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Comparison of in vivo and in vitro activities of alcohol and non-alcohol beer
Abstracts / Toxicology Letters 238S (2015) S56–S383
long palmate alternate, deciduous, petiolate, subcordate which is rough on upper surface, finely wooly on beneath surface. In the transverse section of leaf lamina of shows upper epidermis which is single layered, cells more or less rectangular with outer walls, cuticularized. Both covering and glandular trichomes emerged from the upper epidermal cell. The upper and lower epidermal layers of lamina are continuous over the midrib. However, relatively more trichomes appear on the epidermal layers of the midrib. A patch of vascular bundle is present in the central portion of the midrib. Powder microscopy shows numerous mucilage granules, starch granules, covering, glandular trichomes and stomata which are anomocytic in nature. Stomatal number 6–10, stomatal index value 17.64, palisade ratio 8.12, vein islet number 86.1, and vein termination value 103.5 were determined by standard method. The present research work was undertaken with a view to lay down standards which could be useful to detect the authenticity of this medicinally useful plant. http://dx.doi.org/10.1016/j.toxlet.2015.08.229
P02-003 Binary and tertiary combinations of 3-ADON, 15-ADON and AOH mycotoxins on HepG2 cells: Evaluation of cytotoxic effects and detection of metabolite products A. Juan García ∗ , C. Juan, G. Font, M.-J. Ruiz Leal University of Valencia, Laboratory of Toxicology, Burjassot, Spain Mycotoxins are produced by a number of fungal genera spp as e.g. Aspergillus, Penicillium, Alternaria, Fusarium and Claviceps. Metabolites derived from these mycotoxins are supposing a hazard to animals and human health and it makes necessary to evaluate their effects. This work is focused in studying the cytotoxic effects on HepG2 cells of binary and tertiary combinations of the mycotoxins alternariol (AOH), 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) by the MTT assay, as well as in the identification of the degradation products and/or metabolites originated after treatment by liquid chromatography tandem mass spectrometry (LC–MS/MS) equipment and extracted from culture media. HepG2 cells were treated at different concentrations over 24, 48 and 72 h. IC50 values detected at all times assayed, ranged from 0.8 to >25 M in binary combinations; while in tertiary it ranged from 7.5 to 12 M. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index value as a quantitative measure of the degree of the three mycotoxin interaction. Synergistic, antagonism or additive effect detected in the mixtures of these mycotoxins was different depending on low or high concentration. Among all four mycotoxins combinations assayed, 15-ADON + 3-ADON at 24 h presented the highest toxic potential. Mass spectrometry (MS) scan chromatograms of studied mycotoxins allowed to detect products from: (i) the glutathione conjugate: (ii) sulfuric acid conjugated and (iii) amino group of cysteine conjugate. At all assayed times, recoveries values oscillated depending on the time and combination studied. Acknowledgement: This research has been supported by the Ministry of Science and Innovation (AGL2013-43194-P). http://dx.doi.org/10.1016/j.toxlet.2015.08.230
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P02-004 Biological effects of classic and diet soda drinks assessed in in vivo and in vitro models M. Mateo-Fernández ∗ , V. Moreno-Ariza, M. Balongo-Escobar, L. Luque-Bravo, Z.N. Fernández-Bedmar, M. Martínez-Jurado, S. Demyda-Peyrás, T. Merinas-Amo University of Cordoba, Genetics, Cordoba, Spain Consumption of sugar and diet beverages has increased rapidly during the last couple of decades, carbonated soft drinks having the fourth highest worldwide consumption. In this study we assessed the biological effects of Classic Coca-ColaTM (CCC), Diet Coca-ColaTM (DCC) and fructose syrup (FRU) at five different concentrations using in vitro and in vivo assays to determine their nutraceutical potential. Toxicity, antitoxicity, genotoxicity, antigenotoxicity and their effect on the life and healthspan were established using a Drosophila melanogaster model. In addition, cytotoxicity and DNA fragmentation were analysed using a HL-60 promyelocytic cell line as a hallmark of apoptosis. Our results showed that none of the compounds were toxic at the assayed concentrations. DCC was non-antitoxic at all the tested concentrations as it was not able to reduce the effects of hydrogen peroxide. On the contrary, antitoxic activity was characterised in FRU (100 mM) and in CCC with a negative dose effect. All beverages produced a non-genotoxic effect at all the tested concentrations. While CCC was highly antigenotoxic, this pattern was not observed in DCC. Conversely, the effect of FRU was only increased in combination with hydrogen peroxide. CCC, DCC and FRU increased life expectancy and quality in D. melanogaster at some of the assessed concentrations. Regarding the in vitro assays, CCC and FRU showed a cytotoxic effect in HL-60, as well as DCC although to a lesser extent. CCC and FRU, at high concentrations, induced a weak internucleosomal DNA fragmentation in HL-60 cells. In contrast, this effect was not observed in DCC. In conclusion, the lifespan increase and the lack of toxicity and genotoxicity in the in vivo model (D. melanogaster) results observed in the fructose assessment may mimic those obtained in Classic CocaColaTM complex mixture. Only the antimutagenic inactivity showed by fructose does not agree with the previous assumption. Finally, our results suggested that CCC and Fructose, at the assessed concentrations, could provide more beneficial activity than DCC in the HL-60 model. http://dx.doi.org/10.1016/j.toxlet.2015.08.231
P02-005 Comparison of in vivo and in vitro activities of alcohol and non-alcohol beer T. Merinas-Amo ∗ , M. Martínez-Jurado, R. Merinas-Amo, V. García-Zorrilla, Z.N. Fernández-Bedmar, A. Alonso-Moraga, M. Mateo-Fernández University of Córdoba, Genetics, Cordoba, Spain Much information about the nutritive profile of different types of beer is available on scientific data bases. We have compared blond, stout and non-alcohol Lager beer in order to know if they could be considered as neutraceutical beverages and if different biological activities could be observed among them. In vivo assays were carried out in the Drosophila melanogaster model with the aim of analysing the (i) toxi/antitoxic effects that beers could cause in the flies’ survival percentage; (ii) the geno/antigenotoxic potential that these drinks could provide in this organism by performing the
S66
Abstracts / Toxicology Letters 238S (2015) S56–S383
Somatic Mutation And Recombination Test (SMART); and (iii) the induction of longevity that these beers could exert. Moreover, in vitro assays were performed in HL-60 human leukaemia cell line testing the cytotoxicity and proapoptotic DNA internucleosomal fragmentation potency of these beers. The results showed that all beers exhibited neither toxic nor genotoxic effects, being blond beer healthier than the other ones. Besides they exerted genomic protective effects against hydrogen peroxide. As far as longevity is concerned, the average of lifespan was increased in 10, 7 and 3 days for blond, non-alcohol and stout Lager beer respectively, with respect to their concurrent controls. The in vitro assays showed that the IC50 values of non-alcohol and stout Lager beer were lower than the blond Lager beer value, being the most chemopreventive substances. Nevertheless, the blond beer was the only one that completely inhibited the cellular growth. In conclusion, our results suggest that blond Lager beer is the healthiest one because of the high capacity for protecting against genotoxic oxidative substances, for improving the lifespan extension in Drosophila and the ability to inhibit the growth of HL-60 promyelocytic cells that it exhibits. http://dx.doi.org/10.1016/j.toxlet.2015.08.232
P02-006 The effects of malathion on serum parameters in rat M. Ekremoglu ∗ , H. Pasaoglu, C. Severcan, B. Sen, O.T. Pasaoglu Gazi University, Medical Biochemistry, Ankara, Turkey It is an undeniable fact that malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. However, malathion has many side effects to human health. Our study was designed to evaluate the acute effects of malathion on sera parameters. For this purpose 4 groups were formed. Rats in Group 1 served as control group animals which were only given corn oil. Groups 2–4 were administered 100 mg/kg, 200 mg/kg and 400 mg/kg malathion, respectively, dissolved in corn oil by oral gavage. The rats were sacrificed after 24 h following administration. In order to assess the dose dependent, we studied serum lipase, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), Blood urea nitrogen (BUN), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), ␣-amylase, malondialdehyde (MDA), creatinine and glucose-6-phosphate dehydrogenase (G6PD). The use of malathion inhibited lipase activity. We did not observe lipase activity on Groups 2–4. There were no significant differences of BUN, LDH, MDA and creatinine levels among all groups. We found a significant increase of glucose level in Group 4, compared to Group 2. We found significant increase AST and CK levels of Groups 3 and 4 compared to Groups 1 and 2 and also found significant decrease CK level of Group 4 compared to Group 3. We found a significant increase of ALT level in Group 3, compared to Group 1. We found significant decline ALP levels of Groups 3 and 4 compared to Group 2. We found a significant increase of ␣-amylase level in Group 2, compared to Group 1. At the first, G6PD enzyme activity significantly decrease both of the Groups 2 and 3 and then significantly increase the Group 4 compared to Group 1. http://dx.doi.org/10.1016/j.toxlet.2015.08.233
P02-007 Use of (Q)SAR tools as a first step in a strategy to assign priority to substances migrating from printed paper and board food contact materials based on genotoxic potential M. Van Bossuyt 1,2,∗ , E. Van Hoeck 1 , G. Raitano 3 , N. Ost 4 , G. Ates 2 , G. Schüürman 4 , T. Vanhaecke 2 , E. Benfenati 3 , V. Rogiers 2 , B. Mertens 1 1 Scientific Institute of Public Health, Food, Medicines and Consumer Safety, Brussels, Belgium 2 Vrije Universiteit Brussel, In Vitro Toxicology and Dermato-Cosmetology, Brussels, Belgium 3 Instituto di Ricerche Farmacologiche Mario Negri, Environmental Health Sciences, Milan, Italy 4 Centre for Environmental Research – UFZ, Ecological Chemistry, Leipzig, Germany
In the last decade, several incidents with contaminants leaking from food packaging into food and drinks have raised concerns towards potential adverse health effects caused by exposure to these chemicals (e.g. bisphenol A). In 2011, the European Commission adopted a new regulation on the use of chemicals in plastics intended to come into contact with food. However, to this day, no EU regulation exists on chemicals used in other types of food contact materials (FCM). Consequently, non-plastic FCM are only covered by national legislation or by the general recommendations of the Council of Europe, containing ‘inventory lists’ of additives, monomers, solvents and other compounds that might be present in non-plastic FCM. Since no (recent) safety evaluation has been carried out for over 5000 substances present in these inventory lists, identification of substances requiring further safety data is urgently needed. The current study aims to develop a strategy to assign priority to substances used in printed paper and board. First, information on substances present in the inventory lists of the Council of Europe or from national legislation was assembled in a database. In a second step, their genotoxic potential was evaluated in silico using multiple (Q)SAR models (Toxtree, Derek NexusTM , VEGA Consensus model). An applicability domain assessment was performed to ensure fitting of the test compounds in the models applied. Based on the outcome of the in silico evaluation, substances containing a structural alert for genotoxicity were selected for further more in depth study, combining extensive literature review with in vitro genotoxicity testing. http://dx.doi.org/10.1016/j.toxlet.2015.08.234
P02-008 A short study of deoxynivalenol correlation in diet and urine Y. Rodríguez-Carrasco, G. Font, M.-J. Ruiz-Leal ∗ , B. Houda University of Valencia, Preventive Medicine, Toxicology, Burjassot-Valencia, Spain Cereals are the most important source of food but they are also frequently reported as contaminated by mycotoxins, a heterogeneous group of secondary metabolites of filamentous fungi. The correlation between the widespread contamination of food commodities and the human exposure to mycotoxins makes the monitoring of their levels in urine essential. In this 10-days pilot study the concentrations of deoxynivalenol (DON), one of the main Fusarium mycotoxins, in a survey conducted by one volunteer
long palmate alternate, deciduous, petiolate, subcordate which is rough on upper surface, finely wooly on beneath surface. In the transverse section of leaf lamina of shows upper epidermis which is single layered, cells more or less rectangular with outer walls, cuticularized. Both covering and glandular trichomes emerged from the upper epidermal cell. The upper and lower epidermal layers of lamina are continuous over the midrib. However, relatively more trichomes appear on the epidermal layers of the midrib. A patch of vascular bundle is present in the central portion of the midrib. Powder microscopy shows numerous mucilage granules, starch granules, covering, glandular trichomes and stomata which are anomocytic in nature. Stomatal number 6–10, stomatal index value 17.64, palisade ratio 8.12, vein islet number 86.1, and vein termination value 103.5 were determined by standard method. The present research work was undertaken with a view to lay down standards which could be useful to detect the authenticity of this medicinally useful plant. http://dx.doi.org/10.1016/j.toxlet.2015.08.229
P02-003 Binary and tertiary combinations of 3-ADON, 15-ADON and AOH mycotoxins on HepG2 cells: Evaluation of cytotoxic effects and detection of metabolite products A. Juan García ∗ , C. Juan, G. Font, M.-J. Ruiz Leal University of Valencia, Laboratory of Toxicology, Burjassot, Spain Mycotoxins are produced by a number of fungal genera spp as e.g. Aspergillus, Penicillium, Alternaria, Fusarium and Claviceps. Metabolites derived from these mycotoxins are supposing a hazard to animals and human health and it makes necessary to evaluate their effects. This work is focused in studying the cytotoxic effects on HepG2 cells of binary and tertiary combinations of the mycotoxins alternariol (AOH), 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) by the MTT assay, as well as in the identification of the degradation products and/or metabolites originated after treatment by liquid chromatography tandem mass spectrometry (LC–MS/MS) equipment and extracted from culture media. HepG2 cells were treated at different concentrations over 24, 48 and 72 h. IC50 values detected at all times assayed, ranged from 0.8 to >25 M in binary combinations; while in tertiary it ranged from 7.5 to 12 M. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index value as a quantitative measure of the degree of the three mycotoxin interaction. Synergistic, antagonism or additive effect detected in the mixtures of these mycotoxins was different depending on low or high concentration. Among all four mycotoxins combinations assayed, 15-ADON + 3-ADON at 24 h presented the highest toxic potential. Mass spectrometry (MS) scan chromatograms of studied mycotoxins allowed to detect products from: (i) the glutathione conjugate: (ii) sulfuric acid conjugated and (iii) amino group of cysteine conjugate. At all assayed times, recoveries values oscillated depending on the time and combination studied. Acknowledgement: This research has been supported by the Ministry of Science and Innovation (AGL2013-43194-P). http://dx.doi.org/10.1016/j.toxlet.2015.08.230
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P02-004 Biological effects of classic and diet soda drinks assessed in in vivo and in vitro models M. Mateo-Fernández ∗ , V. Moreno-Ariza, M. Balongo-Escobar, L. Luque-Bravo, Z.N. Fernández-Bedmar, M. Martínez-Jurado, S. Demyda-Peyrás, T. Merinas-Amo University of Cordoba, Genetics, Cordoba, Spain Consumption of sugar and diet beverages has increased rapidly during the last couple of decades, carbonated soft drinks having the fourth highest worldwide consumption. In this study we assessed the biological effects of Classic Coca-ColaTM (CCC), Diet Coca-ColaTM (DCC) and fructose syrup (FRU) at five different concentrations using in vitro and in vivo assays to determine their nutraceutical potential. Toxicity, antitoxicity, genotoxicity, antigenotoxicity and their effect on the life and healthspan were established using a Drosophila melanogaster model. In addition, cytotoxicity and DNA fragmentation were analysed using a HL-60 promyelocytic cell line as a hallmark of apoptosis. Our results showed that none of the compounds were toxic at the assayed concentrations. DCC was non-antitoxic at all the tested concentrations as it was not able to reduce the effects of hydrogen peroxide. On the contrary, antitoxic activity was characterised in FRU (100 mM) and in CCC with a negative dose effect. All beverages produced a non-genotoxic effect at all the tested concentrations. While CCC was highly antigenotoxic, this pattern was not observed in DCC. Conversely, the effect of FRU was only increased in combination with hydrogen peroxide. CCC, DCC and FRU increased life expectancy and quality in D. melanogaster at some of the assessed concentrations. Regarding the in vitro assays, CCC and FRU showed a cytotoxic effect in HL-60, as well as DCC although to a lesser extent. CCC and FRU, at high concentrations, induced a weak internucleosomal DNA fragmentation in HL-60 cells. In contrast, this effect was not observed in DCC. In conclusion, the lifespan increase and the lack of toxicity and genotoxicity in the in vivo model (D. melanogaster) results observed in the fructose assessment may mimic those obtained in Classic CocaColaTM complex mixture. Only the antimutagenic inactivity showed by fructose does not agree with the previous assumption. Finally, our results suggested that CCC and Fructose, at the assessed concentrations, could provide more beneficial activity than DCC in the HL-60 model. http://dx.doi.org/10.1016/j.toxlet.2015.08.231
P02-005 Comparison of in vivo and in vitro activities of alcohol and non-alcohol beer T. Merinas-Amo ∗ , M. Martínez-Jurado, R. Merinas-Amo, V. García-Zorrilla, Z.N. Fernández-Bedmar, A. Alonso-Moraga, M. Mateo-Fernández University of Córdoba, Genetics, Cordoba, Spain Much information about the nutritive profile of different types of beer is available on scientific data bases. We have compared blond, stout and non-alcohol Lager beer in order to know if they could be considered as neutraceutical beverages and if different biological activities could be observed among them. In vivo assays were carried out in the Drosophila melanogaster model with the aim of analysing the (i) toxi/antitoxic effects that beers could cause in the flies’ survival percentage; (ii) the geno/antigenotoxic potential that these drinks could provide in this organism by performing the
S66
Abstracts / Toxicology Letters 238S (2015) S56–S383
Somatic Mutation And Recombination Test (SMART); and (iii) the induction of longevity that these beers could exert. Moreover, in vitro assays were performed in HL-60 human leukaemia cell line testing the cytotoxicity and proapoptotic DNA internucleosomal fragmentation potency of these beers. The results showed that all beers exhibited neither toxic nor genotoxic effects, being blond beer healthier than the other ones. Besides they exerted genomic protective effects against hydrogen peroxide. As far as longevity is concerned, the average of lifespan was increased in 10, 7 and 3 days for blond, non-alcohol and stout Lager beer respectively, with respect to their concurrent controls. The in vitro assays showed that the IC50 values of non-alcohol and stout Lager beer were lower than the blond Lager beer value, being the most chemopreventive substances. Nevertheless, the blond beer was the only one that completely inhibited the cellular growth. In conclusion, our results suggest that blond Lager beer is the healthiest one because of the high capacity for protecting against genotoxic oxidative substances, for improving the lifespan extension in Drosophila and the ability to inhibit the growth of HL-60 promyelocytic cells that it exhibits. http://dx.doi.org/10.1016/j.toxlet.2015.08.232
P02-006 The effects of malathion on serum parameters in rat M. Ekremoglu ∗ , H. Pasaoglu, C. Severcan, B. Sen, O.T. Pasaoglu Gazi University, Medical Biochemistry, Ankara, Turkey It is an undeniable fact that malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. However, malathion has many side effects to human health. Our study was designed to evaluate the acute effects of malathion on sera parameters. For this purpose 4 groups were formed. Rats in Group 1 served as control group animals which were only given corn oil. Groups 2–4 were administered 100 mg/kg, 200 mg/kg and 400 mg/kg malathion, respectively, dissolved in corn oil by oral gavage. The rats were sacrificed after 24 h following administration. In order to assess the dose dependent, we studied serum lipase, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), Blood urea nitrogen (BUN), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), ␣-amylase, malondialdehyde (MDA), creatinine and glucose-6-phosphate dehydrogenase (G6PD). The use of malathion inhibited lipase activity. We did not observe lipase activity on Groups 2–4. There were no significant differences of BUN, LDH, MDA and creatinine levels among all groups. We found a significant increase of glucose level in Group 4, compared to Group 2. We found significant increase AST and CK levels of Groups 3 and 4 compared to Groups 1 and 2 and also found significant decrease CK level of Group 4 compared to Group 3. We found a significant increase of ALT level in Group 3, compared to Group 1. We found significant decline ALP levels of Groups 3 and 4 compared to Group 2. We found a significant increase of ␣-amylase level in Group 2, compared to Group 1. At the first, G6PD enzyme activity significantly decrease both of the Groups 2 and 3 and then significantly increase the Group 4 compared to Group 1. http://dx.doi.org/10.1016/j.toxlet.2015.08.233
P02-007 Use of (Q)SAR tools as a first step in a strategy to assign priority to substances migrating from printed paper and board food contact materials based on genotoxic potential M. Van Bossuyt 1,2,∗ , E. Van Hoeck 1 , G. Raitano 3 , N. Ost 4 , G. Ates 2 , G. Schüürman 4 , T. Vanhaecke 2 , E. Benfenati 3 , V. Rogiers 2 , B. Mertens 1 1 Scientific Institute of Public Health, Food, Medicines and Consumer Safety, Brussels, Belgium 2 Vrije Universiteit Brussel, In Vitro Toxicology and Dermato-Cosmetology, Brussels, Belgium 3 Instituto di Ricerche Farmacologiche Mario Negri, Environmental Health Sciences, Milan, Italy 4 Centre for Environmental Research – UFZ, Ecological Chemistry, Leipzig, Germany
In the last decade, several incidents with contaminants leaking from food packaging into food and drinks have raised concerns towards potential adverse health effects caused by exposure to these chemicals (e.g. bisphenol A). In 2011, the European Commission adopted a new regulation on the use of chemicals in plastics intended to come into contact with food. However, to this day, no EU regulation exists on chemicals used in other types of food contact materials (FCM). Consequently, non-plastic FCM are only covered by national legislation or by the general recommendations of the Council of Europe, containing ‘inventory lists’ of additives, monomers, solvents and other compounds that might be present in non-plastic FCM. Since no (recent) safety evaluation has been carried out for over 5000 substances present in these inventory lists, identification of substances requiring further safety data is urgently needed. The current study aims to develop a strategy to assign priority to substances used in printed paper and board. First, information on substances present in the inventory lists of the Council of Europe or from national legislation was assembled in a database. In a second step, their genotoxic potential was evaluated in silico using multiple (Q)SAR models (Toxtree, Derek NexusTM , VEGA Consensus model). An applicability domain assessment was performed to ensure fitting of the test compounds in the models applied. Based on the outcome of the in silico evaluation, substances containing a structural alert for genotoxicity were selected for further more in depth study, combining extensive literature review with in vitro genotoxicity testing. http://dx.doi.org/10.1016/j.toxlet.2015.08.234
P02-008 A short study of deoxynivalenol correlation in diet and urine Y. Rodríguez-Carrasco, G. Font, M.-J. Ruiz-Leal ∗ , B. Houda University of Valencia, Preventive Medicine, Toxicology, Burjassot-Valencia, Spain Cereals are the most important source of food but they are also frequently reported as contaminated by mycotoxins, a heterogeneous group of secondary metabolites of filamentous fungi. The correlation between the widespread contamination of food commodities and the human exposure to mycotoxins makes the monitoring of their levels in urine essential. In this 10-days pilot study the concentrations of deoxynivalenol (DON), one of the main Fusarium mycotoxins, in a survey conducted by one volunteer