RESUMEN
Irradiation is widely used worldwide to sterilize and kill insects in food, and prevent the germination of agricultural products. However, in Japan, food irradiation is prohibited except to prevent potato sprouting. Herein, 5,6-dihydrothymidine (DHdThd) residue-a damaged nucleoside generated from the thymidine (dThd) residue in DNA contained in food upon irradiation-was used as a detection indicator. Eight dried plant-based food samples were gamma ray-irradiated in the range from 3.2 to 8.3 kGy. Subsequently, DNA was extracted from the irradiated sample and digested into nucleosides by the three enzymes, and the test solution was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Evidently, in all samples, the concentration ratio of DHdThd to dThd in the test solution (DHdThd/dThd) was dependent on the irradiation dose; moreover, during storage under frozen conditions for at least 890 d post-irradiation, this concentration ratio was equal to that immediately after irradiation. The irradiation histories of the eight types of dried plant-based food samples were correctly detected.
Asunto(s)
Irradiación de Alimentos , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , ADN , Timidina/análisis , Irradiación de Alimentos/métodosRESUMEN
Infant formula in liquid for childcare can be stored at room temperature for a certain period of time, reducing the burden of childcare and preparing for disasters. Against this background, domestic manufacturing and sales began in March 2019. AFM1 is a metabolite of aflatoxin B1 (AFB1), a carcinogenic mycotoxin, and is contained in the milk of livestock fed a diet contaminated with AFB1. At present, standard values have not been set for infant formula in liquid as well as prepared infant formula in liquid, and infants consume a large amount of dairy products per body weight, so care must be taken in the intake.In this study, we investigated the actual condition of AFM1 content in dairy products with high intake of infants. As a result of the investigation, the AFM1 of the detected dairy products was 0.001 to 0.005 µg/kg, which was extremely small compared to the AFM1 in the dairy products reported so far. Since infant nutrition depends on dairy products, it is undeniable that they may consume more than adults, so continuous research is needed.
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Aflatoxina M1 , Contaminación de Alimentos , Aflatoxina B1/análisis , Aflatoxina B1/metabolismo , Aflatoxina M1/análisis , Animales , Productos Lácteos , Dieta , Contaminación de Alimentos/análisis , Humanos , Leche/químicaRESUMEN
The quantities of (5S)- and (5R)-5,6-dihydrothymidine (total DHdThd) in frozen beef liver samples were compared between those irradiated by an electron beam (EB) and those subjected to γ-irradiation. DNA extracted from the samples was enzymatically digested to nucleosides and analyzed by liquid chromatography-tandem mass spectrometry for total DHdThd and 2'-deoxythymidine (dThd). Total DHdThd was formed radiospecifically and dose-dependently and the dose-response curves of the ratio of total DHdThd to dThd (total DHdThd/dThd) were similar for both the EB- and γ-irradiated samples. The total DHdThd/dThd was stable after long-term storage (4 months) at -20 °C and the followed heat treatment in a microwave oven. The total DHdThd/dThd could be a robust marker and is equally effective at quantitating both EB- and γ-ray irradiation history. The irradiation doses of raw beef liver samples were estimated using the dose-response curves of the total DHdThd/dThd of other irradiated samples. The ratio of the estimated dose to the actual dose was 0.74-1.30 in the irradiation range of 4.67-7.62 kGy.
RESUMEN
We carried out a collaborative study in six laboratories to confirm the universality of the enhancing effect of co-existing reference pesticides on the GC-MS peak response to a target pesticide (malathion, procymidone, or flucythrinate). First, we confirmed the response enhancement of the target pesticides with increasing numbers of co-existing reference pesticides in solution. Then, using diluted green soybean matrix, we analyzed the target pesticides with two types of matrix-matched calibration, containing the target pesticides or 166 other pesticides. In both cases, the response-enhancing effect of co-existing pesticides was confirmed in all laboratories. The enhancement was reduced by addition of green soybean matrix to the sample and calibration solutions. Our results show that it is necessary to estimate the peak response-enhancing effect of co-existing pesticides in the calibration solution to obtain accurate results with GC-MS determination. The enhancing effect could be reduced by addition of food matrix to the sample and calibration solutions.
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Análisis de los Alimentos/métodos , Residuos de Plaguicidas/análisis , Plaguicidas/análisis , Calibración , Cromatografía de Gases y Espectrometría de MasasRESUMEN
Polyethylene glycol 300 is commonly used as a base material for "analyte protection" in multiresidue pesticide analysis via gas chromatography-mass spectrometry. However, the disadvantage of the co-injection method using polyethylene glycol 300 is that it causes peak instability in α-cyano pyrethroids (type II pyrethroids) such as fluvalinate. In this study, we confirmed the instability phenomenon in type II pyrethroids and developed novel analyte protectants for acetone/n-hexane mixture solution to suppress the phenomenon. Our findings revealed that among the examined additive compounds, three lipophilic ascorbic acid derivatives, 3-O-ethyl-L-ascorbic acid, 6-O-palmitoyl-L-ascorbic acid, and 6-O-stearoyl-L-ascorbic acid, could effectively stabilize the type II pyrethroids in the presence of polyethylene glycol 300. A mixture of the three ascorbic acid derivatives and polyethylene glycol 300 proved to be an effective analyte protectant for multiresidue pesticide analysis. Further, we designed and evaluated a new combination of analyte protectant compounds without using polyethylene glycol or the troublesome hydrophilic compounds. Consequently, we obtained a set of 10 medium- and long-chain saturated fatty acids as an effective analyte protectant suitable for acetone/n-hexane solution that did not cause peak instability in type II pyrethroids. These analyte protectants will be useful in multiresidue pesticide analysis by gas chromatography-mass spectrometry in terms of ruggedness and reliable quantitativeness. Graphical abstract Comparison of effectiveness of the addition of lipophilic derivatives of ascorbic acid in controlling the instability phenomenon of fluvalinate with polyethylene glycol 300.
RESUMEN
We report the collective behavior of numerous plastic bolt-like particles exhibiting one of two distinct states, either standing stationary or horizontal accompanied by tumbling motion, when placed on a horizontal plate undergoing sinusoidal vertical vibration. Experimentally, we prepared an initial state in which all of the particles were standing except for a single particle that was placed at the center of the plate. Under continuous vertical vibration, the initially horizontal particle triggers neighboring particles to fall over into a horizontal state through tumbling-induced collision, and this effect gradually spreads to all of the particles, i.e., the number of horizontal particles is increased. Interestingly, within a certain range of vibration intensity, almost all of the horizontal particles revert back to standing in association with the formation of apparent 2D hexagonal dense-packing. Thus, phase segregation between high and low densities, or crystalline and disperse domains, of standing particles is generated as a result of the reentrant transition. The essential features of such cooperative dynamics through the reentrant transition are elucidated with a simple kinetic model. We also demonstrate that an excitable wave with the reentrant transition is observed when particles are situated in a quasi-one-dimensional confinement on a vibrating plate.
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Purpose: Prenatal exposure to environmental chemicals is a growing concern, because such exposures have been shown to be associated with various diseases. The levels of chemicals and heavy metals in maternal blood, cord blood, maternal urine and amniotic fluid in Japanese pregnant women were investigated. Methods: A total of 145 women, including 14 fetal growth restriction cases, were included in the present study. The levels of phthalates (di[2-ethylhexyl]phthalate and mono[2-ethylhexyl]phthalate), perfluorinated compounds (perfluorooctane sulfonate, perfluorohexanoic acid, perfluorooctanoic acid, and perfluorononanoic acid), pesticides (dimethylphosphate, dimethylthiophosphate, diethylphosphate, diethylthiophosphate, 3-phenoxybenzoic acid, and octachlorodipropyl ether), bisphenol A, nicotine (nicotine, nornicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine), polybrominated diphenyl ethers, and heavy metals were measured. The relationship between fetal growth and the levels of chemicals and heavy metals were investigated. Results: Phthalates, perfluorinated compounds, pesticides, polybrominated diphenyl ethers, and heavy metals were detected in high frequency, whereas nicotine and bisphenol A were almost negative. Phthalates, perfluorinated compounds, and several heavy metals were transferred to the fetus. High perfluorononanoic acid levels in the maternal blood and cord blood, and low perfluorooctanoic acid level in the cord blood were significantly and negatively associated with fetal growth. Conclusions: The present study showed that pregnant women in Japan and their fetuses are exposed to a variety of chemicals and heavy metals.
RESUMEN
A rapid and reliable method for determining irradiation histories of ground beef and prawns was developed on the basis of a method for determining the irradiation history of beef liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of 5,6-dihydrothymidine (DHdThd). Improvements in the method included the following: (1) 50% ethanol precipitation in the DNA extraction step was conducted before the RNase step, (2) snake venom phosphodiesterase I was used for DNA digestion to boost liberation of DHdThd, and (3) a matrix-matched calibration curve was used for determining DHdThd by LC-MS/MS analysis. This method successfully determined irradiation histories of ground beef and prawns. Furthermore, a close correlation between the formation of DHdThd and 2-alkylcyclobutanones, which are an established index of irradiation histories, was observed in ground beef. DHdThd in DNA could be a promising candidate for a new index of irradiation histories of various foods.
Asunto(s)
Cromatografía Liquida/métodos , Decápodos/química , Carne/análisis , Mariscos/análisis , Espectrometría de Masas en Tándem/métodos , Timidina/análogos & derivados , Animales , Bovinos , Decápodos/efectos de la radiación , Irradiación de Alimentos , Carne/efectos de la radiación , Mariscos/efectos de la radiación , Timidina/análisisRESUMEN
Histamine food poisoning is caused by ingestion of spoiled fish containing high levels of histamine. This paper reports cases in which histamine was detected in Osaka prefecture in fiscal year 2015 in a survey of fish and fishery products on the market and the food poisoning. A suspected case of histamine food poisoning was also evaluated to investigate the cause and minimize further problems. Histamine in food was separated on SPE cartridge columns, and analyzed after derivatization with fluorescamine by means of HPLC-FL. Histamine was detected in some fishery products on the market and in food that had caused poisoning. The samples in which histamine was detected were semi-dried whole round herring (Urumeiwashi-maruboshi), mackerel (Saba) and sardine dumpling (Iwashi-tsumire). These foods were the main causes of histamine food poisoning according to the report of the Ministry of Health, Labour and Welfare, Government of Japan.
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Productos Pesqueros/análisis , Peces , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Enfermedades Transmitidas por los Alimentos/etiología , Histamina/análisis , Histamina/aislamiento & purificación , Animales , Cromatografía Líquida de Alta Presión/métodos , Fluorescamina , Histamina/efectos adversos , Humanos , JapónRESUMEN
In multiresidue pesticide analysis using gas chromatography, it has long been recognized that an increase in the number of pesticides present in a standard solution can result in an enhancement of the peak responses of certain pesticides. Despite being widely acknowledged, this phenomenon has been rarely studied and is poorly understood. In this study, the authors have tentatively called this phenomenon the "matrix-like effect" and demonstrated it clearly using gas chromatography with tandem mass spectrometry. Five selected pesticides, namely, omethoate, terbufos, malathion, procymidone, and permethrin, and four internal standard candidates, namely, triphenyl phosphate, naphthalene-d8 , phenanthrene-d10 , and fluoranthene-d10 , were used to evaluate the matrix-like effect following the addition of 58, 108, and 166 other pesticides. With the exception of naphthalene-d8 , the responses of all evaluated pesticides and internal standard candidates were dramatically enhanced by the addition of up to 166 coexisting pesticides. The relative response factors of the five pesticides to each internal standard candidate were not constant under the conditions studied, meaning that these internal standard candidates did not adequately compensate for the matrix-like effect, at least for the five evaluated pesticides. The results revealed that the presence of various mixtures of pesticides in standard solutions might act as an unintentional analyte protectant, that is, some sort of troublesome "quasi-matrix."
RESUMEN
A method for detecting irradiation histories of raw beef livers was developed by measuring 5,6-dihydrothymidine (DHdThd) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liver DNA was extracted using phenol-chloroform extraction followed by precipitation in 50% ethanol. DNA was then enzymatically digested and nucleosides were purified using an OASIS MCX column. DHdThd and thymidine (dThd) contents of resulting test solutions were analyzed using LC-MS/MS. DHdThd was detected specifically after γ-irradiation. Concentration ratios of DHdThd to dThd in the test solutions increased dose-dependently after irradiation at 1.0-11.3kGy, which included the practical dose for sterilization of 2-7kGy. Dose-response curves from beef livers of individual animals almost overlapped. Thus, this method is a candidate for the detection of irradiation histories of foods from which DNA can be extracted.
Asunto(s)
Cromatografía Liquida/métodos , Hígado/efectos de la radiación , Espectrometría de Masas en Tándem/métodos , Timidina/análogos & derivados , Animales , Bovinos , ADN/análisis , Hígado/química , Timidina/análisisRESUMEN
The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an "epimutagen combination" that is effective at low concentrations as detected in maternal peripheral and cord blood.
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Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Mutágenos/toxicidad , Animales , Diferenciación Celular/efectos de los fármacos , Cotinina/toxicidad , Desarrollo Embrionario/efectos de los fármacos , Epigénesis Genética/genética , Éteres/toxicidad , Femenino , Sangre Fetal/efectos de los fármacos , Heterocromatina/efectos de los fármacos , Heterocromatina/genética , Humanos , Mercurio/toxicidad , Ratones , Organofosfatos/toxicidad , Selenio/toxicidadRESUMEN
Quantitative methods using the matrix-matched standard solutions approach are widely used for multi-residue pesticide determination by GC-MS/MS to deal with the issue of matrix effects. However, preparing matrix-matched standard solutions in analyses of many kinds of samples is very time-consuming. In order to solve this problem, a method that employs general matrix standard solutions has been developed using polyethylene glycol (PEG), extract of vegetables-fruit juice (VFJm) and triphenyl phosphate (named the PEG-VFJm method). Here, a validation study for 168 pesticides was performed on three kinds of samples [potato, spinach and apple] at concentrations of 0.010 and 0.050 µg/g. In these three commodities, 144 to 158 pesticides satisfied the required criteria using the matrix-matched method and 129 to 149 pesticides satisfied the same criteria using the PEG-VFJm method. Our results suggest that application of general matrix standard solutions would enable rapid and effective analyses of pesticides.
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Cromatografía de Gases/métodos , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Frutas/química , Residuos de Plaguicidas/análisis , Espectrometría de Masas en Tándem/métodos , Verduras/química , Organofosfatos , Residuos de Plaguicidas/aislamiento & purificación , Polietilenglicoles , SolucionesRESUMEN
A validation study was conducted on a rapid multiresidue method for determination of pesticide residues in vegetables and fruits by LC-MS/MS. Pesticide residues in the vegetables or fruits were extracted with acetonitrile in a disposable tube using a homogenizer, followed by salting out with anhydrous magnesium sulfate and sodium chloride in the presence of citrate salts for buffering. The extract was purified with a double-layered cartridge column (graphite carbon black/primary secondary amine silica gel; GCB/PSA). For citrus fruits a purification step with a C18 column was added (this column was connected to the GCB/PSA column). After removal of the solvent, the extract was resolved in methanol/water and analyzed by means of LC-MS/MS. The method was validated according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan; recovery tests were performed on 8 kinds of vegetables and fruits [cabbage, cucumber, Japanese radish, onion, potato, spinach, Amanatsumikan (a citrus fruit) and apple] by fortification of 161 pesticide residues at the concentrations 0.01 and 0.05 µg/g (each concentration of pesticide residue was extracted from 2 samples on 5 separate days). The trueness of the method for 127 pesticides in all 8 commodities was 70-120% with satisfactory repeatability and within-run reproducibility. This method is concluded to be applicable for determination of pesticide residues in vegetables and fruits.
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Cromatografía Liquida/métodos , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Frutas/química , Residuos de Plaguicidas/análisis , Residuos de Plaguicidas/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos , Verduras/químicaRESUMEN
The concentrations of 10 polybrominated diphenyl ethers (PBDEs) in commercial culture media and protein sources (PSs) for in vitro fertilization (IVF) of human ova were investigated. Samples of 15 IVF media (IVFM), nine sperm preparation media (SPM), and six PSs were analyzed. PBDEs were detected in 10 IVFM, seven SPM, and all PS samples in ranges of 0.6-35, 0.9-31, and 7.5-385pgg(-1), respectively. A dominant PBDE congener BDE-47 was detected in the PS and PS-supplemented samples. Our findings suggested that PS supplementation was the potential cause of PBDE-contamination of IVFM and SPM.
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Medios de Cultivo/análisis , Fertilización In Vitro , Éteres Difenilos Halogenados/análisis , Humanos , Plasma/química , Bifenilos Polibrominados/análisis , ProteínasRESUMEN
If pesticide residues are detected at over the uniform limit (0.01 ppm) in processed foods, the maximum residue limits of pesticides in each ingredient should be considered. For that purpose, analysis of pesticide residues in the ingredients would be required, but in many cases it is difficult to obtain ingredients from the same lot, or the processed foods may have been manufactured overseas. Thus, a means of estimating pesticide concentrations in ingredients before processing would be important. In this study, we estimated the concentration of paclobutrazol, which was detected in LM pickles (LMPs), that would have been present in Takana (LM; mustard leaf) before processing. The LMPs mainly consisted of LM and seasoning liquid (SL), and other ingredients (sesame seeds or red pepper) accounted for less than 0.2% (w/w) of the LMPs. The LM and SL were separated and their paclobutrazol concentrations were determined. The concentration of paclobutrazol in the SL was approximately 1/10 of that detected in LM. Loss of water from LM during processing was taken into account to calculate the content of paclobutrazol in LM before processing, based on the analytical result of processed LM.
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Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Manipulación de Alimentos , Residuos de Plaguicidas/análisis , Triazoles/análisis , Concentración Máxima Admisible , Residuos de Plaguicidas/normasRESUMEN
A rapid multi-residue method for determination of pesticide residues in processed foods manufactured from agricultural products was examined. Five mL water was added to 5 g sample in a polypropylene tube, and the tube was left to stand at room temperature for 30 min. Then, 20 mL acetonitrile was added to the sample. The mixture was homogenized in a high-speed homogenizer, followed by salting out with 1 g NaCl and 4 g anhydrous MgSO4. After centrifugation, the organic layer was purified on a graphitized carbon/PSA cartridge column. After removal of the solvent, the extract was resolved in methanol-water and analyzed with LC-MS/MS. The recoveries of 93 pesticides fortified into 5 kinds of processed foods [Chinese cabbage kimchi, marmalade, raisin, umeboshi (pickled plum) and worcester sauce] were examined at the concentrations of 0.02 and 0.1 µg/g (n=5). The recoveries of 61 pesticides in all foods were 70-120% with relative standard deviation below 20% at both concentrations. Seventy-four processed foods obtained from markets in Japan were examined with this method. Pesticide residues over the maximum residue limit (0.01 µg/g) were detected in 2 processed foods.
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Cromatografía Líquida de Alta Presión/métodos , Productos Agrícolas/química , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Manipulación de Alimentos , Residuos de Plaguicidas/análisis , Espectrometría de Masas en Tándem/métodos , Concentración Máxima AdmisibleRESUMEN
A rapid and simple multi-residue method for determination of pesticides has been applied to drinking water and beverages. To a disposable polypropylene tube containing 10.0 g sample, 20 mL acetonitrile was added and the mixture was shaken vigorously for 1 min to extract pesticides. Then, 1 g sodium chloride and 4 g magnesium sulfate anhydrous were added, followed by vigorous shaking for 1 min and centrifugation to obtain the organic phase. The organic phase was processed with a graphite carbon black/PSA solid phase column. After concentration and reconstitution with 25% methanol containing aqueous solution, the test solution was analyzed with LC-MS/MS. Recovery tests of 91 pesticides fortified (0.02 µg/g) in 35 kinds of drinking water and beverages were conducted. The decline of recoveries in alcoholic beverages is considered to be due to the increase of organic phase volume owing to ethanol included in the alcoholic beverages. A simulation study was carried out with simulated alcoholic beverages, which consisted of 50% grape juice, with various amounts of ethanol and water, to examine pesticides recoveries and volume of the organic phase. The results suggested this method would be applicable both to alcoholic beverages containing less than 10% ethanol and to alcoholic beverages containing over 10% ethanol after dilution with water to below 10% ethanol prior to the addition of acetonitrile. A sample could be processed and analyzed by LC-MS/MS within 2 h. Thus, this method should be useful for monitoring and screening pesticide residues in drinking water and various beverages.
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Bebidas/análisis , Cromatografía Liquida/métodos , Agua Potable/análisis , Residuos de Plaguicidas/análisis , Espectrometría de Masas en Tándem/métodos , Factores de TiempoRESUMEN
In vitro fertilization (IVF) is one of the most important treatments of infertility to provide a chance of conceiving. In IVF treatment, sperm are washed and motile sperm are isolated with sperm washing media (SWM) for the purpose of fertilization; fertilized ova are then incubated for a maximum of 5 or 6d in media for IVF (IVFM). The exposure of fertilized ova to chemicals via such media has not been studied. We determined the concentrations of two contaminants; di(2-ethylhexyl)phthalate (DEHP) and its hydrolyzed product mono(2-ethylhexyl)phthalate (MEHP) in IVFM, SWM, and protein sources (PS: human serum albumin or serum substitute) for IVFM and SWM. The DEHP and MEHP in these media were extracted by a liquid-liquid extraction method and their concentrations determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Fifteen IVFM, nine SWM, and six PS obtained in Japan were examined. The concentrations of DEHP and MEHP in IVFM and SWM were <10-114 and <2.0-263 ng mL(-1), respectively. The concentrations of both DEHP and MEHP were higher in the media containing PS than in those without PS. Either MEHP alone or both DEHP and MEHP were detected in PS. The concentrations of DEHP and MEHP in PS were <10-982 and 47.0-1840 ng mL(-1), respectively. The DEHP and MEHP detected in these media were derived from PS. This is the first study on the chemical contamination of IVFM, SWM, and PS.
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Medios de Cultivo/química , Dietilhexil Ftalato/análogos & derivados , Dietilhexil Ftalato/análisis , Contaminantes Ambientales/análisis , Fertilización In Vitro/estadística & datos numéricos , Dietilhexil Ftalato/toxicidad , Contaminantes Ambientales/toxicidad , Fertilización In Vitro/efectos de los fármacos , HumanosRESUMEN
Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4¢,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.