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

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.
2. P. Grennwald, J. J. Barlow and P. C. Masca, New Eng. J. Med., 1971, 285, 390.
3. G. H. Gass, D. Coats and N. Graham, J. Natl. Cancer Inst., 1964, 33, 971.
4. J. Maddox, Nature (London), 1995, 377, 381.
5. R. G. Glencross, I. B. Munro, B. E. Senior and G. S. Pope, Acta Endocrinol., 1973, 73, 374.
6. R. G. Glencross and G. S. Pope, Anim. Reprod. Sci., 1981, 4, 93.
7. R. G. Glencross, E. C. L. Bleach, S. C. Wood and P. G. Knight, J. Reprod. Fertil., 1994, 100, 599.
8. C. J. M. Arts, P. T. W. Kemperman and H. Van Den Berg, Food Addit. Contam., 1989, 6, 103.
9. C. J. M. Arts and H. Van den Berg, J. Chromatogr., 1989, 489, 225.
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.
12. B. L. Rubin, A. S. Dorfman, L. Black and R. I. Dorfman, Endocrinology, 1951, 49, 429.
13. M. L. Bates, M. J. Warwick and G. Shearer, Food Addit. Contam., 1985, 2, 37.
14. E. A. I. Daeseleire, A. De Guesquiere and C. H. Van Peteghem, J. Chromatogr. Sci., 1992, 30, 409.
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.
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.
17. N. G. Coldham, M. Dave, S. Sivapathasundaram, D. P. McDonnell, C. Connor and M. J. Sauer, Environ. Health Perspect., 1997, 105, 734.
18. S. F. Arnold, M. K. Robinson, A. C. Notides, L. J. Guillette and J. A. McLachlan, Environ. Health Perspect., 1996, 104, 544.
19. S. F. Arnold, D. M. Klotz, B. M. Collins, P. M. Vonier, L. J. Guilette and J. A. McLachlan, Science, 1996, 272, 1489.
20. E. J. Routledge and J. F. Sumpter, Environ. Toxicol. Chem., 1996, 15, 241.
21. K. O. Klein, J. Baron, M. J. Colli, D. P. McDonnell and G. B. Cutler, J. Clin. Invest., 1994, 94, 22475.
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.
23. H. Dobson and P. D. G. Dean, J. Endocrinol., 1974, 61, 479.
24. R. J. Hietzman, Environ. Qual. Safety, 1976, 5, 89 and references cited therein.
25. J. Saumande and A. Lopez-Sebastian, J. Reprod. Fertil., 1982, 66, 411.