Determination of oestrogen concentrations in bovine plasma by a recombinant oestrogen receptor–reporter gene yeast bioassay†

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Graham C. Burdge, Nicholas G. Coldham, Mehul Dave, Maurice J. Sauer and Emma C. L. Bleach


Abstract

A recombinant cell yeast bioassay (RCBA) was applied to the generic measurement of bovine plasma oestrogen concentration. Samples were prepared by diethyl ether extraction of plasma following addition of [3H]17β-oestradiol as internal standard; organic and aqueous phases were separated by freezing (recovery 97.1 ± 0.7%) and dried extract reconstituted in culture medium (recovery 31.4 ± 4.5%). Plasma oestrogen concentrations were measured by incubation of extracts with yeast containing a stable human oestrogen receptor (hER) and a reporter construct comprising an hER response element regulating β-galactosidase expression. The linearity of response for the analysis of spiked plasma samples using the RCBA, following corrections, is described by y = 0.8994x – 0.111 (r2 = 0.9776, P < 0.0001). Inter-assay variation for endogenous oestrogen was 11.5% for >1 pg ml–1. Plasma oestrogen concentrations for intact (n = 5) and castrated (n = 3) males were <0.5 pg ml–1, and 3.7 ± 2.6 pg ml–1 for luteal phase females (n = 10). Analysis by RCBA of sequential samples from heifers during the reproductive cycle failed to detect the pre-ovulatory increase in plasma 17β-oestradiol as determined by radioimmunoassay (RIA) (maximal concentrations 2.09 ± 2.1 pg ml–1 and 32.6 ± 14.6 pg ml–1, respectively). Interestingly, when samples were hydrolysed using Helix pomatia glucuronidase the RCBA gave concentrations (29.5 ± 8.9 pg ml–1) not significantly different to those obtained by RIA. These preliminary findings suggest that a substantial proportion of plasma oestrogen during the pre-ovulatory period may be conjugated. These data indicate the potential of the RCBA to measure biologically active and physiological levels of plasma oestrogens in cattle. One potentially valuable application of this generic oestrogen assay could be in surveillance programmes to detect illegal use of anabolic oestrogens in live-stock where the identity of the analyte may be unknown.


References

  1. A. L. Herbst, H. Ulfelder and D. C. Poskanzer, New Eng. J. Med., 1971, 284, 878 Search PubMed.
  2. P. Grennwald, J. J. Barlow and P. C. Masca, New Eng. J. Med., 1971, 285, 390 Search PubMed.
  3. G. H. Gass, D. Coats and N. Graham, J. Natl. Cancer Inst., 1964, 33, 971 CAS.
  4. J. Maddox, Nature (London), 1995, 377, 381 CrossRef CAS.
  5. R. G. Glencross, I. B. Munro, B. E. Senior and G. S. Pope, Acta Endocrinol., 1973, 73, 374 Search PubMed.
  6. R. G. Glencross and G. S. Pope, Anim. Reprod. Sci., 1981, 4, 93 CrossRef CAS.
  7. R. G. Glencross, E. C. L. Bleach, S. C. Wood and P. G. Knight, J. Reprod. Fertil., 1994, 100, 599 Search PubMed.
  8. C. J. M. Arts, P. T. W. Kemperman and H. Van Den Berg, Food Addit. Contam., 1989, 6, 103 CAS.
  9. C. J. M. Arts and H. Van den Berg, J. Chromatogr., 1989, 489, 225 CrossRef.
  10. D. N. Axelson, D. N. Kirk, R. D. Farrant, G. Cooley, A. M. Lawson and K. D. R. Setchell, Biochem. J., 1982, 201, 353.
  11. W. V. Welshons, G. E. Rottinghaus, D. J. Nonneman, M. Dolan-Timpe and P. F. Ross, J. Vet. Diagn. Invest., 1990, 2, 268 Search PubMed.
  12. B. L. Rubin, A. S. Dorfman, L. Black and R. I. Dorfman, Endocrinology, 1951, 49, 429 Search PubMed.
  13. M. L. Bates, M. J. Warwick and G. Shearer, Food Addit. Contam., 1985, 2, 37 CAS.
  14. E. A. I. Daeseleire, A. De Guesquiere and C. H. Van Peteghem, J. Chromatogr. Sci., 1992, 30, 409 CAS.
  15. R. Renaville, S. Massart, G. Lognay, A. Devolder, M. Sneyers, M. Marlier, M. Severin, A. Burny and D. Portelle, Anim. Prod., 1994, 59, 189 Search PubMed.
  16. D. P. McDonell, Z. Nawaz, C. Densmore, N. L. Weigel, T. A. Pham, J. H. Clark and B. W. O'Malley, J. Steroid Biochem. Mol. Biol, 1991, 39, 291 CrossRef.
  17. N. G. Coldham, M. Dave, S. Sivapathasundaram, D. P. McDonnell, C. Connor and M. J. Sauer, Environ. Health Perspect., 1997, 105, 734 CAS.
  18. S. F. Arnold, M. K. Robinson, A. C. Notides, L. J. Guillette and J. A. McLachlan, Environ. Health Perspect., 1996, 104, 544 CAS.
  19. S. F. Arnold, D. M. Klotz, B. M. Collins, P. M. Vonier, L. J. Guilette and J. A. McLachlan, Science, 1996, 272, 1489 CAS.
  20. E. J. Routledge and J. F. Sumpter, Environ. Toxicol. Chem., 1996, 15, 241 CAS.
  21. K. O. Klein, J. Baron, M. J. Colli, D. P. McDonnell and G. B. Cutler, J. Clin. Invest., 1994, 94, 22475 Search PubMed.
  22. D. S. Tannetta, M. D. Fray, J. H. M. Wrathall, E. C. L. Bleach, R. G. Glencross and P. G. Knight, J. Reprod. Fertil., 1997, 110, 255 Search PubMed.
  23. H. Dobson and P. D. G. Dean, J. Endocrinol., 1974, 61, 479 Search PubMed.
  24. R. J. Hietzman, Environ. Qual. Safety, 1976, 5, 89 Search PubMed and references cited therein.
  25. J. Saumande and A. Lopez-Sebastian, J. Reprod. Fertil., 1982, 66, 411 Search PubMed.
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