title: Metabolic profiling of endocrine-disrupting compounds by on-line cytochrome p450 bioreaction coupled to on-line receptor affinity screening.
abstract: We present a fully automated and hyphenated bioanalytical method for metabolic profiling of potentially harmful xenoestrogens. The system consists of an on-line cytochrome P450 bioreactor coupled to a reversed-phase, gradient high-performance liquid chromatograph. A C18 solid-phase extraction (SPE) unit is used as an interface between the P450 bioreactor and the HPLC column. The HPLC column is linked on-line to a high-resolution screening (HRS)-estrogen receptor alpha affinity detection (ERAD) assay. In effect, the P450 bioreactor produces metabolites that are subsequently trapped on-line by SPE and separated by HPLC. The separated metabolites are then screened on-line, at the moment of elution, for affinity toward estrogen receptor alpha (ERalpha) using the HRS-ERAD assay. The SPE method was optimized with methoxychlor (MXC) and its metabolites mono- and bis-OH-MXC. After optimization, the P450-bioreactor-SPE-HPLC system was made generally applicable to the biocatalysis and trapping of polar to highly apolar compounds. The precision of the P450-bioreactor-SPE-HPLC system is high (relative standard deviation<or=15%), and the HRS-ERAD assay is also very sensitive (having lower limits of detection of 250 ng for bis-OH-MXC and 240 ng for mono-OH-MXC). Finally, bioactivation of 2-hydroxy-4-methoxybenzophenone (benzophenone-3) into ERalpha-binding metabolites by P450 was studied using the validated P450-bioreactor-SPE-HPLC-ERAD system in combination with atmospheric pressure chemical ionization MS. This resulted in the detection of three ERalpha-binding metabolites, of which at least one, a hydroxylated metabolite initially detected only by ERalpha affinity, had not been described previously. The hyphenated P450-bioreactor-SPE-HPLC-HRS-ERAD methodology presented here will be of great interest in on-line research of metabolic activation of endocrine-disrupting compounds.
authors.affiliation: LACDR-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: Sebastiaan M
authors.initials: SM
authors.lastname: Van Liempd
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kool
authors.forename: John H
authors.initials: JH
authors.lastname: Meerman
authors.forename: Hubertus
authors.initials: H
authors.lastname: Irth
authors.forename: Nico P
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D000818:Animals; D001331:Automation; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D052244:Endocrine Disruptors / Q000378:metabolism / Q000633:toxicity*; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D052244:Endocrine Disruptors; D047628:Estrogen Receptor alpha; D003577:Cytochrome P-450 Enzyme System