vol.206 2020

Resumen

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.
Autores: Azqueta Oscoz, Amaya (Autor de correspondencia); Langie, S. A. S.; Boutet-Robinet, E.; et al.

Título: DNA repair as a human biomonitoring tool: Comet assay approaches

Revista: MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH

ISSN: 1383-5742 Vol.781 2019 págs. 71 - 87

Resumen

The comet assay offers the opportunity to measure both DNA damage and repair. Various comet assay based methods are available to measure DNA repair activity, but some requirements should be met for their effective use in human biomonitoring studies. These conditions include i) robustness of the assay, ii) sources of inter- and intra-individual variability must be known, iii) DNA repair kinetics should be assessed to optimize sampling timing; and iv) DNA repair in accessible surrogate tissues should reflect repair activity in target tissues prone to carcinogenic effects. DNA repair phenotyping can be performed on frozen and fresh samples, and is a more direct measurement than genomic or transcriptomic approaches. There are mixed reports concerning the regulation of DNA repair by environmental and dietary factors. In general, exposure to genotoxic agents did not change base excision repair (BER) activity, whereas some studies reported that dietary interventions affected BER activity. On the other hand, in vitro and in vivo studies indicated that nucleotide excision repair (NER) can be altered by exposure to genotoxic agents, but studies on other life style related factors, such as diet, are rare. Thus, crucial questions concerning the factors regulating DNA repair and inter-individual variation remain unanswered. Intra-individual variation over a period of days to weeks seems limited, which is favourable for DNA repair phenotyping in biomonitoring studies. Despite this reported low infra-individual variation, timing of sampling remains an issue that needs further investigation. A correlation was reported between the repair activity in easily accessible peripheral blood mononuclear cells (PBMCs) and intemal organs for both NER and BER. However, no correlation was found between tumour tissue and blood cells. In conclusion, although comet assay based approaches to measure BER/NER phenotypes are feasible and promising, more work is needed to further optimize their application in human biomonitoring and intervention studies.
Autores: Azqueta Oscoz, Amaya (Autor de correspondencia); Muruzábal Gambarte, Damián; Boutet-Robinet, E.; et al.

Título: Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies

Revista: MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS

ISSN: 1383-5718 Vol.843 2019 págs. 24 - 32

Resumen

The comet assay (single cell gel electrophoresis) is widely used as a biomonitoring tool to assess DNA damage - strand breaks, as well as oxidised bases; it can also be adapted to measure DNA repair. It is based on the ability of breaks in the DNA to relax supercoiling, allowing DNA loops to extend from the nuclear core (nucleoid) under an electric field to form a comet-like tail. Most commonly, it is applied to white blood cells. The range of detection is between a few hundred breaks per cell and a few thousand, encompassing levels of damage that can be repaired and tolerated by human cells. Its applications include monitoring various diseases, studying the influence of nutrition on DNA stability, and investigating effects of environmental and occupational mutagens. Here we address the issue of inter-laboratory variation in comet assay results. This variation is largely due to differences in methods. Imposing a standard protocol is not practical, but users should be aware of the crucial parameters that affect performance of the assay. These include the concentration of agarose in which the cells are embedded; the duration of cell lysis, and of enzyme incubation when oxidised bases are being measured; the duration of alkaline unwinding; the duration of electrophoresis and the voltage gradient applied; and the method used to score the comets. Including reference standards in each experiment allows experimental variability to be monitored - and if variation is not extreme, results can be normalised using reference standard values. Reference standards are also essential for inter-laboratory comparison. Finally, we offer recommendations which, we believe, will limit variability and increase the usefulness of this assay in molecular epidemiology.
Autores: Azqueta Oscoz, Amaya; Enciso, J. M.; Pastor Castro, Laura; et al.

Título: Applying the comet assay to fresh vs frozen animal solid tissues: A technical approach

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.132 2019

Resumen

The in vivo comet assay is usually performed in fresh tissues by processing cells immediately after collection, an approach that is not always possible from a logistical point of view. Although the comet assay has been applied to frozen rodent tissue samples on several occasions, there is currently no agreement on the best way to freeze and thaw them. We have tested two different thawing procedures and compared the levels of DNA strand breaks (SBs) and Fpg-sensitive sites in fresh and frozen (for up to year) liver, kidney and lung tissue samples, from untreated and methyl methanosulfonate treated rats. Tissues were snap frozen, stored at - 80 degrees C and processed in such a way that the tissue remained frozen until the cells were in suspension. Our results showed that comparable levels of DNA SBs were detected in fresh and frozen liver and lung samples stored at - 80 degrees C for up to 1 year and 3 months, respectively. In kidney, similar levels of SBs were detected either in fresh or in frozen tissues stored for up to 1 year. However, more studies are needed to control the variability observed in the Fpg-sensitive site levels in this tissue at the different freezing periods.
Autores: Vettorazzi Armental, Ariane (Autor de correspondencia); Pastor Castro, Laura; Guruceaga Martínez, Elisabet; et al.

Título: Sex-dependent gene expression after ochratoxin A insult in F344 rat kidney

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.123 2019 págs. 337 - 348

Resumen

Ochratoxin A (OTA) is a potent rodent nephrocarcinogen; being males more sensitive than females. The objective was to study the response between sexes at gene expression level (whole genome transcriptomics) in kidneys of F344 rats treated with 0.21 or 0.50 mg/kg bw OTA for 21 days. DNA methylation analysis of selected genes was also studied (MALDI-TOF mass spectrometry). OTA-induced response was dose-dependent in males and females, although clearer in males. Females showed a higher number of altered genes than males but functional analysis revealed a higher number of significantly enriched toxicity lists in 0.21 mg/kg treated males. OTA modulated damage, signaling and metabolism related lists, as well as inflammation, proliferation and oxidative stress in both sexes. Eleven toxicity lists (damage, fibrosis, cell signaling and metabolism) were exclusively altered in males while renal safety biomarker and biogenesis of mitochondria lists were exclusively enriched in females. A high number of lists (39) were significantly enriched in both sexes. However, they contained many sex-biased OTA-modulated genes, mainly phase I and II, transporters and nuclear receptors, but also others related to cell proliferation/apoptosis. No biologically relevant changes were observed in the methylation of selected genes.
Autores: Muruzábal Gambarte, Damián; Langie, S, A, S.; Pourrut, B. ; et al.

Título: The enzyme-modified comet assay: Enzyme incubation step in 2 vs 12-gels/slide systems

Revista: MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS

ISSN: 1383-5718 Vol.845 2019

Resumen

The enzyme-modified comet assay is a commonly used method to detect specific DNA lesions. However, still a lot of errors are made by many users, leading to dubious results and even misinterpretations. This technical note describes some critical points in the use of the enzyme-modified comet assay, such as the enzyme concentration, the time of incubation, the format used and the equipment. To illustrate the importance of these conditions/ parameters, titration experiments of formamidopyrimidine DNA glycosylase (Fpg) were performed using the 2 gels/slide and the 12 minigels/slide formats (plus the 12-Gel Comet Assay Unit (TM)). Incubation times of 15 and 30 min, and 1 h were used. Results showed that the 12 minigels/slide system requires a lower volume and concentration of Fpg. A longer time of incubation has a bigger impact when using such format. Moreover, the paper describes how to perform and interpret a titration experiment when using the enzyme. modified comet assay.
Autores: González Peñas, María Elena (Autor de correspondencia); Vettorazzi Armental, Ariane; Lizarraga Pérez, Elena; et al.

Título: Report of the IVth workshop of the spanish national network on mycotoxins and toxigenic fungi and their decontamination processes (MICOFOOD), held in Pamplona, Spain, 29¿31 May 2019

Revista: TOXINS

ISSN: 2072-6651 Vol.11 N° 7 2019
Autores: Vettorazzi Armental, Ariane; Izco, M.; López de Cerain Salsamendi, Adela; et al.

Título: Could Ochratoxin A be a possible etiological factor of Parkinson's disease?

Revista: TOXICOLOGY LETTERS

ISSN: 0378-4274 Vol.314 N° S 2019 págs. S220 - S220
Autores: Sanz Serrano, Julen; Muruzábal Gambarte, Damián; López de Cerain Salsamendi, Adela; et al.

Título: Mutagenicity assessment of a battery of compounds using a miniaturized-Ames test

Revista: TOXICOLOGY LETTERS

ISSN: 0378-4274 Vol.314 N° S 2019 págs. S124 - S124
Autores: Azqueta Oscoz, Amaya; Runden-Pran, E.; Elje, E.; et al.

Título: The comet assay applied to cells of the eye

Revista: MUTAGENESIS

ISSN: 0267-8357 Vol.33 N° 1 2018 págs. 21 - 24

Resumen

The human eye is relatively unexplored as a source of cells for investigating DNA damage. There have been some clinical studies, using cells from surgically removed tissues, and altered DNA bases as well as strand breaks have been measured using the comet assay. Tissues examined include corneal epithelium and endothelium, lens capsule, iris and retinal pigment epithelium. For the purpose of biomonitoring for exposure to potential mutagens in the environment, the eye-relatively unprotected as it is compared with the skin-would be a valuable object for study; non-invasive techniques exist to collect lachrymal duct cells from tears, or cells from the ocular surface by impression cytology, and these methods should be further developed and validated.
Autores: Ferreira, J. ; Ramos, A. A. (Autor de correspondencia); Almeida, T.; et al.

Título: Drug resistance in glioblastoma and cytotoxicity of seaweed compounds, alone and in combination with anticancer drugs: A mini review

Revista: PHYTOMEDICINE

ISSN: 0944-7113 Vol.48 2018 págs. 84 - 93

Resumen

Background: Glioblastomas (GBM) are one of the most aggressive tumor of the central nervous system with an average life expectancy of only 1-2 years after diagnosis, even with the use of advanced treatments with surgery, radiation, and chemotherapy. There are several anticancer drugs with alkylating properties that have been used in the therapy of malignant gliomas. Temozolomide (TMZ) is one of them, widely used even in combination with ionizing radiation. However, the main disadvantage of using these types of drugs in the treatment of GBM is the development of cancer drug resistance. Research of bioactive compounds with anticancer activity has been heavily explored. Purpose: This review focuses on a carotenoid and a phlorotannin present in seaweed, namely fucoxanthin and phloroglucinol, and their anticancer activity against glioblastoma. The combination of natural compounds with conventional drugs is also discussed. Conclusion: Several natural compounds existing in seaweeds, such as fucoxanthin and phoroglucinol, have shown cytotoxic activity in models in vitro and in vivo, acting through different molecular mechanisms, such as antioxidant, antiproliferative, DNA damage/DNA repair, proapoptotic, antiangiogenic and antimetastic. Within the scope of interactions with conventional drugs, there are evidences that some seaweed compounds could be used to potentiate the action of anticancer drugs. However, their effects and mechanisms of action, alone or in combination with anticancer drugs, namely TMZ, in glioblastoma cell, still few explored and require more attention due to the unquestionable high potential of these marine compounds.
Autores: Chaccour Diaz, Carlos Javier; Ngha'bi, K.; Abizanda Sarasa, Gloria María; et al.

Título: Targeting cattle for malaria elimination: marked reduction of Anopheles arabiensis survival for over six months using a slow-release ivermectin implant formulation

Revista: PARASITES AND VECTORS

ISSN: 1756-3305 Vol.11 2018 págs. 287

Resumen

Background: Mosquitoes that feed on animals can survive and mediate residual transmission of malaria even after most humans have been protected with insecticidal bednets or indoor residual sprays. Ivermectin is a widely-used drug for treating parasites of humans and animals that is also insecticidal, killing mosquitoes that feed on treated subjects. Mass administration of ivermectin to livestock could be particularly useful for tackling residual malaria transmission by zoophagic vectors that evade human-centred approaches. Ivermectin comes from a different chemical class to active ingredients currently used to treat bednets or spray houses, so it also has potential for mitigating against emergence of insecticide resistance. However, the duration of insecticidal activity obtained with ivermectin is critical to its effectiveness and affordability. Results: A slow-release formulation for ivermectin was implanted into cattle, causing 40 weeks of increased mortality among Anopheles arabiensis that fed on them. For this zoophagic vector of residual malaria transmission across much of Africa, the proportion surviving three days after feeding (typical mean duration of a gonotrophic cycle in field populations) was approximately halved for 25 weeks. Conclusions: This implantable ivermectin formulation delivers stable and sustained insecticidal activity for approximately 6 months. Residual malaria transmission by zoophagic vectors could be suppressed by targeting livestock with this long-lasting formulation, which would be impractical or unacceptable for mass treatment of human populations.
Autores: Enciso, J. M.; Gutzkow, K. B.; Brunborg, G.; et al.

Título: Standardisation of the in vitro comet assay: influence of lysis time and lysis solution composition on the detection of DNA damage induced by X-rays

Revista: MUTAGENESIS

ISSN: 0267-8357 Vol.33 N° 1 2018 págs. 25-30

Resumen

The alkaline comet assay, in vivo and in vitro, is currently used in several areas of research and in regulatory genotoxicity testing. Several efforts have been made in order to decrease the inter-experimental and inter-laboratory variability and increase the reliability of the assay. In this regard, lysis conditions are considered as one of the critical variables and need to be further studied. Here, we tested different times of lysis (from no lysis to 1 week) and two different lysis solutions in human lymphoblast (TK6) cells unexposed or exposed to X-rays. Similar % tail DNA values were obtained independently of the time of lysis employed for every X-ray dose tested and both lysis solutions. These results, taken together with our previous ones with methyl methanesulfonate and H2O2, which showed clear lysis-time dependence, support that the influence of the lysis time in the comet assay results depends on the type of lesion being detected; some DNA lesions may spontaneously give rise to apurinic or apyrimidinic (AP) sites during the lysis period, which can be converted into strand breaks detectable with the comet assay. Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced. However, the same lysis conditions (i.e. lysis time and lysis solution) should be used when comparing results between different experiments or laboratories.
Autores: Bonilla-Ramirez, L.; Rios, A.; Quiliano, M.; et al.

Título: Novel antimalarial chloroquine- and primaquine-quinoxaline 1,4-di-N-oxide hybrids: design, synthesis, Plasmodium life cycle stage profile, and preliminary toxicity studies

Revista: EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY

ISSN: 0223-5234 Vol.158 2018 págs. 68 - 81

Resumen

Emergence of drug resistance and targeting all stages of the parasite life cycle are currently the major challenges in antimalarial chemotherapy. Molecular hybridization combining two scaffolds in a single molecule is an innovative strategy for achieving these goals. In this work, a series of novel quinoxaline 1,4-di-N-oxide hybrids containing either chloroquine or primaquine pharmacophores was designed, synthesized and tested against both chloroquine sensitive and multidrug resistant strains of Plasmodium falciparum. Only chloroquine-based compounds exhibited potent blood stage activity with compounds 4b and 4e being the most active and selective hybrids at this parasite stage. Based on their intraerythrocytic activity and selectivity or their chemical nature, seven hybrids were then evaluated against the liver stage of Plasmodium yoelii, Plasmodium berghei and Plasmodium falciparum infections. Compound 4b was the only chloroquine-quinoxaline 1,4-di-N-oxide hybrid with a moderate liver activity, whereas compound 6a and 6b were identified as the most active primaquine-based hybrids against exoerythrocytic stages, displaying enhanced liver activity against P. yoelii and P. berghei, respectively, and better SI values than primaquine. Although both primaquine-quinoxaline 1,4-di-N-oxide hybrids slightly reduced the infection of mosquitoes, they inhibited sporogony of P. berghei and compound 6a showed 92% blocking of transmission. In vivo liver efficacy assays revealed that compound 6a showed causal prophylactic activity affording parasitaemia reduction of up to 95% on day 4. Absence of genotoxicity and in vivo acute toxicity were also determined. These results suggest the approach of primaquine-quinoxaline 1,4-di-N-oxide hybrids as new potential dual-acting antimalarials for further investigation.
Autores: Almeida, T. P.; Ferreira, J. ; Vettorazzi Armental, Ariane; et al.

Título: Cytotoxic activity of fucoxanthin, alone and in combination with the cancer drugs imatinib and doxorubicin, in CML cell lines

Revista: ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY

ISSN: 1382-6689 Vol.59 2018 págs. 24 - 33

Resumen

In the present study, we evaluate the in vitro cytotoxicity of fucoxanthin (Fx) on two human leukemia cell lines, K562 and TK6, alone and in combination with the conventional anticancer drugs imatinib (Imat) and doxorubicin (Dox). For the purpose, we assessed the cytotoxic and proliferation effects by cell count, induction of DNA damage by comet assay, and cell death by nuclear condensation, annexin V staining, coupled with propidium iodide uptake, and protein expression of Bax, caspase-3, and Bcl-2 (western blot). Our results show that Imat increased cytotoxicity in TK6 cells and inhibited proliferation in K562 cells, while Dox decreased cell viability and proliferation in both cell lines. Fx per se increased cytotoxicity against K562 cells and decreased cell proliferation of K562 and TK6 cells. The effects were confirmed by phase contrast microscopy. However, the antiproliferative effects are not explained by induction of DNA damage or cell death. In co-incubation, Fx increased antiproliferative effects of both drugs in the cell lines tested, however no differences where observed relative to Fx alone. This study unveiled in vitro cytotoxicity of Fx by inhibition of cell proliferation in both cell lines. Further studies are needed to elucidate the signal transduction pathways and molecular targets involved in that effect.
Autores: Pastor Castro, Laura; Vettorazzi Armental, Ariane (Autor de correspondencia); Enciso, J. M.; et al.

Título: Sex differences in ochratoxin a toxicity in F344 rats after 7 and 21 days of daily oral administration

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.111 2018 págs. 363 - 373

Resumen

Ochratoxin A (OTA) is a potent renal carcinogen in male rats but not in females. The mechanisms underlying these differences are unknown. The sex-dependent response of F344 rats after a repeated OTA oral administration for 7 (0.50 mg/kg bw) or 21 days (0.21 and 0.50 mg/kg bw) was evaluated. General toxicity, sex and thyroid hormones and histopathology were studied. OTA was quantified (HPLC-FLD) in plasma, kidney and liver and the expression of kidney transporters (RT-qPCR) was studied. After 7 days, kidney histopathology showed more pronounced signs of toxicity in males than in females. After 21 days, a higher toxicity was observed but sex differences disappeared. OTA concentration in plasma and tissues was similar in both sexes. Downregulation was the general OTA-induced effect. Oats' downregulation was slow in males and Oat3 did not change in females. Oatp1 was strongly downregulated in males after 21 days. An opposite effect was observed in Bcrp after 21 days: downregulation in males and upregulation in females. Females showed a dose- and time-dependent decrease of progesterone. Despite the sex differences, the final balance in OTA toxicokinetics at renal cell level does not seem to support a higher accumulation of OTA in male kidneys.
Autores: Flores Flores, Myra Evelyn; González Peñas, María Elena (Autor de correspondencia)

Título: Short communication: analysis of mycotoxins in Spanish milk

Revista: JOURNAL OF DAIRY SCIENCE

ISSN: 0022-0302 Vol.101 N° 1 2018 págs. 113 - 117

Resumen

We surveyed the presence of 22 mycotoxins in 191 Spanish cow milk samples. Mycotoxins could be carried over from diet into animal milk and have toxic effects on human and animal health. The interaction of different mycotoxins may be additive or synergetic. Therefore, surveillance of mycotoxin co-occurrence in milk is recommended, Aflatoxins M-1, B-1, B-2, G(1), and G(2), ochratoxins A and B, nivalenol, deoxynivalenol, deepoxy-deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, fusarenon X, T-2 and HT-2 toxins, fumonisins B-1, B-2, and B-3, sterig-matocystin, and zearalenone were analyzed. Samples were treated by liquid-liquid extraction with acidified acetonitrile, followed by an acetonitrile-water phase separation using sodium acetate. The analysis was carried out by HPLC coupled to a triple quadrupole mass spectrometer. None of the analyzed mycotoxins had a concentration level higher than their detection limit (0.05-10.1 mu g/L). The aflatoxin M-1 in the samples never exceeded the level established by the European Union.
Autores: Flores Flores, Myra Evelyn; González Peñas, María Elena (Autor de correspondencia)

Título: Analysis of Mycotoxins in Peruvian Evaporated Cow Milk

Revista: BEVERAGES

ISSN: 2306-5710 Vol.4 N° 2 2018

Resumen

Mycotoxins-toxic secondary fungi metabolites-reach humans through food, producing several effects on their health and economic losses. Mycotoxin co-occurrence is common in food due to the co-presence of different fungi species, each of which may produce different toxins. A survey regarding the presence of 22 mycotoxins (aflatoxins M1, B1, B2, G1, G2; ochratoxins A and B; fumonisins B1, B2 and B3; HT-2 and T-2 toxins; nivalenol; deoxynivalenol; deepoxy-deoxynivalenol; 3 and 15 acetyl-deoxynivalenol; diacetoxyscirpenol; fusarenon X; neosolaniol; sterigmatocystin; and zearalenone) in 30 Peruvian evaporated cow milk samples is presented for the first time. Analysis was carried out by liquid chromatography coupled to tandem mass spectrometry, which was based on two previously validated methods for quantification of these toxic compounds in liquid cow milk, and further validated for the new matrix. The only detected mycotoxin was ochratoxin A, which was found in four samples, although at levels below its limit of quantification (0.2 ng/mL). This initial study indicates that the presence of mycotoxins in evaporated milk is low in Peru. However, we recommend the analysis of more samples and more milk types obtained from urban and rural areas, in order to obtain more data that will allow further risk assessments to be carried out.
Autores: Enciso, José Manuel; López de Cerain Salsamendi, Adela; Pastor Castro, Laura; et al.

Título: Is oxidative stress involved in the sex-dependent response to ochratoxin A renal toxicity?

Revista: FOOD AND CHEMICAL TOXICOLOGY

ISSN: 0278-6915 Vol.116 2018 págs. 379 - 387

Resumen

Ochratoxin A (OTA) is a mycotoxin considered the most powerful renal carcinogen in rodents and classified as a possible human carcinogen. Though its mechanism of action is still unknown, indirect DNA reactivity mediated by oxidative stress has been hypothesized to play an important role. Moreover, large sex-differences have been observed in carcinogenicity studies, male rats being more sensitive than females. Male and female F344 rats were administered (p.o.) with bicarbonate or 0.5 mg OTA/kg b.w. for 7 days; or with bicarbonate, 0.21 or 0.5 mg OTA/kg b.w. for 21 days. Total glutathione (tGSH) and oxidized glutathione (GSSG) levels, glutathione S.transferase (GST) and superoxide dismutase (SOD) activities were analysed in kidneys. The standard alkaline comet assay was used in combination with Formamidopyrimidine-DNA glycosylase (Fpg) to detect oxidized DNA bases in kidney. No biologically relevant sex-differences were observed in all the oxidative-stress related parameters analysed. Indeed, no relevant oxidative-stress related response was observed between treated animals and controls. In accordance with the similar OTA levels and histopathological changes between both sexes observed previously in the same animals, and with other oxidative-stress related parameters measured by others, results support that there are no differences between sexes in the oxidative stress response to OTA.
Autores: Quiliano, M.; Pabon, A.; Moles, E. ; et al.

Título: Structure-activity relationship of new antimalarial 1-aryl-3-susbtituted propanol derivatives: Synthesis, preliminary toxicity profiling, parasite life cycle stage studies, target exploration, and targeted delivery

Revista: EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY

ISSN: 0223-5234 Vol.152 2018 págs. 489 - 514

Resumen

Design, synthesis, structure-activity relationship, cytotoxicity studies, in silico drug-likeness, genotoxicity screening, and in vivo studies of new 1-aryl-3-substituted propanol derivatives led to the identification of nine compounds with promising in vitro (55, 56, 61, 64, 66, and 70-73) and in vivo (66 and 72) antimalarial profiles against Plasmodium falciparum and Plasmodium berghei. Compounds 55, 56, 61, 64, 66 and 70-73 exhibited potent antiplasmodial activity against chloroquine-resistant strain FCR-3 (IC(50)s <0.28 mu M), and compounds 55, 56, 64, 70, 71, and 72 showed potent biological activity in chloroquine-sensitive and multidrug-resistant strains (IC(50)s < 0.7 mu M for 3D7, D6, FCR-3 and C235). All of these compounds share appropriate drug-likeness profiles and adequate selectivity indexes (77 < SI < 184) as well as lack genotoxicity. In vivo efficacy tests in a mouse model showed compounds 66 and 72 to be promising candidates as they exhibited significant parasitemia reductions of 96.4% and 80.4%, respectively. Additional studies such as liver stage and sporogony inhibition, target exploration of heat shock protein 90 of P. falciparum, targeted delivery by immunoliposomes, and enantiomer characterization were performed and strongly reinforce the hypothesis of 1-aryl-3-substituted propanol derivatives as promising antimalarial compounds. (C) 2018 Elsevier Masson SAS. All rights reserved.

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Autores: Assunção, R.; Vettorazzi Armental, Ariane; González Peñas, María Elena; et al.

Título: Climate change and aflatoxins contamination in the Iberian Peninsula

Libro: Encyclopedia of Mycology

ISSN: 978-0-323-85180-0 Vol.2 2021 págs. 168 - 175

Resumen

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?
Autores: Alonso Jauregui, María; López de Cerain Salsamendi, Adela; González Peñas, María Elena; et al.

Título: Combined toxicity of aflatoxins and ochratoxin A

Libro: Aflatoxins: Biochemistry, Toxicology, Public Health, Policies and Modern Methods of Analysis

ISSN: 978-1-53616-785-6 2020 págs. 3 - 28
Autores: Lizarraga Pérez, Elena; Vettorazzi Armental, Ariane; González Peñas, María Elena

Título: Analytical methods for aflatoxin human biomonitoring

Libro: Aflatoxins: Biochemistry, Toxicology, Public Health, Policies and Modern Methods of Analysis

ISSN: 978-1-53616-785-6 2020 págs. 51 - 88
Autores: Vettorazzi Armental, Ariane; Lizarraga Pérez, Elena; González Peñas, María Elena

Título: Review of the Analytical Methodologies and Occurrence Data of Aflatoxins in Cereals and Cereal-Based Foods in Spain

Libro: Aflatoxins: Biochemistry, Toxicology, Public Health, Policies and Modern Methods of Analysis

ISSN: 978-1-53616-785-6 2020 págs. 207 - 243
Autores: González Peñas, María Elena

Título: Yeasts and Molds: Mycotoxins: Classification, Occurrence and Determination

Libro: Reference Module in Food Science

2020 págs. 1-6
Autores: Cobreros Bordenave, Pablo

Título: Supervaluationism, Subvaluationism and the Sorites Paradox

Libro: Solutions to the Sorites Paradox

Vol.1 2019 págs. 38-62
Autores: Azqueta Oscoz, Amaya; Collins, A. R.

Título: Polyphenols and DNA damage: A mixed blessing

Libro: Polyphenols for cancer treatment or prevention

ISSN: 978-3-03842-648-6 2018 págs. 134 - 156

Autores: González Peñas, María Elena (Editor); López de Cerain Salsamendi, Adela (Editor); Vettorazzi Armental, Ariane (Editor); et al.

Título: Special Issue: Detection and Prevention of Food Contaminants

2019

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Título: Micotoxinas y cáncer: estudios de biomonitorización humana y caracterización toxicológica

Código de expediente: PID2021-126026OB-I00

Investigador principal: ARIANE RENATA VETTORAZZI ARMENTAL, MARIA ELENA GONZALEZ PEÑAS.

Financiador: AGENCIA ESTATAL DE INVESTIGACION

Convocatoria: 2021 AEI Proyectos de Generación del Conocimiento

Fecha de inicio: 01-09-2022

Fecha fin: 31-08-2026

Importe concedido: 248.050,00€

Otros fondos: Fondos FEDER
Título: Programa Investigo del Gobierno de Navarra

Código de expediente: 0011-4001-2022-000012

Investigador principal: ARIANE RENATA VETTORAZZI ARMENTAL.

Financiador: GOBIERNO DE NAVARRA

Convocatoria: 2021 GN Investigo

Fecha de inicio: 24-06-2022

Fecha fin: 23-06-2023

Importe concedido: 22.405,94€

Otros fondos: Fondos MRR
Título: Modificaciones del ensayo del cometa para su aplicación en seguridad alimentaria; genotoxicidad de carnes cocinadas y digeridas in vitro

Código de expediente: PID2020-115348RB-I00

Investigador principal: AMAYA AZQUETA OSCOZ, DIANA MARIA ANSORENA ARTIEDA.

Financiador: AGENCIA ESTATAL DE INVESTIGACION

Convocatoria: 2020 AEI PROYECTOS I+D+i (incluye Generación del conocimiento y Retos investigación)

Fecha de inicio: 01-09-2021

Fecha fin: 31-08-2024

Importe concedido: 173.030,00€

Otros fondos: -
Título: Identificación y desarrollo de candidato inhibidor de HDAC6 como tratamiento frente al cáncer de colon (COLON-HDAC6)

Código de expediente: 0011-1411-2021-000097

Investigador principal: ANA GLORIA GIL ROYO.

Financiador: GOBIERNO DE NAVARRA

Convocatoria: 2021 GN PROYECTOS ESTRATEGICOS DE I+D 2021-2024

Fecha de inicio: 01-07-2021

Fecha fin: 31-12-2023

Importe concedido: 412.467,21€

Otros fondos: -
Título: Micotoxinas y Parkinson: el eslabón desconocido?

Código de expediente: 43/2019

Investigador principal: ARIANE RENATA VETTORAZZI ARMENTAL.

Financiador: GOBIERNO DE NAVARRA. DEPARTAMENTO DE SALUD

Convocatoria: 2019 GN Proyectos de Investigación en salud

Fecha de inicio: 19-11-2019

Fecha fin: 18-11-2022

Importe concedido: 80.000,00€

Otros fondos: Fondos FEDER
Título: Multiexposición y toxicidad combinada de micotoxinas en el hombre y animales de granja. Caracterización toxicocinética y metabolismo.

Código de expediente: AGL2017-85732-R

Investigador principal: MARIA ELENA GONZALEZ PEÑAS.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2017 MINECO RETOS INVESTIGACION. PROYECTOS DE I+D+i

Fecha de inicio: 01-01-2018

Fecha fin: 30-09-2021

Importe concedido: 139.150,00€

Otros fondos: Fondos FEDER
Título: Fármacos innovadores para el tratamiento de la Enfermedad de Alzheimer: Estudios preclínicos de eficacia y toxicidad bajo condiciones BPL por vía oral.

Código de expediente: RTC-2017-5994-1

Investigador principal: ANA GLORIA GIL ROYO.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2017 MINECO RETOS COLABORACIÓN

Fecha de inicio: 01-01-2018

Fecha fin: 31-12-2020

Importe concedido: 86.118,00€

Otros fondos: Fondos FEDER
Título: Papel de HDAC5 y SIRT2 en el grado de severidad de la depresión y en la eficacia clínica de los antidepresivos en pacientes de Navarra

Código de expediente: 81/2017

Investigador principal: ROSA MARIA TORDERA BAVIERA.

Financiador: GOBIERNO DE NAVARRA

Convocatoria: 2017 GN SALUD

Fecha de inicio: 16-12-2017

Fecha fin: 31-12-2021

Importe concedido: 90.000,00€

Otros fondos: -
Título: Aplicacion de una nueva estrategia de evaluacion de genotoxicidad en ingredientes funcionales y en frituras de restauracion colectiva

Código de expediente: AGL2015-70640-R

Investigador principal: AMAYA AZQUETA OSCOZ.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2015 MINECO RETOS INVESTIGACION. PROYECTOS I+D+i

Fecha de inicio: 01-01-2016

Fecha fin: 31-12-2020

Importe concedido: 121.000,00€

Otros fondos: Fondos FEDER
Título: Beca RAMÓN Y CAJAL Solicitud RYC-2013-14370

Código de expediente: RYC-2013-14370

Investigador principal: AMAYA AZQUETA OSCOZ.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2014 MINECO Ramón y Cajal

Fecha de inicio: 01-01-2015

Fecha fin: 31-08-2022

Importe concedido: 308.600,00€

Otros fondos: -

Título: PARC_European Partnership for chemicals Risk Assessment

Código de expediente:

Investigador principal: AMAYA AZQUETA OSCOZ

Financiador: COMISIÓN EUROPEA

Convocatoria: HORIZON-HLTH-2021-ENVHLTH-03-01 European partnership for the assessment of risks from chemicals (PARC) Partnerships in Health (2021) (HORIZON-HLTH-2021-ENVHLTH-03)

Fecha de inicio: 01-05-2022

Fecha fin: 30-04-2029

Importe concedido: 431.986,25€

Otros fondos: -

Título: Evaluación de Urea marcada en muestras de plasma de ratón

Investigador principal: MARIA ELENA GONZALEZ PEÑAS

Fecha de inicio: 16-10-2018

Fecha fin: 30-10-2019

Importe: 0

Otros fondos: -
Título: CIEN BIOPRO UV

Investigador principal: PAULA ARANAZ OROZ

Fecha de inicio: 01-11-2017

Fecha fin: 31-12-2021

Importe: 0

Otros fondos: -
Título: CIEN BIOPRO MAT

Investigador principal: PAULA ARANAZ OROZ

Fecha de inicio: 01-11-2017

Fecha fin: 31-12-2021

Importe: 0

Otros fondos: -
Título: CIEN BIOPRO AR

Investigador principal: PAULA ARANAZ OROZ

Fecha de inicio: 01-11-2017

Fecha fin: 31-12-2021

Importe: 0

Otros fondos: -
Título: CIEN BIOPRO MA

Investigador principal: PAULA ARANAZ OROZ

Fecha de inicio: 01-11-2017

Fecha fin: 31-10-2021

Importe: 0

Otros fondos: -
Título: CIEN BIOPRO KM

Investigador principal: PAULA ARANAZ OROZ

Fecha de inicio: 01-11-2017

Fecha fin: 31-12-2021

Importe: 0

Otros fondos: -
Título: CIEN BIOPRO EL

Investigador principal: PAULA ARANAZ OROZ

Fecha de inicio: 01-11-2017

Fecha fin: 31-12-2021

Importe: 0

Otros fondos: -
Título: CIEN BIOPRO AN

Investigador principal:

Fecha de inicio: 30-06-2017

Fecha fin: 31-12-2021

Importe: 0

Otros fondos: -

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