Analytical and Bioanalytical Chemistry

, Volume 385, Issue 5, pp 875–887

SPE-HPLC purification of endocrine-disrupting compounds from human serum for assessment of xenoestrogenic activity

  • Philip Sebastian Hjelmborg
  • Mandana Ghisari
  • Eva Cecilie Bonefeld-Jorgensen
Original Paper

Abstract

Assessment of xenoestrogenic activity in human serum samples requires the removal of endogenous sex hormones to assure that the activity measured originates from xenobiotic compounds only. Serum samples representing high, medium and lower accumulation of persistent organic pollutants (POPs) were extracted using solid-phase extraction (SPE) followed by normal-phase high-performance liquid chromatography (NP-HPLC) for separation of POPs from endogenous hormones. The recovery of polychlorinated biphenyl (PCB) congeners in spiked serum samples was up to 86 %, making the extraction method suitable for the study. MVLN cells, stably transfected with an estrogen receptor (ER) luciferase reporter vector (estrogen response element chemically activated luciferase expression, ERE-CALUX), were exposed to the reconstituted SPE-HPLC extracts for determination of the integrated estrogenic activity. The effects of PCBs were analyzed by direct in vitro exposure of PCBs (nos. 138, 153, 180) and by ex vivo analysis of SPE-HPLC extracts from serum spiked with the PCBs. Similar effects on ER transactivation were observed for the direct in vitro and the ex vivo analysis experiments. The ER transactivation responses determined for actual serum samples were in the linear range of the dose-response curve. 17β-Estradiol titrations showed that the xenoestrogenic effects were mediated via ER. Moreover, our SPE-HPLC-ERE-CALUX assay was demonstrated to elicit high interlaboratory correlation. In the present study the combination of SPE-HPLC purification and the ex vivo estrogenic responses measured by ERE-CALUX was validated and considered to be a valuable tool to assess the combined ER effect of lipophilic serum POPs where additive/synergistic and agonistic/antagonistic effects are integrated giving an overall estimate of exposure and bioactivity.

Keywords

Solid-phase extraction–high-performance liquid chromatography Estrogen response element chemically activated luciferase expression MVLN Endocrine disruption Xenoestrogens 

Abbreviations

BPA

Bisphenol A

BPA-DM

Bisphenol A dimethacrylate

CALUX

Chemically activated luciferase expression

CV

Coefficient of variation

DC-FCS

Dextran-treated fetal calf serum

DDE

Dichlorodiphenyl dichloroethylene

DDT

Dichlorodiphenyl trichloroethane

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

Dimethyl sulfoxide

E1

Estrone

E2

17β-Estradiol

EC40

Concentration exerting 40 % of the effect of the maximal effective concentration

ER

Estrogen receptor

ERE

Estrogen response element

GC

Gas chromatography

HPLC

High-performance liquid chromatography

KHF

Female serum control

KHM

Male serum control

logKow

Logarithm of the octanol–water partitioning coefficient

LTH

Laboratoire de la Toxicologie Humaine

MS

Mass spectrometry

NP

4n-Nonylphenol

PCB

Polychlorinated biphenyl

POP

Persistent organic pollutant

SPE

Solid-phase extraction

tR

Retention time

ɛo

Eluotropic strength

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Philip Sebastian Hjelmborg
    • 1
  • Mandana Ghisari
    • 1
  • Eva Cecilie Bonefeld-Jorgensen
    • 1
  1. 1.Unit of Cellular and Molecular Toxicology, Institute of Public Health, Department of Environmental and Occupational MedicineUniversity of AarhusAarhus CDenmark

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