Human Reproduction Update Advance Access originally published online on December 4, 2007
Human Reproduction Update 2008 14(1):73-82; doi:10.1093/humupd/dmm038
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In vitro bioassays for androgens and their diagnostic applications
1 Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, Uttaranchal, India 2 Endocrinology and Metabolism Unit, Evgenidion Hospital 3 Department of Medical Therapeutics, Alexandra Hospital, Athens University School of Medicine, 80, Vassilios Sofias Avenue, 11528 Athens, Greece 4 Department of Reproductive Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK
5 Correspondence address. Fax: +44-20-75942184; E-mail: ilpo.huhtaniemi{at}imperil.ac.uk
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Abstract |
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 TOP Abstract IntroductionFactors Affecting Circulating...Androgen BioassaysAndrogen In Vitro Bioassays...Clinical Applications of the...Conclusions and Future...References |
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Androgen levels are measured in today's clinical practice almost exclusively by immunoassays. The androgen that is most frequently determined is testosterone (T), but sometimes also the levels of other testicular, ovarian and adrenal androgens such as 5
-dihydrotestosterone, androstenedione, dehydroepiandrosterone and its sulphate may be determined. In many instances, especially when androgen levels are low (as in women and children), the quality of the immunomeasurements is insufficient and the correlation between hormone levels and clinical symptoms is poor. One alternative to improve the clinical relevance of androgen measurements is provided by the recently developed in vitro bioassays of total androgen bioactivity in serum. These assays are not yet ready for routine laboratory diagnostics, but they provide a useful tool for clinical research in disturbances of androgen production. Another application of these assays is the screening for androgenic and antiandrogenic activity in chemical compounds, environmental samples and when suspecting androgen abuse. The purpose of this article is to introduce the current problems of androgen measurement by immunoassays, to describe the novel in vitro bioassay techniques and to review the current information on their application in clinical research.
Key words: androgens / endocrinology / testosterone / immunoassays / bioassays
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Introduction |
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TOPAbstractIntroductionFactors Affecting Circulating...Androgen BioassaysAndrogen In Vitro Bioassays...Clinical Applications of the...Conclusions and Future...References |
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Androgens or male sex hormones are a class of steroid hormones produced in the male by testicular Leydig cells, in the female by ovarian theca cells, and in both sexes by zona reticularis of the adrenal cortex. Structurally they all belong to the 19-carbon androstane series of steroids. The vast number of molecules with biological androgen activity is always based on their ability to bind to and activate the androgen receptor (AR), a ligand activated transcription factor, and to induce thereby androgen-specific effects on gene expression.
Androgen levels are measured in current clinical practice almost exclusively by immunoassays, which are based on an antibody's ability to recognize a specific chemical structure of the steroid molecule. The assays have variable specificity and sensitivity and the immunoreactivity detected does not necessarily correlate with the overall androgenic bioactivity in the sample, because the basis of antigen recognition is structural, not functional, and the antibody does not have the same binding specificity as AR. An alternative to immunoassays is gas chromatography-mass spectrometry (GC-MS), a method that is very specific but more labour-intensive and expensive for routine clinical use. Furthermore, although it measures accurately molecules with specific chemical structure, it does not monitor their bioactivity.
The first hormone assays were based on measurement of hormonal effects in vivo. These assays were insensitive and labour-intensive, but their functional relevance was good. Novel in vitro bioassays are now available for several hormones, and they often manage to combine the practicality and high sensitivity of immunoassays with the functional relevance of bioassays. We will review here the current in vitro methods to determine androgenic and antiandrogenic bioactivities. We first describe briefly the methodology of the assays, then their applicability for the detection of androgenic and antiandrogenic activities of chemical compounds and environmental samples, and finish by describing their use in androgen measurements in clinical situations.
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Factors Affecting Circulating Androgen Bioactivity |
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TOPAbstractIntroductionFactors Affecting Circulating...Androgen BioassaysAndrogen In Vitro Bioassays...Clinical Applications of the...Conclusions and Future...References |
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The most important circulating androgen of gonadal origin is testosterone (T) (Table 1). It is converted in several extragonadal target tissues of androgens (e.g. prostate and hair follicle) to the biologically more active androgen, 5
-dihydrotestosterone (DHT), which however is present at much lower concentration in peripheral serum. Quantitatively the most important adrenal androgens are dehydroepiandrosterone (DHEA) and its sulphate conjugate. Other weaker androgens include the T precursor androstenedione, its reduced metabolite androsterone and the T metabolites 3-androstanediol and 3β-androstanediol. Many androgens are present in circulation in much higher concentrations as their sulphate or glucuronide conjugates. Although the conjugates lack biological activity they may serve through deconjugation as a pool of precursors for biologically active steroids. The overall androgenic activity in circulation is thus the sum of T, DHT and their precursors and metabolites with weaker androgenic bioactivity. The intrinsic bioactivity per molecule of the latter is low, only 0.1–1% of that of T and DHT (Gao et al., 2005
; Jasuja et al., 2005). The picture is complicated further by the fact that some of the androgenic compounds in circulation have antiandrogenic activities upon binding to AR (Chen et al., 2004, 2005). Furthermore, the conversion of weak androgens into their more active metabolites may occur at the end-organ level, such as 5-reduction of T to DHT in prostate (Labrie, 2004). Hence, the measurement by immunoassay of the serum level of a single androgenic hormone, e.g. T, may give an oversimplified picture about the overall level of biologically active androgens.
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All androgens exert their biological activity on their target cells through binding to AR, which interaction triggers the target cell specific responses in gene expression characteristic of androgen actions (Fig. 1). In addition, there is a non-classical non-genomic action that is mediated by putative membrane receptors (Papakonstanti et al., 2003
; Lange et al., 2007), the identity of which has so far remained elusive. T and DHT are by far the most potent androgens, as reflected by their affinity to bind to AR and to trigger functional responses. The other circulating androgens, mainly of adrenal origin, are in this respect much weaker, albeit not negligible. Their role may become functionally significant when the gonadal androgen levels are low, as in children or in post-menopausal women. Some androgen activity is also possessed by other steroids, e.g. progestins, many non-steroidal pharmacological compounds, as well as by a variety of non-hormonal chemical compounds (Gao et al., 2005). Hence, if an individual is exposed to substances with androgenic activity, for instance through medication or exposure to endocrine disrupting chemicals (EDCs), the androgen bioactivity in his/her circulation is the sum of a mixture of endogenous and exogenous hormones or hormone-like compounds, and only a part of them are detected by specific immunoassays for androgenic steroids.
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Another level of complexity is added to the concept of androgen bioactivity by their binding to plasma proteins (Vermeulen, 2004
). The most important of them is the specific sex hormone-binding globulin (SHBG), which binds T and DHT with high affinity, and about 40% of serum T is bound to SHBG. The binding is tight, and because protein-bound T is not able to cross the cell membrane to access the AR, SHBG-bound androgens are not considered biologically active. Another nearly 60% of T is bound to albumin, but because the affinity of this binding is low, T dissociates easily from this protein and can contribute to the bioactive fraction of androgens. Finally, only 
1–3% of T is free, which fraction alone was previously considered biologically active. However, free and albumin-bound T together form apparently the more accurate bioavailable fraction of serum T. The protein binding of the other androgens varies in extent and affinity, which further complicates the composition of bioavailable androgen levels.
Immunoassays for steroid hormones employ antisera generated against steroids (e.g. T) that have been conjugated as haptens to a macromolecule (e.g. thyroglobulin). Owing to the small size of steroid molecules, only the competitive immunoassay principle is possible, since one steroid molecule is too small to bind two antibodies, which is required for the more specific and sensitive immunometric assays. Hence, the current T immunoassays are not fully specific, and their sensitivity, although sufficient for reliable determination of T in male serum, is too low for accurate detection of this hormone in women, children and in male hypogonadism (Matsumoto and Bremner, 2004; Wang et al., 2004; Sikaris et al., 2005; Wheeler, 2006). Measurements of other androgens or their precursors, e.g. DHT, DHEA sulphate or androstenedione, do not improve much the diagnostic relevance. One explanation is that normal serum androgen assays do not take into account the variability in protein binding, and that the total androgenic activity in serum represents in fact the sum of several androgenic steroid molecules with variable bioavailable fractions. The influence of protein binding may be controlled for by the measurement of total T and SHBG and calculation from them of the bioactive fraction of T, referred to as calculated free T, but there are also methods for direct measurement of free T (Vermeulen, 2004). These assays may in some cases improve the clinical relevance of the information, but still leave a large proportion of the measurements equivocal. GC-MS is the golden standard in steroid measurements, but neither does it monitor the bioactivity of steroids nor their dependence on protein binding.
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Androgen Bioassays |
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TOPAbstractIntroductionFactors Affecting Circulating...Androgen BioassaysAndrogen In Vitro Bioassays...Clinical Applications of the...Conclusions and Future...References |
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An alternative to improve the biological relevance of androgen assays is to measure bioactive hormone levels. The original methods for hormone activity measurement were in vivo bioassays, which were insensitive, labour-intensive and expensive, but had good biological relevance. Today the same functional principle can be adapted to the test-tube level in in vitro bioassays with similar sensitivity and practicality as provided by immunoassays. Bioassays monitor simultaneously all compounds with specific hormonal activity in a given sample irrespective of their chemical structure. Such assays can also detect compounds with hormone antagonist action.
Bioassays determine the amount of a biologically active substance in terms of the functional response in relation to that evoked by standards with known bioactivity (Bangham, 1952; Jeffcoate, 1996; Meager, 2006). The unknown sample does not need to be chemically pure or homogeneous, and the activity of all compounds, irrespective of their nature, will contribute to the overall bioactivity. The sample may even be a mixture of agonistic and antagonistic activities, and the result is their combined net effect. Thus a bioassay measures the ‘relative activity’ or ‘potency’ of a substance (or mixture of substances), i.e. its specific capacity to achieve an intended biological effect. It is therefore important to define a measure of the functional response for a substance within a given bioassay, and this is most simply done by defining a ‘unit’ of biological activity (Bangham, 1952; Meager, 2006). The unit is defined as equivalent to the lowest concentration of the substance at which the functional response can be detected and reproducibly measured. Alternatively, bioactivity can be expressed as equivalents of a known bioactive standard, e.g. T, in androgen bioassay. In the most useful in vitro bioassays, the range of the functional response, i.e. the difference between minimum and maximum response (the dynamic range), is large and it covers the practical range of concentrations/dilutions of the substance to be measured, thus allowing the generation of accurate ‘dose–response’ data (Meager, 2006).
In vivo bioassays of androgens
Most of the earlier assays of bioactivity of androgenic compounds were based on in vivo responses, e.g. the growth of capon comb or accessory sex organs of male rats (Dorfman, 1962). The so-called Hershberger assay, widely used in drug and chemical testing, measures androgenic and antiandrogenic activity in castrated mice or rats by monitoring the weight response of their androgen-dependent tissues, such as the levator ani muscle, prostate or seminal vesicles (Hershberger et al., 1953). The higher the androgenic dose of a sample the higher is the weight gain of the organs. These assays are very specific but slow, labour-intensive, expensive and in breach with the current trend of reducing the usage of live animals. When Hershberger assay is used to measure antiandrogenic activity, prepubertal rats are treated with androgen, and the antiandrogenic activity is reflected by the degree of inhibition of the androgen effect (Ashby et al., 2004).
In vitro bioassays of androgens
In vitro bioassays using primary cells or cell lines, often after genetic modification, are a novel alternative for in vivo bioassays. They are used for screening of hormonally active substances, in particular EDCs, either as pure compounds or as biological (e.g. blood, tissues) and environmental (e.g. sewage water) samples (Baker, 2001; Eertmans et al., 2003; Soto et al., 2006). Many assays have been optimized for high-throughput screening of large numbers of samples. The basic principles of some of the most widely used in vitro bioassays are summarized in Table 2. Their main advantages over in vivo assays are their high sensitivity and specificity, larger capacity and better cost-effectiveness. However, the data generated by in vitro assays have limited value in terms of risk assessment, since they cannot fully replicate the complex differences between species. They are, for example, unable to monitor the metabolism of test substances, which is an important determinant of their activity in vivo. This may result, depending on the situation, in either ‘false negative’ or ‘false positive’ responses (Gray et al., 2002). On the other hand, different cell lines may also metabolise the test substances to different extents and possess a different array of AR co-regulators, which may explain why all cell assays do not give identical results. Several studies have summarized and compared the performance of the different in vitro androgen bioassays for chemical compounds (Hartig et al., 2002; Korner et al., 2004; Christiaens et al., 2005; Sonneveld et al., 2006; Soto et al., 2006), but limited data still exist on their biological applications. Owing to the above limitations, in vitro assays may never totally replace in vivo bioassays.
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Receptor binding assays
Receptor binding assays have been developed to measure compounds, including hormones, that are able to bind specifically to receptors. Briefly, the hormone standard or unknown sample is incubated with labelled (usually radiolabelled) ligand hormone in the presence of a receptor preparation (Wong et al., 1995
; Lambright et al., 2000). The extent to which the unknown sample replaces receptor binding of the labelled hormone correlates with its bioactivity. In androgen assays, the receptor preparation can be a cell line expressing an endogenous or transfected AR, the labelled androgen can be [3H]-labelled androgen agonist R1881 and the unknown sample an aliquot of human serum. COS-7 (Christiaens et al., 2005) and MCF-7 (Sonneveld et al., 2006) cells transfected with hAR, or recombinant human (h) AR (Fang et al., 2003; Freyberger and Ahr, 2004; Scippo et al., 2004), have been used in such assays. These assays thus monitor one feature of the androgen activity, i.e. its binding to AR, but they do not take into account the capability of the hormone-receptor complexes to induce functional responses. Therefore, binding assays cannot discriminate between androgen agonists and antagonists; the latter also bind to AR, though without evoking functional responses (Roy and Pereira, 2005).
Cell proliferation assays
This is one of the simplest and most sensitive assays available for estrogenic compounds (Desaulniers et al., 1998; Rasmussen and Nielsen, 2002; Christiaens et al., 2005; Roy and Pereira, 2005; Soto et al., 2006). In this in vitro system on MCF-7 cells, termed E-SCREEN assay, the ability of a test substance to stimulate cell proliferation is measured (Soto et al., 1994).
Only a few cell proliferation assays have been developed for androgens, and most of them use the human prostate cell line LNCaP-FGC developed in the early 1980s (Horoszewicz et al., 1983). Because the AR gene in this cell line has a point mutation in its ligand binding domain that enhances estrogen binding, the assay is not totally specific for androgens. Hence, although this cell line cannot be used for the specific screening of androgens and antiandrogens, it has been used extensively to study androgen effects on cell proliferation and gene expression (Olea et al., 1990; Soto et al., 2006). MCF-7 cells stably transfected with hAR have been developed into an androgen dependent cell proliferation assay, A-SCREEN (Szelei et al., 1997), and used for the screening of environmental chemicals (Korner et al., 2004). The disadvantages of the proliferation assays are the difficulty to control concomitant toxic effects on cells and the batch-to-batch variation of responses (Roy and Pereira, 2005).
Reporter gene assays
These assays measure the level of expression of an androgen-dependent reporter gene in response to hormonal stimulation. They are the only method adapted for the measurement of androgen bioactivity in human serum. In these assays, either mammalian cell lines (e.g. MCF-7, COS-1, CHO and HEK293) or yeast strains (e.g. Saccharomyces cerevisiae) are transfected with plasmids encoding the hormone receptor (if not endogenously expressed) and a reporter gene. The latter consists of a promoter sequence containing one or several hormone response elements (HRE) specific for the receptor to be activated, coupled to a reporter gene (Sohoni and Sumpter, 1998; Hoogenboom et al., 2001; Roy et al., 2004). When the cells are exposed to hormone this will bind to the receptor; then the hormone-receptor complex binds to the HRE of the reporter gene resulting in its activation (Fig. 2). The reporter gene encodes an enzyme such as luciferase, β-galactosidase, green fluorescent protein or chloramphenicol acetyl transferase (CAT), which convert the added specific substrate into a signal that is easily measurable by luminometry, spectrophotometry or fluorimetry (Sohoni and Sumpter, 1998; Roy et al., 2004).
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Reporter gene assays on yeast cells (Routledge and Sumpter, 1996
; Gaido et al., 1997) offer a number of advantages, being easy to culture, ideal for genetic manipulation and possessing metabolic competence. However, the yeast cell walls vary in permeability to chemical compounds and express a different array of AR co-regulators than mammalian cells. Yeast-based assays were originally used for the screening of estrogenic EDCs, but lately they have also been adapted for the measurement of androgens and progestins (Holmes et al., 1998; Michelini et al., 2005). An earlier report exists on an ultrasensitive recombinant yeast cell bioassay for the measurement of estrogen levels in human serum (Klein et al., 1994), but similar assays have not been developed for the measurement of androgens in serum.
Mammalian cell-based reporter gene assays are becoming more common, not only in EDC and drug screening, but also in the analysis of bioactive levels of steroid hormones. A variety of mammalian cell lines have been used in these assays (Fuhrmann et al., 1992; Battmann et al., 1998; Schrader and Cooke, 2000; Terouanne et al., 2000; Blankvoort et al., 2001; Hartig et al., 2002; Wilson et al., 2002; Guth et al., 2004; Roy et al., 2004; Araki et al., 2005; Christiaens et al., 2005; Roy and Pereira, 2005; Sonneveld et al., 2005; Yamada et al., 2005; Xu et al., 2006). A general problem with many of the cell lines used is the endogenous expression of other steroid receptors, e.g. glucocorticoid receptors (GRs) in CHO cells (Roy et al., 2004), and if the sample to be studied contains other steroid hormones, as serum samples do, they can also stimulate reporter gene transcription and reduce the specificity of the measurement of androgen bioactivity. Another confounder is steroid metabolism of the reporter cells, either activating or inactivating bioactive hormones, which will alter the bioactivity readout of the sample. The assays do not take into account the possibility that a part of androgen actions in vivo is mediated through non-genomic mechanisms.
Some features of the existing mammalian cell based bioassays for the measurement of androgens in human peripheral serum are listed in Table 3. The first androgen bioassay suitable for serum measurements was reported by Raivio et al. (2001), using COS-1 cells transiently transfected with the N-terminal and DNA binding fragments of AR, luciferase reporter gene and an AR co-activator, AR-interacting protein 3 (ARIP3), to increase the signal/background ratio. The sensitivity of the assay is 1 nmol/l, and it detects in unextracted serum 25% of the androgen bioactivity that can be measured by total T immunoassay after lipid extraction. Hence, the bioavailable fraction of serum androgen with this method is clearly less than the 60% calculated on the basis of free plus albumin-bound hormone. The reason for the difference may be that the assay only detects T and DHT of the mixture of the numerous androgenic compounds present in peripheral serum, some of which may be relevant in the diagnostics of hyperandrogenism (Paris et al., 2002b; Roy et al., 2006).
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The second in vitro bioassay for human serum androgens (Paris et al., 2002b
) used CHO-K1 cells stably transfected with AR and the luciferase reporter gene. The assay appears sensitive but the presence of endogenous GR in the cell line (Shen et al., 1993; Vinggaard et al., 1999; Roy et al., 2004, 2006), and its involvement in modulating the androgen responses, reduces its specificity. Furthermore, the assay does not take the binding of bioactive steroids by SHBG and other binding proteins into consideration, an effect which has been addressed in detail by others (Raivio et al., 2001; Roy et al., 2006). The CHO cells used also metabolize T to DHT and DHEA to T, thus altering the concentrations of androgens from their steady state in vivo.
The third assay adapted for human serum measurements was reported by Roy et al. (2006), using the human embryonic kidney (HEK), 293 cell line stably transfected with human AR and MMTV-Luc reporter plasmids. This assay has a wide dynamic range and is minimally influenced by other circulating steroids such as estradiol, progesterone and cortisol. It also detects bioactivity of other androgens than T and DHT, though with the expected much lower potency (Fig. 3). The higher proportion of androgen bioactivity/immunoreactivity measured in unextracted serum (50%), when compared with the assay of Raivio et al. (2001) (see above) may reflect the fact that the whole hAR protein recognizes a wider variety of ligands than the truncated AR ligand-binding-domain of the latter assay. An additional advantage of this bioassay is that the 96-well format is suitable for high throughput testing using the bioluminescent detection system. Slightly later, another very similar assay was reported by Chen et al. (2006) using the same cell line and receptor/reporter system. In this assay, the androgen bioactivity levels were 87% of those measured by T immunoassay.
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Despite the good correlations found between androgen bioactivity and immunoreactive T, the ratio of the two activities in the different assays, from 25% to 87%, is the matter of some concern (Table 3). Each assay seems to take the protein binding of androgens into consideration to different extents, apparently due to a variety of differences in experimental protocols. Additional developmental work is clearly needed to be able to measure the biologically most relevant fraction of the free and differently protein-bound forms of serum androgens.
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Androgen In Vitro Bioassays in Monitoring Androgenic and Antiandrogenic Activities of Chemical Compounds and Environmental Samples |
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TOPAbstractIntroductionFactors Affecting Circulating...Androgen BioassaysAndrogen In Vitro Bioassays...Clinical Applications of the...Conclusions and Future...References |
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Hormone bioassays are important in the testing of drugs and other chemical compounds for their expected and unexpected endocrine activities. There is an increasing concern that certain chemicals (EDCs) in the environment are affecting the health of humans and wildlife by disrupting normal endocrine functions, including fertility (Toppari et al., 1996
). Although compounds with estrogenic effects have received most of the attention, any endocrine effect through nuclear receptors, including all steroid hormones, vitamin D and thyroid hormones, is equally possible and in need of monitoring. The vast number of chemical compounds cannot be tested with the labour-intensive and expensive in vivo methods and therefore in vitro bioassays offer a practical, highly sensitive and specific first tier method for their high-throughput screening. Because these assays are used for pre-screening, it is important that they have a low false negative rate. In vitro testing also makes it possible to reduce the usage of experimental animals.
Owing to their high capacity some reports of the in vitro testing comprise hundreds of compounds (Fang et al., 2003; Araki et al., 2005), and the findings in general agree well with existing in vivo data. Unexpected androgenic effects are rare, but antiandrogenic activities seem to be much more common (Roy et al., 2004; Araki et al., 2005); they quite often coincide with estrogenic effects of the same compounds (Sohoni and Sumpter, 1998; Schlumpf et al., 2001; Paris et al., 2002a; Ma et al., 2003). For example, several estrogenic environmental chemicals, such as vinclozolin (a fungicide), p,p'-DDE and methoxychlor (potent metabolites of DDT), linuron (a herbicide), phthalate esters (plasticizers in manufacturing polyvinyl chloride products), pyrethrin (an insecticide), dieldrin and aldrin (common pesticides), were also found to be antiandrogens (Roy et al., 2004).
Pulp and paper mill effluents, as well as municipal waste water, contain androgenic and antiandrogenic activities (Parks et al., 2001; Michelini et al., 2005). Besides alkylphenols, progesterone and its metabolites, as well as the illegal use of anabolic steroids in cattle industry, were suggested as the source of the androgenic activity. Another recent study (Christiaens et al., 2005) on waste water samples from municipal and industrial sources found that almost all samples contained estrogenic activity, and most were androgenic or antiandrogenic. These studies clearly demonstrate the potential of in vitro assays for pre-screening of larger numbers of environmental samples before subjecting the positive ones to more rigorous analysis.
An interesting application of androgen bioassays is their use in the detection of abuse of androgenic-anabolic compounds. Because the assay measures all androgenic activities in the sample, irrespective of their chemical structure, it can also detect previously unknown compounds, like new androgenic doping agents. Also the total activity of cocktails of multiple compounds can be detected; this method is used in doping practice to lower the concentration of individual compounds below the detection limit by structure-based assays (immunoassay and GC-MS). The AR-LUX reporter gene assay has been successfully tested for the detection of hormonal anabolic compounds in cattle urine (Blankvoort et al., 2003).
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Clinical Applications of the Androgen In Vitro Bioassays |
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TOPAbstractIntroductionFactors Affecting Circulating...Androgen BioassaysAndrogen In Vitro Bioassays...Clinical Applications of the...Conclusions and Future...References |
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There are several clinical situations where the assessment of androgen bioactivity rather than simple measurement of T immunoreactivity would be desirable. This need usually occurs in men when the circulating androgen levels are low (e.g. late-onset hypogonadism, LOH) and the decision to treat may arise, and in women and children with clinical signs of androgen excess where the direct proof of hyperandrogenism by immunological assays is missing. The diagnostic accuracy could be facilitated if the information about the total androgen bioactivity in circulation were available.
The few androgen specific in vitro bioassays currently available for the evaluation of serum samples (Table 3) have been tested to some extent in various clinical situations. In particular, the group of Jänne et al. (see below) have collected such information using the transactivation assay that they developed several years ago (Raivio et al., 2001). When serum samples from prepubertal and pubertal boys were analysed, the androgen bioactivity levels showed good correlation with biological signs of androgen action, such as testis volume and stage of puberty. Furthermore, the levels were suppressed in boys with cryptorchidism and/or constitutional delay of puberty (CDP), whereas they increased appropriately after hCG treatment (Raivio et al., 2001). The ratio of androgen bioactivity to T increased as a function of puberty, which is in keeping with the reciprocal change of T and SHBG at puberty (Belgorosky and Rivarola, 1987). In contrast, and unexpectedly, the bioactive/immunoreactive androgen ratio was higher in cryptorchid boys during hCG treatment than in boys with CDP. No correlation of serum androgen bioactivity with serum DHEA, DHT or androstenedione levels was observed in samples with low T levels. This is expected due to the low bioactivity of the other androgens, but not with DHT, which had >10-fold higher bioactivity than T in the assay (Raivio et al., 2001). The authors concluded that the assay provides results that correlate well with clinical signs of androgenicity. They also suggested that in several situations the information provided by the bioassay could be superior to that of conventional immunoassays, especially if androgenic compounds with unknown structure, such as in the case of anabolic steroid abuse, or when antiandrogens are used. In these cases, the findings could even be opposite to immunoassay results.
In another study concentrating on post-natal androgen levels in healthy and cryptorchid boys, it was shown that both androgen bioactivity and immunoassayable T correlated well with the position of testes at 3 months of age (Raivio et al., 2003b). Most of the boys with abnormal testicular descent had unmeasurable androgen bioactivity, and importantly, the score describing testicular localization correlated negatively with androgen bioactivity. These findings suggest that infant boys are exposed to bioactive androgen levels during the post-natal 2–3 month activation of the pituitary-gonadal axis, and that this activity is lower in connection with abnormal testicular descent. The functional significance of the post-natal androgen peak is difficult to determine because of a concomitant rise in serum SHBG levels (Chaussain et al., 1978). In another study monitoring androgen bioactivity in boys with CDP, it was found that it closely correlated with pubertal progression; such correlation was also found in boys treated either with androgen alone or with androgen combined with the aromatase inhibitor letrozole, to delay epiphysial closure (Raivio et al., 2004). It was concluded that androgen bioactivity mediates the tempo of pubertal maturation. All treated boys had supranormal androgen bioactivity and letrozole monotherapy was even able to induce hyperandrogenism and virilization in pubertal boys.
In male patients who present with symptoms of late onset hypogonadism (LOH), the finding of relatively, but not definitively, low circulating levels of androgens does not help in the decision to treat, especially in view of the extreme variability in the normal range of serum T (Matsumoto and Bremner, 2004; Wang et al., 2004). Likewise, if androgen replacement therapy is initiated, T measurements by immunoassay may not be suitable for assessment of therapeutic dose. It is possible, although not yet proven, that androgen bioassays could improve the diagnostics of genuine androgen deficiency in suspected LOH. In another study by Raivio et al. (2002) androgen bioactivity was measured in the serum of men with borderline LOH receiving transdermal DHT treatment (Fig. 4). A 7-fold increase in bioactive androgen was detected during the treatment (from a mean of 3.3 up to 23.6 nmol/l T equivalents). At the same time, T immunoreactivity was suppressed by 70% (from 14.7 to 4.1 nmol/l), and DHT immunoassay measurements documented an increase from 1.5 to 9 nmol/l. It is not possible to decipher from the T and DHT levels whether a therapeutic level of DHT was attained. In contrast, the bioassay indicated that androgen bioactivity in the circulation of these men was elevated from clearly subnormal to the normal male range. This information is important in assessing the therapeutic dose of the hormone replacement therapy.
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The same group examined serum androgen bioactivity in men who had previously undiagnosed prostate cancer, comparing them with age-matched men with benign prostate hyperplasia (BPH) (Raivio et al., 2003a
). They found that whereas immunoreactive T and SHBG levels did not differ between the two groups, the androgen bioactivity levels in the cancer group were slightly but significantly lower than in the BPH group, in particular in those with the well-differentiated and less aggressive forms. No clear explanation could be suggested for this finding, but the possibility of T or AR antibodies in prostate Ca patients, alterations in the levels of androgens other than T (of adrenal origin) or accumulation of antiandrogenic compounds in the circulation of patients were discussed. Curiously, four cancer patients displaying normal immunoreactive T had unmeasurable bioactivity levels, but the reason for this discrepant finding could not be explained. To conclude, these results demonstrated clearly that bioactivity measurements of androgens can yield information differing drastically from that obtained by immuno-T measurements; they may be clinically applicable in prostate cancer when assessing the efficacy of androgen ablation therapy.
In their latest study, the Finnish group used the androgen bioassay to monitor the antiandrogenic activity conferred by flutamide treatment on children with congenital adrenal hyperplasia (Hero et al., 2005). As an experimental approach to improve the control of height velocity and bone maturation of these children, the authors administered a combination of aromatase inhibitor (letrozole) and antiandrogen (flutamide). Serum samples collected from the children both before and during antiandrogen treatment showed undetectable androgen bioactivity. When varying concentrations of T were added to the serum samples, the expected androgen bioactivity was measured in the pretreatment samples, but flutamide (or its metabolites) present in the treatment samples totally inhibited bioactivity of the added T. These results indicate another potentially useful application of these methods, i.e. to allow the titration of the therapeutic concentration of antiandrogen, which is clinically important in the adjustment of correct dose.
A relatively small amount of clinical samples were analysed using the bioassay of Paris et al. (2002b). They reported mean androgen bioactivity levels of 2.1 nmol/l in normal prepubertal boys, 43.1 nmol/l in pubertal boys and 5.9 nmol/l in pubertal girls. These levels appear rather high, which may be due to the reported conversion of T to DHT and DHEA to T by the CHO cells used in the assays. The clinical applicability of this assay may therefore be limited.
Using their androgen in vitro bioassay Roy et al. (2006) measured serum androgen levels in healthy men and women, patients with polycystic ovary syndrome (PCOS) and in ageing men. In all groups, bioactive androgen levels in unextracted serum were 50% of that measured by T immunoassay, whereas after ether extraction, the levels measured with both assays were almost identical. The relative bioactivity of several testicular and adrenal androgens corresponded well with the existing information about their bioactivities (Fig. 3). Of the serum bioassays used (Table 3), this is the one giving results closest to the expected proportion of bioavailable androgen. Despite the limited number of samples, the findings demonstrated the generally good correlation of the bioactivity measurements with the immunoreactive T data.
In a recent study where a new AR mediated luciferase transactivation bioassay was used (Chen et al., 2006), good correlation between immunoreactive and bioactive androgens was found in a small group of adult men. Androgen bioactivity in serum was very similar in oophorectomized and intact post-menopausal women, providing further evidence for the predominantly adrenal origin of these activities. In that study, when the bioassay results were compared with those obtained using other methods, such as GC-MS, good correlation was found. Furthermore, androgen bioactivity appeared to correlate well with biochemical indices of cardiovascular risk, such as triglyceride and insulin levels, thus confirming earlier findings (Sutton-Tyrrell et al., 2005). Of note, such associations were not found with total immunoreactive T measurements. Similar information would be important in women with androgen excess and androgen deficiency conditions, because a role for adrenal androgens in the metabolic syndrome developing in women after menopause has been suggested (Alevizaki et al., 2006). On the other hand, the potential importance of androgen deficiency in women, e.g. in sexual dysfunction, is being increasingly discussed (Wierman et al., 2006).
In the same study (Chen et al., 2006), when women at the menopausal transition were studied, an age group difference was observed in the correlations between immunoreactive and bioactive androgens. The correlation was good in young women and those with androgen excess, such as in PCOS, and also in men, whereas there was no correlation in women immediately after menopause. Fluctuation in SHBG levels during the menopausal transition was considered responsible for this difference. The proportion of androgen bound to SHBG was found highly variable at this age, which could explain the poor correlation between bioactive and immunoassayable androgen levels. The main clinical message of this study was that androgenic bioactivity may be underestimated by the measurement of immunoreactive free T in women, possibly because additional steroids with androgenic activity, not detected by T immunoassay, may contribute to the overall biological effect. Therefore, the in vitro bioassays hold a great promise in improving the accuracy of laboratory diagnostics of female hyperandrogenism. These assays do not provide information about the chemical nature of the androgens in serum, but are unique in providing information about the sum of the biological activities contributed by the entirety of the various androgenic compounds circulating at various concentrations. Furthermore, they may also obviate the need for SHBG and free T measurements in assessing the bioavailable fraction of circulating androgens.
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Conclusions and Future Directions |
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TOPAbstractIntroductionFactors Affecting Circulating...Androgen BioassaysAndrogen In Vitro Bioassays...Clinical Applications of the...Conclusions and Future...References |
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Reliable monitoring of androgen concentrations continues to be a challenge in clinical practice. Although the conventional immunoassays of T measure the levels of this hormone relatively reliably when they are high, as in healthy adult men, they have proven less reliable at low concentrations, as in women, children and ageing men with LOH. The poor correlation between clinical symptoms and androgen levels is also recognized in female hyperandrogenism, such as PCOS, where the contribution of adrenal androgens, not detected by T immunoassays, may be significant. From the conceptual point of view, the weakness of immunoassays is that they measure immunoreactivity of structurally related compounds, but do not take into account the most important feature of a hormone, its biological activity. The classical in vivo bioassays of hormones have excellent functional relevance, but for practical reasons, they are unsuitable for clinical diagnostics. Therefore, the novel in vitro bioassays offer an approach to combine the benefits of the in vivo bioassays and immunoassays, providing good functional relevance with high sensitivity and practicality. Several suitable in vitro bioassays for androgens have been developed recently. They have provided promising results in clinical research settings for the improvement of clinical measurements of androgens. What is now needed is more data on larger numbers of samples to assess whether a real benefit in the clinical diagnostics can be offered by these novel assays. At the moment, they are a valuable novel research tool.
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References |
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TOPAbstractIntroductionFactors Affecting Circulating...Androgen BioassaysAndrogen In Vitro Bioassays...Clinical Applications of the...Conclusions and Future...References |
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Alevizaki M, Saltiki K, Mantzou E, Anastasiou E, Huhtaniemi I. The adrenal gland may be a target of LH action in postmenopausal women. Eur J Endocrinol (2006) 154:875–881.
Araki N, Ohno K, Nakai M, Takeyoshi M, Iida M. Screening for androgen receptor activities in 253 industrial chemicals by in vitro reporter gene assays using AR-EcoScreen cells. Toxicol In Vitro (2005) 19:831–842.[CrossRef][Web of Science][Medline]
Ashby J, Lefevre PA, Tinwell H, Odum J, Owens W. Testosterone-stimulated weanlings as an alternative to castrated male rats in the Hershberger anti-androgen assay. Regul Toxicol Pharmacol (2004) 39:229–238.[CrossRef][Web of Science][Medline]
Baker VA. Endocrine disrupters-testing strategies to assess human hazard. Toxicol In Vitro (2001) 15:413–419.[CrossRef][Web of Science][Medline]
Bangham D. Assays and Standards (1952) New York: Academic Press.
Battmann T, Branche C, Bouchoux F, Cerede E, Philibert D, Goubet F, Teutsch G, Gaillard-Kelly M. Pharmacological profile of RU 58642, a potent systemic antiandrogen for the treatment of androgen-dependent disorders. J Steroid Biochem Mol Biol (1998) 64:103–111.[CrossRef][Web of Science][Medline]
Belgorosky A, Rivarola MA. Changes in serum sex hormone-binding globulin and in serum non-sex hormone-binding globulin-bound testosterone during prepuberty in boys. J Steroid Biochem (1987) 27:291–295.[CrossRef][Web of Science][Medline]
Blankvoort BM, de Groene EM, van Meeteren-Kreikamp AP, Witkamp RF, Rodenburg RJ, Aarts JM. Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor. Anal Biochem (2001) 298:93–102.[CrossRef][Web of Science][Medline]
Blankvoort BM, Aarts JM, Schilt R, Geerdink P, Spenkelink B, Rodenburg RJ. Detection of hormonal anabolic compounds in calf urine and unverified growth-promoting preparations: application of the AR-LUX bioassay for screening and determination of androgenic activity. Analyst (2003) 128:1373–1381.[CrossRef][Medline]
Chaussain JL, Brijawi A, Georges P, Roger M, Donnadieu M, Job JC. Variations of serum testosterone estradiol binding globulin (TeBG) binding capacity in infants during the first year of life. Acta Paediatr Scand (1978) 67:649–653.[Web of Science][Medline]
Chen F, Knecht K, Leu C, Rutledge SJ, Scafonas A, Gambone C, Vogel R, Zhang H, Kasparcova V, Bai C, et al. Partial agonist/antagonist properties of androstenedione and 4-androsten-3beta,17beta-diol. J Steroid Biochem Mol Biol (2004) 91:247–257.[CrossRef][Web of Science][Medline]
Chen F, Knecht K, Birzin E, Fisher J, Wilkinson H, Mojena M, Moreno CT, Schmidt A, Harada S, Freedman LP, et al. Direct agonist/antagonist functions of dehydroepiandrosterone. Endocrinology (2005) 146:4568–4576.
Chen J, Sowers MR, Moran FM, McConnell DS, Gee NA, Greendale GA, Whitehead C, Kasim-Karakas SE, Lasley BL. Circulating bioactive androgens in midlife women. J Clin Endocrinol Metab (2006) 91:4387–4394.
Christiaens V, Berckmans P, Haelens A, Witters H, Claessens F. Comparison of different androgen bioassays in the screening for environmental (anti)androgenic activity. Environ Toxicol Chem (2005) 24:2646–2656.[CrossRef][Web of Science][Medline]
Desaulniers D, Leingartner K, Zacharewski T, Foster W. Optimization of an MCF7-E3 call proliferation assay and effects on environmental pollutants and industrial chemicals. Toxicology In Vitro (1998) 12:409–422.[CrossRef][Web of Science]
Dorfman R. Androgens and Anabolic Agents (1962) New York: Academic Press.
Eertmans F, Dhooge W, Stuyvaert S, Comhaire F. Endocrine disruptors: effects on male fertility and screening tools for their assessment. Toxicol In Vitro (2003) 17:515–524.[CrossRef][Web of Science][Medline]
Fang H, Tong W, Branham WS, Moland CL, Dial SL, Hong H, Xie Q, Perkins R, Owens W, Sheehan DM. Study of 202 natural, synthetic, and environmental chemicals for binding to the androgen receptor. Chem Res Toxicol (2003) 16:1338–1358.[CrossRef][Web of Science][Medline]
Freyberger A, Ahr HJ. Development and standardization of a simple binding assay for the detection of compounds with affinity for the androgen receptor. Toxicology (2004) 195:113–126.[CrossRef][Web of Science][Medline]
Fuhrmann U, Bengtson C, Repenthin G, Schillinger E. Stable transfection of androgen receptor and MMTV-CAT into mammalian cells: inhibition of cat expression by anti-androgens. J Steroid Biochem Mol Biol (1992) 42:787–793.[CrossRef][Web of Science][Medline]
Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier CJ, McDonnell DP. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol Appl Pharmacol (1997) 143:205–212.[CrossRef][Web of Science][Medline]
Gao W, Bohl CE, Dalton JT. Chemistry and structural biology of androgen receptor. Chem Rev (2005) 105:3352–3370.[CrossRef][Web of Science][Medline]
Gray LE Jr, Ostby J, Wilson V, Lambright C, Bobseine K, Hartig P, Hotchkiss A, Wolf C, Furr J, Price M, et al. Xenoendocrine disrupters-tiered screening and testing: filling key data gaps. Toxicology (2002) 181–182:371–382.
Guth SE, Bohm S, Mussler BH, Eisenbrand G. Sensitive in vitro test systems to determine androgenic/antiandrogenic activity. Mol Nutr Food Res (2004) 48:282–291.[CrossRef][Web of Science][Medline]
Hartig PC, Bobseine KL, Britt BH, Cardon MC, Lambright CR, Wilson VS, Gray LE Jr. Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening. Toxicol Sci (2002) 66:82–90.
Hero M, Janne OA, Nanto-Salonen K, Dunkel L, Raivio T. Circulating antiandrogenic activity in children with congenital adrenal hyperplasia during peroral flutamide treatment. J Clin Endocrinol Metab (2005) 90:5141–5145.
Hershberger LG, Shipley EG, Meyer RK. Myotrophic activity of 19-nortestosterone and other steroids determined by modified levator ani muscle method. Proc Soc Exp Biol Med (1953) 83:175–180.[CrossRef][Medline]
Holmes P, Humfrey C, Scullion M. Appraisal of Test Methods for Sex-Hormone Disrupting Chemicals Capable of Affecting the Reproductive Process (1998) UK: Leicester UK. http://www.oecd/ehs/test/monos.htm.
Hoogenboom LAP, de Haan L, Hooijerink D, Bor G, Murk AJ, Brouwer A. Estrogenic activity of estradiol and its metabolites in the ER-CALUX assay with human T47D breast cells. APMIS (2001) 109:101–107.[CrossRef][Web of Science][Medline]
Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA, Murphy GP. LNCaP model of human prostatic carcinoma. Cancer Res (1983) 43:1809–1818.
Jasuja R, Ramaraj P, Mac RP, Singh AB, Storer TW, Artaza J, Miller A, Singh R, Taylor WE, Lee ML, et al. Delta-4-androstene-3,17-dione binds androgen receptor, promotes myogenesis in vitro, and increases serum testosterone levels, fat-free mass, and muscle strength in hypogonadal men. J Clin Endocrinol Metab (2005) 90:855–863.
Jeffcoate S. The role of bioassays in the development, licensing and batch control of biotherapeutics. Trends Biotechnol (1996) 14:121–124.[CrossRef][Web of Science][Medline]
Klein KO, Baron J, Colli MJ, McDonnell DP, Cutler GB Jr. Estrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay. J Clin Invest (1994) 94:2475–2480.[Web of Science][Medline]
Korner W, Vinggaard AM, Terouanne B, Ma R, Wieloch C, Schlumpf M, Sultan C, Soto AM. Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals. Environ Health Perspect (2004) 112:695–702.[Web of Science][Medline]
Labrie F. Adrenal androgens and intracrinology. Semin Reprod Med (2004) 22:299–309.[CrossRef][Web of Science][Medline]
Labrie F, Belanger A, Cusan L, Gomez JL, Candas B. Marked decline in serum concentrations of adrenal C19 sex steroid precursors and conjugated androgen metabolites during aging. J Clin Endocrinol Metab (1997) 82:2396–2402.
Lambright C, Ostby J, Bobseine K, Wilson V, Hotchkiss AK, Mann PC, Gray LE Jr. Cellular and molecular mechanisms of action of linuron: an antiandrogenic herbicide that produces reproductive malformations in male rats. Toxicol Sci (2000) 56:389–399.
Lange CA, Gioeli D, Hammes SR, Marker PC. Integration of rapid signaling events with steroid hormone receptor action in breast and prostate cancer. Annu Rev Physiol (2007) 69:171–199.[CrossRef][Web of Science][Medline]
Ma R, Cotton B, Lichtensteiger W, Schlumpf M. UV filters with antagonistic action at androgen receptors in the MDA-kb2 cell transcriptional-activation assay. Toxicol Sci (2003) 74:43–50.
Matsumoto AM, Bremner WJ. Serum testosterone assays–accuracy matters. J Clin Endocrinol Metab (2004) 89:520–524.
Meager A. Measurement of cytokines by bioassays: theory and application. Methods (2006) 38:237–252.[CrossRef][Web of Science][Medline]
Michelini E, Leskinen P, Virta M, Karp M, Roda A. A new recombinant cell-based bioluminescent assay for sensitive androgen-like compound detection. Biosens Bioelectron (2005) 20:2261–2267.[CrossRef][Web of Science][Medline]
Olea N, Sakabe K, Soto AM, Sonnenschein C. The proliferative effect of "anti-androgens" on the androgen-sensitive human prostate tumor cell line LNCaP. Endocrinology (1990) 126:1457–1463.
Papakonstanti EA, Kampa M, Castanas E, Stournaras C. A rapid, nongenomic, signaling pathway regulates the actin reorganization induced by activation of membrane testosterone receptors. Mol Endocrinol (2003) 17:870–881.
Paris F, Servant N, Terouanne B, Balaguer P, Nicolas JC, Sultan C. A new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity in children. J Clin Endocrinol Metab (2002) a 87:791–797.
Paris F, Servant N, Terouanne B, Sultan C. Evaluation of androgenic bioactivity in human serum by recombinant cell line: preliminary results. Mol Cell Endocrinol (2002) b198:123–129.[CrossRef][Web of Science][Medline]
Parks LG, Lambright CS, Orlando EF, Guillette LJ Jr, Ankley GT, Gray LE Jr. Masculinization of female mosquitofish in Kraft mill effluent-contaminated Fenholloway River water is associated with androgen receptor agonist activity. Toxicol Sci (2001) 62:257–267.
Raivio T, Palvimo JJ, Dunkel L, Wickman S, Janne OA. Novel assay for determination of androgen bioactivity in human serum. J Clin Endocrinol Metab (2001) 86:1539–1544.
Raivio T, Tapanainen JS, Kunelius P, Janne OA. Serum androgen bioactivity during 5alpha-dihydrotestosterone treatment in elderly men. J Androl (2002) 23:919–921.
Raivio T, Santti H, Schatzl G, Gsur A, Haidinger G, Palvimo JJ, Janne OA, Madersbacher S. Reduced circulating androgen bioactivity in patients with prostate cancer. Prostate (2003) a55:194–198.[CrossRef][Web of Science][Medline]
Raivio T, Toppari J, Kaleva M, Virtanen H, Haavisto AM, Dunkel L, Janne OA. Serum androgen bioactivity in cryptorchid and noncryptorchid boys during the postnatal reproductive hormone surge. J Clin Endocrinol Metab (2003) b88:2597–2599.
Raivio T, Dunkel L, Wickman S, Janne OA. Serum androgen bioactivity in adolescence: a longitudinal study of boys with constitutional delay of puberty. J Clin Endocrinol Metab (2004) 89:1188–1192.
Rasmussen TH, Nielsen JB. Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen). Biomarkers (2002) 7:322–336.[CrossRef][Web of Science][Medline]
Routledge E, Sumpter JP. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ Toxicol Chem (1996) 15:241–248.[CrossRef][Web of Science]
Roy P, Pereira BM. A treatise on hazards of endocrine disruptors and tool to evaluate them. Indian J Exp Biol (2005) 43:975–992.[Medline]
Roy P, Salminen H, Koskimies P, Simola J, Smeds A, Saukko P, Huhtaniemi IT. Screening of some anti-androgenic endocrine disruptors using a recombinant cell-based in vitro bioassay. J Steroid Biochem Mol Biol (2004) 88:157–166.[CrossRef][Web of Science][Medline]
Roy P, Franks S, Read M, Huhtaniemi IT. Determination of androgen bioactivity in human serum samples using a recombinant cell based in vitro bioassay. J Steroid Biochem Mol Biol (2006) 101:68–77.[CrossRef][Web of Science][Medline]
Schlumpf M, Cotton B, Conscience M, Haller V, Steinmann B, Lichtensteiger W. In vitro and in vivo estrogenicity of UV screens. Environ Health Perspect (2001) 109:239–244.[Web of Science][Medline]
Schrader TJ, Cooke GM. Examination of selected food additives and organochlorine food contaminants for androgenic activity in vitro. Toxicol Sci (2000) 53:278–288.
Scippo ML, Argiris C, Van De Weerdt C, Muller M, Willemsen P, Martial J, Maghuin-Rogister G. Recombinant human estrogen, androgen and progesterone receptors for detection of potential endocrine disruptors. Anal Bioanal Chem (2004) 378:664–669.[CrossRef][Web of Science][Medline]
Shen P, Xie ZJ, Li H, Sanchez ER. Glucocorticoid receptor conversion to high affinity nuclear binding and transcription enhancement activity in Chinese hamster ovary cells subjected to heat and chemical stress. J Steroid Biochem Mol Biol (1993) 47:55–64.[CrossRef][Web of Science][Medline]
Sikaris K, McLachlan RI, Kazlauskas R, de Kretser D, Holden CA, Handelsman DJ. Reproductive hormone reference intervals for healthy fertile young men: evaluation of automated platform assays. J Clin Endocrinol Metab (2005) 90:5928–5936.
Sohoni P, Sumpter JP. Several environmental oestrogens are also anti-androgens. J Endocrinol (1998) 158:327–339.[Abstract]
Sonneveld E, Jansen HJ, Riteco JA, Brouwer A, van der Burg B. Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. Toxicol Sci (2005) 83:136–148.
Sonneveld E, Riteco JA, Jansen HJ, Pieterse B, Brouwer A, Schoonen WG, van der Burg B. Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities. Toxicol Sci (2006) 89:173–187.
Soto AM, Chung KL, Sonnenschein C. The pesticides endosulfan, toxaphene, and dieldrin have estrogenic effects on human estrogen-sensitive cells. Environ Health Perspect (1994) 102:380–383.[Web of Science][Medline]
Soto AM, Maffini MV, Schaeberle CM, Sonnenschein C. Strengths and weaknesses of in vitro assays for estrogenic and androgenic activity. Best Pract Res Clin Endocrinol Metab (2006) 20:15–33.[CrossRef][Medline]
Sutton-Tyrrell K, Wildman RP, Matthews KA, Chae C, Lasley BL, Brockwell S, Pasternak RC, Lloyd-Jones D, Sowers MF, Torrens JI. Sex-hormone-binding globulin and the free androgen index are related to cardiovascular risk factors in multiethnic premenopausal and perimenopausal women enrolled in the Study of Women Across the Nation (SWAN). Circulation (2005) 111:1242–1249.
Szelei J, Jimenez J, Soto AM, Luizzi MF, Sonnenschein C. Androgen-induced inhibition of proliferation in human breast cancer MCF7 cells transfected with androgen receptor. Endocrinology (1997) 138:1406–1412.
Terouanne B, Tahiri B, Georget V, Belon C, Poujol N, Avances C, Orio F Jr, Balaguer P, Sultan C. A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects. Mol Cell Endocrinol (2000) 160:39–49.[CrossRef][Web of Science][Medline]
Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette LJ Jr, Jegou B, Jensen TK, Jouannet P, Keiding N, et al. Male reproductive health and environmental xenoestrogens. Environ Health Perspect (1996) 104(Suppl_4):741–803.[CrossRef][Web of Science][Medline]
Vermeulen A. Reflections concerning biochemical parameters of androgenicity. Aging Male (2004) 7:280–289.[CrossRef][Medline]
Vinggaard AM, Joergensen EC, Larsen JC. Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals. Toxicol Appl Pharmacol (1999) 155:150–160.[CrossRef][Web of Science][Medline]
Wang C, Catlin DH, Demers LM, Starcevic B, Swerdloff RS. Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab (2004) 89:534–543.
Wheeler MJ. Measurement of androgens. Methods Mol Biol (2006) 324:197–211.[Medline]
Wierman ME, Basson R, Davis SR, Khosla S, Miller KK, Rosner W, Santoro N. Androgen therapy in women: an Endocrine Society Clinical Practice guideline. J Clin Endocrinol Metab (2006) 91:3697–3710.
Wilson VS, Bobseine K, Lambright CR, Gray LE Jr. A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists. Toxicol Sci (2002) 66:69–81.
Wong C, Kelce WR, Sar M, Wilson EM. Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J Biol Chem (1995) 270:19998–20003.
Xu LC, Sun H, Chen JF, Bian Q, Song L, Wang XR. Androgen receptor activities of p,p'-DDE, fenvalerate and phoxim detected by androgen receptor reporter gene assay. Toxicol Lett (2006) 160:151–157.[CrossRef][Web of Science][Medline]
Yamada T, Sumida K, Saito K, Ueda S, Yabushita S, Sukata T, Kawamura S, Okuno Y, Seki T. Functional genomics may allow accurate categorization of the benzimidazole fungicide benomyl: lack of ability to act via steroid-receptor-mediated mechanisms. Toxicol Appl Pharmacol (2005) 205:11–30.[CrossRef][Web of Science][Medline]
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