La plus ça change, la plus c’est la même chose—Will Bioassays Make a Comeback?

William Rosner

Department of Endocrinology St. Luke’s-Roosevelt Hospital Center New York, New York 10019

Address correspondence and requests for reprints to: William Rosner, M.D., Department of Endocrinology, St. Luke’s-Roosevelt Hospital Center, 1000 Tenth Avenue, New York, New York 10019.


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It is fitting that in the early days of the 21st century, we are presented with a new bioassay for androgens. Arguably, a primitive bioassay for androgens may have been the first seed from which the tree of endocrinology grew.

"The date of birth of ‘the science of internal secretion’ is that memorable meeting of the Societé de Biologie of Paris of June 1st 1889, where Brown-Séquard, then 72 yr of age reported on his experiments undertaken to prove his hypothesis by means of subcutaneous injections of testicular juice into himself" (1), quoted in Ref. 2 .

In the days when the general paradigm in endocrinology consisted of observing a biological effect and then finding the hormone that caused it, bioassays were irreplaceable. Once "all the hormones were discovered," bioassays became the bottleneck through which quantitative advances in our knowledge had to flow. As hormones became chemically identified, methods for their assay based on their chemical properties were developed, at least in the case of hormones that were not proteins. Subsequently, immunologically based methods revolutionized not only hormone assays but also the whole of endocrinology, as evidenced by a Nobel Prize to Rosalyn Yalow in 1977. Compared with bioassays in whole animals, immunoassays have the advantage of being inexpensive, rapid, sensitive, specific, and precise. Chemically or immunologically based assays are based on structure rather than function and, if all goes well and the method is as claimed, there is no unintended interference in the assay.

One of the many reasons for advancing beyond bioassays, when assaying mixtures of biological origin, is the ability to assign the response observed in the assay to a chemically homogeneous substance. Specificity is at the core of modern assays. Indeed, in immunologically based assays, substances that cross-react with the material to be measured compromise the assay. In this respect, the cell-based bioassay devised by Raivio et al. (3) deliberately suffers from this flaw. Because this bioassay, as do all others, quantifies an androgenic response, rather than a specific androgen, it measures all of the compounds that are capable of eliciting the response.

The specificity and sensitivity of the current assay are based, respectively, on the binding specificity of the androgen receptor and the Kd that governs the binding. The androgen receptor, when transfected into a cell, behaves just as it does in the intact organism. It recognizes and responds to a variety of structures, including nonsteroidal antiandrogens. Added to this basic principle is an impressive piece of molecular legerdemain that not only allows the assay system to be expressed in cells in culture, but gives it a large dynamic range, with a response at maximum inputs that is over 700-fold that at the sensitivity of the assay. The reporting module of the assay is luminescence, safe, and simple. The detecting module of the assay, binding to the androgen receptor, is precisely parallel in principle to early ligand-binding assays for estrogens that used solubilized receptors from target tissues as central reagents (4, 5). Indeed, a 21st century version of such an assay for estrogens, similar in principle to the one by Raivio et al. (3), was published recently (6). As already implied, these kinds of receptor-based assays have a property that is at the same moment their blight and their blessing—they measure biological activity with small regard to structure.

The Raivio assay is reported in units of testosterone equivalence. What is measured bears a curvilinear relationship to the actual testosterone concentration in the sample, and the absolute values are substantially smaller than the testosterone actually present in the plasma being assayed. Both the nonlinear relationship and the underestimation are strongly influenced by binding to the plasma proteins, sex hormone-binding globulin, and albumin. Hence, the results of the assay in a given sample are determined not only by the concentration of androgen, as is desirable, but by the concentration of the proteins to which they bind. Finally, the reproducibility of the assay, a coefficient of variation of 8.3% within assays and 21% between assays, is excellent for a bioassay but not so good when compared with modern immunoassays. Part of the between assay variation arises from the fact that transient transfections with five different plasmids must be done to create the "reagent." Stably transfected cells might improve this parameter; it certainly would make the assay more easily accessible to those who would use it.

One might get the impression from the foregoing that I find this assay not to be a useful addition to our endocrinologic bag of tricks. Not at all. I like it. However, I do not believe it will find a place in the regular practice of clinical endocrinology. In that setting, we need more accuracy and specificity, not less. Where this assay will find a home is in those settings where bioassays are currently needed, and will be needed in the future. We need androgens that build muscles and bone, but are reproductively inert, and that increase libido without causing hirsutism and alopecia. We need bioassays for bioactive compounds in the environment, so-called endocrine disrupters. We need antiandrogens that deprive only prostate cancer, not other androgen target tissues, of their hormone. To unearth useful compounds in the laboratory, and harmful ones in the environment, bioassays are irreplaceable. This one could find a place in that milieu.

Received March 21, 2001.

Accepted March 21, 2001.


    References
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 Introduction
 References
 

  1. Biedel A.1910 Innere Sekretion. Berlin and Vienna: Urban und Schwarzenberg.
  2. Tausk M.1984 The emergence of endocrinology. In: Parnham MJ, Bruinvels J, eds. Discoveries in pharmacology: haemodynamica, hormones & inflammation. Elsevier; 219–249.
  3. Raivio T, Palvimo JJ, Dunkel L, Wickman S, Jänne OA. 2001 Novel assay for determination of androgen bioactivity in human serum. J Clin Endocrinol Metab. 86:1539–1544.[Abstract/Free Full Text]
  4. Korenman SG. 1969 Comparative binding affinity of estrogens and its relation to estrogenic potency. Steroids. 13:163–177.[CrossRef][Medline]
  5. Korenman SG. 1968 Radio-ligand binding assay of specific estrogens using a soluble uterine macromolecule. J Clin Endocrinol Metab. 28:127–130.[Medline]
  6. Rogers JM, Denison MS. 2000 Recombinant cell bioassays for endocrine disruptors: development of a stably transfected human ovarian cell line for the detection of estrogenic and anti-estrogenic chemicals. In Vitro Mol Toxicol. 13:67–82.[Medline]




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