Abstract
BackgroundEpidemiological documentation of endocrine disruption is complicated by imprecise exposure assessment, especially when exposures are mixed. Even if the estrogenic activity of all compounds were known, the combined effect of possible additive and/or inhibiting interaction of xenoestrogens in a biological sample may be difficult to predict from chemical analysis of single compounds alone. Thus, analysis of mixtures allows evaluation of combined effects of chemicals each present at low concentrations.MethodsWe have developed an optimized in vitro E-Screen test to assess the combined functional estrogenic response of human serum. The xenoestrogens in serum were separated from endogenous steroids and pharmaceuticals by solid-phase extraction followed by fractionation by high-performance liquid chromatography. After dissolution of the isolated fraction in ethanol-DMSO, the reconstituted extract was added with estrogen-depleted fetal calf serum to MCF-7 cells, the growth of which is stimulated by estrogen. After a 6-day incubation on a microwell plate, cell proliferation was assessed and compared with the effect of a 17-beta-estradiol standard.Results and conclusionsTo determine the applicability of this approach, we assessed the estrogenicity of serum samples from 30 pregnant and 60 non-pregnant Danish women thought to be exposed only to low levels of endocrine disruptors. We also studied 211 serum samples from pregnant Faroese women, whose marine diet included whale blubber that contain a high concentration of persistent halogenated pollutants. The estrogenicity of the serum from Danish controls exceeded the background in 22.7 % of the cases, while the same was true for 68.1 % of the Faroese samples. The increased estrogenicity response did not correlate with the lipid-based concentrations of individual suspected endocrine disruptors in the Faroese samples. When added along with the estradiol standard, an indication of an enhanced estrogenic response was found in most cases. Thus, the in vitro estrogenicity response offers a promising and feasible approach for an aggregated exposure assessment for xenoestrogens in serum.
Highlights
Epidemiological documentation of endocrine disruption is complicated by imprecise exposure assessment, especially when exposures are mixed
The compounds were monitored by diode-array detection, and a library based on retention time as well as the UV-spectrum was generated
Recovery studies were performed by comparing the peak area obtained from a supplemented serum sample, extracted and analyzed as described above, with the peak area obtained from direct injection of an equivalent amount of the pure standard
Summary
Epidemiological documentation of endocrine disruption is complicated by imprecise exposure assessment, especially when exposures are mixed. Even if the estrogenic activity of all compounds were known, the combined effect of possible additive and/or inhibiting interaction of xenoestrogens in a biological sample may be difficult to predict from chemical analysis of single compounds alone. Epidemiological research on endocrine disruption is hampered by difficulties in assessment of mixed exposures. A promising approach to assess the combined functional estrogenic response in a serum sample was published by Sonnenschein, Soto and coworkers in 1995 [1]. This approach has been further developed and refined by research groups in the US [2], in Denmark [3] and in Spain [4]. Many xenoestrogens identified so far are generally lipophilic, suggesting that a separation based on lipophilicity may constitute a feasible compromise
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