Does modafinil enhance activity of patients with myotonic dystrophy?

We performed a double-blind placebo-controlled crossover study in 13 patients with myotonic dystrophy to address the question whether modafinil, known to improve hypersomnolence in myotonic dystrophy, may improve levels of activity as well. We used the Epworth Sleepiness Scale as a measure of hypersomnolence and a structured interview of the patient and the partner or housemate as a measure of activity. We additionally used a restricted form of the RAND-36 to relate a possible improvement of activity to perceived general health. We confirmed earlier positive findings of modafinil regarding reduced somnolence (p = 0.015), but no significant effects were seen regarding activity levels (p = 0.2 for patients’ self-reports and 0.5 for partners’ reports).
Introduction
Myotonic dystrophy (MD) is a multi-system disorder, the most well known symptoms being muscle weakness and myotonia. Distressing daytime sleepiness and diminished spontaneous activity, often referred to as inertia, reduced initiative, inactivity or apathy, are frequently reported. These latter symptoms often seem to cause more hindrance in daily life, both to patients and their spouses, than muscle weakness itself. Hypersomnia can even be present when there is virtually no weakness [1]. Recent reports [2, 3, 4] have demonstrated that modafinil has a beneficial effect on daytime sleepiness in MD. Although the relationship between excessive sleepiness and the lack of spontaneous activity is not clear, it seemed reasonable to suppose that increasing vigilance might result in an increase in spontaneous activity. The present study intended to answer the following questions: firstly, do patients with MD undertake more activities when using modafinil; secondly, does any such improvement relate to a change in somnolence or to another disease characteristic?
Patients and methods
Thirteen outpatients (5 males) participated in a randomized double-blind crossover placebo controlled study. Their mean age was 43.5 years (SD: 13.9 years). Age of onset of symptoms was before 12 years of age in 3 patients; all three lived independently at the time of study, although they needed professional social support with respect to their household or daily activities. Three patients were employed in highly responsible jobs. The remaining patients had been considered unfit for normal paid employment for reasons related to their disorder. They were mainly involved in housekeeping. With the exception of two elderly men with considerable weakness of the legs, weakness had little impact on activities of daily living (ADL) functions. Even these two men were ambulant, although they used a wheelchair regularly. Twelve patients had a partner or housemate.
Medication was given during two periods of 14 days, separated by a one-week washout period. The study was preceded by a two-week period free of all drugs with an exception being made for contraception. Patients were randomized for either placebo first or modafinil first. The modafinil dose was 200 mg per day for the first week. The patients were instructed to double the dose during the second week of each period if they perceived an insufficient effect.
The main outcome measure was an increase in spontaneous activity, assessed using a novel structured interview of both the patient and the partner or housemate, if present. These interviews took place at home after the first period, and by telephone after the second one. After the first medication period the patient and the partner/housemate were asked to compare the level of activity with that of the preceding two (-baseline-) weeks. After the second medication period they were asked to compare that period with the baseline weeks.
The following issues were addressed, and scores attributed:
have you, during the past two weeks, been more active than during the baseline period?no (0 points)to some degree (1 point)definitely (2 points)can you give one (1 point), two (2 points) or three (3 points) substantial and observable examples of activities/specific actions you undertook that you would otherwise not have done?
The range of the score was therefore zero to five points. The partner was asked the same questions with respect to the patient. During the interview patients and partners were asked whether they had guessed which medication, modafinil or placebo, the patient had used in the past period. If so, they were asked what made them believe so. The answers were not used in the assessment of medication effects, but served to estimate possible unblinding. The RAND-36 questionnaire was used to measure possible changes in subjective general health [5]. This was filled in before the start and after the end of each medication period. The questionnaire was restricted to issues considered relevant to the study: General Health, Role Limitations by emotional and physical problem, Social Functioning, Vitality, and Mental Health. The Epworth Sleepiness Scale (ESS, [6] was used to assess possible changes in hypersomnolence.
After completion of the trial the remaining capsules in the medication boxes were counted to assess compliance. The institutional Committee of Medical Ethics had approved the study. Patients gave written informed consent after study information was provided orally and in writing at the patients’ home.
Results
All patients completed the trial. Medication compliance was good: only three patients had omitted one dose each. The only reported side effect was slight headache in one patient using modafinil. Ten patients doubled the dose of both modafinil and placebo after the first week, meaning that results largely concern a daily dose of 400 mg modafinil. More often than not both patients (67%) and their partners/housemates (77%) correctly guessed when they had been taking either modafinil or Placebo, usually on the basis of a ‘decreased sleepiness’ and/or ‘increased activity’.
The structured interview regarding activity and actions did not show significant differences between modafinil and placebo (p = 0.2 for patients and p = 0.5 for partners/housemates).
The RAND-36 questionnaires revealed a poor perception of general health for the whole group with a mean value of 29 points out of 100 (range 0–50) on the General Health rating.
The ratings were virtually identical for each patient over the four assessments (p = 1,Wilcoxon test). The perception of Role Limitations varied widely: mean 66 out of 100 (range 0–100). A medication related change was not observed (p = 0.7, Wilcoxon test). This also held for the perception of Social Functioning (p = 0.6), Vitality (p = 0.2) and Mental Health (p = 0.5). The ESS revealed a significant improvement with modafinil, in that the mean score decreased from 10.5 (range: 3–18) to 6.8 points (range: 1–15); for placebo the corresponding values were 10.5 (range: 3–18) and 10.7 (range: 2–17) points (p = 0.015, Wilcoxon test). There was no suggestion of a difference in outcome between patients with high and those with low scores. There was no significant relationship between the increase in activity/actions as perceived by the patient/ partner and indicated by the structured interview, and changes in perceived hypersomnolence as measured by the ESS (p = 0.38, Spearman’s test).
Discussion
The present study confirmed the beneficial effect of modafinil on excessive sleepiness in MD, but did not detect a concomitant effect on spontaneous activity as measured by a structured interview of the patients and their partners. This interview, not formally validated, was designed to reflect a clinically relevant and observable increase in daily activity by asking for specific actions. Examples might be that patients went to the theatre after a busy day, when they would otherwise have postponed such a visit, or cleaning up the shed. By asking for specific actions we hoped to distinguish actions from the mere feeling of being active or the intention to become so.
The study was small, leaving open the possibility that minor changes have been missed. The study was also focused on short-term effects and thus it is not able to detect changes of behaviour that take more time to become manifest, but we believe that a fortnight is long enough to detect relevant improvements in activity as defined above. A further consideration is the unblinding we have observed, which was most probably due to a correct perception of an effect on somnolence. This might have confounding effects on the interpretation of intended double-blind studies of modafinil on symptoms other than hypersomnolence. In the present study this does not seem to have happened, as the effect on hypersomnolence was neither related to perceived improved activity, nor to perceived aspects of general health. That many patients and partners reported more activity in addition to less sleepiness when asked why they thought that modafinil or placebo had been used, might be the result of the expectations implied in the aim of the study as discussed with the participants. The structured interview did not detect this increased activity, which we feel speaks in favour of its validity.
In a previous study of 11 patients modafinil improved excessive daytime sleepiness in MD, measured with the Multiple Sleep Latency Test and the Epworth Sleepiness Scale [2]. Possible effects on spontaneous activity were not considered. MacDonald et al. [3], in a double-blind cross-over placebo-controlled study of 40 patients with a time-scale identical to ours confirmed the reduction of somnolence as measured by Epworth and Stanford Sleepiness Scales. They also found modafinil-induced decreased fatigue-inertia, and increased vigor-activity, as measured by the Profile of Mood States. The latter findings might predict improved observed activity, but the study did not include this issue and our study did not demonstrate such effect. Using the RAND-36 they also observed enhanced measures of energy and perception of health, but no changes in the other items of the test; the former effects were not confirmed in our study. Talbot et al. [4] performed a similar double-blind cross-over study of 19 patients selected for hypersomnolence (ESS 10 or more points), using the ESS, a Modified Maintenance of Wakefulness test (MWT), a steering simulator, the Short Form 36 and an “activity diary” as measures. They found a reduction of sleepiness, especially in the MWT, less convincingly in the ESS. The other tests did not show significant changes. Data from the activity diaries are not given.
It is apparent that the symptoms referred to as inertia, reduced initiative, inactivity or apathy are hard to define and even harder to measure. Recently, van der Werf et al. inferred that the lack of correlation between fatigue scores and sleepiness in MD suggests that different pathophysiological mechanisms underlie these clinical manifestations [7]). We believe that our findings point in the same direction.

modafinil
myotonic dystrophy
hypersomnolence
levels of activity
rand-36


Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.
Introduction
Mycoplasma mycoides subsp. mycoides small colony type (SC) is an extra-cellular pathogen living in close association with host cells. It causes contagious bovine pleuropneumonia (CBPP), an acute, subacute or chronic infection of the respiratory system in cattle with a mortality rate of up to 30%, causing severe losses in livestock production, in particular on the African continent (Provost et al. 1987; Food and Agriculture Organization of the United Nations 2003).
Lipoproteins are usually strongly antigenic membrane proteins known to play a central role in interactions between bacteria and eukaryotic cells, particularly with respect to adhesion, and to stimulate the release of pro-inflammatory cytokines (Mühlradt and Frisch 1994; Herbelin et al. 1994; Brenner et al. 1997; Marie et al. 1999; Calcutt et al. 1999; Belloy et al. 2003). Lipoproteins have been put forward as possible virulence factors of pathogenic mycoplasmas (Dyson and Smith 1997; Vilei et al. 2000; Pilo et al. 2003).
Membrane lipoprotein LppQ is the predominant antigen of M. mycoides subsp. mycoides SC and shows the strongest signal on immunoblots containing total antigen from this pathogen reacted with serum from cattle that have suffered from CBPP. It induces a specific, early and persistent immune response in infected animals (Abdo et al. 2000). LppQ is encoded as a precursor (of 445 amino acids) with a consensus sequence for prokaryotic signal peptidase II and a lipid attachment site (Figure 1). The leader sequence of LppQ shows a typical transmembrane structure with a significant helix formation capacity (Abdo et al. 2000). LppQ was shown to be a membrane protein by Triton X-114 phase partitioning and lipidation was demonstrated by palmitic acid radiolabelling (Abdo et al. 2000). The C-terminal part of LppQ was found to possess repeated integral membrane structures rich in hydrophobic and aromatic amino acids, which have pore formation potential, and immunoblot analysis showed that the C-terminal domain possesses no particular immunogenicity, as serum derived from cattle naturally infected with M. mycoides subsp. mycoides SC did not react against it (Abdo et al. 2000). In contrast, the N-terminal domain of LppQ has three strongly hydrophilic domains and was shown to be the origin of the strong antigenic response against LppQ in naturally infected cattle. From these data, Abdo and collaborators deduced that the N-terminal part of LppQ is exposed at the outer surface of M. mycoides subsp. mycoides SC (Abdo et al. 2000).
Fig. 1Structure of lipoprotein LppQ. (Panel A) LppQ protein sequence. Repeats are indicated by dashed arrows. The symbol # corresponds to the signal peptidase II cleavage site, which allows for the obtainment of mature LppQ protein. (Panel B) The 10 C-terminal repeats rich in hydrophobic and aromatic residues, as identified with the MEME software, which build the transmembrane region of LppQ. Black background indicates conserved residues. The consensus sequence is shown. (Panel C) Model for protein topology of LppQ in the membrane lipid-bilayer of M. mycoides subsp. mycoides SC. Repeat numbers and amino acid positions for the beginning of mature LppQ (28), the beginning of C-terminal portion (193) and the protein end (445) are indicated. The N-terminal part is supposed to be anchored in the lipid-bilayer by the lipid anchor of the Cys28 residue. The three hydrophilic domains of the N-terminal part, which were found with significant scores for coiled-coil tertiary structure (Abdo et al. 2000), are depicted as globular items. The C-terminal part consists of a transmembrane region built up of the 10 repeated integral membrane structures, whose consensus sequence is shown at the bottom
Due to the strong antigenicity of the extracellular N-terminal part of LppQ and its specificity for M. mycoides subsp. mycoides SC, a recombinant peptide comprising amino acids 22-218 of the LppQ precursor protein was used to develop a specific and sensitive ELISA assay for the serological detection of CBPP in cattle (Bruderer et al. 2002). Furthermore, LppQ seems to be involved in the inflammatory processes of M. mycoides subsp. mycoides SC, as cattle immunized with the recombinant peptide showed an increased susceptibility to infection with M. mycoides subsp. mycoides SC and exhibited more severe symptoms of CBPP than unvaccinated animals (Nicholas et al. 2004).
As LppQ is apparently involved in the molecular mechanisms of pathogenicity of M. mycoides subsp. mycoides SC (Nicholas et al. 2004; Pilo et al. 2006), we have investigated the topology of LppQ in M. mycoides subsp. mycoides SC by means of high resolution field emission scanning electron microscopy (SEM), with particular respect to the predicted localization of LppQ on the mycoplasmal cell surface. This technique allows proteins to be localized on mycoplasmas unequivocally and with high accuracy and resolution (Pilo et al. 2005). Here, we provide evidence that the N-terminal part of LppQ is located on the surface of the mycoplasmal cell membrane and is accessible from the extracellular side while the C-terminal part of LppQ is not exposed.
Materials and methods
Bioinformatic analysis
Prediction of membrane topology was carried out by using the softwares TMpred (Hofmann and Stoffel 1993; http://www.ch.embnet.org/software/TMPRED_form.html), TopPred (von Heijne 1992; http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) and Phobius (Käll et al. 2004; http://phobius.cgb.ki.se/). Protein secondary structure was predicted with the MINNOU software available on the web page http://minnou.cchmc.org/ (Cao et al. 2006). Determination of the C-terminal repeats of lipoprotein LppQ was assessed by the MEME software version 3.5.4 at http://meme.sdsc.edu/meme/meme.html (Bailey and Elkan 1994).
M. mycoides subsp. mycoides SC strain and sample preparation
M. mycoides subsp. mycoides SC strain Afadé was used in the present study. This strain was isolated in 1968 at Farcha Laboratory, N’Djamena, Chad, from a cow from Afadé, Northern Cameroon, which suffered from acute CBPP. The strain Afadé is known to be highly virulent (Vilei and Frey 2001) and was used previously for experimental infection studies (Abdo et al. 1998). It was grown in standard mycoplasma medium (Axcell Biotechnologies, St. Genis l’Argentière, France) by inoculation of 20 μl of a frozen stock culture into 3 ml of medium and incubation for 3 days at 37°C. Growth and handling of live M. mycoides subsp. mycoides SC were performed in a biological safety laboratory fulfilling the BSL-3 containment safety standards.
Crude lysates were prepared by resuspending harvested mycoplasmas in TES buffer (10 mM Tris–HCl, 1 mM EDTA, 0.8% NaCl, pH 8.0) at a concentration of approximately 109 cells/ml (Abdo et al. 2000).
Production of antibodies against recombinant LppQ peptides
The polyhistidine-tagged recombinant peptides LppQ-N′ (amino acids 22 to 218) and LppQ-C′ (amino acids 214 to 422) from the N- and C-terminal portions of LppQ, respectively, were produced in Escherichia coli strain BL21(DE3) harbouring the respective plasmids as reported previously (Abdo et al. 2000; Bruderer et al. 2002). Recombinant peptides were polyhistidine-tagged at both their N- and C-termini. Purification of the peptides was performed by Ni2+ chelation chromatography and the purity of the peptides was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Polyclonal monospecific sera directed against LppQ-N′ and LppQ-C′ were obtained by subcutaneous immunization of rabbits with 160–200 μg of purified recombinant polyhistidine-tagged peptides in 500 μl of 50 mM PBS buffer (50 mM Na2HPO4/NaH2PO4, 140 mM NaCl, pH 8.0) mixed with 500 μl of Adjuvant 10 (Gerbu Biotechnik GmbH, Gaiberg, Germany). Booster immunizations with 40 and 20 μg of protein were performed 2- and 4-weeks later, respectively. The rabbits were bled 10 days after the last booster immunization and antisera were stored at −20°C.
Immunoglobulin G (IgG) fractions were purified from the two rabbit antisera with the HiTrap Protein G kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. Immunoglobulins from pre-immune sera were also purified. The purity of all IgGs was analyzed by SDS-PAGE. Protein concentrations were determined by using the method of Bradford (1976).
Immunoblot analysis
Whole cell antigens of strain Afadé were separated by 5–15% gradient SDS-PAGE (Ausubel et al. 1999) and blotted onto a nitrocellulose membrane with a pore size of 0.2 μm (Bio-Rad Laboratories AG, Reinach, Switzerland). The membrane was blocked with 1% skim milk powder in TBS buffer (100 mM Tris–HCl, 150 mM NaCl, pH 7.5) for 60 min at room temperature. Strips were incubated with corresponding IgGs at a concentration of 1–2 μg/ml (diluted 1:2000) overnight at 4°C and washed three times with TBS buffer for 5 min. Serum obtained from a cow that was infected experimentally with M. mycoides subsp. mycoides SC strain Afadé (Abdo et al. 1998; diluted 1:100) or previously prepared (Abdo et al. 2000) rabbit hyperimmune sera directed against LppQ-N′ and LppQ-C′ (diluted 1:1000) were used as controls. The strips were incubated for 90 min at room temperature with alkaline phosphatase-labelled goat anti-rabbit IgG (H + L) (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) diluted 1:2000 or monoclonal antibody anti-bovine IgG (Sigma, Buchs, Switzerland) diluted 1:5000 in skim milk buffer. The strips were washed three times with TBS buffer. The colour reaction was initiated with 0.3 mg/ml nitroblue tetrazolium (NBT) (Roche Diagnostics, Rotkreuz, Switzerland) and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche Diagnostics) in alkaline buffer (7 mM Na2CO3, 3 mM NaHCO3, 1 mM MgCl2, pH 9.6) and stopped with distilled water.
Preparation of M. mycoides subsp. mycoides SC for immunogold labelling
Coverslips that had been coated with gold or platinum and treated with poly-l-lysine (Sigma) to promote cell adhesion were immersed into 3 ml of fresh standard medium in multiwell plates. Thereafter, 100 μl of mycoplasma culture were added and the cultures were incubated for 5 to 6 days at 37°C until stationary phase. Coverslips with adhering mycoplasmas were washed three times with PBS buffer (2.9 mM NaH2PO4, 7.1 mM Na2HPO4, 137 mM NaCl, pH 7.4) at 37°C and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After fixation, samples were washed with PBS buffer and blocked with 1% BSA and 0.2 M glycine in PBS for 15 min at room temperature. Thereafter, mycoplasma cells were incubated with anti-LppQ-N’ or anti-LppQ-C’ IgGs diluted 1:100 (30 μg/ml) in PBS containing 1% BSA overnight at 4°C. After another washing step with PBS buffer for 5–10 min, mycoplasmas were labelled with 15 nm colloidal gold-conjugated goat anti-rabbit antibody (British Biocell International, Cardiff, UK) diluted 1:50 in PBS for 90 min at room temperature. Coverslips were then washed with 0.1 M cacodylate buffer (pH 7.4) and processed for SEM.
Scanning electron microscopy
Coverslips were incubated in 1.33% osmium tetraoxide with 0.11% ruthenium red in 0.13 M cacodylate buffer (pH 7.4) for 15 min, washed with 0.1 M cacodylate buffer, dehydrated through an ascending ethanol series and dried by evaporation of hexamethyldisilazane (Sigma) as described previously (Stoffel et al. 1993; Stoffel et al. 2002; Stoffel and Friess 2002). Finally, they were mounted on metal stubs with a conductive adhesive (Provac AG, Balzers, Liechtenstein).
Samples were examined without further metal coating. Secondary electron micrographs and corresponding backscattered images were obtained with a fully digital field emission scanning electron microscope DSM 982 Gemini (Zeiss, Oberkochen, Germany) at an accelerating voltage of 5 kV, a working distance of 6–8 mm and a magnification from 50,000 to 100,000×.
Control experiments included omission of primary antibody as well as the use of a rabbit anti-calcitonin antibody (Anawa, Zurich, Switzerland) and of IgGs from rabbit pre-immune sera as irrelevant substitutes for the anti-LppQ primary antibodies.
Results
Structure of the C-terminal domain of lipoprotein LppQ
Conventional predictors of membrane topology available online did not envisage the C-terminal domain of LppQ to be membrane-associated. However, determination of repeats in the LppQ protein sequence revealed that its C-terminal domain, represented by the last 250 amino acids, consists of a region built up of 10 repeated units rich in hydrophobic and aromatic residues (Figure 1). These repeat units of 25 amino acids were all found to adopt a particular secondary structure consisting of helices of approximately 10 amino acids flanked upstream and downstream by amino acids forming random coils (not shown).
Monospecificity of IgG preparations
On immunoblots containing total antigen of M. mycoides subsp. mycoides SC strain Afadé, purified IgG preparations directed against the N-terminal and against the C-terminal halves of LppQ each identified a single protein band with an apparent molecular mass of 48 kDa, which corresponds to the calculated size of 49.1 kDa of LppQ (Figure 2). Reference rabbit hyperimmune sera directed against LppQ-N′ and LppQ-C′ collected almost a decade ago gave identical results. Control experiments with IgGs purified from pre-immune sera did not detect any corresponding antigen (Figure 2).
Fig. 2Immunoblot analysis of total antigen of M. mycoides subsp. mycoides SC with anti-LppQ-N′ and anti-LppQ-C′. Approximately 10 μg of total antigen of strain Afadé per lane was separated by 5–15% gradient SDS-PAGE, transferred onto a nitrocellulose membrane and probed with the serum from a cow infected experimentally with strain Afadé (Abdo et al. 1998), or with IgGs from rabbit pre-immune sera or from antisera against LppQ-N′ and LppQ-C′, respectively. Reference rabbit hyperimmune sera directed against LppQ-N′ and LppQ-C′ obtained almost a decade ago (Abdo et al. 2000) were also used. The arrowhead indicates the position of LppQ on the immunoblots
Morphology of the mycoplasmas
Electron microscopy showed cultivated M. mycoides subsp. mycoides SC to be basically spherical in shape. They usually occurred as single cells but pairs or small groups of up to five organisms were also noted. Cell diameter varied between 200 and 500 nm and the cell surface was slightly grainy and irregular (Fig. 3A, C, E). Between fully grown microorganisms, small granular debris were observed.
Fig. 3Scanning electron micrographs of M. mycoides subsp. mycoides SC with anti-LppQ-N′ and anti-LppQ-C′ antibodies. Secondary electron microscope images (A, C, E) reveal the spherical shape and rough cell surface of the mycoplasmas. Corresponding backscattered electron micrographs (B, D, F) show indirect immunolabelling with 15 nm colloidal gold antibodies directed against the N-terminal extracellular domain of LppQ (B). In contrast, LppQ is not detected with anti-LppQ-C′ antibody (D). Labelling is abolished when an anti-calcitonin antibody is used as an irrelevant primary antibody (F)
Immunogold labelling
Labelling of mycoplasmas on coverslips with anti-LppQ-N’ antibodies yielded a SEM signal of moderate to high density at the cell surface (Fig. 3B). Specific labelling of granular debris was also present. In contrast, labelling with anti-LppQ-C′ antibodies was absent in most of the cases (Fig. 3D) and appeared to be insignificant and coincidental in few cases (Table 1), as also observed in the three control experiments whereby labelling was investigated in the absence of primary antibodies and when pre-immune serum or anti-calcitonin antibodies were used (Fig. 3F). Overall, there were significant differences between the number of particles labelled per cell by using anti-LppQ-N’ antibodies and those labelled in the other four experiments (P < 0.0001; Table 1)
Table 1Statistical analysisStatistical dataTreatmentAnti-LppQ-N’Anti-LppQ-C’Anti-calcitoninPre-immune serumNo primary antibodySingle cells investigated2748192023Labelled particlesTotal33317675Min20000Max314332Average per cella12.33 ± 7.580.35 ± 0.840.32 ± 0.820.35 ± 0.810.22 ± 0.52P-valueb<0.0001<0.0001<0.0001<0.0001a± Standard deviationbCalculated by the analysis of variance (anova) method: single factor. Differences between the number of particles labelled per cell by using anti-LppQ-N’ antibodies and those labelled in the other four experiments were analysed
Discussion
The goal of the present study was to discriminate the distribution of extracellular vs. integral membrane domains of lipoprotein LppQ in M. mycoides subsp. mycoides SC at the ultrastructural level. Amino acid sequence analysis for hydrophobicity by the method of Hopp and Woods (1981) revealed the N-terminal portion of LppQ to be particularly hydrophilic (Abdo et al. 2000). Thus, the lipidated N-terminal domain was predicted to be exposed at the extracellular surface of the plasma membrane (Abdo et al. 2000). On the other hand, the C-terminal portion consists of a region built up of 10 hydrophobic repeated units, and not nine as reported previously (Abdo et al. 2000). The repeat units were found to consist each of approximately 10 amino acids forming helices delimited on both sides with random coils, i.e. conformations without a regular secondary structure, and were not predicted to be membrane-associated by bioinformatic analysis. Since transmembrane helices are mostly composed of hydrophobic amino acid and the degree of hydrophobicity of the C-terminal domain of LppQ is clearly significant, the C-terminal portion was expected to be an integral membrane domain (Fig. 1). It has to be noted at this point that lipoproteins generally do not possess additional membrane-spanning domains (MSD) beyond their N-terminal lipid anchors. However, there are relevant examples from Gram-positive bacteria, which contradict this general statement about lipoproteins and substantiate instead the occurrence of such additional MSDs. These examples include lipoproteins CtaC and QoxA of Bacillus subtilis (Bengtsson et al. 1999; Antelmann et al. 2001), as well as the proven lipoprotein of Mycoplasma pneumoniae F0F1 ATPase (Pyrowolakis et al. 1998).
High resolution scanning electron microscopy in combination with immunogold labelling revealed extracellular epitopes with antibodies against LppQ-N′ but no signal above the background of the three negative controls was detected with the antibody against LppQ-C′. These results substantiate the predicted localization of the two domains, as they unambiguously corroborate the accessibility of the N-terminal domain at the extracellular side of the plasma membrane. The failure to immunolabel the C-terminal epitopes is an indication that they may be imbedded in the membrane. However, one cannot rule out the possibility that such epitopes (i) are buried in the centre of a globular protein, (ii) are masked by other surface components or (iii) are not recognized in their native forms by rabbit antibodies produced by inoculation of the recombinant peptide. Beyond providing ultrastructural evidence for the location of the hydrophilic and hydrophobic domains of LppQ, these results also demonstrate that immunogold labelling in scanning electron microscopy allows for subtle topographical discrimination and that selective visualization of peptides being exposed at the outer surface of microorganisms can be achieved with high accuracy and reliability.
The domain analysis of LppQ, which demonstrates the outstanding surface localization of the strongly hydrophilic N-terminal part of the lipoprotein, may explain the strong humoural response elicited by this lipoprotein during infection of cattle by M. mycoides subsp. mycoides SC (Abdo et al. 2000). It has to be demonstrated yet whether LppQ is also able to induce any cell-mediated immune response, as it is more often the case for cytoplasmic proteins. Absence of a cell-mediated immune response would explain the lack of protection in animals immunized with purified recombinant LppQ. In this respect, it should be noted that immunization of cattle with the purified recombinant N-terminal domain of LppQ elicited inflammatory processes of M. mycoides subsp. mycoides SC and exacerbated the clinical signs of CBPP after experimental infection compared to non-immunized animals (Nicholas et al. 2004).
Conclusions
Direct visualization of the N-terminal part of LppQ at the outer surface of M. mycoides subsp. mycoides SC, as demonstrated herein, provides evidence for the contention that LppQ is located to the extracellular side of the plasma membrane of mycoplasmas and that it is easily accessible by the humoural immune system of infected animals upon contact of host cells with M. mycoides subsp. mycoides SC.

contagious bovine pleuropneumonia (cbpp)


ISCEV standard for clinical pattern electroretinography—2007 update

The pattern electroretinogram (PERG) is a retinal response evoked by viewing a temporally alternating pattern, usually a black and white checkerboard or grating. The PERG is important in clinical and research applications because it provides information both about retinal ganglion cell function and, because the stimulus is customarily viewed with central fixation, the function of the macula. The PERG can therefore facilitate interpretation of an abnormal pattern VEP by revealing the retinal responses to a similar stimulus to that used for the VEP. However, practitioners may have difficulty choosing between the different techniques for recording the PERG that have been described in the literature. The International Society for Clinical Electrophysiology of Vision published a standard for clinical PERG recording in 2000 to assist practitioners in obtaining good quality reliable responses and to facilitate inter-laboratory communication and comparison. This document is the scheduled revision of that standard.
Introduction
The pattern electroretinogram (PERG) is a retinal biopotential evoked when a temporally modulated patterned stimulus (checkerboard or grating) of constant mean luminance is viewed. The PERG is most often evoked by alternating contrast-reversal of a black and white square checkerboard pattern with central fixation. It may be altered in dysfunction confined to the macula or to the retinal ganglion cells, which do not significantly affect the a- and b-waves of the conventional full-field ERG, and thus the PERG receives clinical and research attention in both neurological and ophthalmological practice. Clinically, the PERG can be used in a patient with an abnormal visual evoked potential to establish whether a retinal (macular) disorder is present, and thus differentiate between macular and optic nerve dysfunction as a cause for the VEP abnormality. It can also directly demonstrate retinal ganglion cell dysfunction.
However, the PERG is a very small signal, typically in the region of 2–8 μV across a normal population, and PERG recording is technically more demanding than standard flash ERGs. The amplitude values recorded will depend upon stimulus characteristics and the electrodes being used. The recordings published in the literature vary considerably in technique and technical quality; this document is intended to guide new or existing users to the most appropriate techniques for recording a standard clinical PERG.
The International Society for Clinical Electrophysiology of Vision (ISCEV) initially published “PERG Guidelines” [1], intended as a guide to practice and to assist in interpretation of PERGs. Those guidelines were subsequently revised to a PERG standard [2] and the present document is the scheduled revision of that standard. The standard PERG represents a minimum protocol for recording a PERG with straightforward technical procedures that should allow reproducible responses to be recorded under defined conditions. As a minimum, it is intended that the standard method and responses be widely used, but in addition to rather than to the exclusion of other paradigms. Common additional PERG techniques are also briefly described in this document, and individual laboratories are encouraged to use additional stimuli and protocols tailored to their own requirements.
The standard is based upon equipment and analytic capabilities currently available in most neurophysiological or ophthalmological electrodiagnostic clinics. This document addresses recording conditions and technology specific to the PERG, and presumes that the reader already has basic understanding and skills in clinical electrophysiology. Although much of the document will apply equally to adults and children, the standard is not necessarily appropriate to paediatric applications. The standard will be reviewed by ISCEV in a further four years.
Waveform nomenclature and measurement
The waveform of the PERG evoked by pattern-reversal stimuli depends on the temporal frequency of the stimulus. By convention, positivity is displayed upward.
Transient PERG
The ISCEV standard PERG is a transient response i.e. a response that is effectively complete before the next contrast reversal. Transient recording allows separation of the PERG components. At low temporal frequencies (<6 reversals per second (rev/s); equivalent to <3 Hz) transient PERGs are obtained (Fig. 1). The PERG waveform in normal subjects usually consists of a small initial negative component with a peak time of approximately 35 ms, N35, which is followed at 45–60 ms by a much larger positive component (P50). This positive component is followed by a larger negative component at 90–100 ms (N95).
Fig. 1A normal PERG. The amplitude of P50 in a normal subject is usually between 2.0 and 4.0 μV
For the transient PERG, amplitude measurements are made between peaks and troughs: the P50 amplitude is measured from the trough of N35 to the peak of P50. In some patients the N35 is poorly defined; in these cases N35 is replaced by the average baseline between time zero and the onset of P50. The N95 amplitude is measured from the peak of P50 to the trough of N95. It should be recognised that measured in this way, N95 amplitude includes the P50 amplitude and P50 that of N35. It can be argued that baseline to peak measurements may be more meaningful. However, the P50 and N95 components may not be independent. At the time of writing there are no peer-reviewed data attempting to resolve these issues, and given the wealth of data already published using peak-to-peak measures, they remain the standard measures for PERG amplitudes.
The times to peaks in the waveform (implicit time) should be measured from the onset of the contrast-reversal to the peak of the component concerned; it should be noted that the highest absolute amplitude point on a waveform will not always be appropriate for the definition of the peak if there is contamination from muscle activity or other artifacts. The peak should be designated where it would appear on a smoothed or idealised waveform (see Fig. 1). The peak times or implicit times are often (erroneously) referred to as latencies. Correctly, latency refers to the delay prior to the onset of a response, not to the maximum activity of a peak.
Basic technology
Standard equipment for visual stimulus generation, amplification of physiological signals, and the recording and storing of electrophysiological data, is required for PERG testing. Information about the calibration of equipment and measurement of the parameters specified in this standard appears in the ISCEV Calibration Guidelines [3].
Electrodes
Recording electrodes
Clinical ERG electrodes that contact the cornea or nearby bulbar conjunctiva should be used as the active electrodes to record standard PERGs. Electrodes that degrade image quality on the retina (this includes all contact lens electrodes) must not be used. Thin conductive fibres and foils can usually be positioned without topical anaesthesia. Electrode integrity should be checked prior to insertion, to meet guidelines for each electrode type. It is recommended not to measure impedance in situ unless explicitly specified by the particular equipment manufacturer. Electrodes should be carefully positioned to minimise instability (a major source of artifact or interference). Those who perform the test should be aware of possible causes of artifact.Fibre electrodes are best positioned in relation to the upper margin of the lower eyelid. Some place the electrode in the lower conjunctival fornix (under the lower eyelid); such a position may reduce trial-to-trial variability but will also result in a lower amplitude PERG. Optimum stability is achieved by tethering the electrode at the nasal canthus.Foil electrodes should be positioned directly under the centre of the pupil so that there is minimal or no movement of the electrode when the patient blinks. This is best achieved by having the foil curve over the lower eyelashes without contacting them, and then tethering the lead to the cheek. The junction of the electrode and lead should form as straight a line as possible and the junction should not touch the skin.Loop electrodes should be hooked into the lower fornix. Loops should be folded so that the contact windows on otherwise insulated wire are positioned on the bulbar conjunctiva, about 5 mm under the limbus. Loop electrodes should not touch the cornea. To achieve this, the limbs of the loop should diverge widely (15–20 mm) before entering the fornix. The lead is then taped to the cheek.
The appropriate techniques for individual electrode types are very important to achieve stable and reproducible PERG recordings. Additional sources should be consulted in relation to the specific electrode used.
Surface (skin) active electrodes should not routinely be used for recording the standard PERG; a surface electrode positioned on the lower eyelid will record PERGs of lower amplitude than those recorded from an electrode in contact with the eye. Surface electrode recordings may however be useful when a corneal electrode is contraindicated or in paediatric practice. The use of a surface electrode to record the PERG is a variation from the standard and should be noted in the report.
Reference electrodes
Separate surface reference electrodes should be placed on the skin near the ipsilateral outer canthus of each eye. Mastoid, earlobe or forehead locations may result in contamination of the PERG from cortical potentials or the fellow eye. If monocular PERG recording is performed, the electrode in the occluded eye may be used as a reference.
Ground electrodes
A separate surface electrode should be attached and connected to the amplifier “ground input”; the forehead would be a typical location, but other locations are acceptable as the location of the ground electrode should not affect the standard PERG.
Surface electrodes
The skin should be prepared with a suitable cleaning agent, and a suitable conductive paste used to ensure good electrical connection. The impedance between the skin electrodes used for reference and ground, measured on the subject, should be less than 5 kΩ. Since the electrode in the eye will have very low impedance, a low impedance of the reference electrode is also important to obtain recordings as free as possible from mains (line frequency) interference.
Electrode cleaning and sterilisation
Electrodes should be cleaned and sterilised according to local health and safety regulations. The ISCEV Standard for full-field flash ERGs describes the appropriate care of ERG electrodes [4].
Stimulus parameters
This standard specifies the protocol for basic clinical PERG recording. Laboratories may choose to test more conditions or parameters than are described herein.
Field and check size
A black and white reversing checkerboard should be used for the standard PERG. It is not necessary to use a square stimulus field, but the aspect ratio between the width and the height of the stimulus field should not exceed 4:3. The mean of the width and the height of the stimulus field should be 15° (±3°) with a check size of 0.8° (±0.16°).
Luminance
PERGs are difficult to record with low stimulus luminance, and a photopic luminance level for the white areas of greater than 80 cd · m−2 is required. The mean luminance of the stimulus screen must be constant during checkerboard reversals (i.e. no transient luminance change).
Contrast
The contrast between black and white squares should be maximal (close to 100%) for the standard PERG and not less than 80%. The contrast and luminance used should be reported.
Frame rate
Raster-based CRTs are typically used to present the pattern stimuli. The frame rate of the CRT is a significant stimulus parameter for PERGs, and a frequency of 75 Hz or greater should be used.
Background illumination
The luminance of the background beyond the checkerboard field is not critical when using the standard PERG technique providing dim or ordinary room lighting is used; ambient lighting should be the same for all recordings. Care should be taken to keep bright lights out of the subjects’ direct view. Pupil diameter should be recorded.
Reversal rate
The standard transient PERG should be obtained using a reversal rate of 4 rev/s ± 0.8 rev/s (i.e. 2 Hz ± 0.4 Hz).
Calibration
All stimulus parameters including luminance and contrast should be calibrated either locally or by the manufacturer and regular recalibration is advised [3].
Recording equipment
Amplification systems
AC-coupled amplifiers with a minimum input impedance of 10 MΩ should be used. Amplification systems must be electrically isolated from the patient according to the current safety standards for medical recording systems. The frequency response of bandpass amplifiers should include the range from 1–100 Hz; analogue notch filters (that suppress signals at the alternating current line frequency) should not be used. Some users may encounter severe electromagnetic interference from the stimulus display that makes it difficult to obtain satisfactory recordings with these filter settings. Ideally, such interference should be eliminated by shielding or modifying the equipment; rearranging the electrode leads may be of benefit.
Averaging and signal analysis
Signal averaging is necessary because of the small amplitude of the PERG. The analysis period (sweep time) for the standard PERGs should be 150 ms or greater. Some laboratories assess baseline stability by displaying two successive responses on the same trace.
Artefact rejection
Computerised artifact rejection is essential. The limits for rejection should be set at no higher than ± 50 μV, and preferably lower. The amplifiers must return to baseline rapidly following artifactual signals to avoid inadvertent storage of non-physiological data.
Sampling rate
A minimum sampling rate of 1,000 Hz (1 ms per point) is recommended. See the calibration standard (3) for further information.
Data display systems
Display systems must have adequate resolution to represent accurately the characteristics of this small amplitude signal. Ideally the recording system provides simultaneous display of the input signal and the accumulating average. In the absence of a simultaneous display, the system should allow a rapid alternation between displaying the input signal and displaying the current average so that the quality of the input signal can be adequately monitored. Even with a computerised artifact rejection system, it is important that the input signal be continuously monitored for baseline stability and the absence of amplifier blocking.
Clinical protocol
Preparation of the patient
Positioning
The patient should be as comfortable as possible with their head in a stable position against a head-rest. A chin rest is inappropriate and should not be used.
Pupils
The PERG should be recorded without dilatation of the pupils, to preserve accommodation and thus retinal image quality.
Fixation
A fixation mark in the centre of the screen at a node of the checkerboard is essential. If there are any doubts about the quality of fixation in an individual patient, an effective method is to give the patient a (laser) pointer and have them point at the middle of the screen throughout. Excessive blinking during recording should be discouraged, pauses may be advantageous.
Refraction
Because of the nature of the stimulus, PERG examination should be performed with optimal visual acuity at the testing distance. Patients should wear the appropriate optical correction for the test distance.
Monocular and binocular recording
Proper positioning of recording and reference electrodes will permit either monocular or binocular recording of the PERG. Binocular recording is recommended for the standard PERG because it is generally more stable, it reduces examination time and it allows fixation by the better eye in cases of asymmetric visual loss. Monocular stimulation is required to record the PERG and the VEP simultaneously.
Recording
A minimum of 100 artefact-free sweeps should be collected and averaged for a standard PERG. More sweeps, perhaps as many as 300, will be needed when the PERG is small or undetectable or in a “noisy” subject. At least two trials for each stimulus condition should be obtained to confirm reproducibility (i.e. at least one replication). It may be beneficial to superimpose repeated PERG recordings to evaluate quality and reproducibility.
PERG reporting
Reporting
It is recommended that all reports contain measurements of P50 and N95 amplitude (see above), and P50 peak time (the peak of N95 is often rather broad precluding accurate peak time measurement of this component). All reports should also contain the stimulus parameters (luminance, contrast, and field size), and normal ranges for the laboratory concerned. Pupil size should be noted. Whenever practical, reporting of PERG results should include representative waveforms with appropriate amplitude and time calibrations, marks for the N35, P50 and N95 components, and should show replications.
Normative data/reference ranges
At present there are no standard international reference ranges for PERG measurements. Each laboratory should establish normal values for its own equipment and patient population. It should be noted that there are PERG changes with age.
Additional tests
Large field PERGs
For some applications, such as glaucoma assessment, a larger field, such as 30°, may be more appropriate. Further, some practitioners find the improved signal-to-noise ratio obtained with a larger field may have advantages; users may wish to consider recording a large field PERG in addition to the standard response.
Steady-state PERG
At higher temporal frequencies, i.e. above 10 rev/s (5 Hz), the successive waveforms overlap and a “steady-state” PERG is evoked. There are situations in which the steady-state PERG is useful and some laboratories favour it for glaucoma studies. Since little extra time is required, laboratories may wish to consider recording it in addition to the transient response.
For steady-state PERG, a reversal rate of approximately 16 rev/s (8 Hz) is recommended. The steady-state PERG waveform is roughly sinusoidal, and interpretation of steady-state PERGs requires measurement of amplitude and phase shift (relative to the stimulus) of the second harmonic by Fourier analysis. The presence of a significant first harmonic indicates technical problems. For correct interpretation the analysis period must be an integer number of stimulus cycles, preferably greater than 6. Steady-state PERG recording without the capability for such analysis is not recommended.
Reporting of steady-state PERGs should include the amplitude and phase shift of the PERG at the reversal rate (i.e. at the second harmonic of the stimulus rate in Hz).

pattern electroretinogram
electroretinogram
macula
clinical electrophysiology
retina


A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17β-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, of 5α-dihydrotestosterone, methyltrienolone, and 17β-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action.
Introduction
There is concern that chemicals in our food, water, and environment affect human health by disrupting normal endocrine function, possibly leading to reproductive failure in humans and tumors in sensitive tissues [1, 2]. This relates to chemicals with previously unknown hormonal properties, like certain pesticides and plasticizers, but also to compounds used in pharmaceutical preparations, eg., oral contraceptives and tablets for hormone-replacement therapy, the endogenous steroids excreted in urine of man and domestic animals and potentially also compounds used for their growth-promoting properties in animals. Of all endocrine disruptors, environmental estrogens are the most studied [3]. However, recent studies show a crucial involvement of the androgen receptor in abnormal sex development. The presence of pollutants with adverse effects on human androgen receptor (hAR) has been reported from paper-mill effluents and as a result of intensive farming [4, 5]. Xenoandrogenic exposure-related disorders include testicular cancer, hypospadias, cryptorchidism, and poor sperm and very recently prepubertal gynecomastia was linked to both estrogenic and antiandrogenic effects of lavender and tea tree oil [6].
Chemical and immunological methods are commonly used to detect steroid hormones in food, clinical practice, environmental samples, or doping control. Owing to the great variety of chemicals with hormonelike activity, these methods have the drawback that they only quantify the compound of interest and are not able to determine biological activity of unknown compounds and their metabolites, this in contrast to biological assays. Receptor-based transcription activation assays can be used to detect all compounds having affinity for a given receptor [7, 8]. In contrast to receptor binding assays, receptor gene bioassays also include the transactivation step and can distinguish between receptor agonists and receptor antagonists [9]. This feature is very helpful in detecting both known and unknown compounds.
Several assays have been developed for this purpose, using both mammalian and yeast cells. In general, transcription activation assays based on mammalian, or more particular human, cell lines have been shown to be more sensitive than yeast-based assays, and may be able to identify compounds that require human metabolism for activation into their active state. Metabolic conversion can either activate or inactivate some compounds [10], whereas the relatively low metabolic capacity of yeast ensures that the test reflects the activity of the original compound. In addition, yeast-based assays have several other advantages. These include low costs, easy handling, lack of known endogenous receptors that may compete with the receptor activity under investigation (no crosstalk), and the use of media that are devoid of steroids [11–13]. Furthermore, yeast cell assays are extremely robust and survive extracts from dirty sample matrices such as sediments, urine, and feed [14–16]. Especially in the case of androgens, the lack of known endogenous receptors in yeast is a great advantage compared with mammalian cell lines, as androgen responsive elements (AREs) can also be activated by the progesterone receptor and the glucocorticoid receptor. To avoid potential crosstalk in mammalian cell lines, a lot of efforts was expended to construct an ARE that is specific and no longer inducible by the progesterone and glucocorticoid receptor [17–19]. However, up till now such an ARE does not exist and it is doubtful whether it will be found, as the consensus progesterone responsive element/glucocorticoid responsive element is equal to the consensus ARE. Moreover, the glucocorticoid receptor is normally expressed in all mammalian cell types. So far this has resulted in cell lines that are not specific for androgens and that also respond to gestagens or glucocorticoids [20–22].
This paper reports the development of a new yeast androgen bioassay by creating a stably transfected yeast strain that expresses yeast enhanced green fluorescent protein (yEGFP) as a measurable reporter protein in response to androgens. The lack of known endogenous receptors in yeast enabled us to use the strong nonspecific consensus ARE sequence, which is actually a common hormone responsive element that is recognized by the androgen, progesterone and glucocorticoid receptors and can therefore not be used in mammalian cell lines expressing more than one of these receptors. Exposures to 17β-testosterone, 17β-estradiol, progesterone, dexamethasone, and other compounds were performed in 96-well plates in order to demonstrate the suitability and specificity of this new yeast androgen bioassay. Additionally, flutamide and several brominated flame retardants were tested for their antagonistic mode of action and the results were compared with a yeast androgen bioassay expressing β-galactosidase as a reporter protein.
Materials and methods
Chemicals
Chemicals and methods to prepare the growth media, to perform PCR, to isolate DNA, and to transform bacteria and yeast were as described earlier [23]. Corticosterone, dexamethasone, 17α-estradiol, 17β-estradiol, estrone, flutamide, 4-hydroxytamoxifen, medroxyprogesterone 17-acetate, and progesterone were obtained from Sigma (St. Louis, MO, USA). The following compounds were obtained from Steraloids (Newport, RI, USA): 17β-boldenone, diethylstilbestrol, 5α-dihydrotestosterone, 17α-ethynylestradiol, 17β-testosterone and 17β-trenbolone. Tetrahydrogestrinone (THG) was a gift from M. Thevis (DSHS, Cologne, Germany). Copper sulfate and dimethyl sulfoxide (DMSO) were obtained from Merck (Darmstadt, Germany) and methyltrienolone was obtained from PerkinElmer (USA). All restriction endonucleases and corresponding buffers were obtained from New England Biolabs (Hitchin, UK) and the yeast β-galactosidase assay kit was from Pierce Biotechnology (Rockford, IL, USA). 2,4,6-Tribromophenol (TBP), BDE-39, and the hydroxyl derivative 4-OH-BDE-17 were synthesized at the Wallenberg Laboratory (Stockholm University, Sweden).
Yeast strains
The yeast Saccharomyces cerevisiae (CEN.PK 102-5B, K20, URA3−, HIS3−, LEU−) host strain was a gift from H. Silljé (University of Utrecht, The Netherlands). The yeast androgen bioassay with β-galactosidase as a marker was kindly provided by D.P. McDonnell (Duke University, USA).
Plasmids
The p403-GPD and p406-CYC1 yeast expression vectors were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). The pyEGFP3 plasmid was a gift from A.J. Brown (Stanford University, USA).
Construction of the p403-GPD-hAR expression vector
The yeast cells provided by McDonnell were grown overnight and chromosomal DNA was isolated. This DNA was used to serve as a template for the PCR to obtain the complementary DNA (cDNA) of hAR. Full-length hAR cDNA was obtained using the Expand High Fidelity PCR system (Boehringer Mannheim) and an Eppendorf Mastercycler gradient. The sequence of the 5′-primer was 5′-GCTCTAGAATGGAAGTGCAGTTAGGGCTGGG-3′, containing a restriction site for XbaI just before the ATG start codon. The sequence of the 3′-primer was 5′-GCGGATCCTCACTGGGTGTGGAAATAGATGGG-3′, containing a restriction site for BamHI just after the TGA stop codon. This PCR generated a full-length double-stranded (ds) cDNA of 2,763 bp of the hAR gene with a 5′-XbaI and a 3′-BamHI restriction site.
The 2,763-bp full-length hAR PCR product was isolated from a 1% low-melt agarose gel, cleaved with XbaI and BamHI and ligated into the corresponding site of the p403-GPD yeast vector. Plasmid digestion and PCR controls revealed several good clones.
Construction of the p406-ARE2-CYC1-yEGFP reporter vector
A set of complementary oligonucleotides (a and b), each with two consensus ARE sequences (in bold), were synthesized. A solution with both cDNA oligonucleotides, 2.5 μM of each, was heated at 95 °C and cooled down to room temperature in 2 h. This set gave a ds DNA with a 5′-SacI sticky end and a 3′-MscI blunt end.Sa: 5′-AAAGTCAGAACAGCATGTTCTGATCAAATCTAGAAGATCCAAAGTCAGAACAGCATGTTCTGATCAAACTCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGG-3′Sb: 5′-CCATCCACGCTATATATACACGCCTGGCGGATCTGCTCGAGTTTGATCAGAACATGCTGTTCTGACTTTGGATCTTCTAGATTTGATCAGAACATGCTGTTCTGACTTTAGCT-3′.
This ds DNA was cloned into the corresponding site of the p406-CYC1 vector. Subsequently, yEGFP [24] obtained from a HindIII/SalI double digestion of pyEGFP was cloned in the corresponding HindIII/SalI sites of the p406-ARE2-CYC1 reporter construct. Plasmid digestion and PCR controls revealed several good clones.
Transformation of yeast cells
Transformation of yeast K20 host strain (Ura−, His−, and Leu−) was performed by the lithium acetate protocol as described earlier [23]. First, the yeast was transformed with the p406-ARE2-CYC1-yEGFP reporter vector, integrated at the chromosomal location of the uracil gene via homologous recombination. Therefore, prior to transformation, the reporter vector was linearized by cutting with StuI, which has a unique restriction site in the URA3 marker gene. Transformants were grown on minimal medium plates containing l-leucine and l-histidine (MM/L plates). This yeast reporter strain was then transformed with the p403-GPD-hAR expression vector, which was linearized by cleavage with NsiI, which has a unique restriction site in the HIS3 marker gene (histidine). Transformants were grown on MM/L plates and PCR controls were used to select clones that contain the p406-ARE2-CYC1-yEGFP reporter and the p403-GPD-hAR expression construct.
PCR controls
PCR controls were performed on the reporter-receptor transformants. Yeast chromosomal DNA of transformants was isolated and PCR controls were performed. PCR I was performed with a 5′-primer on the backbone of the reporter plasmid and a 3′-primer on the ARE2 sequence. The sequence of the 5′-primer was 5′-AGCGAGTCAGTGAGCGAGGAAG-3′ and the sequence of the 3′-primer was 5′-TGCTGTTCTGACTTTGGATC-3′. PCR II was performed with a 5′-primer on the CYC1 (cytochrome c oxidase) promoter of the reporter plasmid and a 3′-primer on the CYC1 terminator. The sequence of the 5′-primer was 5′-TCTATAGACACACAAACACAA-3′ and the sequence of the 3′-primer was 5′-GGGAGGGCGTGAATGTAAG-3′. PCR III was performed with the primers that were also used to obtain the full length cDNA of the hAR (see “Construction of the p403-GPD-hAR expression vector”).
Streamlined yEGFP assay with the yeast androgen bioassay
The day before running the assay, a single colony from a MM/L agar plate was used to inoculate 10 mL of the selective MM/L medium. This culture was grown overnight at 30 °C with vigorous orbital shaking. At the late log phase, the yeast androgen receptor biosensor was diluted in the selective MM/L medium to an optical density (OD) at 604 nm between 0.08 and 0.12. For exposure, aliquots of 200 μL of this diluted yeast culture were pipetted into each well of a 96-well plate and 2 μL of a 17β-testosterone or other stock solution in DMSO was added. DMSO-only controls were included in each experiment and each sample concentration was assayed in triplicate. Exposure was performed for 24 h at 30 °C and orbital shaking at 125 rpm. Fluorescence was measured at 0 and 24 h directly in a CytoFluor multiwell plate reader (Series 4000, PerSeptive Biosystems) using excitation at 485 nm and measuring emission at 530 nm. The fluorescence signal was corrected with the signals obtained with MM/L containing DMSO solvent only. Densities of the yeast culture were determined by measuring the OD at 630 nm, but this was only done to check whether a sample was toxic for the yeast cells. For the calculation of the relative androgenic potency (RAP) of the compounds in the yeast androgen bioassay, the data of a complete dose–response curve were fitted using the equation  (Slide write Plus, version 6.00). This is equivalent to , where response is the measured fluorescence signal, [agonist] is the concentration of the test compound, and EC50 is the concentration of the test compound giving half-maximum response.
lacZ-based yeast androgen bioassay: β-galactosidase assay
An agar plate containing the selective growth medium, consisting of a yeast nitrogen base with dextrose (2%), lysine (36 mg/L), tryptophan (48 mg/L), uracil (24 mg/L), and adenine (41 mg/L), was inoculated with the yeast androgen receptor cytosensor from a frozen -80 °C stock (20% glycerol v/v). The plate was incubated at 30 °C for 24–48 h and then stored at 4 °C. The day before running the assay, a single colony of the yeast was used to inoculate 10 mL of the selective growth medium. This culture was grown overnight at 30 °C with vigorous orbital shaking at 225 rpm. At the late log phase, the yeast androgen receptor cytosensor was diluted in growth medium to an OD of 0.06 at 604 nm, and CuSO4 (0.05 mM) was added to induce the expression of the hAR. For exposure in 96-well plates, aliquots of 200 μL of this diluted yeast culture were pipetted into each well and 2 μL of stock solutions in DMSO was added. Exposure was performed for 24 h at 30 °C and 125 rpm, and the β-galactosidase activity was measured with a commercial yeast β-galactosidase assay kit from Pierce (Rockford, IL, USA). This kit uses o-nitrophenyl β-d-galactopyranoside as a substrate, and the solution turns yellow upon hydrolysis of β-d-galactopyranoside to o-nitrophenol and galactose. The yellow o-nitrophenol is measured in a Biotek (Winooski, VT, USA) model ELx 808 series ultra microplate reader at 405 nm. Densities of the yeast culture were determined by measuring the OD at 630 nm. The measured response at 405 nm was corrected for the OD at 630 nm.
Results and discussion
A recombinant yeast cell was constructed that expresses the hAR and yEGFP as a reporter protein in response to androgens. Both the receptor construct as well as the reporter construct were stably integrated into the yeast genome by the use of yeast-integrating plasmids. For the construction of the reporter vector the p406-CYC1 plasmid, containing the URA3 marker gene, was used. Two consensus AREs were placed in the SacI/MscI site of the truncated CYC1 promoter in a way that the −254 to −147 XhoI-SphI part of the CYC1 promoter was restored [23]. High expression levels of the androgen receptor were obtained by placing the cDNA of the hAR gene behind the strong constitutive yeast glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter in the p403-GPD plasmid. This plasmid contains the HIS3 marker gene. Transfected strains were checked with PCR. The correct and specific functioning of the yeast androgen bioassay was studied by exposures to 17β-testosterone and other compounds and the results were compared with results obtained with the lacZ-based yeast androgen bioassay provided by McDonnell. In addition, the antiandrogenic properties of several brominated flame retardants were investigated.
PCR controls
A number of different PCR controls were carried out to check the integration of the vectors into the yeast genome. Figure 1 shows the gel electrophoresis results of these PCR controls. PCR I (Fig. 1a) was performed with primers on the backbone of the p406 plasmid and on the ARE2 sequence. As expected, it gave the specific 360-bp band with the p406-ARE2-CYC1-yEGFP reporter vector and the DNA that was isolated from the yeast androgen biosensor. The negative controls, performed with the empty p406-CYC1 plasmid and with the DNA that was isolated from the empty yeast host (the nontransfected yeast cell), showed no PCR bands. PCR II (Fig. 1a) was performed with primers on the CYC1 promoter and the CYC1 terminator. As expected, it gave the specific 873-bp band with the reporter vector and the DNA that was isolated from the biosensor, because both contain the reporter construct with the yEGFP that was ligated between the CYC1 promoter and CYC1 terminator. The negative controls, performed with the empty p406-CYC1 plasmid and with the DNA that was isolated from the empty yeast host, did not show the reporter-specific 873-bp band. However, this PCR generated a 435-bp band with the DNA of the empty yeast host and the biosensor. This 435-bp band corresponds to the CYC gene of the yeast host itself and is therefore also a specific band. PCR III (Fig. 1b) was performed with the primers on the hAR gene. As expected, it gave the specific 2,763-bp band with the p403-GPD-hAR expression vector and the DNA that was isolated from the biosensor. In the negative control, performed with the DNA that was isolated from the yeast host, the receptor-specific 2,763-bp band was not present. These PCR controls demonstrate that all specific PCR bands can be seen, thus demonstrating that our yeast androgen bioassay contains the p403-GPD-hAR expression vector and the p406-ARE2-CYC1-yEGFP reporter vector, both stably integrated in the yeast genome.
Fig. 1PCR controls. The PCR controls were performed as described in “PCR controls.” a Lanes 1, 6 and 11 contain a 100-bp ladder. PCR I was performed with primers on the backbone of the p406 plasmid and on the ARE2 sequence. Lanes 2–5 are PCR I on the p406-ARE2-CYC1-yEGFP reporter vector, the DNA that was isolated from the yeast transformant, the empty p406-CYC1 plasmid, and the DNA that was isolated from the empty yeast host (the nontransfected yeast cells), respectively. PCR II was performed with primers on the CYC1 promoter and the CYC1 terminator. Lanes 7–11 are PCR II on the reporter vector, the DNA from the yeast transformant, the empty p406-CYC1 plasmid, and the DNA from the empty yeast host respectively. b Lane 1 contains a 1-kb ladder. PCR III was performed with the primers on the human androgen receptor gene. Lanes 2–4 are PCR III on the p403-GPD-hAR expression vector, the DNA from the yeast transformant, and the DNA from the empty yeast host, respectively. yEGFP yeast enhanced green fluorescent protein
Dose–response curves obtained with the new yeast androgen bioassay
The dose–response curves for several natural and synthetic androgens are shown in Fig. 2. 5α-Dihydrotestosterone, 17β-testosterone and 17β-boldenone caused a dose-related increase in the production of yEGFP, demonstrating that these compounds are potent androgens. The bioassay showed a limit of detection of 3 nM for 5α-dihydrotestosterone with a dynamic range from 3 to 500 nM and very low standard deviations (les than 3%). The figure also shows that 17β-estradiol and progesterone give a response. The female hormone 17β-estradiol gives a full dose–response curve, but the maximum of the curve is reached at a 500 times higher concentration than that of 17β-testosterone and is less steep. Progesterone gives a response, but the maximum response is only about 35% of that of 17β-testosterone and is reached at a 25 times higher concentration. Both 17β-estradiol and progesterone are known to possess androgenic properties. Progesterone displays low binding to the androgen receptor [25] and shows androgenic effects in vivo [26]. According to [27], 17β-estradiol and progesterone showed androgenic activity in ten out of 11 and seven out of nine mammalian cell reporter gene (MCRG) systems, respectively. The corticosteroids corticosterone and dexamethasone showed no response in our assay.
Fig. 2Response of the yeast androgen biosensor to different substances. Exposure to 17β-testosterone, 5α-dihydrotestosterone, progesterone, dexamethasone, 17β-estradiol, and 17β-boldenone was started by adding to 200 μL of a yeast culture a 2-μL aliquot of a stock solution of the compound in dimethyl sulfoxide (DMSO). Fluorescence was determined after 24 h as described in “Streamlined yEGFP assay with the yeast androgen bioassay.” Fluorescence signals are the mean of a triplicate with the standard deviation (SD). 17β-T 17β-testosterone, DHT 5α-dihydrotestosterone, Prog progesterone, Dex dexamethasone, 17β-E2 17β-estradiol, Bold 17β-boldenone
Table 1 shows the calculated EC50, ie., the concentration giving a half-maximum response, and the RAP, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, for several compounds. The yeast androgen bioassay showed good sensitivity towards all androgens tested, with the following range of potencies: 5α-dihydrotestosterone > 17β-trenbolone > methyltrienolone > tetrahydrogestrinone > 17β-testosterone > 17β-boldenone > medroxyprogesterone acetate > 17β-estradiol > progesterone. Steroids representative for other hormone receptors, like estrone, 17α-estradiol, 17α-ethynylestradiol, and diethylstilbestrol for the estrogen receptor and corticosterone and dexamethasone for the glucocorticoid receptor, showed no agonistic response. Only 17β-estradiol, progesterone and medroxyprogesterone acetate gave a clear agonistic response. However, these compounds are known to exert androgenic effects.
Table 1EC50 concentrations and relative androgenic potencies (RAP) of compounds in the yeast androgen biosensor expressing yeast enhanced green fluorescent protein in response to androgensCompoundQualitative response for AR agonismaCommentsbEC50 (nM) in the yeast androgen bioassaycRAPd17β-Testosterone Positive (11/11)Strong AR agonist761.05α-DihydrotestosteronePositive (21/21)Strong AR agonist, weak ER agonist332.317β-Boldenone5100.1517β-TrenbolonePositiveBinds strongly to AR521.5MethyltrienolonePositive (8/8)AR agonist541.4TetrahydrogestrinoneAR agonist651.217β-EstradiolPositive (10/11)AR agonist and antagonist, strong ER agonist9,0000.0084EstronePositive (2/2)AR agonist, strong ER agonistNRNR17α-EstradiolNegative (1/1)ER agonistNRNR17α-EthynylestradiolNegative (1/1)Strong ER agonistNRNRDiethylstilbestrolNegative (2/2)Strong ER agonistNRNR4-HydroxytamoxifeneNegative (1/1)ER antagonistNRNRProgesteronefPositive (7/9)1,7000.045Medroxyprogesterone acetate Positive (4/4)Weak AR agonist1,5000.051CorticosteroneNegative (1/1)Binds weakly to ARNRNRDexamethasonePositive (3/4)AR agonistNRNRFlutamideNegative (5/5)AR antagonistNRNR2,4,6-Tribromophenol NRNRBDE-19NRNR4-OH-BDE-17NRNRAR androgen receptor, ER estrogen receptor, NR no responseaQualitative response for AR agonism across all mammalian cell reporter gene studies (data obtained from [27])bComments obtained from [27]cThe EC50 is the concentration giving half-maximum response.dThe RAP is defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound.eThis compound was toxic to yeast above 30 μM.fThese compounds reach a maximum response that is lower than 70% of the maximum response obtained with 17β-testosterone. The maxima obtained with 4-androstenedione and progesterone are about 40 and 35%, respectively.
Compared with the NIH publication [27], there are a few discrepancies. According to [27], estrone is an androgen receptor agonist that showed androgenic activity in two out of two MCRG systems. However, some mammalian cells are able to convert estrone into 17β-estradiol and vice versa. This conversion is ascribed to 17β-hydroxysteroid dehydrogenase 3 and this enzyme is responsible for the high relative estrogenic potency (REP) of estrone in the estrogen bioassay with the T47-D breast cancer cells (ER-CALUX test). In that test, the estrogenic potency of estrone was equal to that of 17β-estradiol and a REP of 1.0 was reported for estrone [10]. This probably explains why estrone gave a positive result for androgenic activity in two MCRG systems, but gave a negative result in our yeast androgen bioassay. Yeast is obviously not able to convert estrone into 17β-estradiol. The reported REP of 0.2 in our yeast estrogen biosensor corresponds nicely with the in vivo potency of this compound [28]. Dexamethasone was also described as an androgen receptor agonist in the NIH publication, showing androgenic activity in three out of four mammalian assays, but gave a negative result in our yeast androgen bioassay. However, these MCRG systems use an MMTV-Luc reporter construct or an ARE-Luc reporter construct. Both the MMTV and the ARE sequence are recognized by the glucocorticoid receptor and this means that the response found in these MCRG systems is probably due to crosstalk, as the glucocorticoid receptor is normally expressed in all cell types.
Dose–response curves obtained with the lacZ-based yeast androgen bioassay
Figure 3 shows the dose–response curves for several natural and synthetic androgens obtained with the lacZ-based yeast androgen bioassay. The assay was simplified in our laboratory by scaling it down to a 96-well format and the use of a β-galactosidase assay kit. With increasing β-galactosidase activity, the density of the yeast culture, measured at 630 nm, dropped to about 50% (data not shown). Therefore, the measured â-galactosidase activity was corrected for the OD of the yeast culture. Table 2 shows the calculated EC50 and the corresponding RAP values. The data demonstrate that there is a good correlation between the EC50 values for androgens determined in our laboratory and those reported by Gaido et al. [29] in 1997. It seems that the lacZ-based yeast androgen bioassay, in terms of EC50 values, is 5–10 times more sensitive than our new bioassay. However, there was almost no difference in the limit of detection. Although the curves in the β-galactosidase assay go up at around 0.3 nM and in the yEGFP assay at around 1 nM, the limit of detection is about 3 nM for 5α-dihydrotestosterone in both assay types. This is mainly because the standard deviations are much higher in the β-galactosidase assay. However, the curves in the β-galactosidase assay are much steeper and are thus responsible for the lower EC50 values. The steeper curves are probably due to using an enzyme as a marker, β-galactosidase compared with a yEGFP marker protein, and the expression of the RSP5 cofactor that enhances transcription activation in the lacZ-based yeast androgen bioassay. The dynamic range for 5α-dihydrotestosterone in the McDonnell assay was from 3 to 100 nM and is slightly smaller than the range of the new bioassay (3–500 nM). There were also no great differences in the RAP values determined, although methyltrienolone was more potent in the lacZ-based bioassay. However, the new yEGFP bioassay is less sensitive for 17β-estradiol and progesterone: RAPs of 0.008 and 0.045, respectively, and for the latter no full dose–response curve, compared with RAPs of 0.1 and 0.068 and full dose–response curves in the lacZ-based bioassay. This means that our new bioassay is more specific for detecting compounds with a pure androgenic mode of action. The main difference between the lacZ-based and our yEGFP biosassay is that protein RSP5, which is a counterpart of the mammalian RPF1, is overexpressed in the lacZ-based bioassay in order to enhance transcriptional efficacy. However, this cannot explain the observed difference with methyltrienolone , as according to Imhof and McDonnell [30], this did not alter the potency or specificity of the assay. In addition and in contrast to the lacZ-based yeast androgen bioassay there were no differences in the density of the yeast culture measured at 630 nm upon exposure to different compounds that induced yEGFP expression, which indicates a decreased growth of yeast cells exposed to androgens in the lacZ-based yeast androgen bioassay. The only other known yeast androgen bioassay, one that uses luciferase as a reporter protein, displays similar characteristics in terms of specificity and was 5–10 times more sensitive in terms of EC50 values [15]. However, this assay needs the correction of the same yeast strain that only and stably expressed luciferase as an external control to correct for and normalize the aspecific responses caused by variation in cell vitality due to matrix and analyte toxicity. Unorrected dose–response curves for 17β-testosterone displayed more than 10 times higher EC50 values. An earlier assay described by Lee et al. [31] in 2003 uses β-galactosidase, but only expresses the hinge-ligand binding domain of the androgen receptor.
Fig. 3Response of the McDonnell yeast androgen bioassay to different substances. Exposure to 17β-testosterone, 5α-dihydrotestosterone, progesterone, dexamethasone, 17β-estradiol, and 17β-boldenone was started by adding to 200 μL of a yeast culture a 2-μL aliquot of a stock solution of the compound in DMSO. The β-galactosidase activity was determined after 24 h and corrected for the optical density at 630 nm as described in “lacZ-based yeast androgen bioassay: β-galactosidase assay.” Signals are the mean of a triplicate with the SDTable 2EC50 concentrations and RAPs of compounds in the yeast androgen bioassay expressing β-galactosidase in response to androgensCompoundEC50 (nM)a by Gaido et al. [29]EC50 (nM)b in our laboratoryRAP17β-Testosterone4.711.51.05α-Dihydrotestosterone3.54.92.317β-BoldenoneND700.217β-TrenboloneND130.9MethyltrienoloneND3.73.1TetrahydrogestrinoneND11.51.017β-Estradiol 86.1950.117α-EstradiolNDNRNRProgesterone89.31700.068CorticosteroneNDNRNRDexamethasoneNDNRNRND not determinedaValues obtained by Gaido et al. [29], using the McDonnell yeast androgen bioassaybValues determined in our laboratory, using the McDonnell yeast androgen bioassay (see “Dose–response curves obtained with the lacZ-based yeast androgen bioassay”)
Antiandrogenic activity
The specificity of the new yeast androgen bioassay was further demonstrated by the ability of antiandrogens to suppress the induction of yEGFP. Figure 4 shows the antiandrogenic activity of the known antagonist flutamide and three brominated flame retardants, BDE-39, TBP, and 4-OH-BDE-17. The antagonistic properties were examined by coexposure with a concentration of 5α-dihydrotestosterone that induced a submaximal response (50 nM). None of these four compounds were able to show an agonistic response (Table 1), but Fig. 4 clearly shows that all three were able to inhibit the response induced by 5α-dihydrotestosterone . The IC50 value was about 1 μM for flutamide, and TBP and 4-OH-BDE-17 were about as potent, while BDE-39 was clearly less antiandrogenic. Similar results were obtained with a human cell line. Only TBP was less potent in that test, but cytotoxicity of TBP could not be excluded [32].
Fig. 4Inhibition of a submaximal response obtained by 5α-dihydrotestosterone with flutamide and three brominated flame retardants (BFRs). Coexposure to a concentration of 5α-dihydrotestosterone that induced a submaximal response was started by adding to 200 μL of a yeast culture, 1 μL of a 5α-dihydrotestosterone and 1 μL of the BFR stock solution in DMSO. Fluorescence was determined after 24 h as described in “Streamlined yEGFP assay with the yeast androgen bioassay.” Fluorescence signals are the mean of a triplicate with the SD. TBP 2,4,6-tribromophenol
Conclusions
A recombinant yeast cell was constructed that expresses the hAR and yEGFP as a reporter protein in response to androgens. Compared with other yeast androgen bioassays, this new biosassay showed a similar limit of detection and dynamic range. However, the measurement of the fluorescence (yEGFP) can be followed as a function of incubation time and is easier, quicker, and cheaper than the measurement of the β-galactosidase or the luciferase activity, which needs cell wall disruption and/or the addition of expensive substrates. Owing to the ease of the yEGFP measurement, standard deviations are generally less than 3%. Moreover, the assay seems to be more robust and more specific for detecting compounds with a pure androgenic mode of action.
Brominated flame retardants with suspected antiandrogenic properties were able to inhibit the response obtained with 5α-dihydrotestosterone, the most potent endogenous androgen, demonstrating that this yeast androgen bioassay is suited to detect compounds with both agonistic and antagonistic characteristics.
As all compounds tested were able to show either their agonistic or their antagonistic properties, neither the cell wall nor the cell membrane seemed to be an obstacle. As for the yeast estrogen bioassay, we validated this new yeast androgen bioassay according to international criteria, eg., the determination of the decision limit (CCα) and the detection capability (CCβ). The decision limit is then used to distinguish negative and suspect samples [16] (unpublished results). The assay was proven to be useful to detect the new designer steroid tetrahydrogestrinone in human urine [33] while prohormones with an androgenic mode of action, e.g., dehydroepiandrosterone, are not active in yeast-based bioassays and need metabolic activation before they can be detected [34]. Future work will include the validation of the assay for urine and feed and the screening of prohormones with and without metabolic activation. To mimic the in vivo metabolic activation, liver slices, liver cell lines, liver S9 enzymes, and pure enzymes will be used.

antagonists
receptor
metabolism
crosstalk
brominated flame retardants
saccharomyces cerevisiae