A strategy for the investigation of toxic mechanisms and protection by efflux pumps using Schizosaccharomyces pombe strains: Application to rotenone.

Effects not related with the inhibition of complex I of the mitochondrial electron transport chain are studied in S. pombe, which lacks it. This study aims: First, the use of a strategy with S. pombe strains to investigate the toxicity, mechanisms of action, interactions and detoxication by efflux pumps. Second, to investigate the mechanisms of toxic action of rotenone. In the dose-response assessment, the yeast presented a good correlation with the toxicity in Daphnia magna for 15 chemicals. In the mechanistic study, the mph1Δ strain presented marked specificity to the interaction with microtubules by carbendazim. DNA damage caused by hydroxyurea, an inhibitor of deoxynucleotide synthesis, was identified with marked specificity with the rad3Δ strain. The sty1Δ strain was very sensitive to the oxidative and osmotic stress induced by hydrogen peroxide and potassium chloride, respectively, being more sensitive to oxidative stress than the pap1Δ strain. The protection by exclusion pumps was also evaluated. Rotenone presented low toxicity in S. pombe due to the lack of its main target, and the marked protection by the exclusion transporters Bfr1, Pmd1, Caf5 and Mfs1. Marked cellular stress was detected. Finally, the toxicity of rotenone could be potentiated by the fungicide carbendazim and the antimetabolite hydroxyurea. In conclusion, the use of S. pombe strains is a valid strategy to: a) assess global toxicity; b) investigate the main mechanisms of toxic action, particularly spindle and DNA interferences, and osmotic and oxidative stress not related to complex I inhibition; c) explore the detoxication by efflux pumps; and d) evaluate possible chemical interactions. Therefore, it should be useful for the investigation of adverse outcome pathways.

Gold(I) metallocyclophosphazenes with antibacterial potency and antitumor efficacy. Synergistic antibacterial action of a heterometallic gold and silver-cyclophosphazene.

One of the most important uses of phosphazenes today involves its biomedical applications. They can also be employed as scaffolds for the design and construction of a variety of ligands in order to coordinate them to metallic drugs. The coordination chemistry of the (amino)cyclotriphosphazene ligand, [N3P3(NHCy)6], towards gold(I) complexes has been studied. Neutral complexes, [N3P3(NHCy)6{AuX}n] (X = Cl or C6F5; n = 1 or 2) (1-4), cationic complexes, [N3P3(NHCy)6{Au(PR3)}n](NO3)n (PR3 = PPh3, PPh2Me, TPA; n = 1, 2 or 3) (6-12) [TPA = 1,3,5-triaza-7-phosphaadamantane] and a heterometallic compound [N3P3(NHCy)6{Au(PPh3)}2{Ag(PPh3)}](NO3)3 (13) have been obtained and characterized by various methods including single-crystal X-ray diffraction for 7, which confirms the coordination of gold atoms to the nitrogens of the phosphazene ring. Compounds 1, 4, 6-13 were screened for in vitro cytotoxic activity against two tumor human cell lines, MCF7 (breast adenocarcinoma) and HepG2 (hepatocellular carcinoma), and for antimicrobial activity against five bacterial species including Gram-positive, Gram-negative, and Mycobacteria. Both the median inhibitory concentration (IC50) and minimum inhibitory concentration (MIC) values are among the lowest found for any gold or silver derivatives against the cell lines and particularly against the Gram-positive (S. aureus) strain and the mycobacteria used in this work. Structure-activity relationships are discussed in order to determine the influence of ancillary ligands and the number and type of metal atoms (silver or gold). Compounds 4 and 8 showed not only maximal potency on human cells but also some tumour selectivity. Remarkably, compound 13, with both gold and silver atoms, showed outstanding activity against both Gram-positive and Gram-negative strains (nanomolar range), thus having a cooperative effect between gold and silver, with MIC values which are similar or lower than those of gentamicine, ciprofloxacin and rifampicine. The broad spectrum antimicrobial efficacy of all these metallophosphazenes and particularly of heterometallic compound 13 could be very useful to obtain materials for surfaces with antimicrobial properties that are increasingly in demand.

Potential Application of A Synthetic Organo-funtionalized High Load Expandable Mica as A Drug Carrier for Controlled Release.

BACKGROUND: In this work the cytotoxicity and gastric and gastrointestinal resistance of a high-load synthetic expandable mica, Na-mica-4, is studied for the first time. The hydrophilic character of this clay mineral can be modified by ion exchange reaction between Na+ inorganic cations housed in the interlayer space, and surfactant molecules, resulting in the formation of an organophilic material. This adsorption capability of organic compounds makes them very useful for a wide range of applications, such as their use as drug carriers. Previous studies have shown the high adsorption capacity of organofunctionalized Na-mica-4 of different types of drugs. Objetive: To carry out initial trials aimed at testing the cytotoxicity of a synthetic organofunctional expandable mica and evaluating its resistance to gastric and gastrointestinal digestion. METHODS: A highly charged sodium mica (Na-mica-4) was synthesized and organofunctional by cationic exchange with an alkylamine, primary amine of 18 carbon atoms (C18-mica-4). Both were characterized by X-ray diffraction, field transmission electron microscopy, surface-specific analysis, differential scanning calorimetry, and thermal gravimetric analysis. In addition, screening cytotoxicity trials were conducted on the human intestinal cell line Caco-2 with C18-mica-4 (0-125 µg/ml). RESULTS: Only one of the endpoints evaluated (the reduction of tetrazolium MTS salt by dehydrogenase enzymes) showed a significant decrease in cellular viability after 48h at the highest concentration tested. C18-mica-4 shows structural resistance to both, gastric and gastrointestinal, digestion. CONCLUSION: A successful development of a functionalized mica has been made with a promising potential application as a carrier to the drug.

Plastics in Cyanobacterial Blooms-Genotoxic Effects of Binary Mixtures of Cylindrospermopsin and Bisphenols in HepG2 Cells.

Ever-expanding environmental pollution is causing a rise in cyanobacterial blooms and the accumulation of plastics in water bodies. Consequently, exposure to mixtures of cyanotoxins and plastic-related contaminants such as bisphenols (BPs) is of increasing concern. The present study describes genotoxic effects induced by co-exposure to one of the emerging cyanotoxins-cylindrospermopsin (CYN)-(0.5 µg/mL) and BPs (bisphenol A (BPA), S (BPS), and F (BPF); (10 µg/mL)) in HepG2 cells after 24 and 72 h of exposure. The cytotoxicity was evaluated with an MTS assay and genotoxicity was assessed through the measurement of the induction of DNA double strand breaks (DSB) with the γH2AX assay. The deregulation of selected genes (xenobiotic metabolic enzyme genes, DNA damage, and oxidative response genes) was assessed using qPCR. The results showed a moderate reduction of cell viability and induction of DSBs after 72 h of exposure to the CYN/BPs mixtures and CYN alone. None of the BPs alone reduced cell viability or induced DSBs. No significant difference was observed between CYN and CYN/BPs exposed cells, except with CYN/BPA, where the antagonistic activity of BPA against CYN was indicated. The deregulation of some of the tested genes (CYP1A1, CDKN1A, GADD45A, and GCLC) was more pronounced after exposure to the CYN/BPs mixtures compared to single compounds, suggesting additive or synergistic action. The present study confirms the importance of co-exposure studies, as our results show pollutant mixtures to induce effects different from those confirmed for single compounds.

(Amino)cyclophosphazenes as Multisite Ligands for the Synthesis of Antitumoral and Antibacterial Silver(I) Complexes.

The reactivity of the multisite (amino)cyclotriphosphazene ligands, [N3P3(NHCy)6] and [N3P3(NHCy)3(NMe2)3], has been explored in order to obtain silver(I) metallophosphazene complexes. Two series of cationic silver(I) metallophosphazenes were obtained and characterized: [N3P3(NHCy)6{AgL}n](TfO)n [n = 2, L = PPh3 (2), PPh2Me (4); n = 3, L = PPh3 (3), PPh2Me (5), TPA (TPA = 1,3,5-triaza-7-phosphaadamantane, 6)] and nongem-trans-[N3P3(NHCy)3(NMe2)3{AgL}n](TfO)n [n = 2, L = PPh3 (7), PPh2Me (9); n = 3, L = PPh3 (8), PPh2Me (10)]. 5, 7, and 9 have also been characterized by single-crystal X-ray diffraction, thereby allowing key bonding information to be obtained. Compounds 2-6, 9, and 10 were screened for in vitro cytotoxic activity against two tumor human cell lines, MCF7 (breast adenocarcinoma) and HepG2 (hepatocellular carcinoma), and for antimicrobial activity against five bacterial species including Gram-positive, Gram-negative, and Mycobacteria strains. Both the IC50 and MIC values revealed excellent biological activity for these metal complexes, compared with their precursors and cisplatin and also AgNO3 and silver sulfadiazine, respectively. Both IC50 and MIC values are among the lowest values found for any silver derivatives against the cell lines and bacterial strains used in this work. The structure-activity relationships were clear. The most cytotoxic and antimicrobial derivatives were those with the triphenylphosphane and [N3P3(NHCy)6] ligands. A significant improvement in the activity was also observed upon a rise in the number of silver atoms linked to the phosphazene ring.

Investigation of mechanisms of toxicity and exclusion by transporters of the preservatives triclosan and propylparaben using batteries of Schizosaccharomyces pombe strains.

Triclosan (TCS) and propylparaben (PPB) are antimicrobials widely used. They present many similarities in their applications and also in their human and environmental health risks. In order to investigate the mechanisms of toxic action and the efflux pumps involved in their detoxication, we used a strategy with batteries of Schizosaccharomyces pombe yeast strains, either defective in cell signalling, in detoxification pumps, or in cell surveillance mechanisms. Yeast were exposed up to 20 h in solid medium or in liquid medium in 96-well plates. The mechanisms of action investigated were spindle defects (mph1), stress (pmk1), DNA interference (rad3) or diverse effects (MDR-sup). The efflux pumps investigated were Bfr1, Pmd1, Mfs1 and Caf5 or the Pap1 transcription factor. Here we show that TCS was 75 times more toxic than PPB in the wild type fission yeast. More oxidative stress and less protection by exclusion pumps were observed for TCS than for PPB. The cytotoxicity produced by TCS decreased from bfr1>mfs1>pmd1 > pap1 and caf5A deficient strains. In contrast, cytotoxic concentrations of PPB caused only a mild stress. The protection provided for PPB by the transporters was more marked than for TCS, decreasing from Pmd1, Caf5, Mfs1 and Bfr1. Furthermore, microtubule and DNA interferences were revealed for PPB, according to the cytotoxicity of mph1 and rad3 defective cells, respectively. As both compounds present complex adverse effects at concentrations close to exposure, and their combination clearly causes a strong potentiation, more exhaustive controls and regulations in their use should be considered.

Genotoxic activity of bisphenol A and its analogues bisphenol S, bisphenol F and bisphenol AF and their mixtures in human hepatocellular carcinoma (HepG2) cells.

The use of bisphenol A (BPA) in manufacturing of plastics is being gradually replaced by presumably safer analogues such as bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF). Despite their widespread occurrence in the environment, there is a knowledge gap in their toxicological profiles. We investigated cytotoxic/genotoxic effects as well as changes in the expression of selected genes involved in the xenobiotic metabolism, response to oxidative stress and DNA damage upon exposure to BPs and their mixtures in human hepatocellular carcinoma HepG2 cells. BPS and BPF slightly decreased the viability of HepG2 cells, while BPAF was the most cytotoxic compound tested. BPA, BPF and BPAF induced the formation of DNA double strand breaks determined with γH2AX assay, while BPS was inactive (5-20 µg/mL). All four BPs up-regulated the expression of CYP1A1 and UGT1A1, while BPS up-regulated and BPAF down-regulated also the expression of GST1A. Only BPA up-regulated oxidative stress responsive gene GCLC, while BPAF up-regulated the expression of CDKN1A and GADD45a. At concentrations relevant for human exposure (ng/mL range) BPA and its analogues as individual compounds and in mixtures did not exert genotoxic activity, whereas BPA and BPAF as well as the mixtures up-regulated the expressions of CYP1A1 and UGT1A1.

Integration of fish cell cultures in the toxicological assessment of effluents.

The pollution by industrial and municipal effluents are major sources of concerns. Fish cell cultures were applied in different strategies of the evaluation of effluents, particularly whole toxicity, toxicity identification evaluation and mode of action studies based in adverse outcome pathways. Whole effluent toxicity was evaluated using a battery of five model systems from four trophic levels: Daphnia magna was the most sensitive system, followed by the hepatoma fish cell line PLHC-1, the bacterium Allivibrio fischeri, the fibroblastic fish cell line RTG-2 and the algae Chlorella vulgaris, detecting a risk of eutrofization. The uptake of neutral red was more sensitive than the content of protein assay. The main morphological alterations observed were cell loss, hydropic degeneration, and a general loss of lysosomes and of their perinuclear distribution. The toxicity was characterized in PLHC-1 cells through toxicity identification evaluation, in which a partial reduction with graduation at pH 11, filtration, aeration and addition of thiosulfate or EDTA was shown; on the other hand, a low sorption in solid phase extraction suggested that the main responsible were not organic compounds. Consequently, it was not necessary to apply an effect directed analysis HPLC fractionation. In the chemical identification phase, Zn, Cd, As, Cu and Pb were quantified in decreasing concentrations. In the toxicity confirmation phase, a reconstituted sample and individual solutions, presented decreasing toxicity: Zn > Pb > As+5 > Cd > Cu > As+3, the global toxicity being explained by response addition. In the last step, the mode of action was investigated using five specific biomarkers. While metallothionein and succinate dehydrogenase activity were increased, no changes occurred for lysosomal function, acetylcholinesterase and EROD activities, the responsibility of the toxicity for the elements found being confirmed.

Mutagenic and genotoxic potential of pure Cylindrospermopsin by a battery of in vitro tests.

Cylindrospermopsin (CYN) is a cyanobacterial toxin with an increasing world-wide occurrence. The main route of human exposure is through the ingestion of contaminated food and water. The European Food Safety Authority has identified the need to further characterize the toxicological profile of cyanotoxins and in this regard the genotoxicity is a key toxicological effect. The data available in the scientific literature show contradictory results. Thus, the aim of this study was to investigate the mutagenic and genotoxic effects of pure CYN using a battery of different in vitro assays including: the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test) (0-10 µg/mL), the mammalian cell micronucleus (MN) test (0-1.35 µg/mL and 0-2 µg/mL in absence or presence of S9 fraction, respectively) and the mouse lymphoma thymidine-kinase assay (MLA)(0-0.675 µg/mL) on L5178YTk ±â€¯cells, and the standard and enzyme-modified comet assays (0-2.5 µg/mL) on Caco-2 cells. Positive results were obtained only when the metabolic fraction S9 was employed in the MN test, suggesting pro-genotoxic properties of CYN. Also, DNA damage was not mediated by oxidative stress as CYN did not induced changes in the modified comet assay. These data could contribute to a better risk assessment of this cyanotoxin.

Bioaccessibility and decomposition of cylindrospermopsin in vegetables matrices after the application of an in vitro digestion model.

Research on the human exposure to Cylindrospermopsin (CYN) via consumption of contaminated food is of great interest for risk assessment purposes. The aim of this work is to evaluate for the first time the CYN bioaccessibility in contaminated vegetables (uncooked lettuce and spinach, and boiled spinach) after an in vitro digestion model, including the salivar, gastric and duodenal phases and, colonic fermentation under lactic acid bacteria. The results obtained showed that the digestion processes are able to diminish CYN levels, mainly in the colonic phase, especially in combination with the boiling treatment, decreasing CYN levels in a significant way. Moreover, the potential decomposition products in a pure CYN solution and in CYN-contaminated vegetables were evaluated using UHPLC-MS/MS Orbitrap. Under the conditions assayed, only two diastereoisomers of the same fragment with m/z 292.09617 have been detected in all the analysed samples, with the exception of digested vegetables. Therefore, in terms of risk assessment, the digestion seems to play an important role in reducing the final bioaccesibility of CYN, and the consumption of cooked vegetables (spinach) would be safer in comparison to raw vegetables.

Use of micronucleus and comet assay to evaluate evaluate the genotoxicity of oregano essential oil (Origanum vulgare l. Virens) in rats orally exposed for 90 days.

Essential oils from Origanum spp. exhibit antioxidant and antimicrobial activities making them suitable for use as food additives. The incorporation of oregano essential oil in active food packaging is under study; however, it has been not authorized for this purpose thus far. In order to fulfill the requirements of the European Food Safety Authority (EFSA), the aim of the present study was to determine the genotoxic potential of oregano essential oil using both the micronucleus (MN) test and comet (standard and enzyme-modified) assays in Wistar rats treated with 50, 100, or 200 mg/kg body weight administered daily for 90 days. MN was performed in cells from the bone marrow and standard and enzyme-modified comet assays were conducted in stomach, liver and blood cells. The major compound detected in the analytical study of oregano essential oil from Origanum vulgare L. virens, was carvacrol (55.82%) followed by thymol (5.14%), as well as their precursors, γ-terpinene (16.39%), and ρ-cimne (4.71%). The results obtained in the genotoxicity assays indicated lack of effect in MN and standard comet assay under the conditions tested. Furthermore, no apparent oxidative damage was observed in the enzyme-modified comet assay in any of the tissues examined of rats exposed to oregano essential oil for 90 days. Therefore, this oregano essential oil appears to be safe in Wistar rats and might be considered as a potential active material in food packaging industry.

In vitro toxicity evaluation of new silane-modified clays and the migration extract from a derived polymer-clay nanocomposite intended to food packaging applications.

The clay montmorillonite (Mt) is among the nanofillers more frequently used for food packaging applications. The organomodification of clays with different modifiers, such as silanes, is an important step in the preparation of improved polymer/clay materials known as nanocomposites. However, the toxicological data about these nanofillers is still scarce. In the present study, an in vitro toxicological evaluation in Caco-2 cells of two silane-modified clays based on Mt, Clay3 and Clay4 (0-250µg/ml), was performed. The cytotoxicity, cell death, genotoxicity and oxidative stress produced by both organoclays were evaluated after 24 and 48h of exposure. Moreover, the migration extracts obtained from nanocomposites of polypropylene (PP) + Clay3 and only PP were also investigated. Only Clay4 induced cytotoxicity, showing a reduction of cell viability to 63% of the control, as well as oxidative stress in a concentration-dependent manner. Regarding the PP-Clay3 migration extract, no cytotoxic effects were observed after exposure to the tested concentrations (0-100%). Moreover, significant differences in the presence of Ca, Mg and Si compared to the PP extract were obtained, although migration levels were in accordance with the food contact materials regulation. These findings indicate that a case-by-case toxicological assessment of organoclays should be performed.

Bioaccesibility of Cylindrospermopsin from cooked fish muscle after the application of an in vitro digestion model and its bioavailability.

Humans can be exposed to cyanotoxins through the ingestion of contaminated water, food or beverages. In the present work, the bioaccesibility of Cylindrospermopsin (CYN), one of the most relevant cyanotoxins, was evaluated in a pure CYN solution and cooked CYN-contaminated fish muscles (20 µg/mL). An in vitro digestion model including the salivar, gastric, duodenal and colonic phases was performed, being each fraction analyzed by HPLC-MS-MS to evaluate CYN degradation. Moreover, Caco-2/TC7 cells were exposed to the digested duodenal and colonic phases to elucidate the final bioavailability of CYN in an approximation to the real human exposure scenario. The results revealed that CYN bioaccesibility decreased after the digestive process in all the cooked fish samples. The most drastic reductions were observed after lactic acid bacteria exposure. Thus, the highest bioaccesibility values were obtained in fish cooked by steaming (12.5%) and broiling (10.9%) meanwhile CYN was not detected in the colonic phase after boiling and microwaving. Regarding the duodenal and colonic availability, only in CYN pure digested solution the cyanotoxin was identified. The results obtained showed that digestion processes plays a very important role in the degradation of CYN, which should be considered when preparing a risk assessment of CYN.

Genotoxic potential of the binary mixture of cyanotoxins microcystin-LR and cylindrospermopsin.

Increased eutrophication of water bodies promotes cyanobacterial blooming that is hazardous due to the production of various bioactive compounds. Microcystin-LR (MCLR) is among the most widespread cyanotoxins classified as possible human carcinogen, while cylindrospermopsin (CYN) has only recently been recognized as health concern. Both cyanotoxins are genotoxic; however, the mechanisms of their action differ. They are ubiquitously present in water environment and are often detected together. Therefore, we studied genotoxic potential of the binary mixture of these cyanotoxins. Human hepatoma cells (HepG2) were exposed to a single dose of MCLR (1 µg/mL), graded doses of CYN (0.01-0.5 µg/mL), and their combinations. Comet and Cytokinesis block micronucleus assays were used to detect induction of DNA strand breaks (sb) and genomic instability, respectively, along with the transcriptional analyses of the expression of selected genes involved in xenobiotic metabolism, immediate/early cell response and DNA-damage response. MCLR induced DNA sb that were only transiently present after 4 h exposure, whereas CYN, after 24 h exposure, induced DNA sb and genomic instability. The MCLR/CYN mixture induced DNA sb after 24 h exposure, but to lesser extent as CYN alone. On the other hand, induction of genomic instability by the MCLR/CYN mixture was comparable to that induced by CYN alone. In addition, patterns of changes in the expression of selected genes induced by the MCLR/CYN mixture were not significantly different from those induced by CYN alone. Our results indicate that CYN exerts higher genotoxic potential than MCLR and that genotoxic potential of the MCLR/CYN mixture is comparable to that of CYN alone.

In vitro toxicological assessment of an organosulfur compound from Allium extract: Cytotoxicity, mutagenicity and genotoxicity studies.

Garlic (Allium sativum) and onion (Allium cepa) are being used in the food industry as flavoring but also for their antimicrobial activities. These activities are mainly derived from the organosulfur compounds (OSCs). Propyl propane thiosulfinate (PTS) is an OSC with potential use in the active packaging, but its safety should be guaranteed before being commercialized. The aim of this work was to investigate for the first time the cytotoxicity of PTS as well as its in vitro mutagenic/genotoxic potential using the following battery of genotoxicity tests:(1)the bacterial reverse-mutation assay in S. typhimurium (Ames test, OECD 471, 1997); (2) the micronucleus test (MN, OECD 487, 2016); (3) the mouse lymphoma thymidine-kinase assay (MLA, OECD 476, 2015), and (4) the comet assay (standard and modified with restriction enzymes). The results revealed that PTS was not mutagenic neither in the Ames test nor in MLA. However, genotoxic effects were recorded in the MN test on mammalian cells (L5178YTk+/-cells) after PTS exposure at the highest concentration tested (17.25 µM) without S9, and also its metabolites (+S9, from 20 µM). Moreover, in the comet assay, PTS induced DNA breaks damage in Caco-2 cells at the highest concentration tested (280 µM) but it did not induce oxidative DNA damage.

Genotoxicity evaluation of carvacrol in rats using a combined micronucleus and comet assay.

Genotoxic data of substances which could be incorporated into food packaging are required by the European Food Safety Authority. Due to its antioxidant and antibacterial properties carvacrol is one of these compounds. This work aims to study for the first time the in vivo genotoxic effects produced in rats orally exposed to 81, 256 or 810 mg cavacrol/kg body weight (bw) at 0, 24 and 45 h. A combination of the micronucleus assay (OECD 474) in bone marrow and the standard (OECD 489) and enzyme-modified comet assay was used to determine the genotoxicity on cells isolated from stomach and liver of exposed animals. In addition, a histopathological study was performed on the assayed tissues, and also in the lungs due to the volatility of carvacrol. Direct analytical pyrolysis was used to search for carvacrol in viscera and to ensure that the compound reaches stomach and liver cells. Results from MN-comet assay revealed that carvacrol (81-810 mg/kg bw) did not induce in vivo genotoxicity or oxidative DNA damage in any of the tissues investigated. Moreover, no histopathological changes were observed. Altogether, these results suggest lack of genotoxicity of carvacrol and therefore its good profile for its potential application as food preservative.

Induction of micronuclei and alteration of gene expression by an organomodified clay in HepG2 cells.

Clay2 is an organomodified montmorillonite developed by the Technological Institute of Packaging, Transport and Logistic (ITENE) in order to improve polymeric materials used in food packaging. There is not much known on Clay2 toxic potential, particularly at DNA level, therefore it is mandatory to assess its toxicity prior to its commercialization. In the present study the human hepatoma cell line (HepG2) was exposed to non-cytotoxic concentrations of Clay2 and the genomic stability was studied with the Cytokinesis block micronucleus cytome assay, by determining the formation of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs). Moreover, the expression of various genes involved in the mechanisms of its action using the real-time quantitative PCR was studied. The results obtained provide the evidence that Clay2 is potentially genotoxic as it increased the frequency of micronuclei. In addition it deregulated genes involved in the metabolism, immediate-early response/signaling, DNA damage and oxidative stress showing new valuable information on the cellular response to Clay2. Nonetheless, further studies are highly needed to elucidate the molecular mechanisms of clays toxicity.

Effects of two organomodified clays intended to food contact materials on the genomic instability and gene expression of hepatoma cells.

Globally, food industries have made significant progress in order to increase the shelf-life of food products and have fewer economic losses. In this sense, the use of organomodified clays destined to be incorporated in polymer matrices play a novel role, leading to improved materials named nanocomposites with enhanced technological profiles. Due to the presence of these clays into the package, the safety of the consumers is a main concern. Cloisite(®)30B and Clay1 are two organomodified clays containing quaternary ammonium salts as modifiers, that can be potentially used to reinforce packaging polymers. Available toxicity data about these clays, specifically genotoxicity, is still limited and inconclusive in some aspects. Thus, the purpose of this work was to evaluate both clays ability to induce genomic instability through the cytokinesis block micronucleus cytome assay (CBMN) and for the first time, their influence in the modulation of several genes involved in genotoxicity and cell death mechanisms. Overall, no genotoxicity response was obtained in any case at the conditions tested. On the other hand, significant expression changes were observed on the genes selected. Nevertheless, further studies are highly needed to elucidate and increase the knowledge about the molecular mechanisms of clays toxicity.

Genotoxic potential of montmorillonite clay mineral and alteration in the expression of genes involved in toxicity mechanisms in the human hepatoma cell line HepG2.

Montmorillonite, also known as Cloisite(®)Na(+) (CNa(+)), is a natural clay with a wide range of well-documented and novel applications, such as pharmaceutical products or food packaging. Although considered a low toxic product, the expected increased exposure to CNa(+) arises concern on the potential consequences on human and environmental health especially as its genotoxicity has scarcely been investigated so far. Thus, we investigated, for the first time, the influence of non-cytotoxic concentrations of CNa(+) (15.65, 31.25 and 62.5 µg/mL) on genomic instability of human hepatoma cell line (HepG2) by determining the formation of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) with the Cytokinesis block micronucleus cytome assay. Further on we studied the influence of CNa(+) on the expression of several genes involved in toxicity mechanisms using the real-time quantitative PCR. The results showed that CNa(+) increased the number of MNi, while the numbers of NBUDs and NPBs were not affected. In addition it deregulated genes in all the groups studied, mainly after longer time of exposure. These findings provide the evidence that CNa(+) is potentially genotoxic. Therefore further studies that will elucidate the molecular mechanisms involved in toxic activity of CNa(+) are needed for hazard identification and human safety assessment.

In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays.

Currently, antimicrobial additives derived from essential oils (Eos) extracted from plants or spices, such as Origanum vulgare, are used in food packaging. Thymol and carvacrol, the major EO compounds of O. vulgare, have demonstrated their potential use as active additives. These new applications use high concentrations, thereby increasing the concern regarding their toxicological profile and especially their genotoxic risk. The aim of this work was to investigate the potential in vitro genotoxicity of thymol (0-250 µM) and carvacrol (0-2500 µM) at equivalent doses to those used in food packaging. The micronucleus (MN) test and the mouse lymphoma (MLA) assay on L5178Y/Tk(±) mouse lymphoma cells were used. The negative results for thymol with the MN with and without the S9 fraction and also with the MLA assay reinforce the view that this compound is not genotoxic in mammalian cells. However, carvacrol presented slight genotoxic effects, but only in the MN test at the highest concentration assayed (700 µM) and in the absence of metabolic activation. The lack of genotoxic response in the MLA assay after 4 and 24h of exposure indicates a low genotoxic potential for carvacrol. Alternatively, the general negative findings observed in both assays suggest that the MN results of carvacrol are marginal data without biological relevance. These results can be useful to identify the appropriate concentrations of these substances to be used as additives in food packaging.