Revistas
Revista:
NANOMATERIALS
ISSN:
2079-4991
Año:
2023
Vol.:
13
N°:
1
Págs.:
3
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.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2023
Vol.:
38
N°:
1
Págs.:
51 - 57
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.
Revista:
TOXINS
ISSN:
2072-6651
Año:
2022
Vol.:
14
N°:
6
Págs.:
400
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.
Revista:
NANOMATERIALS
ISSN:
2079-4991
Año:
2022
Vol.:
12
N°:
6
Págs.:
914
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:
Arce-Lopez, B.; Alvarez-Erviti, L.; De Santis, B.; et al.
Revista:
TOXINS
ISSN:
2072-6651
Año:
2021
Vol.:
13
N°:
7
Págs.:
477
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).
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2021
Vol.:
153
Págs.:
112237
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.
Revista:
FOODS
ISSN:
2304-8158
Año:
2021
Vol.:
10
N°:
2
Págs.:
339
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.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2021
Vol.:
156
Págs.:
112494
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.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2021
Vol.:
152
Págs.:
112164
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.
Revista:
INTERNATIONAL JOURNAL OF GASTRONOMY AND FOOD SCIENCE
ISSN:
1878-450X
Año:
2021
Vol.:
26
Págs.:
100432
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.
Revista:
TOXINS
ISSN:
2072-6651
Año:
2021
Vol.:
13
N°:
10
Págs.:
734
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.
Revista:
TOXINS
ISSN:
2072-6651
Año:
2021
Vol.:
13
N°:
3
Págs.:
177
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.
Revista:
ARCHIVES OF TOXICOLOGY
ISSN:
0340-5761
Año:
2021
Vol.:
95
N°:
8
Págs.:
2825 - 2838
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.
Revista:
TOXICOLOGY LETTERS
ISSN:
0378-4274
Año:
2020
Vol.:
330
Págs.:
108 - 117
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.
Revista:
TOXICOLOGY LETTERS
ISSN:
0378-4274
Año:
2020
Vol.:
335
Págs.:
98 - 98
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
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.
Revista:
TOXINS
ISSN:
2072-6651
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2019
Vol.:
123
Págs.:
337 - 348
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.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2018
Vol.:
116
Págs.:
379 - 387
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:
Bonilla-Ramirez, L.; Rios, A.; Quiliano, M.; et al.
Revista:
EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY
ISSN:
0223-5234
Año:
2018
Vol.:
158
Págs.:
68 - 81
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.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2018
Vol.:
111
Págs.:
363 - 373
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:
Enciso, J. M.; Gutzkow, K. B.; Brunborg, G.; et al.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2018
Vol.:
33
N°:
1
Págs.:
25-30
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:
Quiliano, M.; Pabon, A.; Moles, E. ; et al.
Revista:
EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY
ISSN:
0223-5234
Año:
2018
Vol.:
152
Págs.:
489 - 514
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.
Autores:
Benito, A.; Ibáñez, C.; Moncho, W.; et al.
Revista:
EFSA SUPPORTING PUBLICATIONS
ISSN:
2397-8325
Año:
2017
Vol.:
14
N°:
8
Págs.:
1274E
Revista:
JOURNAL OF MEDICAL MICROBIOLOGY
ISSN:
0022-2615
Año:
2017
Vol.:
66
N°:
7
Págs.:
946 - 958
Purpose. The aim of this study was to develop an immunogenic protective product against Shigella flexneri by employing a simple and safe heat treatment-based strategy. Methodology. The physicochemical characteristics of naturally produced (OMV) and heat-induced (HT) outer-membrane vesicles from S. flexneri were examined, including a comparison of the protein content of the products. Toxicological and biodistribution studies, and a preliminary experiment to examine the protective effectiveness of HT in a murine model of S. flexneri infection, were also included. Results. This method simultaneously achieves complete bacterial inactivation and the production of the HT vaccine product, leading to a safe working process. The obtained HT complex presented a similar morphology (electron microscopy) and chemical composition to the classical OMV, although it was enriched in some immunogens, such as lipoproteins, OmpA or OmpC, among others. The HT formulation was not toxic and biodistribution studies performed in mice demonstrated that the vaccine product remained in the small intestine after nasal administration. Finally, a single dose of HT administered nasally was able to protect mice against S. flexneri 2a. Conclusion. The convenient and safe manufacturing process, and the preliminary biological evaluation, support the use of the self-adjuvanted HT complex as a new vaccine candidate to face shigellosis. Further development is required, such as additional immune analyses, to evaluate whether this new subunit vaccine can be useful in achieving full protection against Shigella.
Revista:
INTERNATIONAL JOURNAL OF PHARMACEUTICS
ISSN:
0378-5173
Año:
2017
Vol.:
517
N°:
1 - 2
Págs.:
67 - 79
he main concerns with drugs designed for oral administration are their inactivation or degradation in the harsh conditions of the gastrointestinal tract, their poor solubility through the gastrointestinal mucus gel layer, the poor intestinal epithelium permeability that limits their absorption, and their toxicity. In this context, poly(anhydride) nanoparticles are capable of protecting the drug from the harsh environment, reduce the drug's toxicity and, by virtue of surface modification, to enhance or reduce their mucus permeability and the bioadhesion to specific target cells. The copolymer between methyl vinyl ether and maleic anhydride (commercialized as Gantrez® AN 119) are part of the poly(anhydride) nanoparticles. These biocompatible and biodegradable nanoparticles (NPs) can be modified by using different ligands. Their usefulness as drug carriers and their bioadhesion with components of the intestinal mucosa have been described. However, their toxicity, genotoxicity and mucus permeation capacity has not been thoroughly studied. The aim of this work was to evaluate and compare the in vitro toxicity, cell viability and in vitro genotoxicity of the bioadhesive empty Gantrez® AN 119 NPs modified with dextran, aminodextran, 2-hydroxypropyl-ß-cyclodextrin, mannosamine and poly-ethylene glycol of different molecular weights. Results showed that, in general, coated NPs exhibit better mucus permeability than the bare ones, those coated with mannosamine being the most permeable ones. The NPs studied did not affect cell metabolism, membrane integrity or viability of Caco-2 cells at the different conditions tested. Moreover, they did not induce a relevant level of DNA strand breaks and FPG-sensitive sites (as detected with the comet assay).
Revista:
INTERNATIONAL JOURNAL OF PHARMACEUTICS
ISSN:
0378-5173
Año:
2017
Vol.:
530
N°:
1-2
Págs.:
187 - 194
Gantrez (R) AN 119-based NPs have been developed as oral drug carriers due to their strong bioadhesive interaction with components of the gastrointestinal mucosa and to their adaptable surface. The use of mannosamine to coat Gantrez (R) AN 119-based NPs results in a high mucus-permeable carrier, able to reach the gastrointestinal epithelium. Although their efficacy to transport a therapeutic agent has been demonstrated, their safety has not yet been thoroughly studied. They have proved to be non-cytotoxic, non-genotoxic and non-mutagenic in vitro; however, the in vivo toxicity profile has not yet been determined. In this study, the in vivo genotoxic potential of Gantrez (R) AN 119 NPs coated with mannosamine (GN-MA-NP) has been assessed using the in vivo comet assay in combination with the enzyme formamidopyrimidine DNA glycosylase in mice, following the OECD test guideline 489. To determine the relevant organs to analyse and the sampling times, an in vivo biodistribution study was also carried out. Results showed a statistically significant induction of DNA strand breaks and oxidized bases in the duodenum of animals exposed to 2000 mg/kg bw. However, this effect was not observed at lower doses (i.e. 500 and 1000 mg/kg which are closer to the potential therapeutic doses) or in other organs. In conclusion, GN-MA-NP are promising nanocarriers as oral drug delivery systems.
Revista:
INTERNATIONAL JOURNAL OF PHARMACEUTICS
ISSN:
0378-5173
Año:
2017
Vol.:
523
N°:
1
Págs.:
418 - 426
In the last years, the development of nanomaterials has significantly increased due to the immense variety of potential applications in technological sectors, such as medicine, pharmacy and food safety. Focusing on the nanodevices for oral drug delivery, poly(anhydride) nanoparticles have received extensive attention due to their unique properties, such as their capability to develop intense adhesive interactions within the gut mucosa, their modifiable surface and their biodegradable and easy-to produce profile. However, current knowledge of the possible adverse health effects as well as, toxicological information, is still exceedingly limited. Thus, we investigated the capacity of two poly(anhydride) nanoparticles, Gantrez (R) AN 119 -NP (GN-NP) and Gantrez (R) AN 119 covered with mannosamine (GN-MA-NP), and their main bulk material (Gantrez (R) AN 119-Polymer), to induce DNA damage and thymidine kinase (TK+/-) mutations in L5178Y TK+/- mouse lymphoma cells after 24 h of exposure. The results showed that GN-NP, GN-MA-NP and their polymer did not induce DNA strand breaks or oxidative damage at concentrations ranging from 7.4 to 600 mu g/mL. Besides, the mutagenic potential of these nanoparticles and their polymer revealed no significant or biologically relevant gene mutation induction at concentrations up to 600 mu g/mL under our experimental settings. Considering the non-genotoxic effects of GN-NP and GN-MA-NP, as well as their exceptional properties, these nanoparticles are promising nanocarriers for oral medical administrations. (C) 2017 Elsevier B.V. All rights reserved.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2017
Vol.:
106
Págs.:
477 - 486
In the last years, casein nanoparticles have been proposed as carriers for the oral delivery of biologically active compounds. However, till now, no information about their possible specific hazards in vivo was available. The aim of this work was to assess the safety of casein nanoparticles when administered orally to animals through a 90 days dose-repeated toxicity study (OECD guideline 408), that was performed in Wistar rats under GLP conditions. After 90 days, no evidences of significant alterations in animals treated daily with 50,150 or 500 mg/kg bw of nanoparticles were found. This safety agrees well with the fact that nanoparticles were not absorbed and remained within the gut as observed by radiolabelling in the biodistribution study. After 28 days, there was a generalized hyperchloremia in males and females treated with the highest dose of 500 mg/kg bw, that was coupled with hypernatremia in the females. These effects were related to the presence of mannitol which was used as excipient in the formulation of casein nanoparticles. According to these results, the No Observed Adverse Effect Level (NOAEL) could be established in 150 mg/kg bw/day and the Lowest Observed Effect Level (LOEL) could be established in 500 mg/kg bw/day. (C) 2017 Published by Elsevier Ltd.
Autores:
Marín de Mas, I.; Marín, S.; Pachón, G.; et al.
Revista:
FRONTIERS IN MOLECULAR BIOSCIENCES
ISSN:
2296-889X
Revista:
INTERNATIONAL JOURNAL FOR PARASITOLOGY, DRUGS AND DRUG RESISTANCE
ISSN:
2211-3207
Año:
2016
Vol.:
6
N°:
3
Págs.:
184 - 198
Synthesis of new 1-aryl-3-substituted propanol derivatives followed by structure-activity relationship, in silico drug-likeness, cytotoxicity, genotoxicity, in silico metabolism, in silico pharmacophore modeling, and in vivo studies led to the identification of compounds 22 and 23 with significant in vitro anti-plasmodial activity against drug sensitive (D6 IC50 <= 0.19 mu M) and multidrug resistant (FCR-3 IC50 <= 0.40 mu M and C235 IC50 <= 0.28 mu M) strains of Plasmodium falciparum. Adequate selectivity index and absence of genotoxicity was also observed. Notably, compound 22 displays excellent parasitemia reduction (98 +/- 1%), and complete cure with all treated mice surviving through the entire period with no signs of toxicity. One important factor is the agreement between in vitro potency and in vivo studies. Target exploration was performed; this chemotype series exhibits an alternative antimalarial mechanism. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2016
Vol.:
98
Págs.:
169 - 178
Ochratoxin A (OTA) is a mycotoxin that contaminates foodstuffs. The most relevant concern is its high kidney carcinogenicity in male rats and its unclear mechanism of action. It has been hypothesized that variations in transport mechanisms in kidney cells may be the reason of different sex-dependent sensitivities towards OTA. The aim of this study was to analyze, by RT-qPCR, renal transporters expression in 15-week-old male (M) and female (F) F344 rats at basal level and after single oral OTA administration (0.50 mg/kg bw). Temporal profiles (24h, 48h, 72h, 96h, 1 and 2 months) were studied per sex and transporter. The reference gene for all comparisons was Ppia. At basal level, sex differences were confirmed for Oatp1, Bcrp (M>F) and Oat2 (F>M). OTA tended to inhibit the expression of almost all transporters in both sexes, but clearly induced the expression of Oat2 in males. Regarding time profiles, the highest sex differences involved Oat (Slc22) transporters: Oat2, Oat3 and Oat5 expression showed a significant increase in males (24h) while Oat1, Oat2 and Oat5 level decreased in females (48h). Overall, basal sex differences in F344 rats and the specific sex-dependent response to OTA of Oat2 might contribute to high kidney damage in male rats. (C) 2016 Elsevier Ltd. All rights reserved.
Revista:
JOURNAL OF MEDICINAL CHEMISTRY
ISSN:
0022-2623
Año:
2016
Vol.:
59
N°:
24
Págs.:
10929 - 10945
Chagas disease is a neglected tropical disease with 6-7 million people infected worldwide, and there is no effective treatment. Therefore, there is an urgent need to continue researching in order to discover novel therapeutic alternatives. We present a series of arylaminoketone derivatives as means of identifying new drugs to treat Chagas disease in the acute phase with greater activity, less toxicity, and a larger spectrum of action than that corresponding to the reference drug benznidazole. Indexes of high selectivity found in vitro formed the basis for later in vivo assays in BALB/c mice. Murine model results show that compounds 3, 4, 7, and 10 induced a remarkable decrease in parasitemia levels in acute phase and the parasitemia reactivation following immunosuppression, and curative rates were higher than with benznidazole. These high antiparasitic activities encourage us to propose these compounds as promising molecules for developing an easy to synthesize anti-Chagas agent.
Revista:
JOURNAL OF ALZHEIMERS DISEASE
ISSN:
1387-2877
Año:
2016
Vol.:
54
N°:
3
Págs.:
1085 - 1094
Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by the presence of aggregates of the amyloid-ß peptide (Aß) that are believed to be neurotoxic. One of the purposed damaging mechanisms of Aß is oxidative insult, which eventually could damage the cellular genome. Stress and associated increases in glucocorticoids (GCs) have been described as a risk factor for the development of AD, although the purported genotoxic effects of GCs have not been fully characterized. Therefore, it is possible to speculate about purported synergistic effects of GCs on the Aß-driven genotoxic damage. This in vitro study addresses the single and combined cyto/genotoxic effects of Aß and GCs in SH-SY5Y cells. Cytotoxicity was determined by the MTT assay, and the genotoxic effects were studied using the comet assay. A comet assay derivation allows for measuring the presence of the FPG-sensitive sites (mainly 8-oxoguanines) in the DNA, apart from the DNA strand breaks. Treatment with Aß (10 ¿M, 72¿h) induced cytotoxicity (35% decrease in cell viability) and DNA strand breaks, but had no significant effect on oxidative DNA damage (FPG sites). Corticosterone showed no effect on cell viability, genotoxicity, or reparation processes. Corticosterone was unable to neither reverse nor potentiate Aß driven effects. The present results suggest the existence of alternative mechanisms for the Aß driven damage, not involving oxidative damage of DNA. In addition, could be suggested that the interaction between Aß and GCs in AD does not seem to involve DNA damage.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2015
Vol.:
30
N°:
1
Págs.:
139 - 146
Nutrient excess and unbalanced diets can result in overproduction of reactive oxygen species (ROS), which are associated with oxidative stress. Cocoa extract contains antioxidants that inhibit the harmful effects of ROS. This trial analysed the effect of cocoa extract consumption integrated as a bioactive compound into ready-to-eat meals, on oxidative stress at the level of DNA in overweight/obese subjects. Fifty volunteers [57.26(5.24) years, 30.59(2.33)kg/m(2)] participated in a 4-week double-blind, randomised, placebo-controlled parallel nutritional intervention. Half of the volunteers received meals supplemented with 1.4 g/day cocoa extract, while the other half received control meals, both within a 15% energy restriction diet. Lymphocytes were isolated and endogenous strand breaks, oxidised bases and resistance to H2O2-induced damage were measured by the comet assay. The intake of ready-to-eat meals supplemented with cocoa extract did not show relevant changes in the oxidative status of DNA. However, in the cocoa group, oxidised bases negatively correlated with methyl epicatechin-O-sulphate (r = -0.76; P = -0.007) and epicatechin sulphate (r = -0.61; P = -0.046). When volunteers of both groups were analysed together, a marginal decrease (P = 0.072) in oxidised bases was observed, which attributed to weight loss. Subjects who started the intervention with higher levels of damage showed a greater reduction in oxidised bases after 4 weeks (P = 0.040) compared to those who had lower baseline levels. In conclusion, even if 1.4 g of cocoa supplementation for 4 weeks did not show notable changes in terms of antioxidant status of DNA, the energy restriction showed a slightly decrease in oxidised bases and this was seen to a greater extent in subjects who started the intervention with higher levels of damage. On the other hand, the inverse associations found between oxidised bases and some cocoa-derived metabolites suggest that a protective effect might be seen in a longer period of time or in subjects with higher baseline DNA damage.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2015
Vol.:
30
N°:
1
Págs.:
21 - 28
The alkaline comet assay is now the method of choice for measuring different kinds of DNA damage in cells. Several attempts have been made to identify and evaluate the critical points affecting the comet assay outcome, highlighting the requirement of arriving at a standardised protocol in order to be able to compare the results obtained in different laboratories. However, reports on the effect of modifying the time of lysis are lacking. Here we tested different times of lysis (from no lysis to 1 week) in control HeLa cells and HeLa cells treated with different concentrations of methyl methanesulfonate (MMS) or H2O2. We also tested different times of lysis in the comet assay combined with formamidopyrimidine DNA glycosylase (FPG) in untreated and Ro 19-8022 plus light-treated HeLa cells. The same DNA damage levels were detected in the absence of lysis or after 1h of lysis when the standard comet assay was used to detect the MMS- and H2O2-induced lesions; the response increased when longer lysis was used, up to at least 1 week. When FPG was used, a minimum lysis period of 5 min was necessary to allow the enzyme to reach the DNA; the same DNA damage levels were detected after 5 min or 1h of lysis and the response increased up to 24h. In conclusion, the time of lysis can be varied depending on the sensitivity needed in both versions of the assay, and a constant time of lysis should be used if results from different experiments or laboratories are to be compared.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2015
Vol.:
76
Págs.:
116 - 124
Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are genotoxic mycotoxins that can contaminate a variety of foodstuffs, the liver and the kidney being their target organs, respectively. The micronucleus (MN) assay (bone marrow) and the comet assay (liver and kidney) were performed simultaneously in F344 rats, treated with AFB1 (0.25 mg/kg b.w.), OTA (0.5 mg/kg b.w.) or both mycotoxins. After AFB1 treatment, histopathology and biochemistry analysis showed liver necrosis, focal inflammation and an increase in Alanine Aminotransferase and Aspartate Aminotransferase. OTA alone did not cause any alteration. The acute hepatotmdc effects caused by AFB1 were less pronounced in animals treated with both mycotoxins. With regard to the MN assay, after 24 h, positive results were obtained for AFB1 and negative results were obtained for OTA, although both toxins caused bone marrow toxicity. In the combined treatment, OTA reduced the toxicity and the number of MN produced by AFB1. In the comet assay, after 3 h, positive results were obtained for AFB1 in the liver and for OTA in the kidney. The combined treatment reduced DNA damage in the liver and had no influence in the kidney. Altogether, these results may be indicative of an antagonistic relationship regarding the genotoxicity of both mycotoxins
Revista:
INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION
ISSN:
0963-7486
Año:
2015
Vol.:
66
N°:
Supl. 1
Págs.:
S13 - S21
The growing presence of products on the market with added value in terms of health makes essential their regulation and harmonization in critical aspects such as safety. The toxicology applied to the bioactive compounds should demonstrate the absence of toxic effects at doses advised for consumption, as well as evaluate the potential toxic effects in the assumption that the products are used in quantities superior to those recommended. The specific strategy should be defined case by case; therefore, prior to any toxicological development, it is essential to study all the information regarding the bioactive compounds (BACs) characterization, nutridynamics and nutrikinetics, that is available. In this guideline, a general strategy to be applied in the development of BACs is proposed. It includes a first in vitro phase to discard genotoxicity and endocrine effects and a second in vivo phase with different possibilities regarding the duration and the extension of the studies.
Autores:
Godschalk, R. W.; Ersson, C.; Stepnik, M.; et al.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2014
Vol.:
29
N°:
4
Págs.:
241 - 249
This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).
Autores:
Ersson, C.; Moller, P.; Forchhammer, L.; et al.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2013
Vol.:
28
N°:
3
Págs.:
279 - 286
The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found doseresponse relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.
Revista:
JOURNAL OF BIOMEDICAL NANOTECHNOLOGY
ISSN:
1550-7033
Año:
2013
Vol.:
9
N°:
11
Págs.:
1891 - 1903
The use of bioadhesive polymers as nanodevices has emerged as a promising strategy for oral delivery of therapeutics. In this regard, poly(anhydride) nanoparticles have shown great potential for oral drug delivery and vaccine purposes. However, despite extensive research into the biomedical and pharmaceutical applications of poly(anhydride) nanoparticles, there are no studies to evaluate the interaction of these nanoparticles at a cellular level. Therefore, the main objectives of this study were to evaluate the cytotoxicity as well as the cell interaction of different poly(anhydride) nanoparticles: conventional (NP), nanoparticles containing 2-hydroxypropyl-beta-cyclodextrin (NP-HPCD) and nanoparticles coated with poly(ethylene glycol) 6000 (PEG-NP). For this purpose, nanoparticles were prepared by solvent displacement method and labelled with BSA-FITC. Nanoparticles displayed a size about 175 nm with negative surface charge. Cytotoxicity studies were developed by MTS and LDH assays in HepG2 and Caco-2 cells. Results showed that only in HepG2 cells, NP and NP-HPCD induced significant cytotoxicity at the highest concentrations (1 and 2 mg/mL) and incubation times (48 and 72 h) tested. Studies to discriminate between cytoadhesion and cytoinvasion were performed at 4 degrees C and 37 degrees C in Caco-2 cell line as intestinal cell model. Nanoparticles showed cytoadhesion to the cell surface but not internalization; PEG-NP was the most bioadhesive followed by NP-HPCD and NP as demonstrated by flow cytometry. Finally, cellular localization of particles by fluorescence confocal microscopy confirmed the association of these nanoparticles with cells. Thus, this study demonstrated the safety of NP, NP-HPCD and PEG-NP at cellular level and its bioadhesive properties within cells.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2013
Vol.:
28
N°:
3
Págs.:
271 - 277
The alkaline comet assay, when employed as a genotoxicity test, has relatively low sensitivity because it fails to detectuat non-cytotoxic concentrationsuknown genotoxins that do not induce breaks or alkali-labile sites. We demonstrate that this limitation is overcome by incorporating in the assay the DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) to convert damaged bases to breaks. We tested 11 chemicals in human TK-6 cells: three non-cytotoxicud-mannitol, Tris and EDTA; two cytotoxicuTriton X-100 and fluometuron; and six genotoxicumethylmethanesulphonate (MMS), methylnitrosourea (MNU), cyclophosphamide, benzo(a)pyrene, 4-nitroquinoline-1-oxide (4NQO) and etoposide. At concentrations of MMS, MNU, benzo(a)pyrene or 4NQO causing little or no cytotoxicity and few if any DNA breaks, FPG substantially enhanced the cellular response. Etoposide increased breaks but not FPG-sensitive sites. Cyclophosphamide, a DNA cross linker, gave a response without FPG at 1 M, but there was no increase with FPG. Triton X-100-induced breaks were secondary to cytotoxicity. The remaining compounds induced no damage. Thus, FPG enhances sensitivity of the comet assay without compromising selectivity.
Autores:
Forchhammer, L.; Ersson, E.; Loft, S.; et al.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2013
Vol.:
28
N°:
2
Págs.:
241
Correction to Forchhammer et al. 27 (6): 665
Revista:
FOOD CONTROL
ISSN:
0956-7135
Año:
2013
Vol.:
32
N°:
1
Págs.:
63 - 68
The co-occurrence of ochratoxin A (OTA) and its five analogs (OTB, OTC, MeOTA, MeOTB and EtOTB) in 96 red wine samples from Mediterranean countries has been demonstrated, for the first time, in this study.
OTA was detected in 99% of the samples (<LOD - 455 ng L-1). This mycotoxin appeared simultaneously with OTB (2.05-119 ng L-1) in all the samples and in 89.6% of them OTC (<LOD - 31.5 ng L-1) also accompanied both. OTB appears at comparable levels and incidence just like OTA does, and OTC median concentration is approximately 10% of that of OTA. A high statistical association was found between the concentrations of OTA OTB and OTA OTC. MeOTA, MeOTB and EtOTB were detected in 62.5, 83.3 and 83.3% of the samples, respectively. In 44.8% of the wines, the 6 ochratoxins appeared simultaneously.
There was no evidence for ochratoxin A levels being greater in wines from Southern Europe than those described from North Europe. Samples from North Africa presented statistically the highest values for OTA, OTB, OTC and EtOTB.
Autores:
Gonda, M.; Marcos, N.; Nunes, E.; et al.
Revista:
MEDCHEMCOMM
ISSN:
2040-2503
Año:
2013
Vol.:
4
N°:
3
Págs.:
595 - 607
Phenazine-5,10-dioxides have been identified as prodrugs for antitumour therapy that undergo hypoxic-selective bioreduction, in the solid tumour cells, to form cytotoxic species. We investigated structural modifications of the phenazine-5,10-dioxide scaffold attempting to find new selective hypoxic cytotoxins with additional ability to inhibit DNA topoisomerase II. Four series of new phenazine-5,10-dioxides aryl-substituted connected by different linkers were prepared. The clonogenic survivals of V79 cells on aerobic and anaerobic conditions were determined, and studies of oxic DNA-interaction and hypoxic DNA topoisomerase II-inhibition, for the most relevant derivatives, were performed. Four new hypoxic-selective cytotoxins were identified at the assayed doses. In some of them were operative the DNA-interaction and/or the inhibition of DNA topoisomerase II. For one of the unselective cytotoxin biotransformation studies were performed on aerobic and anaerobic conditions, explaining the lack of selectivity.
Revista:
BIOANALYSIS
ISSN:
1757-6180
Año:
2013
Vol.:
5
N°:
3
Págs.:
289 - 305
Background: IFN-alpha 5 has been demonstrated to induce stronger signaling and higher expression of antiviral genes than IFN-alpha 2, which is the current treatment in chronic viral hepatitis. However, there is no specific and validated quantification method in order to conduct kinetic studies as part of the preclinical and clinical evaluation for regulatory purposes. Results: A novel integration of an antiviral assay against the cytopathic effect of the encephalomyocarditis virus in HeLa cells with a very sensitive method for assay processing - the Vialight (R) Plus assay - is presented for IFN-alpha 5 activity quantification. The bioassay has been validated in macaque and human serum and it has been demonstrated to be selective, precise and accurate. Conclusion: The validated bioassay meets suitable acceptance criteria for these types of biological assays.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2012
Vol.:
50
N°:
3-4
Págs.:
989-995
Polyphenols are characterized by the presence of phenol units in the molecules. These compounds may show antioxidant ability by scavenging reactive oxygen species (ROS) of the free radical type. A polyphenol enriched cocoa extract (PECE) was obtained from cocoa seeds with 28% of procyanidins which were mainly epicatechin oligomers. PECE was very active as free radical scavenger against 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl (HNTTM) radicals; and the tris(2,3,5,6-tetrachloro-4-nitrophenyl)methyl (TNPTM) assay showed that the PECE might not be pro-oxidant. Thus it was considered a good candidate to be tested in in vitro models. It showed mild cytotoxic power on Hep G2 cells and induced ROS in a dose-dependent manner being weak oxidant only at high concentrations near the limit of solubility. The antioxidant properties were assayed in Hep G2 treated with the mycotoxins ochratoxin A (OTA) and/or aflatoxin B1 (AFB1). The PECE was not effective against AFB1 but it increased the cell viability and reduced significantly the amounts of ROS in cells treated with OTA or mixtures of AFB1 + OTA. These results are coherent with the role of oxidative pathways in the mechanism of OTA and indicate that polyphenols extracted from cocoa may be good candidates as antioxidant agents. (C) 2011 Elsevier Ltd. All rights reserved.
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2012
Vol.:
50
N°:
10
Págs.:
3440 - 3446
Humans are exposed to the hepatotoxic aflatoxin B1 (AFB1) and nephrotoxic ochratoxin A (OTA) through diet. However, kinetic and toxicological data after their co-administration are scarce. In this study, a single oral dose of AFB1 (0.25 mg/kg bw) + OTA (0.5 mg/kg bw) was administered to fasted F344 rats. Blood, liver and kidney were harvested at different timepoints for mycotoxins quantification, relative weight calculation, clinical biochemistry and histopathology analysis. Toxicity parameters pointed to acute toxicity in liver due to AFB1. No remarkable toxicity was observed in kidneys or immunological organs. Maximum observed concentrations in plasma (C-max) were at 10 min and 2 h for AFB1 and OTA, respectively. AFB1 plasma concentration could indicate a rapid absorption/metabolism of the mycotoxin; and AFB1 liver and kidney concentrations were lower than LOQ and LOD, respectively. For OTA, C-max was 4326.2 mu g/L in plasma. In kidney and liver C-max was reached at 8 h and concentrations were very similar between both organs at all timepoints. Due to the low levels of AFB1, the effect of OTA on AFB1 kinetics could not be assessed. However, AFB1 seems not to affect OTA kinetics, as its profile seems very similar to kinetic studies performed only with OTA in similar conditions. (C) 2012 Elsevier Ltd. All rights reserved.
Revista:
FOOD CHEMISTRY
ISSN:
0308-8146
Año:
2012
Vol.:
132
N°:
1
Págs.:
35-42
One-hundred and twenty-three barley samples from a region of Spain (Navarra) were analysed in order to evaluate the possible co-occurrence of aflatoxins (AFB1, AFG1, AFB2 and AFG2), ochratoxin A (OTA) and zearalenone (ZEA). The results indicated that 80% of the samples presented detectable, although very low levels, of two or more mycotoxins. The most frequent combinations were AFB1 and OTA; AFB1, ZEA and OTA; and AFB1 and ZEA. In general, the statistical study did not show significant differences between levels or incidence for the mycotoxins in different years of harvest, variety of barley, farming or origin. The calculated values for daily intake were low and the risk to consumers could be assumed to be very low. However, the co-occurrence of several mycotoxins, and therefore synergic or additive effects, should be taken into account when determining permitted levels or risk assessment. (C) 2011 Elsevier Ltd. All rights reserved.
Revista:
FOOD CONTROL
ISSN:
0956-7135
Año:
2012
Vol.:
28
N°:
2
Págs.:
295-298
In this paper, a statistical overview of the simultaneous presence of 14 mycotoxins in Spanish barley is presented. The co-occurrence of more than two mycotoxins has been observed in 95% of the samples. From a descriptive standpoint, the results show that the most common combinations were AFB1, OTA and DON (29%), and AFB1, OTA, DON and ZEA (26%), although other combinations could also occur. However, the statistical study determined that other associations had a higher impact or importance. Four factors or combinations of variables could explain the 63% of variability found for the samples. These factors were the associations of: type-B trichothecenes and DAS; type-A trichothecenes and NIV; AFB1, AFG1 and ZEA; and AFB2 and AFG2. The toxin-producing fungi and environmental conditions have been essential in explaining the associations between the toxins. (C) 2012 Elsevier Ltd. All rights reserved.
Revista:
FOOD CONTROL
ISSN:
0956-7135
Año:
2012
Vol.:
25
N°:
1
Págs.:
81-88
In this survey, 123 barley samples from a northern region of Spain (Navarra) have been analyzed for co-occurrence of eight type-A and type-B trichothecenes (DON, NIV, 3-ADON, 15-ADON, FUS-X, T-2, HT-2 and DAS). Samples were classified according to place and year of harvest (2007 and 2008), type of farming (organic or traditional) and variety of barley. The rains during anthesis had a great influence on the trichothecene levels, observing higher contaminations in samples collected during 2008. In addition, type-A trichothecenes tend to be more present in cooler areas, while type-B appears more often in warmer regions. The type of farming has not led to significant differences in mycotoxins levels, although a trend toward higher incidence and contamination in traditional samples has been observed. On the other hand, it was observed that Pewter, the favorite barley variety for the malting industry in Spain, has been the most susceptible to contamination with trichothecenes. (c) 2011 Elsevier Ltd. All rights reserved.
Autores:
Forchhammer, L.; Ersson, C.; Loft, S.; et al.
Revista:
MUTAGENESIS
ISSN:
0267-8357
Año:
2012
Vol.:
27
N°:
6
Págs.:
665 - 672
There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.
Revista:
FOOD CONTROL
ISSN:
0956-7135
Año:
2012
Vol.:
27
N°:
1
Págs.:
139 - 145
Ochratoxin A (OTA), B (OTB) and their methyl (MeOTA, MeOTB) and ethyl (OTC, EtOTB) esters were evaluated in 51 red wine samples from Navarra (Spain). Detectable levels of OTA and OTB were found in 100% of the samples, and 71% showed the presence of OTC. The six ochratoxins appeared simultaneously in 18% of the samples. Results indicated that OTC is hydrolyzed to OTA in red wine. Therefore, ochratoxin intake from wine can be underestimated when only assessed by OTA analysis. Analyzed Navarra wines are scarcely contaminated with ochratoxins and their contribution to human intake is low, with the worst case being 4.7% and 6.6% of the provisional tolerable weekly intake (PTWI) for OTA and for the sum of ochratoxins, respectively. No significant differences were generally found between vintages. With the exception of OTA, no significant differences were observed between organic and traditional farming. Levels of ochratoxins were positively correlated with temperature and inversely correlated with humidity and rainfall.
Revista:
PHARMACEUTICAL RESEARCH
ISSN:
0724-8741
Año:
2012
Vol.:
29
N°:
9
Págs.:
2615 - 2627
To evaluate the acute and subacute toxicity of poly(anhydride) nanoparticles as carriers for oral drug/antigen delivery.
Three types of poly(anhydride) nanoparticles were assayed: conventional (NP), nanoparticles containing 2-hydroxypropyl-beta-cyclodextrin (NP-HPCD) and nanoparticles coated with poly(ethylene glycol) 6000 (PEG-NP). Nanoparticles were prepared by a desolvation method and characterized in terms of size, zeta potential and morphology. For in vivo oral studies, acute and sub-acute toxicity studies were performed in rats in accordance to the OECD 425 and 407 guidelines respectively. Finally, biodistribution studies were carried out after radiolabelling nanoparticles with (99m)technetium.
Nanoparticle formulations displayed a homogeneous size of about 180 nm and a negative zeta potential. The LD50 for all the nanoparticles tested was established to be higher than 2000 mg/kg bw. In the sub-chronic oral toxicity studies at two different doses (30 and 300 mg/kg bw), no evident signs of toxicity were found. Lastly, biodistribution studies demonstrated that these carriers remained in the gut with no evidences of particle translocation or distribution to other organs.
Poly(anhydride) nanoparticles (either conventional or modified with HPCD or PEG6000) showed no toxic effects, indicating that these carriers might be a safe strategy for oral delivery of therapeutics.
Autores:
Benítez, Diego; Cabrera, Mauricio; Hernández, Paola; et al.
Revista:
JOURNAL OF MEDICINAL CHEMISTRY
ISSN:
0022-2623
Año:
2011
Vol.:
54
N°:
10
Págs.:
3624 - 3636
Revista:
FOOD CONTROL
ISSN:
0956-7135
Año:
2011
Vol.:
22
N°:
12
Págs.:
1949 - 1955
Forty-six breakfast cereal samples from the Spanish market have been analyzed for the occurrence of aflatoxins (AFB1, AFG1, AFB2 and AFG2), ochratoxin A (OTA) and zearalenone (ZEA). According to the results, 9% of the samples were contaminated with AFB1 although no sample exceeded the LOQ (0.2 mu g kg(-1)), and no sample presented detectable levels of the other aflatoxins (AFB2, AFG1 and AFG2). Zearalenone and OTA contaminated 48 and 39% of the samples, respectively, with mean values of the samples having quantification levels of 25.40 and 0.37 mu g kg(-1), respectively. The co-occurrence of OTA and ZEA was observed in 28% of the samples. Aflatoxin B1 appeared only in the corn-based breakfast cereals, whereas ZEA and OTA showed the highest contamination rates in the samples containing wheat and wheat and rice, respectively. No sample of high-fiber content was contaminated with AFB1, whereas OTA and ZEA occurred with higher incidence in high-fiber content samples. Moreover, the daily exposure to AFB1, OTA and ZEA is discussed.
Autores:
Cabrera, Mauricio; López, Gloria V.; Gómez, Luis E.; et al.
Revista:
DRUG AND CHEMICAL TOXICOLOGY
ISSN:
0148-0545
Año:
2011
Vol.:
34
N°:
3
Págs.:
285 - 293
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2011
Vol.:
49
N°:
9
Págs.:
1935 - 1942
The impact of age and gender on Ochratoxin A (OTA) distribution in kidney and liver were studied. OTA was quantified in kidney and liver of young and mature rats of both sexes. Data was fit simultaneously using the population approach with NONMEM program. Fed and fasted mature males showed a 30% decrease and an 11% increase in relative bioavailability, respectively, in comparison with the rest of the groups. The OTA concentrations reached in kidney and liver were very similar between both organs. The models that best fit to data were the ones that considered that distribution of OTA to kidney and liver occurs from the central compartment and that elimination occurs mainly from the liver compartment. The kinetic analysis revealed that both, the apparent volume of distribution of the central compartment (V/F) and the apparent volume of distribution of the liver and kidney compartments (V(L,K)/F) increased significantly with body weight. Thus, the sex differences observed in organs distribution are a reflection of the differences in relative bioavailability observed in adult males, as a consequence of the fed and fasted conditions and to the significant higher body weight of mature males which directly affected the V/F and V(L,K)/F.
Revista:
ISRN PHARMACOLOGY
ISSN:
2090-5165
Año:
2011
Vol.:
2011
Págs.:
314209 -
Revista:
FOOD AND CHEMICAL TOXICOLOGY
ISSN:
0278-6915
Año:
2011
Vol.:
49
N°:
11
Págs.:
2883 - 2889
Mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) can be present together in food commodities. These food contaminants are considered to be genotoxins, acting by different mechanisms. The aim of this work was to characterize combined genotoxic in vitro effects of both mycotoxins in Hep G2 cells. For this purpose, cytotoxicity was first determined in isolated and combined treatments in order to determine the dose range of genotoxicity studies. Co-exposure of cells to OTA + AFB1 for 24 h resulted in additive effects. Genotoxicity was determined in Hep G2 cells by the modified comet assay with restriction enzymes (endo ill and FPG). Significant reactive oxygen species formation was detected in both single and combined treatments. AFB1 was genotoxic after 3 h with external metabolic activation (S9 mix) and after 24 h without metabolic activation. Co-exposure to OTA significantly decreased DNA damage induced by AFB1, not only in breaks and apurinic sites but also in FPG-sensitive sites. The apparent contradiction between additive cytotoxic effects and antagonic genotoxic effects may be explained if AFB1 and OTA compete for the same CYPs, yielding more ROS but less AFB1 adducts.
Revista:
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
ISSN:
1570-0232
Año:
2011
Vol.:
879
N°:
26
Págs.:
2733 - 2740
A rapid and simple method for the simultaneous quantification of AFB1 and OTA in rat plasma, liver and kidney by UHPLC-FLD has been successfully validated according to the following criteria: selectivity, stability, linearity, precision, accuracy, recovery, robustness and limits of quantification and detection. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (100 mu L) were extracted with acetonitrile:formic acid mixture (99:1) (300 mu L). Chromatographic separation was performed with a mixture of water and acetonitrile:methanol (50:50), both acidified with 0.5% of formic acid using a gradient profile. The method avoids the use of immunoaffinity columns and allows reduction of sample and solvent volumes as well as toxic wastes. The detection is based on a photochemical reaction which enhances the AFB1 response without affecting the OTA signal before reaching the fluorescent detector. The mycotoxin recovery for each matrix was very efficient, between 93% and 96% for AFB1 and between 94% and 96% for OTA. For both mycotoxins the LOQs were 2 mu g/L in plasma and 8 mu g/kg in liver and kidney. The method has successfully been applied to rat samples after a single oral administration of a mixture of AFB1 and OTA and it could be a useful tool in toxicokinetic and toxicological studies.
Revista:
Bioorganic & Medicinal Chemistry
ISSN:
0968-0896
Año:
2010
Vol.:
18
N°:
9
Págs.:
3125 - 3132
Revista:
Food and Chemical Toxicology
ISSN:
0278-6915
Año:
2010
Vol.:
48
N°:
11
Págs.:
3159 - 3166
Autores:
Cabrera, M.; Lavaggi, M. L.; Croce, F.; et al.
Revista:
Bioorganic & Medicinal Chemistry
ISSN:
0968-0896
Año:
2010
Vol.:
18
N°:
14
Págs.:
5391 - 5399
Revista:
TOXINS
ISSN:
2072-6651
Año:
2010
Vol.:
2
N°:
5
Págs.:
1054 - 1064
Revista:
JOURNAL OF DRUG DELIVERY SCIENCE AND TECHNOLOGY
ISSN:
1773-2247
Año:
2010
Vol.:
20
N°:
5
Págs.:
353 - 359
Autores:
Lavaggi, M. L.; Nieves, M.; Cabrera, M.; et al.
Revista:
European Journal of Medicinal Chemistry
ISSN:
0223-5234
Año:
2010
Vol.:
45
N°:
11
Págs.:
5362 - 5369
Autores:
Lavaggi, M. L.; Cabrera, M.; Aravena, M. A.; et al.
Revista:
Bioorganic & Medicinal Chemistry
ISSN:
0968-0896
Año:
2010
Vol.:
18
N°:
12
Págs.:
4433 - 4440
