Fernández Mariana F, Rivas Ana, Olea-Serrano Fátima, Cerrillo Isabel, Molina-Molina José M, Araque Patricia, Martínez-Vidal José L, Olea Nicolas
Laboratory of Medical Investigations, Hospital Clínico, University of Granada, 18071 Granada, Spain.
Anal Bioanal Chem. 2004 May;379(1):163-70. doi: 10.1007/s00216-004-2558-5. Epub 2004 Mar 13.
Test systems to screen for estrogenicity and appropriate biomarkers of human exposure are required for epidemiological studies of endocrine disruption. We addressed these issues by developing and standardising a method to assess the total estrogenic xenobiotic burden in human adipose tissue. In this study, which is the continuation of a previous work, we have improved the protocol for extensive fractionation of a higher number of tissue samples in order to investigate bioaccumulated xenoestrogens that are candidates for estrogenicity and to assess their combined estrogenic effect. This was achieved by extensive HPLC separation of xenoestrogens from endogenous hormones followed by testing of individual fractions in the E-Screen test for estrogenicity. Organochlorine pesticides, PCBs and halogenated bisphenols and alkylphenols were collected in the most lipophilic fractions, followed by progestins, androgens and estradiol esters, and then by steroidal estrogens; phyto- and myco-estrogens were collected around the end of the run. These results were confirmed by exhaustive chemical analysis. In 458 human adipose tissue samples, the total effective xenoestrogen burden was positive in 75% of samples in the pooled fraction that contained organohalogenated xenoestrogens (mean 515.3 pM Eeq/g lipid; range 0-14.5 nM) and in 82% of samples in the pooled fraction where natural estrogens eluted (mean 696.6 pM Eeq/g lipid; range 0-12.9 nM). Organochlorine pesticides emerged as candidate chemicals for the estrogenicity of the first pooled fraction, because DDT and derivatives were present in 98.3% of the samples. However, no correlation was found between the concentration of any single chemical and the estrogenicity determined in the bioassay. There may be several reasons for this lack of concordance: (i) the estrogenic effects depicted in the E-Screen bioassay are a consequence of the combined effect of several organohalogens or (ii) the proliferative effect is due to other chemicals not measured. Because additive, synergistic or antagonistic mechanisms may account for the final effect observed in the pooled fractions, the approach proposed in this work is more appropriate for exposure assessment in epidemiological studies than the determination of individual chemicals in human samples.
对于内分泌干扰的流行病学研究而言，需要用于筛查雌激素活性以及人类暴露的合适生物标志物的测试系统。我们通过开发和标准化一种评估人体脂肪组织中总雌激素性外源性物质负荷的方法来解决这些问题。在这项作为先前工作延续的研究中，我们改进了方案，以便对更多组织样本进行广泛分级分离，从而研究作为雌激素活性候选物的生物累积性外源性雌激素，并评估它们的联合雌激素效应。这是通过高效液相色谱法从内源性激素中广泛分离外源性雌激素，然后在E-Screen试验中对各个级分进行雌激素活性测试来实现的。有机氯农药、多氯联苯、卤代双酚和烷基酚集中在最具亲脂性的级分中，其次是孕激素、雄激素和雌二醇酯，然后是甾体雌激素；植物雌激素和霉菌雌激素在运行接近尾声时被收集。这些结果通过详尽的化学分析得到了证实。在458份人体脂肪组织样本中，在含有有机卤代外源性雌激素的合并级分中，75%的样本总有效外源性雌激素负荷呈阳性（平均515.3 pM Eeq/g脂质；范围0 - 14.5 nM），在天然雌激素洗脱的合并级分中，82%的样本呈阳性（平均696.6 pM Eeq/g脂质；范围0 - 12.9 nM）。有机氯农药成为第一个合并级分雌激素活性的候选化学物质，因为98.3%的样本中存在滴滴涕及其衍生物。然而，在生物测定中未发现任何单一化学物质的浓度与雌激素活性之间存在相关性。这种不一致可能有几个原因：（i）E-Screen生物测定中所描述的雌激素效应是几种有机卤化物联合效应的结果，或者（ii）增殖效应是由于未测量的其他化学物质引起的。由于相加、协同或拮抗机制可能解释了在合并级分中观察到的最终效应，因此本研究中提出的方法比测定人体样本中的单个化学物质更适合用于流行病学研究中的暴露评估。
