丙烯酰胺对睾丸间质细胞R2C活性及孕酮合成功能的影响

目的:研究丙烯酰胺(acrylamide,AA)对体外培养的睾丸间质细胞R2C生长及孕酮合成功能的影响。方法:用浓度为0.25、0.5、0.75、1、2、4、6 mmol/L的AA作用于R2C细胞24 h,四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法获得IC25、IC50及IC75的AA作用浓度。用以上3种浓度的AA处理R2C细胞24 h,观察细胞形态。AA作用于R2C细胞4 h后,通过彗星实验(Comet assay)检测细胞DNA的损伤程度;放射免疫法(radioimmunoassay,RIA)测定AA处理R2C细胞4 h和24 h后的孕酮合成量。结果:AA能抑制R2C细胞活性,其IC25、IC50、IC75分别为1.140、1.925、3.250 mmol/L;3种浓度的AA能不同程度地影响R2C细胞生长形态;作用4 h对R2C细胞DNA有损伤作用;各浓度组作用24 h后均能降低R2C细胞的孕酮合成量。结论:AA能影响R2C细胞增殖及孕酮合成能力。     

重金属Cd对大鼠睾丸间质瘤细胞R2C孕酮合成的影响

探讨了Cd对体外大鼠睾丸间质瘤细胞R2C合成孕酮通路的影响,用MTT(噻唑蓝)比色法检测CdSO_4对R2C细胞生长抑制率的影响,通过数据分析,确定CdSO_4作用于R2C细胞的IC_(25)、IC_(50)、IC_(75)浓度值,用IC_(25)、IC_(50)、IC_(75)3种浓度的CdSO_4溶液培养R2C细胞,再采用碘标放射免疫法、流式细胞分析仪、ROS检测试剂盒和Western Blot法测定IC_(25)、IC_(50)、IC_(75)对孕酮合成、线粒体膜电位、ROS和孕酮合成相关蛋白St AR、CYP11A1的影响。结果表明,CdSO_4可以显著抑制R2C细胞的增殖,且CdSO_4作用于R2C细胞的IC_(25)、IC_(50)、IC_(75)浓度值分别为(24.93±0.023 5)、(44.80±0.047 9)、(80.51±0.035 7)μmol/L;IC_(25)、IC_(50)、IC_(75)3种浓度均能影响R2C孕酮合成;CdSO_4可以增加R2C细胞ROS的产生和线粒体膜电位的损伤比率;CdSO_4可以下调St AR、CYP11A1蛋白的表达,且对St AR蛋白的影响更为显著。重金属Cd可刺激R2C产生过量的ROS、下调St AR、CYP11A1蛋白的表达,进而影响线粒体的正常作用以及阻碍孕酮合成通路,最终影响孕酮的合成。     

油脂微波加热模型中3-氯-1,2-丙二醇脂肪酸酯的形成

通过建立油脂微波加热模型,研究不同的油脂微波加热后3-氯-1,2-丙二醇(3-chloropropane-1,2-diol,3-MCPD)脂肪酸酯含量的变化情况以及NaCl溶液、pH值、时间、不连续微波和金属离子等因素对3-MCPD脂肪酸酯形成的影响。结果表明:1)熟榨植物油微波加热后3-MCPD脂肪酸酯含量增加均超过9 mg/kg,远超过其他植物油。2)3-MCPD脂肪酸酯含量随NaCl的质量浓度增加而增加,随NaCl溶液体积分数的增加呈先增加后减少趋势。3)酸性环境促进3-MCPD脂肪酸酯形成。4)微波加热10 min内,3-MCPD脂肪酸酯含量与时间呈正相关。5)微波总时间一定的情况下,不连续微波产生的3-MCPD脂肪酸酯明显少于连续微波。6)金属离子作为催化剂参与活性中间物的形成,能明显地促进3-MCPD脂肪酸酯的形成。上述结果可为食品微波加工处理过程中危害物3-MCPD脂肪酸酯的控制研究提供数据支持。     

3-氯-1,2-丙二醇脂肪酸酯的代谢及毒理学研究进展

3-氯-1,2-丙二醇脂肪酸酯(3-MCPD酯)主要是植物油脱臭过程中产生的一类过程污染物, 其在生物体内经脂肪酶水解会生成3-氯-1,2-丙二醇(3-MCPD), 3-MCPD具有神经、肾脏、遗传毒性以及潜在的致突变性, 是国际公认的食品污染物。近年来国内外学者对于3-MCPD酯的毒理学研究逐渐增多, 研究发现3-MCPD酯的毒性作用范围与游离3-MCPD的毒性作用范围相同, 这支持酯键断裂形成游离3-MCPD, 并且主要由3-MPCD施加毒性的观点。虽然3-MCPD酯相比游离3-MCPD的急性毒性较低, 但2种化合物都产生广泛的肾组织病理学包括肾小球损害和肾小管上皮细胞增生及睾丸毒性。本文主要对3-MCPD酯的生物代谢及毒性研究进行了概述, 旨在给国内相关毒理学研究提供参考, 并有助于后续健康风险评估工作的开展。     

食品中3-氯-1,2-丙二醇脂肪酸酯研究进展

3-氯-1,2-丙二醇(3-MCPD)作为一种公认的食品污染物已有数十年之久随着对3-MCPD的深入研究,在越来越多的食品中检测出较高含量的3-MCPD.最近的研究发现3-MCPD更多地以酯化形式存在于咖啡、婴幼儿配方奶粉及食用油等很多食品中这些存在于食品中的3-MCPD脂肪酸酯(3-MCPD酯)在一定条件下能够释放3-MCPD,从而引起对机体的毒性作用 目前已有研究报道了3-MCPD棕榈酸单酯在动物体内的急性毒性作用和细胞毒性作用,因此,进一步深入研究3-MCPD酯的毒性显得尤为重要本文对3-MCPD酯在食品中的存在及影响因素、检测方法、代谢转化以及毒性研究进行了综述.     

食品污染物3-氯-1,2-丙二醇毒理作用的研究进展

氯丙醇类化合物3-氯-1,2-丙二醇(3-MCPD)是目前国际上广为关注的食品污染物,最初发现在酸水解植物蛋白加工而成的食品中.近年来,许多食品特别是精制油中发现大量3-MCPD脂类,其能够在体内通过代谢途径产生3-MCPD.研究发现3-MCPD在啮齿类动物中存在明显的毒性作用,且具有种属和位点特异性.结合国内外毒理学研究成果,概述3-MCPD体内代谢途径,详细综述其急性毒理、致癌性以及遗传、生殖、免疫、神经毒性机制的研究进展,并展望了3-MCPD毒理学评价进一步研究的方向.     

羧甲基赖氨酸在方便面中含量及其对雄性生殖和胚胎发育毒性的研究

采用酶联免疫吸附测定法(ELISA)对市售20个品牌方便面中游离态和结合态羧甲基赖氨酸(CML)含量进行了定量分析,并以体外细胞培养评价了CML的雄性生殖毒性和胚胎发育毒性。市售方便面中,游离态CML质量分数为0.39~0.80 mg/kg,结合态CML质量分数为19.42~34.99 mg/kg。CML作用于睾丸间质细胞R2C细胞24 h,浓度在1 mmol/L以下对细胞存活率没有显著抑制作用,浓度在2 mmol/L以下对孕酮分泌量没有显著的抑制作用;CML作用于胚胎干(ES)细胞D3和成纤维细胞3T3 10 d,浓度在4 mmol/L以下对3T3细胞存活率没有显著影响,浓度在0.5 mmol/L以下对D3细胞存活率没有显著影响。根据市售方便面中的CML浓度评估CML的摄入量,常规食用方便面摄入的CML不会对这几种细胞的存活率和R2C细胞的孕酮分泌量产生显著抑制作用,可认为不会产生雄性生殖和胚胎发育毒性。     

甘油与氯化钠模型热反应中生成3-氯-1,2-丙二醇的研究

以甘油、氯化钠、水为原料建立热反应模型,结合气相色谱-质谱法(gas chromatography-mass spectrometry,GC-MS)分析,对其生成的3-氯-1,2-丙二醇(3-MCPD)进行了研究.通过正交实验,考察了反应时间、温度和各原料用量对3-MCPD生成量的影响,同时研究了在热反应模型中分别加入不同种类单糖和氨基酸后3-MCPD生成量的变化规律,并对其反应机理进行了探讨.结果表明,反应时间2h、反应温度140℃、甘油用量0.05g(占原料总量0.31％)、NaCl用量1.0g(6.23％)、水用量15g(93.46％),3-MCPD生成量最少;葡萄糖、果糖、木糖、甘露糖、半乳糖、缬氨酸、精氨酸、赖氨酸、丝氨酸、亮氨酸、苯丙氨酸、苏氨酸、组氨酸使3-MCPD的生成量减少;谷氨酸、蛋氨酸、丙氨酸、天冬氨酸、甘氨酸使3-MCPD的生成量增加;半胱氨酸、酪氨酸、脯氨酸、色氨酸对3-MCPD的生成无明显作用.     

气相色谱法检测植物油中3-氯丙醇酯

目的建立气相色谱(GC)检测植物油中3-氯-1,2-丙二醇酯(3-氯丙醇酯,3-MCPD酯)含量的方法。方法不同浓度的3-MCPD标准溶液中加入苯硼酸(PBA)进行衍生化反应,正己烷萃取,进气相色谱检测,由3-MCPD的量和峰面积对应关系做标准曲线。测定4种食用油中3-MCPD酯含量。结果该法线性相关性良好,最低检出限0.025 mg/kg,定量限0.086 mg/kg。线性范围0.1~0.8 mg,回收率在79%~108%之间,精密度试验相对标准偏差(RSD)均小于5。市场上4种植物油中3-氯丙醇酯含量由低到高依次为花生油、大豆油、棕搁油、菜籽油,其3-MCPD酯的含量范围是3020~51520μg/kg。结论该分析方法操作简单,灵敏度高,能满足一般油脂中3-MCPD酯检测的要求。     

动植物油脂中3-氯-1,2-丙二醇酯的研究进展

随着对氯丙醇的深入研究和检测技术的不断更新,在越来越多的食品中检测出高含量的氯丙醇。目前,有许多关于在动植物油脂中检测出3-氯-1,2-丙二醇(3-MCPD)酯的报道,并得出油脂精炼过程是产生3-MCPD酯的主要途径,精炼工艺中的脱臭工序是3-MCPD酯形成的关键工序。假设3-MCPD酯的含量按照100%转化,则转化生成的3-MCPD将远远超过每日最大耐受量(TDI)2μg/kg。因此,本文对3-MCPD酯的来源及影响因素、检测方法和其在动植物油脂中的发展趋势进行了综述。     

Change of fatty acid esters of MCPD and glycidol during restaurant deep frying of fish nuggets and their correlations with total polar compounds

This study examined the fatty acid esters of 2-monochloropropane-1,2-diol (2-MCPD), 3-monochloropropane-1,2-diol (3-MCPD) and glycidyl esters (GEs) in frying oils during fish nuggets deep frying. The 3-MCPD esters significantly increased in the first 12 h and then decreased, whereas 2-MCPD esters increased to a maximum at 24 h (0.69–0.81 mg kg⁻¹) and then decreased. The GEs decreased with frying time and degraded to approximately 0.05 mg kg⁻¹ after 36 h in all frying oils. The correlation results showed that the 3-MCPD esters had a positive correlation with 2-MCPD esters (R = 0.910), so 3-MCPD esters could be an indicator of the presence of MCPD esters in frying oils. Strong correlations were found between MCPD esters and the peroxide values and p-anisidine values, indicating that changes in MCPD esters may be related to oil oxidation. These results may improve our understanding of MCPD esters and GEs in frying oils and find ways to control them during frying.     

Toxicological assessment of 3-monochloropropane-1,2-diol (3-MCPD) as a main contaminant of foodstuff in three different in vitro models: Involvement of oxidative stress and cell death signaling pathway

3-Monochloropropane-1,2-diol (3-MCPD) as a main source of food contamination has always been known as a carcinogenic agent. Kidney, liver, testis, and heart seem to be the main target organs for 3-MCPD. Because oxidative stress and mitochondrial dysfunction have been realized to be involved in 3-MCPD-induced cytotoxicity, the present study aimed to investigate the probable toxicity mechanisms of 3-MCPD in isolated mitochondria, HEK-293 cell line, and cell isolated from the rats’ liver and kidney through measuring multiparametric oxidative stress assay. Based on the data indicating no significant difference between 3-MCPD-treated groups and control group, metabolites of 3-MCPD have a key role in organ toxicity caused by them. To further investigating the suggested hypothesis, the effect of 3-MCPD toxicity on HEK-293 cell line was examined. Although the proliferation declined after exposure to a low dose of 3-MCPD (10 to 200 µM), controversial responses in higher concentration (2 to 10 mM) have led to studies on the effect of oxidative stress and cell death signaling on isolated kidney and liver cells. Treatment of the isolated kidney and liver cells with 3-MCPD resulted in an increase in the level of reactive oxygen species (ROS), the collapse of mitochondrial membrane potential (MMP), and activation of cell death signaling without creating any significant difference in the amount of reduced glutathione. In fact, 3-MCPD can disrupt the mitochondrial electron transfer in isolated cells, which is correlated with the impairment of mitochondrial oxidative phosphorylation system, the rise of ROS level, and the failure of MMP, leading to the release of cytochrome c from mitochondria to cytosol and finally the activation of cell death signaling.     

我国市售酱油氯丙醇污染研究:地区间污染水平的比较

采用稳定性同位素稀释气相质谱法定量测定多组分氯丙醇,调查了我国市售酱油氯丙醇的污染水平。调查分3次进行,所抽查的629份市售酱油产于2003~2004年间,来自我国11个省、市。各地区样品3-氯-1,2-丙二醇(3-MCPD)的检出率均较高,在90%~100%之间,3-MCPD水平在0.003~189 mg/kg之间,几何均数为21.1μg/kg;2-氯-1,3-丙二醇(2-MCPD)的检出率在14.3%~83.8%之间;1,3-二氯-2-丙醇(1,3-DCP)、2,3-二氯-1-丙醇(2,3-DCP)的检出率分别在0%~24%、0~18.9%之间,全部样品的几何均数低于定量限。总体上,我国市售酱油氯丙醇污染水平有明显下降,但以各地有限的抽查样品评价,污染的地区差异明显,来自河南、广西、浙江样品污染水平以及检出率相对较高,而福建、江苏、北京以及上海样品污染水平较低。     

The effects of domestic cooking on the levels of 3-monochloropropanediol in foods

The results are reported of a study to determine the effect of domestic cooking procedures on the level of 3-monochloropropane-1,2-diol (3-MCPD) in selected foods. Samples of 23 foods comprising stock cubes, gravies, a cake mix, batters, breads, cheese and meats were subjected to a range of cooking procedures including grilling, toasting and microwaving. The resulting levels of 3-MCPD were compared with those present in the foods before cooking. Grilling and toasting produced substantial increases in the 3-MCPD content of bread, forming up to 0.3mg/kg, and of most cheeses, resulting in levels of up to about 0.1mg/kg. Microwave cooking produced elevated 3-MCPD levels in some cheeses. Frying laboratoryproduced batters increased 3-MCPD levels to about 0.1mg/kg whereas a retail batter contained no detectable 3-MCPD when fried. The remaining foods showed little or no discernible increase on cooking.     

Fatty acid esters of 3-chloropropane-1,2-diol in edible oils.

A series of 25 virgin and refined edible oils, obtained from retailers, was analyzed for levels of free 3-chloropropane-1,2-diol (3-MCPD) and 3-MCPD released from esters with higher fatty acids (bound 3-MCPD). Oils containing free 3-MCPD ranging from <3 microg kg-1 (LOD) to 24 microg kg-1. Surprisingly, bound 3-MCPD levels were much higher and varied between <100 (LOD) and 2462 microg kg-1. On average, virgin oils had relatively low levels of bound 3-MCPD, ranging from <100 (LOD) to <300 microg kg-1 (LOQ). Higher levels of bound 3-MCPD were found in oils from roasted oilseeds (337 microg kg-1) and in the majority of refined oils (<300-2462 microg kg-1), including refined olive oils. In general, it appears that the formation of bound 3-MCPD in oils is linked to preliminary heat treatment of oilseeds and to the process of oil refining. Analysis of unrefined, de-gummed, bleached, and deodorized rapeseed oil showed that the level of bound MCPD decreased during the refining process. However, additional heating of seed oils for 30 min at temperatures ranging from 100 to 280 degrees C, and heating at 230 degrees C (260 degrees C) for up to 8 h, led to an increase in bound 3-MCPD levels. On the other hand, heating of olive oil resulted in a decrease in bound 3-MCPD levels. For comparison, fat isolated from salami was analyzed for intact fatty acid esters of 3-MCPD. This fat contained bound 3-MCPD at a level of 1670 microg kg-1 and the fatty acid esters of 3-MCPD mainly consisted of 3-MCPD diesters; monoesters of 3-MCPD were present in smaller amounts. The major types of 3-MCPD diesters (about 85%) were mixed diesters of palmitic acid with C18 fatty acids (stearic, oleic, linoleic acids). These diesters were followed by 3-MCPD distearate (11%) and 3-MCPD dipalmitate (4%). Generally, very little 3-MCPD existed as the free compound (31 microg kg-1).     

Updated occurrence of 3-monochloropropane-1,2-diol esters (3-MCPD) and glycidyl esters in infant formulas purchased in the United States between 2017 and 2019

ABSTRACT

Fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD) and glycidol are potentially carcinogenic and/or genotoxic processing contaminants that are formed during the process of edible oil refining. Because of their toxicological properties, the presence of these compounds in refined oils and foods containing these oils, particularly infant formula, poses a potential food safety concern. For this reason, recent research efforts have focussed on the development of methods for the analysis of MCPD and glycidyl esters in infant formula in order to estimate levels of exposure. This work presents occurrence data for 3-MCPD and glycidyl esters in 222 infant formulas purchased in the United States between December 2017 and January 2019. The results of this study show a wide range of contaminant concentrations across four different manufacturers, with average bound 3-MCPD concentrations ranging from 0.035 µg g^?1 to 0.63 µg g^?1 and average bound glycidol concentrations ranging from 0.019 µg g^?1 to 0.22 µg g^?1. The data suggest that manufacturers B and C source palm oil produced with mitigation measures, leading to reduced amounts of 3-MCPD and glycidyl esters in their infant formulas. Additionally, comparison with a previously published study in our laboratory of the occurrence of 3-MCPD and glycidyl esters in infant formula purchased in the U.S. between 2013 and 2016 revealed that, since 2016, contaminant concentrations have decreased in products produced by manufacturers A, B, and C, while contaminant amounts in formulas from manufacturer D have slightly increased.     

3-MCPD: A Worldwide Problem of Food Chemistry

3-Monochloropropane-1,2-diol (3-MCPD) is a heat-induced food contaminant that has been widely investigated for decades. This paper presents an overview of current knowledge about 3-MCPD, including its formation routes, occurrence in various foodstuffs, analytical approach, toxicological aspects, and future research perspectives. So far, 3-MCPD was determined in its free and bound form in thermally treated foods, edible oils and fats, and infant foods including human breast milk. Contaminants in infant foods and human breast milk were highlighted in this paper as a serious problem as they can pose a potential hazard for infants. The analytical approach of 3-MCPD determination has been modified for over a decade. Nowadays, the method based on determining the derivative of this compound by using gas chromatography and mass spectrometry is widely used. However, there is still a big need for developing new methods that would produce repeatable results. Some of the toxicologic aspects associated with 3-MCPD still remain unknown. A number of studies on the carcinogenicity and genotoxicity of 3-MCPD were carried out on rodents; however, no clinical studies on humans have been reported so far. Moreover, both detrimental effect on kidneys and antifertility activity have been widely reported. The knowledge of 3-MCPD absorption into body fluids and tissues and its metabolic pathways is based on sometimes conflicting data derived from different studies. In conclusion, although a lot of research has been carried out on 3-MCPD, there is still a need for further research in this area.     

Influence of composition,moisture, pH and temperature on the formation and decay kinetics of monochloropropanediols in wheat flour dough

A study of the formation and decay kinetics of 3-chloropropane-1,2-diol (3-MCPD) and 2-chloropropane-1,3-diol (2-MCPD) in model dough systems is reported. The influence of ingredient composition, moisture, pH and temperature were assessed. It was shown that white flour, salt and water alone were sufficient to generate 2-MCPD and 3-MCPD. The formation of 3-MCPD agreed with zero order kinetics and occurred most readily in dry, full recipe dough; below 15%, the exact moisture content was not critical. The formation of 2-MCPD followed that of 3-MCPD but at approximately one fifth of the rate, irrespective of dough type. The decay of 3-MCPD was consistent with first order kinetics and the overall rate in dough was slower than that in pure water. The decay reaction was inhibited by a decrease in moisture content and a drop in pH seen at high temperatures in the cereal systems investigated. One of the products of 3-MCPD decay in dough was 2-chloropropane-1,3-diol (2-MCPD). Data obtained from the model system studies showed good agreement with 3-MCPD levels formed in real foods. Possible mechanisms of decay and formation in dough are discussed.     

Bestimmung von Dichlorpropanolen und Monochlorpropandiolen in Würzen und würzehaltigen Lebensmitteln

Zusammenfassung Es wird eine Methode zur Bestimmung von Dichlorpropanolen (DCP) und Monochlorpropandiolen (MCPD) in Würzen und würzehaltigen Lebensmitteln beschrieben. Nach Adsorption an einer Extrelut-Säule werden die DCP und MCPD mit Ethylacetat extrahiert. Saure Begleitsubstanzen werden durch eine schwache Alkalisierung in der wäßrigen Phase zurückgehalten. Im eingeengten Ethylacetat-Extrakt werden die DCP und MCPD durch Capillargaschromatographie mit massenselektiver Detektion (EI, SIM-Modus) bestimmt, wobei die Nachweisgrenzen unter 0,05 mg/kg für die DCP bzw. 0,1 mg/kg für die MCPD liegen. Mittels GC/MS konnte in Würzen neben 3-MCPD auch das isomere 2-MCPD identifiziert werden. Es wurde daher in die Untersuchungen mit einbezogen. Dabei zeigte sich, daß in manchen Proben der 2-MCPD-Gehalt höher lag als der 3-MCPD-Gehalt. Über die Untersuchungsergebnisse wird berichtet.

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Determination of dichloropropanols and monochloropropandiols in seasonings and in foodstuffs containing seasonings

Summary A method is described for the determination of dichloropropanols (DCP) and monochloropropandiols (MCPD) in seasonings and in foodstuffs containing seasonings. After adsorption onto an Extrelut column DCP and MCPD are extracted using ethyl acetate. Due to a weak alkalisation, acid compounds remain in the water phase. The ethyl acetate extract is concentrated and DCP and MCPD are determined by capillary gas chromatography with mass selective detection (Ei, SIM mode). The detection limits are below 0.05 mg/kg for DCP and 0.1 mg/kg for MCPD. In seasonings, 3-MCPD and its isomer, 2-MCPD, were identified by GC/MS analysis. Therefore 2-MCPD was included in the investigations. In some samples 2-MCPD was the predominating isomer. The results of the investigations are reported.