A binary screening assay for pro-oestrogens in food: metabolic activation using hepatic microsomes and detection with oestrogen sensitive recombinant yeast cells.
An assay, employing microsomes prepared from rat liver and a recombinant cell bioassay (RCBA) expressing the human oestrogen receptor (alpha) linked to a reporter gene, was evaluated for the detection of pro-oestrogens in food using methoxychlor and mestranol as model compounds. Bio-activation of the hop phytoestrogen isoxanthohumol to the potent oestrogen 8-prenylnaringenin was also investigated. The oestrogenic potency values for reference standards determined with the RCBA (17beta-oestradiol = 100%) were: methoxychlor 0.0025%, mestranol 1.3%, isoxanthohumol 0.001%, and for their potential respective metabolites were: bishydroxymethoxychlor 0.015%, 17alpha-ethynyl oestradiol 69% and 8-prenylnaringenin 0.4%. Incubation of methoxychlor and mestranol (10 microM) with microsomes prepared from the liver of rats treated with Aroclor 1254 significantly increased (p < 0.001) their oestrogenic potency from 0.0021 and 2.4% to 0.015 and 8.3%, respectively. In contrast, the potency of the hop phytoestrogen isoxanthohumol was unchanged. Metabolites were identified by UV-HPLC-MS/MS as monohydroxy methoxychlor and HPTE from methoxychlor, and the major metabolite of mestranol was 17alpha-ethynyl oestradiol. There was no evidence for the metabolism of isoxanthohumol. Mestranol was also activated by microsomes induced with saline (control), beta-napthoflavone, 3-methylcholantherene, isoniazid or pregnenolone-16alpha-carbonitrile, but not phenobarbitone. These studies demonstrate the principle for use of a binary assay system for the detection of pro-oestrogens and indicate the potential value for risk assessment of endocrine disrupting chemicals.
Coldham
N G
NG
Department of Risk Research, Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB, UK. n.g.coldham@vla.defra.gsi.gov.uk
Horton
R
R
Byford
M F
MF
Sauer
M J
MJ
D000818:Animals; D001681:Biological Assay / Q000379:methods; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D045166:Estradiol Congeners / Q000032:analysis* / Q000493:pharmacokinetics; D047628:Estrogen Receptor alpha; D005504:Food Analysis / Q000379:methods*; D005506:Food Contamination / Q000032:analysis*; D017930:Genes, Reporter; D006801:Humans; D007306:Insecticides / Q000032:analysis; D008656:Mestranol / Q000032:analysis; D008731:Methoxychlor / Q000032:analysis; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D011960:Receptors, Estrogen / Q000235:genetics; D015003:Yeasts / Q000187:drug effects
D045166:Estradiol Congeners; D047628:Estrogen Receptor alpha; D007306:Insecticides; D011960:Receptors, Estrogen; D008656:Mestranol; D008731:Methoxychlor