Biological Effects of Food Coloring in In Vivo and In Vitro Model Systems

(1) Background: The suitability of certain food colorings is nowadays in discussion because of the effects of these compounds on human health. For this reason, in the present work, the biological effects of six worldwide used food colorings (Riboflavin, Tartrazine, Carminic Acid, Erythrosine, Indigotine, and Brilliant Blue FCF) were analyzed using two model systems. (2) Methods: In vivo toxicity, antitoxicity, and longevity assays using the model organism Drosophila melanogaster and in vitro cytotoxicity, DNA fragmentation, and methylation status assays using HL-60 tumor human cell line were carried out. (3) Results: Our in vivo results showed safe effects in Drosophila for all the food coloring treatments, non-significant protective potential against an oxidative toxin, and different effects on the lifespan of flies. The in vitro results in HL-60 cells, showed that the tested food colorings increased tumor cell growth but did not induce any DNA damage or modifications in the DNA methylation status at their acceptable daily intake (ADI) concentrations. (4) Conclusions: From the in vivo and in vitro studies, these results would support the idea that a high chronic intake of food colorings throughout the entire life is not advisable

Toxicological and Epigenetic Studies of Two Types of Ale Beer, Tyrosol and Iso-Alpha Humulone

Although many benefits drawn from beer consumption are claimed, the epidemiological records are contradictory with respect to cancer prevention. The purpose of this study was to investigate the possible health-related activities involving genome safety and the ageing processes of two types of lyophilised ale beers (blond and stout), as well as two of their bioactive compounds (tyrosol and iso-alpha humulone). A multipurpose trial set of in vivo toxicity, antitoxicity, mutagenicity, antimutagenicity, lifespan and healthspan assays using Drosophila melanogaster were used. In parallel, several in vitro assays were designed using the cancer cell line HL-60 in order to establish the possible chemopreventive activity of the selected substances, where epigenetic modulation of DNA methylation changes, clastogenic activity and tumour cell inhibition growth were evaluated. The safety of the four substances was confirmed: lyophilised blond ale beer (LBAB), lyophilised stout ale beer (LSAB), tyrosol and iso-alpha humulone were neither toxic nor genotoxic. Moreover, all substances, except tyrosol, revealed the ability to protect individual genomes against oxidative radicals and to exert antimutagenic activity against the genotoxin hydrogen peroxide. With respect to the degenerative process indicators of lifespan and healthspan, tyrosol was the only compound that did not exert any influence on the life extension of Drosophila; LBAB induced a significant lifespan extension in D. melanogaster; LSAB and its distinctive compound iso-alpha humulone induced a reduction in longevity. The in vitro assays showed the cytotoxic activity of LBAB, LSAB and tyrosol against HL-60 cells. Moreover, proapoptotic DNA fragmentation or DNA strand breakage was observed for both types of beers and iso-alpha humulone at different concentrations. Furthermore, the lyophilised ale beers and tyrosol exhibited an increasing genome-wide methylation status, while iso-alpha humulone exhibited a demethylation status in repetitive cancer cell sequences. Although the biological activities assigned to beer consumption cannot be linked to any specific molecule/element due to the complexity of the phenolic profile, as well as the multifactor brewing process, the results obtained let us propose lyophilised ale beers as safe potential nutraceutical beverages when consumed in moderate amounts. The prevention of toxicity and genetic oxidative damage, as well as the induction of tumor cell death and modulation of the methylation status, are the key activities of beer that were shown in the present research

Toxicological Studies of Czech Beers and Their Constituents

Background: Czech beers are unique because they are brewed using specific technology at a particular latitude and for being entirely produced in the area of the Czech Republic. The purpose of this work is the evaluation of toxicological e ects of a variety of freeze-dried Czech beers, their raw materials (malts, hops and yeast) and processed-beer (wort, hopped wort and young beer). Methods: In vivo assays to evaluate the safety and protective e ects in the Drosophila melanogaster eukaryotic system, and the in vitro evaluations of chemopreventive and DNA damage activity using the HL-60 tumour human cell line were carried out. Results: The safe e ects for all the analysed substances and general protective e ects against H2O2 were shown both at the individual and genomic level in the Drosophila animal model, with some exceptions. Moreover, all the substances were able to inhibit the tumour cell growth and to induce DNA damage in the HL-60 cells at di erent levels (proapoptotic, single/double strands breaks and methylation status). Conclusions: The promising e ects shown by freeze-dried Czech beers due to their safety, protection against a toxin, chemopreventive potential and the induction of DNA damage in tumour cells, allow the proposition of Czech beer as a beverage with nutraceutic potentia

Role of food additives in the modulation of degenerative processes: in vivo and in vitro studies

La dieta es uno de los agentes medioambientales más importantes que ejercen efectos sobre la salud y por tanto está relacionada con el riesgo de adquirir enfermedades. En el presente trabajo hemos evaluado los efectos biológicos que distintos aditivos alimentarios ejercen en modelos animales y celulares, añadiendo un nuevo cuerpo de datos a la ciencia en el campo de la nutracéutica. Para llevar a cabo nuestro objetivo, se han usado ensayos a diferentes niveles (molecular, celular, individual y poblacional) para analizar la seguridad y el posible efecto protector frente a una toxina oxidativa en el organismo modelo Drosophila melanogaster, así como el potencial quimiopreventivo y la capacidad de inducir daño en el ADN en células tumorales de leucemia humana HL-60. Todos estos estudios consisten en un conjunto de ensayos de detección de actividad biológica donde se pueden verificar varios puntos relacionados con los procesos degenerativos. La metodología usada se resume a continuación: i) Los ensayos in vivo usando el modelo Drosophila han permitido estudiar: - Toxicidad y Antitoxicidad: se evalúa la seguridad y la capacidad protectora a nivel individual midiendo el porcentaje de individuos supervivientes con respecto a su control negativo o positivo cuando Drosophila es tratada con diferentes concentraciones de aditivos alimentarios de forma simple y/o combinados con una toxina oxidativa (H2O2). - Genotoxicidad y Antigenotoxicidad: evaluación de la seguridad genómica y el potencial protector a nivel cromosómico mediante el análisis de mutaciones y recombinaciones somáticas en células en expansión clonal de alas de Drosophila tratadas en su estado larvario con diferentes concentraciones de los aditivos alimentarios en estudio de forma simple o combinada con H2O2. - Longevidad: evaluación de los efectos sobre la extensión y calidad de vida en poblaciones de Drosophila alimentadas durante toda su vida con los aditivos alimentarios a diferentes concentraciones. ii) Los ensayos in vitro usando el modelo celular promielocítico humano HL-60 nos han permitido evaluar: - Citotoxicidad: capacidad de los aditivos alimentarios para inhibir el crecimiento tumoral a diferentes concentraciones. - Daño en el ADN: potencial clastogénico de los aditivos alimentarios para inducir fragmentación proapoptótica del ADN y/o daño en el ADN en células tumorales individuales induciéndoles cometas. - Estado de metilación: capacidad de los aditivos alimentarios de modular el epigenoma de células tumorales mediante el estudio de los patrones del estado de metilación de amplias secuencias genómicas (secuencias repetitivas Alu, LINE y Satélite). Basándonos en los resultados obtenidos tanto en los ensayos in vivo del organismo modelo Drosophila como en los ensayos in vitro en la línea celular HL-60, sobre la seguridad, potencial nutracéutico y potencial quimiopreventivo de nuestros aditivos, las conclusiones más destacables para la concentración correspondiente a la Ingesta Diaria Admisible (IDA) de los aditivos estudiados en la presente tesis quedan reflejadas a continuación: 1.- Los aditivos alimentarios tienen un carácter seguro. Ninguno de los aditivos ensayados alcanza la DL50, ni tampoco induce mutaciones genéticas cuando Drosophila melanogaster es alimentada a distintas concentraciones. 2.- Cuatro de los aditivos mostraron capacidad protectora frente a un agente tóxico (Nitrato de Sodio, Tartrazina, Indigotina y Glutamato); cuatro mostraron resultados perjudiciales (Eritrosina, Azul Brillante FCF, Nitrito de Potasio y Nitrito de Sodio); el resto no mostraron efectos significativos, tanto a nivel de individuo como genómico. 3.- La mayoría de los aditivos mostraron un potencial nutracéutico bien significativamente positivo (Tartrazina, Azul Brillante FCF, Glutamato, Ácido Cítrico y Nitrato de Sodio) o bien neutro sobre la expansión de vida de Drosophila, siendo solo tres de ellos (Ácido Carmínico, Eritrosina y BHA) los que acortaron significativamente la longevidad . 4.- Tan solo resultó tener potencial quimiopreventivo uno de los compuestos estudiados (Nitrato de Potasio); el resto de aditivos indujo crecimiento celular y no fue capaz de ejercer daño genómico en las células de leucemia humana HL-60 tratadas. De forma general, podemos destacar el escaso potencial nutracéutico y el casi nulo potencial quimiopreventivo que los aditivos ensayados muestran, para las concentraciones IDA extrapolables establecidas, en nuestros ensayos in vivo e in vitro. Por todo lo anterior comentado, además de hacer un uso y consumo responsable de alimentos que contengan dichos compuestos, y continuar con las evaluaciones periódicas por los diferentes comités alimentarios, también se necesitan más estudios que nos permitan dilucidar las actividades biológicas y el potencial nutracéutico de los aditivos alimentarios estudiados con mayor claridad. Por ejemplo, se deben usar células no tumorales para compararlas con nuestros estudios in vitro. Ensayos clínicos en humanos también se podrían realizar como último paso.Diet is one of the most important environmental agents that exert a significant effect on health and consequently related to disease risk. In this work, the biological effects that food additives show on animal and cellular model systems have been evaluated, adding a new data corpus to science in the nutraceutical area. In order to carry out our goal, multilevel assays (molecular, cellular, individual and population) were used to analyse the safety and the possible protective effects against an oxidative toxin in the Drosophila melanogaster model organism, as well as the chemopreventive potential and the induction of DNA damage ability in the human leukaemia tumour HL-60 cells. Here, we present a set of biological activity screening assays where several points related to degenerative processes can be checked. Briefly, the used methodology is below mentioned: i) In vivo assays using the Drosophila model allowed to study: - Toxicity and Antitoxicity: the safety and protective ability at individual level measuring the survival percentage of individuals with respect to their concurrent negative or positive controls is evaluated when Drosophila is treated either with different concentrations of a single food additive or it is combined with an oxidative toxin (H2O2). - Genotoxicity and Antigenotoxicity: the genomic safety and protective potential at chromosomal level measuring the somatic mutation and recombination wing spot test of Drosophila treated at larval stage with different concentrations of a single or a combined food additive with H2O2 is evaluated. - Longevity: evaluation of effects on the life extension and quality of life of Drosophila populations feed over time with different concentrations of food additives. ii) In vitro assays using the HL-60 promyelocytic human cell model allowed us to evaluate: - Cytotoxicity: ability of food additives to inhibit the tumour cells growth treated with different concentrations. - DNA damage: clastogenic potential of food additives to induce proapoptotic DNA fragmentation and/or DNA damage in single tumour cell by inducing comets. - Methylation status: capacity to modulate epigenome by studying the methylation status pattern of genome wide sequences (Alu, LINE and Satellite repetitive elements) by treating tumour cells with food additives. According to the obtained results, both in the in vivo assays with the model organism Drosophila and the in vitro assays with the HL-60 cell line, about the safety, nutraceutical potential and chemopreventive potential of our additives, the most highlighted conclusions from the present thesis are bellow mentioned: 1.- Food additives are safe. None of the additives tested reaches the lethal dose 50 (LD50), nor does it induce genetic mutations when Drosophila melanogaster is fed at different concentrations. 2.- Four of the additives showed protective capacity against a toxic agent (Sodium Nitrate, Tartrazine, Indigotine and Glutamate); four showed harmful results (Erythrosine, Brilliant Blue FCF, Potassium Nitrite and Sodium Nitrite); the rest did not show significant effects, both at the individual and genomic level. 3.- Most of the additives showed a significantly positive nutraceutical potential (Tartrazine, Brilliant Blue FCF, Glutamate, Citric Acid and Sodium Nitrate) or neutral on the life expansion of Drosophila, being only three of them (Carminic Acid, Erythrosine and BHA) which significantly shortened longevity. 4.- Only one of the compounds studied (Potassium Nitrate) turned out to have chemopreventive potential; the rest of the additives induced cell growth and were not able to exert genomic damage in the treated HL-60 human leukaemia cells. In general, it can be highlighted the low nutraceutical potential and the almost zero chemopreventive potential that the additives tested show, for the established extrapolated Acceptable Daily Intake (ADI) concentrations, in our in vivo and in vitro tests. For all the foregoing, in addition to making a responsible use and consumption of foods that contain these compounds, and continuing with periodic evaluations by the different food committees, more studies are also needed that allow us to elucidate the biological activities and nutraceutical potential of the food additives studied more clearly. For instance, non-tumour cell must be used to be compared with our in vivo studies. When appropriate, clinic assays should also be performed

Biological Effects of Food Coloring in In Vivo and In Vitro Model Systems

(1) Background: The suitability of certain food colorings is nowadays in discussion because of the effects of these compounds on human health. For this reason, in the present work, the biological effects of six worldwide used food colorings (Riboflavin, Tartrazine, Carminic Acid, Erythrosine, Indigotine, and Brilliant Blue FCF) were analyzed using two model systems. (2) Methods: In vivo toxicity, antitoxicity, and longevity assays using the model organism Drosophila melanogaster and in vitro cytotoxicity, DNA fragmentation, and methylation status assays using HL-60 tumor human cell line were carried out. (3) Results: Our in vivo results showed safe effects in Drosophila for all the food coloring treatments, non-significant protective potential against an oxidative toxin, and different effects on the lifespan of flies. The in vitro results in HL-60 cells, showed that the tested food colorings increased tumor cell growth but did not induce any DNA damage or modifications in the DNA methylation status at their acceptable daily intake (ADI) concentrations. (4) Conclusions: From the in vivo and in vitro studies, these results would support the idea that a high chronic intake of food colorings throughout the entire life is not advisable